ORIGINAL ARTSCLE
`
`
`Endocrine Research
`
`Ciinical and Biochemical Consequences of CYP17A1
`Inhibition with Abiraterone Given with and without
`
`Exogenous Glucocorticoids in castrate Men with
`Advanced Prostate Cancer
`
`Gerhardt Attard, Alison H. M. Reid, Richard J. Auchus, Beverly A. Hughes,
`Amy Mulick Cassidy, Emilda Thompson, Nikhil Babu Oommen, Elizabeth Folkerd,
`Mitch Dowsett, Wiebke Arit,* and Johann S. de Sono*
`
`The Institute of Cancer Research (GA, A.H.M.R., A.M.C., E.F., ¥\/|.D., J.S.d.B.), Sutton, Surrey SM2 SNG,
`United Kingdom; The Royal Marsden National Health Service Foundation Trust (GA, A.H.M.R., E.T.,
`N.B.O., E.F., MD, J.S.d.B.), Sutton, Surrey SM2 SPT, United Kingdom; Division of Metabolism,
`Endocrinology, and Diabetes (R.}.A.), Department oi‘ internal Medicine, University of Michigan, Ann
`Arbor, Michigan 48209; and Centre for Endocrinology, Diabetes, and Metaboiism {B.A.H,, WA), School
`of Clinical and Experimental Medicine, University of Birmingham, Birmingham 8&5 T22, United Kingdom
`
`Context: Abiraterone acetate is a sma|i~moiecule cytochrome P1350 17A1 (CYP17A1) inhibitor that
`is active in castration—resistant prostate cancer.
`
`Objective: Our objective was to determine the impact of abiraterone with and without dexameth-
`asone treatment on in viva steroidogenesis.
`
`Design and Methods: We treated 42 castrate, castration-resistant prostate cancer patients with
`continuous, daily abiraterone acetate and prospectiveiy collected blood and urine before and
`during abiraterone treatment and after addition of dexamethasone 0.5 mg daily.
`
`Results: Treatment with singie~agent abiraterone acetate was associated with accumulation of
`steroids with mineralocorticoid properties upstream of CYPTIA1. This resulted in side effects,
`including hypertension, hypokalernia, and fluid overload, in 38 of 42 patients that were generally
`treated effectively with eplerenone. importantly, serum and urinary androgens were suppressed
`by more than 90% from baseline. Urinary metabolites of 17—hydroxypregne-nolone and l7-hy-
`droxyprogesterone downstream of 17a-hydroxylase remained unchanged. However, 3o:5or~17-
`hydroxypregnanolone, which can be converted via the backdoor pathway toward 5a~dihydrotes~
`tosterone, increased significantly and correlated with ievels of the major Sordihydrotestosterone
`metabolite androsterone. In contrast, urinary metaboiites of 11-deoxycortisol and active gluco-
`corticoids declined significantly. Addition of dexamethasone to abiraterone acetate significantiy
`suppressed ACTH and endogenous steroids, including 3a5a-17-hydroxypregnanolone.
`
`Conclusion: CYP17A1 inhibition with abiraterone acetate is characterized by significant suppres~
`sion ofandrogen and cortisol synthesis. The iatter is associated with a rise in ACTH that causes raised
`
`rnineralocorticoids, leading to side effects and incomplete 17a-hydroxyiase inhibition. Concomi»
`tant inhibition of 17,20~iyase resuits in diversion of trhydroxyprogesterone metabolites toward
`androgen synthesis via the backdoor pathway. Addition of dexamethasone reverses toxicity and
`could further suppress androgens by preventing a rise in substrates of backdoor androgen
`synthesis. (J Clin Endocrinol Metab 97: 507-516, 2012)
`
`
`
`
`iS5N Print G621-972K ISSN Orrline 1945-7197
`Printed in US A.
`Copyright © 2012 by the Endocrine Society
`doi: 10_i2iO!;c.20I1-2189 Received July 30, 201l.A-zcepted October 31. 2011.
`First Published Online December 14, 2011
`
`* WA and J So 5. are jolitt senior authors.
`Abbreviations: An, Androsterone; AR, androgen receptor, CRPC, castration-resistant pros-
`tate cancer; CYPWA3 , cytochrome P450 17A1, DHEA, dehydroepaandrosterone,‘ DHEAS,
`dehydroepiandrosterone sulfate,‘ DOC,
`ibdeoxycortrcosterone; 3a:5a:—‘i"iHP, 3a5av
`17OH—pregnanolone. LGMSIMS,
`iiquid chromatograpnyftandern mass spectrometry;
`17OHP, 17-hydroxyprogesierone; PSA, prostate-specific antigen; THALDO, 3a,S,B—tetra—
`hydroaldosterone; THS, tetranydro-1 1-deoxycortisol,
`
`J Ciin Endocrinoi Metab, February 2012, 97{2):5D7—516
`
`jcem.endojournais.org
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`

`508
`
`Attard er an‘.
`
`CYP17A1 inhibition by Abiraterone
`
`JC|in Endocrinoi Metab, February 2012, 97(2).507w516
`
`rostate cancer is the second most common cause of male
`P cancer—related death in the Western world (1). Treat-
`ment—naive prostate cancer is usually a hormone-driven dis-
`ease, with a response to castration observed in more than
`90% of patients. The median duration of response is 18
`months. Overwhelming evidence now confirms that in a sig-
`nificant proportion of patients, relapse with castration-resis-
`tant prostate cancer (CRPC) occurs secondary to reactiva-
`tion of androgen receptor (AR) signaling, including by serum
`androgens from nongonadal sources (2). Cytochrome P450
`17A1 (CYP17A1 ) is a key enzyme in cortisol synthesis via its
`17a-hydroxylase activity and plays a central role in androgen
`biosynthesis with its 1 7,20—lyase activity catalyzing the conver-
`sion of 17—hydroxypregnenolone to the main androgen precur-
`sor dehydroepiandrosterone (DHEA) (3, 4). CYP17A1 is ex-
`pressed in the gonads but also at extragonadal sites including
`the prostate (5-«7) where it might contribute to intracrine
`hormone synthesis.
`Abiraterone is a rationally designed, srnall—molecule in-
`hibitor of CYP17A1 (8, 9). The specificity of abiraterone
`for inhibition of 17,20-lyase us. 170:-byclroxylase is low
`(1.4-fold, IC53 1" 2.9 nM compared with 4 mi) (10), and
`treatment with abiraterone acetate would therefore be ex-
`
`pected to cause a rise in ACTH with a consequent increase
`in llwdeoxycorticosterone (DOC) and corticosterone,
`mimicking the effects observed in patients with congenital
`adrenal hyperplasia due to inactivating CYP17A1 muta-
`tions (3). To date, it has proven difficult to develop a small-
`moiecule therapeutic that specifically inhibits only the
`17,20-lyase activity of CYP17A1 (11).
`When administered to noncastrate men, abiraterone ac-
`etate resulted in suppression of testosterone with a subse-
`quent LH surge that overcame inhibition of gonadal testos«
`terone synthesis
`(12). We subsequently reported that
`continuous inhibition of CYP17A1 with oral abiraterone ac-
`
`etate in chemotherapy-naive, CRPC patients was safe and
`significantly suppressed serum androgens and estrogens (6,
`13). Importantly, we and others reported significant antitu—
`mor activity in phase I/ll trials with single-agent abiraterone
`acetate after multiple previous lines of hormone therapy, in-
`cluding ketoconazole, and in chemotherapy-treated patients
`(13-15 ). Recently, abiraterone acetate was given regulatory
`approval for the treatment of men with advanced prostate
`cancer progressing after docetaxel after a phase ill study
`showed that combined treatment with abiraterone acetate
`
`and prednisone conferred a survival benefit when compared
`with prednisone alone (16).
`We and others previously used RIA to measure serum
`androgens. Androstenedione and DHEA sulfate (DHEAS)
`were suppressed to below the lower limits of detection (2
`rigid! and 15 ,u.g/dl, respectively), and DHEA declined
`3-fold; however, because cross-reactivity with abiraterone
`
`was observed in this assay, the detection of DHEA in pa-
`tients on abiraterone acetate could be artifactual (6, 15).
`
`A liquid chromatographyltandem mass spectrometry (LC-
`MS/MS) assay was used to measure serum testosterone
`levels that declined to below the lower limit of sensitivity
`
`(1 ng/dl) in all patients (6, 15 ). However, these studies have
`incompletely dissected the biochemical consequences of
`treatment with abiraterone acetate. We here report the
`first detailed, mass spectrometry-based analysis of the ste-
`roidogenic effects of CYP17A1 inhibition in samples
`taken from medically castrated patients treated with sin-
`gle-agent abiraterone acetate and with the combination of
`abiraterone acetate and dexamethasone.
`
`Patients and Methods
`
`Patients
`
`All the patients included in this prospectively planned analysis
`were enrolled into the continuous daily study of abiraterone acetate
`performed at the Royal Marsden Hospital (RMH), London, UK
`(COU-001, www.clinicaltrials.gov identifier NCTO0473512). All
`patients were castrate (serum testosterone < 50 ng/dl) for the du-
`ration of the study (ongoing treatment with a LHRH analog), had
`an Eastern Co-operative Oncology Group {ECOG) performance
`status of 0 or 1, had not received previous treatment with chemo-
`therapy or radionuclides, and had progressive disease as defined by
`prostate-specific antigen (PSA) Working Group I (17). The ethics
`reviewcommittees ofthe RMI-I approved this study, and all patients
`gave informed consent. The antitumor activity and safety data ob-
`served in this study were reported previously (6, 13).
`
`Treatment and procedures
`Abiraterone acetate powder was administered in four 250-rng
`capsules to fasted patients daily in 28-Cl cycles. Safety evaluations
`were conducted at baseline, weekly for the first two cycles and
`every cycle thereafter and included a physical examination and
`complete blood count, clotting, serum creatinine, electrolytes,
`and liver function tests. All adverse events were graded according
`to the U.S. National Cancer Institute common toxicity criteria
`version 3.0. Toxicity related to elevated mineralocorticoid levels
`was managed with the selective mineralocorticoid receptor an-
`tagonist eplerenone (50~200 mg/d), and treatment with gluco-
`corticoids to suppress ACTH was only used if mineralocorticoid
`antagonism did not reverse these toxicities. Spironolactone was
`not used because it is an agonist for wild-type AR (18). Abi-
`raterone acetate was continued until PSA progression as defined
`by PSA Working Group I (17). Patients were then given the op-
`tion to continue abiraterone acetate in combination with dexa-
`
`rnethasone 0.5 mg daily, which as the standard glucocorticoid
`preparation for treating CRPC at our institution (19) was used
`in preference to other steroids to allow the evaluation of antitu-
`rnor activity. Eplerenone was discontinued after initiation of
`dexamethasone when toxicities related to mineralocorticoid ex-
`cess resolved. Patients continued treatment on abiraterone ace-
`tate and dexamethasone until PSA, radiological (20), or clinical
`progression; withdrawal of consent; or death (6, 13).
`
`

`

`J Clin Endocrinol Metab, February 2012, 97(2):507—516
`
`jcern.endojourna|5.org
`
`509
`
`Patients were given the option to consent to additional blood
`draws for the evaluation of serum androgens and plasma ACTH
`weekly for the first two cycles, on d 1 every cycle thereafter until
`progression on single-agent abiraterone acetate and after addi-
`tion ofdexarnethasone to abiraterone acetate, weekly for the first
`cycle and monthiy thereafter. Patients were also given the option
`to consent to and provide a 24-h urine sample for steroid me-
`tabolite analyses before treatment, after a minimum of 28 cl con-
`tinuous dosing (cl 1, cycie 2 or 3) and on cl 1, cycie 6 or 7 (Sup-
`plementai Fig. 1, published on The Endocrine Society’sjournals
`Online web site at http://jcernendojournais.org).
`
`Measurement of ACTH and steroids
`
`Plasma ACTH was measured by the RMH Academic Bio-
`chemistry Laboratories with a solid-phase two—site sequential
`chemiluminescent immunometric assay (LKAC1) using an IM-
`MULITF. 1000 autoanaiyzer (Siemens Heathcare Diagnostics
`Products Ltd., Surrey, UK). The analytical sensitivity was 9 pg]
`ml. The intraassay precision and interassay precision was 6.1 and
`9.4%, respectively, at 5] pg/ml. Serum testosterone, DHEAS,
`and androstenedione were measured using a modified LC-
`MS/MS assay developed by Esoterix (Calabasas Hiils, CA) with
`lower limits of sensitivity of 0.05 ng/dl for testosterone, 0.1 ng/dl
`for DHEAS, and 0.1 rig/dl for androstenedione.
`Twenty-seven urinary steroids were measured by gas chro-
`matography/rnass spectrometry in seiected-ion-monitoring
`mode after solid-phase extraction and derivatization as de~
`scribed previously (21, 22). For simplification, the ]7-deoxy-
`21-carbon steroids that represent metabolites of DOC, cor-
`ticosterone, and 18-hydroxycorticosterone are described as
`mineralocorticoid precursor metabolites. Similariy, the 17—hy-
`droxy-21-carbon steroids derived from active glucocorticoids
`are summarized as glucocorticoid metabolites. The sum of the
`urinary glucocorticoid metabolites or-corrol, )3-cortol,
`ix-cor-
`tolone, and B—cortolone is reported as cortols plus cortolones.
`The 19—carbon steroids represent the sum of androgen and an-
`drogen precursor metabolites (Table 1).
`
`Statistics
`
`The median time on treatment, defined as the time from start
`until discontinuation of abiraterone acetate treatment or addition
`
`of dexamethasone, for the intention—to—treat population with cen-
`soring of patients who did not have progressive disease was calcu-
`lated using the Kaplan-Meier method. The significance of the dif-
`ference between pretreatment us. on-treatment urinary steroid
`levels and lcvcis on~treatmcnt vs. after addition of dcxamethasonc
`
`was determined using the sign test (calculated using Stata version
`10.1). This nonparametric method tested the null hypothesis that
`the median of the differences of paired data is 0. Correlations of
`nonparametric data are defined by the Spearman correiation coef-
`ficient (r) calculated using Prism statistical software (version 5;
`GraphPad, San Diego, CA). An r of 1 is a perfect correlation. All P
`values are two sided, and a resuit was considered significant when
`the P value was <0.05.
`
`Results
`
`Sing|e—agent abiraterone acetate is associated
`with a syndrome of secondary rnineralocorticoid
`excess
`
`This cohort was presented previously when five patients
`continued on treatment (13), but now none of these pa-
`tients remain on single-agent abiraterone acetate. Abi-
`
`raterone acetate was discontinued prematurely because of
`rapid disease progression in four patients and for other
`reasons in five patients (two for deranged liver function
`tests, one due to acute-onset idiopathic poiyneuropathy,
`
`another due to worsening asthma and cosinophilia, and
`the fifth for a diagnosis of coiorectal carcinoma requiring
`chemotherapy). Three patients required addition of dam-
`rnethasone to abiraterone acetate due to side effects of
`
`secondary mineralocorticoid excess. The remaining 30 pa-
`tients continued treatment on abiraterone acetate until a
`
`confirmed rise in PSA, at which time dexarnethasone was
`
`added to evaluate reinduction of sensitivity as reported
`
`previously (Suppicmental Fig. 1) (6, 13). The median time
`on treatment calculated using the Kaplan~l\/Ieier method
`was 231 cl (95% confidence interval 2 122-307).
`
`We here present in detail the clinical manifestations and
`management of side effects attributable to mineralocorti—
`coid excess secondary to CYP17A1 inhibition in patients
`receiving abiraterone acetate without exogenous gluco-
`corticoids. Four of 42 patients received abiraterone ace-
`tare with no clinical evidence of mineralocorticoid excess
`
`(and no rise in PSA) for 38, 65, 253, and 392 d. Dexa—
`
`methasone was added before an attempt with eplerenone
`due to intractable migrainous headaches in two patients:
`in one on d 72 in the presence ofgrade 3 hypokalemia (2.8
`mmollliter), grade 2 hypertension, and grade 1 lower limb
`edema and in the other on d 58 in the presence of grade 1
`
`hypokalemia. The other 36 patients deveioped ciinical ev-
`idence of a syndrome of mineralocorticoid excess and
`were treated with cplerenonc that was initiated after a
`median of 28 d (range, 6-387) (Fig. 1A). Thirty-five pa-
`
`tients had hypokaietnia at initiation of eplerenone (Fig.
`1B), whereas one had normal serum potassium, grade 1
`hypertension, and grade 1 lower limb edema (Fig. 1C). in
`
`addition to hypoltalernia, 10 patients had a raised biood
`pressure, five patients had a raised blood pressure and
`grade 1 lower limb edema, and five patients had grade 2
`lower limb edema. One patient had grade 3 lower iimb
`edema, grade 3 pulmonary edema, and a serum potassium
`of 3.4 mmol/iiter (Fig. 1C) that did not resolve with epler—
`enone and required addition of dexamethasone 0.5 mg
`daily to controi his symptoms. Eplerenone up to a maxi-
`mum daily dose of 200 mg (Fig. 1D) resolved the ciinical
`syndrome of mineralocorticoid excess in the other 35
`patients.
`
`Continuous daily abiraterone acetate leads to
`ACTH—driven rriineralocorticoid excess
`
`Forty—two patients were treated for a totai of 10,359 d
`(1479 wk) with single—agent 1000—mg abiratcrone acetate.
`
`We and others have previously used RIA to identify a
`significant increase in serum DOC, corticosterone, and
`
`

`

`510
`
`Attard et an‘.
`
`CYP17A1 Inhibition by Abiraterone
`
`J Clin Endocrinol Metab, February 2012, 97(2):507+516
`
`Percent change in urinary steroid metabolite excretion with continuous daily abiraterone acetate and
`TABLE 1.
`after addition of dexamethasone 0.5 mg once daily
`
`Baseline vs. single-agent
`abiraternne acetate (n = 21)
`
`Abiraterone acetate vs. after
`addition of dexamethasone (n = 7)
`
`% change from
`baseline
`
`% change from single
`agent abiraterone
`
`Range
`
`Median P value
`
`Range
`
`Median
`
`P value
`
`249 to 4400
`
`842
`
`<0.0001
`
`-99 to -77
`
`381 to 6106
`
`1919
`
`<0.0001
`
`-100 to -62
`
`342 to 8769
`-63 to 3535
`662 to 8091
`1 159 to 9658
`349 to 4581
`659 to 14222
`
`3069
`882
`2997
`4488
`$819
`3317
`
`<0.0001
`<0.0001
`<0.0001
`<0.0001
`<0.0001
`<0.0001
`
`+99 to +67
`+100 to +61
`+100 to +62
`-100 to +62
`+99 to -64
`-100 to +84
`
`-93
`
`-91
`
`+93
`-92
`-95
`-94
`-96
`+99
`
`0.0156
`
`0.0156
`
`00156
`0.0156
`0.0156
`0.0156
`0.0156
`0.0156
`
`Urinary 17-deoxy—21-carbon steroids
`Pregnenolone metabolites
`Pregnenediol (5-PD)
`Progesterone metabolites
`Pregnanediol (PD)
`Mineralocorticoid precursor metabolites
`Tetrahydro-DOC (TH-DOC)
`So:-Tetrahydro-DOC (50:-TH-DOC)
`Tetrahydrocorticosterone (THE)
`Tetrahydro—1 1-dehydrocorticosterone (THA)
`5a—Tetra-1 1—dehydrocorticosterone (50:-THA)
`5or-Tetrahydrocorticosterone (Ba-THB)
`Mineralocorticoid metabolites
`
`3cz5B—tetrahydro-aldosterone (THALDO)
`
`-100 to 49
`
`+40
`
`00118
`
`+92 to 267
`
`-21
`
`>0.9999
`
`Urinary 17-hydroxy-21—carbon steroids
`‘!7—Hydroxypregneno|one metabolites
`5-Pregnenetriol (5-PT)
`17—hydroxyprogesterone (1 7OHP)
`metabolites
`Pregnanetriol {P11
`17-OH-Pregnanolone (17HP)
`30:50:-17-OH-pregnanolone(30:50:-17HP)
`Pregnanetriolone
`1 1-Deoxycortisol metabolites
`Tetrahyciro—11-deoxycortisol (THS}
`Glucocorticoid metabolites
`Tetrahydrocortisoi ("?HF)
`Soc-Tetrahydrocortisol (5ar~THF)
`Cortisol
`Cortols and cortolones
`Tetrahydrocortisone (THE)
`
`Urinary 19—car'oon steroids
`Metabolites of androgens and androgen
`precursors
`DHEA
`Androsterone (An)
`Etiocholanolone (ETIO)
`
`-87 to 330
`
`-15
`
`0.1892
`
`-99 to -68
`
`-95
`
`0.0156
`
`+85 to 215
`+75 to 314
`-60 to 589
`-75 to 800
`
`+36
`9
`100
`29
`
`0.3833
`>0.9999
`0.0002
`0.1892
`
`+98 to -53
`+99 to 0
`-100 to -50
`-100 to -33
`
`+93 to 71
`
`- 73
`
`<0.0001
`
`-100 to - 59
`
`-97 to 2
`-96 to 27
`+100 to 138
`-89 to -30
`-96 to 16
`
`-86
`-85
`+44
`-68
`-80
`
`<0.0001
`<0.0001
`0.0015
`<0.0001
`<0.0001
`
`-99 to -84
`-100 to -87
`-100 to -100
`-100 to -80
`-99 to -83
`
`-82
`+81
`-99
`M80
`
`--90
`
`-94
`-97
`-100
`-94
`-95
`
`0.0156
`0.0313
`0.0156
`0.0156
`
`0.0156
`
`0.0156
`0.0156
`0.0313
`0.0156
`0.0156
`
`-99 to -35
`-98 to -39
`-100 to 179
`
`-97
`-94
`-65
`
`<0.0001
`<0.0001
`00.0118
`
`-100 to 633
`-94 to 100
`-7510 90
`
`-43
`-39
`25
`
`>0.9999
`>0.9999
`0.6875
`
`A significance in the difference between metabolites is denoted by a Pvalue < 0.05 calculated using the sign test.
`
`11-deoxycortisol levels after treatment with abiraterone
`acetate (6, 15). This increase was associated with a sig-
`nificant rise in ACTH (median, 660% increase; range,
`283-1416”/o increase; P value < 0.0001, sign test) from a
`median of 17 pg/ml (range, <9+50 pgfml) before treat-
`
`ment to 124 pg/ml (range, 46-370 pg/ml) on treatment
`(n = 26). To further study the effect of abiraterone acetate,
`we have used gas chromatography/mass spectrometry to
`comprehensively study urinary steroid metabolites in 24-h
`urine collections before treatment and after one or two
`
`cycles (28-56 d) of single—agent abiraterone acetate.
`Twenty-one patients consented to these analyses and pro-
`vided 24-h urine samples. All urinary mineralocorticoid
`
`precursor metabolites (upstream of CYP17A1) increased
`markedly on treatment; however, the metabolite of alde-
`
`sterone, 3a,5B-tetrahydroaldosterone (THALDO) de-
`clined (Fig. 2A). The median of the sum of urinary min-
`eralocorticoid metabolites excluding THALDO rose 26-
`
`fold after one or two cycles of treatment from 847 p.g/24
`h (range, 388-1503 p.g/24 h) to 22,752 pg/24 h (range,
`7729-75535 p.g/24 h). Changes in individual metabolites
`and their significance calculated using the sign test are
`reported in Table 1. In contrast, and explaining the rise in
`ACTH, all metabolites of active glucocorticoids declined
`significantly (Table 1) with a 5-fold decrease in the median
`
`of the sum of metabolites from 9086 pg/24 h (range,
`
`

`

`J Ciin Endocrinol Metab, February 2012, 97(2):SO7~S15
`
`;cern.endojourna|5.org
`
`511
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`
`2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5
`
`Numberofpatients
`
`12
`10
`
`0NA0')O0
`
`50
`
`100
`
`150
`
`200
`
`Days (L092)
`
`Serum potassium (mrnollL)
`
`Eplemmme dose (mg)
`
`
`
`
`
`C
`
`“vP°*a'*-W
`Raised biood pressure
`
`Grade ‘H2 tower limb edema
`
`Grade 3 fluid retention
`
`B 91011 1213I41516¥?‘EB19202122232425262?2E293-03132333435163738
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`
`
`HG. 1. Syndrome of mineralocorticoid excess in patients (n : 38) treated with singEe—agent abiraterone acetate. A, Scatter piot showing number
`of patients and days after starting singie-agent abiraterone acetate that eplerenone was commenced (n : 36; vertical red line marks median value
`of 28 d) (|og2 scale); 3, scatter piot showing number of patients and serum potassium level at initiation of epierenone (n : 36); C, clinical
`manifestation of rnineralocorticoid excess at start of eplerenone (n = 36) or dexamethasone in 2 2). Each column represents a patient, and
`shaded boxes signify presence of toxicity. Patient numbers are random, Patients 37 and 38 (red box) represent the two patients administered
`dexamethasone before a trial of eplerenone, and patient 14 (red arrow) represents the one patient who required dexarnethasone despite a trial
`with epierenone. D, Number of patients and dose of epierenone that resolved the clinicai syndrome of mineralocorticoid excess (n 2 35).
`
`4645-17633 pg/24 h) before treatment to 2154 pg/24 h
`(range, 426-7416 ,u.g/24 ii) after one or two cycles of abi-
`
`three on-treatment samples from 32 patients were avail-
`able for analysis. Serum testosterone declined to a median
`
`raterone treatment (Fig. 213). A one—way ANOVA showed
`no evidence of a difference in mean urinary excretion of min~
`eralocorticoid precursor metabolites and the dose of cpler—
`enone required to controi toxicity [F(_5,15, == 027; P =
`0.9214]. We also evaluated urinary steroid metabolites
`from nine patients who Continued singie-agent abi-
`raterone acetate for at least five or six cycles (140-—l 68 d)
`
`and did not observe a significant difference in urinary me-
`tabolites compared with aftcr one or two cycles (Fig. 2).
`
`Abiraterone significantiy suppresses urinary
`
`androgen metaboiites and serum androgens
`The median of the sum of urinary 19~carbon steroids,
`zle. androgen metabolites, in castrate men before starting
`
`abiraterone acetate was 735 pg/24 E1 (range, 127-6755
`pig/24 h). After 28 ~56 d treatment with single—agent abi-
`raterone acetate, urinary androgen metabolites were all
`significantly suppressed (Fig. 2C and Table 1) with a 20-
`
`foid decrease to 3'7 p.g/24 h (range, 6-896 ug/24 it). There
`was no significant further change from after one or two
`cycles to after five or six cycles of single—agent abiratcronc
`acetate (:1 m 9) (Fig. 2C).
`We then measured circulating androgen levels using an
`ultrasensitivc LC—MS/MS assay {Esoterix) in samples col-
`lected before and on treatment and at progression. Ninety-
`
`of 0.26 ng/dl with a range of <0.05—0.90 ng/dl (decline to
`<0.05 ng/dl in five of 32 patients). Serum DHEAS do
`clined to a median of 0.2. ng/dl with a range of <0.1—9.4
`ng/dl (<0.1 ng/dl in 14 of 32 patients). Serum androstene—
`dione declined to a median of 0.32 ng/dl with a range of
`<0.1—1.58 ng/dl (<0.1 ng/di in three of19 patients). Im-
`portantly, there was no rise in serum testosterone or
`
`DHEAS in the 1 1 patients evaluated at disease progression
`on abiraterone acetate using these ultrasensitive assays, in
`contrast to previously reported changes at progression on
`lcetoconazole (23).
`
`CYP17A1 inhibition with abiraterone is associated
`with increased substrates of the backdoor
`
`pathway of DHT synthesis
`
`Urinary metaboiites of 1”7—hydroxypregncnolone and
`17—hydroxyprogesterone (1701-ii’) did not change signif-
`icantly with abiratcrone acetate, but there was a signifi-
`
`cant increase in the 1'/OHP metabolite 30:5 a-17OH—preg—
`nanolone (3a5a:—17PIP) from a median of 4 pg/24 h
`(range, 1—~13.6 pg/24 h) to 8 pg/24 h (range, 1.7—36.1
`pig/24 h) on abirateronc acetate (rnedian intra-patient
`
`change = 100%, P value for significance of rise 3 0.0002,
`sign test) (Table 1 and Fig. 2B). Interestingly, 3a5a—l 7i-IP
`ievels on abiraterone acetate showed a significant corre-
`
`

`

`512
`
`Attard et al.
`
`CYP1'/‘A1 inhibition by Abiraterone
`
`J Clin Endocrinol Metab, February 2012, 97(2):507w~516
`
`_\ C
`
`Androsterone
`
`10
`
`hours)
`(Log101.19124
`
`
`1
`
`10
`
`100
`
`30501-17HP (Log1O ;.igI24 hours)
`FIG. 3. Scatter plot showing correlation between urinary An and
`urinary 3aSoz-17HP (n 2 30).
`
`lation with androsterone (An), the major metabolite of 5a~
`reduced androgens including DHT (Spearrnan r 2 0.73;
`95% confidence interval 2: 0.49-0.87; P < 0.0001) (Fig. 3).
`In contrast, tetrahydro-1Ldeoxycortisol (THS), the main
`
`metabolite of 11—deoxycortisol, declined significantly in all
`patients from a median of 117 pg/24 h (range, 62-238 pg/24
`h) before treatment to 29 ).ig!24 h (range, 6-158 rig/24 h)
`(median intra-patient change = -73%; P value for signifi-
`
`cance of decline < 0.0001, sign test) (Table 1).
`
`Addition of exogenous glucocorticoids to
`abiraterone acetate reverses mineralocorticoid
`excess and is associated with increased
`
`suppression of glucocorticoid precursor
`metabolites
`
`Addition of oral dexarnethasone (0.5 mg/d) to single-
`agent abiraterone acetate resulted in a decrease (within
`<14 cl) in plasma ACTH to below the lower limit of sen-
`
`sitivity (10 pg/ml) in all six patients studied, consistent
`with previous reports of resolution of side effects of min-
`eralocorticoid excess on addition of exogenous glucocor-
`
`ticoids (6, 13-15). Twenty-four hour urine samples for
`evaluation of steroid metabolite excretion after addition
`of dexamethasone to abiraterone acetate were available
`
`from nine patients. The median of the sum of urinary min-
`
`eralocorticoid precursor metabolites declined to 555
`peg/24 h (range, 135-4343 ,u.g/24 h) after addition of dexa—
`methasone, which was lower than pretreatment levels.
`However, the sum of urinary mineralocorticoid precursor
`metabolites remained higher than before treatment in
`three of nine patients (Fig. 2A). All endogenous gluc0cor-
`ticoid precursor metabolites were significantly suppressed
`after addition of dexamerhasone including 3a5cx—17I-IP
`that declined to a median of 1 pug/24 h (range, 0-3.4 pg/2.4
`h) (Fig. 2B and Table 1}.
`
`The median of the sum of androgen metabolites after
`addition of dexamethasone (nine patients) was slightly
`
`mm
`
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`Metabolitos of 17-dooxy.21-carbon steroids
`
`..C
`
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`
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`
`Metahoiies of 17~hydroxy,21-carbon steroids
`
`case-an and prior 8: starter
`abiratarona seams
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`i abirabrunu anemia
`abirmamno acetate
`; Amr1681o196daysnt
`
`Aim 24 to 43 says ofabiraheroria
`canals and daxamethasona
`
`
`
`Metabolites 0119-carbon steroids
`
`FIG. 2. Changes in urinary mineralocorticoid metabolites (aldosterone
`metabolite separated from mineralocorticoid precursor steroids) (A),
`glucocorticoids (active glucocorticoid metabolites separated from
`glucocorticoid precursors) (B), and androgen metabolites (C) in castrate
`men with advanced prostate cancer pretreatment on treatment with
`abiraterone acetate (during cycle 2 or 3 and cycle 6 or 7) and after
`addition of dexamethasone. Mean and 5th and 95th percentile values
`are shown. Abbreviations for urinary steroids are given in Table 1 and
`Ref. 22. A significant difference between levels before treatment vs. on
`abiraterone acetate and between on abiraterone acetate vs. after
`addition of dexamethasone for each metaboiite as reported in Table 3
`is denoted by asterisks: *, Pvalue 0.05-0.01; **, Pvalue 0.009—0.001;
`***, Pvalue < 0.001.
`
`

`

`J Clin Endocrinoi Metab, February 2012, 9”/(2):507—S16
`
`jcem.endojourna|s.org
`
`513
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`
`III Pretreatment Q On abiraterone acetate
`
`Afteradding dexamethasone
`
`FIG. 4. Decline in serum testosterone (rig/dl) on treatment with abiraterone acetate and after addition of dexamethasone due to PSA progression.
`Testosterone concentration is presented as a log10 scale and is interrupted at I ng/dl. The lower limit oi sensitivity of the assay is 0.05 ngldl. Pt,
`Patient.
`
`lower than on abiraterone acetate alone (25 pg/24-h;
`range, 5—~62 ug/24 h), but the changes in individual me-
`tabolites were not significant (Table 1). Serum testoster-
`one and DHEAS were also measured in eight patients tak-
`ing combination abiraterone acetate and dexamethasone.
`Serum testosterone was lower in all patients on addition of
`dexamethasone compared with on single-agent abi-
`raterone acetate: on addition of dexamethasone, serum
`
`testosterone declined to below 0.05 ng/dl in four of eight
`patients (Fig. 4). Serum DHEAS was below 0.1 ng/dl in five
`of eight patients on abiraterone acetate alone, and in the
`other three patients, a decline was observed on addition of
`dexamethasone.
`
`Discussion
`
`In this report, we present a planned analysis of prospec-
`tively collected samples on the clinical and biochemical
`consequences of CYP17A1 inhibition. To comprehen-
`sively evaluate changes in mineralocorticoid, glucocorti-
`
`coid, and androgen production in men with CRPC treated
`with abiraterone acetate, we used mass spectrometry-
`based measurements of urinary steroids and serum andro-
`gens. We confirmed a marked rise in urinary metabolites
`
`rnineralocorticoid activity (Figs. 2A and 5A). These ste-
`roids also have weak giucocorticoid properties, and due to
`their high levels, abiraterone acetate can be safely admin-
`
`istered as a single agent with no clinical manifestations of
`
`glucocorticoid deficiency (6, 24). Urinary aldosterone me-
`
`tabolite excretion was unchanged, which is in keeping
`
`with our previous finding of unchanged serum aldoste—
`
`tone levels employing a RIA (6); this could be explained by
`
`suppression of the renin—angiotensin—aldosterone system
`and consequently down—regulated aidosterone synthase
`expression due to the abiraterone-induced accumulation
`
`of steroids with rnineralocorticoid properties. Contrary to
`
`previous reports by us and others of raised serum 1 1-de-
`
`oxycortisol (6, 15), we here report 4-fold suppression of
`
`THS, the urinary metabolite of 11-deoxycortisol. It is
`
`likely that previously used RIA gave falsely high readings
`
`in the presence of highly up—regulated levels of steroid
`
`precursors such as DOC due to cross—reactivity issues.
`Predictably, all urinary 21-carbon steroids, mineralo-
`
`corticoids and glucocorticoids alike, were suppressed on
`
`addition of oral dexarnethasone 0.5 mgld to abiraterone
`acetate due to loss of ACTH drive (Fig. 5B). However, as
`
`shown in Fig. 2A, mineralocorticoid levels in some pa-
`tients on abiraterone actetate and dexamethasone were
`
`of steroids upstream of CYl’17A1 that are abie to exert
`
`higher than before treatment. This observation could ex-
`
`

`

`514
`
`Attard et ai.
`
`CYP1 7A1 inhibition by Abiraterone
`
`J Ciin Endocrinoi Metab, February 2012, 97(2):SO7—51 5
`
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`
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`
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