U.S. patent application number 14/613881 was filed with the patent office on 2015-06-04 for cancer targeting using carbonic anhydrase isoform ix inhibitors.
This patent application is currently assigned to STICHTING MAASTRICHT RADIATION ONCOLOGY "MAASTRO- CLINIC". The applicant listed for this patent is STICHTING MAASTRICHT RADIATION ONCOLOGY "MAASTRO- CLINIC", Claudiu Supuran, UNIVERSITE MONTPELLIER 2 Sciences et Techniques. Invention is credited to Philippe Lambin, Claudiu Supuran, Jean-Yves Winum.
Application Number | 20150150849 14/613881 |
Document ID | / |
Family ID | 45507845 |
Filed Date | 2015-06-04 |
United States Patent
Application |
20150150849 |
Kind Code |
A1 |
Lambin; Philippe ; et
al. |
June 4, 2015 |
CANCER TARGETING USING CARBONIC ANHYDRASE ISOFORM IX INHIBITORS
Abstract
The present invention concerns novel carbonic anhydrase IX
inhibitors comprising a nitroimidazole moiety and their use in
therapy of hypoxic conditions, in particular cancer treatment,
especially chemotherapy and radiotherapy. The compounds of the
invention have an increased specificity for the carbonic anhydrase
IX enzyme compared to the art. The present invention relates to
novel nitroimidazole derivates represented by formula (1), wherein
R.sub.1, R.sub.2, and Z are as defined herein:
Inventors: |
Lambin; Philippe; (Genappe
Bousvalle, BE) ; Winum; Jean-Yves; (Saint Andre de
Sangonis, FR) ; Supuran; Claudiu; (Florence,
IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Supuran; Claudiu
STICHTING MAASTRICHT RADIATION ONCOLOGY "MAASTRO- CLINIC"
UNIVERSITE MONTPELLIER 2 Sciences et Techniques |
Florence
Maastricht
Montpellier |
|
IT
NL
FR |
|
|
Assignee: |
STICHTING MAASTRICHT RADIATION
ONCOLOGY "MAASTRO- CLINIC"
Maastricht
NL
UNIVERSITE MONTPELLIER 2 Sciences et Techniques
Montpellier
FR
Supuran; Claudiu
Florence
IT
|
Family ID: |
45507845 |
Appl. No.: |
14/613881 |
Filed: |
February 4, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13997378 |
Jun 24, 2013 |
8980932 |
|
|
PCT/NL2011/000083 |
Dec 21, 2011 |
|
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14613881 |
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Current U.S.
Class: |
514/158 ;
514/398 |
Current CPC
Class: |
C07D 233/91 20130101;
A61K 31/4164 20130101; C07D 233/88 20130101; A61K 31/635 20130101;
C07D 233/95 20130101; A61P 35/00 20180101 |
International
Class: |
A61K 31/4164 20060101
A61K031/4164; A61K 31/635 20060101 A61K031/635 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 24, 2010 |
NL |
1038473 |
Claims
1-35. (canceled)
36. A method of treating a subject afflicted with a disease
characterized by overexpression of one or more carbonic anhydrase
enzymes, comprising administering to the subject an effective
amount of a compound, or a pharmaceutically acceptable salt
thereof, of formula (1): ##STR00004## wherein Z is Z.sub.1 with
formula (2a): ##STR00005## or Z is Z.sub.2 with formula (2b):
(CH.sub.2)nCH.sub.2X, where X=sulfonamide, sulfamate or sulfamide;
or or Z is Z.sub.3 with formula (2c): ##STR00006## wherein R.sub.1
and R.sub.2 are, each independently, H, alkyl, alkenyl, alkynyl,
cycloalkyl, heterocyclic, aryl, cyano or halogen atom; R.sub.3,
R.sub.4, R.sub.6 and R.sub.7 are, each independently H, alkyl,
alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, halogen atom,
cyano, alkoxy, sulfonamide, sulfamate or sulfamide; R.sub.5 is
sulfonamide, sulfamate or sulfamide; Y=O or S; and n=0, 1, 2, 3, 4
or 5.
37. A method of treating a subject afflicted with a disease
characterized by overexpression of one or more carbonic anhydrase
enzymes, comprising administering to the subject an effective
amount of a compound, or a pharmaceutically acceptable salt
thereof, of formula (1), ##STR00007## wherein the compound is a
compound of formula (3a) ##STR00008## a compound of formula (3b)
##STR00009## wherein X=sulfonamide, sulfamate or sulfamide; or a
compound of formula (3c) ##STR00010## wherein R.sub.1 and R.sub.2
are, each independently, H, alkyl, alkenyl, alkynyl, cycloalkyl,
heterocyclic, aryl, cyano or halogen atom; R.sub.3, R.sub.4,
R.sub.6 and R.sub.7 are, each independently H, alkyl, alkenyl,
alkynyl, cycloalkyl, heterocyclic, aryl, halogen atom, cyano,
alkoxy, sulfonamide, sulfamate or sulfamide; R.sub.5 is
sulfonamide, sulfamate or sulfamide; Y=O or S; and n=0, 1, 2, 3, 4
or 5.
38. The method according to claim 36, wherein the disease is a
pre-cancerous or cancerous disease, wherein said disease is
characterized by overexpression of MN/CA IX protein.
39. The method according to claim 38, wherein the cancerous disease
is a cancer of breast, brain, kidney, colorectal, lung, head and
neck or bladder.
40. The method according to claim 38, wherein the cancer is
colorectal cancer.
41. The method according to claim 36, wherein R.sub.1 and R.sub.2
are, each independently, H or CH.sub.3.
42. The method according to claim 36, wherein
R.sub.1=R.sub.2=H.
43. The method according to claim 36, wherein
R.sub.3=R.sub.4=R.sub.7.
44. The method according to claim 36, wherein
R.sub.3=R.sub.4=R.sub.7=H.
45. The method according to claim 36, wherein R.sub.5 or R.sub.6 is
sulfonamide.
46. The method according to claim 36, wherein n=0, 1 or 2.
47. The method according to claim 36, wherein R.sub.1=H and
R.sub.2=CH.sub.3.
48. The method according to claim 36, wherein n=1.
49. The method according to claim 36, wherein Z is Z.sub.2 with
formula (2b), wherein X is sulfamate or sulfamide.
50. The method according to claim 36, wherein Y=S.
51. A method of treating a subject afflicted with a disease
characterized by overexpression of one or more carbonic anhydrase
enzymes, comprising administering to the subject a pharmaceutical
composition comprising an effective amount of a compound or salt of
claim 36 and a pharmaceutically acceptable carrier.
Description
FIELD OF THE INVENTION
[0001] The present invention concerns novel carbonic anhydrase IX
inhibitors comprising a nitroimidazole moiety including their use
in cancer treatment, especially radiotherapy.
BACKGROUND
[0002] Cancer is a leading cause of death and accounts for
approximately 13% of all deaths in the world. Most cancers form
solid tumors in tissues like head and neck, colon, breast, lung,
liver and stomach, and are often characterized by low oxygen
concentrations (hypoxia) and acidification of the microenvironment
surrounding the tumor cells. Hypoxia and acidification of the
extratumoral environment are both associated with aggressive tumor
growth, metastasis formation and poor response to radiotherapy,
surgery and/or to anticancer chemotherapy. The most important
pathway that acts on changes in oxygen concentration is the
`Hypoxia Inducible Factor-1 pathway` (HIF-1 pathway). Under hypoxic
conditions, the transcription factor HIF-1.alpha. is stabilized and
binds to HIF-1.beta.. The formed complex can translocate to the
nucleus and bind to the hypoxic-responsive elements (HRE's) of
genes involved in anaerobic metabolism, pH regulation,
angiogenesis, cell proliferation and survival.
[0003] Carbonic anhydrases (CAs) form a large family of ubiquitous
zinc metalloenzymes of great physiological importance. As catalysts
of reversible hydration of carbon dioxide to bicarbonate and
protons (CO.sub.2+H.sub.2OH.sup.++HCO.sub.3.sup.-), these enzymes
participate in a variety of biological processes, including
respiration, calcification, acid-base balance, bone resorption,
formation of aqueous humor. To date, 16 isozymes are characterized
from which 15 present in humans. CAs in humans are present in
several tissues (e.g. GI tract, reproductive tract, skin, kidneys,
lungs, eyes, . . . ) and are localized in different parts of the
cell. Basically, there are several cytosolic forms (CA I-III, CA
VII), four membrane-bound isozymes (CA IV, CA IX, CA XII and CA
XIV), one mitochondrial form (CA V) as well as a secreted CA
isozyme, CA VI.
[0004] It has been shown that some tumor cells predominantly
express only some membrane-associated CA isozymes, such as CA IX
and CA XII. CAs show considerable diversity in their tissue
distribution, levels, and putative or established biological
functions. Some of the CAs are expressed in almost all tissues (CA
II), whereas the expression of others appears to be more restricted
(e.g., CA VI and CA VII in salivary glands).
[0005] Furthermore, it is conceivable that CA activity might also
be well exploited in tumors, since tumors often display a reversed
pH gradient across the plasma membrane when compared with normal
tissues, indicating the contribution of CAs in providing protons
for acidification of the extracellular environment and bicarbonate
ions to maintain a neutral intracellular milieu. In addition to the
acidifying effect on the extracellular pH, it can influence the
uptake of anticancer drugs and modulate the response of tumor cells
to conventional therapy, such as radiation therapy. One of the CA
isozymes, CA IX, shows restricted expression in normal tissues, but
is tightly associated with different types of tumors. CA IX,
originally detected in human carcinoma HeLa cells as a cell
density-regulated antigen (Pastorekova et al, 1992), is strongly
induced by tumor hypoxia, through a transcriptional activation by
the HIF-1 pathway. Strong association between carbonic anhydrase CA
IX expression and intratumoral hypoxia has been demonstrated in
carcinomas. CA IX distribution is often examined in relation to the
extent of necrosis as an indicator of severe hypoxia and to
microvascular density as a measure of angiogenesis. Furthermore, CA
IX associated with worse relapse-free survival and overall survival
in patients with invasive tumors. CA IX is also a significant
prognostic indicator of overall survival and metastasis-free
survival after radiotherapy and chemoradiotherapy.
[0006] Hypoxia is linked with acidification of extracellular
environment that facilitates tumor invasion and CA IX is believed
to play a role in this process via its catalytic activity (Svastova
et al, 2004). CA IX has a very high catalytic activity with the
highest proton transfer rate among the known CAs, has been shown to
acidify the extracellular environment and is therefore an
interesting target for anticancer therapy, preferably in
combination with conventional treatment schedules. Targeting CA IX
would be preferred above targeting HIF-1, the master regulator of
the transcriptional response of mammalian cells to oxygen
deprivation, since controversial results have been reported
depending on the cell type used, the subunit targeted, the site of
tumor and the timing of HIF-1 inhibition (early or later in tumor
growth). Furthermore, because most of the small-molecules
inhibitors of HIF-1 affect multiple signalling pathways and/or
targets indirectly associated with HIF, assessment of their
activity as HIF inhibitors cannot be based on therapeutic efficacy,
which might be unrelated to HIF inhibition (Melillo, 2006).
[0007] Recently, it has emerged that carbonic anhydrase inhibitors
(CAIs) could have potential, besides the established role as
diuretics and anti-glaucoma drugs, as novel anti-obesity,
anti-cancer and anti-infective drugs. There are 2 main classes of
carbonic anhydrase inhibitors: the metal complexing anions and the
unsubstituted sulfonamides and their derivatives, which bind to the
Zinc ion of the enzyme either by substituting the nonprotein zinc
ligand or add to the metal coordination sphere (Supuran, 2008).
However, the critical problem in designing these inhibitors is the
high number of isozymes, the diffuse localization in tissues and
the lack of isozyme selectivity of the presently available
inhibitors.
[0008] All six classical CAIs (acetazolamide, methazolamide,
ethoxzolamide, dichlorophenamide, dorzolamide, and
dichlorophenamide) used in clinical medicine or as diagnostic
tools, show some tumor growth inhibitory properties. Most of the
clinically used sulfonamides mentioned above are systemically
acting inhibitors showing several undesired side effects due to
inhibition of many of the different CA isozymes present in the
target tissue/organ (15 isoforms are presently known in humans).
Therefore, many attempts to design and synthesize new sulfonamides
were recently reported, in order to avoid such side effects.
Isozymes associated to cell membranes (CA IV, CA IX, CA XII and CA
XIV), with the enzyme active site generally oriented
extracellularly, provide a rational basis for targeting. CA IX and
CA XII are both extracellularly located on hypoxic tumor cells and
are therefore the best candidates.
[0009] The ideal characteristics for specific CA IX inhibitors
should demonstrate a relatively low inhibition constant (Ki in the
nanomolar range) and should be relatively specific over the
cytosolic enzymes CA I and CA II. A number of aromatic sulfonamides
has been presented (see e.g. WO2004/048544) that specifically bind
to the extracellular components of the in particular CAIX enzyme,
which show a higher specificity than those hitherto known in the
art. Therapeutic and diagnostic sulfonamide agents are described in
WO2006/137092. In WO2008/071421 it was shown that the inhibitory
effect of heterocyclic sulfonamides can be further increased by
oxidative substituents, in particular nitrosated or nitrosylated
substituents, since such groups may increase the acidity of the
zinc binding groups and as such being beneficial for the carbonic
anhydrase inhibitory properties. Sulfonamide-based metal chelate
complexes for imaging are described in WO2009/089383. However, a
large variation is reported in CA IX inhibitory constants for the
sulfonamides as well as variation in the selectivity of the
inhibitors. Sulfamate and sulfamide inhibitors have also been
proposed as candidates (Winum et al, 2009).
[0010] Traditional anticancer therapy like surgery, irradiation and
chemotherapy are used to treat cancer patients as a combined or
single treatment. The basic principle of irradiation is to damage
the cancer cells to such an extent that they will die. Free
radicals are formed and damage the DNA immediately or they react
with oxygen, creating reactive oxygen species which damage the cell
and more specific the DNA in the cell. However when no or little
oxygen is present, what is the case in hypoxic tumors, less
reactive oxygen species are formed and the irradiation is not as
effective. It has been shown that a 3 fold higher radiation dose is
required to kill the same amount of hypoxic cells as compared under
normal oxygen concentrations. The concept of radiosensitization of
hypoxic cells emerged when certain compounds were able to mimic
oxygen and thus enhance radiation damage. The first compounds which
demonstrated radiosensitization were nitrobenzenes, followed by
nitrofurans and 2-nitroimidazoles, such as misonidazole (see e.g.
WO2006/102759). Although in experimental tumor models enhanced
radiation damage was observed, most of the clinical trials using
misonidazole were unable to demonstrate a significant improvement
in radiation response, although benefit was seen in certain
subgroups of patients (Overgaard, 1989). The most likely
explanation is the fact that the misonidazole doses were too low,
limited by the risk of neurotoxicity. Alternative, better
radiosensitizing drugs, such as etanidazole and pimonidazole, were
synthesized and tested, but clinical results did not result in a
significant therapeutic benefit. Less toxic drugs, such as
nimorazole, could theoretically achieve lower sensitizing ability
compared with misonidazole, but due to it far lower toxicity, much
higher, clinically relevant doses can be obtained. Only clinical
studies in patients with supraglottic and pharyngeal carcinomas
(DAHANCA 5) resulted in highly significant benefit in terms of
improved loco-regional tumor control and disease-free survival
(Overgaard et al, 1998). More specific targeting towards the
hypoxic tumor cell using lower doses is therefore an important
requisite for new compounds.
BRIEF SUMMARY OF THE INVENTION
[0011] It now has surprisingly been found that the compounds of the
invention, as represented by formula (1):
##STR00001##
in which Z is Z1 represented by formula (2a):
##STR00002##
[0012] or Z is Z2 represented by formula (2b): (CH2).sub.nCH2X
[0013] or Z is Z3 represented by formula (2c):
##STR00003##
[0014] R1 and R2 can be, each independently, H, alkyl, alkenyl,
alkynyl, cycloalkyl, heterocyclic, aryl, cyano or halogen atom,
[0015] R3, R4, R6 and R7 can be, each independently H, alkyl,
alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, halogen atom,
cyano, alkoxy, sulfonamide, sulfamate or sulfamide,
[0016] R5 can be sulfonamide, sulfamate or sulfamide,
[0017] X=sulfonamide, sulfamate or sulfamide,
[0018] Y=O or S,
[0019] and n=0, 1, 2, 3, 4 or 5, demonstrate not only a higher
specificity for CA IX than any compound known in the art, but also
have a much increased radiosensitizing effect. This is the more
unexpected as the stereochemical orientation of the various active
groups in the compounds of the invention is quite different from
that of the compounds known in the art be it sulfonamides or
nitroimidazoles. In other words, it is surprising that the
radiosensitizing activity of the nitroimidazoles has been retained
although the group now is part of a larger entity and the compounds
are less neurotoxic.
[0020] Therefore the compounds of the invention have a
significantly improved overall profile for treating solid tumours,
such as tumours of the breast, brain, kidney, colorectal, lung,
head and neck, bladder etc. compared to carbonic anhydrase
inhibitors known in the art. Also other therapeutic fields such as
treating eye disorders in particular, glaucoma, ocular
hypertension, age-related macular degeneration, diabetic macular
edema, diabetic retinopathy, hypertensive retinopathy and retinal
vasculopathies, epilepsy, high-altitude disorders and neuromuscular
diseases fall within the range of applications of the compounds of
the invention.
[0021] Another finding is that the compounds of the invention also
show an unexpected positive effect on radiosensitivity.
Extracellular acidosis has been thought to be the result of excess
production of lactic acid. However, glycolytic deficient cells
(cells in which lactic acid production is hampered) result in
tumors with a similar extent of extracellular acidosis, indicating
other involved players aside lactic acid. Several studies have been
shown that extracellular acidosis makes tumours less sensitive to
irradiation treatment (Brizel et al, 2001; Quennet et al, 2006).
Carbonic anhydrase inhibiting sulfonamides are able to reduce the
extracellular acidosis in tumors and are therefore a possible tool
to improve the sensitivity to irradiation of tumours. In addition,
the fact that CA IX expression is limited in normal healthy tissue,
while highly overexpressed in tumours, makes the carbonic anhydrase
isozyme IX an attractive target within the concept.
[0022] On the other hand, hypoxic conditions in tumours make them
less sensitive to the ionising radiation commonly used in
radiotherapy (Thomlinson & Gray, 1955). Attracting the CA
inhibitory compounds towards hypoxic cells, would greatly increase
the possible therapeutic effect. This can be done using
nitroimidazoles which are trapped in hypoxic cells after a two-fold
electron reduction upon low oxygen conditions.
[0023] In other words, on the one hand there is a need to increase
the anti-acidic, antitumorigenic effects and specificity of CA IX
inhibiting sulf(on)amides and on the other hand there is a need to
target specifically hypoxic cells using substituted nitroimidazoles
to make compounds more suitable for radiosensitizing therapy.
[0024] These needs are met by the present invention which provides
multifunctional dual CAIX targeting drug compounds and preparations
for the treatment of cancer in a patient in need thereof comprising
compounds of formula 1a-c above.
[0025] Further objects of the present invention are also
pharmaceutical compositions containing at least a compound of the
present invention of formula (1a-c) together with non toxic
adjuvants and/or carriers usually employed in the pharmaceutical
field.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The invention will now be described in more detail below,
with reference to the figures in which
[0027] FIG. 1 shows the synthesis of compounds 4a-d;
[0028] FIG. 2 shows the synthesis of compounds 6a-d;
[0029] FIG. 3 shows a preferred compound (7) of the invention;
[0030] FIG. 4 shows another preferred compound (9) of the
invention;
[0031] FIGS. 5a-b show the effect of compounds (6a, 6c, 7 and 9) of
the invention on acidosis in an in vitro tumor cell culture
model.
[0032] FIGS. 6a-k show the effect of compounds of the invention in
combination with chemotherapeutic agents or radiation in an in vivo
tumor model.
[0033] FIG. 7 depicts formula (1) according to the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0034] FIG. 1 shows scheme 1.
[0035] Reagents and conditions: (i) 1 equiv. of 2-nitroimidazole, 1
equiv. of tert-butyl bromoacetate, 4 equiv. of potassium carbonate,
MeCN, RT, 1 night; (ii) cocktail of trifluoroacetic
acid/water/thioanisole 95/2.5/2.5 v/v, room temperature, 1 night;
(iii) 1 equiv. of 4-dimethylaminopyridine (DMAP), 1 equiv. of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC),
N,N-dimethylacetamide (DMA), room temperature, 2 days.
[0036] FIG. 2 shows scheme 2.
[0037] Reagents and conditions: (i) 1 equiv. of
1-(2-aminoethyl)-2-methyl-5-nitroimidazole dihydrochloride
monohydrate, 1 equiv. SCN-Ph-SO.sub.2NH.sub.2, 2 equiv. of
triethylamine, MeCN, room temperature, 1 hour.
[0038] FIG. 3 shows the scheme of the preferred compound of the
invention.
[0039] Reagents and conditions: (i) 1 equiv. of
1-(2-aminoethyl)-2-methyl-5-nitroimidazole dihydrochloride
monohydrate, 4 equiv. of triethylamine, 1 equiv. of
chlorosulfonylisocyanate, 1 equiv. of tert-butanol,
CH.sub.2Cl.sub.2, rt, 1 hour; (ii) trifluoroacetic acid
CH.sub.2Cl.sub.2 7/3, rt, 6 hours.
[0040] FIG. 4 shows the scheme of the second preferred compound of
the invention.
[0041] Reagents and conditions: (i) 1 equiv. of
2-methyl-5-nitro-1-imidazolylethanol, N,N-dimethylacetamide, 3
equiv. sulfamoyl chloride, rt, 1 night.
[0042] The compounds of the present invention can be synthesised
according to the following procedures. All reagents and solvents
were of commercial quality and used without further purification,
unless otherwise specified. All reactions were carried out under an
inert atmosphere of nitrogen. TLC analyses were performed on silica
gel 60 F.sub.254 plates (Merck Art.1.05554). Spots were visualized
under 254 nm UV illumination, or by ninhydrin solution spraying.
Melting points were determined on a Buchi Melting Point 510 and are
uncorrected. .sup.1H and .sup.13C NMR spectra were recorded on
Bruker DRX-400 spectrometer using DMSO-d.sub.6 as solvent and
tetramethylsilane as internal standard. For .sup.1H NMR spectra,
chemical shifts are expressed in .delta. (ppm) downfield from
tetramethylsilane, and coupling constants (J) are expressed in
Hertz. Electron Ionization mass spectra were recorded in positive
or negative mode on a Water MicroMass ZQ. It is referred to the
attached FIGS. 1 and 2.
tert-butyl-(2-nitro-imidazol-1-yl) acetate (2)
[0043] To a mixture of 2-nitroimidazole 1 (17.7 mmol, 1 equiv.) and
anhydrous potassium carbonate (70.74 mmol, 4 equiv.) in 20 mL of
acetonitrile is added dropwise a solution of tert-butylbromoacetate
(17.7 mmol, 1 equiv.) in 10 ml of acetonitrile. The mixture is
stirred one night at room temperature and concentrated under
vacuum. The residue is purified by chromatography on silica gel
using a mixture CH2Cl2/MeOH 95/5 as eluent to give the expected
compound as white powder in 71% yield. mp 95-97.degree. C.; .sup.1H
NMR (DMSO-d6, 400 MHz) .delta. 1.46 (s, 9H), 4.99 (s, 2H), 7.06 (d,
1H, J=1.01 Hz), 7.17 (d, 1H, J=1.01 Hz). MS (ESI.sup.+/ESI.sup.-)
m/z 226.15 [M-H].sup.-, 262.13 [M.sup.+Cl].sup.-, 250.20
[M+H].sup.+
(2-nitro-imidazol-1-yl) acetic acid (3)
[0044] Compound 2 (2.5 g) is dissolved in 20 mL of a cocktail of
TFA, water, thioanisole 95-2.5-2.5 and stirred at room temperature
for one night. The mixture is then concentrated under vacuum and
co-evaporated several times with diethyl ether until formation of a
powder. After filtration, the precipitate is washed with
dichloromethane and acetonitrile to give quantitatively the
expected product. Mp 143.degree. C. (decomposition); .sup.1H NMR
(DMSO-d.sub.6, 400 MHz) .delta. 5.21 (s, 2H), 7.21 (d, 1H, J=1.01
Hz), 7.64 (d, 1H, J=1.01 Hz). .sup.13C (DMSO-d.sub.6, 101 MHz)
.delta. 50.65, 127.69, 128.44, 168.56, 168.57; MS
(ESI.sup.+/ESI.sup.-) m/z 170.12 [M-H].sup.-, 341.05 [2M-H].sup.-,
194.14 [M+Na].sup.+.
[0045] General Procedure for the Preparation of Compounds
(4a-d)
[0046] To a solution of compound 3 (1.17 mmol, 1 equiv.) in 8 mL of
N,N-dimethylacetamide was added the aminoalkylbenzene sulfonamide
(1.17 mmol, 1 equiv.), 4-dimethylaminopyridine (1.17 mmol, 1
equiv.) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (1.17
mmol, 1 equiv.). The mixture was stirred two days at room
temperature, then diluted with ethyl acetate and washed three times
with water. The organic layer was dried over anhydrous magnesium
sulfate, filtrated and concentrated under vacuum. The residue was
then purified by chromatography on silica gel using methylene
chloride--methanol 98-2 v-v as eluent.
2-(2-nitro-imidazol-1-yl)-N-(4-sulfamoylphenyl)acetamide (4a,
DH296)
[0047] Yield: 68%; mp 163-165.degree. C.; .sup.1H NMR
(DMSO-d.sub.6, 400 MHz) .delta. 5.36 (s, 2H), 7.24 (d, 1H, J=1.01
Hz), 7.28 (s, 2H), 7.67 (d, 1H, J=1.01 Hz), 7.70 (d, 2H, J=8.9 Hz),
7.77 (d, 2H, J=8.9 Hz), 10.7 (s, 1H); .sup.13C (DMSO-d.sub.6, 101
MHz) .delta. 52.20, 118.70, 126.81, 127.57, 128.84, 138.76, 141.18,
144.79, 165.02; MS (ESI.sup.+/ESI.sup.-) m/z 324.09 [M-H].sup.-,
359.92 [M+Cl].sup.-, 649.15 [2M-H].sup.-, 685.01 [2M+Cl].sup.-,
348.14 [M+Na].sup.+.
2-(2-nitro-imidazol-1-yl)-N-(3-sulfamoylphenyl)acetamide (4b,
DH304)
[0048] Yield: 79%; mp 195-197.degree. C.; .sup.1H NMR
(DMSO-d.sub.6, 400 MHz) .delta. 5.35 (s, 2H), 7.24 (d, 1H, J=1.01
Hz), 7.38 (s, 2H), 7.52 (m, 2H), 7.65 (m, 1H), 7.67 (d, 1H, J=1.01
Hz), 8.14 (s, 1H), 10.81 (s, 1H); .sup.13C (DMSO-d.sub.6, 101 MHz)
.delta. 52.16, 116.10, 120.72, 121.92, 127.59, 128.86, 129.67,
138.67, 144.72, 164.87, 167.75; MS (ESI.sup.+/ESI.sup.-) m/z 324.24
[M-H].sup.-, 360.18 [M+Cl].sup.-, 685.13 [2M+Cl].sup.-,
326.24[M+H].sup.+, 348.07 [M+Na].sup.+, 364.17 [M+K].sup.+, 673.18
[2M+Na].sup.+.
2-(2-nitro-imidazol-1-yl)-N-(4-sulfamoylbenzyl)acetamide (4c,
DH305)
[0049] Yield: 83%; mp 181-183.degree. C.; .sup.1H NMR
(DMSO-d.sub.6, 400 MHz) .delta. 4.37 (d, 2H, J=5.7 Hz), 5.22 (s,
2H), 7.18 (d, 1H, J=1.01 Hz), 7.35 (s, 2H), 7.43 (d, 2H, J=8.4 Hz),
7.67 (d, 1H, J=1.01 Hz), 7.76 (d, 2H, J=8.4 Hz), 9.15 (t, 1H,
J=6.06 Hz); .sup.13C (DMSO-d.sub.6, 101 MHz) .delta. 41.81, 51.55,
106.87, 125.58, 127.39, 138.91, 142.66, 142.99, 156.82, 165.88; MS
(ESI.sup.+/ESI.sup.-) m/z 338.15 [M-H].sup.-, 374.22 [M+Cl].sup.-,
713.16 [2M+Cl].sup.-, 340.15 [M+H].sup.+, 362.17 [M+Na].sup.+.
2-(2-nitro-imidazol-1-yl)-N-[2-(4-sulfamoylphenyl)ethyl]acetamide
(4d, DH302)
[0050] Yield: 89%; mp 139-141.degree. C.; .sup.1H NMR
(DMSO-d.sub.6, 400 MHz) .delta. 2.80 (t, 2H, J=6.9 Hz), 3.16 (m
2H), 5.07 (s, 2H), 7.18 (d, 1H, J=1.01 Hz), 7.30 (s, 2H), 7.40 (d,
2H, J=8.2 Hz), 7.61 (d, 1H, J=1.01 Hz), 7.74 (d, 2H, J=8.2 Hz),
8.46 (t, 1H, J=5.6 Hz); .sup.13C (DMSO-d.sub.6, 101 MHz) .delta.
34.59, 51.49, 125.64, 127.38, 128.74, 129.09, 142.05, 143.39,
144.87, 165.57; MS (ESI.sup.+/ESI.sup.-) m/z 352.19 [M-H].sup.-,
388.07 [M+Cl].sup.-, 354.12 [M+H].sup.+, 376.09 [M+Na].sup.+,
729.21 [2M+Na].sup.+.
[0051] General Procedure for the Preparation of Compounds
(6a-d):
[0052] To a solution of the commercially available compound 5 (0.76
mmol, 1 equiv.) in 10 mL of acetonitrile, was added the
corresponding isothiocyanate (0.76 mmol, 1 equiv.). The reaction
was stirred for one hour at room temperature and then filtered. The
filtrate was concentrated under vacuum, and the residue obtained
purified by chromatography on silica gel using methylene
chloride-methanol 95-5 as eluent.
N-(4-sulfamoylphenyl)-N-((2-aminoethyl)-2-methyl-5-nitroimidazolyl)thioure-
a (6a, DH307)
[0053] Yield: 72%; mp 186-188.degree. C.; .sup.1H NMR
(DMSO-d.sub.6, 400 MHz) .delta. 2.42 (s, 3H), 3.91 (m, 2H), 4.5 (t,
2H, J=5.68 Hz), 7.3 (s, 2H), 7.46 (d, 2H, J=8.7 Hz), 7.71 (d, 2H,
J=8.7 Hz), 8.05 (s, 2H), 10.0 (s, 1H); .sup.13C (DMSO-d.sub.6, 101
MHz) .delta. 13.81, 42.87, 44.86, 122.46, 126.30, 133.19, 138.66,
139.14, 141.88, 151.35, 180.88; MS (ESI.sup.+/ESI.sup.-) m/z 385.17
[M+H].sup.+, 407.07 [M+Na].sup.+, 769.22 [2M+H].sup.+, 383.21
[M-H].sup.-, 419.18 [M+Cl].sup.-, 767.16. [2M-H].sup.-.
N-(3-sulfamoylphenyl)-N-((2-aminoethyl)-2-methyl-5-nitroimidazolyl)thioure-
a (6b, DH309)
[0054] Yield: 75%; mp 66-68.degree. C.; .sup.1H NMR (DMSO-d.sub.6,
400 MHz) .delta. 2.43 (s, 3H), 3.91 (m, 2H), 4.49 (t, 2H, J=5.68
Hz), 7.39 (s, 2H), 7.51 (m, 1H), 7.55 (s, 1H), 7.57 (m, 1H), 7.81
(s, 1H, 1H), 7.93 (m, 1H), 8.04 (s, 1H), 9.94 (s, 1H); .sup.13C
(DMSO-d.sub.6, 101 MHz) .delta. 13.81, 42.82, 44.99, 120.29,
121.39, 126.62, 129.09, 133.18, 138.65, 139.51, 144.35, 151.37,
181.28; MS (ESI.sup.+/ESI.sup.-) m/z 385.23 [M+H].sup.+, 406.94
[M+Na].sup.+, 791.19 [2M+Na].sup.+, 383.12 [M-H].sup.-, 419.09
[M+Cl].sup.-, 767.26 [2M-H].sup.-.
N-(4-sulfamoylbenzyl)-N-((2-aminoethyl)-2-methyl-5-nitroimidazolyl)thioure-
a (6c, DH310)
[0055] Yield: 82%; mp 67-69.degree. C.; .sup.1H NMR (DMSO-d.sub.6,
400 MHz) .delta. 2.37 (s, 3H), 2.87 (m, 2H), 4.43 (t, 2H, J=5.18
Hz), 4.70 (br s, 2H), 7.32 (s, 2H), 7.34 (d, 2H, J=8.4 Hz), 7.68
(s, 1H), 7.75 (d, 2H, J=8.4 Hz), 8.03 (s, 1H), 8.15 (s, 1H);
.sup.13C (DMSO-d.sub.6, 101 MHz) .delta. 13.84, 30.64, 42.70,
45.44, 125.52, 127.23, 133.17, 138.61, 142.48, 151.41, 181.44; MS
(ESI.sup.+/ESI.sup.-) m/z 399.23 [M+H].sup.+, 421.16 [M+Na].sup.+,
797.08 [2M+H].sup.+, 819.26 [2M+Na].sup.+, 397.10 [M-H].sup.-,
433.09 [M+Cl].sup.-, 795.33 [2M-H].sup.-.
N-(4-sulfamoylphenylethyl)-N-((2-aminoethyl)-2-methyl-5-nitroimidazolyl)th-
iourea (6d; DH308)
[0056] Yield: 86%; mp 75-77.degree. C.; .sup.1H NMR (DMSO-d.sub.6,
400 MHz) .delta. 2.35 (s, 3H), 2.83 (m, 2H), 3.62 (m, 2H), 3.82 (m,
2H), 4.41 (m, 2H), 7.31 (s, 2H), 7.37 (d, 2H, J=8.2 Hz), 7.52 (s,
1H), 7.63 (s, 1H), 7.74 (d, 2H, J=8.2 Hz), 8.03 (s, 1H); .sup.13C
(DMSO-d.sub.6, 101 MHz) .delta. 13.78, 30.64, 45.4, 125.65, 129.05,
133.18, 138.54, 142.03, 143.45, 151.42, 180.83; MS
(ESI.sup.+/ESI.sup.-) m/z 413.06 [M+H].sup.+, 435.02 [M+Na].sup.+,
825.09 [2M+H].sup.+, 847.21 [2M+Na].sup.+, 411.06 [M-H].sup.-,
447.20 [M+Cl].sup.-, 822.99 [2M-H].sup.-, 859.26 [2M+Cl].sup.-.
Preferred Compounds (7) and (9)
N-[2-(2-methyl-5-nitro-imidazol-1-yl)ethyl]sulfamide (7; DH348)
[0057] To a solution of 5 (3.83 mmol, 1 equiv.) and triethylamine
(30.63 mmol, 4 equiv.) in 10 mL of methylene chloride was added a
solution of chlorosulfonyl isocyanate (4.59 mmol, 1.2 equiv),
tert-butanol (4.59 mmol, 1.2 equiv.) in 2 mL of methylene chloride
(prepared ab-initio). The mixture was stirred at room temperature
for one hour, then diluted with ethyl acetate and washed with
water. The organic layer was dried over anhydrous sodium sulfate,
filtered and concentrated under vacuum. The residue was purified by
chromatography on silica gel using methylene chloride-methanol 98-2
as eluent. This intermediate was then diluted in a solution of
trifluoroacetic acid in methylene chloride (30% volume), and
stirred at room temperature for 6 hours. The mixture was then
concentrated under vacuum and co-evaporated several times with
diethyl ether to give the expected sulfamide as a white powder.
Overall Yield: 70%; mp 122.degree. C.; .sup.1H NMR (DMSO-d.sub.6,
400 MHz) .delta. 2.52 (s, 3H), 3.26 (m, 2H), 4.37 (t, 2H, J=5.81
Hz), 6.65 (s, 2H), 6.86 (s, 1H), 8.1 (s, 1H); .sup.13C
(DMSO-d.sub.6, 101 MHz) .delta. 14.03, 41.8, 46.0, 132.68, 138.26,
151.65; MS (ESI.sup.+/ESI.sup.-) m/z 250.19 [M+H].sup.+, 272.34
[M+Na].sup.+, 499.32 [2M+H].sup.+, 249.09 [M-H].sup.-, 284.12
[M+Cl].sup.-, 533.14 [2M+Cl].sup.-.
N-[2-(2-methyl-5-nitro-imidazol-1-yl)ethyl]sulfamate (9; DH338)
[0058] To a solution of the commercially available compound 8 (1.75
mmol, 1 equiv.) in N,N-dimethylacetamide, was added sulfamoyl
chloride (5.25 mmol, 3 equiv.). The mixture was stirred at room
temperature for one night, then diluted with ethyl acetate, and
washed three times with water. The organic layer was dried over
anhydrous magnesium sulphate, filtered and concentrated under
vacuum. The residue was purified by chromatography on silica gel
using methylene chloride-methanol 9-1 as eluent. Yield: 81%; mp
166-168.degree. C.; .sup.1H NMR (DMSO-d.sub.6, 400 MHz) .delta.
2.45 (s, 3H), 4.35 (t, 2H, J=5.05 Hz), 4.61 (t, 2H, J=5.05 Hz),
7.57 (s, 2H), 8.06 (s, 1H); .sup.13C (DMSO-d.sub.6, 101 MHz)
.delta. 14.04, 44.98, 57.21, 133.10, 138.32, 151.82; MS
(ESI.sup.+/ESI.sup.-) m/z 250.3 [M+H].sup.+, 272.32 [M+Na].sup.+,
521.30 [2M+Na].sup.+,770.16 [3M+Na].sup.+.
[0059] In Vitro Experiments
[0060] The compounds of the invention were tested for their effects
on CA inhibition and the resulting effect on extracellular acidosis
using classical chemistry and biology assays.
[0061] CA Inhibiting Activity
[0062] The compounds of the invention were tested on their
inhibitory activity on carbonic anhydrase in the following
experiment:
[0063] The inhibition constants (K.sub.i) the compounds for four CA
isozymes, CA I, II, IX and XII were determined. An Applied
Photophysics (Oxford, UK) stopped-flow instrument has been used for
assaying the CA-catalyzed CO.sub.2 hydration activity (Khalifah,
1971). Phenol red (at a concentration of 0.2 mM) has been used as
indicator, working at the absorbance maximum of 557 nm, with 10 mM
Hepes (pH 7.5) as buffer, 0.1 M Na.sub.2SO.sub.4 (for maintaining
constant the ionic strength), following the CA-catalyzed CO.sub.2
hydration reaction for a period of 10-100 s. The CO.sub.2
concentrations ranged from 1.7 to 17 mM for the determination of
the kinetic parameters and inhibition constants. For each inhibitor
at least six traces of the initial 5-10% of the reaction have been
used for determining the initial velocity. The uncatalyzed rates
were determined in the same manner and subtracted from the total
observed rates. Stock solutions of inhibitor (1 mM) were prepared
in distilled-deionized water with 10-20% (v/v) DMSO (which is not
inhibitory at these concentrations) and dilutions up to 0.1 nM were
done thereafter with distilled-deionized water. Inhibitor and
enzyme solutions were preincubated together for 15 min at room
temperature prior to assay, in order to allow for the formation of
CA IX-inhibitor complex. The inhibition constants were obtained by
non-linear last-squares methods using PRISM 3 and represent the
mean from at least three different determinations.
[0064] The results are listed in table 1 below.
TABLE-US-00001 TABLE 1 K.sub.I (nM) Compounds hCA I hCA II hCA IX
hCA XII 9 (DH 338) 4435 33.8 8.3 8.9 7 (DH 348) 9576 10.1 20.4 8.1
6a (DH307) 105 5.5 7.3 8.0 6d (DH308) 84 6.6 7.8 7.6 6b (DH309) 483
7.4 7.2 7.7 6c (DH310) 79 2.9 8.3 8.5 4a (DH296) 3203 330 70 64 4d
(DH302) 101 3.8 7.3 8.0 4b (DH304) 107 37 7.9 8.1 4c (DH305) 79 4.8
8.0 6.7
[0065] Acidosis
[0066] The effects of compounds of the invention on acidosis in
tumor cells was measured in the following experiment.
[0067] Aim of the in vitro experiments was to assess the efficacy
of 4 compounds (7, 9, 6a and 6c) in reducing the extracellular
acidification upon hypoxia.
[0068] The experiment was set up as previously described (Dubois et
al, 2007). A colorectal (HT-29) (FIG. 5a) and a cervical (HeLa)
(FIG. 5b) carcinoma cell line were tested in normoxic (ambient
oxygen concentration) and hypoxic (0.2% oxygen) conditions. HT-29
cells are known to be constitutive hypoxia inducible CA IX (CA IX
expression under normoxia and increased CA IX expression upon
hypoxia) expressing cells. Therefore, compounds were added after 1
h of hypoxic exposure (to ensure active CA IX) and incubation was
done for another 23 h (total of 24 h hypoxia). HeLa cells are
hypoxia inducible (at lower density) CA IX expressing cells.
Therefore, HeLa were first incubated for 24 h hypoxia to ensure CA
IX expression in the first place (time point assessed in time
series experiments). Afterwards, compounds were added and cells
were incubated for another 24 h.
[0069] Experiments were performed in triplicate for each condition.
The described conditions were done both for HT-29 and HeLa under
normoxic and hypoxic conditions.
[0070] Following conditions were assessed:
[0071] Blank 1 mM or 0.1 mM (no addition of compound, only
DMSO/PBS)
[0072] DH307 1 mM or 0.1 mM
[0073] DH310 1 mM or 0.1 mM
[0074] DH338 1 mM or 0.1 mM
[0075] DH348 1 mM or 0.1 mM
[0076] S=AEBS (4-(2-aminoethylbenzenesulfonamide); Sigma) 1 mM or
0.1 mM (known to reduce extracellular pH as described by (Svastova
et al, 2004))
[0077] Cells were seeded in 6 cm dishes (HT-29: 10e6; HeLa:
4.times.10.sup.5: to compensate for cell size) in 5 ml of DMEM
supplemented with 10% FCS. The day after, medium was replaced by
3.6 ml of freshly prepared DMEM/FCS 10%, from which the pH was
measured=pH at incubation), after which dishes were placed in the
hypoxic chamber (normoxic dishes remained at ambient air in the
incubator: 37.degree. C., 95% humidity, 5% CO.sub.2). Compounds
were added (1 h for HT-29 or 24 h for HeLa after start hypoxic
exposure) to have a final concentration of 1 mM or 0.1 mM, by
adding 400 .mu.l to the dishes, starting from a 10 or 1 mM stock
(DMSO final concentration 0.1%). Blanc controls received DMSO/PBS
without compound. The pH of the medium was measured after 24 h
(HT-29) or 48 h (HeLa) inside the hypoxic chamber after calibration
of the electrode to reduced oxygen concentrations. Delta pH (pH at
end hypoxic exposure-pH of replacement medium before incubation)
were calculated and shown in the graph of FIGS. 5a and 5b.
[0078] The important results are described below:
[0079] Hypoxia results into an extracellular acidification. The
medium is more acid in blank conditions upon hypoxia compared with
normoxia.
[0080] The control compound (S) effectively reduced the
extracellular acidification upon hypoxia at 1 mM concentration
(less at 0.1 mM) for both cell lines. No effect was seen upon
normoxic exposure
[0081] All compounds were able to reduce the extracellular
acidification. For HT-29 cells, DH348 (compound 7) and DH338
(compound 9) give the best results (1 mM). At 0.1 mM DH348 gives an
additional reduction compared with the other compounds. For HeLa,
all compounds effectively reduced the extracellular acidification.
DH310 even made the medium alkaline (1 mM) and also 0.1 mM caused a
strong reduction. Since this effect was not seen in HT-29,
interpretation should be done carefully. DH348 demonstrated the
best results in HeLa, and confirms the obtained data for HT-29.
[0082] In Vivo Experiments
[0083] Aim of these experiments was to assess the in vivo
therapeutic effect of DH348 in combination with conventional
treatment modalities such as radiotherapy and chemotherapy. We
hypothesize that blocking CA IX may both decrease extracellular
acidosis, and thus increase the effect of irradiation, as well as
increase the uptake of the weak base doxorubicin, and therefore its
therapeutic effect. Furthermore, a clinically approved sulfonamide
Acetazolamide (AZA)--a known general carbonic anhydrase inhibitor
(meaning no preference for one CA)--was used to prove the CA IX
specificity of the investigated compounds.
[0084] Two experimental setups were used. The first experiments
were carried out on parental HT-29 xenografts. The second were done
on HT-29 xenografts harbouring a knock-down for CA IX. A shRNA
construct against CA IX was introduced in the HT-29 cells using the
pRETRO-super vector. After selection and screening, cells with a
95% efficient knock-down for CA IX were selected and designated KD
cells. As a control, a scrambled shRNA construct was used and those
cells were designated EV cells. These cells still demonstrate CA IX
mRNA and protein expression. Tumor xenografts were produced by
injecting the colorectal carcinoma cells (1,5 10e6) subcutaneously
into the lateral flank of NMRI-nu mice (28-32 g). Tumor growth was
monitored 3.times./week by measuring the tumor dimensions in 3
orthogonal directions. Measurements were corrected for skin
thickness (-0.5 mm) and tumor volumes were calculated using the
formula A.times.B.times.C.times.pi/6, were A, B and C represent the
orthogonal diameters. At an average tumor volume of 250 mm.sup.3,
DH348 was injected intravenously (5.times.5 mg/kg intravenously)
using the lateral tail vein. At day 3 animals were anaesthetized
using Sodium Pentobarbital (Nembutal, 0.1 ml/100 g body weight) and
positioned in the irradiation field using a custom-built jig and
subjected to irradiation with a single dose of 10 Gy (15 meV
electron beam) using a linear accelerator (Siemens). Another group
of animals started at day 3 with doxorubicin treatment (5 mg/kg
i.v. IX/week for 3 weeks). Tumor growth and potential treatment
toxicity was monitored (3.times./week), by daily evaluation of the
body weight. When tumors reached four times the treatment starting
volume, animals were sacrificed and tumors excised for further
histopathological evaluation. In summary the treatment groups are
arranged as follows, with the results presented in the Figures:
[0085] 6a: HT-29 parental tumors (WT): effect of irradiation 10 Gy
alone compared to no treatment (controls).
[0086] 6b: HT-29 parental tumors (WT): effect of combination
treatment (DH348+irradiation 10Gy) compared with DH348, irradiation
or no treatment.
[0087] 6c: Effect of irradiation 10 Gy alone compared to no
treatment on HT-29 shscrambled (EV) and shCA IX (KD) tumors.
[0088] 6d: Effect of DH348 treatment compared to no treatment on EV
and KD tumors
[0089] 6e: Effect of AZA treatment compared to no treatment on EV
and KD tumors.
[0090] 6f: Effect of combination treatment with DH348 and
irradiation 10 Gy compared to the single treatment modalities on EV
tumors.
[0091] 6g: Effect of combination treatment with DH348 and
irradiation 10 Gy compared to the single treatment modalities on KD
tumors.
[0092] 6h: Effect of combination treatment with AZA and irradiation
10 Gy compared to the single treatment modalities on EV tumors.
[0093] 6i: Effect of combination treatment with AZA and irradiation
10 Gy compared to the single treatment modalities on KD tumors.
[0094] 6j: HT-29 parental tumors (WT): effect of doxorubicin
treatment compared to no treatment (controls).
[0095] 6k: HT-29 parental tumors (WT): effect of combination
treatment (DH348+doxorubicin) compared with the single treatment
modalities.
[0096] The tumor growth curves are shown in FIGS. 6a-6d. Tumor
volumes are normalized to start treatment=100 mm.sup.3). No
significant treatment toxicity was observed as monitored by total
body weight changes during treatment. Treatment with the compound
of this invention results in a growth delay compared with no
treatment. When combined with conventional clinically available
treatment schedules, it results in a sensitization for radiotherapy
and to a smaller extent for chemotherapy. This is indicated by the
enhanced growth delay for the combination treatment, compared with
the individual treatment arms. Furthermore, we have demonstrated
similar results for the parental and scrambled shRNA bearing
tumors. More important, we have shown a specific effect of the
compounds on CA IX, since (1) the compound had no therapeutic
effect on the CA IX shRNA (KD) tumors and (2) no enhanced growth
delay was observed when combined with irradiation compared with
irradiation alone. The specificity of the compound for CA IX was
further confirmed by evaluation of the general CA inhibitor AZA,
which demonstrated also an effect on the KD tumors. Since several
other CA isozymes then CA IX are present in healthy tissues and AZA
has inhibitory properties against those, normal tissue toxicity in
prolonged treatments can be expected, a phenomenon what will be
greatly reduced using the CA IX specific compounds of this
invention.
[0097] Pharmaceutical Preparations
[0098] The daily dose of active ingredient can be administered to a
host in a single dose or it can be an effective amount divided into
several smaller doses that are administered throughout the day. The
pharmacotherapeutic regimen for treating the aforementioned
diseases with a compound of the invention and/or with the
pharmaceutical compositions of the present invention will be
selected in accordance with a variety of factors, including for
example age, body weight, sex and medical condition of the patient
as well as severity of the disease, route of administration,
pharmacological considerations and eventual concomitant therapy
with other drugs. In some instances, dosage levels below or above
the aforesaid range and/or more frequent may be adequate, and this
logically will be within the judgment of the physician and will
depend on the disease state. The compounds of the invention may be
administered orally, parenterally, rectally or topically, by
inhalation spray o aerosol, in dosage unit formulations containing
conventional non-toxic pharmaceutically acceptable carriers,
adjuvants and vehicles as desired. Topical preparations can be
administered as solutions, suspensions or emulsions (dispersions)
in an acceptable vehicle.
[0099] Topical administration may also involve the use of
transdermal administration such as transdermal patches or
iontophoresis devices. The term "parenteral" as used herein
includes subcutaneous injections, intravenous, intramuscular,
intrasternal injection or infusion techniques. Injectable
preparations, for example, sterile injectable aqueous or oleaginous
suspensions may be formulated according to known art using suitable
dispersing or wetting agents and suspending agents. The sterile
injectable preparation may also be a sterile injectable solution or
suspension in a non-toxic parenterally acceptable diluent o
solvent. Among the acceptable vehicles and solvents are water,
Ringer's solution and isotonic sodium chloride solution.
[0100] Suppositories for rectal administration of the drug can be
prepared by mixing the active ingredient with a suitable
non-irritating excipient, such as cocoa butter and polyethylene
glycols.
[0101] Solid dosage forms for oral administration may include
capsules, tablets, pills, powders, granules and gels. In such solid
dosage forms, the active compound may be admixed with at least one
inert compound such as sucrose, lactose or starch. Such dosage
forms may also comprise, as in normal practice, additional
substances, e.g. lubricating agents such as magnesium stearate. In
the case of capsules, tablets and pills, the dosage forms may also
comprise buffering agents. Tablets and pills can additionally be
prepared with enteric coatings. Liquid dosage forms for oral
administration may include pharmaceutically acceptable emulsions,
solutions, suspensions, syrups and elixirs containing inert
diluents commonly used in the art, such as water. Such compositions
may also comprise adjuvants such as wetting agents, emulsifying and
suspending agents, and sweetening, flavouring and the like.
[0102] It is further contemplated that the compounds of the present
invention can be used with other medicaments known to be useful in
the treatment of carcinomas.
[0103] The above examples are only to be considered as an
illustration of the invention and do not limit the scope of the
invention in any way. Hence, obvious variations and variants of the
invention will be apparent to one skilled in the art.
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