Patent Application: US-56723804-A

Abstract:
the present invention is related to the treatment of demyelinating and autoimmune diseases , more particularly with the treatment of multiple sclerosis . the treatment consists of the administration of p2x purinergic receptors antagonist substances which cause a remission of the symptoms common to these types of diseases . this is demonstrated in in vitro cell models as well as in animal models .

Description:
the first aspect of the invention refers to the use of p2x purinergic receptor antagonists for the treatment of demyelinating and autoimmune diseases . autoimmunity requires the activation of a precise cascade of processes in cells of the immune system . one part of these cells , the macrophages and the lymphocytes , express p2x1 , p2x2 , p2x5 and p2x7 receptors , and the activation of the latter causes the release of pro - inflammatory cytokines such as tumour necrosis factor - α ( tnf - α ) and il - 1β as well as apoptosis by mechanisms which are still not characterised ( burnstock , 2002 , arteriorscler thromb vasc biol . 22 , 364 - 373 ). however , the exact functions which mediate the p2x receptors in the immune system are still not well understood . it is this expression of p2x receptors in cells of the immune system which makes the use of the p2x receptor antagonists suitable for the treatment of autoimmune diseases . a preferred embodiment of the invention contemplates treating a disease such as multiple sclerosis . among the p2x receptor antagonists there are some which are called wide spectrum owing to the fact that they are capable of binding themselves to several of the family of p2x receptors , although with different affinities to each one of them ; and others which are selective to a group of receptors from the p2x family . the following formulas represent some of these wide spectrum p2x receptor antagonists : ppads ( tetrasodium salt of pyridoxalphosphate - 6 - azophenyl - 2 ′, 4 ′- disulfonic acid ) ( i ) iso - ppads ( tetrasodium salt of pyridoxalphosphate - 6 - azophenyl - 1 ′, 4 ′- disulfonic acid ) ( ii ) suramin ( hexasodium salt of 8 , 8 ′-[ carbonylbis [ imino - 3 , 1 - phenylenecarbonylimino ( 4 - methyl - 3 , 1 - phenylene ) carbonylimino ]] bisnaphthalene - 1 , 3 , 5 - trisulphonic acid ( iii ) evans blue ( tetrasodium salt of 6 , 6 ′-[( 3 , 3 ′- dimethyl [ 1 , 1 ′- biphenyl ]- 4 , 4 ′- diyl ) bis [ 4 - amino - 5 - hydroxy - 1 , 3 - naphthalenedisulfonic acid ]) ( iv ) nf023 ( hexasodium salt of 8 , 8 ′-[ carbonylbis [ imino - 3 , 1 - phenylenecarbonylimino ] bis - 1 , 3 , 5 - naphthalenetrisulfonic acid ] ( v ) nf279 hexasodium salt of ( 8 , 8 ′-[ carbonylbis ( imino - 4 , 1 - phenylenecarbonylimino - 4 , 1 - phenylenecarbonylimino )) bis ( 1 , 3 , 5 - naphthalenetrisulfonic acid ] ( vi ) cbb - g ( coomassie brilliant blue g )( vii ) nf449 ( octasodium salt of 4 , 4 ′, 4 ″, 4 ′″-( carbonylbis ( imino - 5 , 1 , 3 - benzenetriylbis ( carbonylimino ))) tetrakis - benzene - 1 , 3 - disulfonic acid ) ( viii ) o - atp ( sodium salt of adenosine 5 - triphosphate , oxidised with periodate ) ( ix ) kn - 62 ( ester of 4 -[( 2s )- 2 -[( 5 - isoquinolinylsulfonyl ) methylamino ]- 3 - oxo - 3 -( 4 - phenyl - 1 - piperazinyl ) propyl ] phenyl isoquinolinesulfonic acid ) ( x ) ppnds ( tetrasodium salt of pyridoxal - 5 ′- phosphate - 6 -( 2 ′- naphthylazo - 6 ′- nitro - 4 ′, 8 ′- disulfonate ) ( xi ) rb2 1 - amino - 4 -[[ 4 -[[ 4 - chloro - 6 -[[ 3 ( or 4 )- sulfophenyl ] amino ]- 1 , 3 , 5 - triazin - 2 - yl ] amino ]- 3 - sulfophenyl ] amino ]- 9 , 10 - dihydro - 9 , 10 - dioxo - 2 - anthracenesulfonic acid ) ( xii ) besides those mentioned previously there are other wide spectrum antagonists such as mrs2220 ( cyclic pyridoxine - α4 , 5 - monophosphate - 6 - azo - phenyl - 2 ′, 5 ′ disulfonate ), ip51 ( pentapotassium salt of p 1 , p 5 - diinosine - 5 - pentaphosphate ) o el tnp - atp ( monolithium salt of 2 ′, 3 ′- o - 2 , 4 , 6 - trinitrophenyladenosine 5 ′- triphosphate ), as well as selective ones such as , for example , hma ( 5 -( n , n hexamethylene ) amiloride ). the ic 50 of some of the previous compounds in relation to the different px2 receptor subgroups are set out in table 1 . in a preferred embodiment , one of the aforementioned antagonists is a p2x7 selective receptor antagonist , o - atp . in studies carried out by the inventors ( see example further on ) this compound has been shown to be specially suitable for the treatment of multiple sclerosis owing to the relative importance of the presence of p2x7 receptors in oligodendrocytes compared to the other p2x receptors . another aspect of the invention refers to a pharmaceutical composition which comprises of at least one p2x purinergic receptor antagonist , either wide spectrum or selective of a receptor subgroup , along with at least one pharmaceutically accepted excipient . the pharmaceutically accepted excipients will be those excipients of the technique which allow the suitable formulation of the pharmaceutical composition of the invention . this formulation could be formulated for its oral , intravenous , topical , rectal , subdermal , etc ., administration . that is to say , it can be presented in the form of solutions , pills , capsules , implants , etc . likewise , this formulation can be of immediate release or controlled release . the wide spectrum inhibitors can be selected from among the previously mentioned compounds . a preferred realisation contemplates a pharmaceutical composition which contains at least o - atp , a selective antagonist of p2x7 receptors . in the following example the studies carried out by the inventors are detailed which illustrates the basis of the invention . the cell cultures were carried out from the optic nerve of the perinatal rat ( p12 ) following established protocols , which were adapted and introduced into the laboratory according to a recent description ( matute et al , 1997 , proc . natl . acad . sci . usa 94 , 8830 - 8835 ). the electrophysiological recordings were carried out in 2 to 5 day cultures , and according to the guidelines indicated in previous works ( patneau et al 1994 , neuron 12 : 357 - 371 ). the cells were recorded in a chamber which allowed the composition of the extracellular medium to vary by means of a constant flow ( 0 . 5 - 1 ml / min ). the recording electrodes were glass capillaries which contained specific solutions compatible with the cytoplasm ion concentrations . the study of the responses mediated by the purinergic receptors was carried out using the “ whole - cell patch - clamp ” technique , measuring the currents generated by the external application of selective agonists and antagonists of the aforementioned receptors . the concentration of cytosol calcium was determined by the method of grynkiewikcz et al ( 1985 ; j . biol . chem . 260 , 3440 - 3450 ). the oligodendrocytes were loaded with 5 mm of fura - 2 / am , and they were then washed and studied in a zeiss inverted microscope equipped with a monochromator , 40 × immersion objective , an orca high resolution digital camera , and aquacosmos software ( hamamatsu photonics ). the changes in cytosol calcium levels in response to agonists and antagonists , in the presence and absence of extracellular calcium , were studied . the calibration was performed at the end of the studies by means of the successive application of ionomycin and egta , and the calcium concentration was estimated using the measurement of the 340 / 380 nm ratio . the nerves were isolated from young adult rats , and were perfused for 30 min in artificial cerebrospinal fluid ( acsf ) saturated in oxygen by bubbling with 95 % oxygen and 5 % co2 , under conditions comparable to those described for oligodendrocytes in culture ( fern and möller , 2000 , j . neurosci . 20 : 34 - 42 ). next , they were incubated with purinergic agonists and antagonists for different times . later , the nerves were perfused for 1 to 24 hours with normal acsf saturated with oxygen . after this time passed the damage was evaluated histologically as we have described in vivo ( matute , 1998 , proc . natl . acad . sci . usa 95 : 10229 - 10234 ), and the biochemical changes which are underlying to this damage were analysed . immunochemical methods in oligodendrocyte cultures , optic nerve and nerve tissue of experimental animals commercial antibodies were used for the study of the presence of oligodendroglial lineage markers , components of myelin , astrocytes and microglia . the techniques included immunocytochemistry , immunohistochemistry and immunoblotting ( western blot ), all these are described in detail ( see for example , domercq et al , 1999 , eur . j . neurosci . 11 , 2226 - 2236 ) the experiments on the optic nerve were carried out in rabbits ( new zealand white ) which , due to their size , enable better manipulation in experimental surgery . the procedure used was that described previously ( matute , 1998 , proc . natl . acad . sci . 95 , 10229 - 10234 ). the purinergic agonists were applied using osmotic micropumps which released small quantities of solute for a determined time . later , the effect of this application on the nerve was evaluated by means of a panel of oligodendrocyte markers and their progenitors , myelin , axonal integrity , astrogliosis y microgliosis . lewis rats were used which were immunised subcutaneously with basic myelin protein in the back paws ( 100 micrograms / animal in 100 microlitres ) and freund &# 39 ; s adjuvant with 5 . 5 mg / ml of tuberculosis mycobacterium h37ra . the spinal cord was extracted when the animals had symptoms of the disease ( 12 - 14 days post - immunisation ) and the expression of the purinergic receptors was analysed using immunochemical techniques ( immunoblotting and inmunohistochemical ). atp ( 1 mm ) induces an input current which does not desensitise in the majority of oligodendrocytes examined ( 77 . 3 %± 7 . 9 ; n = 47 ; fig1 a ). the atp analogue , 2 ′, 3 ′- o -( 4 - benzoyl - 4 - benzoyl ) ( bzatp , 100 μm ), which is a wide spectrum p2x agonist but with a higher affinity for the p2x7 receptor ( ralevic & amp ; burnstock , 1998 ), also induced similar responses ( fig1 a ). on the other hand , α , β - methylene - atp ( α , β - me - atp , 100 μm ), a selective p2x1 , p2x3 and p2x2 / 3 heteromers agonist , did not generate currents in oligodendrocytes . it was observed that the amplitude of currents generated by atp and bzatp depends on the concentration of the corresponding agonist ( fig1 a ) ( ec 50 = 8 . 77 mm and 0 . 52 mm respectively ). likewise , it could be established that the absence of mg 2 + and ca 2 + , which increase the concentration of atp 4 − , the active form of the p2x receptors , increase the responses by 4 - 10 fold ( fig1 a ). the ppads wide spectrum antagonist ( 100 μm ), for its part , completely blocked the currents induced by atp ( fig1 ). for its part , oxidised atp ( o - atp ), a preferential antagonist of the p2x7 receptors , partially blocks the atp currents . for its part , cu 2 + ( 1 mm ) which is a selective inhibitor of the p2x7 receptors ( virginio et al , 1997 ), reduces atp currents , while propofol ( 60 μm ), a potentiator of the p2x4 receptors , does not alter these currents . these results indicate that the p2x receptors present in oligodendrocytes have electrophysiological properties compatible with a predominance of the p2x7 sub - unit . the activation of p2x receptors increase the cytosol ca 2 + levels [ ca 2 + ] i was monitored after applying atp and bzatp with the objective of characterising the effects of the activation of p2x receptors on oligodendrocytes . these cells respond to atp ( 10 μm ) with a rapid increase in basal cytosol [ ca 2 + ] i ( 250 ± 65 nm ) to 1200 ± 468 nm ( fig2 a ). these responses are repressed in the presence of ppads ( 50 μm ) and with the absence of ca 2 + in the incubation solution . these results indicate that the [ ca 2 + ] i increases are due to the entrance of ca 2 + across the plasma membrane and not due to the liberation of this from intracellular deposits . bz - atp ( 0 . 01 - 1 mm ) also activates the entrance of ca 2 + into oligodendrocytes in a dose dependent way ( fig2 b , d ). this effect disappears in the absence of extracellular ca 2 + and is blocked by ppads ( fig2 b , d ). these results suggest that the p2x receptors which contain the p2x7 sub - unit are the principal mediators of the response to atp . in agreement with this idea , the o - atp p2x7 selective antagonist ( 1 mm ) ( fernández et al , 2001 ), reduces the increase in [ ca 2 + ] i induced by bz - atp by 63 ± 8 ( fig2 d , e ). for its part , propofol ( 60 μm ), which triggers the responses mediated by p2x4 ( tomioka et al ., 2000 ), promotes the increase of [ ca 2 + ] i generated by 0 . 1 and 1 mm atp in 60 %± 22 and 77 %± 34 respectively ( fig2 c , d ). therefore , the p2x native receptors which contain p2x4 also contribute to the entrance of calcium induced by atp in oligodendrocytes . the activation of p2x receptors induce ca 2 + dependent oligodendroglial death at all atp concentrations ( 0 . 01 - 1 mm ) tested , death was produced in 15 - 27 % of oligodendrocytes which is inhibited in the presence of 50 μm ppads and after removing ca 2 + from the culture medium ( fig3 a ). in the same way the bz - atp agonist caused a toxicity similar to atp ( fig3 b ). other purinergic agonists such as atp - γ - s , which is a more stable analogue than atp , and α , β - meatp , are also toxic for the oligodendrocytes , which exclude the possibility that the metabolites of atp might be the agents causing the toxicity after activating receptors different to the p2x ones . overall , the toxicity tests showed that the oligodendrocytes are vulnerable to the activation of p2x receptors by atp and its analogues . the analysis of the expression p2x receptors using immunohistochemistry with specific antibodies in cultures of differentiated oligodendrocytes ( galc + / mbp + ) demonstrates that these cells mainly have the p2x2 , p2x4 y p2x7 subunits ( see table 2 ). this expression profile is consistent with the electrophysiological properties and toxicity characteristics observed in these cultures . also , the pattern of the subunits observed in vitro also corresponds with that observed in situ in optic nerve ( table 3 ) by means of double marking of the subunits and antibodies specific to the oligodendroglial and astroglial lineage ( fig5 ). to determine if atp is toxic to the oligodendrocytes in a preparation of nerve tissue without dissociating , entire optic nerves isolated from adult rats were perfused with artificial cerebrospinal fluid with atp ( 100 μm ) for 3 h . under these conditions an increase of & gt ; 3 times the number of cells which showed nuclear condensation as compared to the control nerves perfused without atp was produced ( fig5 ). the damaged cells are located in the longitudinal axis of the nerve and make up part of interfascicular oligodendrocyte rows . stimulation with atp in the presence of ppads ( 10 μm ) prevents the death of the oligodendrocytes . next , the atp - γ - s and bzatp agonists were infused over the optic nerve using osmotic pumps which released very small quantities of solute for 3 days . the histological examination of the nerves 7 days after starting the application showed tissue damage in an area restricted to the proximity of the cannula ( fig6 ). also , this zone had intense gliosis , lack of myelin and axonal damage ( fig6 ). overall these results indicate that the activation of p2x kills oligodendrocytes in situ and that the lesions in vivo share properties common to multiple sclerosis plaques . blocking of p2x improves the motor symptoms of acute and chronic eae the effects of the wide spectrum antagonist ppads and the more selective o - atp in the triggering off and on the course of eae induced by the immunisation of lewis rats with basic myelin protein were investigated . the immunised rats showed signs of motor deficits around 10 days post - injection , and they reached a maximum at 14 days ( fig7 ). the treatment with ppads ( 30 mg / kg , two times per day ) from 7 to 14 days post - injection did not improve the symptoms or the course of the disease . on the other hand the application of o - atp ( 1 and 5 mg / kg , every 12 h ) for the same period reduced or prevented the appearance of symptoms common to eae ( fig7 ). later , the efficacy of o - atp in improving the symptoms of eae was evaluated in a chronic - recurrent - remittent model . for this , da rats were immunised with syngeneic spinal cord , the appearance of severe neurological deficits being observed 7 - 9 days post - injection , and which reached their first peak around 11 days . treatment with o - atp ( 2 . 5 mg / kg , every 12 h ), once the maximum intensity of the symptoms were established , reduced the symptoms and also eliminated those common to the chronic phase ( fig8 ). with the objective of understanding the mechanism of action by which o - atp improves the prognosis of eae , the levels of p2x7 receptors over which the preferred form of this drug acts on the lumbar - sacral spinal cord , the region most affected in this experimental disease , was evaluated using western blot . we found that the levels of this subunit were reduced by half in animals subjected to eae , and that these levels returned to those of the controls in those animals with eae treated with o - atp ( fig9 ). these results indicate that treatment with o - atp protects the cells which express p2x7 from dying , and consequently , the oligodendrocytes , which are the main type of cells which express this subunit in the spinal cord . the results shown previously demonstrate for the first time that the oligodendrocytes have p2x receptors . likewise , the electrophysiological , pharmacological and molecular properties of these receptors , as well as their increased permeability to calcium , are given in detail . this latter property results in that the oligodendrocytes may be vulnerable to intense and / or prolonged stimulus mediated by these receptors , as has been demonstrated with glutamergic receptors in this cell population ( matute et al , 2001 , trends neurosci 24 , 224 - 230 ). the vulnerability of the oligodendrocytes to the signals mediated by the p2x receptors is one of the causes of nervous tissue damage which underlies the experimental disease , eae , a model of multiple sclerosis . finally , blocking of the p2x receptors until triggering off the disease drastically reduces the neurological symptoms in acute eae , and improves the outcome and prognosis in chronic eae once the symptoms are established . the invention described herein constitutes a means for the treatment of multiple sclerosis , a disease which lacks efficient treatments which slow down or check its progression . the routes of intervention which have resulted in the development of drugs in clinical trial phase or for use as drugs in the treatment of multiple sclerosis have mechanisms of action which regulate the functioning of the immune system . the fact that the blocking of the p2x may prevent the symptoms of acute eae , a model of ms which mimics the inflammatory / autoimmune phase of the disease , indicates that these drugs can in fact be powerful immunomodulatory agents which may prevent the autoimmunity which triggers off ms and other diseases . finally , the p2x receptor antagonists on being protector agents of the death of oligodendrocytes , the cell population which suffers most damage in ms , have great therapeutic potential in the neurodegenerative phase of this disease , a phase which is prolonged for decades and in which patients suffer a progressive deterioration which continues its course with motor and sensory disorders causing invalidity . abbracchio , m . p . and burnstock , g . 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