U.S. patent application number 12/747589 was filed with the patent office on 2010-12-23 for use of phthalimide and/or sulphonamide derivatives in the treatment of diseases which require reducing the tnf-alpha levels and an exogenous source of nitric oxide, phthalimide derivatives, sulphonamide derivatives, and a method for obtaining a sulphonamide derivative.
This patent application is currently assigned to UNIVERSIDADE ESTADUAL DE CAMPINAS - UNICAMP. Invention is credited to Chin Chung Man, Jean Leandro Dos Santos, Fernando Ferreira Costa, Carolina Lanaro, Lidia Moreira Lima.
Application Number | 20100324107 12/747589 |
Document ID | / |
Family ID | 40755919 |
Filed Date | 2010-12-23 |
United States Patent
Application |
20100324107 |
Kind Code |
A1 |
Dos Santos; Jean Leandro ;
et al. |
December 23, 2010 |
USE OF PHTHALIMIDE AND/OR SULPHONAMIDE DERIVATIVES IN THE TREATMENT
OF DISEASES WHICH REQUIRE REDUCING THE TNF-alpha LEVELS AND AN
EXOGENOUS SOURCE OF NITRIC OXIDE, PHTHALIMIDE DERIVATIVES,
SULPHONAMIDE DERIVATIVES, AND A METHOD FOR OBTAINING A SULPHONAMIDE
DERIVATIVE
Abstract
The present invention refers to the use of phthalimide and/or
sulphonamide derivatives with nitric oxide donor properties, which
have important activities in increasing the gamma-globin gene
expression and anti-inflammatory and analgesic activities,
effective in the treatment of hematologic diseases which require
reducing the TNF-.alpha. levels and an exogenous source of nitric
oxide. More particularly, the present invention describes the use
of such phthalimide and/or sulphonamide derivatives for the
treatment of sickle-cell disease. The invention also has as a novel
characteristic the disclosure of new functionalized phthalimide
derivatives designed from the prototypes thalidomide and
hydroxyurea, and designed rationally through the strategy of
molecular hybridization for the treatment of said diseases. The
invention still discloses a new method for obtaining a specific
sulphonamide derivative which can be used in the preparation of a
drug for the treatment of diseases which require reducing the
levels of the TNF-.alpha. factor and an exogenous source of nitric
oxide.
Inventors: |
Dos Santos; Jean Leandro;
(em Franca, BR) ; Chung Man; Chin; (em Araraquara,
BR) ; Moreira Lima; Lidia; (Rio de Janeiro, BR)
; Ferreira Costa; Fernando; (em Campinas, BR) ;
Lanaro; Carolina; (em Campinas, BR) |
Correspondence
Address: |
THE NATH LAW GROUP
112 South West Street
Alexandria
VA
22314
US
|
Assignee: |
UNIVERSIDADE ESTADUAL DE CAMPINAS -
UNICAMP
Campinas, SP
BR
UNIVERSIDADE FEDERAL DO RIO DE JANEIRO - UFRJ
Rio de Janeiro, RJ
BR
UNIVERSIDADE ESTADUAL PAULISTA " J LIO DE MESQUITA FILHO " -
UNESP
|
Family ID: |
40755919 |
Appl. No.: |
12/747589 |
Filed: |
December 12, 2008 |
PCT Filed: |
December 12, 2008 |
PCT NO: |
PCT/BR08/00386 |
371 Date: |
September 7, 2010 |
Current U.S.
Class: |
514/417 ;
548/476; 548/477; 564/86 |
Current CPC
Class: |
A61P 31/00 20180101;
A61P 7/06 20180101; A61P 29/00 20180101; A61K 9/0019 20130101; A61P
7/00 20180101; A61K 31/4196 20130101 |
Class at
Publication: |
514/417 ;
548/477; 548/476; 564/86 |
International
Class: |
A61K 31/4035 20060101
A61K031/4035; C07D 209/48 20060101 C07D209/48; C07C 311/48 20060101
C07C311/48; A61P 7/06 20060101 A61P007/06; A61P 29/00 20060101
A61P029/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 12, 2007 |
BR |
PI0705396-7 |
Claims
1. Use of a compound of general formula (I) ##STR00015## Where:
W.dbd.H, halogen, NO.sub.2, NH.sub.2, OH, C.sub.1-C.sub.6 alcoxy,
C.sub.1-C.sub.6 haloalcoxy, C.sub.1-C.sub.6 haloalkyl; R
corresponds to C.sub.1-C.sub.7 alkyl, 2-phenyl, 3-phenyl, 4-phenyl,
2-benzyl, 3-benzyl, 4-benzyl, 2-ethylbenzyl, 3-ethylbenzyl,
4-ethylbenzyl, benzyl, thiophene, furan, pyrrole, 2-pyridine,
3-pyridine, 4-pyridine, pyrazine, pyrimidine, benzothiophene,
benzofuran, indole, quinoline, isoquinoline, naphthalene,
CH.sub.2-2-thiophene, CH.sub.2-3-thiophene, CH.sub.2-2-furan,
CH.sub.2-3-furan, CH.sub.3CH.sub.2-2-thiophene,
CH.sub.3CH.sub.2-3-thiophene, CH.sub.3CH.sub.2-2-furan,
CH.sub.3CH.sub.2-3-furan; R' corresponds to O--NO.sub.2.sup.- or
SO.sub.2NHOH or furoxan or any pharmaceutically-acceptable salt
thereof, characterized by being for the preparation of a drug for
the treatment of diseases which require reducing the levels of the
TNF-.alpha. factor and an exogenous source of nitric oxide.
2. Use according to claim 1, characterized by the compound being of
formula (IA) ##STR00016##
3. Use according to claim 1, characterized by the compound being of
formula (IB) ##STR00017##
4. Use according to claim 1, characterized by the compound being of
formula (IC) ##STR00018##
5. Use according to claim 1, characterized by the compound being of
formula (ID) ##STR00019##
6. Use according to claim 1, characterized by the compound being of
formula (IE) ##STR00020##
7. Use of a compound of general formula (II)
W--R.sub.1--SO.sub.2NHR.sub.2 II wherein W.dbd.H, halogen,
NO.sub.2, NH.sub.2, OH, C.sub.1-C.sub.6 alcoxy, C.sub.1-C.sub.6
haloalcoxy, C.sub.1-C.sub.6 haloalkyl , R.sub.1 corresponds to
2-phenyl, 3-phenyl, 4-phenyl, 2-benzyl, 3-benzyl, 4-benzyl,
2-ethylbenzyl, 3-ethylbenzyl, 4-ethylbenzyl, benzyl, thiophene,
furan, pyrrole, 2-pyridine, 3-pyridine, 4-pyridine, pyrazine,
pyrimidine, benzothiophene, benzofuran, indole, quinoline,
isoquinoline, naphthalene, CH.sub.2-2-thiophene,
CH.sub.2-3-thiophene, CH.sub.2-2-furan, CH.sub.2-3-furan,
CH.sub.3CH.sub.2-2-thiophene, CH.sub.3CH.sub.2-3-thiophene,
CH.sub.3CH.sub.2-2-furan, CH.sub.3CH.sub.2-3-furan; R.sub.2
corresponds to OH, H, C(.dbd.O)NHOH, C(.dbd.S)NHOH, C(.dbd.O)NOH
(C.sub.6H.sub.5); or any pharmaceutically acceptable salt thereof,
characterized by being for the preparation of a drug for the
treatment of diseases which require reducing the levels of the
TNF-.alpha. factor and an exogenous source of nitric oxide.
8. Use according to claim 7, characterized by the compound being of
formula (IIA) ##STR00021##
9. Use according to claim 1, characterized by being for the
treatment of sickle-cell disease.
10. Pharmaceutical composition for the treatment of diseases which
require reducing the levels of the TNF-.alpha. factor and an
exogenous source of nitric oxide characterized by comprising the
compound as defined in claim 1 in a pharmaceutically acceptable
carrier.
11. Method for obtaining the compound of formula (IIA) ##STR00022##
Characterized in that it comprises the following steps: a) mixing,
in a suitable container, hydroxylamine hydrochloride, sodium
bicarbonate and water; b) adding ethanol to the mixture obtained in
step a; c) adding 4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)
benzenesulphonyl chloride to the mixture obtained in step b.
12. Method for obtaining according to claim 11, characterized in
that the addition of ethanol in step b) occurs only after the
elimination of the CO.sub.2 released in step a).
13. Method for obtaining according to claim 11, characterized in
that after step c), the solvent is evaporated and the obtained
product is washed with hot dichloromethane.
14. Compound characterized by being of formula (IC)
##STR00023##
15. Compound characterized by being of formula: ##STR00024##
Description
[0001] The present invention describes the use of to phthalimide
derivatives with nitric oxide donor properties, which have
important activities in increasing the gamma-globin gene expression
and anti-inflammatory and analgesic activities, effective in the
treatment of hematologic diseases which require reduced TNF-.alpha.
levels and an exogenous source of nitric oxide. More particularly,
the present invention describes the use of such phthalimide
derivatives for the treatment of sickle-cell disease.
DESCRIPTION OF THE PRIOR ART
[0002] The sickle-cell disease is the most prevalent hematologic
genetic disease known, and is characterized by a point mutation in
the .beta.-globin gene, more specifically a single nucleotide
change (GTG into GAG) in the sixth codon of the .beta.-globin gene,
resulting in the substitution of a glutamic acid with valine on the
surface of the .beta.-globin chain variant (.beta..sup.s-globin)
(SAFO, M. K et al. J. Med. Chem. v. 47, pp. 4665-4676, 2004).
[0003] The substitution of glutamate with a valine has major
consequences on the three-dimensional structure of hemoglobin.
Glutamic acid is negatively-charged and valine is a neutral amino
acid, thereby allowing the approximation of hemoglobin molecules,
and, consequently, the polymerization, when deoxygenized. In the
deoxy conformation of sickle-cell hemoglobin (Hb S), valine, which
is present in the chain, carries out hydrophobic interactions with
the pocket, comprised of hydrophobic amino acids, from a
neighboring Hb S molecule, which is not possible in the oxygenated
state of hemoglobin, since the hydrophobic pocket is inaccessible
in this condition (ADACHI, K. et al. J. Biol. Chem. v. 263, n. 12,
pp. 5607-5610, 1988).
[0004] These interactions lead to the polymerization of deoxy-Hb S
at low oxygen pressures, a typical situation of capillary beds in
metabolically-active tissues (AVILA, C. M. et al. Bioorg. Med.
Chem. v. 14, pp. 6874-6885, 2006).
[0005] The polymerization of Hb S is the central process of
vaso-occlusion, a characteristic of the sickle-cell disease (BUNK,
H. F. N Engl J Med v. 337, pp. 762-769, 1997; KAUL D. K. et al.
Blood Rev. v. 10, pp. 29-44, 1996; a) FERRONE, F. A. et al. J. Mol.
Biol. v. 183, pp. 591-610, 1985. b) FERRONE, F. et al. J. Mol.
Biol. v. 183, pp. 611-631, 1985; SAMUEL, R. E., et al. Blood. v.
82, pp. 3474-3481, 1993).
[0006] Due to the intracellular polymerization of hemoglobin, on
account of the oxygenation-deoxygenation cycles, the cells
containing Hb S take on a sickle shape.
[0007] The sickling of red blood cells is associated with the
reversible changes of the membrane. With repeated
sickling/desickling cycles, the aberrations in the membrane
function and structure become increasingly pronounced, culminating
in the membrane being fixed in the sickled shape (LEE, G. R. et al.
Vol. I Manole, 1998, pp. 1161-1163.)
[0008] The sickle red blood cells showed a normal adherence to the
vascular endothelium, monocyte, and macrophages (DUITS, A. J. et
al. Clin Immunol Immunopathol v. 81, pp. 96-98, 1996; OKPALA, I. et
al. J. Eur. J. Haematol. v. 69, pp. 135-144, 2002.)
[0009] This property of the sickle blood is given by the deformable
sickle cells, but not by the irreversibly sickled cells, perhaps
because the rigid cells are not able to form multiple surface
contacts with the endothelial cells. This fact denotes a strong
positive correlation with the frequency and severity of the pain
crises. The turbulence areas in the capillaries are the prevailing
sites for adherence.
[0010] Vascular occlusion is the main event responsible for the
clinical picture of sickle-cell disease, being the cause of pain
crises and organ failure. Vaso-occlusive crises initiate at the
venular microcirculation, as the sickle cells become trapped. The
primary event that is critical for vaso-occlusion includes the
adhesion of red blood cells (reticulocytes and deformed dense
cells) to the venular endothelium. This adhesion leads to the
formation of heterocellular aggregates (white blood cells and
sickle cells), which also contribute to obstruction, resulting in
local hypoxia, increase on formation of Hb S polymers, and
propagation of the occlusion of the neighboring vasculature.
Neutrophil transmigrations through endothelial gap junctions
increase the inflammation in the microvasculature (OKPALA, I. et
al. Eur. J. Haematol. v. 69, pp. 135-144, 2002.; OKPALA, I. Blood
Rev. v. 18, pp. 65-73, 2004).
[0011] Sickle red blood cell masses repeatedly clog the vessels of
the microcirculation, leading to painful vascular occlusion crises.
5% to 10% among children or young adults with sickle-sell disease
show, owing to the clogging of the microcirculation vessels,
symptomatic pictures of stroke, effusion or hemorrhage resulting
from stenosis or aneurismal dilatation of important cerebral
arteries.
[0012] It has been reported that the increase of fetal hemoglobin
is beneficial to patients with sickle-cell disease, increasing its
survival and reducing pain episodes (CHARACHE, S. et al. Blood. v.
79, pp. 2555-2565, 1992).
[0013] Recently it has been reported that patients with sickle-cell
disease show a significant increase on the circulating levels of
cytokines, including the tumor necrosis factor-alpha (MALAVE, I. et
al. Acta Haematol. v. 90, pp. 172-176, 1993; FRANCIS, R. Jr., et
al. J. Natl. Med. Assoc. v. 84: 611-615, 1992.; BUCHANAN et al.
Hematology. pp. 35-47, 2004), the increased expression of which is
directly associated with different pathologies of inflammatory
origin (MAKHATADZE, N. J. Hum. Immunol. v. 59, pp. 571-579,
1998).
[0014] TNF-.alpha. exerts pro-inflammatory effects, increasing the
chemiotactic properties, the adherence of neutrophils to the
vascular endothelium, due to the increase of adhesion molecules,
stimulating the production of free radicals and the synthesis of
other inflammatory mediators, such as IL-1 and PGE2. TNF-.alpha.
also induces changes on the coagulation and anticoagulation
properties and increases the hepatic synthesis of some acute-phase
reagents. Furthermore, it is an important mediator of septic
syndrome and endotoxic shock, being able to suppress the
biosynthesis of lipoprotein lipases and lipogenic enzymes in
adipose tissue, impairing the storage of lipids on adipocytes.
[0015] The ability of TNF-.alpha. to change the anticoagulation
properties of the vascular endothelium and to induce the
pro-coagulation activity on the cellular surface of endothelium,
stimulating the production of the platelet-activating factor (PAF),
and increasing the leukocyte adhesion to the vascular endothelium
cells, results in a increase of the resistance to the blood flow,
making circulation difficult and, thus, aggravating the
microvascular stasis and the deoxygenation of Hb S.
[0016] Accordingly, an increase on the TNF-.alpha. blood levels in
patients with sickle-cell disease may aggravate vaso-occlusive
crises and also lead to the occurrence of infectious and
inflammatory episodes (MALAVE, I. et al. Acta Haematol. v. 90, pp.
172-176, 1993).
[0017] In fact, Malave et al (MALAVE, I. et al. Acta Haematol. v.
90, pp. 172-176, 1993), reported an interesting inverse correlation
between the percentage of fetal hemoglobin (Hb F) and the serum
concentration of TNF-.alpha.. These authors showed that patients
having high plasma levels of TNF-.alpha. exhibit a consequent
reduction on Hb F levels. Taking into account that Hb F has a
beneficial effect, improving the tissue oxygenation and reducing
the polymerization of Hb S, such inverse correlation increases the
risk of strokes concurrently with symptoms associated with
sickle-cell disease. In addition, TNF-.alpha. has a major role on
peripheral hyperalgesia, and its inhibition has been associated
with the reduction of chronic and acute pain, which accounts for
the analgesic effect of thalidomide, the first anti-TNF-.alpha.
drug introduced in therapeutics (RIBEIRO, R. A. et al. Eur. J.
Pharmacol., v. 391, pp. 97-103, 2000).
[0018] For these reasons, the inhibition of TNF-.alpha. has been
shown as an important strategy for preventing vascular and
inflammatory complications related with sickle-cell disease.
[0019] Various substances have been reported to have direct action
on the inhibition of TNF-.alpha.. These substances include tumor
necrosis factor-alpha converting enzyme (TACE) inhibitors,
neutralizing antibodies (infliximab), and drugs structurally
related with thalidomide.
[0020] Many laboratories and research groups have been reported the
anti-inflammatory and immunomodulatory properties of thalidomide,
demonstrating its therapeutic potential against the treatment of
pathologies such as multiple myeloma, cachexia, tuberculosis,
arthritis, among others (MIYACHI, H. et al. Bioorg. Med. Chem. v.
5, n. 11, pp. 2095-2102, 1997.)
[0021] In this regard, the development of new thalidomide analogs,
containing the main pharmacophores for the inhibitory activity of
TNF, and free of toxicophoric moieties, responsible for the
teratogenicity, constitutes a unique aim for the development of new
therapeutic possibilities in the treatment of pathologies
associated with or aggravated by the increase on the TNF plasma
levels, as in the case of sickle-cell disease.
[0022] Nevertheless, there is no specific treatment for sickle-cell
disease so far. The treatment of this genetic disease is based on
the use of drugs which minimize or fight against the symptomatology
of sickle-cell disease. Drugs which are useful to the symptomatic
treatment available in the market include desferrioxamine
(Desferal.RTM.), anti-pneumococcal vaccines, prophylactic
penicillin, folic acid (daily doses), and hydroxyurea
(Hydrea.RTM.).
[0023] Hydroxyurea (HU) is a known inhibitor of the synthesis of
ribonucleotide reductase, an enzyme responsible for converting
ribonucleotides into deoxyribonucleotides, interfering with DNA
synthesis, and thus, limits the DNA synthesis (YARBRO, J. M. Semin.
Oncol. v. 19, pp. 1-10, 1992; HANFT, V. N. et al. Blood. v. 95, n.
11, 3589-3593, 2000).
[0024] Although HU is the main drug available for the treatment of
sickle-cell disease approved by the Food and Drug Administration
(FDA) agency, various adverse effects are associated with its
prolonged use, many of which are due to its ability to interrupt
the cell cycle in S and G1 phases (BUCHANAN, G. R. et al.
Hematology pp. 35-47, 2004; STUART, M. J. and NAGEL, R. L. Lancet.
v. 364, pp. 1343-1360, 2004.), which actions characterize it as a
cytotoxic and antineoplastic agent.
[0025] Recent studies have showed that therapy with hydroxyurea
(HU) reduces deaths associated with sickle-cell disease by 40%. The
therapeutic benefit of HU is based on the increase of the levels of
fetal hemoglobin (Hb F), a genetically distinct hemoglobin that
inhibits the polymerization of deoxygenated sickle-cell hemoglobin
(Hb S), preventing or hindering the occurrence of symptoms related
with this pathology (STEINBERG, M. H. et al. JAMA, v. 289, pp.
1645-1651, 2003; CHARACHE, S. et al., Medicine v. 75, pp. 300-326,
1996).
[0026] Besides inhibiting the ribonucleotide reductase, HU also
exerts its action mechanism as a nitric oxide (NO) donator drug, an
important mediator in maintaining the normal blood flow and
pressure. It is known that HU reacts with oxy- and deoxyhemoglobin
to form methemoglobin, which then reacts with another HU molecule
in order to form iron-nitrosyl-hemoglobin (HbNO). The formation of
HbNO involves a number of reactions of the hydroxylamine moiety in
order to form NO (COKIC, V. P. et al. Blood. v. 108, n. 1. pp.
184-191, 2006).
[0027] The benefit of nitric oxide (NO) in the treatment of
sickle-cell disease is based on its ability to stimulate the
production of fetal hemoglobin (Hb F) through the soluble guanylate
cyclase (sGC) pathway. The activation of sGC increases the
expression of .gamma.-globin in erythroleukemic cells and primary
human erythroblasts. The inhibition of sGC prevents this increase,
which suggests that the sGC pathway regulates the expression of
.gamma.-globin, and consequently, the synthesis of fetal hemoglobin
(Hb F). Works have been demonstrating this hypothesis, showing that
HU activates sGC and also induces the expression of mRNA
.gamma.-globin, increasing the levels of fetal hemoglobin (Hb F) in
K562 erythroleukemic cells and human progenitor cells (CONRAN, N.
et al. Br. J. Haematol. v. 124, pp. 547-554. 2004).
[0028] These results suggest that the induction of Hb F mediated by
NO induces the activation of sGC and support the therapeutic
strategy based on nitric oxide for patients with sickle-cell
disease.
[0029] Furthermore, NO has vasodilator effects, which aggregates
beneficial effects in physiopathology and in the treatment of
sickle-cell disease. (KING, S. B. Free Rad. Biol. Med. v. 37, n 6,
pp. 737-744, 2004).
[0030] The present invention relates to the novel use of some
phthalimide derivatives and sulphonamide derivatives in the
preparation of alternative drugs for the treatment of diseases
which involve the need of reducing the levels of the TNF-.alpha.
factor and the need of an exogenous source of nitric oxide. The
invention described herein discloses a solution for the major
limitations associated with the drug therapy of diseases which
involve the need of reducing the levels of the TNF-.alpha. factor
and the need of an exogenous source of nitric oxide, providing an
alternative for the reduction of side and adverse effects of
commonly-used compounds.
[0031] This invention also provides two new phthalimide derivatives
which are used in the preparation of drugs for the treatment of
said diseases, as well as a new process for obtaining a specific
sulphonamide derivative also used in the preparation of drugs for
the treatment of diseases which involve the need of reducing the
levels of the TNF-.alpha. factor and the need of an exogenous
source of nitric oxide. In a more particular aspect, the present
invention overcomes the problems related with the major limitations
and complications associated with the drug therapy conventionally
used for the treatment of sickle-cell disease, thus improving the
quality of life of the patient with sickle-cell disease.
BRIEF DESCRIPTION OF THE INVENTION
[0032] The present invention provides alternatives for the
treatment of diseases in which there is an involvement of the
increase of the TNF-.alpha. levels and the need of an exogenous
source of nitric oxide for treatment.
[0033] In an aspect of the invention, the major limitations and
complications associated with the drug therapy usually employed in
the treatment of sickle-cell disease could be overcome or minimized
with the use of the nitric oxide donor and TNF-.alpha. modulatory
phthalimide and sulphonamide derivatives, thus improving the
quality of life of the patient with sickle-cell disease.
[0034] The present invention refers to the use of a compound of
general formula (I)
##STR00001##
[0035] wherein W.dbd.H, halogen, NO.sub.2, NH.sub.2, OH,
C.sub.1-C.sub.6 alcoxy, C.sub.1-C.sub.6 haloalcoxy, C.sub.1-C.sub.6
haloalkyl; R corresponds to C.sub.1-C.sub.7 alkyl, 2-phenyl,
3-phenyl, 4-phenyl, 2-benzyl, 3-benzyl, 4-benzyl, 2-ethylbenzyl,
3-ethylbenzyl, 4-ethylbenzyl, benzyl, thiophene, furan, pyrrole,
2-pyridine, 3-pyridine, 4-pyridine, pyrazine, pyrimidine,
benzothiophene, benzofuran, indole, quinoline, isoquinoline,
naphthalene, CH.sub.2-2-thiophene, CH.sub.2-3-thiophene,
CH.sub.2-2-furan, CH.sub.2-3-furan, CH.sub.3CH.sub.2-2-thiophene,
CH.sub.3CH.sub.2-3-thiophene, CH.sub.3CH.sub.2-2-furan,
CH.sub.3CH.sub.2-3-furan; corresponds to O---NO2.sup.2 or
SO.sub.2NHOH or furoxan; or any pharmaceutically acceptable salt
thereof, in the preparation of a drug for the treatment of diseases
which require reducing the levels of the TNF-.alpha. factor and an
exogenous source of nitric oxide.
[0036] The invention also refers to the use of a compound of
general formula (II)
W--R.sub.1--SO.sub.2NHR.sub.2 (II)
[0037] wherein W.dbd.H, halogen, NO.sub.2, NH.sub.2, OH,
C.sub.1-C.sub.6 alcoxy, C.sub.1-C.sub.6 haloalcoxy, C.sub.1-C.sub.6
haloalkyl, R.sub.1 corresponds to 2-phenyl, 3-phenyl, 4-phenyl,
2-benzyl, 3-benzyl, 4-benzyl, 2-ethylbenzyl, 3-ethylbenzyl,
4-ethylbenzyl, benzyl, thiophene, furan, pyrrole, 2-pyridine,
3-pyridine, 4-pyridine, pyrazine, pyrimidine, benzothiophene,
benzofuran, indole, quinoline, isoquinoline, naphthalene,
CH.sub.2-2-thiophene, CH.sub.2-3-thiophene, CH.sub.2-2-furan,
CH.sub.2-3-furan, CH.sub.3CH.sub.2-2-thiophene,
CH.sub.3CH.sub.2-3-thiophene, CH.sub.3CH.sub.2-2-furan,
CH.sub.3CH.sub.2-3-furan; R.sub.2 corresponds to OH, H,
C(.dbd.O)NHOH, C(.dbd.S)NHOH, C(.dbd.O)NOH (C.sub.6H.sub.5); or any
pharmaceutically acceptable salt thereof, in the preparation of a
drug for the treatment of diseases which require reducing the
levels of the TNF-.alpha. factor and an exogenous source of nitric
oxide.
[0038] The invention still refers to a pharmaceutical composition
for the treatment of diseases which require reducing the levels of
the TNF-.alpha. factor and an exogenous source of nitric oxide
comprising said composition, said compound being selected among
those resulting from formulae I and/or II or combinations thereof
in a pharmaceutically acceptable carrier.
[0039] The invention also refers to a method for obtaining the
compound of formula IIA
##STR00002##
[0040] comprising the following steps of:
[0041] a) mixing, in a suitable container, hydroxylamine
hydrochloride, sodium bicarbonate and water
[0042] b) adding ethanol to the mixture obtained in step a
[0043] c) adding 4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)
benzenesulphonyl chloride to the mixture obtained in step b
[0044] The invention still refers to a compound of formula
(IC):
##STR00003##
[0045] and also to a compound of formula (IE)
##STR00004##
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1--Effect of derivatives (300 .mu.mol/Kg), via i.p, in
a mouse ear edema assay induced by capsaicin. Values represent the
mean and standard error of the average of 5 animals. (*p<0.05
was considered significant at the 95% confidence level using
Student's t test)
[0047] FIG. 2--Effect of derivatives (300 .mu.Mol/Kg), administered
orally, in a peritonitis assay induced by 3% thioglycolate in
mouse. Values represent the mean and standard error of the average
of 4 animals. (*p<0.05 was considered significant at the 95%
confidence level using Student's t test)
[0048] FIG. 3--Expression of gamma-globin mRNA in the presence of
example 7 at different concentrations, in the absence of hemin, at
times of 24 h, 48 h, 72 h and 96 h.
[0049] FIG. 4--Dose-response curve of compound IIA at
concentrations of 5 .mu.M, 30 .mu.M, 60 .mu.M and 100 .mu.M at
times 24 h, 48 h, 72 h and 96 h in the absence of hemin.
[0050] FIG. 5--Cell viability of the designed compounds.
[0051] FIG. 6--Dosing of nitric oxide by indirect pathway
(nitrite)
DETAILED DESCRIPTION OF THE INVENTION
[0052] Currently, there is no specific treatment for hematologic
diseases of genetic origin, but there are on the market drugs which
are useful to the symptomatic treatment, which improve the quality
of life of patients bearing these diseases.
[0053] The present invention has as its main novel characteristic
the use of functionalized phthalimide and/or sulphonamide
derivatives in the preparation of drugs for the treatment of
diseases which require reduced levels of the TNF-.alpha. factor and
an exogenous source of nitric oxide. The invention also has as a
novel characteristic the disclosure of new functionalized
phthalimide derivatives designed from the prototypes thalidomide
and hydroxyurea, and designed rationally through the strategy of
molecular hybridization for the treatment of said diseases. The
invention also comprises, as another novel characteristic, a new
method for obtaining a specific sulphonamide derivative which can
be used in the preparation of a drug for the treatment of diseases
which require reducing the levels of the TNF-.alpha. factor and an
exogenous source of nitric oxide.
[0054] The new derivative was obtained with good to excellent
chemical yields, by employing a methodology characterized by having
a few synthetic steps, from commercially-available compounds, which
qualifies this methodology for industrial use.
[0055] The present invention refers to the use of a compound of
general formula (I)
##STR00005##
[0056] wherein W.dbd.H, halogen, NO.sub.2, NH.sub.2, OH,
C.sub.1-C.sub.6 alcoxy, C.sub.1-C.sub.6 haloalcoxy, C.sub.1-C.sub.6
haloalkyl; R corresponds to C.sub.1-C.sub.7 alkyl, 2-phenyl,
3-phenyl, 4-phenyl, 2-benzyl, 3-benzyl, 4-benzyl, 2-ethylbenzyl,
3-ethylbenzyl, 4-ethylbenzyl, benzyl, thiophene, furan, pyrrole,
2-pyridine, 3-pyridine, 4-pyridine, pyrazine, pyrimidine,
benzothiophene, benzofuran, indole, quinoline, isoquinoline,
naphthalene, CH.sub.2-2-thiophene, CH.sub.2-3-thiophene,
CH.sub.2-2-furan, CH.sub.2-3-furan, CH.sub.3CH.sub.2-2-thiophene,
CH.sub.3CH.sub.2-3-thiophene, CH.sub.3CH.sub.2-2-furan,
CH.sub.3CH.sub.2-3-furan; R' corresponds to O--NO.sub.2.sup.- or
SO.sub.2NHOH or furoxan; or any pharmaceutically-acceptable salt
thereof, in the preparation of a drug for the treatment of diseases
which require reducing the levels of the TNF-.alpha. factor and an
exogenous source of nitric oxide. Preferably, the compound of
general formula I described is used in the preparation of a drug
for the treatment of sickle-cell disease.
[0057] In a preferred embodiment of the invention, the compound
designated by (1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl
nitrate is used in the preparation of a drug for the treatment of
diseases which require reducing the levels of the TNF-.alpha.
factor and an exogenous source of nitric oxide. Said compound has
the structural formula (IA):
##STR00006##
[0058] and is preferably used in the preparation of a drug for the
treatment of sickle-cell disease.
[0059] In another preferred embodiment of the invention, the
compound designated by
2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl nitrate is used in
the preparation of a drug for the treatment of diseases which
require reducing the levels of the TNF-.alpha. factor and an
exogenous source of nitric oxide. Said compound has the structural
formula shown as follows (IB):
##STR00007##
[0060] and is preferably used in the preparation of a drug for the
treatment of sickle-cell disease.
[0061] Another preferred embodiment of the invention uses the
compound designated by
3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)benzyl nitrate in the
preparation of a drug for the treatment of diseases which require
reducing the levels of the TNF-.alpha. factor and an exogenous
source of nitric oxide. Said compound has the structural formula
(IC):
##STR00008##
[0062] and is preferably used in the preparation of a drug for the
treatment of sickle-cell disease.
[0063] In still another preferred embodiment of the invention, the
compound designated by
4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)benzyl nitrate is used in
the preparation of a drug for the treatment of diseases which
require reducing the levels of the TNF-.alpha. factor and an
exogenous source of nitric oxide. Said compound has the structural
formula (ID):
##STR00009##
[0064] and is preferably used in the preparation of a drug for the
treatment of sickle-cell disease.
[0065] In another preferred embodiment of the invention, the
compound designated by
2-[4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)phenyl]ethyl nitrate
is used in the preparation of a drug for the treatment of diseases
which require reducing the levels of the TNF-.alpha. factor and an
exogenous source of nitric oxide. Said compound has the structural
formula (IE):
##STR00010##
[0066] and is preferably used in the preparation of a drug for the
treatment of sickle-cell disease.
[0067] The present invention also refers to the use of a compound
of general formula (II)
W--R.sub.1--SO.sub.2NHR.sub.2 (II)
[0068] wherein W.dbd.H, halogen, NO.sub.2, NH.sub.2, OH,
C.sub.1-C.sub.6 alcoxy, C.sub.1-C.sub.6 haloalcoxy, C.sub.1-C.sub.6
haloalkyl , R.sub.1 corresponds to 2-phenyl, 3-phenyl, 4-phenyl,
2-benzyl, 3-benzyl, 4-benzyl, 2-ethylbenzyl, 3-ethylbenzyl,
4-ethylbenzyl, benzyl, thiophene, furan, pyrrole, 2-pyridine,
3-pyridine, 4-pyridine, pyrazine, pyrimidine, benzothiophene,
benzofuran, indole, quinoline, isoquinoline, naphthalene,
CH.sub.2-2-thiophene, CH.sub.2-3-thiophene, CH.sub.2-2-furan,
CH.sub.2-3-furan, CH.sub.3CH.sub.2-2-thiophene,
CH.sub.3CH.sub.2-3-thiophene, CH.sub.3CH.sub.2-2-furan,
CH.sub.3CH.sub.2-3-furan; R.sub.2 corresponds to OH, H,
C(.dbd.O)NHOH, C(.dbd.S)NHOH, C(.dbd.O)NOH (C.sub.6H.sub.5); or any
pharmaceutically-acceptable salt thereof, in the preparation of a
drug for the treatment of diseases which require reducing the
levels of the TNF-.alpha. factor and an exogenous source of nitric
oxide. Preferably, the compound of general formula previously
described is used in the preparation of a drug for the treatment of
sickle-cell disease.
[0069] In a preferred embodiment of the invention, the compound
designated by 4-amino-N-hydroxybenzenesulphonamide is used in the
preparation of a drug for the treatment of diseases which require
reducing the levels of the TNF-.alpha. factor and an exogenous
source of nitric oxide. Said compound has the structural formula
(IIA):
##STR00011##
[0070] and is preferably used in the preparation of a drug for the
treatment of sickle-cell disease.
[0071] The subject invention still refers to pharmaceutical
compositions for the treatment of diseases which require reducing
levels of the TNF-.alpha. factor and an exogenous source of nitric
oxide comprising said compositions, said compounds being selected
among those resulting from formulae I and/or II or combinations
thereof. The compositions described in the subject invention
comprise compounds of general formula I and/or II or
pharmaceutically acceptable salts thereof, in association with a
pharmaceutically acceptable excipient.
[0072] The pharmaceutical compositions of the present invention may
be administered in a variety of dosage forms, such as orally, in
the form of tablets, capsules, sugar or tablets covered with a
film, liquid solutions or suspensions; rectally in the form of
suppositories; parenterally, i.e., intramuscularly, or by infusion
or intravenous and/or intrathecal and/or intraspinal injection.
[0073] The pharmaceutical compositions to which this invention
relates are usually prepared according to conventional methods and
administered in a suitable pharmaceutical form.
[0074] Solid oral pharmaceutical forms may contain, together with
the active compound, different diluents, such as lactose, dextrose,
saccharose, cellulose, corn starch, potato starch, or other
suitable diluents; lubricants, such as silica, talc, stearic acid,
magnesium or calcium stearate, and/or polyethylene glycols or other
pharmaceutically acceptable lubricants; binding agents such as
starches, gum arabic, gelatin, methylcellulose,
carboxymethylcellulose, polyvinylpyrrolidone, or other suitable
binding agents; disaggregating agents, such as starch, alginic
acid, alginates or starch or sodium glycolate, or other suitable
disaggregating agents; effervescent mixtures; dyes; sugary
materials; wetting agents such as lectin, polysorbates,
laurylsulphates; and, generally, non-toxic pharmacologically
inactive substances used in pharmaceutical formulations.
Preparations of said pharmaceutical compositions may be performed
in a known way, such as by means of mixture, granulation, pressing
into tablets, sugar covering, film coating processes or other
suitable processes.
[0075] Liquid dispersions for oral administration may include, for
example, syrups, emulsions and suspensions. Syrups may contain, as
a carrier, for example, saccharose or saccharose with glycerine
and/or mannitol and/or sorbitol or another pharmaceutically
acceptable carrier. Suspensions and emulsions may contain, as a
carrier, among others, a natural gum, agar, sodium alginate,
pectin, methylcellulose, carboxymethylcellulose, polyvinyl alcohol
or other suitable carriers.
[0076] Suspensions or solutions for intramuscular injection may
contain, together with the active compound, a pharmaceutically
acceptable carrier, i.e., sterile water, olive oil, ethyl oleate,
glycols, i.e., polyethylene glycol, or other pharmaceutically
acceptable carrier, and, if desired, a suitable amount of lidocaine
hydrochloride. Solutions for intravenous injections or infusions
may contain, as a carrier, for example, sterile water, or
preferably, they may be in the form of sterile salt, aqueous or
isotonic solutions, or may contain, as a carrier, propylene glycol
or another pharmaceutically acceptable carrier.
[0077] The suppositories may contain, together with the active
compound, a pharmaceutically acceptable carrier, such as cocoa
butter, polyethylene glycol, sorbitan polyoxyethylene, fatty acid
ester surfactant, lecithin, or other pharmaceutically suitable
carriers.
[0078] The present invention also refers to a novel method for
obtaining the compound designated by
4-amino-N-hydroxybenzenesulphonamide of formula (IIA)
##STR00012##
[0079] through the following steps of:
[0080] a) mixing, in a suitable container, hydroxylamine
hydrochloride, sodium bicarbonate and water
[0081] b) adding ethanol to the mixture obtained in step a
[0082] c) adding 4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)
benzenesulphonyl chloride to the mixture obtained in step b
[0083] In a preferred embodiment of the invention, the synthesis of
4-amino-N-hydroxybenzenesulphonamide is carried out by adding, into
a 10 mL round-bottom flask, 21.6 mg of hydroxylamine hydrochloride
(0.31 mmol), 26.1 mg of sodium bicarbonate (0.31 mmol), and 0.1 ml
of distilled water. Upon ceasing the elimination of CO.sub.2, 2 mL
of ethanol was added. Next, 100 mg of
4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) benzenesulphonyl
chloride (0.31 mmol) were added. The reaction was observed by thin
layer chromatography (TLC) (eluent: 100% Dichloromethane) until the
end of reaction was indicated. After 45 minutes under reaction, the
solvent is evaporated under reduced pressure, and the obtained
product is washed with hot dichloromethane (so as not to remove the
4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) benzenesulphonyl
chloride which did not react in order to give approximately 82 mg
(80%) of 4-amino-N-hydroxybenzenesulphonamide as a white powder,
with a melting range higher than 275.degree. C.
(C.sub.14H.sub.8ClNO.sub.4S ; PM=318.306). The compound obtained
(4-amino-N-hydroxybenzenesulphonamide) through the described method
is used, as detailed previously, in the treatment of diseases which
require reducing the levels of the TNF-.alpha. factor and an
exogenous source of nitric oxide. Preferably, the obtained compound
(4-amino-N-hydroxybenzenesulphonamide) is used in the preparation
of a drug for the treatment of sickle-cell disease.
[0084] The present invention still refers to a new phthalimide
derivative designated by
3-(1-3-dioxo-1,3-dihydro-2H-isoindol-2-yl)benzyl nitrate
represented by the formula shown as follows (IC)
##STR00013##
[0085] and used, as described previously, in the preparation of a
drug for the treatment of diseases which require reducing the
TNF-.alpha. levels and an exogenous source of nitric oxide.
[0086] The invention also refers to another phthalimide derivative
designated by
2-[4-(1-3-dioxo-1,3-dihydro-2H-isoindol-2-yl)phenyl]ethyl nitrate
of general formula shown as follows (IE)
##STR00014##
[0087] and used, as described previously, in the preparation of a
drug for the treatment of diseases which require reducing the
TNF-.alpha. levels and an exogenous source of nitric oxide.
[0088] The compounds described in the present invention were
subjected to a number of tests in order to ensure the intended
activities in the treatment of diseases which require reducing the
TNF-.alpha. levels and an exogenous source of nitric oxide.
Particularly, the compounds were subjected to tests in order to
ensure their activity as auxiliary agents in the treatment of
symptoms of sickle-cell disease. The tests carried out and the
results obtained are described in the following.
[0089] Mutagenic Activity test
[0090] Firstly, the compounds were evaluated with the AMES test in
order to identify a possible mutagenicity. This test is important
to obtain compounds having a lower genotoxic profile, and also
guides molecular changes to obtain more safe compounds. The tests
were conducted with the previously described compounds of general
formula IA, IB, IC, ID, IE, and IIA and are shown in tables 1, 2
and 3 below.
TABLE-US-00001 TABLE 01 Mutagenic evaluation in Salmonella
typhimurium TA100 and TA102 strains in the presence and absence of
metabolic activation (S9) of the compound IA. concentration TA 100
TA 102 Compound nmol/plate +S9 -S9 +S9 -S9 IA 0 129.3 .+-. 8.1
136.7 .+-. 12.4 213.5 .+-. 15.5 197.33 .+-. 16.01 7.25 154 .+-.
14.2 (1.19) 140.5 .+-. 17 (1.03) 298 .+-. 11.3 (1.4) 263.7 .+-. 15
(1.33) 14.5 155 .+-. 5.9 (1.19) 161.7 .+-. 11 (1.18) 320.8 .+-. 17
(1.5) 249.7 .+-. 12 (1.26) 29 140.3 .+-. 2 (1.08) 122.7 .+-. 3.06
(0.9) 385 .+-. 21.8 (1.8) 261.3 .+-. 20 (1.32) 56 126.3 .+-. 8.1
(0.97) 234 .+-. 39.5 (1.71) 499 .+-. 8.9 (2.34) 153 .+-. 21 (0.77)*
112 133.3 .+-. 11 (1.03) 335 .+-. 15.7 (2.45) 351.2 .+-. 12 (1.64)*
141 .+-. 18 (0.71)* *cell death
TABLE-US-00002 TABLE 02 Mutagenic evaluation in Salmonella
typhimurium TA100 and TA102 strains in the presence and absence of
metabolic activation (S9) of compounds IB, IC and IIA.
concentration TA 100 TA 102 Compound .mu.mol/plate +S9 -S9 +S9 -S9
IB 0 104 .+-. 7.4 115 .+-. 13.2 219 .+-. 8.9 323 .+-. 10.2 0.01 354
.+-. 6.9 (3.4) 171 .+-. 48.9 (1.49) 249 .+-. 11.7 (1.13) 394 .+-.
30 (1.22) 0.021 335 .+-. 10.8 (3.22) 200 .+-. 23.3 (1.74) 269 .+-.
10.6 (1.22) 452 .+-. 23 (1.55) 0.042 397 .+-. 25.9 (3.8) 223 .+-.
45.04 (1.94) 239 .+-. 22.5 (1.09) 502 .+-. 14 (1.25) 0.085 395 .+-.
40.2 (3.8) 206 .+-. 30.2 (1.8) 247 .+-. 21.4 (1.12) 405 .+-. 24
(1.34) 0.17 261 .+-. 11 (2.5)* 165 .+-. 11.1 (1.43)* 214 .+-. 32
(0.97)* 433 .+-. 35.4 (1.54) IC 0 129 .+-. 8.1 179 .+-. 8.72 372
.+-. 27.5 254.7 .+-. 14.6 0.224 165 .+-. 13.2 (1.27) 216.7 .+-.
10.2 (1.21) 272 .+-. 26 (0.73) 281 .+-. 25 (1.10) 0.488 153 .+-.
13.1 (1.18) 224.5 .+-. 24.6 (1.25) 378 .+-. 6.1 (1.01) 286 .+-. 4.2
(1.12) 0.896 146 .+-. 5 (1.12) 266.3 .+-. 8.4 (1.48) 405 .+-. 11
(1.09) 303 .+-. 9.2 (1.19) 1.8 163 .+-. 22.5 (1.26) 239.7 .+-. 11.3
(1.34) 399.3 .+-. 40 (1.07) 395.7 .+-. 9 (1.55) 3.58 158.3 .+-. 9.3
(1.22) 323.7 .+-. 10.2 (1.8) 431 .+-. 17.9 (1.15) 387 .+-. 9.7
(1.52) IIA 0 129.3 .+-. 8.14 146.7 .+-. 12.1 372.3 .+-. 27.5 197.3
.+-. 16.1 0.98 160 .+-. 12.4 (1.24) 146.3 .+-. 5.1 (0.99) 385 .+-.
33.1 (1.03) 222 .+-. 12 (1.12) 1.96 175.7 .+-. 2 (1.36) 180.5 .+-.
7.8 (1.23) 323 .+-. 5.54 (0.87) 241 .+-. 13 (1.22) 3.92 185.3 .+-.
15 (1.43) 179 .+-. 11.3 (1.22) 423.7 .+-. 22 (1.13) 251 .+-. 19.2
(1.27) 7.85 206 .+-. 13.6 (1.59) 172.7 .+-. 12.5 (1.17) 387 .+-.
14.4 (1.03) 232 .+-. 13.1 (1.17) 15.7 354 .+-. 6.5 (2.74) 175 .+-.
2.8 (1.19) 300 .+-. 4.9 (0.8)* 210 .+-. 23 (1.06) *cell death
TABLE-US-00003 TABLE 03 Mutagenic evaluation in Salmonella
typhimurium TA100 and TA102 strains in the presence and absence of
metabolic activation (S9) of compounds ID and IE. concentration TA
100 TA 102 Compound .mu.mol/plate +S9 -S9 +S9 -S9 ID 0 129.3 .+-.
8.1 179 .+-. 8.7 372.3 .+-. 27.5 260.1 .+-. 11.6 0.224 157 .+-. 13
(1.21) 228 .+-. 18 (1.27) 405.7 .+-. 13 (1.09) 340.3 .+-. 9.7
(1.31) 0.488 184.7 .+-. 16 (1.42) 239 .+-. 11.5 (1.33) 434.7 .+-.
21 (1.16) 373.5 .+-. 21 (1.43) 0.896 216.7 .+-. 18 (1.67) 231 .+-.
20.2 (1.29) 370 .+-. 6 (0.99) 391.2 .+-. 6.3 (1.5) 1.8 192.7 .+-.
16 (1.49) 212.3 .+-. 17 (1.18) 397.3 .+-. 9.3(1.06) 486.8 .+-. 20
(1.87) 3.58 263 .+-. 12.3 (2.03) 349 .+-. 5.6 (1.94) 427.3 .+-. 5
(1.14) 420 .+-. 8.6 (1.62)* IE 0 129.3 .+-. 8.1 136.7 .+-. 12.4
372.3 .+-. 27.5 197.3 .+-. 16.1 0.12 363 .+-. 25.2 (2.8) 143.3 .+-.
5 (1.04) 335.3 .+-. 11 (0.9) 256 .+-. 6.9 (1.29) 0.25 478.7 .+-.
4.22 (3.7) 135.3 .+-. 12 (0.99) 295.3 .+-. 28 (0.8) 262 .+-. 19.1
(1.32) 0.5 628 .+-. 40.8 (4.86) 136.3 .+-. 13 (0.99) 406 .+-. 14.1
(1.09) 266.7 .+-. 18 (1.35) 1 739 .+-. 25.4 (5.72) 121.5 .+-. 14
(0.89) 465 .+-. 10.6 (1.25) 265.7 .+-. 10 (1.34) 2 750 .+-. 13.6
(5.79) 177 .+-. 23.6 (1.30) 447 .+-. 19.8 (1.2) 201 .+-. 4.7
(1.02)* *cell death
[0091] The compound of formula IA showed mutagenicity ratios (RM)
of 2.45 and 2.34; in the TA100 strain, the absence of metabolic
activation (112 nmol/plate), and in the TA102 strain, in the
presence of metabolic activation (56 nmol/plate), respectively. At
concentrations higher than 56 nmol in TA 102 (+S9), toxicity can be
observed, with a reduction on the number of revertants per plate
(Table 01). Compound IA is an alkyl derivative which has reactive
methylene carbon, i.e., the carbon atom has a positive partial
charge by removal of the electron density due to the more
electronegative a moieties. The presence of this methylene carbon
promotes the attack by bionucleophiles, leading to elimination of
the nitrate, which readily decomposes into a radical species
generating a number of detrimental effects in the DNA of the
prokaryote, which does not have a repair system as efficient as
that of eukaryotes. Further, there may occur addition of the
bionucleophile into the reactive methylene carbon, resulting in the
formation of a covalent adduct, irreversibly modifying the original
structure of said bionucleophile.
[0092] The compound of formula IB shows, at the concentrations
used, mutagenicity in TA100 strain in the presence of metabolic
activation, at all tested concentrations and with the following
mutagenicity ratios (MR): 0.01 .mu.mol (3.4); 0.021 .mu.mol (3.22);
0.042 .mu.mol (3.8); 0.085 .mu.mol (3.8) e 0.17 .mu.mol (2.5)
(Table 02). Among the alkyl series tested, that was the one which
showed the highest MR. When compound IB is compared with compound
IA, it can be clearly seen that the latter has a lower hindrance to
attacks on the methylene carbon, facilitating the access of the
bionucleophile. The hindrance in compound IA is higher due to the
presence of bulky moieties at positions .alpha.. This could account
for the higher mutagenicity ratio of compound IB, and allows us to
predict that methylene compounds will be less mutagenic than
ethylene compounds. This hypothesis is better analyzed through the
results of tests with compounds ID and IE.
[0093] Compound IC did not show mutagenicity at the concentrations
used, although in the test with the TA100 strain in the absence of
metabolic activation and at the concentration of 3.58
.mu.mol/plate, it showed a mutagenicity ratio of 1.8; that is,
signs of mutagenicity (Table 01). When compound IA is compared with
compound IC, it can be observed that the latter--an interphenylene
derivative--has a lower mutagenicity since the sign has appeared
only at 3.58 .mu.mol/plate, while in compound IA, mutagenicity has
occurred at 112 nmol/plate with a MR of 2.45 (TA100; -S9). Also,
when the compound IC is compared with the compound IB, it can be
observed a lower mutagenicity of the interphenylene derivative.
These data suggest that the aryl derivatives, that is, those having
an aromatic ring bonded to the phthalimide moiety (compounds IC,
ID, IE and IIA), have a lower mutagenicity than the alkyl
derivatives, that is, those in which the alkyl chain is directly
bonded to the phthalimide moiety (compounds IA and IB); supporting
the hypothesis that steric factors hinder the access of the
bionucleophile to the reactive site, modulating the mutagenicity of
the synthesized derivatives.
[0094] The compound IIA is a sulphonamide derivative which does not
have the nitrate moiety, common to all other compounds. The
literature reports that hydroxylamine derivatives, or hydroxamic
acid derivatives, show mutagenicity largely due to the major
toxicophoric contribution from this moiety (ZHU, X. et al. Mut.
Res. v. 425, pp. 153-167, 1999). For a long time, the hydroxylamine
moiety has been pointed out as one of the main metabolites,
generated in the reduction of the nitro group, responsible for the
mutagenic activity of nitro compounds (e.g., chloramphenicol,
metronidazole, and nitrofurans). Nevertheless, it has been found
that they are radical species formed in steps prior to reductions
which generate mutagenic products rather than the hydroxylamine
derivative itself (TOCHER, J. H. Gen. Pharmac. v. 28, n. 4. pp.
485-487, 1997).
[0095] Thus, the compound IIA containing the hydroxylamine moiety
was evaluated in order to verify the toxicophoric contribution from
this moiety in the synthesized compound. This derivative was
subsequently reacted with phthalic anhydride to obtain a
phthalimide derivative.
[0096] At the concentration of 15.7 .mu.mol/plate in the TA100
strain, with metabolic activation, the compound IIA exhibited a
mutagenicity ratio of 2.74. Above 15.7 .mu.mol/plate, there is a
reduction on the number of revertants per cell toxicity. The
possible mutagenesis of compound IIA, which occurs only in the
presence of metabolic activation, could be assigned to the
formation of a radical and/or oxidized derivative from this
compound. When comparing the concentration of compound IIA used in
the test with the aryl derivatives (compounds IC, ID and IE), it
can be seen that, although there is mutagenesis, it is observed
only at high concentrations, being up to 125 times higher, in
number of moles, than that found for compound IE (0.12
.mu.mol/plate).
[0097] The compound ID showed in the TA100 strain, in the presence
of metabolic activation and at a concentration of 3.58
.mu.mol/plate, a mutagenicity ratio of 2.03; while in the absence
of metabolic activity, at this same concentration, it showed
mutagenicity signs with MR values of 1.94. At a concentration of
1.8 .mu.mol/plate in the absence of S9, in the TA102 strain, it
showed mutagenicity signs, with a MR of 1.87 (Table 03). When
comparing the compound ID, a regioisomer of the compound IC, we
observed a discrete profile of higher mutagenicity and/or a
mutagenicity sign of the compound ID with respect to compound
IC.
[0098] The compound IE, an interphenylene derivative of compound
IB, in the TA100 strain and in the presence of metabolic
activation, showed, as well as compound IB, mutagenesis at
concentrations of 0.12; 0.25; 0.5; 1 and 2 .mu.mol/plate, with MR
values of 2.8 ; 3.7; 4.86; 5.72 e 5.79, respectively (Table 03).
Although MR values are higher in compound IE with respect to
compound IB, the latter is at a lower molar concentration. The need
for higher concentrations for compound IE to show mutagenesis
confirms, in structural terms, what had already been observed
between compounds IA and IC: the presence of phenyl bonded to the
phthalimide moiety reduces the mutagenicity of the compounds.
[0099] When comparing compound IE with compound ID, both
para-substituted, we could also confirm, as seen between compounds
IA and IB, that the ethyl spacing increases mutagenesis, when
compared to methyl spacing. As discussed previously, this factor is
probably related, besides the electron factor, mainly to the steric
factor, due to the better access by nucleophiles to the carbon a to
the nitrate moiety.
[0100] When relating the obtained compounds with thalidomide and
hydroxyurea patterns, we observed a sensitivity of the AMES test in
responding to the examples, when compared to HU and thalidomide.
This observation, although it suggests a higher mutagenic activity
of the synthesized compounds regarding the patterns of structural
planning, can not be conclusive, and more tests are required for
such statement. In addition, thalidomide, HU and synthesized
compounds are structurally distinct and, due to this chemical
particularity, could show a differentiated mutagenic profile.
[0101] From the sets of results obtained in the AMES test, we could
infer that:
[0102] Alkyl derivatives (compounds IA and IB) show higher
mutagenicity expressed by the average of the number of
revertants/plate than aryl derivatives (compounds IIA, IC and
ID);
[0103] Derivatives with ethylene spacing show higher mutagenesis
than methylene compounds;
[0104] This set of results allows us to conclude that the benzyl
spacing is more suitable in order to obtain compounds with lower
mutagenicity.
[0105] Further, even for mutagenic compounds, a mutagenicity ratio
of 2 is considered low if compared to drugs used in therapeutics,
such as metronidazole, which has a MR of 14.9 when tested at 58.4
.mu.mol in TA 100 without metabolic activation (SILVA, A. T. A. et
al. Mini Rev. Med. Chem., v. 5, pp. 893-914, 2005). This allows us
to conclude that although there are signs of mutagenicity for the
compounds, it is too low, and these results may not reflect in
eukaryotic cells.
[0106] Mouse Ear Edema Assay Induced by Capsaicin
[0107] This assay is characterized by an acute inflammatory
response of the ear, with development of edema, and it was
performed in order to evaluate the anti-inflammatory activity of
the synthesized compounds.
[0108] In this assay, indomethacin was used as a control at 100
.mu.mol/Kg, and the phthalimide derivatives were firstly evaluated
at 300 .mu.mol/Kg via i.p. From table 4, it can be observed that
compounds IC and IE show an ear edema inhibition percentage higher
than 64%. Compound IC showed an inhibition percentage of about
64.09% in the performed assay. The other compounds, however, show a
similar activity when compared to indomethacin (FIG. 01), taking
into consideration the standard error. These results suggest that
the synthesized compounds show anti-inflammatory activity in the
acute phase, probably due to the inhibition of the cytokine
TNF.alpha., since it is known that phthalimide derivatives have
this activity.
TABLE-US-00004 TABLE 4 Ear edema assay induced by capsaicin (i.p.)
Compound Compound Compound Compound Compound Compound +Control IA
IB IC IIA ID IE N 5 5 5 5 5 5 5 Average -- 53.75 46.55 64.09 49.24
37.18 76.58 inhibition % EPM -- 8.66 3.49 6.44 10.58 19.38 5.32 *
Animals showed higher values than controls
[0109] Peritonitis Assay
[0110] In a second assay, the number of total leukocytes
(10.sup.6/mL) was evaluated in order to evaluate the ability to
inhibit their infiltration in the inflammatory process. All
phthalimide derivatives showed inhibition activity of the leukocyte
infiltrate (FIG. 2; Table 5) with a similar activity profile,
considering the standard error. These results demonstrate the
anti-inflammatory potential of these compounds.
TABLE-US-00005 TABLE 5 Results of the peritonitis assay Compound
Compound Compound Compound Compound Compound +Control IA IB IC IIA
ID IE N 3 3 3 3 3 3 3 Inhibition % 12.5 51.25* 42.85 28.57 32.14
26.25 Cell 5.6 4.9 2.73 3.2 4.0 3.8 4.13 Number avg (.times.106)
EPM 0.20 0.7 1.0 0.8 0.30 0.2 1.16 # The animal has bleed *P <
0.05 (Student's T test)
[0111] Abdominal Writhing Induced by Acetic Acid
[0112] In order to evaluate the peripheral analgesic activity of
the compounds, an abdominal writhing assay induced by acetic acid
(Table 6) was carried out. In this assay, we could infer that
compound ID has an important analgesic activity, inhibiting
abdominal writhing by 66%. Other compounds, such as IA, IIA and IE,
also have significant inhibitions of the abdominal writhing induced
by acetic acid, demonstrating the analgesic potential of these
compounds. The analgesic activity may be related to the ability of
these compounds to inhibit the cytokine TNF.alpha., since it is
known that this would be one of the mechanisms that explain the
analgesia of molecules such as thalidomide.
TABLE-US-00006 TABLE 6 Abdominal writhing assay induced by acetic
acid Compound Compound Compound Compound Compound +Control IA IB
IIA ID IE Average 47.54 28.6 39.2 30.4 16.2 Inhibition % -- 39.83
23.22 36.05 66.03 33.95 EPM 3.39 9.25 8.65 7.83 5.24 6.60
[0113] Assays for evaluating the increase of gamma-globin by PCR in
K562 cell culture
[0114] From results obtained in the evaluation of the gene
expression induced by compounds in a culture of K562
erythroleukemic cells, and quantified by Real Time PCR, we can
conclude that:
[0115] Compound IE has activity in this model, increasing the gene
expression of gamma-globin, in the presence or absence of
hemin;
[0116] In the presence of hemin, compound IE did not show a
significantly higher activity than in the absence of hemin, when
compared to the control (FIG. 3);
[0117] Apparently, compound IE has an effect on the expression of
gamma-globin at low concentration (5 .mu.M e 30 .mu.M);
[0118] The compound IE showed high percentages of cell viability
(higher than 90%) in assays with and without hemin, demonstrating
the absence of toxic effects at the concentrations used;
[0119] Compound IIA showed higher activity than the control in the
expression of gamma-globin (FIG. 4).
[0120] The increase on the expression of gamma-globin induced by
compound IIA is higher than the increase provided by compound IE,
suggesting that compound IIA is more efficient in increasing the
gene expression of gamma-globin.
[0121] When comparing the compound IIA with HU data in the
literature, it was observed that the compound IIA shows activity at
5 .mu.M in 48 hours, whereas for HU to produce a comparable
activity, it is used at 10 .mu.M in the same time.
[0122] The cell viability at 0 h was 97%, and this pattern was
maintained during the realization of the assay, demonstrating the
absence of toxicity of the compound IIA (FIG. 5).
[0123] Methodologies used for pharmacological assays:
[0124] Procedures for evaluating the mutagenic activity (AMES
Test)
[0125] The procedure was firstly developed by (MARON, D. M. and
AMES, B. N. Mut. Res. v. 113, pp. 173-215, 1983)
[0126] Strains Used in the Assay:
[0127] There are many genetically modified strains of Salmonella
typhimurium in order to detect a prevalent type of mutation, which
include: TA97, TA98, TA100 and TA102. TA100 and TA102 detect
mutations which cause base pair substitutions, while TA 98 and TA
97 detect changes where there is a gap in the DNA reading frame
(MARON & AMES, 1983).
[0128] For this assay, Salmonella typhimurium TA100 and TA102
strains from the mutagenicity laboratory of the "Faculdade de Ci
ncias Farmac uticas--UNESP Araraquara" were used. Such strains have
the following characteristics: (AMES, 1983)
[0129] 1--Are auxotrophic with respect to histidine;
[0130] 2--Have various mutations in the histidine operon, which are
target for reverse mutation;
[0131] 3--Detect many mutagenic agents which cause shift in the DNA
reading frame, which restore the correct reading frame for
histidine synthesis;
[0132] 4--Mutation in hisG46 gene, in the reading frame of the hisG
gene, which encodes the first enzyme for histidine synthesis, TA
100-specific;
[0133] 5--Mutation in gene hisD3052, constituted by 8 repeated
residues of -GC--, near the shift mutation site in the reading
frame of the hisD gene, which encodes the TA 98-specific histidinol
dehydrogenase enzyme;
[0134] 6--Mutation (rfa), which causes partial loss of the
lipopolysaccharide barrier, increasing the permeability of the
bacterial cell wall, facilitating the diffusion of large molecules
into the cell;
[0135] 7--Mutation (urvB), which causes damage in the repair system
by excision, resulting in an increase on the detection sensitivity
of various mutagenic agents. It also causes the bacterium to become
dependant on biotin to grow;
[0136] 8--Plasmid pKM101, which enhances the resistance to
ampicillin, and also increases the spontaneous and chemical
mutagenesis by stimulating the error-prone DNA repair system.
[0137] Maintenance and Storage of Strains
[0138] Salmonella typhimurium strains were stored in a freezer at
-80.degree. C., in flasks for freezing with 0.9 mL of culture and
0.1 mL of DMSO as a cryoprotector agent, so as to maintain all
their genetic characteristics unchanged.
[0139] Before freezing, all strains had their genotypes confirmed
(histidine auxotrophy, rfa mutation, pKM101 plasmid, uvrb deletion,
and spontaneous reversion rate).
[0140] Preparation of Culture Media and Solutions
[0141] Vogel-Bonner Medium E (VB)
[0142] 0.25 g of magnesium sulphate, 2.5 g of citric acid, 12.5 g
of dibasic potassium phosphate, and 4.375 g of sodium and ammonium
phosphate were dissolved into 16.75 mL of distilled water at
45.degree. C. (amounts enough for 25 mL of VB solution). The
solution was sterilized in an autoclave for 15 minutes at
121.degree. C.
[0143] 40% Glucose
[0144] 50 mL of a 40% glucose solution were prepared, which was
sterilized in an autoclave for 15 minutes at 121.degree. C.
[0145] Glycosylated Minimum Agar (GMA)
[0146] 7.5 g of agar was dissolved into 465 mL of distilled water,
and then the solution was sterilized in an autoclave for 15 minutes
at 121.degree. C.
[0147] Subsequently, a sterile laminar flow, 10 mL of VB, and 25 mL
of 40% glucose were added.
[0148] Top Agar
[0149] 0.5 g of sodium chloride and 0.6 g of agar were dissolved
into 100 mL of distilled water. The solution was sterilized in an
autoclave for 15 minutes at 121.degree. C.
[0150] 0.05 mM biotin/histidine solution (10 mL/100 mL of top
agar)
[0151] 0.00123 g of biotin and 0.00096 g of histidine were
dissolved into 10 mL of distilled water. The solution was
sterilized in an autoclave for 15 minutes at 121.degree. C.
[0152] Oxoid Nutrient Broth n.2.
[0153] 0.75 g of Oxoid medium was dissolved into 30 mL of distilled
water. The solution was sterilized in an autoclave for 15 minutes
at 121.degree. C.
[0154] Positive and Negative Controls
[0155] The negative control is the solvent used to dissolve the
sample, using as a standard volume, the highest volume of the
tested sample 100 .mu.L, which is also the amount that is required
to dissolve the maximum used concentration of the drug.
[0156] The positive controls are mutagenic compounds specific for
each strain and test condition, with 25 .mu.L/plate of sodium azide
(1.25 .mu.g/plate) and 100 .mu.L of mitomycin (0.5 .mu.g/plate)
being the controls for TA100 and TA102, respectively, in the
absence of metabolic activation. For assays with metabolic
activation, the positive control for TA100 is 50 .mu.L 2-antramine
(1.25 .mu.g/plate) and for TA102 it is 50 .mu.L 2-aminofluorene
(1.25 .mu.g/plate).
[0157] Assay Procedure Without Metabolic Activation System
(-S9)
[0158] The preincubation method was used.
[0159] 1.sup.th Day
[0160] All solutions and culture media previously described were
prepared. In laminar flow, 10 mL of VB solution and 25 mL of 40%
glucose solution (previously prepared) were added to the sterile
material (GMA), followed by homogenization, and about 25 mL of AMG
were distributed to each plate.
[0161] The GMA distributed to the plates was left under rest for 48
hours in an oven at 37.degree. C. for subsequent use.
[0162] 2.sup.th Day
[0163] In laminar flow, Salmonella typhimurium (TA100 and TA 102)
strains were inoculated individually with a platinum loop, in the
respective nutrient broths and maintained at 37.degree. C., under
constant stirring (160 rpm) during 14 hours, in order to achieve a
density of 1 to 2.times.10.sup.9 bacteria/mL.
[0164] 3.sup.th Day
[0165] Different concentrations of the compounds were added into
100 .mu.L of 0.2M phosphate buffer pH 7.4 (or 500 .mu.L of the
mixture S9 in metabolic activation assays) and incubated for 20-30
minutes at 37.degree. C. Solutions containing the compounds had
DMSO as the solvent. Thereafter, 2 mL of top agar supplemented with
traces of histidine and biotin was added, homogenized and plated in
glycosylated minimum medium. After solidification of the top agar,
the plates were incubated for 48 hours at a 37.degree. C. Then, the
counting of the number of revertant colonies per plate was
performed. All tested concentrations, positive and negative
controls were run in triplicate.
[0166] 5.sup.th Day
[0167] After 48 hours, the revertant colonies were counted
manually, and the protoCOL Colony Counter Version 3.15.630
(1998-2001) SYNBIOSIS LTD system was used for the positive
control.
[0168] Evaluating and Interpreting the Results
[0169] The final data obtained from the assay was analyzed using
the statistical software Salanal (Salmonella Assay Analysis)
version 1.0 from the Research Triangle Institute, RTP, North
Carolina, USA. Such software allows the dose-response effect to be
evaluated by means of analysis of variance (ANOVA-test F)
computations between the measurement of the number of revertants at
the different tested concentrations (doses) and the negative
control, followed by a linear regression. The software model chosen
for analyzing the data was Bernstein's (BERNSTEIN, L. et al. Mutat.
Res. v. 97, p. 267-281, 1982.). The slope of the straight line from
the linear portion of the dose-response curve is also provided by
this software and corresponds to the number of revertants induced
per measurement unit of the analyzed sample.
[0170] From the results, the mutagenicity ratio (MR) to was
computed for each analyzed dose from each compound. MR is given by
the following equation:
MR=average number of revertants per test plate (spontaneous+induced
revertants)
[0171] average number of revertants per plate of the negative
control (spontaneous revertants)
[0172] The spontaneous growing means that the number of revertants
which developed on the plate, regardless of being induced or not,
wherein values higher or equal to 2 are considered as a positive
response (VALENT, G. V. et al. Env. Toxicol. Water Quality. v. 8,
p. 371-381, 1993.).
[0173] Assay Procedure with Metabolic Activation System (+S9)
[0174] The mutagenicity test with metabolic activation system was
performed with a microsomal fraction S9 (S9 mix) prepared from a
liver homogenizate from Sprague Dawley rats, previously treated
with Aroclor 1254, acquired in the freeze-dried form.
[0175] 50 mL of S9 mix were prepared using the following solutions
shown in Table 7 below:
TABLE-US-00007 TABLE 7 Solutions used for the preparation of S9
mix. Sterile water 19.75 mL Phosphate buffer 0.2M 25 mL NADP 0.1M 2
mL (freezer) G-6-P 1M 250 .mu.L (refrigerator) MgCl 0.4M 500 .mu.L
(refrigerator) KCl 1.65M 500 .mu.L S9 Fraction Dissolved into 2 mL
of sterile miliQ water
[0176] The procedure for this assay is the same, however, instead
of the buffer, 500 .mu.L of the S9 mixture should be added.
[0177] The S9 mixture has a viability of 4 hours from preparation
when put on ice. The plates are then incubated for 48 hours at
37.degree. C. After the required time has elapsed, the counting of
the revertant colonies was carried out. All tested concentrations,
positive and negative controls were run in triplicate.
[0178] Procedures for Assay in K562 Cell Culture
[0179] The human leukemia cell line K562 ATCC (American Type
Culture Collection), Philadelphia, Pa., USA was used. The cells
were cultured in DMEM medium (Dulbecco's Modified Eagle Medium,
Invitrogen, USA) containing 10% of fetal bovine serum and
glutamine. The cells were maintained at 37.degree. C. under a 5%
CO.sub.2 atmosphere. For the experiments, the cells were incubated
at a density of 1.times.10.sup.5 cells/mL. In order to carry out
the culture with hemin (30 uM), it was added 72 hours before the
beginning of the experiment with the desired compound.
[0180] The 0-hour time consisted in removing non-treated K562
cells. From this point, the respective compounds were added at the
desired concentrations (5, 30, 60 and 100 uM), the cells were then
maintained for 7 days under culture, without a to new addition of
any compound or substitution of the culture medium. Cell
collections were performed at the following times: 0, 24, 48, 72
and 96 hours. The morphology of the cells was analyzed at these
points through cytospin slides stained with Leishman and the cell
viability was performed by staining with trypan blue in a
Neuberger's chamber.
[0181] RNA Extraction
[0182] The extraction method with TRIzol reagent (Gibco-BRL,
Gaithersburg, Md.) according to the manufacturer's instructions was
used in order to obtain the RNA from K562. The sample containing
K562 and TRIzol was incubated for 5 minutes at room temperature in
order to achieve complete dissociation of the nucleoproteic
complexes, 200 .mu.L of chloroform (CHCl.sub.3) was added and
thoroughly stirred, and incubation was again performed by 5 minutes
at room temperature. After centrifugation for 15 minutes at 19,000
g at a temperature of 4.degree. C., the supernatant was obtained
and stored in another tube, proceeding immediately to the step of
precipitation with 500 .mu.L of cold isopropanol. After
homogenization, a new incubation was carried out for 10 minutes at
room temperature, followed by centrifugation for 10 minutes at
19,000 g at 4.degree. C. The supernatant was disposed of and 800
.mu.l of 70% cold ethanol was added to the precipitate, with a new
centrifugation being carried out for 5 minutes at 14,000 at
4.degree. C. Finally, the supernatant was disposed of and the RNA
precipitate was left to dry for 10 minutes at room temperature, and
then resuspended in sterile water containing diethyl pyrocarbonate
(DEPC) and incubated at 55.degree. C. for 10 minutes and
subsequently put on ice for total solubilization of the RNA.
[0183] The sample was checked as to its integrity by
electrophoresis on a 1.2% denaturing agarose gel. The samples
having a suitable amount of RNA showed integrity on both ribosomal
subunits: 18S e 28S. After electrophoresis, the RNA samples were
stored in a freezer at -80.degree. C.
[0184] Complementary DNA (cDNA) Synthesis
[0185] The RNA samples obtained were subjected to the complementary
DNA (cDNA) synthesis using the Superscript III RTTM kit
(Invitrogen, Life Technologies). After reading in a
spectrophotometer (Gene Quant-Pharmacia, USA) and quantification, 3
.mu.g of RNA were treated with the enzyme DNase I (Invitrogen, Life
Technologies), for removal of contaminant DNA. 1.0 .mu.L of 1
u/.mu.L DNase I, 1.0 .mu.L of 10.times. DNase I Reaction Buffer
(200 mM Tris-HCl, 20 mM MgCl2, 500 mM KCl2) and water sufficient
for a final volume of 10.0 .mu.L of reaction were added. The
reaction was carried out for 15 minutes at room temperature and
stopped with 1.0 .mu.L of 25 mM EDTA, and incubated for 10 minutes
at 65.degree. C.
[0186] For cDNA synthesis, 1.0 .mu.L of 50 .mu.M oligo (dT) 20 and
1.0 .mu.L of 10 mM dNTP's were then added. The samples were
incubated for 5 minutes at 65.degree. C., followed by 1 minute at
4.degree. C. To each sample, 10.0 .mu.L of the following reaction
mixture were added: 2 .mu.L of 10.times. RT buffer, 4.0 .mu.L of 25
mM MgCl2, 2.0 .mu.L of 0.1 M DTT, 1.0 .mu.L of 40 U/.mu.L RNase
OUTTM and 1.0 .mu.L of 200 U/.mu.L, Superscript III RTTM. The
reaction occurred for 50 minutes at 50.degree. C., followed by 5
minutes at 85.degree. C. Thereafter, 1.0 .mu.l of 2 U/.mu.L E. coli
RNase H was added for 20 minutes at 37.degree. C.
[0187] Verification of Complementary DNA Synthesis
[0188] The verification of cDNA synthesis was made by means of PCR
for amplification of the beta-actin (BAC) gene. Reactions were
carried out with: 5.0 .mu.L of 10.times. PCR buffer (20 mM
Tris-HCl, 500 mM KCl), 1.5 .mu.l of 50 mM MgCl2, 1.0 .mu.L of 10 mM
dNTP's, 1.0 .mu.L of 10 mM of BACF primer
(5'-AAGAGATGGCCACGGCTGCT-3'), 1.0 .mu.L, of 10 mM of BACR primer
(5'- TCGCTCCAACCGACTGCTGT-3'), 0.5 .mu.L of Taq DNA polymerase, 1.0
.mu.l of cDNA and 39 .mu.L of water, for a final volume of 50
.mu.L. The program was started for 2 minutes at 94.degree. C.,
followed by 35 cycles: 94.degree. C./30 seconds-58.degree. C./45
seconds-72.degree. C./40 seconds, being terminated by 72.degree.
C./7 minutes. The products were subjected to electrophoresis on 1%
agarose gel to verify the amplification of 640 pb.
[0189] Design of Primers for Quantitative Real-Time PCR
Reaction
[0190] The primers used in Quantitative Real-Time PCR reactions
were designed with the software "Primer Express" (Applied
Biosystems), analyzed in the program Blast
(www.ncbi.nlm.nih.gov/blast) to verify the conditions for formation
of structures, such as hairpins and dimers.
[0191] Standardizations Required for Quantitative Real-Time PCR
[0192] Primer Concentration
[0193] The optimum concentration of primer to be used in
quantitative real-time PCR should be sufficiently low to allow
duplication of all copies of the gene present in the sample. Using
the same amount of sample, reactions containing each of the primers
(sense and antisense) were performed at the final concentration of
150 nM, 300 nM, 600 nM e 900 nM. The cycle in which fluorescence is
detected above the established threshold is called threshold cycle
or Tc. Since the same amount of sample was used in all reactions,
the Tc should not change. If the increase on the primer
concentration caused a reduction in Tc, so the amount of this
reagent in the reaction was still insufficient. Thus, the optimum
concentration chosen was the minimum, associated with the lower
Tc.
[0194] The sequence and length of the amplified fragments from each
primer pair used in the amplification of the genes studied in the
quantitative real-time PCR technique is shown in table 8.
TABLE-US-00008 TABLE 8 Sequence and length of the amplified
fragments Length of the amplified Gene Primer Sequence fragment
Gamma-Glob-F 5'-CCAGCTGAGTGAACTGCACTGT-3' 81 bp Gamma-Glob-R
5'-ACGGTCACCAGCACATTTCC-3' .beta.-actin-F 5'-AGGCCAACCGCGAGAAG-3'
79 bp .beta.-actin-R 5'-ACAGCCTGGATAGCAACGTACA-3' GAPDH-F
5'-GCACCGTCAAGGCTGAGAAC-3' 89 bp GAPDH-R
5'-CCACTTGATTTTGGAGGGATCT-3'
[0195] The primer concentrations used in the amplification of the
studied genes and the amplification efficiency obtained is shown in
Table 9. The concentrations were defined by the amplification
efficiency generated under the tested conditions.
TABLE-US-00009 TABLE 9 Primer concentrations Primer Used
concentration Primer efficiency Gamma-glob 150 nM 100% .beta.-actin
300 nM 100% GAPDH 300 nM 100%
[0196] Reaction Efficiency
[0197] In order that the real-time PCR reaction is is reliable and
reproducible, optimum reaction conditions are required, i.e., the
amplifications must show 100% amplification efficiency at every
cycle, occurring sample duplication. The amplification efficiency
is obtained from formula 10 (-1/slope), wherein slope means the
slope value of the curve. Optimization occurs using the optimum
primer concentration with 7 known sample amounts, in logarithmic
scale: 2 ng (2.times.100), 6.32 ng (2.times.100.5), 20 ng
(2.times.101), 63.2 ng (2.times.101.5), 120 ng, 200 ng
(2.times.102) e 632 ng (2.times.102.5). The results are used to
construct a standard curve Tc versus sample amount.
[0198] Quantitative Real-Time PCR
[0199] After reading in a spectrophotometer (Gene Quant-Pharmacia,
USA) and quantification, cDNA aliquots were used as template in
quantitative real-time PCR reactions. The technique consists in
optically monitoring the fluorescence emitted during the PCR
reaction, by means of the binding of a is specific probe or dye to
the newly synthesized strand.
[0200] The reactions, always run in duplicate, were performed using
the reagent SYBERGreen PCR Master Mix.RTM. (Applied Biosystems),
which in addition to containing all reagents required for PCR
(dNTP's, MgCL2, buffer, Taq Ampli-Gold), also contains the
SYBERGreen dye, a double-stranded intercalating agent necessary for
the detection of the reaction from cycle to cycle. Further, cDNA
samples and specific primers for the analyzed gene were used.
[0201] The real-time amplification detection was performed in the
apparatus ABI 5700 Sequence Detector System.RTM. (Applied
Biosystems) in fluorescence versus cycle number graphs. The higher
the expression of a gene, that is, the more copies there are at the
beginning of the reaction, the earlier will occur amplification,
and hence, the lower will be the Tc.
[0202] The reactions carried out contained 12.5 .mu.L of the
reagent SYBERGreen PCR Master Mix.RTM., 25 ng of cDNA sample and
the optimum determined primer concentration, making up a final
volume of 25 .mu.L. In all cases, negative controls were made
containing sterile water in place of the sample. The reactions were
prepared in 96-well plates (Sorenson, BioScience Inc.) with plastic
caps which allow the passage of light. The program was started at
95.degree. C./10 minutes, followed by 45 cycles: 95.degree. C./15
seconds-60.degree. C./1 minute. At the end of a normal
amplification, a degradation step is added during which the
temperature rises gradually from 60.degree. C. to 95.degree. C. As
the products generated by PCR denaturate with the temperature rise,
the florescence signal of SYBR Green decreases. The resulting graph
allows verifying if there is one or more PCR products present in
each reaction, due to melting temperature differences between
amplified PCR products, this difference being caused by the number
and composition of bases of each product.
[0203] Analysis of Real-Time Data
[0204] The expression of the genes of interest was determined in a
relative way, being normalized with respect to genes called
calibrators; in this study, we used .beta.-actin and GAPDH, which
are genes whose expression is supposedly constitutive, that is,
they show little variation between various conditions. However,
some studies have been demonstrating that the expression of these
genes may change substantially. From the Tc values obtained, the
arithmetic mean of Tc duplicates was computed. Subsequently, the
amount of expression (Q) was obtained by means of the formula
Q=EdeltaTc, where E=reaction efficiency and delta Tc=lowest Tc
observed-Tc of the sample. Thus, the expression was related with
the sample which exhibited the highest expression (Lowest Tc
observed), which received a value Q=1. The Q values of the
calibrator genes of to each sample were subjected to the Gnorm
program, which computes the geometric mean between the same, which
value is called sample Normalization Factor. The normalized
expression of a given gene of interest in a certain sample is given
by the ration between value Q of the gene of interest of the sample
and the sample Normalization Factor. The obtained data is expressed
in arbitrary units or absolute expression value.
[0205] Abdominal Writhing Assay Induced by Acetic Acid
[0206] The antinociceptive profile was evaluated through the
abdominal writhing assay induced by acetic acid in mice. In this
assay, Swiss mice from both genders were used, weighing from 21 to
28 grams, fasted for a time period of about 8 hours. The test
substance was administered orally and had, as a carrier, 5% gum
arabic. After 1 hour, the administration of acetic acid 0.1N (0.1
mL/10 g weight) was performed in the peritoneal cavity of the
animals. Ten minutes after the injection of acetic acid, writhings
were counted during 20 minutes. Controls were made for the carrier
(gum arabic) and it does not show pharmacological activity.
[0207] Mouse Ear Edema Assay Induced by Capsaicin
[0208] This assay was performed using Swiss mice from both genders
weighing from 18 to 30 grams. The animals were fasted for 8 hours
with free access to water. This assay consists in locally
administering (right ear) 20 .mu.l of a capsaicin solution (250
.mu.g/ear) diluted in acetone, 1 hour after the i.p. administration
(diluted in 0.5% gum arabic). The left ear (control) received the
carrier in which capsaicin was diluted (acetone) and the right ear
received capsaicin. This assay is characterized by an acute
inflammatory response of the ear, with development of edema. The
animals were sacrificed and their ears were weighted to obtain the
inflammation index. A biopsy (8 mm in diameter) of the ear was
carried out. Next, the weights of the inflamed ears were compared
against the weights of the contralateral ear (control ear) which
were not treated with the phlogistic agent. The edema inhibition
percentage was calculated by subtracting the ear treated with the
carrier by that treated with capsaicin from each group of animals
treated with the test substances, and then it was divided by the
difference between the groups of the irritating agents and the
control groups. The result was subtracted by 1 and multiplied by
100, being shown in Table 4.
[0209] Peritonitis Assay
[0210] The mice were treated with the substances being analyzed or
carrier and after 1 h of oral administration, they were
simultaneously subjected to the peritonitis assay, by administering
intraperitoneally 1 ml of a 3% thioglycolate solution. 4 h after
the administration of thioglycolate, the peritoneal cavity was
washed with 3 ml of a HANKS solution (Balanced salt solution, free
of Ca.sup.2+ and Mg.sup.2+). Next, the peritoneal wash was analyzed
and the total counting of white blood cells was made in a
Newbauer's chamber under an optical microscope with a 40.times.
objective. Results are shown in Table 5.
[0211] Statistical Analysis
[0212] The significance levels between experimental groups and the
control were generated using the Student's T Test. The values were
considered significant when *P<0.05. Results were expressed as
mean .+-.standard error of the mean, as indicated in the legends of
figures.
[0213] Citotoxicity Assay in Mouse Peritoneal Macrophages
[0214] The determination of cell viability (FIG. 5) to was
performed by suspending peritoneal macrophages in a RPMI solution,
set at a concentration of 5.times.10.sup.6 cells/mL, 100 .mu.L of
which were added to each cavity of 96-cavity tissue culture plates,
being incubated with drugs and prodrugs at concentrations of
10.sup.-4, 10.sup.-5, 10.sup.-6, 10.sup.-7, 10.sup.-8 mM during 24
hours at 37.degree. C. and 5% of CO.sub.2. The absorbance reading
was performed in a UV/Visible spectrophotometer at 540 nm with
reference filter at 620 nm (Microplate Reader-Model
550-BIORAD).
[0215] Obtainment and Culture of Peritoneal Exudate Cells:
[0216] Mice were previously inoculated with 3.0 mL of 3%
thioglycolate intraperitoneally in order to stimulate the
macrophages of this cavity. After 3 days of stimulation, the
animals were sacrificed and the peritoneal macrophages collected.
The cells were then washed from 2 to 3 times per centrifugation at
358 g (Centrifuge Fanem Excelsa II 206MP) during 5 minutes in
sterile PBS and, then, resuspended in 1 mL of RPMI-1640 for
counting in a Neubauer's chamber. After counting, the concentration
was set at 5.times.10.sup.6 cells/ml and these cells were
distributed to disposable sterile plates with 96 cavities. The
plates thus containing the suitable concentration of cells were
taken to incubation for 24 hours in a oven at 37.degree. C.,
containing 95% of moisture and 5% of CO.sub.2 in the presence of
the anti-inflammatory agents and taurine derivatives at the
respective concentrations: 10.sup.-8, 10.sup.-7, 10.sup.-6,
10.sup.-5 and 10.sup.-4 mM or even in the presence of the RPMI-1640
medium only. LPS was used as a positive control and the RPMI-1640
medium as a cell control. The culture supernatants, upon the end of
the incubation period, were collected in order to determine the
nitric oxide levels thereof. The RPMI-1640 used during this whole
process was supplemented with 2 mM /-glutamine, penicillin (100
U/ml), streptomycin (100 ug/ml), 5% of bovine fetal serum and
2-mercaptoethanol 5.times.10.sup.-2M(RPMI-1640-C).
[0217] Nitric Oxide Dosage
[0218] After obtaining the supernatants from macrophage cultures,
as described above, the concentration of nitric oxide was
evaluated. This evaluation was made by measuring the concentration
of accumulated nitrite (a stable degradation product of nitric
oxide) through a diazotization reaction with the Griess reagent (1%
sulfanilamide, 0.1% naphthylenediamine dihydrochloride, in 5% of
phosphoric acid), according to the method of Green et al. (1982).
To do so, 50 .mu.l of the culture supernatant was incubated with
the same volume o Griess reagent at room temperature (10 minutes)
and, thereafter, absorbances were measured at 550 nm in a ELISA
reader (Microplate Reader-Model 550-BIORAD). Nitrite concentrations
were obtained from a standard curve previously prepared with known
NaNO.sub.2 molar concentrations. The tests were run in triplicate
and the values expressed in micromole of NO.sup.2-/5.times.10.sup.5
cells. Results are shown in FIG. 6.
* * * * *