`
`Endocrine Research
`
`Clinical 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, Eiizabeth Folkerd,
`Mitch Dowsett, Wiebke Arlt,* and Johann S. de Bono*
`
`The Institute of Cancer Research (GA, A.H.M.R., A.M.C.. E.F., M.D., J.S.d.B.), Sutton, Surrey SM2 SNG,
`United Kingdom; The Royal Marsden Nationai Health Service Foundation Trust (G.A., A.H.M.R., E.T.,
`N.B.O., E.F., M.D., J,S.d.B.), Sutton, Surrey SM2 5PT. United Kingdom; Diviszon of Metabolism,
`Endocrinology, and Diabetes (R.J.A.), Department of Internal Medlcme, University of Michigan, Ann
`Arbor, Michigan 48109; and Centre for Endocrinology, Diabetes, and Metabolism (B.A.H., W.A.), School
`of Clinical and Experimentai Medicine, University of Birmingham, Birmingham 315 T22, United Kingdom
`
`Context: Abiraterone acetate is a small~molecule cytochrome P450 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 vivo steroidogenesis.
`
`Design and Methods: We treated 42 castrate, castration-resistant prostate cancer patients with
`continuous, daily abiraterone acetate and prospectively collected blood and urine before and
`during abiraterone treatment and after addition of dexamethasone 0.5 mg daily.
`
`Results: Treatment with single-agent abiraterone acetate was associated with accumulation of
`steroids with mineralocorticoid properties upstream of CYP17A1. This resulted in side effects,
`including hypertension, hypokalemia, 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—hydroxypregnenolone and 17—hy-
`droxyprogesterone downstream of 17a-hydroxylase remained unchanged. However, 3a5ar-17-
`hydroxypreg nanolone, which can be converted via the backdoor pathway toward Sci-dihydrotes‘
`tosterone, increased significantly and correlated with levals of the major Sa-dihydrotestosterone
`metabolite androsterone. In contrast, urinary metabolites of 11-deoxycortisol and active giuco-
`corticoids declined significantly. Addition of dexamethasone to abiraterone acetate significantly
`suppressed ACTH and endogenous steroids, including BaSa—t7-hydroxypregnanolone.
`
`
`
`Conclusion: CYP17A1 inhibition with abiraterone acetate is characterized by significant supprEs-
`sion ofandrogen and cortisol synthesis. The latter is associated with a rise in ACTH thatcauses raised
`mineralocorticoids, leading to side effects and incomplete 17a—hydroxylase inhibition. Concomi-
`tant inhibition of 17,20-lyase results in diversion of 17‘hydroxyprogesterone 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)
`ISSN UnlinE 1945-7197
`' WA and J S d B are gomt senior authors.
`Abbreviations: An, Anorostemne: AR, androgen receptor; C RFC. castration-resistant pros-
`tate cancer; CYPlMi, cytochrome P450 17M, DHEA, dehydroepianorosterone; DHEAS,
`dehydroepiandrosterone sulfate; DOC,
`ll-deoxycorucosterone; BrzScr‘lTHF‘, 3.25a-
`170H-pregnanoione. LC—MSIMS,
`liquid chromatography/tandem mass spectrometry;
`I7OHP. U-hydrnxyprngeslerone; FSA, prostate-specific antigen; THALDO, Basil-tetra—
`hydroaldosierone, THS, :etrahydro-‘l l-deoxycortisoi,
`
`ESSN Print 0021-372X
`Printed in U.S A.
`Copyright Ct 2012 by The Endocrine Society
`doi: 10.12101102011-2189 Received July 3D. 2011. Accepted October 31. 2011.
`First Published Online December 14, 2011
`
`J Clin Endocrinol Metab. February 2032, 97(2):507-516
`
`ycemendojournaisorg
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`CYP17A1 Inhibition by Abiraterone
`
`J Clin Endocrinol Metab, February 2012. 97(2) 507—51 6
`
`restate 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
`17A] (CYPl 7A1) 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—
`SlOn 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 prosrate (5—7) where it might contribute to intracrine
`hormone synthesis.
`Abiraterone is a rationally designed, small-molecule in-
`hibitor of CYP17A1 (8, 9). The specificity of abiraterone
`for inhibition of 17,20-lyase US. l7a-hydroxylase is low
`(IA—fold, 1C50 = 2.9 are compared with 4 nM) (10), and
`treatment with abiraterone acetate would therefore be ex~
`
`pected to cause a rise in ACTH with a consequent increase
`in 11-deoxycorticosterone (DOC) and corticosterone,
`mimicking the effects observed in patients with congenital
`adrenal hyperplasia due to inactivating CYPI7A1 muta-
`tions (3). To date, it has proven difficult to develop a small-
`molecule 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 III! 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
`ng/cll and ‘15 pig/d1, respectively), and DHEA declined
`3-fold; however, because cross-re activity with abiraterone
`
`was observed in this assay, the detection of DHEA in pa—
`tients on abiraterone acetate could be artifactual (6, 15).
`
`A liquid chromatography/tandem mass spectrometry (LC-
`MS/MS) assay was used to measure serum testosterone
`levels that declined to below the lower limit of sensitivity
`
`(1 ngldl) 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 CYi’17A1 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 Matsden Hospital (RMH), London, UK
`(CCU-001, www.clinicaltrials.gov identifier NCT00473512). All
`patients were castrate (serum testosterone < 50 ngfdl) 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
`rcvicwcommittees ofthe RMl-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—d 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, cloning, 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 (SOwZDO mgld), and treatment with gluco-
`corticoids to suppress ACTH was only used if mineralocorticoid
`antagonism did not reverse these toxicities. Spironoiactone was
`not used because it is an agonist for wild-type AR (18). Abi-
`ratcrone 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-
`methasone 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 (2.0), or clinical
`progression, withdrawal of consent; or death (6, 13).
`
`
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`item endojournalserg
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`509
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`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 of dexamethasone to abiraterone acetate, weekly for the first
`cycle and monthly thereafter. Patients were also given the option
`to consent to and provide a 24-h urine sample for steroid me-
`tabolite analyses before treatment, aftera minimum of 28 d con~
`tinuous dosing (d 1, cycle 2 or 3) and on d 1, cycle 6 or 7 (Sup—
`plemental Fig. 1, published on The Endocrine Society’s journals
`Online web site at http://jcemendoiournals.org).
`
`Measurement of ACTH and steroids
`
`Plasma ACTH was measured by the RMH Academic Bio-
`chemistry Laboratories with a SDlid~phase two-site sequential
`chemiluminescent immunometric assay (LKACI) using an IM-
`MULITE 1000 autoanalyzer (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] pglml. Serum testosterone, DHEAS,
`and androstenedione were measured using a modified LC-
`MS/MS assay developed by Esoterix (Calabasas Hills, CA) with
`lower limits of sensitivity of 0.05 ng/dl for testosterone, 0.1 ng/dl
`for DI-lEAS, and 0.1 ngldl for androstenedione.
`Twenty~seven urinary steroids were measured by gas chro—
`matographylmass spectrometry in seiecred-ion—monitoring
`mode after solid-phase extraction and derivatization as de
`scribed previously (21, 22). For simplification, the 17-deoxy—
`21—carbon steroids that represent metabolites of DOC, cor—
`ticosterone, and lS-hydroxycorticostetone are described as
`mineralocorticoid precursor metabolites. Similarly, the 17-hy—
`droxy~21-carbon steroids derived from active glucocorticoids
`are summarized as glucoconicoicl metabolites. The sum of the
`urinary glucocorticoid metabolites ot-cortol, B—cortol, a-cor-
`tolone, and Bvcortolone 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 leveis cit-treatment us. after addition of dcxamethasone
`was determined using the sign test (calculated using Stats 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 correlation 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 result was considered significant when
`the P value was <0.05.
`
`Results
`
`Single-agent abiraterone acetate is associated
`with a syndrome of secondary mineralocorticoid
`excess
`
`Forty—two patients were treated for a total of 10,35 9 d
`(1479 wk) with single-agent 1000—mg abiraterone acetate.
`
`This cohort was presented previously when five patients
`continued on treatment (13), but now none of these pa~
`ticnts remain on single-agent abiraterone acetate. Abi—
`ratcrone 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 clue to worsening asthma and eosinophilia, and
`the fifth for a diagnosis of colorectal carcinoma requiring
`chemotherapy). Three patients required addition of dexa-
`methasone 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 dexamethasone was
`
`added to evaluate reinduction of sensitivity as reported
`previously (Supplemental Fig. 1) (6, 13). The median time
`on treatment calculated using the Kaplan-Meier method
`was 231 d (95% confidence interval = 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-
`tate 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 of grade 3 hypoitalemia (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 developed clinical ev-
`idence of a syndrome of mineraiocorticoid excess and
`were treated with cpicrenone that was initiated after a
`median of 28 01 (range, 6—387) (Fig. 1A). Thirty-five pa~
`tients had hypokalemia 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 hypokalemia, 10 patients had a raised blood
`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 limb
`edema, grade 3 pulmonary edema, and a serum potassium
`of 3.4 mmollliter (Fig. 1C) that did not resolve with epler-
`enone and required addition of dexamethasone 0.5 mg
`daily to control his symptoms. Eplerenone up to a maxi—
`mum daily dose of 200 mg (Fig. 1D) resolved the clinical
`syndrome of mineralocorticoid excess in the other 35
`patients.
`
`significant increase in serum DOC, corticosterone, and
`
`Continuous daily abiraterone acetate leads to
`ACTH-driven mineraiocorticoid excess
`
`We and others have previously used RIA to identify a
`
`MYLAN PHARMS. INC. EXHIBIT 1086 PAGE 3
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`Attard etal.
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`CYP17A1 Inhibition by Abiraterone
`
`J Clin Endocrinol Metab, February 2012, 97(2):SO7~516
`
`
`
`TABLE 1. Percent change in urinary steroid metabolite exc
`after addition of dexamethasone 0.5 mg once daily
`
`retion with continuous daily abiraterone acetate and
`
`w B
`
`aseline vs. single-agent
`abiraterone
`acetate (11 = 21)
`
`Abiraterone acetate vs. after
`addition of dexamethasone (n = 7)
`
`% change from single
`% change from
`baseline
`agent abiraterone
`
`Range
`Median P value
`Range
`Median
`P value
`
`Urinary 17—deoxy-21-carbon steroids
`Pregnenolone metabolites
`Pregnenediol (5—PD)
`Progesterone metabolites
`Pregnanediol (PD)
`Mineralocorticoid precursor metabolites
`Tetrahydro-DOC (THOOC)
`er-Tetrahydro-DOC (5m-TH~DOC)
`Tetrabydrocorticosterone (THE)
`Tetrahydro~1 1-dehydrocorticosterone (THA)
`Sa-Tetra-l 1-dehydrocorticosterone (So-THA)
`5a—Tetrahydrocorticosterone (SwTHB)
`Mineralocorticoid metabolites
`3uSB-tetrahydro-aidosterone (THALDO)
`
`Urinary 17-hydroxy-21-carbon steroids
`17—Hydroxypregnenolone metabolites
`S-Pregnenetriol (S-PT)
`17-hydroxyprogesterone (17OHP)
`metabolites
`Pregnanetriol (PT)
`17-OH-Pregnanoione(17l—iP)
`3a5a—17~OH—pregnanolone (3a5a—1 7HP)
`Pregnanetriolone
`1 1-Deoxycortisol metabolites
`Tetrahydro—i 1-deoxycortisol (THS)
`Glucocorticoid metabolites
`Tetrahydrocortisol (THF)
`Sa—Tetrahydrocortisol (SchHF)
`Cortisol
`Cortols and cortolones
`Tetrahydrocortisone (THE)
`
`249 to 4400
`
`842
`
`<00001
`
`~99 to ~77
`
`381 to 6106
`
`1939
`
`<00001
`
`~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
`1819
`3317
`
`<0.0001
`<0.0001
`<00001
`<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
`
`0.0155
`0.0156
`0.0156
`0.0156
`0.0155
`0.0156
`
`~100 to 49
`
`~40
`
`0.01 18
`
`~92 to 267
`
`~21
`
`>0.9999
`
`~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
`
`~99 to ~84
`<0.0001
`— 100 to ~87
`(0.0001
`0.0015 ~10010 ~100
`<0.0001
`~100 to ~80
`<0.0001
`~99 to ~83
`
`~82
`~81
`~99
`~80
`
`~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
`
`Urinary 19-carbon steroxds
`Metabolites of androgens and androgen
`precursors
`>0.9999
`~43
`~100 to 633
`<0.0001
`~97
`~99 to ~35
`DHEA
`>0.9999
`~39
`~94 to 100
`<0.0001
`~94
`~98 to ~39
`Androsterone (An)
`
`Eti0cholanolone (ETlO) 0.6875 ~100 to 179 ~65 00.01 18 ~75 to 90 25
`
`
`
`
`
`
`A significance in the difference between metabolites is denoted by a Pvalue < 0.05 calculated using the sign test.
`
`11-de0xycortisol levels after treatment with abiraterone
`acetate (6, 15). This increase was associated with a sig-
`nificant rise in ACTH (median, 660% increase; range,
`283441696 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—5 6 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 ng24
`h (range, 388-1503 pg/24 h) to 22,752 )1.ng4 h (range,
`7729—75535 pug/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 “3/24 h (range,
`
`
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`MYLAN PHARMS. INC. EXHIBIT 1086 PAGE 4
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`J Clin Endocrinol Metab, February 2012, 97(2);507—S16
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`icemendojournalsorg
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`511
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`
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`
`2.5 2.6 2,7 2.5 2.9 3.0 3.1 3.2 3.3 3.4 3.5
`
`Days(LogZ)
`
`Serum potassium (mmolIL)
`
`so
`
`100
`
`150
`
`200
`
`Eplerenone dose (mg)
`
`
`
`C
`
`Hypoltalemia
`
`fiaisad blood pressure
`
`Grade 1r: towerlimb edema
`
`Grade 3 fluid retention
`
`i2 3
`
`4 56 T
`
`1
`fl91011t213M1516lTlBlEN212223242526272829303132333‘3535
`
`
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`
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`
`
`
`HG. 1. Syndrome of mineralocorticoid excess in patients (n = 38) treated with Single-agent abiraterone acetate A, Scatter plot showing number
`of patients and days after starting single-agent abiraterone acetate that eplerenone was commenced in = 36; vertical red line marks median value
`of 28 d) (IogZ scale); 8‘ scatter plot snowing number of patients and serum potassium level at initiation of eplerenone (n = 36); C, clinical
`manifestation of mineralocorticoid excess at start of eplerenone (n = 36) or dexamethasone (n = 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 dexamethasone despite a trial
`with eplerenone. D, Number of patients and dose of eplerenone that resolved the clinical syndrome of mineralocorticoid excess (n = 35).
`
`4645—17633 rig/24 h) before treatment to 2154 rig/24 h
`(range, 426—741 6 pig/24 h} after one or two cycles of abi-
`raterone treatment (Fig. 2B). A one-way ANOVA showed
`no evidence of a difference in mean urinary excretion of min—
`eralocorticoid precursor metabolites and the dose of epler—
`enone required to control toxicity [Ffi‘m : 0.27; P a
`0.9214}. We also evaluated urinary steroid metabolites
`From nine patients who continued single—agent abi-
`raterone acetate for at least five or six cycles (140—168 d)
`and did not observe a significant difference in urinary me-
`tabolites compared with after one or two cycles (Fig. 2).
`
`Abiraterone significantly suppresses urinary
`androgen metabolites and serum androgens
`The median of the sum of urinary 19—carbon steroids,
`Le. androgen metabolites, in castrate men before starting
`abiratcronc acetate was 735 pg124 h (range, 127-6755
`rig/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-
`lold decrease to 37 tag/24 h (range, 6—896 irag/24 b). There
`was no significant further change from after one or two
`cycles to after five or six cycles of single—agent abirateronc
`acetate (n = 9) (Fig. 2C).
`We then measured circulating androgen levels using an
`ultrasensitive LC-MS/MS assay (Esoterix) in samples col-
`lected before and on treatment and at progression. Ninety-
`
`rhree (in-treatment samples from 32 patients were avail-
`able for analysis. Scrum testosterone declined to a median
`of 0.26 ng/dl with a range of <0.05—0.9O ng/dl (decline to
`<O.US ng/dl
`in five of 32 patients). Scrum DHEAS dc—
`clined to a median of 0.2 rig/d1 with a range of <0.l—9.4
`ngfdl (<01 rig/d1 in 14 of 32 patients). Serum androstene-
`dione declined to a median of 0.32 rig/d1 with a range of
`<0.1—1.58 ng/dl (<O.'.l ng/dl in three of 19 patients). lm-
`portantly, there was no rise in serum testosterone or
`DHEAS in the 11 patients evaluated at disease progression
`on abiratcrone acetate using these ultrasensitive assays, in
`contrast to previously reported changes at progression on
`kctoconazole (23).
`
`CYP17A1 inhibition with abiraterone is associated
`with increased substrates of the backdoor
`
`pathway of DHT synthesis
`Urinary metabolites of 17—hydroxypregnenolone and
`l7-hydroxyprogestcrone (170HP) did not change signif—
`icantly with abiraterone acetate, but there was a signifi-
`cant increase in the 17OHP metabolite 3a5ct-17OH‘preg—
`nanolonc (3a5a-17HP) from a median of 4 tag/24 h
`(range, 1—13.6 [1.3124 11) to 8 pg/24 h (range, 1.7—36.1
`rig/24 h] on abiraterone acetate (median intra-paticnt
`change 2 100%, P value for significance of rise 7» 0.0002,
`sign test) (Table 1 and Fig. 23). Interestingly, 3a5a-17HP
`levels on abiratcrone acetate showed a significant corre—
`
`
`
`MYLAN PHARMS. INC. EXHIBIT 1086 PAGE 5
`
`
`
`512
`
`Attard et ai.
`
`CYP17A1 inhibition by Abiraterone
`
`J Clin Endocrinoi Metal). February 2012, 97(2):507—516
`
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`305u-17HP (Log10 tigl'zat hours)
`FIG. 3. Scatter plot showing correlation between urinary An and
`urinary 3a5rx—17HP (n 2 30).
`
`iation with androsterone (An), the major metabolite of 5&-
`reduced androgens including DHT (Spearman r z 0.73;
`95% confidence interval == 0.49—0.87; P < 0.0001) (Fig. 3).
`In contrast, tetrahydro-l1—deoxycortisol (Tl-IS), the main
`metabolite of 11-deoxycortisol, declined significantly in all
`patients from a median of 117 1.1.3124 h (range, 62—238 rig/24
`h) before treatment to 29 rig/24 h (range, 6-158 pig/24 h}
`(median intro-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 dexamethasonc (0.5 mgld) 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 ofurinary min-
`eralocorticoid precursor metabolites declined to 555
`pgl24 h (range, 135—4343 tag/24 h} after addition of desca-
`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 glucocor-
`ticoid precursor metabolites were significantly suppressed
`after addition of dexamethasone including 3o5a-1 "fl-1P
`that declined to a median of 1 ngl24 11 (range, 0—3.4 rag/24
`h) (Fig. 2B and Table 1).
`The median of the sum of androgen metabolites after
`addition of dexamcthasone (nine patients) was slightly
`
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`FIG. 2. Changes in urinary mineralocorticoid metabolites (aldosterone
`metabolite separated from mineralocorticoid precursor steroids) (A),
`giucocol‘ticoids (active giucocorticoid 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 alter
`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 v5. on
`abiraterone acetate and between on abiraterone acetate vs. after
`addition of dexamethasone for each metabolite as reported in Table l
`is denoted by asterisks: *. Pvalue 0.05—0.01: **, Pvaiue 0.0094001:
`h“, P vaiue < 0.001.
`
`MYLAN PHARMS. INC. EXHIBIT 1086 PAGE 6
`
`
`
`J Clin Endocrinol Metab, February 2012, 97(2):507~516
`
`jcemendojournalsorg
`
`513
`
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`
`III Pretreatment a 0n abiraterone acetate
`
`After adding dexamethasona
`
`FIG. 4. Decline in serum testosterone (ng/dl) on treatment with abiraterone acetate and after addition of dexamethasone due to PSA progression
`Testosterone concentration 15 presented as a log10 scale and is interrupted at I ngfdl. The lower limit of sensitivity of the assay is 0.05 ngr‘dl. Pt,
`Patient.
`
`lower than on abiratcrone acetate alone (25 pg/24—h;
`range, 5-62 ugl24 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 testosrerone 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 rig/d] 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 CYPI7A1 inhibition. To comprehen—
`sively evaluate changes in mineraloeorticoid, glucocorti—
`cold, 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
`of steroids upstream of CYP17A1 that are able to exert
`
`mincralocorticoid activity (Figs. 2A and 5A). These ste-
`roids also havr: weak glucocorticoid properties, and due to
`their high levels, abiratcrone acetate can be safely admin»
`istered as a single agent with no clinical manifestations of
`glucocorticoid deficiency (6, 2.4}. Urinary aldosterone me-
`tabolite excretion was unchanged, which is in keeping
`with our previous finding of unchanged serum aldostc-
`rone levels employinga RIA (6); this could be explained by
`suppression of the renin~angiotensin—aldostcrone system
`and consequently down-regulated aldosterone synthase
`expression due to the abiraterone-induced accumulation
`
`of steroids with mineralocorticoid properties. Contrary to
`previous reports by us and others of raised serum 11~de—
`
`oxycortisol (6, 15), we here report 4-fold suppression of
`“THE, the urinary metabolite of ll-deoxycornsol. 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 lecarbon steroids, mineralo~
`
`Corticoids and glucocorticoids alike, were suppressed on
`addition of oral dexamethasone 0.5 mgld to abiraterone
`acetate due to loss of ACTH drive (Fig. SB). However, as
`shown in Fig. 2A, mineralocorticoid levels in some pa—
`tients on abiraterone actetate and dexamethasone were
`
`higher than before treatment. This observation could ex-
`
`MYLAN PHARMS. INC. EXHIBIT 1086 PAGE 7
`
`
`
`514
`
`Attard et at.
`
`CYP1?A1 Inhibition by Abiraterone
`
`J Clin Endocrinoi Metal), February 2012, 97(2):507—51 6
`
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