Patent Application: US-62827700-A

Abstract:
the present invention is directed to methods for determining the androgenic or anti - androgenic activity of a sample and to a cell useful in these methods . to determine androgenic activity , the sample is contacted and incubated with a cell comprising i ) a luciferase reporter plasmid , ii ) a fusion protein comprising a ligand - binding domain of the androgen receptor and a gal4 dna - binding domain , said fusion protein being able to bind to binding sites in said luciferase reporter plasmid , iii ) a fusion protein comprising an n - terminal region of the androgen receptor and a transcriptional activation domain , and iv ) an androgen receptor - interacting protein 3 . the cell is then lysed and the luciferase activity of the lysate is measured . the measured luciferase activity is compared to that obtained by repeating the above method except that a control is added instead of the sample , to give the relative luciferase activity of the sample . the relative luciferase activity is used to detect or quantify an active androgen in the sample .

Description:
the assay according to the present invention has appeared useful for quantitation of circulating androgen bioactivity in human serum , also in pediatric patients . thus we expect that the assay will have wide ramifications in clinical endocrinology . according to another embodiment , we believe that the assay according to the present invention is very useful for screening test compounds with respect to their androgenic activity . in this case , instead of a serum sample , a solution of the test compound is contacted with the transfected cell . according to a further embodiment , this method is also applicable for measuring anti - androgenic activity of a test compound in solution , or a serum sample . in this ease , the luciferase activity emitted from a first sample comprising a first compound having androgenie activity is measured . next , the luciferase activity emitted from a second sample comprising said first compound having androgenic activity and either i ) a second compound which shall be tested in respect of anti - androgenic activity , or ii ) serum is measured . the luciferase activities obtained from the two measurements are compared and the decreased luciferase activity emitted from said second sample is used to detect or quantify the anti - androgenic activity of the second compound or serum . in case the androgenic activity of a serum sample shall be studied , it is preferably to treat the sample in order to release the androgen from binding proteins before the sample is contacted with the cell . such treatment can , for example , be extraction with diethylether . the cell to be transfected shall be a cell which is not androgen sensitive . suitable cells are mammalian cells although the invention is not restricted hereto . for example , yeast cells fulfilling this condition may also be employed . a suitable mammalian cell is the cos - 1 cell ( atcc no . crl1650 ). according to a preferable embodiment , the the luciferase reporter plasmid is pg5 - luc , the vector cloned with the ligand - binding domain of the human androgen receptor is pm - har , comprising the amino acids 657 - 919 , the vector cloned with the n - terminal region of the rat androgen receptor is pvp16 - rar , comprising the amino acids 5 - 538 , and the vector cloned with the androgen receptor - interacting protein 3 is pflag - arip3 . the bioassay according to the present invention , based on arip3 - facilitated interaction between the lbd and n - terminal region of ar , has many advantages over known methods . as the assay is based on the use of a transfected cell , the preparation of the probe can be carried out just by cultivating the cell . the assay is very sensitive and specific . it has a wide dynamic range , and exhibits minimal cross reactivity to estradiol . for example , in boys , serum androgen bioactivity levels and testosterone concentrations measured by ria correlated strongly , but bioactivity levels were lower , which probably reflects binding of androgens to shbg . the invention will be illuminated by the following non - restrictive experimental section . we have developed a mammalian cell ( cos - 1 ) bioassay , which can measure androgen bioactivity directly from a small amount ( 10 μl ) of human serum . the recombinant assay is based on androgen - dependent interaction between the ligand - binding domain and the n - terminal region of androgen receptor ( ar ), which were fused to gal4 dna - binding domain of saccharomyces cerevisiae and transcriptional activation domain of herpes simplex vp16 protein , respectively . the interaction is amplified by coexpressing ar - interacting protein 3 ( arip3 ) in the cells . the reporter plasmid contained five gal4 binding sites upstream of luciferase gene ; luciferase activity in cell lysates is derived from androgen bioactivity in human serum . saturating concentration of testosterone in fetal calf serum ( fcs ) induced & gt ; 700 - fold induction in relative luciferase activity . the sensitivity was & lt ; 1 . 0 nmol / l of testosterone in fcs . the intra - and interassay coefficient of variations were 8 . 3 % and 25 %, respectively . interaction between the ar termini was blocked by nonsteroidal antiandrogens , and the assay exhibited minimal cross - reactivity with estradiol . serum androgen bioactivity was studied in 23 boys with constitutional delay of puberty ( cdp ; aged , 13 . 9 to 16 . 8 yrs ), and in 9 prepubertal boys with cryptorchidism ( aged , 1 . 0 to 6 . 4 yrs ). androgen bioactivity was detectable in 15 boys with cdp , and in all boys with cryptorchidism during treatment with hcg ( range , 1 . 0 - 14 . 5 testosterone equivalents ). serum androgen bioactivity measured by the bioassay correlated strongly to serum testosterone concentration ( r = 0 . 93 , p & lt ; 0 . 0001 , n = 22 ), but not to dihydrotestosterone , dhea , or androstenedione levels . plasmids . all plasmid constructs have been reported previously ( 4 , 6 - 7 ), and only a short description of each is given here . the lbd of human ar ( containing amino acids 657 - 919 ) ( 7 ), and the n - terminal region of rat ar ( 5 - 538 ) ( 4 ) were created by pcr and the products were cloned into the pm and vp16 vectors ( clontech laboratories inc ., palo alto , calif . ), respectively . the luciferase reporter pg5 - luc contains five gal4 - binding sites in front of the minimal tata box . pflag - arip3 has been described ( 6 ). cell culture and transfection . cos - 1 cells ( american type culture collection ) were maintained in phenol red - free dulbecco &# 39 ; s minimal essential medium ( dmem ; gibco brl , santa clara , calif .) containing penicillin ( 25 units / ml ), streptomycin ( 25 units / ml ), and 10 % ( vol / vol ) fetal calf serum ( fcs ; gibco brl , paisley , uk ). twenty - four hours before transfection , the cells were divided onto a 96 - well plate ( nunc , roskilde , denmark ) at a density of 1000 cells / well . the plates were incubated overnight at 37 ° c . in a humidified athmosphere of 5 % co 2 / air . three hours before transfection , the cell culture medium was replaced by dmem containing 8 % of charcoal - stripped fcs . the cells were transfected using fugene reagent ( roche molecular biochemicals , mannheim , germany ) according to the instructions provided by the manufacturer . each well received a total of 49 ng dna ( pg5 - luc , 18 ng ; pm - har ( 657 - 919 ), 9 ng ; pflag - arip3 , 9 ng ; pvp16 - rar ( 5 - 538 ), 9 ng ; and pcmvβ4 ng ). twenty - four hours after transfection , medium in each well was replaced by 90 μl of phenol red - free dmem without fcs , and 10 μl of testosterone - containing fcs in triplicate ( standard ) or 10 μl human serum ( unknown sample ) in duplicate was added . after an overnight incubation at 37 ° c . humidified atmosphere of 5 % co 2 / air , the wells were aspirated empty , the cells were lysed in 30 μl of diluted reporter lysis buffer ( promega , madison , wis . ), and 10 μl of cell lysates were transferred to 96 - well plates for measurements of β - galactosidase ( 8 ) and luciferase ( 9 ) activities . sex steroids and nonsteroidal antiandrogens . dehydroepiandrosterone ( dhea ; 3β - hydroxy - 5 - androsten - 17 - one ), androstenedione ( 4 - androstene - 3 , 17 - dione ), and 5α - dihydrotestosterone ( dht ; 17β - hydroxy - 5α - androstan - 3 - one ), obtained from steraloids inc . ( wilton , n . h . ), were dissolved and serially diluted in ethanol , and added to charcoal - stripped fcs to yield the following concentrations : 6 . 13 nmol / l , 25 nmol / l , and 100 nmol / l . dht was diluted further in fcs to result a serum concentration of 0 . 78 nmol / l . these steroids were tested in the bioassay in parallel with testosterone standard curve . to investigate the transactivation potential of estradiol , 17β - estradiol was dissolved in charcoal - stripped fcs to result serum concentration of 500 nmol / l . the nonsteroidal antiandrogens casodex (( 2rs )- 4 ′- cyano - 3 -( 4 - fluorophenylsulfony )- 2 - hydroxy - 2 - methyl - 3 ′( trifluoromethyl - propionanilide ), and hydroxyflutamide ( 4 - hydroxy - α , α , α - trifluoro - 2 - methyl - 4 ′- nitro - m - propionotoluidide ) were obtained from zeneca pharmaceuticals ( macclesfield , uk ) and schering corp . ( bloomfeld , n . j . ), respectively . antiandrogens were serially dissolved in ethanol , and added to charcoal - stripped fcs containing 10 nmol / l of testosterone . the highest antiandrogen concentrations in the resulting fcs were 1 μmol / l of hydroxyflutamide and 10 μmol / l of casodex ; the measurements were carried out in quadruplicate in one transfection . preparation of standards and patient sera for the bioassay . testosterone ( steraloids inc ., wilton , n . h .) was dissolved , serially diluted in ethanol , added to charcoal - stripped fetal calf serum ( fcs ; hyclone , logan , utah ), and divided in aliquots , which were stored at − 70 ° c . for future use as standards in the bioassay . sixty microliters of serum from each boy ( see below ) was centrifuged briefly , filtered through a 0 . 22 μm spin - x centrifuge filter unit ( corning costar corporation , new york , n . y . ), and stored at − 70 ° c . until used . ether extraction . testosterone was added to pooled serum of 10 prepubertal ( age range , 1 . 0 - 8 . 0 yr ) boys with cryptorchidism ( 13 ). the serum pool was divided in 300 μl aliquots in glass tubes , followed by 300 μl of diethylether ( merck , darmstadt , germany ). the tubes were vortexed briefly , centrifuged for 10 minutes in 4 ° c ., and placed in dry ice - ethanol (− 70 ° c .) bath to freeze the water phase , after which the organic phase was transferred to a new glass tube . freezing was repeated once , diethylether was evaporated , and the samples were reconstituted in 300 μl of charcoal - stripped fcs ( 5 tubes ). the tubes were shaken gently overnight in 4 ° c ., and filtered through a 0 . 22 μm spin - x centrifuge filter unit . 30 - 60 μl of serum from each tube was taken for testosterone ria ( see below ). serum sex hormone - binding globulin ( shbg ) level in the pooled sera of the boys ( and in charcoal stripped fcs ) was measured using the assay described below . subjects . thirty - two boys , aged 1 . 0 - 16 . 8 yr , were investigated . twenty - three boys , aged 13 . 9 - 16 . 8 yr , had constitutional delay of puberty ( cdp ). clinical data , together with serum hormone levels on 19 boys have been published previously ( 10 ). the boys were in early puberty ( 18 boys were at tanner stage g2 , and 5 at stage g3 ), and had no underlying diseases that could have accounted for the delay in puberty . sixty - five per cent had a history of pubertal delay in the family . the boys were clinically examined , puberty was staged according to tanner ( 11 ), the testes were measured with a ruler , testicular volume was calculated from the formula length × width 2 × 0 . 52 ( 12 ), and a single blood sample was drawn . twenty - two boys have been followed up ≧ 12 months ; puberty has progressed in each subject . another study group consisted of nine boys aged 1 . 0 - 6 . 4 yr with cryptorchidism ( clinical data and serum hormone levels have been published previously ; 13 ). these boys were treated with human chorionic gonadotropin ( hcg ; 1500 - 5000 iu i . m ., three times with one week interval ). blood samples were drawn immediately before the treatment and on the fourth day after the last hcg injection . the blood samples were allowed to clot , serum was separated by centrifugation and stored at − 70 ° c . until required . the study protocol was accepted by the ethical committee of the hospital for children and adolescents , university of helsinki . informed consents were obtained from the guardian , and in addition from the boys with cdp . immunoassays . serum testosterone concentrations were measured using a commercially available ria kit ( orion diagnostica , espoo , finland ; 10 , 13 ). according to the manufacturer , the assay has a 4 . 5 % cross - reactivity with 5α - dihydrotestosterone , and minimal cross reactions to other steroid hormones . serum dht , androstenedione , and dhea concentrations were measured in boys with cdp after separation of steroid fractions on lipidex - 5000 microcolumn ( packard - becker , groninger , the netherlands ) as previously described ( 14 ). serum shbg concentrations in twenty - two boys with cdp were measured using time - resolved fluoroimmunoassay ( wallac oy , turku , finland ). according to the manufacturer , the sensitivity of the shbg assay is better than 0 . 5 nmol / l , inter - and intraassay coefficients of variation are both & lt ; 5 %. data analysis . relative luciferase activities were calculated by dividing luciferase acitivities by β - galactosidase activities to correct for differences in transfection efficiency . standard curves for the bioassay were fitted with a 4 parameter weighted equation using the assayzap program ( biosoft inc ., cambridge , uk ); the results are expressed in nmol / l testosterone equivalents . pearson &# 39 ; s correlation coefficient was calculated between paired variables to investigate their relationship . mean values of different parameters were tested by paired and unpaired t tests , when appropriate . all mean values are expressed ± sd . statistical significance was accepted for p & lt ; 0 . 05 . the principle of the bioassay for quantitating the response of mammalian cells to androgens in human serum is presented in fig1 . androgens from the serum enter cos - 1 cells and induce the interaction between the lbd and n - terminal region of ar . this interaction is enhanced by arip3 ( 6 ). the complexes bind to gal4 binding sites , located in pg5 - luc and the vp16 transcriptional activation domains are tethered to the luciferase gene promoter , leading to activation of luciferase gene transcription . luciferase activity in cell lysates corresponds to androgen bioactivity in human serum . dose response . different amounts of testosterone were added to charcoal - stripped fcs , and the resulting dose response curves are shown in fig2 . values are presented as units of relative luciferase activity ( luciferase activities divided by β - galactosidase activities obtained for each well ). the steepest increase in relative luciferase activity occurred at testosterone concentrations in fcs below 10 nmol / l . the median of maximal fold induction ( calculated as the ratio of relative luciferase activity induced by 100 nmol / l testosterone to activity induced by charcoal - stripped fcs without added testosterone ) from 5 different assay runs was 745 . biopotency of androgens and estrogen . to investigate the biopotencics of different naturally occurring androgens , dht , androstenedione , and dhea were added to charcoal - stripped fcs . dihydrotestosterone was the most active androgen ; fcs containing 0 . 78 nmol / l of dht induced relative luciferase activity equal to 10 . 0 nmol / l testosterone equivalents . only the highest concentration of androstenedione ( 100 nmol / l in fcs ) induced a signal equal to 1 . 3 nmol / l of testosterone equivalents . dhea did not activate luciferase gene expression at any dose . the relative luciferase activity induced by fcs containing a high concentration of estradiol ( 500 nmol / l ) was & lt ; 0 . 1 % of that achieved with fcs containing saturating testosterone concentration ( 100 nmol / l ). inhibition by nonsteroidal antiandrogens . the effect of antiandrogens on the interaction between the fragments of ar was investigated by first adding testosterone to charcoal stripped fcs to yield a subsaturating concentration ( 10 nmol / l ). then , increasing amounts of hydroxyflutamide and casodex were added to aliquots of the testosterone - containing fcs , and 10 μl of each dilution was subjected to measurement in the bioassay . fcs containing 100 ( hydroxyflutamide ) or 1000 ( casodex ) ( i . e . clinically achievable concentrations of antiandrogens ) times the molar amount of testosterone , suppressed relative luciferase activities to a level corresponding ˜ 5 % of the activity achieved by testosterone - containing fcs without added antiandrogens . sensitivity and precision . the sensitivity of the bioassay was defined as mean + 2 standard deviations of multiple luciferase activities induced by charcoal - stripped fcs without added testosterone ; it was below the signal induced by fcs containing 1 . 0 nmol / l of testosterone ( cell culture medium containing 0 . 1 nmol / l of testosterone ). intraassay coefficient of variation ( cv ) was defined as repeated measurement of the same human serum sample . at 4 . 9 nmol / l testosterone equivalents , the intraassay cv was 8 . 3 %. interassay cv was 25 % ( as determined from 5 assay runs ). patient data . serum androgen bioactivity levels were above the assay sensitivity in 15 boys with cdp , and in all 9 prepubertal boys with cryptorchidism during treatment with hcg ( androgen bioactivity levels before the hcg treatment were below the detection limit of the assay ). the mean of androgen bioactivity levels above the assay sensitivity was 4 . 3 ± 3 . 2 nmol / l , and range 1 . 0 - 14 . 5 nmol / l testosterone equivalents ( n = 24 ). these values were on the rising part of the dose response curve , and correlated strongly to serum testosterone levels measured by ria ( r = 0 . 93 , p & lt ; 0 . 0001 , n = 22 ; fig3 ). when expressed as a function of serum total testosterone , the average serum androgen bioactivity levels were 26 ± 3 . 7 % and 33 ± 9 % in boys with cdp or cryptorchidism , respectively ( p & lt ; 0 . 05 ). in boys with cdp , this percentage and testis volume correlated positively ( r = 0 . 49 , n = 13 , p = 0 . 09 ). in boys with cdp , serum androgen bioactivity levels did not correlate to serum dhea ( range , 4 . 7 - 21 . 1 nmol / l ), dht ( 0 . 3 - 3 . 4 nmol / l ), or androstenedione ( 0 . 9 - 4 . 4 nmol / l ) concentrations . when investigating the relationship between bioactivity levels and shbg , relative luciferase activities below the sensitivity of the bioassay were set equal to the detection limit of the bioassay . one boy had clearly higher serum shbg level ( 250 nmol / l ) than the others with cdp ( range , 36 - 125 nmol / l ), and this value was excluded from all correlation analyses . androgen bioactivity levels correlated negatively to serum shbg concentrations ( r = 0 . 44 , p & lt ; 0 . 05 , n = 21 ), and positively to testicular volumes ( r = 0 . 77 , p & lt ; 0 . 0001 , n = 23 ) ether extraction of human serum . to investigate the relationship between serum androgen bioactivity and testosterone levels further , testosterone was added to pooled sera of 10 prepubertal boys with cryptorchidism to yield serum concentration of 21 nmol / l . in this pool , serum testosterone concentration , measured with ria , was 20 . 6 ± 0 . 9 nmol / l , and androgen bioactivity level , measured with the bioassay , was 5 . 7 ± 0 . 6 nmol / l testosterone equivalents ( fig4 ). concentration of shbg in this serum pool was 135 nmol / l . we next extracted the pooled sera with diethylether , which releases steroid hormones from their binding proteins ; the steroid - containing phase was reconstituted in charcoal stripped fcs ( without measurable shbg ). this should render free plus initially protein - bound androgens in human serum available to the cells of the bioassay . indeed , after the procedure , serum androgen bioactivity in the pool increased from 5 . 7 ± 0 . 6 nmol / l to 13 . 0 ± 1 . 6 nmol / l testosterone equivalents ( fig4 ; p & lt ; 0 . 005 ). the actual rise in serum androgen bioactivity was likely to be even higher , as not all testosterone was recovered even by the ria ; the mean testosterone level measured after reconstitution was 14 . 1 ± 1 . 3 nmol / l ( mean recovery 69 %; fig4 ). the earliest assays for measuring bioactivity of androgenic compounds were based on androgen - dependent responses of living organisms , like the growth of the capon comb or accessory sex organs of the male rat ( 15 ). development of cell culture and recombinant dna techniques have enabled more sensitive and precise assays for evaluating biological responses of living cells to sex steroids . nevertheless , the only assay so far that measures sex steroid bioactivity in human serum has been the recombinant ultrasensitive bioassay for estrogens , which utilizes yeast cells ( 16 ). yeast cells were not suitable for the current bioassay since , probably due to permeability properties of the cell wall , nonsteroidal antiandrogens did not show any antagonistic activity in yeast cells ( 7 ). to our knowledge the current mammalian cell assay , based on arip3 - enhanced interaction between the fragments of ar , is the first to measure androgen bioactivity in human serum . the sensitivity of the assay was better than the signal induced by 1 . 0 nmol / l of testosterone in the 10 μl fcs aliquot , corresponding to 0 . 1 nmol / l of testosterone in cell culture medium . this is of the same order of magnitude as the k d of ar for testosterone ( 17 , 18 ), which casts some doubt on the possibility to enhance the assay sensitivity without additional manipulation of the sample significantly . although ether extraction and subsequent concentration of the sample in fcs increased the relative sensitivity of the bioassay , the assay without the extraction procedure has the benefit of directly measuring androgen bioactivity in human serum . in boys , serum androgen bioactivity and testosterone concentrations correlated strongly . testosterone in serum is bound with high affinity to shbg and with low affinity to albumin ; only ˜ 2 - 3 % of total testosterone is free ( 18 , 19 ). the amount of biologically active testosterone has been suggested to be a function of free testosterone ( 20 ). the androgen bioactivity levels we found , however , were obviously too high to represent merely the free testosterone fraction ( 21 ). indeed , in the course of the assay , sera were diluted to cell culture medium in a ratio of 1 to 10 , which should lead to dissociation of the weakest protein - steroid complexes . thereafter , both the free plus initially albumin bound testosterone fractions in serum , which together are often referred to as the bioavailable testosterone ( 20 , 23 - 25 ), should be available for the cells of the bioassay . on the other hand , because of the high androgenic potential of dht in fcs , one would expect to find a positive correlation between serum androgen bioactivity and dht levels in boys with cdp . lack of relationship between these variables may , however , reflect the fact that the affinity of dht to shbg is three times that of testosterone ( 26 ), which may render the low amounts of circulating dht in boys biologically inert . moreover , adding of testosterone to shbg - containing serum pool of prepubertal boys , resulted to androgen bioactivity levels which were approximately one fourth of the testosterone levels measured with ria . in similar vein , extraction of human serum with diethylether and subsequent reconstitution of the sample to charcoal - stripped fcs increased bioactivity levels . taken together , although we can not exclude the existence of yet unidentified factor ( s ) in human serum that inhibit sex steroid entry or action within the target cells , these findings suggest that shbg - bound steroids are not available to cos - 1 cells of the bioassay . serum androgen bioactivity levels and testis volumes correlated strongly , which probably reflects increased testicular testosterone production towards adulthood . the ratio of serum androgen bioactivity to testosterone also tended to increase as a function of puberty , which would be expected , because of the reciprocal changes in serum testosterone and shbg levels during adolescence ( 27 , 28 ). on the other hand , this ratio was higher in prepubertal boys with cryptorchidism during hcg treatment than in the older boys with cdp . this was not expected , because prepubertal subjects have higher serum shbg levels ( 27 ) than boys in early puberty . the androgen - dependent interaction between the fragments of ar exhibits minimal cross reactivity to estradiol ( 3 , 5 ). this was also observed in the present study . moreover , nonsteroidal antiandrogens casodex and hydroxyflutamide suppressed relative luciferase activity , suggesting that the current assay is applicable to screen rapidly compounds for both androgenic and antiandrogenic activity . however , caution is required in the interpretation of these results , since in the assays employing fragments of ar or full - length ar , some synthetic compounds may act differently ( 5 ). nevertheless , our unpublished results indicate that androgen bioactivity levels in boys measured with the current assay correlate strongly with those measured by an assay based on the use of full - length ar . it will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments , only a few of which are disclosed herein . it will be apparent for the expert skilled in the field that other embodiments exist and do not depart from the spirit of the invention . thus , the described embodiments are illustrative and should not be construed as restrictive . 1 . mangelsdorf d j , evans r m . 1995 the rxr heterodimers and orphan receptors . cell . 83 : 841 - 850 . 2 . langley e z , zhou x , wilson e m . 1995 evidence for an anti - parallel orientation of the ligand - activated human androgen receptor dimer . j biol chem . 270 : 29983 - 29990 . 3 . doesburg p , kuil c w , berrevoets c a , et al . 1997 functional in vivo interaction between the amino - 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