Patent Application: US-63628784-A

Abstract:
a method is provided for recognizing and diagnosing diseases or conditions associated with immune system dysfunction and loss of integration of brain function , particularly a subset of alzheimer &# 39 ; s disease , and for the in vitro testing to determine efficacy of possible therapeutic agents . the method involves measurement of immunological parameters on peripheral blood immunocytes .

Description:
laboratory evaluation of new therapeutic agents by in vitro tests on peripheral blood immunocytes patients consisted of 22 individuals with mild cerebral atrophy on cat scan diagnosed as ad patients by the global deterioration scale ( gds ) for assessment of primary degenerative dementia ( reisberg , et al ., 1982 ). controls consisted of 22 age - matched ( ages 58 - 91 ) elderly healthy individuals , and 18 disease controls whose symptoms mimicked ad , but resulted from other causes ( e . g ., parkinsonism , cerebrovascular accident , etc .). immunocytes of patients and disease controls were 1a - 2p tested in vitro both with and without ( 10 - 3 - 10 - 7 m ). oral ingestion of 1a , 2p ( 800 mg / day ) 1a - 2p in tablet form ( 2 tablets 3 times a day ) with meals , or switched to placebo after 6 months ( upon deterioration , some were switched back ) constituted the in vivo drug therapy . table 1 l shows representative clinical values of several immunological parameters before and after 1a - 2p therapy . therapeutic benefits from 1a - 2p therapy in interactive t cell - depressed subset of ad patients paralleled by correction of immunodepression table 1 illustrates interactive t - cell monitoring and 1a - 2p therapy in selected patients . patients mab , whr , and bfp evinced interactive t - cell depression , in vitro correction by 1a - 2p , and encouraging clinical improvement after in vivo administration of 1a - 2p . additionally , they showed the appropriate in vivo and in vitro changes when switched to a placebo . patients mlh , vsb , and e . s . evinced normal interactive t - cell levels ( but other immunological abnormalities ), in vitro correction of said abnormalities in patients mlh and vsb , and lack of clinical improvement despite 1a - 2p administration . generally speaking , no improvement occurred in ad patients with a normal interactive t - cell level prior to 1a - 2p treatment , and no change in interactive t - cell levels was seen in aged , healthy controls after administration of 1a - 2p . table 1__________________________________________________________________________laboratory values and disease course from representativeindividual patients . sup . a demonstrating interactivet - cell monitoring and 1a - 2p therapy__________________________________________________________________________ in vitro correctionstage of lab abnormality by 1a - 2p given 1a - 2p ; name disease . sup . b date yes no yes no if so , date__________________________________________________________________________m . a . b . iv 06 / 17 / 83 inter - ( to 100 %) 08 / 21 / 83 ( 1 ) active t - cells ( 50 % of controlw . m . r . v 09 / 14 / 83 inter - ( to 100 %) 09 / 19 / 83 ( 2 ) active t - cells ( 48 % of controlb . f . p . iv 06 / 28 / 83 inter - ( to 100 %) 07 / 25 / 83 ( 3 ) active t - cells ( 60 % of controlm . l . h . iv 06 / 28 / 83 nk cells inter - ( to 100 %) inter - 07 / 25 / 83 ( 4 ) ( 20 % of active active control ) t - cells t - cells normal normalv . s . b . iv 08 / 23 / 83 il - 1 inter - il - 1 inter - 07 / 25 / 83 ( 5 ) ( 40 % of active normal - active control ) t - cells ized t - cells normal normale . s . ii 01 / 25 / 83 il - 2 pro - inter - il - 2 not 06 / 20 / 83 ( 6 ) duction active corrected ( 40 % of t - cells t - cells control ) normal normal__________________________________________________________________________correction in vivo changed to placebo change reinstitutedname in vitro date ( stage ) yes date no in vitro in vivo on 1a - 2p__________________________________________________________________________m . a . b . yes 01 / 25 / 83 yes yes 02 / 18 / 84 interactive stage not done ( 1 ) inter - ( ii ) t - cells ivactive dropped ( 60 % t - cells of normalnormalw . m . r . yes 02 / 28 / 84 yes yes 02 / 13 / 84 not done stage not done ( 2 ) inter - ( ii ) ivactivet - cellsnormalb . f . p . yes 10 / 03 / 83 yes yes 11 / 07 / 83 interactive stage 02 / 06 / 84 ( 3 ) nk cells ( ii ) t - cells iv normalizenormal dropped ( 50 % in vitro of normal and almost in vivo ( stage i ) m . l . h . yes 11 / 15 / 83 no yes 11 / 16 / 83 no ( 4 ) change change in in symp - symp - toms tomsv . s . b . yes 09 / 31 / 83 still yes 01 / 03 / 84 yes no ( 5 ) ( iv ) il - 1 change dropped in symp - ( 35 %; inter - toms active t - cells still normale . s . no 08 / 17 / 83 no yes 09 / 13 / 83 no change no change ( 6 ) correc - correc - ( still stagetion tion , ii ) still ( ii ) __________________________________________________________________________ . sup . a patients 1 - 3 show response to 1a2p in vitro and in vivo and demonstrate importance of interactive tcell test ; patients 4 - 6 suggest irrelevance of in vitro correction of other tests in presence of normal interactive tcells . . sup . b v = worst ; i = mild . the full scale ranges up to vii . peripheral blood lymphocytes ( pbl ) may be obtained by venipuncture from individuals for the desired functional immunocyte studies . preferred protocols for a number of studies are given below . the level of interactive t cells is depressed in that subset of ad patients who showed clinical improvement upon treatment with an agent which restored interactive t - cell levels in vitro . immunoglobulin synthesis , il - 1 and il - 2 activity , and 2 - deoxyglucose uptake were also abnormal in some ad patients , but restoration of normality by piracetam treatment was not accompanied by clinical improvement . levels of active ( active t - cells are concerned with mediator production and are a measure of the total afferent limb of cell - mediated immunity ) and total t - cells may be measured , fundenberg , wybran and robbins ( 1975 ). lif production after cona and pha stimulation , oppenheim and rosenstreich ( 1976 ), were measured when indicated using methods described in arala - chaves , silva , porto , picoto , ramos and fudenberg ( 1977 ). interactive t - cells are those which bind to established human b lymphoid cell lines to form rosettes . they seem to be smaller than normal t - cells , typically 10 - 100 cubic microns rather than 100 - 200 cubic microns . these cells may be the human equivalent of mouse contrasuppressor cells . we employ the raji b lymphoid cell line for this assay , goust and fudenberg ( 1983 ). the percent of rosettes formed between normal peripheral blood lymphocytes and raji cells is 27 . 8 ± 5 . 3 mean ± s . d at a 20 : 1 ( pbl : raji ) ratio . to perform the assay , ficoll - hypaque isolated pbl were suspended in rpmi - 1640 medium supplemented with 1 % bovine serum albumin ( bsa ) and adjusted to a concentration of 2 × 10 7 cells / ml . raji cells were washed twice in same medium and adjusted to a concentration of 1 × 10 6 cells / ml . 50 - ul aliquots of the two cell suspensions are then mixed ( 20 : 1 ratio of pbl to raji ), centrifuged at 150 g for 5 minutes at 4 ° c ., and incubated for 1 hour in an ice water bath . after gentle resuspension , the percent of raji cells surrounded by three or more lymphocytes ( rosettes ) were determined . 3 . natural killer ( nk ) cells the becton dickinson hnk1 and / or leu - 11 monoclonal antibodies may be used for enumeration of natural killer cells ( nk ); cells from the erythroleukemic cell line k562 may be used as target cells . effector cells were prepared from pbl by nylon wool column separation of t - and b - cells and followed by srbc rosette formation . monocyte depleted , nylon wool non - adherent cells are mostly nk and k cells . 100 ul of nylon wool nonadherent cells were added with 100 ul of 51 cr - labeled target cells ( 1 × 10 4 ) in the wells of a flat - bottomed microtest plate at a ratio of 50 : 1 or 25 : 1 . target cells incubated without effector cells are used to determine the level of spontaneous lysis . maximum lysis was determined by adding 10 % triton x - 100 solution ( no effector cells ) instead of medium to the well . the percentage cytotoxicity was calculated as : ## equ1 ## each assay is performed in triplicate . il - 2 activity in microculture may be measured as follows : 4 × 10 3 cells of a cloned il - 2 dependent t - cell line ( ctll ) are washed extensively and incubated in 30 ul of supernatant in a total of 100 ul culture medium with 5 % fetal calf serum at 37 ° c . in flat - bottomed microwells . after 24 hours , 2 uci methyl -[ 3 h ]- thymidine ( 2 . 0 ci / mmole ) is added for a further 4 - 5 hours . cells are then harvested onto filter paper disks using an automated cell harvester and counted in a liquid scintillation counter . isolated pbls were suspended in complete rpmi at 2 × 10 6 cells / ml and pwm was added at a final concentration of 1 : 50 . one half ml of the cell suspension was placed in each well of a 24 - well flat bottom microtiter plate and incubated at 37 ° c . in a 5 % co 2 incubator for 8 days . for cultures containing 1a - 2p , drug was added at day zero at a final concentration of 10 - 3 m . cultures were supplemented daily with 80 ul of nutritional cocktail as described by mishell and dutton ( 1967 ). the culture supernates were recovered following incubation and assayed for igm and igg content by the immunosorbent assay ( elisa ) as described by khansari , fudenberg and merler , immunobiol 164 : 12 , # 74 ( 1983 ). a quantity ( 0 . 1 ml ) of one tenth of one ml lymphocyte suspension ( 5 × 10 6 cells / ml ) was placed in each well of a 96 - well flat bottom microtiter plate . pwm ( gibco , grand island biological co ., grand island , ny ) was added to each well at a final concentration of 1 : 50 . a therapeutic agent , such as 1a - 2p , may be added to the cells at the concentration of 10 - 3 m and followed by incubation at 37 ° c . in a 5 % co 2 for 96 hours . the cultures were fed with 0 . 5 uci of 3 h - thymidine ( new england nuclear , boston , ma ) for additional 18 hours of incubation . after incubation , the cells were harvested and radioactivity was counted in a scintillation counter . the control cultures were treated similarly but without 1a - 2p . while specific assays for these parameters are described above , it should be understood that this invention is not limited to any specific assay method , and that no specific number of assays is required . furthermore , this invention is not limited to the three immunological parameters listed above . nor is it necessary that all of the aforementioned parameters be monitored in the course of testing a specific therapeutic agent or diagnosing a particular clinical condition . this invention embraces the immunological testing of new neuropsychotropric or nootropic agents without limitation to those enumerated , and of agents having nootropic as well as other psychotropic or other drug action . in addition to 1a - 2p , other pyrrolidone derivatives and analogs , including but not limited to those listed earlier , and other b cell mitogens , are potential or recognized nootropic agents which may be tested or used therapeutically , in accordance with the teachings herein . in vitro immunological testing of pramiracetam suggests that it is a more potent nootropic agent than 1a , 2p . from the foregoing description , one skilled in the art can easily ascertain the essential characteristics of this invention and , without departing from the spirit and scope thereof , modify the invention to adapt to various usages and conditions . the disclosures of the references cited below are incorporated by reference herein . 1 . whitehouse , p . j ., price , d . l ., strubb , r . g ., clark , a . w ., coule , j . t ., and delong , m . r . alzheimer &# 39 ; 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