Patent Application: US-201514925350-A

Abstract:
the compound - bisoxy ) carbonyl ) oxy ) methyl ) 1 , 1 ′- adipoylbis possesses physicochemical properties suitable for pharmaceutical development and is capable of generating - 1 -- 2 - carboxy - pyrrolidin - 1 - yl ]- 6 - oxo - hexanoyl ] pyrrolidine - 2 - carboxylic acid in quantities capable of depleting serum amyloid p component efficiently following oral administration , making it useful in the treatment of amyloidosis , alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis .

Description:
the following terms are intended to have the meanings presented herewith below and are useful in understanding the description and the scope of the present invention . “ pharmaceutically acceptable ” means approved or approvable by a regulatory agency of the federal government of the united states of america or the corresponding agency in countries other than the united states of america ( such as the ema , the european medicines agency ), or that is listed in the united states pharmacopeia or european pharmacopoeia ( ph . eur .). “ therapeutically effective amount ” means the amount of a compound that , when administered to a subject for treating a disease , is sufficient to effect such treatment for the disease . the term “ antibody ” is used in the broadest sense to refer to molecules with an immunoglobulin - like domain and includes monoclonal , recombinant , polyclonal , chimeric , humanised , human , bispecific and heteroconjugate antibodies ; and antigen binding fragments . an antibody according to the present invention activates the human complement system and / or results in regression of amyloid deposits . it will be appreciated that reference to “ treatment ” includes acute treatment or prophylaxis as well as the alleviation of established symptoms and / or retardation of progression of the disease , and may include the suppression of symptom recurrence in an asymptomatic patient . the term “ anti - sap ” in relation to an “ antibody ”, i . e . an “ anti - sap antibody ”, means an antibody that binds to human sap with no or insignificant binding to any other proteins , including closely related molecules such as c - reactive protein ( crp ). the term “ cdr ” means complementarity determining region . a cdr of an antibody as used herein means a cdr as determined using any of the well known cdr numbering methods , including kabat , chothia , abm and contact methods . by “ circulating sap ” is meant sap that is present in free form in the plasma in vivo and in vitro and is not associated with amyloid deposits in the tissues . in order to use the compound of formula ( i ) in therapy , it will normally be formulated into a pharmaceutical composition in accordance with standard pharmaceutical practice . the invention therefore provides a pharmaceutical composition , which comprises a therapeutically effective amount of a compound of formula ( i ) and optionally one or more pharmaceutically acceptable carriers and / or excipients . the present invention also provides a process for preparing a pharmaceutical composition , the process comprising admixing a therapeutically effective amount of a compound of formula ( i ) and optionally one or more pharmaceutically acceptable carriers and / or excipients . the present invention also provides a dosage form comprising the pharmaceutical composition of the invention . a pharmaceutical composition of the invention , which may be prepared by admixture , suitably at ambient temperature and atmospheric pressure , is usually adapted for oral administration and , as such , may be in the form of tablets , capsules , oral liquid preparations , powders , granules , or lozenges . in one embodiment , there is provided the pharmaceutical composition of the invention for oral administration . tablets and capsules for oral administration may be in unit dose form , and may contain conventional excipients , such as binding agents ( e . g . pregelatinised maize starch , polyvinylpyrrolidone or hydroxypropyl methylcellulose ); fillers ( e . g . lactose , microcrystalline cellulose or calcium hydrogen phosphate ); tabletting lubricants ( e . g . magnesium stearate , talc or silica ); disintegrants ( e . g . potato starch or sodium starch glycollate ); and acceptable wetting agents ( e . g . sodium lauryl sulphate ). the tablets may be coated according to methods well known in normal pharmaceutical practice . oral liquid preparations may be in the form of , for example , oily suspension , non - aqueous solutions , emulsions , syrups or elixirs , or may be in the form of a dry product for reconstitution with suitable aqueous or non - aqueous vehicle immediately prior to administration . such liquid preparations may contain conventional additives such as suspending agents ( e . g . sorbitol syrup , cellulose derivatives or hydrogenated edible fats ), emulsifying agents ( e . g . lecithin or acacia ), non - aqueous vehicles ( which may include edible oils e . g . almond oil , oily esters , ethyl alcohol or fractionated vegetable oils ), preservatives ( e . g . methyl or propyl - p - hydroxybenzoates or sorbic acid ), and , if desired , conventional flavourings or colorants , buffer salts and sweetening agents as appropriate . preparations for oral administration may be suitably formulated to give controlled release of the active compound . in another embodiment , the dosage form is a tablet or a capsule . the composition may contain from 0 . 1 % to 99 % by weight , preferably from 10 to 60 % by weight , of the active material , depending on the method of administration . the dose of the compound used in the treatment of the aforementioned disorders will vary in the usual way with the seriousness of the disorders , the weight of the sufferer , and other similar factors . however , as a general guide suitable unit doses may be 0 . 05 to 5000 mg , 1 . 0 to 1000 mg , or 100 to 600 mg , for example 100 , 200 or 300 mg , and such unit doses may be administered more than once a day , for example two or three times a day . such therapy may extend for a number of days , weeks , months or years . the invention further provides the compound of formula ( i ) or a pharmaceutical composition as herein described , as for use in therapy . the compound of formula ( i ) is therefore of use in the treatment of diseases wherein sap depletion would be beneficial . the invention further provides the compound of formula ( i ) or a pharmaceutical composition as herein described , for use in depletion of sap . in a further aspect there is provided a method of treatment of a disease or disorder in a subject wherein sap depletion would be beneficial , which method comprises administration of a therapeutically effective amount of the compound of formula ( i ). in a further aspect there is provided a method of treatment of a disease or disorder in a subject wherein sap depletion would be beneficial , which method comprises administration of a therapeutically effective amount of a pharmaceutical composition as herein described . the invention further provides a method of depletion of sap in a subject , which method comprises administering to the subject a therapeutically effective amount of the compound of formula ( i ). the invention further provides a method of depletion of sap in a subject , which method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition as herein described . many forms of transmissible spongiform encephalopathy ( prion diseases ) are associated with amyloid deposits in the brain , and the present invention therefore relates to all these diseases or disorders , including variant creutzfeldt - jakob disease in humans , creutzfeldt - jakob disease itself , kuru and the various other forms of human prion disease , and also bovine spongiform encephalopathy , chronic wasting disease of mule - deer and elk , and transmissible encephalopathy of mink . the invention further provides a method of treatment of a disease or disorder in a subject , wherein the disease or disorder is selected from the group consisting of amyloidosis , alzheimer &# 39 ; s disease , type 2 diabetes mellitus , osteoarthritis , bacterial infections , invasive candiasis , transmissible spongiform encephalopathy , variant creutzfeld - jakob disease in humans , creutzfeld - jakob disease , kuru , other human prion diseases , bovine spongiform encephalopathy , chronic wasting disease of mule - deer and elk , and transmissible encephalopathy of mink , which method comprises administering to the subject a therapeutically effective amount of the compound of formula ( i ). the invention further provides a method of treatment of a disease or disorder in a subject , wherein the disease or disorder is selected from the group consisting of amyloidosis , alzheimer &# 39 ; s disease , type 2 diabetes mellitus , osteoarthritis , bacterial infections , invasive candiasis , transmissible spongiform encephalopathy , variant creutzfeld - jakob disease in humans , creutzfeld - jakob disease , kuru , other human prion diseases , bovine spongiform encephalopathy , chronic wasting disease of mule - deer and elk , and transmissible encephalopathy of mink , which method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition as herein described . the invention further provides a method of treatment of a disease or disorder in a subject , wherein the disease or disorder is selected from the group consisting of amyloidosis , alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis , which method comprises administering to the subject a therapeutically effective amount of the compound of formula ( i ). the invention further provides a method of treatment of a disease or disorder in a subject , wherein the disease or disorder is selected from the group consisting of amyloidosis , alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis , which method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition as herein described . the invention further provides a method of treatment of a disease or disorder in a subject , wherein the disease or disorder is selected from the group consisting of alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis , which method comprises administering to the subject a therapeutically effective amount of the compound of formula ( i ). the invention further provides a method of treatment of a disease or disorder in a subject , wherein the disease or disorder is selected from the group consisting of alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis , which method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition as herein described . the invention further provides a method of treatment of a disease or disorder in a subject , wherein the disease or disorder is amyloidosis , which method comprises administering to the subject a therapeutically effective amount of the compound of formula ( i ). the invention further provides a method of improvement of efficacy of human dna vaccines , which method comprises administering to the subject a therapeutically effective amount of the compound of formula ( i ). the invention further provides a method of improvement of efficacy of human dna vaccines , which method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition as herein described . the invention further provides the use of the compound of formula ( i ) in combination with a dna vaccine . the invention further provides for the compound of formula ( i ) for use in the improvement of the efficacy of human dna vaccines . the invention further provides for a pharmaceutical composition as herein described for use in the improvement of the efficacy of human dna vaccines . the invention further provides for the use of the compound of formula ( i ) in the improvement of the efficacy of human dna vaccines . the invention further provides for the use of a pharmaceutical composition as herein described in the improvement of the efficacy of human dna vaccines . the invention further provides the use of the compound of formula ( i ) in the manufacture of a medicament for use in the improvement of the efficacy of human dna vaccines . by “ improvement in efficacy of human dna vaccines ” is meant enabling the dna vaccine to induce immunoprotective immune responses against the immunogens encoded by the human dna vaccine . the invention further provides a method of treatment of a disease or disorder in a subject , wherein the disease or disorder is selected from the group consisting of transmissible spongiform encephalopathy , variant creutzfeldt - jakob disease in humans , creutzfeldt - jakob disease , kuru , bovine spongiform encephalopathy , chronic wasting disease of mule - deer and elk , and transmissible encephalopathy of mink , which method comprises administering to the subject a therapeutically effective amount of the compound of formula ( i ). the invention further provides a method of treatment of a disease or disorder in a subject , wherein the disease or disorder is selected from the group consisting transmissible spongiform encephalopathy , variant creutzfeldt - jakob disease in humans , creutzfeldt - jakob disease , kuru , bovine spongiform encephalopathy , chronic wasting disease of mule - deer and elk , and transmissible encephalopathy of mink , which method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition as herein described . the term “ amyloidosis ” encompasses both systemic amyloidosis ( including , but not limited to , al - type amyloidosis , aa - type amyloidosis , dialysis amyloidosis , attr ( transthyretin ) amyloidosis , hereditary systemic amyloidosis ) and local amyloidosis ( including , but not limited to cerebral amyloid angiopathy ). in another aspect , the invention provides for the compound of formula ( i ) or a pharmaceutical composition as herein described for use in the treatment of a disease or disorder wherein sap depletion would be beneficial . in another aspect , the invention provides for the compound of formula ( i ) or a pharmaceutical composition as herein described for use in the depletion of sap . in one embodiment , the invention provides for the compound of formula ( i ) or a pharmaceutical composition as herein described for use in the depletion of sap in vivo . in another aspect , the invention provides for the compound of formula ( i ) for use in the treatment of a disease or disorder selected from the group consisting of amyloidosis , alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis . in another aspect , the invention provides for a pharmaceutical composition as herein described for use in the treatment of a disease or disorder selected from the group consisting of amyloidosis , alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis . in one embodiment , the invention provides for the compound of formula ( i ) for use in the treatment of a disease or disorder selected from the group consisting of alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis . in one embodiment , the invention provides for the compound of formula ( i ) for use in the treatment of amyloidosis . in another aspect , the invention provides for a pharmaceutical composition as herein described for use in the treatment of a disease or disorder selected from the group consisting of alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis . in another aspect , the invention provides for a pharmaceutical composition as herein described for use in the treatment of amyloidosis . in another aspect , the invention provides for the use of a compound of formula ( i ) or one or more pharmaceutical compositions herein described in the treatment of a disease or disorder wherein sap depletion would be beneficial . in another aspect , the invention provides for the use of a compound of formula ( i ) or one or more pharmaceutical compositions herein described in the depletion of sap . in another aspect , the invention provides for the use of a compound of formula ( i ) in the treatment of a disease or disorder selected from the group consisting of amyloidosis , alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis . in another aspect , the invention provides for the use of a pharmaceutical composition herein described in the treatment of a disease or disorder selected from the group consisting of amyloidosis , alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis . in another aspect , the invention provides for the use of a compound of formula ( i ) in the treatment of a disease or disorder selected from the group consisting of alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis . in another aspect , the invention provides for the use of a compound of formula ( i ) in the treatment of amyloidosis . in another aspect , the invention provides for the use of a pharmaceutical composition herein described in the treatment of a disease or disorder selected from the group consisting of alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis . in another aspect , the invention provides for the use of a pharmaceutical composition herein described in the treatment of amyloidosis . in another aspect , the invention provides for the use of a compound of formula ( i ) in the manufacture of a medicament for use in the treatment of a disease or disorder wherein sap depletion would be beneficial . in another aspect , the invention provides for the use of a compound of formula ( i ) in the manufacture of a medicament for use in the depletion of sap . in another aspect , the invention provides for the use of a compound of formula ( i ) in the manufacture of a medicament for use in the treatment of a disease or disorder selected from the group consisting of amyloidosis , alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis . in another aspect , the invention provides for the use of a compound of formula ( i ) in the manufacture of a medicament for use in the treatment of a disease or disorder selected from the group consisting of alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis . in another aspect , the invention provides for the use of a compound of formula ( i ) in the manufacture of a medicament for use in the treatment of amyloidosis . when used for the treatment of amyloidosis , the compounds of formula ( i ) may be administered with an anti - sap antibody . in an embodiment , the anti - sap antibody binds to the a face of human sap . in an embodiment , the anti - sap antibody comprises the heavy chain complementarity determining regions ( cdrs ) present within seq id no : 28 ( seq id no : 1 ) and the light chain cdrs present within seq id no : 35 ( seq id no : 2 ) in wo 11 / 107480 . in an embodiment the anti - sap antibody comprises a heavy chain variable region of seq id no : 28 ( seq id no : 1 ) and a light chain variable region of seq id no : 35 ( seq id no : 2 ) in wo 11 / 107480 . in an embodiment , the anti - sap antibody comprises a human iggi or igg3 human constant domain . in an embodiment the anti - sap antibody consists of a heavy chain of seq id no : 62 ( seq id no : 3 ) and a light chain of seq id no : 64 ( seq id no : 4 ) in wo 11 / 107480 . in an embodiment , the anti - sap antibody comprises the heavy and light chain cdrs described within wo 11 / 107480 as seq id no : 1 ( seq id no : 5 ), seq id no : 2 ( seq id no : 6 ) and seq id no : 3 ( seq id no : 7 ), seq id no : 4 ( seq id no : 8 ), seq id no : 5 ( seq id no : 9 ) and seq id no : 6 ( seq id no : 10 ). therefore , in a further aspect , the invention provides for a method of treatment of amyloidosis which method comprises administering to a subject a therapeutically effective amount of a compound of formula ( i ) or a pharmaceutical composition as herein described in co - administration with an anti - sap antibody . in one embodiment , the administration of the compound of formula ( i ) in co - administration with an anti - sap antibody is sequential . such sequential administration may be close in time ( eg . simultaneously ) or remote in time . furthermore , it does not matter if the compounds are administered in the same dosage form , e . g . one compound may be administered intravenously and the other compound may be administered orally . in a further embodiment , the compound of formula ( i ) is administered first . in a further embodiment , the anti - sap antibody is administered when the level of circulating sap in the subject has been reduced to a level of less than 2 mg / l . in one embodiment , the level of circulating sap has been reduced to a level of 1 mg / l or less . in a further embodiment the level of circulating sap has been reduced to a level of 0 . 5 mg / l or less . the level of circulating sap can be measured using a commercially available elisa ( enzyme - linked immunosorbent assay ) kit ( e . g . hk331 human sap elisa kit from hycult biotech ). in a further embodiment , the compound of formula ( i ) is administered for 5 - 8 days or until the level of the sap circulating in the subject has been reduced to a level of less than 2 mg / l whichever is longer . in one embodiment , the level of the sap circulating in the subject has been reduced to 1 mg / l or less . in a further embodiment , the level of sap circulating in the subject has been reduced to 0 . 5 mg / l or less . in a further embodiment administration of the compound of formula ( i ) is continued while a single dose of 200 - 2000 mg ( preferably 250 - 1000 mg , more preferably 250 - 600 mg ) of anti - sap antibody is administered , and for 4 - 6 days thereafter . this constitutes a ‘ therapeutic course ’— patients may require several courses to achieve the desired therapeutic effect . they are also likely to require intermittent repeat treatment . in one embodiment , the therapeutic course is repeated at least once at 3 - 6 week intervals as required . in a further embodiment , the therapeutic course is repeated at least once at 3 - 6 week intervals followed by at least one therapeutic course at 6 - 12 month intervals as required . in a further aspect there is provided a method of treatment of a disease or disorder in a subject wherein sap depletion would be beneficial , which method comprises administration of a therapeutically effective amount of the compound of formula ( i ) in co - administration with an anti - sap antibody . in a further aspect there is provided a method of treatment of a disease or disorder in a subject wherein sap depletion would be beneficial , which method comprises administration of a therapeutically effective amount of a pharmaceutical composition as herein described in co - administration with an anti - sap antibody . in a further aspect there is provided a method of depletion of sap , which method comprises administration of a therapeutically effective amount of the compound of formula ( i ) in co - administration with an anti - sap antibody . in a further aspect there is provided a method of depletion of sap , which method comprises administration of a therapeutically effective amount of a pharmaceutical composition as herein described in co - administration with an anti - sap antibody . the invention further provides a method of treatment of a disease or disorder in a subject , wherein the disease or disorder is selected from the group consisting of amyloidosis , alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis , which method comprises administering to the subject a therapeutically effective amount of a compound of formula ( i ) in co - administration with an anti - sap antibody . the invention further provides a method of treatment of a disease or disorder in a subject , wherein the disease or disorder is selected from the group consisting of amyloidosis , alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis , which method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition as herein described in co - administration with an anti - sap antibody . in another aspect , the invention provides for the compound of formula ( i ) or one or more pharmaceutical compositions herein described in co - administration with an anti - sap antibody for use in the treatment of a disease or disorder wherein sap depletion would be beneficial . in another aspect , the invention provides for the compound of formula ( i ) or one or more pharmaceutical compositions herein described in co - administration with an anti - sap antibody for use in the depletion of sap . in another aspect , the invention provides for the compound of formula ( i ) in co - administration with an anti - sap antibody for use in the treatment of a disease or disorder selected from the group consisting of amyloidosis , alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis . in another aspect , the invention provides for the compound of formula ( i ) in co - administration with an anti - sap antibody for use in the treatment of amyloidosis . in one embodiment , the invention provides for the compound of formula ( i ) in co - administration with an anti - sap antibody for use in the treatment of systemic amyloidosis . in one embodiment , the invention provides for the compound of formula ( i ) in co - administration with an anti - sap antibody for use in the treatment of al - type amyloidosis . in one embodiment , the invention provides for the compound of formula ( i ) in co - administration with an anti - sap antibody for use in the treatment of aa - type amyloidosis . in one embodiment , the invention provides for the compound of formula ( i ) in co - administration with an anti - sap antibody for use in the treatment of dialysis amyloidosis . in one embodiment , the invention provides for the compound of formula ( i ) in co - administration with an anti - sap antibody for use in the treatment of attr ( transthyretin ) amyloidosis . in one embodiment , the invention provides for the compound of formula ( i ) in co - administration with an anti - sap antibody for use in the treatment of hereditary systemic amyloidosis . in one embodiment , the invention provides for the compound of formula ( i ) in co - administration with an anti - sap antibody for use in the treatment of local amyloidosis . in one embodiment , the invention provides for the compound of formula ( i ) in co - administration with an anti - sap antibody for use in the treatment of cerebral amyloid angiopathy . in another aspect , the invention provides for a pharmaceutical composition as herein described in co - administration with an anti - sap antibody for use in the treatment of a disease or disorder selected from the group consisting of amyloidosis , alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis . in another aspect , the invention provides for a pharmaceutical composition as herein described in co - administration with an anti - sap antibody for use in the treatment of amyloidosis . in one embodiment , the invention provides for a pharmaceutical composition as herein described in co - administration with an anti - sap antibody for use in the treatment of systemic amyloidosis . in one embodiment , the invention provides for a pharmaceutical composition as herein described in co - administration with an anti - sap antibody for use in the treatment of al - type amyloidosis . in one embodiment , the invention provides for a pharmaceutical composition as herein described in co - administration with an anti - sap antibody for use in the treatment of aa - type amyloidosis . in one embodiment , the invention provides for a pharmaceutical composition as herein described in co - administration with an anti - sap antibody for use in the treatment of dialysis amyloidosis . in one embodiment , the invention provides for a pharmaceutical composition as herein described in co - administration with an anti - sap antibody for use in the treatment of attr ( transthyretin ) amyloidosis . in one embodiment , the invention provides for a pharmaceutical composition as herein described in co - administration with an anti - sap antibody for use in the treatment of hereditary systemic amyloidosis . in one embodiment , the invention provides for a pharmaceutical composition as herein described in co - administration with an anti - sap antibody for use in the treatment of local amyloidosis . in one embodiment , the invention provides for a pharmaceutical composition as herein described in co - administration with an anti - sap antibody for use in the treatment of cerebral amyloid angiopathy . in another aspect , the invention provides for the use of a compound of formula ( i ) or a pharmaceutical composition as herein described in co - administration with an anti - sap antibody in the treatment of a disease or disorder wherein sap depletion would be beneficial . in another aspect , the invention provides for the use of a compound of formula ( i ) or a pharmaceutical composition as herein described in co - administration with an anti - sap antibody for use in the depletion of sap . in another aspect , the invention provides for the use of a compound of formula ( i ) in co - administration with an anti - sap antibody in the treatment of a disease or disorder selected from the group consisting of amyloidosis , alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis . in another aspect , the invention provides for the use of a compound of formula ( i ) in co - administration with an anti - sap antibody in the treatment of amyloidosis . in one embodiment , the invention provides for the use of a compound of formula ( i ) in co - administration with an anti - sap antibody in the treatment of systemic amyloidosis . in one embodiment , the invention provides for the use of a compound of formula ( i ) in co - administration with an anti - sap antibody in the treatment of al - type amyloidosis . in one embodiment , the invention provides for the use of a compound of formula ( i ) in co - administration with an anti - sap antibody in the treatment of aa - type amyloidosis . in one embodiment , the invention provides for the use of a compound of formula ( i ) in co - administration with an anti - sap antibody in the treatment of dialysis amyloidosis . in one embodiment , the invention provides for the use of a compound of formula ( i ) in co - administration with an anti - sap antibody in the treatment of attr ( transthyretin ) amyloidosis . in one embodiment , the invention provides for the use of a compound of formula ( i ) in co - administration with an anti - sap antibody in the treatment of hereditary systemic amyloidosis . in one embodiment , the invention provides for the use of a compound of formula ( i ) in co - administration with an anti - sap antibody in the treatment of local amyloidosis . in one embodiment , the invention provides for the use of a compound of formula ( i ) in co - administration with an anti - sap antibody in the treatment of cerebral amyloid angiopathy . in another aspect , the invention provides for the use of a pharmaceutical composition as herein described in co - administration with an anti - sap antibody in the treatment of a disease or disorder selected from the group consisting of amyloidosis , alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis . in another aspect , the invention provides for the use of a pharmaceutical composition as herein described in co - administration with an anti - sap antibody in the treatment of amyloidosis . in one embodiment , the invention provides for the use of a pharmaceutical composition as herein described in co - administration with an anti - sap antibody in the treatment of systemic amyloidosis . in one embodiment , the invention provides for the use of a pharmaceutical composition as herein described in co - administration with an anti - sap antibody in the treatment of al - type amyloidosis . in one embodiment , the invention provides for the use of a pharmaceutical composition as herein described in co - administration with an anti - sap antibody in the treatment of aa - type amyloidosis . in one embodiment , the invention provides for the use of a pharmaceutical composition as herein described in co - administration with an anti - sap antibody in the treatment of dialysis amyloidosis . in one embodiment , the invention provides for the use of a pharmaceutical composition as herein described in co - administration with an anti - sap antibody in the treatment of attr ( transthyretin ) amyloidosis . in one embodiment , the invention provides for the use of a pharmaceutical composition as herein described in co - administration with an anti - sap antibody in the treatment of hereditary systemic amyloidosis . in one embodiment , the invention provides for the use of a pharmaceutical composition as herein described in co - administration with an anti - sap antibody in the treatment of local amyloidosis . in one embodiment , the invention provides for the use of a pharmaceutical composition as herein described in co - administration with an anti - sap antibody in the treatment of cerebral amyloid angiopathy . in another aspect , the invention provides for the use of a compound of formula ( i ) and an anti - sap antibody in the manufacture of a medicament for use in the treatment of a disease or disorder wherein sap depletion would be beneficial . in another aspect , the invention provides for the use of a compound of formula ( i ) and an anti - sap antibody in the manufacture of a medicament for use in the depletion of sap . in another aspect , the invention provides for the use of a compound of formula ( i ) and an anti - sap antibody in the manufacture of a medicament for use in the treatment of a disease or disorder selected from the group consisting of amyloidosis , alzheimer &# 39 ; s disease , type 2 diabetes mellitus and osteoarthritis . in another aspect , the invention provides for the use of a compound of formula ( i ) and an anti - sap antibody in the manufacture of a medicament for use in the treatment of amyloidosis . in one embodiment , the invention provides for the use of a compound of formula ( i ) and an anti - sap antibody in the manufacture of a medicament for use in the treatment of systemic amyloidosis . in one embodiment , the invention provides for the use of a compound of formula ( i ) and an anti - sap antibody in the manufacture of a medicament for use in the treatment of al - type amyloidosis . in one embodiment , the invention provides for the use of a compound of formula ( i ) and an anti - sap antibody in the manufacture of a medicament for use in the treatment of aa - type amyloidosis . in one embodiment , the invention provides for the use of a compound of formula ( i ) and an anti - sap antibody in the manufacture of a medicament for use in the treatment of dialysis amyloidosis . in one embodiment , the invention provides for the use of a compound of formula ( i ) and an anti - sap antibody in the manufacture of a medicament for use in the treatment of attr ( transthyretin ) amyloidosis . in one embodiment , the invention provides for the use of a compound of formula ( i ) and an anti - sap antibody in the manufacture of a medicament for use in the treatment of hereditary systemic amyloidosis . in one embodiment , the invention provides for the use of a compound of formula ( i ) and an anti - sap antibody in the manufacture of a medicament for use in the treatment of local amyloidosis . in one embodiment , the invention provides for the use of a compound of formula ( i ) and an anti - sap antibody in the manufacture of a medicament for use in the treatment of cerebral amyloid angiopathy . in a further embodiment the disease or disorder is systemic amyloidosis . in a further embodiment the disease or disorder is al - type amyloidosis . in a further embodiment the disease or disorder is aa - type amyloidosis . in a further embodiment the disease or disorder is dialysis amyloidosis . in a further embodiment the disease or disorder is attr ( transthyretin ) amyloidosis . in a further embodiment the disease or disorder is hereditary systemic amyloidosis . in a further embodiment the disease or disorder is cerebral amyloid angiopathy . in one embodiment the disease or disorder is type 2 diabetes mellitus . in another embodiment of the invention , an article of manufacture , or “ kit of parts ”, containing one or more unit doses of an anti - sap antibody and one or more unit doses of the compound of formula ( i ), useful for the treatment of amyloidosis , is provided . in one embodiment the kit of parts comprises a unit dose of an anti - sap antibody and one or more unit doses of the compound of formula ( i ). suitably the kit of parts is formulated for the separate or sequential administration of the one or more unit doses of compound of formula ( i ) and the unit dose of the anti - sap antibody . in one embodiment , the kit of parts comprises a container comprising one or more unit doses of the compound of formula ( i ) or a pharmaceutical composition as herein described and the unit dose of the anti - sap antibody . in another embodiment , the kit of parts comprises a first container comprising one or more unit doses of the compound of formula ( i ) or a pharmaceutical composition as herein described and a second container comprising the unit dose of the anti - sap antibody . the kit of parts may further comprise directions for the administration of the one or more unit doses of compound of formula ( i ) and the unit dose of the anti - sap antibody for treating or preventing amyloidosis . in an alternative embodiment of the invention , an article of manufacture , or “ kit of parts ”, containing one or more unit doses of the compound of formula ( i ) or a pharmaceutical composition as herein described and one or more unit doses of a dna vaccine is provided . suitable containers include , for example , bottles , vials , syringes and blister packs , etc . wo2011 / 139917 discloses anti - transthyretin ( anti - ttr ) antisense oligonucleotides potentially useful in the modulation of expression of transthyretin and in treating , preventing , delaying or ameliorating transthyretin amyloidosis . in another aspect , the invention provides for a method of treatment of attr ( transthyretin ) amyloidosis , which method comprises i ) administering to a subject a therapeutically effective amount of a compound of formula ( i ) or a pharmaceutical composition as herein described in co - administration with an anti - sap antibody , and ii ) administering to a subject a therapeutically effective amount of an anti - ttr antisense oligonucleotide . in one embodiment of the invention , the anti - ttr antisense oligonucleotide is isis 420915 . in one embodiment , steps i ) and ii ) are carried out sequentially . wo2009040405 discloses agents for stabilising the tetrameric form of transthyretin useful in the treatment or prevention of transthyretin amyloidosis . therefore , in another aspect , the invention provides for a method of treatment of attr ( transthyretin ) amyloidosis , which method comprises i ) administering to a subject a therapeutically effective amount of a compound of formula ( i ) or a pharmaceutical composition as herein described in co - administration with an anti - sap antibody , and ii ) administering to a subject a therapeutically effective amount of an agent as described in wo2009040405 . in one embodiment , steps i ) and ii ) are carried out sequentially . the compound of formula ( i ) may be synthesised substantially according to reaction scheme 1 . the compounds of formula ( i ) can be prepared by reaction of the chloromethyl carbonate of formula ( ii ) with cphpc in the presence of a solvent ( e . g . dioxane ), a base ( e . g . potassium carbonate ) and catalytic amounts of tbai ( tetrabutylammonium iodide ). the chloromethyl carbonate of formula ( ii ) can be prepared by reaction of chloromethyl carbonochloridate with tetrahydro - 2h - pyran - 4 - ol in the presence of a solvent ( e . g . diethyl ether or dichloromethane ) and a base ( e . g . pyridine or dimethylaminopyridine ). chloromethyl carbonochloridate ( also known as chloromethyl chloroformate ) and tetrahydro - 2h - pyran - 4 - ol are commercially available ( aldrich ). alternatively , the compounds of formula ( i ) can be synthesised substantially according to reaction scheme 2 starting from d - proline (( r )- pyrrolidine - 2 - carboxylic acid ). the following example illustrates the invention . this example is not intended to limit the scope of the present invention , but rather to provide guidance to the skilled artisan to prepare and use the compound , compositions , and methods of the present invention . while particular embodiments of the present invention are described , the skilled artisan will appreciate that various changes and modifications can be made without departing from the spirit and scope of the invention . all publications , including but not limited to patents and patent applications , cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth . the following intermediates and examples illustrate the preparation of the compound of the invention . 1 h and 13 c nmr spectra were recorded on a bruker 300 or 400 mhz . chemical shifts are reported in parts per million ( ppm , units ). high - resolution mass spectra were recorded on a micromass lct ( tof ) spectrometer coupled to analytical high performance liquid chromatography ( hplc ). hplc was conducted on a waters x - terra ms c18 column ( 3 . 5 μm 30 × 4 . 6 mm id ) eluting with 0 . 01m ammonium acetate in water ( solvent a ) and 100 % acetonitrile ( solvent b ), using the following elution gradient 0 - 0 . 5 minutes 5 % b , 0 . 5 - 3 . 75 minutes 5 % to 100 % b , 3 . 75 - 4 . 5 100 % b , 4 . 5 - 5 100 % to 5 % b , 5 - 5 . 5 5 % b at a flow rate of 1 . 3 ml / minute at 40 ° c . the mass spectra ( ms ) were recorded on a waters lct mass spectrometer using electrospray positive ionisation [ es + ve to give mh + molecular ions ] or electrospray negative ionisation [ es - ve to give ( m − h ) − molecular ions ] modes . analytical hplc was conducted on a xselect xp c18 column ( 2 . 5 μm 30 × 4 . 6 mm id ) eluting with 0 . 1 % formic acid in water ( solvent a ) and 0 . 1 % formic acid in acetonitrile ( solvent b ), using the following elution gradient 0 - 3 . 2 minutes : 5 % to 100 % b , 3 . 2 - 4 . 0 minutes 100 % b , at a flow rate of 1 . 8 ml / minute at 40 ° c . the mass spectra ( ms ) were recorded on a waters zq mass spectrometer using electrospray positive ionisation [ es + to give mh + molecular ions ] or electrospray negative ionisation [ es − to give ( m − h ) − molecular ions ] modes . to a solution of tetrahydro - 2h - pyran - 4 - ol ( 40 g , 392 mmol ) in diethyl ether ( 500 ml ) was added pyridine ( 38 . 0 ml , 470 mmol ) and the solution was cooled to 0 ° c . chloromethyl carbonochloridate ( 41 . 8 ml , 470 mmol ) was added dropwise leading to the formation of a white solid . the reaction mixture was stirred at rt for 18 h . the reaction mixture was washed with water ( 200 ml ), with hcl 0 . 5n ( 200 ml ), and then with a saturated solution of nahco 3 ( 200 ml ). the organic phase was dried over na 2 so 4 and concentrated under reduced pressure . toluene was added and the solution was concentrated under reduced pressure ( to remove chloromethyl carbonochloridate in excess ). chloromethyl ( tetrahydro - 2h - pyran - 4 - yl ) carbonate ( intermediate 1 ) was obtained as a colourless oil ( 70 g , 360 mmol , 92 % yield ). 1 h nmr ( 400 mhz , cdcl 3 ) δ ppm 5 . 75 ( s , 2h ), 4 . 92 ( m , 1h ), 3 . 95 ( m , 2h ), 3 . 56 ( m , 2h ), 2 . 02 ( m , 2h ), 1 . 80 ( m , 2h ). to a solution of chloromethyl carbonochloridate ( 5 g , 38 . 8 mmol ) in dcm ( 50 ml ) was added tetrahydro - 2h - pyran - 4 - ol ( 3 . 96 g , 38 . 8 mmol ) and the solution was cooled to 0 ° c . dmap ( 4 . 97 g , 40 . 7 mmol ) was added then the reaction mixture was stirred at rt for 18 h . the reaction mixture was diluted with water and extracted with dcm ( 3 × 100 ml ). the organic phases were combined , dried over na 2 so 4 and concentrated under reduced pressure . the pale yellow oil was purified by chromatography eluting with 10 % etoac in cyclohexane . the appropriate fractions were combined and concentrated in vacuo to give the required product as a colorless oil ( 2 . 2 g , 11 . 3 mmol , 29 . 2 % yield ). 1 h nmr ( 300 mhz , cdcl 3 ) δ ppm 5 . 76 ( s , 2h ), 4 . 92 ( m , 1h ), 3 . 94 ( m , 2h ), 3 . 57 ( m , 2h ), 2 . 02 ( m , 2h ), 1 . 80 ( m , 2h ). solution a : potassium carbonate ( 29 . 8 g , 216 mmol ) was added to a stirred suspension of ( 2r , 2 ′ r )- 1 , 1 ′- adipoylbis ( pyrrolidine - 2 - carboxylic acid ) ( 35 g , 103 mmol ) in 1 , 4 - dioxane ( 1 l ) and the reaction mixture was stirred at 80 ° c . for 30 min . solution b : tbai ( 7 . 60 g , 20 . 57 mmol ) was added to a solution of chloromethyl ( tetrahydro - 2h - pyran - 4 - yl ) carbonate ( 42 . 0 g , 216 mmol ) in dioxane ( 50 ml ) and the mixture was stirred at rt for 15 min . the solution b was added to the solution a . the reaction mixture was stirred at 80 ° c . for 18 h . the reaction mixture was filtered and concentrated under reduced pressure . the residue was taken up in etoac ( 400 ml ) and washed with an aq . solution of nahco 3 ( 2 × 100 ml ), an aq . solution of sodium thiosulfate ( 50 ml ) and with 0 . 5n hcl ( 100 ml ). the organic layer was dried over anhydrous na 2 so 4 , filtered and concentrated in vacuo . the yellow oil was solubilized in 2 - methf ( 100 ml ) and sonicated until crystallization occurred . the mixture was left to stand for 1 h at rt . the precipitate was filtered and washed with a mixture 2 - methf / ipr 2 o 70 / 30 to afford ( 2r , 2 ′ r )- bis ((((( tetrahydro - 2h - pyran - 4 - yl ) oxy ) carbonyl ) oxy ) methyl ) 1 , 1 ′- adipoylbis ( pyrrolidine - 2 - carboxylate ) ( example 1 ) as an off white powder ( 42 g , 64 . 0 mmol , 62 . 2 % yield ). the product was dried under reduced pressure ( 5 mbar ) and 35 ° c . for 12 h . 1 h nmr ( 400 mhz , cdcl 3 ) δ ppm 5 . 88 ( d , j = 5 . 5 hz , 2h ), 5 . 73 ( d , j = 5 . 5 hz , 2h ), 4 . 87 ( m , 2h ), 4 . 50 ( m , 2h ), 3 . 93 ( m , 4h ), 3 . 65 ( m , 2h ), 3 . 55 ( m , 6h ), 2 . 42 - 1 . 90 ( m , 16h ), 1 . 84 - 1 . 60 ( m , 8h ). some minor peaks were observed due to the presence of rotamers . ( 2r , 2 ′ r )- bis ((((( tetrahydro - 2h - pyran - 4 - yl ) oxy ) carbonyl ) oxy ) methyl ) 1 , 1 ′- adipoylbis ( pyrrolidine - 2 - carboxylate ) ( 170 g , 259 mmol ) was suspended in 2 - methf and heated to 90 ° c . until complete dissolution . the solution was filtered when still hot and allowed to cool to rt . the precipitate was filtered and washed with a mixture of 2 - methf / ipr 2 o 70 / 30 to afford ( 2r , 2 ′ r )- bis ((((( tetrahydro - 2h - pyran - 4 - yl ) oxy ) carbonyl ) oxy ) methyl ) 1 , 1 ′- adipoylbis ( pyrrolidine - 2 - carboxylate ) ( 130 g , 198 mmol , 76 % yield ) as an off white crystalline solid . the product was dried under reduced pressure ( 5 mbar ) and 35 ° c . for 24 h . 1 h nmr ( 400 mhz , cdcl 3 ) δ ppm 5 . 87 ( d , j = 5 . 5 hz , 2h ), 5 . 71 ( d , j = 5 . 5 hz , 2h ), 4 . 86 ( m , 2h ), 4 . 49 ( m , 2h ), 3 . 91 ( m , 4h ), 3 . 63 ( m , 2h ), 3 . 54 ( m , 6h ), 2 . 43 - 1 . 85 ( m , 16h ), 1 . 84 - 1 . 59 ( m , 8h ). some minor peaks were observed due to the presence of rotamers . 13 c nmr ( 100 mhz , cdcl 3 ) 171 . 69 , 170 . 85 , 153 . 12 , 82 . 26 , 77 . 36 , 77 . 05 , 76 . 72 , 73 . 94 , 65 . 02 , 58 . 41 , 46 . 95 , 34 . 11 , 31 . 47 , 29 . 02 , 24 . 80 , 24 . 17 . hrms : m / z calculated for c 30 h 45 n 2 o 14 [ m + h ] + 657 . 2870 . found 657 . 2883 . xrpd data were acquired on a panalytical x &# 39 ; pert pro powder diffractometer , model pw3040 / 60 using an x &# 39 ; celerator detector . the acquisition conditions were : radiation : cu kα , generator tension : 40 kv , generator current : 45 ma , start angle : 2 . 0 ° 2θ , end angle : 40 . 0 ° 2θ , step size : 0 . 0167 ° 2θ , time per step : 31 . 75 seconds . the sample was prepared by mounting a few milligrams of sample ( compound of formula ( i )) on a silicon wafer ( zero background plate ), resulting in a thin layer of powder . the xrpd spectrum of the crystalline solid form of compound of formula ( i ) is presented in fig1 . characteristic xrpd angles and d - spacings for the compound of formula ( i ) are recorded in table 1 . the margin of error is approximately ± 0 . 1 ° 2θ for each of the peak assignments . peak intensities may vary from sample to sample due to preferred orientation . in a further aspect , the invention provides for a compound of formula ( i ) as a crystalline solid characterised by an xrpd spectrum that is substantially as shown in fig1 . in a further aspect , the invention provides for a compound of formula ( i ) as a crystalline solid characterised by an xrpd spectrum comprising the peaks of table 1 . the data hereinafter reported compares the compound of formula ( i ) with ( 2r , 2 ′ r )- bis ( 1 -( pivaloyloxy ) ethyl ) 1 , 1 ′- adipoylbis ( pyrrolidine - 2 - carboxylate ) ( comparator compound ). ( 2r , 2 ′ r )- bis ( 1 -( pivaloyloxy ) ethyl ) 1 , 1 ′- adipoylbis ( pyrrolidine - 2 - carboxylate ) ( also known as ( r )- 1 -( 6 -{( r )- 2 -[ 1 -( 2 , 2 - dimethyl - propionyloxy )- ethoxycarbonyl ]- pyrrolidin - 1 - yl }- 6 - oxo - hexanoyl )- pyrrolidine - 2 - carboxylic acid 1 -( 2 , 2 - dimethyl - propionyloxy )- ethyl ester ) can be synthesised according to the experimental protocol disclosed in wo2003 / 051836 . the compound of formula ( i ) is obtainable as a crystalline solid . by contrast , comparator compound is obtained as an oil ( see wo2003 / 051836 ). a known quantity of the compound of formula ( i ) was weighed into a suitable vessel ( e . g . a screw capped clear glass vial ) and a known volume of the required media added ( e . g . simulated gastric fluid ph 1 . 6 [ sgf ], simulated fed state intestinal fluid ph 6 . 5 [ fessif ], simulated fasted state intestinal fluid ph 6 . 5 [ fassif ], water [ purified water ], or britton - robinson buffer ). the compound was wetted with the media by vortex mixing for 30 seconds to 1 minute . the sample was then visually observed to ensure that undissolved solid remained present . the sample was transferred to a gentle mixer ( such as a roller mixer ) and allowed to agitate until the desired time point was reached . at appropriate times the sample was reassessed visually . if all the solid had dissolved the solubility was recorded as & gt ; x mg / ml where x is the known weight used divided by the volume added . if undissolved solid remained a portion of the sample was taken and centrifuged to remove the solid leaving a clear supernatant . the supernatant was diluted volumetrically with a suitable diluent to provide an analytical sample of suitable concentration for analysis . this diluted sample was then analysed by a suitable method such as hplc against a standard ( s ) of known concentration . the solubility of the compound can then be calculated using a knowledge of the concentration of the standard , the relative response ( e . g . peak areas ) of the standard and the analytical sample described , and the dilution of the analytical sample . the solubility of the compound of formula ( i ) in various aqueous media are shown below in table 2 . the assay was designed to evaluate inhibition on cytochrome p450 ( cyp ) 3a4 , 2c9 , 2c19 , 1a2 and 2d6 enzymes from bactosomes source using fluorogenic substrates . compound ( compound of formula ( i ) or comparator compound ) was dissolved in methanol at 1 . 65 mm . daughter solutions were prepared in methanol at 660 , 264 , 106 , 42 , 17 , 6 . 8 , 2 . 7 , 1 . 1 and 0 . 43 μm . nadph cofactor was prepared with glucose - 6 - phosphate ( 7 . 8 mg ), glucose - 6 - phosphate dehydrogenase ( 6 units ), nadp ( 1 . 7 mg ) per 1 ml in nahco 3 2 %. 7 - methoxy - 4 - trifluoromethyl coumarin - 3 - acetic acid ethyl ester ( fca ): 12 . 5 mm in acetonitrile ethoxyresorufin ( er ): 0 . 05 mm in acetonitrile 4 - methylaminomethyl - 7 - methoxy coumarin ( mmc ): 2 . 5 mm in methanol 3 - butyryl - 7methoxy coumarin ( bmc ): 2 . 5 mm in dmso 7 - benzyloxyquinoline ( 7bq ): 2 . 5 mm in acetonitrile diethoxyfluorescein ( def ): 0 . 1 mm in acetonitrile bactosomes ( cypex source ) at the concentration of 10 mg of protein per ml were diluted in phosphate buffer 50 mm ph7 . 4 : 0 . 33 ml of bactosomes cyp2c9 with 23 . 8 ml of buffer and 0 . 11 ml of fca 0 . 33 ml of bactosomes cyp1a2 with 23 . 6 ml of buffer and 0 . 275 ml of er 0 . 33 ml of bactosomes cyp2d6 with 23 . 8 ml of buffer and 0 . 11 ml of mmc 0 . 33 ml of bactosomes cyp2c19 with 23 . 8 ml of buffer and 0 . 11 ml of bmc 0 . 33 ml of bactosomes cyp3a4h with 23 . 6 ml of buffer and 0 . 275 ml of 7bq 0 . 33 ml of bactosomes cyp3a4l with 23 . 6 ml of buffer and 0 . 275 ml of def pre - incubation consisted in mixing 5 μl of compound solution with 220 μl diluted bactosomes and warming it at 37 ° c . for 10 min . incubation was started with the addition of 25 μl of nadph . then fluorescence of substrate metabolite was read in a safire instrument ( from tecan ) for 5 min : fca ( 2c9 ): excitation at 410 nm and emission at 510 nm er ( 1a2 ): excitation at 530 nm and emission at 590 nm mmc ( 2d6 ): excitation at 410 nm and emission at 485 nm bmc ( 2c19 ): excitation at 410 nm and emission at 465 nm 7bq ( 3a4h ): excitation at 410 nm and emission at 530 nm def ( 3a4l ): excitation at 485 nm and emission at 530 nm plotting of inhibition of substrate production against compound concentration allowed the determination of ic 50 values . in fluorescence based screening assays using recombinant human cyp 450 , the compound of formula ( i ) and its mono - ester derivative did not demonstrate significant inhibition of the major human liver cytochrome p450s ( cypex ): cyp3a4 - def and 3a4 - 7bq with ic 50 & gt ; 33 μm . the other isoforms were also not significantly inhibited by both compounds with ic 50 & gt ; 33 μm ). by contrast , comparator compound demonstrated significant inhibition of cyp3a4 - def and cyp3a4 - 7bq . for a compound where the systemic concentration is going to be low , only cyp3a4 inhibition is relevant as only intestinal inhibition will be relevant ( cyp3a is present in enterocytes and is responsible for enterocyte first pass ). the assay was designed for determining physical stability of compound in buffer at various phs . compound of formula ( i ) was dissolved in dmso at 1 mg / ml . phosphate buffer ( pbs ) was prepared by mixing k 2 hpo 4 50 mm and kh 2 po 4 50 mm solutions to obtain 4 buffers at ph 6 . 0 , 7 . 0 , 7 . 5 , 8 . 0 . stability studies were conducted at room temperature , and were started by the addition of 8 μl of dmso solution to 792 μl of pbs 50 mm at ph 6 . 0 , 7 . 0 , 7 . 5 or 8 . 0 ( 1 % dmso ). 75 μl of mixture was taken at 0 , 1 , 2 , 4 and 24 h and 225 μl acetonitrile containing internal standard was added . 2 μl of samples were injected into the liquid chromatography system and eluted with a ascentis c18 column ( 50 × 2 . 1 mm id , 2 . 7 μm ) and with 0 . 1 % formic acid in water ( a ) and 0 . 1 % formic acid in acetonitrile ( b ), using the following elution 2 min gradient : 5 to 95 % b over 1 . 2 min , 95 % b over 0 . 6 min and 0 . 1 min for re - equilibrate column , at 0 . 5 ml / min at 50 ° c . samples were analysed by mass spectrometry with an electrospray source and in positive mode and with following mass transitions : compound of formula ( i ): 657 to 384 monoester of cphpc : 499 to 226 cphpc : 341 to 226 data showing in vitro physical hydrolysis of the compound of formula ( i ) in phosphate buffered saline at ph 6 , 7 , 7 . 5 and 8 at various time points is presented in fig2 . by “ monoester of cphpc ” is meant the compound ( r )- 1 -( 6 - oxo - 6 -(( r )- 2 -(((((( tetrahydro - 2h - pyran - 4 - yl ) oxy ) carbonyl ) oxy ) methoxy ) carbonyl ) pyrrolidin - 1 - yl ) hexanoyl ) pyrrolidine - 2 - carboxylic acid of formula ( iii ). the compound of formula ( i ) seemed to be less sensitive to hydrolysis for a ph below 7 . 5 . less than 20 % of the compound of formula ( i ) was hydrolysed after 24 h in an acidic environment ( ph less than 7 . 0 ). the assay was designed for determining stability of compound in microsomal matrix . compound ( compound of formula ( i )) was dissolved in dmso at 1 mg / ml . daughter solution was prepared in methanol / water ( 50 / 50 ) at 30 . 3 ng / ml . microsomes ( from xenotech ) were diluted at 0 . 625 mg proteins per ml in phosphate buffer 50 mm ph 7 . 4 . pre - incubation consisted of warming microsomal solution 395 μl with 100 μl nahco 3 ( 2 %) at 37 ° c . for 7 min . incubation was started with the addition of 5 μl of daughter solution . 50 μl aliquots of mixture were taken at 0 , 3 , 6 , 12 and 30 min and quenched with 150 μl acetonitrile containing internal standard . after 10 min centrifugation at 4000 rpm , 2 μl of samples were injected into the liquid chromatography system and eluted on a ascentis c18 column ( 50 × 2 . 1 mm id , 2 . 7 μm ) with 0 . 1 % formic acid in water ( a ) and 0 . 1 % formic acid in acetonitrile ( b ), using the following 2 minute elution gradient : 5 to 95 % b over 1 . 2 min , 95 % b over 0 . 6 min and 0 . 1 min for re - equilibrate column , at 0 . 5 ml / min at 50 ° c . samples were analysed by mass spectrometry with an electrospray source , in positive mode and with following mass transitions : compound of formula ( i ): 657 to 384 monoester of cphpc : 499 to 226 cphpc : 341 to 226 controls were made to calculate percentage of disappearance of parent but also appearance of suspected metabolite , i . e . monoester and diacidic form . data showing in vitro intestinal microsomal stability of compound of formula ( i ) in human microsomes is presented in fig3 . “ 0 / 5 / 10 / 30 / 60 min ” refers to the time ( in minutes ) that the sample was taken from the mixture for analysis . as can be seen from fig3 , the compound of formula ( i ) exhibited a low rate of hydrolysis in human intestinal microsomes , even after 60 minutes , suggesting that the compound of formula ( i ) will not be unduly unstable in the gut and so be available for absorption . from the intestine , the compound of formula ( i ) is transported through the intestinal wall and is transported to the bloodstream and liver , both sites of circulating sap . the assay was designed for determining stability of compound in fresh blood . compound ( compound of formula ( i )) was dissolved in dmso at 1 mg / ml . daughter solution was prepared in dmso at 100 μg / ml . fresh blood was diluted 1 / 2 in isotonic buffer ph 7 . 4 . pre - incubation consisted of warming 792 μl of blood at 37 ° c . for 7 min . incubations were started with the addition of 5 μl of daughter solution . 50 μl of mixture were taken at 0 , 5 , 15 , 30 and 60 min . 50 μl of water was add to sample and then quenched with 300 μl acetonitrile containing internal standard . after 10 min centrifugation at 4000 rpm , 2 μl of samples were injected into the liquid chromatography system and eluted with a ascentis c18 column ( 50 × 2 . 1 mm id , 2 . 7 μm ) and with 0 . 1 % formic acid in water ( a ) and 0 . 1 % formic acid in acetonitrile ( b ), using the following elution gradient over 2 minutes : 5 to 95 % b over 1 . 2 min , 95 % b over 0 . 6 min and 0 . 1 min for re - equilibrate column , at 0 . 5 ml / min at 50 ° c . samples were analysed by mass spectrometry with an electrospray source , in positive mode and with following mass transitions : compound of formula ( i ): 657 to 384 monoester of cphpc : 499 to 226 cphpc : 341 to 226 controls were made to calculate percentage of disappearance of parent but also apparition of suspected metabolite , i . e . monoester and diacidic form . data showing in vitro blood stability of compound of formula ( i ) in human blood is presented in fig4 . “ 0 / 5 / 10 / 30 / 60 min ” refers to the time ( in minutes ) that the sample was taken from the mixture for analysis . in vitro liver microsomal activity of was assessed using the protocol detailed above ( intestine and liver microsomal assay protocol ). data showing in vitro liver microsomal stability of compound of formula ( i ) in human liver microsomes is presented in fig5 . “ 0 / 5 / 10 / 30 / 60 min ” refers to the time ( in minutes ) that the sample was taken from the mixture for analysis . in human liver microsomes , a high rate of hydrolysis of compound of formula ( i ) to cphpc was observed ( approximately 50 % conversion to cphpc was achieved within 30 minutes ), suggesting that compound of formula ( i ) is capable of being cleaved to active cphpc once absorbed . 1 . tennent , g . a ., lovat , l . b . and pepys , m . b . 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