Synthesis and pharmacokinetic profile of a quaternary ammonium derivative of chlorambucil, a potential anticancer drug for the chemotherapy of chondrosarcoma

Article abstract of DOI:10.1016/j.bmc.2003.09.006

As a part of our targeting program based on the affinity of the quaternary ammonium moiety for cartilage, our objective was to develop more selective anticancer drugs towards chondrosarcoma that would concentrate in this malignant cartilaginous tissue and so improve the therapeutic index through a reduction of side effects. For this purpose we have synthesized and labeled with ¹⁴C a quaternary ammonium (QA) derivative of chlorambucil. Biological studies performed in rats showed that [¹⁴C]-CQA and [ ¹⁴C]-chlorambucil exhibited different pharmacokinetic profiles. The blood elimination of [¹⁴C]-CQA was faster than that of parent compound. [¹⁴C]-CQA was principally excreted by the fecal way. However, until 15 min after administration, levels of radioactivity were measured in cartilaginous tissues of rats given [¹⁴C]-CQA which was not the case for the rats which had received [¹⁴C]-chlorambucil. Although rates of radioactivity were quantified only during 15 min, these results prove that the functionalization of chlorambucil by a quaternary ammonium group allows the molecule to be carried selectively to cartilaginous tissues.

Full text of DOI:10.1016/j.bmc.2003.09.006

Bioorganic & Medicinal Chemistry 11 (2003) 5007–5012
Synthesis and Pharmacokinetic Profile of a Quaternary
Ammonium Derivative of Chlorambucil, a Potential Anticancer
Drug for the Chemotherapy of Chondrosarcoma
Maryse Rapp,* Isabelle Giraud, Jean-Claude Maurizis and Jean-Claude Madelmont
INSERM UMR 484, Rue Montalembert, BP 184, 63005 Clermont-Ferrand Cedex, France
Received 16 May 2003; accepted 1 September 2003
Abstract—As a part of our targeting program based on the affinity of the quaternary ammonium moiety for cartilage, our objective
was to develop more selective anticancer drugs towards chondrosarcoma that would concentrate in this malignant cartilaginous tissue
and so improve the therapeutic index through a reduction of side effects. For this purpose we have synthesized and labeled with 14C a
quaternary ammonium (QA) derivative of chlorambucil. Biological studies performed in rats showed that [14C]-CQA and [14C]-
chlorambucil exhibited different pharmacokinetic profiles. The blood elimination of [14C]-CQA was faster than that of parent com-
pound. [14C]-CQA was principally excreted by the fecal way. However, until 15 min after administration, levels of radioactivity
were measured in cartilaginous tissues of rats given [14C]-CQA which was not the case for the rats which had received [14C]-chlor-
ambucil. Although rates of radioactivity were quantified only during 15 min, these results prove that the functionalization of
chlorambucil by a quaternary ammonium group allows the molecule to be carried selectively to cartilaginous tissues.
# 2003Elsevier Ltd. All rights reserved.
Introduction
the selectivity of the drugs for the cartilaginous tumors
and consequently to increase the therapeutic benefit.
Chondrosarcoma is a malignant cartilaginous tumor of
bad prognosis, representing the second most common
bone tumor after osteosarcoma.1À3 The only effective
therapy of this neoplasm remains adequate surgery.
Radiotherapy does not appear to affect the course of the
disease, as well as chemotherapy, which is furthermore
highly toxic.3À6
To initiate this project, we have decided to conjugate the
QA moiety with chlorambucil (1) and melphalan (2)
(Chart 1).14,15 We were interested in the selection of
these two drugs because their chemical structure lent
itself well to a binding with our QA carrier through the
carboxylic acid function, and also to a convenient
[14C]-radiolabeling thanks to the chloroethyl alkylating
moieties: in fact, radiolabeled analogues were required
to perform the in vivo biodistribution studies aimed at
the checking of the cartilage targeting. Thus, we have
developed a program based on the design of QA deri-
vatives of chlorambucil and melphalan, CQA (3) and
MQA (4) respectively (Chart 1), and on the evaluation
of their pharmacological activity as well as their biodis-
tribution.14,15 We have shown that not only the QA
functionalization does not alter the cytotoxic potency of
the QA derivatives, which is even improved in the
chondrosarcoma cell line, but also allows the derivative
of melphalan to be carried selectively to cartilaginous
tissues.14,15
An alternative way to potentially improve the chemo-
therapy of chondrosarcoma may consist in the develop-
ment of anticancer agents possessing an enhanced
affinity for the cartilaginous tumors. Precisely, the tar-
geting of cartilage is a research program investigated by
our laboratory,7À15 since we have discovered that the
introduction of a quaternary ammonium (QA) entity in
the structure of compounds could be a means to increase
their affinity for cartilaginous tissues.16À18 In the context
of the improvement of the chemotherapy of chon-
drosarcoma, our strategy consists of the binding of anti-
cancer agents to the QA carrier in order to strengthen
In this paper, we report the syntheses explored for the
preparation of [14C]-labeled CQA and the pharmaco-
*Corresponding author. Tel.: +33-4-7315-0812; fax: +33-4-7315-
0801; e-mail: rapp@inserm484.u-clermont1.fr
0968-0896/$ - see front matter # 2003Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.09.006

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M. Rapp et al. / Bioorg. Med. Chem. 11 (2003) 5007–5012
Chart 1.
kinetic study in rats of [14C]-CQA in comparison with
[14C]-chlorambucil.
For the preparation of [14C]-CQA from [14C]-chlor-
ambucil (1), the synthetic sequence was based on the
syntheses developed for cold compounds.14 Thus,
[14C]-chlorambucil (1) was conjugated with 3-(dimethyl-
amino)propylamine after its preliminary conversion to
acid chloride by treatment with thionyl chloride to
afford the corresponding butyramide derivative (8) in
85% yield after chromatographic purification. The last
step consisted of quaternarization of the free tertiary
amine of 8 by methyl iodide in ethanol to provide the
target quaternary ammonium derivative (3) in 91%
yield. Finally, [14C]-CQA (3) was isolated with a specific
activity of 329 MBq/mmol (8.9 mCi/mmol) and a
radiochemical purity determined to be better than 99%
by thin layer chromatography analysis. The overall
radiochemical yield of the labelling sequence was 32%
based on [1,2-14C]ethylene oxide.
Chemistry
The radiolabeling was introduced in the chloroethyl
groups by treatment of the appropriate arylamine with
[1,2-14C]ethylene oxide. This radiolabeling position was
chosen because it offered the possibility to follow the
becoming of the biological active entities of the com-
pound and to compare the biological results between
[14C]-CQA and [14C]-chlorambucil. With the aim to
optimize the radiochemical preparation of [14C]-CQA,
two reversed reaction paths, differing in the order of
introduction of the nitrogen mustard group and of the
spacer carrying the QA moiety, were explored with
unlabelled materials (route A and route B—Scheme 1).
As these two chemical sequences were found to provide
similar overall yield, route A was finally chosen for the
[14C]-radiosynthesis of CQA. Route A is one radio-
labeled step shorter for the preparation of
[14C]-chlorambucil and [14C]-CQA as compared to route
B (Table 1).
Results and Discussion
Results of the in vivo biodistribution experiments with
[14C]-CQA and the parent molecule chlorambucil in rats
are shown in Table 2. For both compounds radio-
activity was distributed in many tissues especially in the
metabolizing and eliminating organs, liver and kidney.
Radioactivity rates in blood of rats given [14C]-chlor-
ambucil were higher than those given [14C]-CQA. From
15 min after injection, no measurable radioactivity level
was found in blood of rats receiving [14C]-CQA which
indicates a rapid blood clearance. The high radioactivity
levels measured in the kidney and liver proves an
important and fast biliary elimination. Until 15 min
after administration, significant levels of radioactivity
were detected in cartilaginous tissues of rats given
[14C]-CQA when no measurable radioactivity was found
in rats which had received [14C]-chlorambucil. Figure 1
shows an example of a whole-body autoradiograph
performed 5 min after administration of [14C]-CQA to
rat. This image shows a radioactivity level in articular
and intercostal cartilages, and in the intervertebral
disks, higher than one of surrounding tissues, bone and
muscle, that confirms the affinity of CQA for cartilagi-
nous tissues. As in previous studies done in our laboratory
According to route A, the two first steps consisted of the
preparation of methyl 4-(4-aminophenyl)butyrate (6)
from commercially available 4-(4-nitrophenyl)butyric
acid (5) and were carried out with a quantitative yield as
usually described.19,20 Then, the route used for the pre-
paration of [14C]-chlorambucil (1) from compound 6
relied on an adaptation of the syntheses reported in the
literature for the use of radioactivity.19,20 First, com-
pound 6 was reacted with [1,2-14C]ethylene oxide in 1 N
acetic acid to provide, after a purification on silica gel,
the corresponding 4-[bis(2-hydroxy-[1,2-14C]ethyl)]ami-
nophenyl derivative (7) in 46% radiochemical yield. The
treatment of compound 7 with phosphorus oxychloride
in toluene and subsequently with 1 N hydrochloric acid
gave [14C]-chlorambucil (1) in 90% yield. According to
these conditions, [14C]-chlorambucil was isolated with a
specific activity of 329 MBq/mmol (8.9 mCi/mmol) and
a radiochemical purity of 97.3% as determined by thin
layer chromatography analysis.

M. Rapp et al. / Bioorg. Med. Chem. 11 (2003) 5007–5012
5009
Scheme 1.
Table 1.
[
14C]-Radiolabeling of CQA
[
14C]-Radiolabeling of chlorambucil
Total of radiolabeled steps
Route A
Route B
Four steps from compound 6
Three steps from compound 10
Achieved in the preparation of [14C]-CQA
Two additional steps from compound 6
4
5
on other QA derivatives,7,8,10,12,15 these results also show
that the QA derivative of chlorambucil exhibits an affinity
for cartilaginous tissues. However, [14C]-CQA is more
quickly eliminated from cartilage than the QA derivative
of the melphalan, for which rates of radioactivity having
been detected in the cartilaginous tissues up to 1 h after
injection.15 Table 3 presents the radioactivity excreted in
urine, feces and expired air after injection to rats of
[14C]-chlorambucil and [14C]-CQA. Few radioactivity
was found in expired air for the two compounds. The

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M. Rapp et al. / Bioorg. Med. Chem. 11 (2003) 5007–5012
Table 2. Comparative biodistribution of [14C]-chlorambucil (CHLO) and [14C]-CQA (CQA) in male Sprague–Dawley rats following intravenous
injectiona
Time
Compd
Tissue
Liver
Kidney
Cartilage
Blood
Spleen
Lung
b
2 min
5 min
10 min
15 min
30 min
1 h
CHLO
CQA
98.15Æ8.42
238.14Æ51.22
101.13Æ7.92
21.42Æ2.89
13.37Æ1.49
84.24Æ4.68
31.56Æ2.12
131.80Æ14.29
374.22
22.90Æ5.86
41.06
b
CHLO
CQA
68.75Æ7.50
95.55Æ24.32
67.10Æ12.07
12.86Æ2.51
53.95Æ10.66
Æ5.73
111.58Æ18.51
284.61Æ116.12
14.32Æ3.54
12.59
10.08Æ0.9312.61
b
CHLO
CQA
57.95Æ6.34
83.43Æ6.56
102.17
133.28Æ19.7310.40
48.16Æ1.31
11.28Æ3.55
31.61Æ7.41
Æ1.73
Æ1.86
6.28
9.81Æ1.7310.26
b
CHLO
CQA
62.51Æ2.35
41.04Æ2.65
65.95Æ6.58
53.62Æ1.91
46.22Æ2.36
9.69Æ0.34
30.42Æ3.18
8.93Æ0.70
b
7.04Æ0.62
15.58Æ1.50
b
CHLO
CQA
51.20Æ7.22
73.33Æ7.22
Æ5.90
40.91Æ6.48
8.56Æ1.4329.74
10.48Æ1.038.45
Æ6.20
Æ0.71
b
b
27.14Æ2.8348.42
b
b
CHLO
CQA
54.43Æ1.9385.17
19.9Æ1.79
Æ15.48
26.76Æ0.99
5.30
16.70Æ2.52
27.01Æ1.96
b
46.33Æ4.89
7.95
b
b
2 h
CHLO
CQA
28.67Æ2.02
11.46Æ1.59
58.24Æ7.51
34.53Æ6.75
16.81Æ.409.635.1313
Æ1.58
b
7.09Æ1.26
8.38Æ1.29
b
b
6 h
CHLO
CQA
16.84Æ1.35
8.78Æ1.71
41.56Æ12.16
24.22Æ3.97
10.18Æ0.79
4.41
4.97Æ0.94
8.03Æ0.81
4.78Æ1.17
b
b
b
b
b
b
24 h
CHLO
CQA
5.08Æ0.30
14.07Æ1.37
b
5.29Æ0.76
20.18Æ3.74
4.27Æ0.79
3.27Æ0.83
aResults are expressed as nmol drug equivalent per gram of tissue and correspond to the meanÆstandard deviation for the quantification of six
whole-body sections of one animal for each point.
bNon detectable.
amounts of radioactivity measured in lung can be
explained by the strong vascularization of this organ.
Whereas 56% of the radioactivity was excreted in the
urine of rats given [14C]-chlorambucil in the first 24 h,
only 26% of the injected dose was recovered in the urine
for [14C]-CQA. Though mainly urinary, the excretion of
the QA conjugate of melphalan was only 55% at 72 h.15
Our previous works showed that QA compounds were
mainly eliminated by urinary route (more than 85%) as
unchanged molecule, the introduction of the polar qua-
ternary ammonium function leading to a decrease of the
lipophilicity.10,12 The difference of urinary excretion of
the QA conjugates of chlorambucil and melphalan
Figure 1. An example of whole-body autoradiography of serial long-
itudinal section of rat 5 min after injection of [14C]-CQA.
could be related to their partition coefficient, which is
strongly negative for MQA (log P=À1.12Æ0.14) which
explains why MQA is a more hydrophilic compound
than CQA.14 Moreover, higher percentage of radio-
activity was found in the feces of rats given [14C]-CQA
than in those of rats given [14C]-chlorambucil. These
results let us assume different metabolic pathways for
[14C]-CQA, [14C]-MQA and their parent compounds. It
is possible that [14C]-CQA was more metabolized than
[14C]-MQA and especially more quickly, explaining a
weaker concentration in blood and cartilaginous tissues
than for the QA conjugate of melphalan.
Table 3. Cumulative radioactivity excreted in urine feces and expired
air after intravenous administration of [14C]-chlorambucil (CHLO)
and [14C]-CQA (CQA) in male Sprague–Dawley Ratsa
Time
(h)
Urine
Feces
14CO2
Total
excretion
CHLO
CQA
0–24
0–48
0–72
56.94Æ2.26
58.41Æ2.11
59.90Æ1.95
21.86Æ1.70
26.15Æ1.02
26.57Æ1.04
2.37
2.59
2.74
89.21
0–24
0–48
0–72
25.75Æ4.96
27.47Æ5.37
28.33Æ5.39
41.68Æ6.38
42.53Æ6.25
43.34Æ5.69
1.25
1.49
73.16
Conclusion
aResults are expressed as the percentage of the injected dose and cor-
respond to the meanÆstandard deviation for five animals for each
point, except for 14CO2 (one rat studied).
The binding of the QA carrier to chlorambucil was part
of our cartilage targeting program based of the known

M. Rapp et al. / Bioorg. Med. Chem. 11 (2003) 5007–5012
5011
affinity of the quaternary ammonium moiety for carti-
lage. We already used this property to specifically carry
different active principles towards cartilaginous tis-
sues.8,10,14,15 Previous works carried out with the mel-
phalan series showed the affinity of the QA conjugate of
melphalan towards cartilaginous tissue. Cartilage tar-
geting by [14C]-CQA was obtained as expected but
unfortunately weaker than that of MQA. We also
demonstrated in cell culture that the cytotoxic activity
of chlorambucil was not affected by the QA binding,
and even enhanced on chondrosarcoma line.14 Studies
were undertaken concerning the evaluation of the in
vivo antitumor activity of the QA conjugate of chlor-
ambucil and chiefly with that of melphalan on animals
bearing chondrosarcoma. The results will be reported
elsewhere.
329 MBq/mmol (8.9 mCi/mmol). The radiochemical
purity was 97.4% as determined by TLC analysis (Rf
0.30, TLC silica, ethyl acetate). Compound 7 was found
by NMR and TLC to be identical to the unlabeled
reference compound.20
4-{4-[Bis(2-chloro-[1,2-14C]ethyl)amino]phenyl}butyric
acid ([14C]-chlorambucil, 1). To a stirred solution of 7
(710 mg, 2.52 mmol) in toluene (5 mL) at 0 ꢀC, was
added, under an inert atmosphere, phosphorus oxy-
chloride (2 mL). The reaction mixture was refluxed for 2
h and then evaporated under reduced pressure. The
residue was treated with 37% hydrochloric acid (5 mL)
and heated to reflux for 2 h. The mixture was then
diluted and extracted with diethyl ether. The organic
fractions were combined, dried over magnesium sulfate,
filtered and evaporated under reduced pressure.
[14C]-Chlorambucil (1) was isolated as a white solid in
90% yield (691 mg, 830 MBq (22.4 mCi)) and with a
specific activity of 329 MBq/mmol (8.9 mCi/mmol). The
radiochemical purity was 97.3% as determined by TLC
analysis (Rf 0.45, TLC silica, cyclohexane/ethyl acetate
2/1; Rf 0.80, TLC silica, dichloromethane/ethanol 9/1).
Compound 1 was found by NMR and TLC to be iden-
tical to the unlabeled reference compound.20
Experimental
General comments
4-(4-Nitrophenyl)butyric acid (5) was purchased from
Aldrich and [1,2-14C]ethylene oxide from NEN. All sol-
vents and reagents obtained from commercial sources
were used without further purification. Nuclear mag-
netic resonance spectra (1H NMR and 13C NMR) were
performed on a Bruker AM 200 (4.5 T) spectrometer.
Analytical thin layer chromatography (TLC) was con-
ducted on precoated silica gel plates (SDS, 60 F254, 0.25
mm thick) or on precoated aluminium oxide plates
(Merck, 60 F254, neutral type E, 0.20 mm thick) with
both detection by ultraviolet light and visualisation with
iodine. Silica gel 60 (Chromagel, 35–60 mm, SDS) was
used for medium pressure chromatography using the
indicated solvent mixture expressed as volume/volume
ratios. Radiochemical purity was determined by scan-
ning the TLC plates with an Ambis 4000 detector. Spe-
cific activity of compounds was measured in a Wallac
Winspectral 1414 liquid scintillation counter.
N - [3 - (Dimethylamino)propyl] - 4 - {4 - [bis(2 - chloro -
[1,2-14C]ethyl)amino]phenyl}-butyramide (8). To a solu-
tion of [14C]-chlorambucil (1) (350 mg, 1.15 mmol) in
dichloromethane (5 mL), at 0 ꢀC, was added, under an
inert atmosphere, thionyl chloride (0.88 mL). The mix-
ture was stirred at 4 ꢀC for 16 h and then the excess of
thionyl chloride was evaporated under reduced pres-
sure. To the resulting oil dissolved in dichloromethane
(5 mL) was added, under an inert atmosphere, a solu-
tion of 3-(dimethylamino)propylamine (0.16 mL, 1.27
mmol) in the same solvent (5 mL). This mixture was
stirred for 1 h at room temperature, then treated with a
second amount of diamine (0.16 mL, 1.27 mmol) and
stirred for another 4 h at room temperature. After
washing with 1 N sodium hydrogen carbonate and then
with water, the organic layer was dried over magnesium
sulfate, filtered and evaporated under reduced pressure.
The residue was purified by column chromatography on
silica gel (eluent: gradient of ethanol in dichloromethane
from 0 to 50% and then dichloromethane/ethanol/
ammonium hydroxide 50/49/1). Compound 8 was
obtained in 85% yield [381 mg, 322 MBq (8.7 mCi)]
with a specific activity of 329 MBq/mmol (8.9 mCi/
mmol). The radiochemical purity was 99.5% as deter-
mined by TLC analysis (Rf 0.70, TLC silica, dichloro-
methane/ethanol/ammonium hydroxide 50/47/3; Rf
0.77, TLC aluminium oxide, dichloromethane/ethanol
9/1). Compound 8 was found by NMR and TLC to be
identical to the unlabeled reference compound.14
Methyl 4-{4-[bis(2-hydroxy-[1,2 -14C]ethyl)amino]phe-
nyl}butyrate (7). The reaction was performed in a
vacuum manifold by the transfer of [1,2-14C]ethylene
oxide (1850 MBq (50.0 mCi), specific activity: 503MBq/
mmol (13.6 mCi/mmol)) on a solution of methyl 4-(4-
aminophenyl)butyrate (6)19,20 (775 mg, 4.01 mmol) in
1 N acetic acid (7 mL) cooled into liquid nitrogen. After
completing the transfer, the reaction mixture was pro-
gressively warmed to 5 ꢀC, stirred at this temperature
for 1 h and then for another 16 h at room temperature.
After this time, the solution was cooled again to 5 ꢀC,
treated with cold ethylene oxide (1.8 mL, 36 mmol),
stirred for 5 h at room temperature and then evaporated
under reduced pressure. The resulting oil was dissolved
in diethyl ether and this organic phase was washed with
1 N sodium carbonate and with water, dried over mag-
nesium sulfate and evaporated under reduced pressure.
The residue was purified by column chromatography on
silica gel (eluent: gradient of ethyl acetate in cyclohex-
ane from 0 to 20%). Compound 7 was isolated as a
clear oil with a radiochemical yield of 46% [733 mg,
858 MBq (23.2 mCi)] and with a specific activity of
{3-[(4-{4-[Bis(2-chloro-[1,2-14C]ethyl)amino]phenyl}buta-
noyl)amino]propyl}trimethyl-ammonium iodide ([14C]-
CQA, 3). To a solution of 8 (260 mg, 0.67 mmol) in
ethanol (7 mL), was added, under an inert atmosphere,
methyl iodide (63 mL, 1.01 mmol). The mixture was
stirred at room temperature for 3h and then evaporated
under reduced pressure. The resulting oil was dissolved

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M. Rapp et al. / Bioorg. Med. Chem. 11 (2003) 5007–5012
in the minimum amount of methanol and poured into
diethyl ether (150 mL). The resulting precipitate was
then filtered, washed with diethyl ether and dried to
afford 3as a white solid [323mg, 201 MBq (5.4 mCi),
91%] with a specific activity of 329 MBq/mmol (8.9
mCi/mmol). The radiochemical purity was 99.5% as
determined by TLC analysis (Rf 0.05, TLC silica, di-
chloromethane/ethanol/ammonium hydroxide 50/47/3;
Rf 0.30, TLC aluminium oxide, dichloromethane/etha-
nol 9/1). Compound 3 was found by NMR and TLC to
be identical to the unlabeled reference compound.14
Gold cocktail in the Wallac Winspectral 1414 liquid
scintillation spectrometer. Radioactivity of dried feces
was measured after combustion in a Packard 306 Oxi-
dizer (Packard Instrument SA, Rungis, France).
Acknowledgements
This work was supported by Adir, Servier (France).
References and Notes
Tissue distribution study
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Springfield, D. S.; Rosenberg, A. E.; Jennings, L. C. J. Bone
Joint Surgery Am. 1999, 81, 326.
4. Healey, J. H.; Lane, J. M. Clin. Orthop 1986, 204, 119.
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6. Springfield, D. S.; Gebhardt, M. C.; McGuire, M. H. Intr.
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Rapp, M.; Ollier, M.; Renard, P.; Caignard, D.-H. French
Patent, 9,908,020, 1999; PCT/FR 00/01731, 2000.
8. Nicolas, C.; Verny, M.; Giraud, I.; Ollier, M.; Rapp, M.;
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9. Giraud, I.; Rapp, M.; Dupuy, J.-M.; Maurizis, J.-C.;
Madelmont, J.-C. J. Labelled Compd. Rad. 2000, 43, 297.
10. Giraud, I.; Rapp, M.; Maurizis, J.-C.; Madelmont, J.-C.
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Tissue distribution analyses were performed in 5-week-old
male Sprague–Dawley rats weighing 100–120 g (Iffa-
Credo, L’Arbresle, France) after intravenous administra-
tion of each [14C]-labeled test compound [22.5 mmol/kg,
890 kBq (24 mCi) in 200 mL of physiological saline for
CQA or 0.1 M N-methyl-d-glucamin (Meglumin) solu-
tion for chlorambucil]. Animals were euthanasied by
carbon dioxide inhalation 2, 5, 10, 15 and 30 min and 1,
2, 6 and 24 h after administration and rapidly frozen by
immersion in liquid nitrogen. They were then embedded
in a 2% carboxymethyl cellulose gel. The resulting car-
boxyl methylcellulose block was sagittally sectioned at
À22 ꢀC with a Reichert-Jung Cryopolycut cryomicro-
tome (Heidelberg, Germany). When section surfaces of
interest appeared, the corresponding 40-mm-thick slices
were taken using no. 810 Scotch band tape (3M, Saint
Paul, MN, USA) and dried for 48 h at À22 ꢀC. These
selected slices (n=6 for each time studied) were subse-
quently analyzed with the Ambis 4000 detector
(B. Braun Sciencetec), which allows visualization and
quantification of the radioactivity distribution in whole-
body sections. The quantification leads to a surface
activity (cpm/mm2) which, because of a calibration, is
directly converted into nCi per gram of tissue.21 The
final results are expressed as nanomoles drug equivalent
per gram of tissue. To obtain more defined images for
identification of zones of interest, tissue slices were also
applied to hyperfilm bmax (Amersham).22
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Excretion study
The excretion study was conducted in 5-week-old male
Sprague–Dawley rats weighing 100–120 g given each
[14C]-labeled test compound as described above and
housed in metabolic cages (Iffa-Credo, l’Arbresle,
France) enabling separate collection of feces and urine.
Urine and feces were collected 24, 48, 72 h after admin-
istration. For the collection of expired [14C] CO2, ani-
mals (n=1 for each test compound) were housed in an
apparatus designed by us to trap [14C]CO2 with a flask
containing ethanolamine/methanol (20:80 v/v). Radio-
activity of urine and ethanolamine/methanol samples
was directly measured after addition of Packard Ultima