Patent Application: US-55981404-A

Abstract:
use of an agent capable of inhibiting sap ligand binding activity or depleting sap from the plasma of a subject for the production of a medicament for treatment or prevention of osteoarthritis in the subject .

Description:
in order to identify compounds that inhibit ligand binding of sap it is first necessary to have methods for showing such binding . for the purposes of this invention , the binding of sap to test ligands can be demonstrated directly by allowing sap , provided either by whole human or animal serum , or in isolated purified form , to contact the solid phase ligand . contact takes place in physiological buffered saline containing sufficient free calcium ions ( about 2 mmol / l ), which are essential for ligand binding by sap . in the case of isolated human sap , the buffer must also contain 40 g / l of human or bovine serum albumin , in order to keep the sap in solution ; at lower albumin concentrations isolated human sap rapidly autoaggregates and precipitates in the presence of calcium ( 18 , 19 ). suitable ligands to which sap shows its typical calcium dependent binding include agarose ( 24 ), phosphoethanolamine ( 25 ), dna ( 26 ), chromatin ( 27 ), and amyloid fibrils ( 12 , 28 ). they are immobilized on particles , such as agarose , acrylamide , polystyrene , latex , cellulose , or other beads , or on membranes , filters , or plastic or other solid surfaces such as microtitre plates or individual tubes , or may be integral components of solid particles , such as bacteria or agarose gel , that can be used directly . immobilization of soluble ligands , or secondary immobilization of particulate ligands for purposes of convenience , may be by direct non - specific adherence of the ligand , or by covalent attachment via amino , hydroxyl , or other chemical groups on the ligand molecules being coupled directly or via spacer linkers to the solid phase material . after contacting the solid or immobilised ligands , sap that has not bound is washed away with the same buffer in which binding took place , and the presence of sap bound to the ligands is detected and quantified . washing involves phase separation , such as centrifugation of solid particles , or immersion , flow through or flow over of solid surfaces such as membranes , filters , and plastic surfaces . bound sap may be detected directly if the source of sap contains sap that has been labelled with a detectable marker . such markers include gamma - emitting isotopes such as 125 i or 131 i for detection in a gamma counter ; beta - emitting isotopes such as 14 c or 3 h for detection in a beta or scintillation counter ; fluorochromes for detection in a fluorimeter , flow cytometer , or fluorescence activated cell sorter ; enzymes such as peroxidase or alkaline phosphatase for detection by their specific catalytic activity . in all of these cases it is essential to demonstrate that the process of directly labelling the sap does not alter its physiological binding properties . this is done by comparing the binding of labelled and unlabelled sap to an immobilised solid phase ligand , such as phosphoethanolamine attached using a carbodiimide to carboxyhexyl - sepharose ™. binding of sap can also be demonstrated directly by immunochemical assay showing depletion of sap from the offered source of sap , and recovery of the bound sap when , after first washing with calcium containing buffer , the ligand material is eluted with buffer containing edta to chelate calcium ions . alternatively , bound sap may be detected indirectly , using antibodies raised in rabbits , sheep , goats , rats , mice , guinea pigs or other animals , specific for the sap of the species being tested . for this purpose the anti - sap antibodies may themselves be directly labelled with a radioactive isotope , enzyme , fluorochrome or other detectable marker , or the binding of anti - sap antibodies to bound sap may be detected using a second antibody directed against the immunoglobulin of the species of the primary anti - sap reagent . in addition to detection and counting in instruments appropriate for the marker used , binding of sap to micro - organisms or their components may be visualised directly or indirectly using light , fluorescence or electron microscopy . enzyme labelled sap or anti - sap antibodies can be used for light or electron microscopy , fluorochrome labelled reagents for fluorescence microscopy , and gold ( or other electron dense particle ) labelling for electron microscopy . an alternative approach to demonstration of ligand binding by sap is to immobilize the sap on a solid phase and then allow it to bind ligands that are either directly labelled or that can be detected , for example using specific antibodies directed against these ligands . thus isolated purified sap from man or other animals can be immobilized on beads , particles , membranes , filters , or plastic or other solid surfaces , by direct non - specific adherence or by covalent coupling , or by trapping with specific anti - sap antibodies immobilized on the solid phase ( 29 ). using the conditions specified in 1 ] above , suitable ligands , can then be contacted to the immobilized sap and allowed to be bound by it . any of the methods set out in 1 ] and 2 ] above for showing ligand binding by sap can be used to test the capacity of compounds to inhibit such binding . however the speed and ease of use of the different techniques vary greatly , as well as their suitability for different purposes . thus for screening large numbers of compounds , high throughput methods , such as those based on microtitre plates , are essential . a typical method of this type involves having ligand immobilized on the plates , and offering to each well an amount of radiolabelled sap under conditions such that about 40 % of it is bound . compounds to be tested are added to the wells and preincubated in them before addition of the labelled sap , and the effect of their presence on subsequent binding of sap is monitored . in another configuration , the compounds to be tested are preincubated with the labelled sap before the mixture is added to the plates . the reverse configuration , in which the sap is immobilized , is also informative . here the test compounds are preincubated with the immobilized sap before the detectable ligand is added . these different approaches enable detection of compounds that block ligand binding by sap by different mechanisms , and help to distinguish between those that are themselves specific ligands for sap , those that affect the sap molecule in other ways , and those that interact with the ligand to prevent its recognition by sap . surface plasmon resonance ( spr ) is a low throughput but powerfully informative method to identify compounds that are bound by , or themselves bind to sap , either in a calcium dependent fashion or independently of calcium , for the purposes of the present invention . for example , low molecular weight compounds that inhibit binding of sap to its macromolecular ligands , including target ligands relevant to the present invention , can be detected by their effect on the signal generated by fluid phase sap binding to ligand immobilised on the solid phase ( example a ). furthermore , when sap itself is immobilised on the solid phase in an spr instrument , the interaction with it of test molecules provided in the fluid phase generates a signal that can be detected and quantified . purified sap immobilized within an spr instrument gives a quantifiable signal when it is exposed to another molecule that forms a complex with the sap , and this is distinct from the absence of such a signal if no complex is formed . this technique allows compounds to be screened for their capacity to interact with sap , and does not depend on any specific mode of interaction with sap , in particular involving the calcium dependent ligand binding site of sap , so it detects molecules that might not be found in test systems that require calcium dependent sap binding . another low throughput but powerful direct method is isothermal calorimetry that measures the heat of interaction in solution between sap and test compounds according to the present invention . the binding affinity can be measured precisely as a guide to potential efficacy . typical results by this method for the dissociation constants , k d , between sap and various compounds are as follows : the latter two compounds are from ep - a - 915088 , in which ro - 63 - 8695 is ( r )- 1 -[ 6 -[( r )- 2 - carboxy - pyrrolidin - 1 - yl ]- 6 - oxo - hexanoyl ] pyrrolidine - 2 - carboxylic acid , and ro - 64 - 2856 is ( r )- 1 -[[ 4 -[ 2 -[( r )- 2 - carboxy - pyrrolidin - 1 - yl ]- 2 - oxo - ethoxy ]- phenoxy ]- acetyl ]- pyrrolidine - 2 - carboxylic acid . effects on sap in vivo of compounds that block sap - ligand binding in vitro . for the purpose of the present invention , compounds that block binding of sap to target ligands are tested in vivo in transgenic mice expressing human sap , as described elsewhere ( 23 ) for their effects on plasma sap concentrations and the turnover and catabolism of sap . having established by the administration of graded doses that the compounds are not intrinsically toxic , various doses are administered to the human sap transgenic mice . serum is taken at regular intervals for immunochemical assay of sap . in addition , trace radiolabelled human sap may be injected intravenously at different times in relation to the drug dosage , and both whole body counting and blood sampling are performed to monitor the plasma half life and whole body clearance of sap ( 14 , 30 ). in addition the tracer sap is preincubated with different amounts of the compounds being tested , and then injected into mice not receiving any drug , in order to test the effect drug binding has on sap clearance . according to the present invention , compounds that accelerate sap clearance in vivo and / or lower plasma sap concentration , thereby reducing in vivo availability of sap , are likely to be of therapeutic value . compounds that have undergone formal toxicity testing and found to be acceptable for administration in man , are evaluated for their effects on plasma sap concentration , half life , turnover and catabolism . isolated human sap is trace radiolabelled with 125 i and / or 123 i and injected intravenously , followed by plasma turnover studies and whole body scintigraphic imaging , as described elsewhere ( 15 , 23 , 31 ). use of surface plasmon resonance to detect inhibition of calcium dependent ligand binding by sap synthetic aβ - 42 amyloid fibrils ( 28 ) were covalently immobilised on the reactant surface of the fisons iasys surface plasmon resonance instrument , and then exposed to isolated sap ( 32 ) in solution in tris - buffered physiological saline in the absence of calcium ( shown as tn / n in the figures ). no binding of sap occurred and thus no signal was generated . introduction of calcium allowed the sap to bind specifically to the immobilised amyloid fibrils , generating a readily detectable signal ( fig1 ). 1 -[( s )- 3 - mercapto - 2 - methylpropionyl ]- d - proline ( designated on the figures as ro - 3479 ) is a specific inhibitor of calcium dependent ligand binding by sap . addition of 1 -[( s )- 3 - mercapto - 2 - methylpropionyl ]- d - proline ( ro - 3479 ) at 500 μmol / l in tris - buffered physiological saline containing calcium ( tc in the figures ), completely reversed the binding of sap and the corresponding signal ( fig1 ). after addition of sap in the absence of calcium , followed by ro - 3479 at 100 μmol / l , and then allowing equilibration of the system , subsequent addition of calcium to enable specific calcium dependent ligand binding by sap was followed by no signal ( fig2 ), indicating that ro - 3479 not only dissociates sap binding but also inhibits it . the solid phase ligand was regenerated for further calcium dependent ligand binding , by washing it with edta in tris - buffered physiological saline ( te in the figures ), and then re - exposed to sap in tn buffer followed by calcium . the typical signal reflecting ligand binding by sap was observed again , and then completely reversed by addition of ro - 3479 at 50 μmol / l ( fig3 ). screening for inhibitors of binding of 125 i radiolabelled sap to neisseria meningitidis organisms immobilized in microtitre plates a suspension of heat killed neisseria meningitidis at 1 × 10 8 organisms per ml in pbs was dispensed to polystyrene microtitre plates at 50 μl volumes per well , and left overnight at 4 ° c . all wells were then washed three times with 200 μl volumes of pbs containing 0 . 05 % v / v tween 20 , prior to equilibration for 2 min with 0 . 01m tris buffered 0 . 14m nacl / 0 . 002m cacl 2 at ph 8 . 0 ( tc buffer ), containing 4 % w / v bsa and 0 . 05 %/ v tween 20 ( tcbt buffer ). the wells were then emptied before adding to each one the following reagents . for control uninhibited maximal binding : 35 μl tcbt buffer ( containing 2 . 3 mm cacl 2 , 5 . 72 % w / v bsa , 0 . 072 % v / v tween 20 ), 10 μl tc and 5 μl sap radiolabelled with 125 i in 0 . 01m tris buffered 0 . 14m nacl at ph 8 . 0 ( tn buffer ), to provide final concentrations of bsa , 4 %; ca 2 + , 2 mm ; tween 20 , 0 . 05 %. for background , non - specific , non calcium dependent , binding in the presence of edta : 5 μl radiolabelled sap in tn and 45 μl 0 . 01m tris buffered 0 . 14m nacl at ph 8 . 0 containing 11 . 1 mm edta , 4 . 4 % w / v bsa and 0 . 06 % w / v tween 20 ( tebt buffer ) to provide final concentrations of edta , 10 mm ; bsa , 4 %; tween 20 , 0 . 05 %. for testing of inhibitors : 35 μl of tcbt ( containing 2 . 3 mm ca 2 + , 5 . 72 % bsa and 0 . 072 % tween 20 ), 10 μl tc containing test compounds at 10 mm , 1 mm , 100 μm , 10 μm or 1 μm , and 5 μl of radiolabelled sap in tn . all wells were then incubated at room temperature for 2 h before being washed three times with 200 μl volumes of tcbt , allowed to dry for 1 h at room temperature , and bound radiolabelled sap was then counted . the compounds tested in the experiment shown here were a family of molecules developed as inhibitors of sap binding to amyloid fibrils , following identification of an initial lead molecule during high throughput screening of a large compound library according to u . s . pat . no . 6 , 126 , 918 . the original hit was 1 -[( s )- 3 - mercapto - 2 - methylpropionyl ]- d - proline , ( ro - 15 - 3479 ), and a dimer of one of its diastereoisomers , ( r )- 1 -[( s )- 3 -[( s )- 3 -[( r )- 2 - carboxy - pyrrolidin - 1 - yl ]- 2 - methyl - 3 - oxo - propyldisulfanyl ]- 2 - methyl - propionyl ]- pyrrolidine - 2 - carboxylic acid ( ro - 63 - 3300 ) was found to be much more potent . the other two diastereoisomers did not inhibit ligand binding by sap . a chemistry programme then produced , ( r )- 1 -[ 6 -( r )- 2 - carboxy - pyrrolidin - 1 - yl ]- 6 - oxo - hexanoyl ] pyrrolidine - 2 - carboxylic acid ( ro - 63 - 8695 ) ( example 8b of ep - a - 915088 ) and a family of related molecules , that were tested here . their chemical names and coded designations are listed below : ro - 64 - 4383 : ( r )- 1 -[[ 2 , 5 - dihydroxy - 4 -[ 2 -[( r )- 2 - carboxy - pyrrolidin - 1 - yl ]- 2 - oxo - ethyl ]- phenyl ]- acetyl ]- pyrrolidine - 2 - carboxylic acid ro - 642856 : ( r )- 1 -[[ 4 -[ 2 -[( r )- 2 - carboxy - pyrrolidin - 1 - yl ]- 2 - oxo - ethoxy ]- phenoxy ]- acetyl ]- pyrrolidine - 2 - carboxylic acid ro - 63 - 3300 : ( r )- 1 -[( s )- 3 -[( s )- 3 -[( r )- 2 - carboxy - pyrrolidin - 1 - yl ]- 2 - methyl - 3 - oxo - propyldisulfanyl ]- 2 - methyl - propionyl ]- pyrrolidine - 2 - carboxylic acid ro - 15 - 3479 : 1 -[( s )- 3 - mercapto - 2 - methylpropionyl ]- d - proline ro - 64 - 2848 : ( r )- 1 -[[ 3 -[ 2 -[( r )- 1 - carboxy - pyrrolidin - 1 - yl ]- 2 - oxo - ethoxy ]- 2 - methyl - phenoxy ]- acetyl ]- pyrrolidine - 2 - carboxylic acid ro - 64 - 2668 : ( r )- 1 -[[ 3 -[ 2 -[( r )- 2 - carboxy - pyrrolidin - 1 - yl ]- 2 - oxo - ethyl ]- phenyl ]- acetyl ]- pyrrolidine - 2 - carboxylic acid ro - 64 - 2845 : ( r )- 1 -[[ 2 -[ 2 -[( r )- 2 - carboxy - pyrrolidin - 1 - yl ]- 2 - oxo - ethoxy ]- 3 - methoxy - phenoxy ]- acetyl ]- pyrrolidine - 2 - carboxylic acid ro - 64 - 2600 : ( r )- 1 -[ cis - 4 -[( r )- 2 - carboxy - pyrrolidine - 1 - carbonyl ]- cyclohexanecarbonyl ]- pyrrolidine - 2 - carboxylic acid ro - 64 - 5607 : ( r )- 1 -[[ 4 -[ 2 -[( r )- 2 - carboxy - pyrrolidin - 1 - yl ]- 2 - oxo - ethyl ]- naphthalen - 1 - yl ]- acetyl ]- pyrrolidine - 2 - carboxylic acid ro - 64 - 5445 : ( r )- 1 -[[ 5 -[ 2 -[( r )- 2 - carboxy - pyrrolidin - 1 - yl ]- 2 - oxo - ethoxy ]- naphthalen - 1 - yloxy ]- acetyl ]- pyrrolidine - 2 - carboxylic acid ro - 63 - 8593 : ( r )- 1 -[[ 2 -[ 2 -[( r )- 2 - carboxy - pyrrolidin - 1 - yl ]- 2 - oxo - ethoxy ]- phenoxyl ]- acetyl ]- pyrrolidine - 2 - carboxylic acid ro - 63 - 7777 : ( r )- 1 -[[ 4 -[ 2 -[( r )- 2 - carboxy - pyrrolidin - 1 - yl ]- 2 - oxo - ethyl ]- phenyl ]- acetyl ]- pyrrolidine - 2 - carboxylic acid the capacity of the compounds tested to inhibit binding of sap to meningococci is expressed as the percentage by which sap binding was reduced compared to binding in the absence of any inhibitor ( table 1 ). all binding was inhibited by edta , confirming the specific , calcium dependent , nature of the interaction . phosphoethanolamine , a well known ligand of sap ( 25 , 32 ), produced inhibition at high concentration , but only modest effects when diluted . phosphocholine , the specific ligand of c - reactive protein , the other human plasma pentraxin protein closely related to sap , had virtually no effect , as expected since there is no other evidence that sap recognises and binds to phosphocholine under physiological conditions . the ro - 63 - 8695 family of molecules were potent inhibitors with ic 50 values in the sub - micromolar range in several cases . these compounds are thus candidates for further testing according to the present invention . a solution containing 100 μg / ml chicken erythrocyte native long chromatin ( 27 ), in pbs adjusted to ph 9 , was dispensed to microtitre plates containing n - oxysuccinimide - activated surfaces , at 50 μl volumes per well and left at room temperature for 1 h . all wells were then washed three times with 200 μl volumes of pbs , ph 7 . 4 , containing 0 . 05 % v / v tween 20 ( pbst ) and unreacted active sites were blocked by the addition of 50 μl of 2 % w / v bsa in pbs , ph 7 . 4 , to each well , for 30 min at room temperature . wells were then washed three times with 200 μl volumes of pbst prior to equilibration for 2 min with 0 . 01m tris buffered 0 . 14m nacl / 0 . 002m cacl 2 at ph 8 . 0 ( tc buffer ), containing 4 % w / v bsa and 0 . 05 % v / v tween 20 ( tcbt buffer ). the wells were then emptied before adding to each one the following reagents . for control uninhibited maximal binding : 35 μl tcbt buffer ( containing 2 . 3 mm cacl 2 , 5 . 72 % w / v bsa , 0 . 072 % v / v tween 20 ), 10 μl tc and 5 μl sap radiolabelled with 125 i - in 0 . 01m tris buffered 0 . 14m nacl at ph 8 . 0 ( tn buffer ), to provide final concentrations of bsa , 4 %; ca 2 + , 2 mm ; tween 20 , 0 . 05 %. for background , non specific , non calcium dependent , binding in the presence of edta : 5 μl radiolabelled sap in tn and 45 μl 0 . 01m tris buffered 0 . 14m nacl at ph 8 . 0 containing 11 . 1 mm edta , 4 . 4 % w / v bsa and 0 . 06 % w / v tween 20 ( tebt buffer ) to provide final concentrations of edta , 10 mm ; bsa , 4 %; tween 20 , 0 . 05 %. for testing of inhibitors : 35 μl of tcbt ( containing 2 . 3 mm ca 2 + , 5 . 72 % bsa and 0 . 072 % tween 20 ), 10 μl tc containing test compounds at 10 mm , 1 mm , 100 μm , 10 μm or 1 μm , and 5 μl of radiolabelled sap in tn . all wells were then incubated at room temperature for 2 h before being washed three times with 200 μl volumes of tcbt , allowed to dry for 1 h at room temperature , and bound radiolabelled sap was then counted . the compounds tested in the experiment shown here were the same as those specified in example b above . the capacity of the compounds tested to inhibit binding of sap to chromatin is expressed as the percentage by which sap binding was reduced compared to binding in the absence of any inhibitor ( table 2 ). all binding was inhibited by edta , confirming the specific , calcium dependent , nature of the interaction . binding to control wells without chromatin and just blocked with bsa was at the same background level as seen with complete inhibition by edta or the specific inhibitors . phosphoethanolamine , a well known ligand of sap ( 25 , 32 ), produced inhibition at high concentration , but only modest effects when diluted . phosphocholine , the specific ligand of c - reactive protein , the other human plasma pentraxin protein closely related to sap , had virtually no effect , as expected since there is no other evidence that sap recognises and binds to phosphocholine under physiological conditions . the ro - 63 - 8695 family of molecules were potent inhibitors with ic 50 values in the micromolar range in several cases . these compounds are thus candidates for further testing according to the present invention , although interestingly this assay was less sensitive than that using meningococci as the immobilised ligand . a concentrated suspension of purified influenza virus a / shanghai / 24 / 90 at 10 mg protein per ml in pbs was diluted 1 : 50 in pbs and then dispensed to polystyrene microtitre plates at 50 μl volumes per well , and left overnight at 4 ° c . all wells were then emptied before blocking by addition to each well of 200 μl of 2 % w / v bsa in pbs and incubation at room temperature for 1 h . all wells were then washed three times with 200 μl volumes of pbs containing 0 . 05 % v / v tween 20 , prior to equilibration for 2 min with 0 . 01m tris buffered 0 . 14m nacl / 0 . 002m cacl 2 at ph 8 . 0 ( tc buffer ), containing 4 % w / v bsa and 0 . 05 % v / v tween 20 ( tcbt buffer ). the wells were then emptied before adding to each one the following reagents . for control uninhibited maximal binding : 35 μl tcbt buffer ( containing 2 . 3 mm cacl 2 , 5 . 72 % w / v bsa , 0 . 072 % v / v tween 20 ), 10 μl tc and 5 μl sap radiolabelled with 125 i ( 32 ) in 0 . 01m tris buffered 0 . 14m nacl at ph 8 . 0 ( tn buffer ), to provide final concentrations of bsa , 4 %; ca 2 + , 2 mm ; tween 20 , 0 . 05 %. for background , non - specific , non calcium dependent , binding in the presence of edta : 5 μl radiolabelled sap in tn and 45 μl 0 . 01m tris buffered 0 . 14m nacl at ph 8 . 0 containing 11 . 1 mm edta , 4 . 4 % w / v bsa and 0 . 06 % w / v tween 20 ( tebt buffer ) to provide final concentrations of edta , 10 mm ; bsa , 4 %; tween 20 , 0 . 05 %. for testing of inhibitors : 35 μl of tcbt ( containing 2 . 3 mm ca 2 + , 5 . 72 % bsa and 0 . 072 % tween 20 ), 10 μl tc containing test compounds at 10 mm , 1 mm , 100 μm , 10 μm or 1 μm , and 5 μl of radiolabelled sap in tn . all wells were then incubated at room temperature for 2 h before being washed three times with 200 μl volumes of tcbt , allowed to dry for 1 h at room temperature , and bound radiolabelled sap was then counted . the compounds tested in the experiment shown here were the same as those specified in example b above . the capacity of the compounds tested to inhibit binding of sap to the immobilised influenza virus is expressed as the percentage by which sap binding was reduced compared to binding in the absence of any inhibitor ( table 3 ). all binding was inhibited by edta , confirming the specific , calcium dependent , nature of the interaction . binding to control wells without virus and just blocked with bsa was at the same background level as seen with complete inhibition by edta or the specific inhibitors . phosphoethanolamine , a well known ligand of sap ( 25 , 32 ), produced inhibition at high concentration , but only modest effects when diluted . phosphocholine , the specific ligand of c - reactive protein , the other human plasma pentraxin protein closely related to sap , had virtually no effect , as expected since there is no other evidence that sap recognises and binds to phosphocholine under physiological conditions . the ro - 63 - 8695 family of molecules were potent inhibitors with ic 50 values in the sub - micromolar , high nanomolar , range in several cases . these compounds are thus candidates for further testing according to the present invention , and interestingly this assay was more sensitive than those using either whole meningococci or native long chromatin as the immobilised ligand . accumulation of labelled sap was found in joints of patients without dialysis amyloidosis who were suffering from other forms of arthritis : 5 individuals with osteoarthritis ( fig4 ), 12 with rheumatoid arthritis , and one subject with a traumatic effusion . in each of these patients , labelled sap was detected in all joints with a significant effusion , regardless of aetiology . this in itself is not surprising since it is known that plasma proteins enter joint effusions . however , in 20 out of 30 patients with various different arthropathies we have also observed uptake of labelled sap into some joints that did not have clinically detectable effusions . fig4 shows a scintigraphic image of the hands of a patient with osteoarthritis 24 h after intravenous injection of 123 i - labelled sap . uptake and retention of sap is indicated in both carpal areas , in the second metacarpophalangeal joints , and some interphalangeal joints . one possible mechanism for localisation of sap from the blood to a diseased joint may be the presence within the joint of amyloid deposits in articular and peri - articular structures . there is indeed extensive evidence for the widespread presence of microscopic amyloid deposits in the synovium , articular cartilage and / or joint capsules of elderly individuals ( 33 - 44 ). however the overall impression from these observational studies of autopsy and / or resection specimens is that the amyloid deposits are mainly associated with increasing age of the subjects and not particularly with extent or severity of clinical or pathological manifestations of osteoarthritis . alternatively , or in addition , sap may be binding to ligands on structures other than amyloid fibrils that are present in inflamed or damaged joints . the calcium - dependent binding of sap to glycosaminoglycans in vitro has been reported ( 45 ), but was specific for heparan and dermatan sulphates , rather than the chrondroitin sulphate and hyaluronic acid that are most abundant in cartilage and synovial fluid respectively . nevertheless , glycosaminoglycans are ubiquitous in connective tissue and may be abnormally exposed and thereby provide ligands for sap in and around diseased joints . another ligand to which sap binds avidly , in vivo as well as in vitro , is dna ( 26 , 27 ), both free and within chromatin when this is exposed by cell death ( 46 ), and sap also binds to apoptotic cells in vivo ( 47 ). increased cell death in inflamed joints , whether by apoptosis or by necrosis , exposing chromatin , may provide an abnormal density of ligands and thus a focus for sap deposition . in order to test whether sap may be binding to ligands in diseased joints , we first compared the distribution of radiolabelled human serum albumin with that of sap in two patients . in one with proven dialysis associated amyloid , the joint uptake of sap was very much greater than that of albumin , showing that the sap localisation was specific for amyloid . in contrast , in a patient with active rheumatoid arthritis and multiple affected joints with effusions , the localisation of albumin and sap were generally comparable , suggesting a similar non - specific process of effusion into the synovial space for both proteins , although occasional joints showed greater retention of sap than of albumin . furthermore , in one patient whose knee joint effusions were aspirated to dryness 24 h after injection of radiolabelled sap , there remained strong localisation of sap in the joints . secondly , we measured the synovial fluid and serum concentrations of sap and other plasma proteins in paired samples from 15 patients with joint effusions of different aetiologies , and calculated the synovial fluid : serum ratios . there is a well known inverse linear relationship between this ratio and the relative molecular mass of the proteins , reflecting their relative ease of access by diffusion from the circulation into the joint space . however , the ratio was remarkably and substantially lower for sap than expected for a protein of its molecular mass ( fig5 ). this indicates that sap , which evidently can gain access to the synovial fluid , as shown by our studies with labelled sap , is probably binding to structure ( s ) within the joint and is therefore not available for detection and assay in the synovial fluid itself . fig5 shows the ratio of the concentration of various plasma proteins in the synovial fluid and serum from patients with various forms of arthritis causing effusions . there is a linear relationship , r = 0 . 62 , between the relative molecular mass and the synovial fluid / serum concentration ratio for all the proteins shown here except sap , for which the synovial fluid concentration is markedly lower than predicted from its molecular mass and serum concentration . ( key : α 1 ag , α 1 - acid glycoprotein ; alb , albumin ; trf , transferrin ; cer , ceruloplasmin ; α 2 m , α 2 - macroglobulin .) the very common and widespread microscopic amyloid deposits in aged and osteoarthritic joints have not hitherto been thought to contribute to the pathogenesis or symptoms of osteoarthritis . it is also not clear how binding of sap to either amyloid fibrils or other structures in joints might be pathogenetic in osteoarthritis . although artificially aggregated sap can activate the complement system ( 48 ), and could thereby be pro - inflammatory , the binding of sap to any of its known ligands not only does not activate complement , but actually inhibits complement activation by the substrate itself ( 49 , 50 ). also , there is no evidence for complement activation either locally or systemically in patients with osteoarthritis . nevertheless , in the light of our observations of sap localisation to joints and the unexpectedly low concentration of sap free in synovial fluid , we tested whether sap might be involved in osteoarthritis . 1 ) rj , a 64 year old retired general practitioner living in new zealand , has a long history of bone and joint injuries , starting as a child on a farm and continuing as a teenager and then adult playing rugby and skiing . from the age of 42 he has suffered from pain and swelling of previously damaged joints following manual work and especially in cold weather . the proximal interphalangeal joints of the left middle and right fourth fingers , the right elbow and the mid - thoracic intervertebral joints have been most affected . symptoms and signs have progressively worsened over the past 22 years , so that his capacity for physical work had become very restricted and during winter he has required frequent or continuous treatment with non - steroidal anti - inflammatory drugs . these are all typical manifestations of osteoarthritis . in 2000 the discovery of impaired renal function led eventually to a second , and completely separate diagnosis of hereditary systemic amyloidosis caused by a mutation in the gene for fibrinogen a α - chain . this type of amyloidosis does not affect the joints and its pathogenesis and clinical manifestations are completely unrelated to osteoarthritis . he started experimental treatment for his amyloidosis on 9 oct . 2001 with ( r )- 1 -[ 6 -[( r )- 2 - carboxy - pyrrolidin - 1 - yl ]- 6 - oxo - hexanoyl ] pyrrolidine - 2 - carboxylic acid , 10 mg b . d . by subcutaneous injection . in january 2002 , after three months on this potent sap depleting drug and with his plasma sap concentration consistently reduced by over 95 %, he first noted that his symptoms of arthritis were less troublesome than before . this improvement was sustained and increased so that by april 2002 , the autumn in new zealand , it was impressively beyond doubt . throughout that winter , for the first time in many years , he no longer required treatment with non - steroidal anti - inflammatory drugs , despite increased physical activity . he has continued treatment with ( r )- 1 -[ 6 -[( r )- 2 - carboxy - pyrrolidin - 1 - yl ]- 6 - oxo - hexanoyl ] pyrrolidine - 2 - carboxylic acid up to the present ( june 2003 ) and all his symptoms of osteoarthritis have , remarkably , remained in remission . over the past few months he has been doing regular hard physical work on the land , fencing and landscaping , including lifting and carrying significant weights , without suffering any of the pain and swelling that previously severely affected him even without the stress of unusual physical activity . 2 ) cd , a 49 year old service worker from wales , had a 6 - 8 year history of pain and reduced function in several joints . her shoulders were painful , particularly when reaching up or behind her head , making drying her hair and some household tasks painful and difficult . she had mid - thoracic back pain and bilateral ankle aches on most days , usually caused by prolonged standing . she also experienced pain in her right wrist when performing some tasks , particularly opening jars , a manoeuvre that she found difficult . these symptoms are all compatible with osteoarthritis . cd has hereditary systemic amyloidosis caused by a mutation in the gene for apolipoprotein ai , diagnosed in october 2001 during investigation of chronic renal impairment . this type of amyloidosis does not affect the joints or cause arthritic symptoms . she started experimental treatment for her amyloidosis on 22 oct . 2001 with ( r )- 1 -[ 6 -[( r )- 2 - carboxy - pyrrolidin - 1 - yl ]- 6 - oxo - hexanoyl ] pyrrolidine - 2 - carboxylic acid , 15 mg b . d . by subcutaneous injection . in december 2001 , after two months on this potent sap depleting drug and with her plasma sap concentration consistently reduced by over 95 %, she first noted that her joint symptoms were significantly less troublesome than before . this improvement was sustained and increased so that by january 2002 she was free of pain and had normal function of her joints . treatment with ( r )- 1 -[ 6 -[( r )- 2 - carboxy - pyrrolidin - 1 - yl ]- 6 - oxo - hexanoyl ] pyrrolidine - 2 - carboxylic acid finished in october 2002 and her remarkable remission has continued to the present ( june 2003 ). 1 . brion , p . h . and kalunian , k . c . 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