Patent Application
20070010458
The present invention relates to the use of drugs for the arthritis therapy.
Arthritis pathological conditions are characterized by a progressive articulation damage due to the cartilaginoid matrix degradation. With arthritic diseases, it is generally meant diseases affecting articulations. Specifically rheumatoid arthrites, osteoarthrites, etc. can be mentioned.
The arthritis represents one of the most common medical problems and it is one of the main causes of disability. For example in the United States about 20 millions people result affected by arthritis. The factors which can cause the disease onset are various. Among these articulation traumas, obesity, or diseases modifying the cartilage structure or functionality, such for example rheumatoid arthritis, hemochromatosis, gout or Paget's disease, can be mentioned. Other factors are the age and sex. Generally the disease incidence is higher in women.
The arthritic process pathophysiology is progressive and the symptomatology is gradual and initially starts with the ache onset. The disease evolution determines damages to articulations, to tendons and can compromise leg/arm functionality.
The drugs used at present in the treament of arthritis are divided into two groups having different modes of action. The drugs of the first group, such as NSAIDs, provide symptomatic relief, but have no influence on the progress of the disease. The drugs belonging to the second group, have different chemical structures from the former and are effective on the course of the disease. For instance they can prevent irreversible joint damage. Said latter drugs are called disease-modifying agents. Presently the use in therapy of disease modifying agents is limited by their toxicity (Martindale, 31st Ed. 1996 pages 11-13).
At present specific therapies which intervene on the disease course reducing the degenerative effects on the cartilaginoid matrix, with side effects of small entity, so that the drugs can be used for the long term treatments which are generally required, do not exist.
The existing therapies are directed both to the ache treatment, administering analgesics such for example paracetamol, non steroidal antiinflammatory drugs (NSAIDs), and to the maintenance of the articulation functionality by the intraarticular application of drugs such for example corticosteroids or ialuronic acid, or parenteral such for example perdiacerine, sulfasalazine and penicillamine.
Among the above drugs used to treat the painful symptomatology, paracetamol is known to cause damages to liver and its assumption is contraindicated when other drugs are used. The NSAIDs cause even serious gastric damages and recent studies have shown that they can also accelerate the arthritic disease Rashad S., Lancet 1989, 519-522. The sulfasalazine can cause nausea, head-ache and skin rash. The penicillamine is bad tolerated and gives side effects, for example anorexia, nausea.
It is also known to use particular non steroidal antiinflammatory drugs having a 2-oxo-1H-indolic structure such, for example, Tenidap. This drug differently from the other NSAIDs is effective in arthritis interacting in the cytokine form ation, which are endogenous factors responsible for the inflammation and for the degradation of the cartilaginoid matrix. However Tenidap causes damages at hepatic and also renal level. See Martindale XXXIth Ed., pages 99-100.
Recently several studies have been directed to explain the arthritis etiopathology. These researches have shown that some inflammatory factors such for example cytokines, chemokines, etc. are involved in the activation of a cascade of catabolic and degenerative events determining the cartilaginoid matrix degradation.
It is known in the prior art that a group of growth factors, TGF-β proteins (TGF=transforming growth factor) in particular TGF-β1, play an important role in the articular cartilage reparation, promoting both the chondrocyte formation and the regeneration process of the bony tissue (osteoclastogenesis) (N. Felisaz et Al. Osteoarthritis and Cartilage (1999) 7 255 267).
The need was felt to have available compounds capable to induce the expression of the TGF-β proteins, so to be used in the arthritis treatment, without showing the side effects of the prior art drugs.
The Applicant has surprisingly and unexpectedly found compounds capable to solve the above technical problem.
An object of the invention is the use for the arthritis therapy as disease-modifying drugs of compounds or salts thereof having general formula:
A-(B)b0—(C)c0—N(O)s (I)
wherein:
s is an integer and is equal to 1 or 2, preferably 2;
c0 is an integer and is 0 or 1;
b0 is an integer and is 0 or 1; with the proviso that at least one between c0 and b0 is different from zero;
A=R−T1-, wherein
The compounds whose formulas have been indicated above are prepared according to known methods of the prior art, for example described in “The Merck Index”, 12a Ed. (1996), herein incorporated by reference. When available, the corresponding isomers and optical isomers can be used.
When b0=c0=1 the bonds between the drug radical and X2 and between X2 and Y can be, independently the one from the other, of ester, thioester, amide type; when b0=0 and c0=1 the bond between the drug radical and Y is of ester, thioester, amide type.
The radical R of non steroidal antiinflammatory drugs or antiinflammatory analgesic as above defined is selected from the following groups:
wherein:
R1 is H or —OCOR3; wherein R3 is methyl, ethyl or C3-C5 linear or branched alkyl, or the residue of an heterocycle with only one ring having 5 or 6 atoms which can be aromatic, partially or totally hydrogenated, containing one or more heteroatoms independently selected from O, N and S;
R2 is hydrogen, hydroxy, halogen, C1-C4 linear or branched when possible alkyl, C1-C4 linear or branched when possible alkoxyl; a C1-C4 linear or branched when possible perfluoroalkyl, for example trifluoromethyl; nitro, amino, mono- or di(C1-4)alkylamino;
with the proviso that in formula Ia) R1 and R2 cannot be contemporaneously H, preferably when R1=H R2=OH;
preferably in the compounds of formula Ia) T1=—CO— and:
The compounds of formula (I) can be obtained as described in WO 95/30641, WO 00/61537, WO 01/12584.
Preferably Y3 is selected from the following bivalent radicals:
Preferred of Y3 are the following: (Y12), having the two free valences in the ortho positions with respect to the nitrogen atom; (Y16) with the two valences linked to the two heteroatoms; Y1 (pyrazol) 3,5-disubstituted; Y16 is particularly preferred.
The compounds according to the present invention, when at least one functional group salifiable with acids, for example an aminic group, is present, can be transformed into the corresponding salts. For example one way to form the salts is the following: when one basic nitrogen atom is present in the molecule, it is reacted in an organic solvent such for example acetonitrile, tetrahydrofuran with an equimolecular amount of the corresponding organic or inorganic acid.
Examples of organic acids are: oxalic, tartaric, maleic, succinic, citric, trifluoroacetic acids.
Examples of inorganic acids are: nitric, hydrochloric, sulphuric, phosphoric acids.
When the precursor compounds usable in the present invention have one or more chiral centres, they can be in racemic form or as diastereoisomer mixtures, as single enantiomers or single diastereoisomers; if they show a geometric asymmetry the compounds can be used in the cis or trans form.
The compounds of the present invention are prepared in the corresponding pharmaceutical compositions, even at belated release, for parenteral, oral and topical use, such for example sublingual, inhalatory, suppository, transdermal, enema, according to the well known techniques in the field, together with the usual excipients; see for example the volume “Remington's Pharmaceutical Sciences 15th Ed.”
The amount on a molar basis of the active principle in these compositions is generally the same, or lower, compared with that of the corresponding precursor drug.
The daily administrable doses are those of the precursor drugs, or optionally lower. The daily precursor doses can be found in the publications of the field, such for example “Physician's Desk Reference”.
Among the invention compounds those preferred are the following:
It is surprising that the invention compounds are capable to promote the formation of the TGF-beta growth factor since it is known that the corresponding precursor compounds have no efficacy in reducing or preventing the cartilage degeneration process in the arthritic disease. Besides the Applicant has found that the NSAIDS precursor compounds have no effect on the formation of said growth factors.
Furthermore the present invention compounds have no side effects at gastric level and show an improved hepatic tolerability compared with the precursors. As an example, the Applicant has shown that the paracetamol nitroxybutylester has much more limited effects on the transaminase and bilirubin plasmatic levels compared with the paracetamol precursor.
Therefore the present invention compounds can be used in the arthritis therapy to prevent the cartilaginoid matrix degeneration, i.e. as curative and not only symptomatic drugs, combined with improved general tolerability.
The present invention compounds can be used also in the bony metabolism disease therapy, for example growth illness, characterized by an accelerated loss of the bony tissue, such as for example in old people.
It is known that the progressing of arthritic disease is due to the imbalance between pro-inflammatory (like IL-6, TNF-α) and anti-inflammatory (like TGF-β for example) mediators in different cells involved in the inflammation process, like monocytes, lymphocytes, chondrocytes, etc.
IL-6 (interleukin-6) is a potent pro-inflammatory cytokine and has been recognized to be implicated in rheumatoid arthritis (Choy E. H. et al., Arthritis Rheum. 46, 3143, 2002).
TNFα (Tumor necrosis factor α) has been shown to exert inflammatory changes in chondrocytes, such as decreased cell proliferation and decreased proteogycan synthesis. Overall these effects can be considered as signs of cartilage degradation and be implicated in the pathogenesis of arthritis.
Thus the effectiveness of a compound to inhibit TNFα induced-inflammatory changes in chondrocytes can be considered as a measure of the activity on arthritis, since the pharmacological action is to maintain the cartilage matrix integrity.
The compounds of the present invention are effective in reducing or eliminating the imbalance above said. They increase the formation of the anti-inflammatory mediators and decrease of the production of pro-inflammatory mediators.
Thus they have a more favourable pharmacotherapeutic profile than single cytokine-neutralizing agents (anti-TNF, etc) that must be given at very high doses, thus resulting in toxicity.
In rheumatoid arthritis disease a vast majority of patients have intermittent relapses and remissions of the disease. Unlike conventional NSAIDs administration of the drugs of the present invention can prevent disease relapses.
The following Examples are for illustrative purposes and are not limitative of the invention.
Chondrocytes have been isolated from calf cartilage as described in Benya P. D., Biochemistry 1977; 16; 865-872, and used as primary cultures. The primary cultures have been kept in a DMEM culture medium (Dulbecco's modified Eagle medium) (high glucose) containing bovine fetal serum (10% vol.) and antibiotics at 37° C. and in air/CO2 atmosphere (95%/5% vol.) until reaching the culture confluence. A cell sample is kept as a control and not treated with the tested compounds. The tested compounds are added to the other cellular cultures at the concentration 10−5 M and the so treated cultures have been incubated for 24 hours. The compounds have been previously dissolved in a DMSO amount such that the final concentration in the medium is 0.1%. The control has been treated only with DMSO.
The used compounds have been the following:
The following precursor compounds have been contemporaneously tested: aspirin and flurbiprofen.
At the end the cells have been washed 3 times with a medium free from serum and added with BSA (bovine serum albumin, 200 μg/ml) for 5, 30 and 60 minutes respectively and then incubated in a medium devoid of serum (1 ml) for further 6 hours. The conditioned medium has been collected, centrifuged and kept at −70° C. until the use.
Before the experiment, 0.5 ml of cellular culture supernatant have been acidified with HCl (0.1 ml, 1 N) and incubated at room temperature for 10 min, then neutralized with NaOH/HEPES (0.1 ml NaOH 1.2N/0.5 M).
CCL-64 cellular cultures lines in a proliferative state have been prepared as described in Jennings J. C., J. Cell. Physiol. 1988, 137, 167-72, sowing 2×104 cells/well and incubating in the presence of FCS-medium (10% vol.).
After 24 hours the cells have been washed with the medium free from serum and incubated for 24 hours, respectively, with 0.5 ml of conditioned condrocyte medium, prepared as above and with increasing concentrations of TGF-β1 to determine a cellular growth inhibition reference curve, since the growth of said cellular lines is inhibited by the presence of TGF-β1.
At the twentieth hour 3H-timidine (0.5 μCi/ml), a cellular proliferation marker, which is incorporated in the DNA of the new cells has been added to the cultures. The cultures have then been incubated for 4 hours.
At the end the cells have been cold fixed (5° C.) with trichloroacetic acid 5% v/v, washed with the same solution and dissolved in NaOH (0.1 N). On the cells the count in liquid scintigraphy has been carried out to measure the marked timidine incorporated in the samples and in the standards treated with increasing amounts of TGF-β1. From the amount of incorporated timidine it is shown the amount of TGF-β1. The data reported in Table 1 are expressed in percentage of TGFβ1 produced in the samples treated with the tested compounds, compared with the untreated control. The data show that the tested compounds induce in the chondrocytes a significant increase of the TGFβ1 production compared with the untreated controls and the precursor compounds, and that the present invention compounds can therefore be used to prevent or reduce the articular tissue degradation.
Hepatic Tolerability of Paracetamol v. the Corresponding Nitrooxybutylester (NO Paracetamol)
The nitrooxybutylester of paracetamol (NO-paracetamol) has been prepared as decribed in Example F1.
Groups of No. 10 rats have been treated i.p. with NO-paracetamol (1.4 g/Kg i.p.) or with paracetamol (1.16 g/Kg) or with the carrier (0.9% w/v NaCl containing 20% v/v di tween-20) (control group).
After 6 hours from the administration, the animals have been sacrificed, the blood has been collected and the plasma analyzed to determine the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and bilirubin concentrations. The results are reported in Table 2 and have been expressed in pecentage with respect to the values obtained in the control group (100%).
The results show that the paracetamol administration causes hepatic damage since there is an increase of the transaminase and bilirubin values with respect to the controls.
The NO-paracetamol administration does not cause ALT increase while the AST and bilirubin plasmatic levels are much lower than those of the groups treated with paracetamol, and as order of magnitude comparable with those of the controls.
Effect of NO-Flurbiprofen and of Flurbiprofen on Interleukin (IL)-6 Release in Human Monocytes (Ex-Vivo Study)
IL-6 is a potent pro-inflammatory cytokine and has been recognized to be implicated in rheumatoid arthritis (Choy E. H. et al., Arthritis Rheum. 46, 3143, 2002).
Twenty-four healthy subjects of both sexes were enrolled and randomised into three groups of 8 subjects each. Each group was administered as it follows:
Monocytes from whole blood samples obtained before and 4 hours after the last treatment were prepared. Monocytes were extracted by positive selection using paramagnetic beads loaded with anti-CD11 antibody. Cells were then incubated with 10 μg/ml endotoxin for 24 hours, and IL-6 released in cell supernatant measured by ELISA assay.
Results are reported in Table 3. Results are given as % in the confront of IL-6 release obtained in the placebo group.
The Table shows that oral subacute treatment of NO-flurbiprofen, but not of flurbiprofen, markedly suppressed IL-6 release in monocytes
Effect of Flurbiprofen, NO-Flurbiprofen, Indomethacin, NO-Indomethacin (Indomethacin (3-Nitrooxymethyl)Phenyl Ester) on Interleukin (IL)-6 and TGF-β Release in Mouse Spleen Lymphocytes (In Vitro Study)
Spleen lymphocytes were prepared as it follows. Mice were killed by an overdose of ether, and spleens were collected and maintained in a sterile RPMI medium (Sigma-Aldrich) containing 0.5% (vol/vol) L-glutamine and 0.5% (vol/vol) sterile endotoxin-free fetal calf serum (FCS). The spleens were opened and the content (whole cells) collected and diluted with RPMI.
After repeated washings, cells were suspended in 10 ml of RPMI containing 1% (vol/vol) streptomycin and 1% (vol/vol) penicillin. The suspension was then incubated at 37° C. for 24 hours, in an O2/CO2 atmosphere (95%/5% v/v). Monocytes were eliminated by adhesion, and lysis of red cells was obtained by suspension in a solution 0.15 mol/liter NH4Cl and 1 mmol/liter KHCO3. The resulting lymphocytes were resuspended in RPMI-FCS, incubated for 30 minutes at 37° C. with anti-FAS, anti-FASL, or anti-IL2 receptor monoclonal antibodies, and then washed twice with RPMI-FCS. Cells were then incubated with the FITC-conjugated secondary antibody for 30 mins at 4° C., washed twice, and resuspended in PBS/formaldehyde (0.5%). Control samples were treated with the FITC-conjugated secondary antibody only. Stained cells were analysed on a flow cytofluorimeter. Cells were gated using forward vs side scatter to exclude dead cells and debris.
Cells were transferred in plate and then 10 μg/ml endotoxin and each of the following compounds at a concentration of 50 μM added:
The results obtained are reported in Table 4.
The Table shows that both NO-flurbiprofen and NO-indomethacin inhibit the relase of IL-6 and potentiate the release of TGF-β.
Effect of Flurbiprofen, NO-Flurbiprofen, Ibuprofen, NO-Ibuprofen on Human Chondrocytes and Proteoglycan Synthesis (In Vitro Study)
Human chondrocytes were isolated by collagenase digestion from knee cartilage collected from patients undergoing knee replacement surgery. Only primary culture was used to avoid phenotype change of human chondrocytes. TNFα (80 ng/ml) was added to all but control cells. Test compounds were dissolved at a concentration 0.02% (w/v) in DMSO (vehicle).
The following compounds were tested:
The test compounds were incubated with cells at a 100 μM concentration for 24 hours.
Cell proliferation was determined by measuring [3H]-thymidine incorporated into newly synthesized DNA. Cell viability was assessed by MTS assay kit.
Proteoglycan synthesis was determined by [35S]-sulfate incorporation. Cells and supernatant were extracted with 4M guanidinium chloride and purified by Sephadex colums chromatography. The amount of [35S]-sulfate was measured by liquid scintillation counter. Results were normalized by the amount of Dna in the sample and expressed as CPM/μg DNA (CPM=count per minute).
The results are reported in Table 5 and are expressed as % cell growth/proteoglycan synthesis with respect to the control group.
The Table shows that NO-flurbiprofen and NO-ibuprofen reversed the decrease of cell proliferation induced by TNFα. No effect on cell viability was found. Both NO compounds reversed the decrease in proteoglycan synthesis induced by TNFα. The parent NSAIDs did not affect TNFα-induced effects on cell proliferation and proteoglycan synthesis. In both experiments the activity of the parent compounds was almost the same as that of the vehicle.
Effect of Flurbiprofen, NO-Flurbiprofen, Paracetamol and NO-Paracetamol on the Expression of TGF-β Type II Receptor.
Type II collagen and TGF-β type II receptor (TβRII) expression have been reported as agents playing a crucial role in osteoarthritis (OA) physiopathology. Indeed, in experimental models of OA it was found that the physiological levels of said agents are dramatically decreased. This could be one of the main reasons why OA cartilage erosion continues irreversibly (Osteoarthritis and Cartilage, 1998, 6, 146-149).
The steady-state levels of mRNA for type II collagen and TGF-β type II receptor (TβRII) was evaluated in human articular chondrocytes (HAC), cultured in hypoxia (5% v/v O2). The cells were treated or not with interleukin-1β (IL-1β) an agent favouring OA pathology, and NO-NSAIDs, or the corresponding NSAIDs at 10−5M for 48 h.
The following compounds were tested:
It was found that NO-flurbiprofen increased type II collagen mRNA levels (more than 100%) whereas flurbiprofen had no significant effect.
Furthermore NO-paracetamol and NO-flurbiprofen strongly increased TβRII (more than 100%) whereas their corresponding NSAIDS had no effect.
The nitrooxy derivatives according to the present invention stimulate the expression of TGF-β receptor type II and therefore delay the onset or evolution of OA.
| Number | Date | Country | Kind |
|---|---|---|---|
| MI2002A000773 | Apr 2002 | IT | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP03/03183 | 3/27/2003 | WO | 9/13/2006 |
