In vivo targeting of tumor-associated carbonic anhydrases using acetazolamide derivatives

Article abstract of DOI:10.1016/j.bmcl.2009.06.022

We describe the synthesis and characterization of two acetazolamide derivatives containing either a charged fluorophore or an albumin-binding moiety, which restrict binding to carbonic anhydrase IX and XII present on tumor cells. In vivo studies showed th

Full text of DOI:10.1016/j.bmcl.2009.06.022

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4851–4856
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
In vivo targeting of tumor-associated carbonic anhydrases using
acetazolamide derivatives
Julia K. J. Ahlskog a, , Christoph E. Dumelin b, , Sabrina Trüssel a, Jessica Mårlind a, Dario Neri a,
*
a Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Wolfgang-Pauli-Str.10, CH-8093 Zurich, Switzerland
b Philochem AG, c/o ETH Zurich, Wolfgang-Pauli-Str.10, CH-8093 Zurich, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe the synthesis and characterization of two acetazolamide derivatives containing either a
charged fluorophore or an albumin-binding moiety, which restrict binding to carbonic anhydrase IX
and XII present on tumor cells. In vivo studies showed the preferentially targeting of tumor cells by
the fluorescent acetazolamide derivative and the ability of the albumin-binding acetazolamide derivative
to cause tumor retardation in a SK-RC-52 xenograft model of cancer.
Received 8 April 2009
Revised 4 June 2009
Accepted 5 June 2009
Available online 13 June 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Carbonic anhydrase
CA IX
CA XII
Acetazolamide
Tumor targeting
Virtually all tumors require new blood vessels, which provide
nutrients and oxygen to the cancer cells, thus sustaining growth
and offering an avenue for metastatic spread.1 However, not all
cells within the tumor mass receive a sufficient oxygen supply
and survival advantage to tumor cells exposed to the hypoxic
and acidic microenvironment.11
The overexpression of CA IX in breast cancer has been found to
be associated with reduced survival.12 Furthermore, renal cell car-
cinoma (RCC) frequently carry a loss-of-function mutation in the
VHL gene, the product of which is responsible for targeting the oxy-
and cancer cells at a distance of 100–200 lm from the nearest
blood vessel become hypoxic.2 This pattern is particularly pro-
nounced not only in experimental rodent models of cancer, but
also in aggressive human solid tumors such as glioblastoma multi-
forme, where hypoxic cells form a ‘palisade’, delineating a border
between cancer cells in proliferation around pseudoglomerular
blood vessels and necrotic areas.3
gen sensor HIF-1a for proteasomal degradation, thus leading to in-
creased CA IX levels on all tumor cells, due to HIF-1
a
stabilization.13 Indeed, CA IX has been used in the past as antigen
for the generation of tumor targeting monoclonal antibodies14–18
and the chimeric antibody cG250 is currently being studied by
Wilex in Phase III clinical trials for the therapy of RCC.
Carbonic anhydrase IX (CA IX), a membrane-anchored enzyme,
is one of the most prominent proteins, which become over-ex-
pressed in hypoxic conditions.4,5 In addition oxygen availability
also regulates the catalytical activity of CA IX to reversible converse
CO2 to carbonic acid, thereby leading to an acidification of the
extracellular tumor milieu under hypoxic conditions.6,7 Addition-
ally, it has been proposed that transport of bicarbonate into the
Cells may express a number of different carbonic anhydrases.
Carbonic anhydrase I, II, III, VA, VB, VII and XIII are expressed intra-
cellularly, while carbonic anhydrase IV, IX, XII, XIV and XV are
membrane-bound.19 In tumors, not only CA IX but also CA XII is
strongly over-expressed. Indeed, recent RNAi experiments indicate
that only a simultaneous blockade of CA IX and CA XII leads to tu-
mor growth retardation, as a result of impaired acidification of the
tumor environment.11 Furthermore expression as well as activa-
tion of CA IX is required for the successful inhibition of the catalytic
activity which only occurs under hypoxic conditions.20
Based on these considerations, it is not surprising that the
development of carbonic anhydrase inhibitors has been a matter
of intense medicinal chemical research, in an attempt to design
selective inhibitors for the different CA isoforms.19 As CA IX and
CAXII are the most relevant carbonic anhydrases for the develop-
ment of anti-cancer strategies, the use of bioreductive prodrugs21
cytoplasm of tumor cells through a Clꢀ/HCO3 exchanger leads to
ꢀ
the increase of the intracellular pH of cancer cells,6 resulting in
the generation of alternatively spliced components of the extracel-
lular matrix,8,9 which are now frequently used as targets for anti-
body-mediated pharmacodelivery strategies.10 Furthermore, by
regulating the intracellular pH, CA IX and CA XII confer a growth
* Corresponding author. Tel.: +41 44 6337401; fax: +41 44 6331358.
E-mail address: neri@pharma.ethz.ch (D. Neri).
These authors contributed equally to the article.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.022

4852
J. K. J. Ahlskog et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4851–4856
or of membrane-impermeable glycoconjugates22 and CA inhibi-
tors23 has been proposed. Checchi et al. demonstrated in vitro that
the conjugation of sulfonamides to charged moieties displayed a
decreased membrane permeability and selective inhibition of CA
IX, thereby leading to a reversion of the acidification under hypoxic
conditions.23 To our knowledge, none of these therapeutic ap-
proaches has been tested in vivo so far.
Dennis et al. have recently described a general strategy for the
reduction of blood clearance of therapeutic proteins (such as tu-
mor-specific antibody fragments) by means of a C-terminal albu-
min-binding peptide.24 Interestingly, the longer circulatory half-
life of anti-HER2-neu antibodies in stable non-covalent association
with mouse serum albumin (MSA) led to a homogenous decoration
of tumor cells in vivo, which could not be obtained with the same
antibody (Trastuzumab) in Fab or IgG format, that were confined to
perivascular tumor cells.25 In principle, the same strategy could be
used for the modification of small organic enzyme inhibitors, with
the additional advantage of preventing internalization, thus
restricting their scope to membrane-bound and secreted enzyme.
Recently, our lab has described derivatives of 2-amino-6-(4-(4-
iodophenyl)butanamido)hexanoic acid as a general class of stable
and portable albumin-binding moieties, which can be conjugated
to a variety of different drugs and which prolong serum half-lives
in vivo.26
azolamide as targeting moiety for pharmacodelivery applications
is attractive since most tumors express CA IX and CA XII either at
sites of hypoxia or on pVHL-defective tumor cells. Small organic li-
gands may be advantageous for tumor targeting applications, com-
pared to monoclonal antibodies, in view of their rapid
extravasation and homogenous tissue distribution properties.27
A charged fluorophore moiety or the stable non-covalent bind-
ing to serum albumin prevents the internalization of the deriva-
tives, thus restricting binding of the acetazolamide moiety to the
membrane-bound carbonic anhydrase isoforms CA IX and CA XII.
Figure 1A presents two-color fluorescence microscopy images of
tumor sections, showing CA IX staining in green and blood vessels
in red. Two extreme situations are presented. On one hand, virtu-
ally all cancer cells express CA IX in SK-RC-52 renal tumor cells
due to a defective pVHL tumor suppressor.28 By contrast, CA IX is
only detectable at sites of hypoxia (i.e., at >100 lm from tumor
blood vessels) in the LS174T human colorectal adenocarcinoma
xenograft model.
The structures of the two acetazolamide derivatives investi-
gated in this study are depicted in Figure 1B and C. FAM-acetazol-
amide
[2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-5-(5-sulfamoyl-
1,3,4-thiadiazol-2-ylcarbamoyl)benzoic acid] is an amide deriva-
tive of the green fluorophore 5-carboxyfluorescein (FAM), and is
negatively charged at neutral pH. The carboxylic acid of 5-carboxy-
In this Letter, we describe the synthesis and characterization
(in vitro and in vivo) of a novel bispecific acetazolamide derivative,
capable of simultaneous binding to carbonic anhydrases and serum
albumins (‘Albu-acetazolamide’). We used acetazolamide as a
building block, since this compound is able to inhibit a broad spec-
trum of carbonic anhydrases in the low nanomolar range, including
CA IX and CA XII.19 Furthermore, we investigated the conjugation
of acetazolamide to a charged fluorophore (‘FAM-acetazolamide’)
as an alternative avenue to prevent internalization. FAM-acetazol-
amide binding to tumor cells was confirmed by fluorescence-acti-
vated cell sorting, while preferential in vivo tumor targeting was
assessed by fluorescence microscopy analysis of tissue sections,
1 h and 2 h after intravenous (i.v.) administration. The use of acet-
fluorescein (266
(NHS, 292
mol) and N-(3-dimethylaminopropyl)-N0-ethylcarbodi-
imide hydrochloride (EDC-HCl, 292 mol) in 3 ml of dry N,N-
dimethylformamide (DMF) and stirred for 4 h at 25 °C. The reaction
mixture was then added to 5-amino-1,3,4-thiadiazole-2-sulfon-
lmol) was activated with N-hydroxysuccinimide
l
l
amide (Ramidus AB, 319 lmol) and triethylamine (3.2 mmol) dis-
solved in 3 ml of dry DMF and stirred protected from light at
25 °C for 16 h followed by addition of Tris/Cl, pH 8 (1.05 mmol).
After stirring for 30 min at 25 °C protected from light, HPLC purifi-
cation was performed running a linear gradient from 40–60% ace-
tonitrile. The eluting FAM-acetazolamide was detected at
k = 470 nm, the desired fractions were collected, dried under vac-
uum and the concentration was determined by UV/VIS spectrome-
A
B
HO
COOH
O
O
O
N
S
N
HN
O
S
NH2
O
‘FAM-acetazolamide’
C
H
N
H
N
O
O
N
HO
O
O
NH
I
S
N
O
S
O
NH2
‘Albu-acetazolamide’
Figure 1. (A) Immunofluorescence analysis performed on human RCC SK-RC-52 (upper panel) and human colorectal adenocarcinoma LS174T (lower panel) xenografted
tumor tissue sections. Overlay of red (endothelial cells, i.e., anti-CD31 staining) and green (anti-CA IX staining) fluorescence. In the pVHL-defective SK-RC-52 model CA IX is
expressed constitutively, whereas in the LS174 T model the expression occurs in hypoxic regions in distance to blood vessels. Scale bar = 100 lm. (B) Structure of FAM-
acetazolamide, used in this study for biodistribution analysis. (C) Structure of Albu-acetazolamide. This bispecific acetazolamide derivative is able to simultaneously bind to
carbonic anhydrases and serum albumin, thereby preventing its internalization and restricting it to the extracellular space. In this study, Albu-acetazolamide was used for
therapy experiments in tumor-bearing mice.

J. K. J. Ahlskog et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4851–4856
4853
try at 495 nm (e
= 72,000 Mꢀ1 cmꢀ1). ESI-MS m/z 538.71 ([M+H+],
A
FAM-acetazolamide B
FAM
100%), calcd: 538.03 Da. Albu-acetazolamide [6-(4-(4-iodo-
phenyl)butanamido)-2-(4-oxo-4-(5-sulfamoyl-1,3,4-thiadiazol-2-
ylamino)butanamido)hexanoic acid] is a bispecific compound con-
taining the acetazolamide moiety linked to the 2-amino-6-(4-(4-
iodophenyl)butanamido)hexanoic acid moiety, which confers sta-
ble albumin binding and a slow blood clearance profile to the mol-
ecule.26 5-amino-1,3,4-thiadiazole-2-sulfonamide (Ramidus AB,
100
80
60
40
20
0
100
80
60
40
20
0
0 µM
0 µM
0.26 µM
3 µM
0.26 µM
3 µM
611 lmol) was stirred with succinic anhydride (611 lmol) in
5
5
2
3
4
2
3
4
010 10 10 10
FITC-A
010 10 10 10
FITC-A
1 ml of dry N,N-dimethylformamide (DMF) for 24 h at 50 °C fol-
lowed by HPLC purification running a linear gradient from 0% to
100% acetonitrile. After collection of the desired fractions, solvents
and buffer were removed under vacuum. The obtained intermedi-
polyclonal anti-CA IX
C
100
80
60
40
20
0
0 µM
5 µM
ate
acid (366
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluoro-
phosphate (HBTU, Novabiochem, 366 mol) were stirred for
10 min at 25 °C in 3 ml of dry DMF followed by addition of 4-(-p-
iodophenyl)butyl lysine (366 mol) and triethylamine (732 mol)
4-oxo-4-(5-sulfamoyl-1,3,4-thiadiazol-2-ylamino)butanoic
l
mol), N-ethyldiisopropylamine (DIPEA, 366 mol) and
l
l
l
l
in dry DMF. The reaction was stirred for 24 h at 30 °C and purified
by HPLC running a linear gradient from 40% to 60% acetonitrile. The
eluting product Albu-acetazolamide was collected and solvents
and buffer were removed under vacuum. ESI-MS m/z 680.86
([M+H+], 100%), calcd: 680.06 Da. 1H NMR (DMSO-d6): 7.61 (d,
8.3 Hz, 2H), 7.00 (d, 8.3 Hz, 2H), 4.14 (m, 1H (aH)), 3.01 (m, 2H)
2.74 (t, 6.6 Hz, 2H), 2.52 (m, 4H), 2.05 (m, 2H), 1.81–1.50 (m,
4H), 1.43–1.21 (m, 4H) (Bruker 400 avance instrument).
5
2
3
4
010 10 10 10
FITC-A
Figure 2. FACS histogram plots of the LS174T cell line. (A) FACS histogram plots of
cells stained with different concentrations of FAM-acetazolamide (open gray and
black curves) in comparison to non-stained cells (solid curve). (B) FACS histogram
plots of cells stained with different concentrations of FAM (open gray and black
curves) in comparison to non-stained cells (solid curve). (C) FACS histogram plot of
cells stained with a polyclonal anti-CA IX (open curve). The solid curve represents
the plot where the antiserum was omitted.
The ability of FAM-acetazolamide to bind to tumor cells was
assessed by fluorescence-activated cell sorting, revealing a >10-
fold average fluorescence increase upon incubation of LS174T
cells with 3
to evaluate whether this compound was also able to reach tumor
cells in vivo, 680 g (1.26 mol) FAM-acetazolamide were in-
l
M FAM-acetazolamide solution (Fig. 2). In order
In order to demonstrate that the stable binding of Albu-aceta-
zolamide to serum albumin prevents internalization of the drug
into tumor cells, we used mass-spectrometric methodologies to
compare drug uptake in non-transfected HEK EBNA 293, full-
length CA IX (aa 1-459)-transfected HEK EBNA 293 and in
l
l
jected in the tail vein of Balb/c nu/nu mice bearing LS174T tu-
mors of a size of ꢁ500 mg grafted subcutaneously. Figure 3
depicts fluorescence microscopy images of 10
lm-thick tissue
LS174T tumor cells, in the absence or presence of 900
l
M HSA.
sections, obtained from mice sacrificed 1 h and 2 h after i.v.
injection. A strong and heterogeneous tumor uptake was ob-
served at both time points, in agreement with the immunohisto-
chemical staining patterns presented in Figure 1A. At 1 h
negligible background fluorescence was detected for heart, lung,
spleen and muscle, while organs involved in the clearance of the
compound (kidney, liver and intestine) were homogenously
bright. As expected, fluorescence signals in all organs but tumors
substantially decreased at 2 h.
The ability of Albu-acetazolamide to display high-affinity bind-
ing to both CA IX and human serum albumin (HSA) was first con-
firmed by isothermal titration calorimetry (Supplementary Fig. 1).
Albu-acetazolamide exhibited dissociation constants Kd = 3.2 nM
towards the recombinant catalytic domain of human CA IX and
Kd = 820 nM towards HSA. These values are comparable to the Kd
constants of the individual binding moieties,19,26 thus confirming
that the construction of the bispecific molecule did not adversely
affect binding.
To show that Albu-acetazolamide was able to inhibit the enzy-
matic activity of CA IX alone and in the presence of serum albumin,
we used a colorimetric assay based on the conversion of 4-nitroph-
enylacetate.29 The albumin-binding moiety displays comparable
affinities towards both human and murine serum albumin26 and
we used MSA in this assay, in view of the subsequent in vivo cancer
therapy studies in tumor-bearing mice. As expected, Albu-aceta-
zolamide displayed a comparable inhibitory activity when used
Upon the incubation of 5 ꢂ 106 cells with 500
lM of Albu-acetazol-
amide, followed by tandem mass-spectrometric quantitation of the
internalized fraction, a dramatic reduction of drug uptake in the
presence of albumin was observed for all three cell lines (Fig. 4).
We assessed the therapeutic activity of Albu-acetazolamide in
mice bearing subcutaneously grafted SK-RC-52 or LS174T human
tumors. While SK-RC-52 is a human RCC cell line, which displays
a homogenous overexpression of CA IX due to the loss of pVHL,
LS174T is a human colorectal cancer in which CA IX expression is
confined to the membrane of hypoxic cells at a given distance to
tumor blood vessels (Fig. 1A). In view of the over-expression of sur-
face-associated CA, both colorectal cancer and renal cell carcinoma
appear to be promising indications for the study of CA inhibitors.
Furthermore, the LS174T tumor model has recently been used to
assess the anti-cancer activity of RNAi-based invalidation of CA
IX and CA XII.11
Figure 5 shows the results of therapy experiments performed in
the two tumor models, using Albu-acetazolamide alone or in com-
bination with other drugs. We used 5-fluorouracil (5-FU) for the
therapy of LS174T tumors, since this drug is the mainstay of colo-
rectal cancer in most clinical protocols,30 while we used sunitinib
(N-[2-(diethylamino)ethyl]-5-[(Z)-(5-fluoro-1,2-dihydro-2-oxo-
3H-indol-3-ylidine)methyl]-2,4-dimethyl-1H-pyrrole-3-carbox-
amide) for the therapy of SK-RC-52, since this compound is used as
first-line therapy in kidney cancer.31 For both therapy experiments,
a dose of 340 lg (17 mg/kg or 0.5 lmol) of Albu-acetazolamide
alone or in the presence of 10
lM MSA with Ki values of 18 nM
was used for each injection, which was found to be well tolerated
and 8.8 nM, respectively. The Ki values of FAM-acetazolamide
and acetazolamide were determined to be 220 and 230 nM, respec-
tively (Supplementary Fig. 2).
in preliminary mouse treatment studies (data not shown) and
which corresponds to a 250
lM concentration in blood immedi-
ately at the end of the bolus i.v. injection (Supplementary Fig. 3).

4854
J. K. J. Ahlskog et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4851–4856
Figure 3. Biodistribution analysis with FAM-acetazolamide. LS174T xenograft-bearing nude mice were injected i.v. with FAM-acetazolamide (A–H) 1 h or (I–P) 2 h prior to
sacrifice. The accumulation of the fluorescent acetazolamide derivative in (A, I) tumor, (B, J) muscle, (C, K) heart, (D, L) lung, (E, M) intestine, (F, N) spleen, (G, O) kidney and
(H, P) liver is shown in gray scale. Exposure times were 900 ms for (A–H) and 1100 ms for (I–P). Scale bar = 100 lm.
**
***
of tumor growth, which however was not statistically significant,
compared to the control group of mice treated with vehicle alone.
5-FU, which was administered at the 25 mg/kg/day dose com-
monly used in rodent therapy studies32,33 yielded only a modest
tumor growth retardation, yet at the expense of a substantial tox-
icity (Fig. 5D). The combination of Albu-acetazolamide with 5-FU
showed no evidence of cumulative toxicity, but unfortunately did
not offer a therapeutic synergy.
- HSA
+ HSA
3
2
1
*
In summary, we have described the synthesis and properties of
two novel acetazolamide derivatives, which preferentially target
membrane-associated carbonic anhydrases on the surface of tumor
cells.
tHEK EBNA 293 HEK EBNA 293
LS174T
Figure 4. Cell internalization assay with tandem mass-spectrometric quantitation
of Albu-acetazolamide. Three different cell lines [full-length CA IX-transfected HEK
EBNA 293 (tHEK EBNA 293), HEK EBNA 293 and LS174T] were incubated with a
For pharmacodelivery applications, small organic molecules are
increasingly being considered as suitable delivery vehicles, in par-
allel to the more extensively characterized antibody-based ap-
proaches.34,35 However, while human monoclonal antibodies can
be raised against virtually any antigen of choice,36 there are only
few small organic molecules which can be used as portable tu-
mor-targeting moieties for pharmacodelivery applications. In our
study, the combined use of Albu-acetazolamide with standard che-
motherapeutic agents led to substantial tumor growth retardation,
yet without a substantial improvement compared to chemother-
apy alone. It is not clear at present whether this situation may re-
flect a lack of functional activity of carbonic anhydrases for the
tumors which we have investigated or rather an inability for the
albumin-bound acetazolamide derivative to homogenously dis-
tribute within the tumor mass. RNAi-based therapy studies in sta-
bly-transfected LS174T tumors have shown that the simultaneous
invalidation of both CA IX and XII is needed in order to achieve an
85% tumor growth retardation.11 However, in that experimental
setting, all tumor cells were genetically equivalent and pharmaco-
kinetic considerations did not apply. Albumin-binding antibody
fragments have been shown to homogenously reach tumor cells,
500
900
l
M
solution of Albu-acetazolamide, both in the absence and presence of
lM HSA. The assay was performed in triplicate and internalization is expressed
as % of the initial amount of compound subjected to the cells. Significant differences
are based on Student’s t test (two-tailed, two-typed), with *P < 0.0005, **P < 0.0001
and ***P < 0.00005.
In the SK-RC-52 therapy experiment, using a 5-weekly injection
schedule for three consecutive weeks, we observed only a partial
inhibition of tumor growth with Albu-acetazolamide (Fig. 5A).
Sunitinib treatment showed a stronger inhibition of tumor growth.
Tumor stabilizations for at least 20 days could be achieved with a
combination of the two compounds. Both sunitinib and Albu-acet-
azolamide, alone and in combination, were well tolerated as re-
flected by the mouse weights, without evidence of cumulative
toxicity. Only mice in the control treatment group lost weight,
when the tumor burden became greater than 500 mg (Fig. 5B).
The therapeutic results in the LS174T model were less favor-
able. This tumor model grows much more rapidly, thus forcing a
treatment regimen of only two weeks (Fig. 5C). In this model,
Albu-acetazolamide used as single agent led to a slight increase

J. K. J. Ahlskog et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4851–4856
4855
A
C
15% DMSO/ H2O
Albu-acetazolamide
sunitinib
B
100
800
600
400
200
sunitinib + Albu-acetazolamide
95
90
85
10 12 14 16 18 20 22 24 26 28
10 12 14 16 18 20 22 24 26 28
D
15% DMSO/ H2O
Albu-acetazolamide
5-FU
1000
800
600
400
200
100
95
90
85
5-FU + Albu-acetazolamide *
16
6
8
10
12
14
6
8
10
12
14
16
days after tumor cell implantation
Figure 5. Therapy experiments with Albu-acetazolamide alone or in combination with standard of care chemotherapeutics. (A) Tumor growth curve expressed as mean
tumor volume SE of SK-RC-52 in Balb/c nude mice after treatment with saline containing 15% (v/v) DMSO (i.v.), 340 g Albu-acetazolamide (i.v.), 20 mg/kg sunitinib (i.p.) or
340 g Albu-acetazolamide (i.v.) + 20 mg/kg sunitinib (i.p.) (n = 5). (B) Monitoring of the weight loss during therapy for the SK-RC-52 model expressed as% of body weight on
day 1 of treatment. (C) Tumor growth curve expressed as mean tumor volume SE of LS174T in Balb/c nude mice after treatment with saline containing 15% (v/v) DMSO (i.v.),
340 g Albu-acetazolamide (i.v.), 25 mg/kg 5-FU (i.p.) or 340 g Albu-acetazolamide (i.v.) + 25 mg/kg 5-FU (i.p.) (n = 5). The combination subgroup comprised only n = 4 for
l
l
l
l
*
the LS174T study, due to the unexpected death of one mouse ( ). (D) Monitoring of the weight loss during therapy for the LS174T model expressed as % of body weight on day
1 of treatment. Arrows indicate days of treatment. Boxed arrows indicate 5-FU treatment, which was only administered in the first of the two consecutive weeks, due to its
negative effect on the body weight.
while the parental IgG molecule was trapped on perivascular tu-
mor cells.25 Similar localization studies are more difficult to per-
form with Albu-acetazolamide and will require either microau-
toradiographic investigations with radiolabeled analogues or the
use of trifunctional acetazolamide derivatives, capable of albumin
binding and carrying, in addition, a suitable detection agent (e.g.,
a fluorophore, biotin). In spite of these considerations, it was reas-
suring to observe that Albu-acetazolamide treatment was well tol-
erated at doses up to 17 mg/kg/d and led to stabilizations of
tumors, which are difficult to cure. This warrants future preclinical
therapy studies, using different doses and schedules.
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Financial support from the ETH Zürich, the Swiss National Sci-
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