Carbamoylphosphonates control tumor cell proliferation and dissemination by simultaneously inhibiting carbonic anhydrase IX and matrix metalloproteinase-2. Toward nontoxic chemotherapy targeting tumor microenvironment(1)

Article abstract of DOI:10.1021/jm300981b

Carbamoylphosphonates (CPOs) have been identified as inhibitors of matrix metalloproteinases (MMPs) and as orally active, bioavailable, and safe antimetastatic agents. In this article, we focus on the direct antitumor activity of the CPOs. We discovered t

Full text of DOI:10.1021/jm300981b

Article
pubs.acs.org/jmc
Carbamoylphosphonates Control Tumor Cell Proliferation and
Dissemination by Simultaneously Inhibiting Carbonic Anhydrase IX
and Matrix Metalloproteinase-2. Toward Nontoxic Chemotherapy
Targeting Tumor Microenvironment1
Reuven Reich,‡ Amnon Hoffman,‡ Ainelly Veerendhar,‡ Alfonso Maresca,§ Alessio Innocenti,§
Claudiu T. Supuran,*,§ and Eli Breuer*,‡
‡Institute for Drug Research, The Hebrew University of Jerusalem, School of Pharmacy, P.O. Box 12065, Jerusalem IL 91120, Israel
§Universita degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019
Sesto Fiorentino (Florence), Italy
S
* Supporting Information
ABSTRACT: Carbamoylphosphonates (CPOs) have been identified as
inhibitors of matrix metalloproteinases (MMPs) and as orally active, bioavailable,
and safe antimetastatic agents. In this article, we focus on the direct antitumor
activity of the CPOs. We discovered that CPOs also inhibit carbonic anhydrases
(CAs), especially the IX and XII isoforms identified as cancer promoting factors.
Thus, CPOs can be regarded as novel nontoxic drug candidates for tumor
microenvironment targeted chemotherapy acting by two synergistic mechanisms,
namely, inhibiting CAs and MMPs simultaneously. We have also demonstrated
that the ionized CPO acid is unable to cross the cell membrane and thus limited
to interact with the extracellular domains of isozymes CAIX and CAXII. Finally, applying CPOs against cancer cells in hypoxic
conditions resulted in the dose dependent release of lactate dehydrogenase, confirming the direct interaction of the CPOs with
the cancer related isozymes CAIX and XII and thereby promoting cellular damage.
and mostly also ADAMs. Consequently, current efforts are
focused on finding selective inhibitors, those that do not bind
zinc at all,15,16 or phosphonates.17,18
Our work has been focused on metastasis formation in which
MMPs (especially MMP-2) have a vital role by mediating tumor
cell dissemination. Efficient inhibition of MMPs is, therefore, an
important therapeutic target, which has attracted considerable
attention within academia and industry for the last two decades
or so.19,20
In recent years, we have reported in a series of papers that
carbamoylphosphonates (CPOs) possess zinc binding ability21
and are able to act both in vitro and in vivo as inhibitors of
MMPs.22−26
In this article, we wish to report our discovery that the above-
mentioned CPOs also inhibit carbonic anhydrases (CAs), a
family of about 16 enzymes that catalyze the interconversion of
carbon dioxide and bicarbonate to maintain the required pH in
biological fluids. CAs are involved in many physiological and
pathological processes, including respiration and transport of
CO2 and bicarbonate between metabolizing tissues and lungs;
pH and CO2 homeostasis; electrolyte secretion in various tissues
and organs; biosynthetic reactions (such as gluconeogenesis,
lipogenesis, and ureagenesis); bone resorption; calcification; and
INTRODUCTION
■
Matrix metalloproteinases (MMPs) are a family of about 26 zinc
endopeptidases, which collectively have the capacity to degrade
all the major components of the extracellular matrix and have
crucial roles in inflammation, cancer dissemination, and other
indications.2 Although the MMPs play a crucial role in
physiological tissue remodeling and repair, their overexpression
has been linked with a variety of chronic diseases including
cancer, arthritis,3 osteoporosis,4 multiple sclerosis,5 arterio-
sclerosis,6 restenosis,5 meningitis,7 congestive heart failure,8
chronic obstructive pulmonary disease, chronic wounds , liver
cirrhosis,10 cerebral ischemia,11 and others. After more than
twenty years of worldwide research, significant advances have
been made in the clarification of the mechanism of inhibition, yet
no clinically useful inhibitor has been identified. The main reason
for the failure of these trials to reach their end-points was that
severe painful side effects, e.g., the musculoskeletal syndrome,
necessitated interruption of the clinical trials. Other reasons were
that, while some MMPs promote pathology, others have a
protective effect.12 Also, previous attempts to consider MMPs as
targets were burdened by the lack of information on which MMP
does what and where in physiology and pathology. Nowadays, we
have a much better understanding of the individual roles of the
various orthologs.13,14 In addition, all first generation com-
poundsand most of the second generation oneswere
hampered by lack of specificity, i.e., they targeted all MMPs
9
7
a
Received: July 7, 2012
© XXXX American Chemical Society
A
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tumorigenicity.27 Many of the CA isozymes involved in these
processes are important therapeutic targets with the potential to
be inhibited to treat a range of disorders including edema,
glaucoma, obesity, cancer, epilepsy, and osteoporosis.28
In mammals, 16 α-CA isozymes or CA-related proteins have
been described with different catalytic activity, subcellular
localization, and tissue distribution. There are five cytosolic
forms (CA I, CA II, CA III, CA VII, and CA XIII), five membrane
bound isozymes (CA IV, CA IX, CA XII, CA XIV, and CA XV),
two mitochondrial forms (CA VA and CA VB), and a secreted
CA isozyme (CA VI).
Table 1. Inhibition Constants of Alkyl- and Cycloalkyl-CPOs
on MMP2, hCAI, hCAII, hCAIX, and hCAXII
a
In the present article, we report inhibition constants of 21
CPOs on four CAs: CAI, CAII, CAIX, and CAXII. The latter two
isozymes, CAIX and XII, are membrane-type isoforms with
extracellularly exposed active sites, which are induced by the
transcription factor, Hypoxia Induced Factor 1α (HIF-1α).29
They are present in the membranes of tumor cells and are critical
for their proliferation. Because they catalyze the reversible
hydration of carbon dioxide leading to bicarbonate and proton
formation, their upregulation results in decrease of extracellular
pH, and it is strongly associated with cancer cell survival and
malignant progression.30,31 Using various breast cancer cell
lines,32 it has been recently demonstrated that CAIX is essential
to the survival of tumor cells under hypoxic condition of breast
tumor, and its activity contributes to metastasis generation, and
could serve as a specific biomarker for this kind of tumor. The
necessity of CAIX to tumor and metastasis formation has also
been recently demonstrated by using knock-down approach.33
Further, inhibition of CAIX has been shown to result in
regression or growth inhibition of mouse and human breast
tumors as well as inhibition of cancer metastasis formation.32
Both CAs and the MMPs are zinc enzymes that reside and
function in the extracellular environment; therefore, it is not
surprising that they are inhibited by the same zinc-binding, ionic,
and water-soluble inhibitors. From this, it follows that the
anticancer and antimetastatic properties that we have observed
over the last eight years in the in vivo examination of CPOs, as
reported in our previous papers,22,23,25,26 have originated from
the simultaneous inhibition of both CA and MMPs enzymatic
systems.
a
n.d. = not determined.
compounds mentioned above. Among the compounds in Table
1, an in-depth study was devoted to cyclopentyl-CPO (3,
CPCPA),23 which revealed significantly inhibited cellular
invasion and capillary formation in vitro. Further, both i.p. or
oral administration of the compound significantly reduced lung
metastasis formation and s.c. tumor growth in a murine
melanoma model.
Table 2 shows aminoalkyl- and aminocycloalkyl-CPOs.
Earlier, we reported that the two acyclic aminoalkylCPOs 12
and 13, as well as the alicyclic cis-ACCP (15), were able to reduce
TNFα secretion to a level equivalent to the reduction caused by
the steroid drug, budesonide. The reduction in TNFα levels was
Therefore, it was of interest to examine in detail CPOs (aka
phosphonoformamides) as CA inhibitors. The results of these
studies are described in this paper.
RESULTS
■
Carbamoylphosphonates (CPOs) Inhibit Carbonic
Anhydrases. The CA inhibition constants of representative
CPOs on four isozymes, human carbonic anhydrases I, II, IX, and
XII (hCAI, hCAII, hCAIX, and hCAXII) are reported in three
tables. Beside the CA inhibition constants, the CPOs’ MMP-2
inhibition constants have also been added in a separate column.
The results for aliphatic and alicyclic compounds are listed in
Table 1. It is evident from this table that there is no structure−
activity relationship apparent in the obtained results. All IC50
values are of the same order of magnitude, whether they are chain
compounds (1 and 2) or ring compounds (3−7). Neither the
aliphatic nor aromatic nature of the rings has any significant effect
(compare 3−6 vs 7). Furthermore, the pair of enantiomers, 8 and
9, also show similar inhibition constants, in contrast to their
differing inhibition constants on MMPs.24 Finally, Table 1 also
contains 1,4-butanebiscarbamoylphosphonic acid and its tet-
raethyl ester (compounds 10 and 11, respectively),34 both of
which show similar inhibition characteristics to the mono-CPO
Table 2. Inhibition Constants of Aminoalkyl- and
AminocycloalkylCPOs on MMP-2, hCAI, hCAII, hCAIX, and
hCAXII
B
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a
Table 3. Inhibition Constants of Arylsulfonamido- and CarboxamidoCPOs onMMP-2, hCAI, hCAII, hCAIX, and hCAXII
#
symbol
structure
MMP-2 IC50μM IC50 μM hCAI IC50 μM hCAII IC50 μM hCAIX IC50 μM hCAXII
16
17
18
19
20
21
JS-268
JS-325
JS-343
JS-389
JS-403
JS-294
4-PhOC6H4SO2NH(CH2)2NHCOPO3H2
4-PhOC6H4SO2NH(CH2)3NHCOPO3H2
4-PhOC6H4SO2NH(CH2)4NHCOPO3H2
4-PhOC6H4SO2NH(CH2)5NHCOPO3H2
4-PhOC6H4SO2NH(CH2)6NHCOPO3H2
4-PhOC6H4CONH(CH2)2NHCOPO3H2
15
10
7
0.98
0.71
0.75
0.52
0.38
0.62
0.82
0.58
0.1
7.0
6.8
6.1
6.3
7.0
9.9
6.9
6.4
5.6
5.7
6.2
8.6
4
0.31
0.10
0.61
4
20
a
Compounds 16−20 have been reported in ref 26. Compound 21 has been reported in ref 36.
Table 4. In Vivo Results of Dual MMP-2 and CA Inhibitors
CPO #
symbol
drug dose mg/kg
model
Melanoma
mode of drug introduction
% decrease in (lung) metast, of control
ref
3
YK-96 CPCPA
YK-96 CPCPA
YK-96 CPCPA
YK-162 cis-ACCP
YK-162 cis-ACCP
YK-162 cis-ACCP
JS-389
50
IP
86
80
??
22
23
23
25
25
25
26
26
26
26
26
26
26
3
50
Melanoma
Oral
IP
3
50
Subcutan. Tumor
Melanoma
15
15
15
19
19
19
19
20
20
20
50
IP
70
95
∼90
57
76
90
80
82
90
50
50
Melanoma
Oral
IP
50
Orthotopic H. Prostate
Melanoma
12.5
25
IP
JS-389
Melanoma
IP
JS-389
50
Melanoma
IP
JS-389
50
Melanoma
oral
IP
JS-403
12.5
25
Melanoma
JS-403
Melanoma
IP
JS-403
50
Melanoma
Oral
Figure 1. Structures of the three fluorescent probes used in this work.
not accompanied by cytotoxicity, nor did it inhibit cell
proliferation.35 Among the compounds in this table, we singled
out 15 (cis-ACCP) for an in-depth examination, which included
two in vivo models and a pharmacokinetic study.25 Compound
15 (cis-ACCP) was evaluated in vitro and in two in vivo cancer
metastasis models. It reduced metastasis formation in mice by
∼90% when administered by a repetitive once daily dosing
regimen of 50 mg/kg via oral or intraperitoneal routes and was
nontoxic up to 500 mg/kg, following intraperitoneal admin-
istration daily for two weeks. Pharmacokinetic investigation of
15, cis-ACCP, in rats revealed distribution restricted into the
extracellular fluid, which is the site of action for the antimetastatic
activity.25
Finally, Table 3 lists a series of five 4-phenoxybenzenesulfo-
namidoalkylCPOs11 and one 4-phenoxybenzamidoalkylCPO,
compound 21 (JS-294). These sulfonamides, having varying
lengths of polymethylene chains, have been evaluated previously
as MMP inhibitors and showed gradual improvement in their
MMP-2 inhibitory ability with the lengthening of the chain. In
Table 3, we see, in contrast, that the CA inhibitory ability does
C
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not depend on the chain length at all. While the compounds
listed in Tables 1 and 2 have only carbamoylphosphonates as
zinc-binding-groups, in Table 3 we see, for the first time,
molecules having sulfonamide groups near the carbamoylphos-
phonic groups. Yet, the addition of the latter does not lead to
more potent CAIs than the CPOs lacking sulfonamides.
Furthermore, comparing compounds 16 and 21 (JS-294),
which have the same methylene chain lengths, we see that the
replacement of the sulfonamide group of 16 by a carboxamide in
2136 keeps the inhibition profiles in the same order of magnitude
indicating that carbamoylphosphonates are potent CAIs in their
own right, without requiring contribution of sulfonamides.
Table 4 contains a summary of the in vivo results obtained
from tested CPOs mentioned earlier and originally were
attributed to MMP inhibition. This view now needs to be
modified with the inclusion of the effects of CAIX and CAXII.
The last column contains references to the papers containing the
original in vivo results.
Membrane Impermeability of Carbamoylphospho-
nates. The question of carbamoylphosphonate anions’ cell
membrane permeability is one of the critical issues regarding the
scope of action of these inhibitors. Inhibitors that are able to
penetrate into the cells will inhibit the cytosolic CA I, II, III, VII,
and XIII and the mitochondrial CA VA and VB, while those that
cannot enter cells will only inhibit the four membrane-associated
isozymes CA IV, IX, XII, and XIV, the catalytic domains of which
protrude to the extracellular medium. Among the latter four, the
most important isoform is CAIX, which is closely relevant to
tumor cell proliferation.
ification of the tumor microenvironment. These enzymes confer
a survival advantage to tumor cells growing in a hypoxic and
acidic microenvironment.
If an effective CAIX inhibitor would interact with a tumor cell
expressing CAIX in condition of hypoxia, it should block the
CAIX supported tumor cell survival. Seeking evidence for
establishing the direct interaction of representative CPOs with
CAIX, we turned to a similar experiment under hypoxia
conditions. In the first step, we tested the feasibility with two
kinds of tumor cells, namely, HT-1080 and HeLa cells.
Treatment of these two kinds of cells, both of which express
CAIX, with 50 μM inhibitor 19 (JS-389) caused in HeLa cells a
1.3-fold increase in lactate dehydrogenase (LDH), while the
same treatment induced in HT1080 cells a 2.3-fold increase,
respectively, relative to control. Since the formation of LDH
reflects cell damage caused by the CAI, these preliminary results
indicate that HeLa cells are less sensitive than HT1080 cells.
Therefore, in the second experiment we tested dose response of
LDH formation only in HT1080 cells using three doses of two
inhibitors 19 (JS-389) and 20 (JS-403). The results appear in
Table 5. From this table, it appears that the damage to the tumor
cell caused by 19 (JS-389) is larger.
Table 5. Effect of Carbamoylphosphonate CAIs on LDH
Secretion under Hypoxic Conditions
HT-1080 cells
CA inhibitor
LDH
control
conc. μM
CAI JS-389 19
CAI JS-403 20
arbitrary value
We sought the answer to the question of the CPO anions’ cell
membrane permeability by observing the behavior of fluorescein-
bound inhibitor molecule C and to two other control molecules
A and B. The structures of the three fluorescent molecules are
displayed in Figure 1.
50
10
1
2.3
1.5
1
1.9
1.3
1
1
1
1
Fluorescein derivative A was chosen as positive control
because it is known as a molecule easily permeable into cells.37
Molecule B is a carbamoylphosphonic acid diisopropyl ester
linked to a fluorescein molecule. Finally, molecule C is a
fluorescein bound carbamoylphosphonic acid (pKa1 < 2 and pKa2
≈ 5−5.5), which should be doubly ionized at physiological pH.
Fluorescein derivative A had been synthesized according to the
method of Supuran and co-workers.37 Fluorescein derivatives B
and C have been synthesized in this work as described in the
Supporting Information.
The membrane permeabilities of compounds A, B, and C have
been examined on two cell lines, (1) HT-1080 and (2) MDA-
231. These cells had been exposed to the three compounds for 20
and 120 min at 37 °C. Following the incubation period, the cells
were washed three times with PBS and visualized by a fluorescent
inverted microscope. As shown in SI Figures 2S and 3S on HT-
1080 and MDA-231cells, compound A penetrated quite rapidly
even at 20 min. Compound B showed a slower penetration
profile and at 120 min distributed evenly inside the cells. In
contrast, compound C accumulated pericellularly and did not
penetrate the cells.
DISCUSSION
■
In the course of our previous work, studying carbamoylphosph-
onates (CPOs) as MMP inhibitors, several CPOs have shown
higher potency in vivo than that could be expected from the in
vitro results. This indicated that CPOs may have one or more
additional targets. The in vitro tests used for CPOs were specific
to MMPs, while the in vivo test involved B16F10 or other tumor
cells, which potentially express additional metalloenzymes.
One in vitro test is the determination of enzyme kinetics using
recombinant MMP isozymes, while the other is an in vitro
invasion assay, or chemoinvasion across a reconstituted base-
ment membrane by tumor cells, which is dependent on the
presence and activity of certain MMPs, mainly MMP-2 and
MMP-9.39
Another in vitro test used was capillary formation, which has
been shown to be dependent on expression of certain MMPs.
New blood vessel formation is a critical step in the expansion and
in dissemination of a given tumor. This experiment has become
an accepted model of angiogenesis in vitro.40
On the other hand, the in vivo test used most frequently in our
group is the B16F10 melanoma which is relatively convenient to
carry out and yields clear results in three weeks. Another aspect of
B16F10 cells is that they express CA IX, which makes them a
convenient platform for testing CPOs as potential anticancer
CAIs.41 An additional coincidence is that both MMPs and CAs
are zinc enzymes functioning by similar mechanisms; therefore, it
is not surprising that identical or similar molecules would inhibit
them.
Hypoxia. Tumor cells alter their metabolism when in a
hypoxic microenvironment to survive, proliferate, and metasta-
size. CAs catalyze the reversible hydration of carbon dioxide
−
(CO2) to generate a bicarbonate anion (HCO3 ) and a proton
(H+).38 Carbonic anhydrase isozymes CA IX and CA XII are
embedded in the membranes of tumor cells that express them,
maintain a pHi range compatible with cell viability and
proliferation, while they contribute to the extracellular acid-
D
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Detailed in vivo anticancer and antimetastatic results for
selected carbamoylphosphonates (Table 4) have been described
mainly in three publications,23,25,26 in which the biological
activities were attributed to the compounds MMP-2 inhibitory
activity. This view now needs a revision following the discovery
of the dual nature of CPOs and that the observed anticancer and
antimetastatic activities are the results of the dual inhibition!
While inhibition of MMP-2 blocks the breakdown of the
basement membranes, angiogenesis, and the dissemination of
metastases, inhibition of CAIX and CAXII targets hypoxia,
elevates the pH in the tumor microenvironment, and obstructs
the proliferation of the cancer cells.32 These simultaneous actions
are made possible by the similarities of the two kinds of enzymes.
Both MMPs and CAs are zinc enzymes that function in the
extracellular environment through similar mechanisms; there-
fore, ideally they are inhibited by water-soluble zinc binding
inhibitors. The ionized compounds such as CPOs (pK1 ≤ 2; pK2
≈ 5−5.5) fulfill this requirement. As it is demonstrated in this
paper, they are unable to penetrate cells, therefore remain in the
extracellular fluid, and can be expected to be nontoxic inhibitors.
Many metal binding anions, including various phosphorus
acids, have also been recognized as CA isozyme inhibitors. Thus,
orthophosphate anions, carbamoyl phosphate, phosphonofor-
mate (foscarnet), and formate have been reported as weak CAIX
inhibitors (KI’s in the range of 0.56−3.67 mM), whereas mono
and dihydrogen phosphates are even weaker (having KI’s 3.67
and 21.1 mM, respectively).42
Carbamoylphosphonates, also called phosphonoformamides,
are orders of magnitude more potent CA inhibitors relative to
their parent phosphonoformate. This might be the result of the
electron donating effect of the nitrogen lone pair in the R
NHCO moiety, polarizing the carbamoyl CO group, the
effect of which might even extend to the phosphorus and
strengthen its interactions with electrophiles on the enzyme. The
X-ray results of foscarnet bound to a CA support such an
assumption. X-ray crystallography of CA inhibited by a CPO will
answer this question. Interestingly, examination of our results
shows that the nature, steric hindrance, chirality, and even the
esterification of the phosphonic groups in an R−NH−C(O)-
PO3H2 molecule did not appear to influence the extent of the CA
inhibition. This is in sharp contrast to MMPs whose inhibition
constants are sensitive to every kind of difference in the
structures of the N-substituents in the N-substituted CPOs. The
degree of esterification of the −PO3H2 group is also critical for
MMP inhibition. Only the completely free dibasic phosphonic
acids showed MMP inhibition, in contrast to CAs, which are
inhibited equally by diester and by diacid alike. This
phenomenon can perhaps be exploited for designing CA
inhibitors for intracellular purposes after having shown that the
diisopropyl ester is able to penetrate across the cell membrane.
Other dual MMP-CA inhibitors based on hydroxamates as
zinc-binding groups have also been described.43 However, the
utility of these compounds is doubtful, considering the failure of
hydroxamates as clinically useful MMP inhibitors. Most
hydroxamate-based MMP inhibitors are hydrophobic molecules
which have non-ionizable functional groups that enable them to
cross lipidic cell membranes into cells, leading to musculoske-
letal44 side effects. Another shortcoming of hydroxamates is their
very strong inhibition constants and nonselective binding of all
transition metals in the body, especially iron, which is present in
somewhat larger quantities than zinc. The 106- to 1010-fold
higher stability constants of hydroxamate−iron complexes than
those containing with zinc21,45 is also likely to be the source of
some of the toxic side effects.46 CPOs do not cause
musculoskeletal side effects and are relatively weak transition
metal ligands; thus, they are free of the above-mentioned toxicity.
Our attempts to determine stability constants for CPO-iron(III)
complexes failed due to precipitation.21
In recent years, weak bonds and weak interactions are being
seen and discussed more and more frequently in virtually every
branch of science. Biology and its branches are no exception. One
can see a statement like, “for a high degree of specificity the
contact or combining spots on the two particles must be multiple
and weak”47 or “a multitude of weak, or transient, biological
interactions (dissociation constant: Kd > μM), either working
alone or in concert, occur frequently throughout biological
systems”.48
While medicinal chemists usually strive to discover the most
potent drug possible, in some cases the advantage of a weak
interaction between a drug and its receptor had been recognized
and preferred.49
The first carbamoylphosphonate MMP inhibitors we synthe-
sized were lacking the diphenyl ether affinity groups, and they
showed MMP inhibition of short duration. Our most recent
inhibitors having the diphenyl ether as a putative S1′ affinity
group inhibited MMPs for as long as they were monitored (3 h),
indicating higher affinity to the enzymes, yet their inhibition
constants remained in the single-digit μM range, similar to the
magnitude of CAIX’s and CAXII’s inhibition constants. This
appears as the ideal situation for a double (or multiple) target
inhibitor in which both targets promote the same disease (cancer
and metastasis) and are inhibited by a common drug. Assuming
that an inhibitor would have several orders of magnitude stronger
binding constant to one target than to the other, it seems
reasonable that close to 100% of the drug would bind to the
stronger attracting target and the benefit from having a powerful
inhibitor for two targets would be lost. In summary, there is an
advantage in having a relatively weakly binding drug to two or
multiple targets.
The CPOs cell impenetrability substantiates the pharmacoki-
netic results obtained in our previous paper. The reported large
apparent volume of distribution of 19 (JS-389) at steady-state
Vss ∼1650 mL.kg−1 may indicate that the drug is distributed into
both the extracellular and intracellular fluids and tissues. Our
visually confirmed finding regarding the impermeability of the
CPOs into cells is in accord with their high polarity, because of
which they are unlikely to permeate through biological
membranes. Therefore, the alternative explanation put forward
earlier, namely, the possibility of CPO’s adsorption onto bone is
gaining support. This assumption was tested on hydroxyapatite
(HAP) as a model for bone.26,50 There was found 44%
absorption of 19 (JS-389) in 17 h, and 59% of the absorbed
drug was resorbed in 15 h. This affinity of CPOs to bones is in
accordance with a large Vss, as found in this case, making this
compartment a depot providing reversible residence for the
CPOs as shown by the long biological half-life (9 h, following
intravenous administration). The extended half-life means that
such a drug, in the clinical setting, which can be taken once daily,
is the most convenient drug administration regimen for chronic
therapy.
EXPERIMENTAL SECTION
■
Purity of Tested Compounds. All carbamoylphosphonates tested
for carbonic anhydrase inhibition and reported in this article had been
synthesized and purified to at least 95% as confirmed by combustion
E
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analyses. These carbamoylphosphonates appear in the papers cited in
Tables 1, 2, 3, and 4.
Chemical Syntheses. The fluorescent probes’ syntheses are
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described in the Supporting Information.
Carbonic Anhydrase Inhibition. Assaying the CA-catalyzed CO2
hydration activity was done by using an Applied Photophysics stopped-
flow instrument as described previously.51
Permeability Experiments. Compounds A, B, and C have been
dissolved in 1 mL of water in the presence of 15 mg sodium bicarbonate
and tested at 2 mM final concentration.
HT-1080 and MDA-231 cells were exposed to the three compounds
for 20 and 120 min at 37 °C. Following the incubation period, the cells
were washed three times with PBS and visualized by a fluorescent
inverted microscope. The results can be found in the Supporting
Information.
Hypoxia. To induce the oxygen deprivation insult, HT-1080 and
HeLa cells were introduced into a hypoxic device.52 Briefly, the hypoxic
device is composed of two connected chambers, maintained at 37 °C by
circulating hot water into which Petri-dishes with HT-1080 and HeLa
cells are placed. The air in the chambers is replaced with a mixture of
99% N2 and 1% O2 bubbled from a tank through a cylinder containing
water, and passed through the system at a reduced flow rate to prevent
evaporation of the medium. The oxygen level within the device is 1% (as
measured online by an oxygen analyzer), representing a hypoxic insult.
Control cells were kept in the humidified chamber in an atmosphere
containing 21% O2 (normoxia). The cells were treated with various
concentrations of CPOs for 24 h (the whole oxygen deprivation period).
Cell death/damage was measured at the end of the hypoxia period by
quantifying the activity of released LDH into the medium, using a
commercial kit (Pointe, Canton, MI). LDH activity was determined
spectrophotometrically at 340 nm by following the rate of conversion of
oxidized nicotinamide adenine dinucleotide (NAD+) to the reduced
form (NADH). The results can be found in Table 5.
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Control cells, kept in normoxic conditions were not affected by the
addition of CPOs to the culture media (up to 100 uM). LDH activity in
the culture media increased in both cell lines in a dose dependent
manner. (2.4-fold and 1.3-fold respectively).
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ASSOCIATED CONTENT
* Supporting Information
■
S
Table of kinetic parameters, permeability results, structures of
fluorescent probes, cancer cells in the presence of fluorescent
probes at 20 min and 2 h, as well as syntheses of the fluorescent
probes used. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail addresses: elib@ekmd.huji.ac.il, eli.breuer@huji.ac.il.
■
(11) Romanic, A. M.; White, R. F.; Arleth, A. J.; Ohlstein, E. H.;
Barone, F. C. Matrix metalloproteinase expression increases after
cerebral focal ischemia in rats: inhibition of matrix metalloproteinase-9
reduces infarct size. Stroke 1998, 29, 1020−1030.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(12) Konstantinopoulos, P. A.; Karamouzis, M. V.; Papatsoris, A. G.;
Papavassiliou, A. G. Matrix metalloproteinase inhibitors as anticancer
agents. Int. J. Biochem. Cell Biol. 2008, 40, 1156−1168.
This work was supported in part by the Grass Center for Drug
Design and Synthesis of Novel Therapeutics to E.B., A. H., and
R.R. A.H., R.R., and E.B. are affiliated with the David R. Bloom
Center of Pharmacy and the Brettler Center for Pharmacology,
in the School of Pharmacy, Faculty of Medicine, Hebrew
University of Jerusalem. The authors thank Professor Philip
Lazarovici and Ms. Adi Lahiani for their help with the hypoxia
studies.
(13) Sang, Q.-X. A.; Jin, Y.; Newcomer, R. G.; Monroe, S. C.; Fang, X.;
Hurst, D. R.; Lee, S.; Cao, Q.; Schwartz, M. A. Matrix metalloproteinase
inhibitors as prospective agents for the prevention and treatment of
cardiovascular and neoplastic diseases. Curr. Topics Med. Chem 2006, 6,
289−316.
(14) Kruger, A.; Kates, R. E.; Edwards, D. R. Avoiding spam in the
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proteolytic internet: Future strategies for anti-metastatic MMP
inhibition. Biochim. Biophys. Acta 2010, 1803, 95−102.
(15) Engel, C. K.; Pirard, B.; Schimanski, S.; Kirsch, R.; Habermann, J.;
Klingler, O.; Schlotte, V.; Weithmann, K. U.; Wendt, K. U. Structural
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