`
`.3
`
`'-
`
`Contents lists available at
`
`Biochemical and Biophysical Research Communications
`
`journal homepage:
`
`
`
`Specificity of anti—prostate cancer CYPl7Al inhibitors on androgen
`biosynthesis
`
`- CrossMark
`
`Sameer S. Udhane '
`
`, Bernhard Dick
`
`, Qingzhong Hu '
`
`, Rolf W. Hartmann '
`
`,
`
`'
`Amit V. Pandey '
`a Pediatric Endocrinology, Diabetalogy and Metabolism, Department of Pediatrics, University Children's Hospital Bern, 3010 Bern, Switzerland
`'3 Department of Clinical Research, University of Bern, 3010 Bern, Switzerland
`° Department of Nephrology. Hypertension and Clinical Phannacology, University Hospital of Bern, Bem, Switzerland
`‘l Pltarmaceuncal and Medicinal Chemistry. Sanrland University. Campus C23, Saarbriicken. Germany
`9 Helmholtz institute for Pliannaceutical Research Saarland (HIPS). Campus E 8.l, E6123 Soarbriicken, Germany
`
`ARTICLEINFO
`
`ABSTRACT
`
`Article history:
`Received 26 June 2016
`Accepted 4 July 2016
`Available online 6 July 2016
`
`§:,3;,":_:_’R'3:S"
`Ormmnei
`Abimmmm
`Cvpzmz
`pcog
`Premature adrenatche
`Castration resistant prostate cancer
`
`The orteronel. abiraterone and galeterone. which were developed to treat castration resistant prostate
`cancer. inhibit 17,20 lyase activity but little is known about their effects on adrenal androgen biosyn-
`thesis. We studied the effect of several inhibitors and found that orteronel was selective towards 17.20
`lyase activity than abiraterone and galeterone. Gene expression analysis showed that galeterone altered
`the expression of HSD3B2 but orteronel did not change the expression of HSDSBZ. CYPl7Al and Al(Rl C3.
`The CYPIEIAI activity was not inhibited except by compound [V which lowered activity by 23%. Sur-
`prisingly abiraterone caused complete blockade of O(P2lA2 activity. Analysis of steroid metabolome by
`gas chromatography — mass spectrometry revealed changes in steroid levels caused by different in-
`hibitors. We can conclude that orteronel is a highly specific inhibitor of 17.20 lyase activity. The discovery
`of these specific drug actions on steroidogenic enzyme activities would be valuable for understanding
`the regulation of androgens.
`
`to 2016 Elsevier Inc. All rights reserved.
`
`1. Introduction
`
`Androgens are essential for sexual differentiation and repro-
`duction in both male and female. Androgen production in humans
`occurs mainly in the testis, ovaries and the adrenal glands. The
`initial steps of steroidogenesis in both the adrenal and the gonads
`are same and use similar enzymes for steroid biosynthesis path-
`ways
`. Both the adrenal and the ovary produce dehydroepian-
`drosterone (DHEA) as
`the key precursor of androgens and
`estrogens. Premature adrenatche and polycystic ovary syndrome
`(PCOS) are common hyperandtogenic disorders. During adrenatche
`(functional activation of adrenal zona reticularis) production of
`adrenal androgens (D1-IEA and DHEA sulfate) is at a higher level
`
`* Corresponding author. Pediatric Endocrinology, Diabetology & Metabolism.
`l(|I<L C837, University Children's Hospital Bern. Freiburgstrasse 15. Cl-l-3010 Bern.
`Switzerland.
`(A.V. Pandey).
`E—mail address:
`' Current address: Department of Chemistry. University of Cambridge, Lensfield
`Road, Cambridge. CB2 IEW‘ U.K.
`
`0006-291 Xl© 2015 Elseviet Inc. All rights reserved.
`
`In case of premature adrenatche. the concentration of circulating
`adrenal androgens increases at an earlier age. Several studies link
`premature adrenatche with post pubertal adrenal and ovarian
`hyperandrogenism
`. Premature adrenatche is also linked to the
`hyperandrogenism in PCOS
`_. in hyperandrogenic women with
`PCOS, androgen production from both the ovaries and the adrenals
`is higher
`Changes
`in cytochrome P450 l7Al {P450cl7.
`CYPl7Al) activities could explain the increase in androgen secre-
`tion that causes hyperandrogenic disorders
`.
`The CYP17Al plays a vital role in regulating adrenal androgen
`production (
`). CYPl7Al
`is localized in the endoplasmic
`reticulum and responsible for 17:1-hydroxylase and 17,20 lyase re-
`actions. The 17,20 lyase activity regulates androgen production and
`is dependent on cytochrome P450 oxidoreductase (POR)
`, cyto-
`chrome b5 (CYB5)
`and phosphorylation
`-
`. Treatment of
`androgen dependent prostate cancer is by androgen deprivation
`therapy. androgen receptor (AR) antagonists or anti androgen
`agents and CYPl7Al inhibitors that block androgen production
`Hormonal
`therapy such as using a gonadotrophin hormonal
`analogue for treatment of hyperandrogenic disorders may not be
`
`
`
`

`

`1006
`
`5,5. Urlhane et ai. / Biocheinitai and Biophysical Research Communications 477 (2016) 1005 -1010
`
`the extra-gonadal and the gonadal
`specific enough to control
`androgen production. Drugs targeting androgen production are
`being used for treatment of prostate cancer and to decrease the
`androgen levels in hyperandrogenic women
`.
`-
`. But anti
`androgen treatment can have side effects on sexuality of women
`with hyperandrogenism
`. One example is flutamide. a non-
`steroidal anti androgen drug used to treat hyperandrogenic
`women 1
`§. Flutamide has many side effects like diarrhea,
`gynecomastia, muscle cramps, hematological alterations, and
`hepatotoxicity
`.6
`the treatment of the
`CYP17A1 inhibitors are designed for
`androgen dependent prostate cancer patients ( 2
`). Abir-
`aterone prolonged the survival of castration-resistant prostate
`cancer (CRPC) patients in phase III clinical trials
`. Abiraterone
`also has modest affinity towards the androgen receptor
`. In the
`hyperandrogenic disorders like premature or exaggerated adre-
`narche and PCOS, use of total CYP17A1 inhibitors will also block the
`1’/‘oc—hydroxylase activity. New inhibitors of CYP1”/‘A1. orteronel and
`galeterone were also designed for treatment of castration-resistant
`prostate cancer '
`. Effect of abiraterone, orteronel and gale-
`terone on adrenal androgen production is not clear.
`Here we are reporting the effects of CYP17A1 inhibitors on the
`adrenal androgen production. We compared CYP17A1 inhibitors
`orteronel, galeterone. abiraterone and recently reported com-
`pounds I and IV and studied the specificity of their action. We found
`that orteronel is a potent inhibitor of 17,20 lyase activity with no
`impact on other cytochrome P450 enzymes involved in steroid
`hormone biosynthesis. We also analyzed the impact of these in-
`hibitors on the steroid rnetabolome, focusing on C19 steroids. Use
`of specific 17,20 lyase inhibitors can be a treatment option for
`hyperandrogenic disorders for the control of androgen production.
`
`2. Materials and methods
`
`2.1. Cell culture and treatment
`
`Human adrenocortical NCl—H295R cells obtained from American
`Type Culture Collection (ATCC; CRL—2128) were cultivated in
`DMEM,'Ham’s F-12 medium with additives
`as described in
`.
`.
`2
`.
`. Orteronel, galeterone and abiraterone
`were purchased from the Selleckchem (Houston, TX, USA).
`CYP17A1 inhibitors compound I and IV were described earlier
`
`2.2. Steroid profiling
`
`Steroid profiling was performed with the cells grown on 6 well
`plates by adding 100,000 cpm [3H] pregnenolone (preg) to the
`culture medium for 90 min. To study specific CYPT/A1 enzyme
`activities. cells were first treated with 1 1.1M or 2 |.lM CYP17A1 in-
`hibitors. After 24 h of inhibitor treatment, cells were treated with
`1 l.llVl trilostane (a specific blocker of HSD3B) for 90 min before
`adding [3H] preg. For CYP21 A2 activity, cells were treated with
`1 uM of different inhibitors for 24 h and then labeled with [3H] 170:-
`hydroxyprogesterone (17—OH Prog) for 60 min to monitor the
`conversion of 11 -deoxycortisol. Steroids were extracted from cell
`supernatants and separated by thin layer chromatography (TLC) as
`previously described =
`1. Steroid conversion was calculated as
`percentage of
`total
`radioactivity incorporated into specific
`products.
`
`2.4. CYP19/ll enzyme activity using titrated water release assay
`
`Human Placental JEG3 cells were grown on 12 well plates and
`treated with CYPl7A1 inhibitors for 24 h before aromatase activity
`was assayed. Aromatase activity was assayed with 3H labeled an-
`drostenedione ([1B-3H(N)]-androstene-3,17-dione: —100.000 cpmi
`well). Androstenedione was added to the treated cells for 6 h.
`Aromatase activity was then assessed by the titrated water release
`assay as previously described 1
`
`2.5. GC—MS measurement of steroid metabolites
`
`The measurement of steroid hormone metabolites in human
`adrenal H295R cells was performed by gas chromatography — mass
`spectrometry (CC-MS) as previously described
`. The cells were
`grown in 10 cm plates in normal growth medium for 24 h, then
`medium was replaced, and cells were treated with 1 uM ofCYP17A1
`inhibitors in medium without NU-I serum for 24 h. After 12 h of
`
`treatment, 1 pM of pregnenolone was added. At the end, superna-
`tant was collected and concentrated samples were used for steroid
`analysis by GC—MS. All measurements were performed in the
`steroid laboratory of the Department of Nephrology. Hypertension
`and Clinical Pharmacology at the University Hospital of Bern,
`Switzerland.
`
`3. Results and discussion
`
`3.1. CYP17/11 inhibitors modulate the adrenal androgen production
`
`CYP1'/‘A1 is required for androgen production in humans and
`control of its enzyme activity is essential for treatment of hyper-
`androgenic disorders. Abiraterone, galeterone and orteronel are
`inhibitors of CYP17A1 (
`). Abiraterone is an effective
`active site-directed inhibitor, designed for the treatment of CRPC
`patients to suppress the androgen production by inhibition of
`CYP17A1 activities -
`-
`.
`. The galeterone and orteronel were
`designed to selectively inhibit 17.20 lyase activity of CYP17A1 for
`the treatment of CRPC
`'. There have been suggestions that
`galeterone is more specific towards the 17.20 lyase activity of
`CYP17A1 than abiraterone but this effect is not clear -
`. Func-
`
`in viva (cynomolgus
`tional studies on the effects of orteronel
`monkey) and in vitro (human adrenal tumor cells) showed that
`orteronel is a potent inhibitor of 17.20 lyase activity =
`=
`.In addition
`to these well-known inhibitors, Hartmann group synthesized
`several
`inhibitors of steroid biosynthesis
`. They observed
`compound IV was a more potent inhibitor of 17,20 lyase activity
`compared to its effects on 1'/‘oi-hydroxylase activity
`. However,
`the effect of these drugs on adrenal androgen production remains
`unknown.
`
`We used the steroidogenic human adrenal cortex H295R cells to
`study the effect of CYP1‘/‘A1
`inhibitors (orteronel, galeterone,
`compound l and IV and abiraterone). We found that orteronel and
`galeterone drastically decreased the DHEA and androstenedione
`production (
`A). Compounds 1 and IV decreased DHEA.
`androstendione and 11-DOC,i11-deoxycortisol production.
`in the
`cells treated with abiraterone. we only observed the conversion of
`preg to prog and other products in the pathway were completely
`blocked (
`-B). This suggests that abiraterone inhibits CYP17A1
`and possibly other enzymes that are involved in steroidogenesis.
`
`2.3. Quantitative real time PCR (qRT-PCR)
`
`The H295R cells were treated with inhibitors for 72 h. After the
`
`3.2. Orteronel is a specific inhibitor of the CYP17/11 17,20 lyase
`activity
`
`treatment, total RNA was isolated and qRT-PCR was done as pre-
`viously described
`. Details are in
`‘
`
`We studied the effect of CYP17A1 inhibitors in more detail for
`the specificity of the inhibition of CYP17A1 enzyme activities. Cells
`
`

`

`5.5. Udhone at of. / Bfoeheuriicol and Biophysical Research Communications 477 (2016) lI‘J05—l'I‘Jl‘0
`
`1007
`
`Prog
`
`65'
`
`'3
`
`*
`
`50
`45
`
`E 40
`3
`“ -E
`‘D
`:3
`§ 25
`U
`-'3
`20
`
`15
`10
`
`5U
`
`32
`
`n
`
`_
`1.
`
`_
`
`5
`
`M .
`D53?
`.2:
`17—OH Prog
`17-OH Preg
`
`'
`
`1 1 oeoxycortisol Q
`
`Orleronel
`Galelerone
`
`—
`_
`
`9'
`Q
`
`I
`
`a
`
`+
`_
`
`'
`
`"'
`
`El ouso
`I 1pM Compound I
`I 1pM Compound [V
`D tulvi Ablraterone
`
`,
`
`_
`
`. .
`
`"
`
`..
`
`.
`
`__
`
`.,
`
`.
`
`,_ k
`
`.
`
`-
`
`50
`3.
`E 4
`E
`E 3
`"ti
`§
`'5 29
`33
`_. G
`
`fl
`
`i
`“ '
`Q
`*5
`
`Frog
`M I
`preg
`DHEA
`‘17—C>H Prog
`17‘-OH Preg
`
`'
`
`
`
`__
`
`11-deoxycortisol
`
`5'
`
`u
`
`o
`
`no
`
`I
`
`[3 DMSO
`
`I 1 “M Orleronel
`n 1 an Galeterone
`
`
`
`Prod
`
`DA
`
`Preg
`
`DHEA
`
`17-OH
`P799
`
`17-OH 11-deoxycortisol
`preg
`
`°°'“"°""° '
`Compound IV
`Abiralerone
`
`“
`-
`«
`
`+
`—
`-
`
`’
`+
`A
`
`‘
`—
`"'
`
`Pros
`
`DA
`
`Preg
`
`DHEA
`
`17-on
`Pm
`9
`
`1?-on
`Pm
`9
`
`11—ueoxycomso|
`
`0
`
`_
`
`.
`
`.
`
`Fig. 1. Effect of the CYP‘l7Al inhibitors on adrenal steroid profile. For the steroid profiling H29SR cells were treated with 1 pm orteronel, galeterone. compound I and [V and
`abiraterone for 24 h. Steroid production was labeled with [3H] preg for 90 min. Steroids were extracted and resolved by TLC, A) Steroid profiling of adrenal HZEJSR cells treated with
`DMSO and 1 uM of orteronel or galeterone B) Steroid profiling of adrenal l-{29SR cells treated with 1 pm of compound I, compound IV and abiraterone. Representative TLCs are
`depicted on the left. summaries and quantifications of the results are given on the right. Prog. progesterone; A-1A, androstenedione; Preg, pregnenolone; DHEA. dehytlroepian—
`drosterone; 17-OH Preg. ‘l7m-hydroxypregnenolone: 17—0H Prog_ 17o.—hydroxy progesterone: Data are presented as mean ¢ SD of three independent experiments. *p < 0.05,
`“p < 0.01.
`
`*
`
`:3 DMSO
`I1 pM Orteronel
`I1 uM Galeierone
`
`0
`
`,
`
`L: DMSO
`-1pM Orteronel
`-1uM Galeterone
`
`H3933?
`
`CYP17A1
`
`AKR1C3
`
`..
`“LOH preg
`
`160
`.5140
`V’
`E120
`100
`E 80
`DDMSO
`E
`#1 pM Compound I
`I 1 uM Compound IV E 50-
`-'
`_
`1 urn Abiraterone
`E 40
`20
`0
`
`X0
`
`E
`‘
`L ,
`DHEA
`
`r-
`
`4-1»
`
`preg
`
`\
`
`k \
`
`§
`
`it
`
`*
`
`Preg
`
`DHEA
`
`*
`
`" “ ..
`17-OH preg
`
`
`
`A
`
`Preg
`DHEA
`
`“'0” 9'99
`Oneronel
`Galeterone
`
`B
`
`we
`
`3
`_
`_
`
`100
`g.
`gt ‘ ‘E80
`3 an
`E
`"" 0
`'g4
`:20
`
`_
`+
`
`0
`
`‘
`+
`_
`
`100
`
`€30
`._
`*5 S60
`"D
`E 40
`§
`‘ES
`as 20
`0
`
`-
`_
`+
`
`I
`-
`Dlargg
`17-OH preg i
`Compound I
`_
`Compound IV —
`Abiraterone -
`
`i
`
`-
`+
`~
`
`+
`—
`—
`
`Fig. 2. Effects of I'.'YP17A1 inhibitors on NC!-H295 cells. A and B . Effect of inhibitors on EYPWA1 enzyme activity. The enzyme activities of CYP17A1 were checked in HZQSR
`cells treated with control [DMSO) and 1 plvl oforteronel. galeterone, compound I, compound IV and abiraterone. CYP17Al activities were assessed by the conversion of [3H] preg to
`17-OH preg for l7:t—hydroxylase activity and 17-OH preg to DHEA for 17.20 lyase activity. Representative TLCs are depicted on the left, summaries and quantifications of the results
`are given on the right. Data are given as mean 3 SD of three independent experiments. Preg. pregnenolone; DHEA, dehydroepiandrosterone: ll’-OH Preg.170:-hydroxypregnenolone.
`‘p < 0.05,‘*p < 0.01. C. Impact of CYPITM inhibitors on genes involved in androgen hiosynthesis. I-l295R cells were treated with control (DMSO) or 1 pM ortetonel and
`galeterone for 72 h and then RNA was isolated and transcribed to CDNA, Results of qRTvPCR validation of l-lSD3B2, CYPl7Al and AKRIC3 genes relative to the housekeeping gene
`cyclophilin A. Expression of the genes was analyzed by SYBR Green real—time PCR. Analysis of relative gene expression was determined by the TM” method. *p < 0.05.
`
`

`

`1008
`
`5.5. Udhone er al. /Biochemical and Biophysical Research Communications 477 {20l‘6) 10054010
`
`A
`
`14
`120
`
`72
`E. 10
`8
`E 80
`‘E
`OJ
`E 60
`‘L
`40
`20
`
`0
`
`Aromatase activity
`
`..
`
`Control
`
`Orteronel
`
`.
`Galeterone Abiraterone Compoundlcornpound IV
`
`21 -Hydroxylasa activity
`
`3'
`
`an
`
`3"
`
`“
`
`4!
`
`1'.’vOH Prcig
`
`‘
`
`.
`
`‘
`
`120
`
`3
`
`. 11-deoxycortisol
`
`100~
`
`were treated with inhibitors and then with 1 uM trilostane (a
`specific blocker of HSD3B) for 90 min before labeling with [3H]
`preg. We found that the orteronel decreased only DHEA production,
`suggesting selectivity of inhibition towards the 17.20 lyase activity
`of CYPl7Al (
`A). Treatments of galeterone. abiraterone, com-
`pound I and IV showed a decrease in the conversion from preg to
`17-OH preg and to DHEA which suggests these compounds
`inhibited both the hydroxylase as well as lyase activity of CYPi7A1
`(
`A—B). These data indicate that orteronel is a specific inhibitor
`of 17,20 lyase activity, as observed in previous studies i
`
`3.3. Effect of CYPI7/‘ll inhibitors on genes involved in androgen
`biosynthesis
`
`Abiraterone and galeterone are steroidal structures that also act
`as androgen receptor (AR) antagonist. Orteronel is a non-steroidal
`CYPl7Al
`inhibitor. Abiraterone has been reported to alter the
`androgen regulated gene expression in prostate cancer cells
`through the AR—mediated signaling
`. Since. both the orteronel
`and galeterone have shown potential as potent CYPl7Al inhibitors.
`we conducted further experiments to find out if these inhibitors
`have any impact on gene transcription. The H295R cells were
`treated with control (DMSO) or 1 uM orteronel and galeterone for
`72 h. Gene expressions were studied by relative quantification PCR
`(qRT-PCR). We studied the expression of three key genes CYPi7A1,
`HSD3B2 and Al<Rl C3 that are involved in androgen biosynthesis.
`Interestingly, we found a slight increase in HSD3B2 gene expression
`upon treatment with galeterone (
`C). In presence of orteronel
`no significant changes in gene expression were observed (
`C).
`
`£117-OH Frog
`
`I11-deoxycortisol
`
`up
`
`32 20
`
`U B
`
`E" an
`‘D
`so
`
`409
`‘E
`
`S E
`
`3.4.
`
`influence of Cl/Pl7Al inhibitors on aromatose activity
`
`Control Orteronel Galeterond Abi‘
`ralerone
`
`rcompounld Cornpolund
`I
`N
`
`To further study the specificity of the CYPl'/‘Al inhibitors, we
`determined their impact on other related steroidogenic enzyme
`activities. First, we studied the effect of inhibitors on aromatase
`(CYPIQA1) activity. The activity and expression of aromatase differs
`between different cell types depending on the need of cells for
`estrogens. Aromatase is localized in endoplasmic reticulum and
`catalyzes the last step of estrogen biosynthesis from androgens
`{converts androstenedione to estrone and testosterone to estra-
`diol). The orteronel. galeterone. compound I and abiraterone
`showed no effect of on aromatase activity (
`A). Compound IV
`treatment resulted in a decrease of aromatase activity by 23%
`{
`A), which was in accordance with its reported modest inhi-
`bition of aromatase in vitro (lC5g r 228 nM)
`
`3.5. Abiraterone severely inhibits 2I—hydroxylose (CYPZIAZ)
`activity
`
`We then studied the effect of these inhibitors on the 21-
`hydroxylase (CYP2lA2) enzyme activity. The 21-hydroxylase plays
`a role in producing glucocorticoids (cortisol) and mineralocorticoid
`(aldosterone). Cortisol helps maintain blood sugar levels. protects
`the body from stress and suppresses inflammation. Maintaining the
`cortisol level is required for the gonadal function. as disruption in
`cortisol production may affect
`fertility by the regulation of
`hypothalamic—pituitary—adrenal (HPA) and gonadal (HPG] axis ;
`CYPZIA2 activities were assessed by checking the conversion of
`[3H] 17—OH Prog to l1—deoxycortisol. Orteronel. galeterone, com-
`pound l and 1V treatment did not change ll-deoxycortisol pro-
`duction. suggesting no effect on CYP2lA2 activity. Surprisingly.
`abiraterone was found to completely inhibit the CYP2‘I A2 activity
`(
`B). This suggests that abiraterone is not a specific inhibitor of
`CYPl7Al and can also inhibit CYP2lA2 activity. Protein sequence
`comparison of CYPl7Al and CYPZIA2 shows an identity of around
`
`Fig. 3. Influence of CYPITAI inhibitors on other steroid metabolizing cytochrome
`P450 activity. The effect of inhibitors on P450 arornatase (CYPIQAI) and 21-
`hydroxylase (CYP2lA2). A) Effect of inhibitors on aromatase. Effect of inhibitors
`was studied on arornatase activity in human placental JEC.-3 cells. Cells were treated
`with control (DMSO) and 1 uM of orteronel, galeterone, abiraterone, compound I and
`IV for 24 h before aromatase activity was tested. Aromatase activity was assayed with
`androstenedione using 2 uM cold substrate and 31-! labeled androstenedione [[113-
`3H(N)]—androstene—3,l7—dione;
`-100,000 cpmfwell) as
`radioactive tracer. Andro-
`stenedione was added to the treated cells for 6 h. Aromatase activity was measured by
`observing the conversion of 3H—androstenedione to estrone using the titrated water
`release assay. Data are presented as mean 3 SEM of two independent experiments. B)
`Effect on EYPZIM activity. The 21-hydroxylase activity was checked in HZQSR cells
`treated with control (DMSO) and 1 uM of orteronel, gaieterone, abiraterone, compound
`I and compound [V for 24 h. CYP2lA2 activities were measured by observing the
`conversion of[3H] 17‘—OH prog to ll-deoxycortisol. Representative TLCs are depicted in
`the upper panel, summaries and quantilications of the results are given in the lower
`panel, Data are presented as mean 3; SD of three independent experiments. “[3 < (101.
`
`28% but structures of CYPl7A1 and CYP21A2 have many similarities
`and may retain similar binding sites. The inhibition of CYP2‘lA2
`could be a serious problem since using this drug inhibits whole
`adrenal androgen biosynthesis. Use of abiraterone for androgen
`lowering effects. where inhibition of CYP2lA2 is not desired. will
`create additional complications.
`
`impact of CYPl‘7AI inhibitors on the metabolomics of C19
`3.6.
`steroids by GC-MS
`
`inhibitors orteronel, galeterone and abiraterone
`The CYPl7Al
`showed significant decrease in potent androgens like testosterone
`and dihydrotestosterone (
`'-
`). Galeterone lowered all classical
`and backdoor pathway metabolites of androgens in human adrenal
`cells. However, orteronel and abiraterone treatment lead to an in-
`crease of androgen metabolite androsterone, which is involved in
`
`

`

`5.5. Uclhane et al. / Biochemical and Biophysical Research Communications 477 (20:16) 1005 1010
`
`1009
`
`Table 1
`The table shows the GC—MS measurement of secreted steroid metabolites in the human adrenal H295R cells in the untreated vs the treated condition. The change in me-
`tabolites are indicated based on untreated levels which are set as hundred percent compared to CYl"l7Al inhibitor treatments. *nd: Not determined; +: increased metabolite.
`
` Metabolites Untreated Drteroncl Galeterone Abiraterone
`
`
`
`
`Androgen metabolites
`Androsterone
`Etiocholanolonc
`Androstenediol
`llii-Hydroxy-Androsterone
`11B-Hydroxy-Etiocholanolone
`Dehydroepiandrosteronc
`5:1 androstendiol
`16a-l-Iydroxy-DHEA
`Sat androstenetriol
`5—Pregnenetrio1
`Testosterone
`Sm-Dihydrotestosterone
`Estrogen metabolites
`Estriol
`17-0 Estradiol
`Progesterone metabolites
`17'a—0H—Pregnanolone
`Pregnanetriol
`Corticosterone metabolites
`Tetrahydrocorticosterone
`5-2-Tetrahydrocorticosterone
`Cortisol-metabolites
`Cortisone
`Tetrahydrocortisone
`o:—Corto|one
`B-Cortolone
`20¢-Dihydrocortisone
`2(l|3-Dihydrocortisone
`Cortisol
`TH—Cortisol
`Sm-Tetrahydrocortisol
`a—Cortol
`B—Cortol
`200:-Dihydrocortisol
`
`100
`100
`100
`100
`rid
`100
`100
`100
`100
`100
`100
`100
`
`100
`nd
`
`T00
`100
`
`nd
`nd
`
`nd
`100
`100
`nd
`nd
`nd
`100
`100
`100
`nd
`nd
`nd
`
`443
`3915
`328
`104
`-l
`4
`80
`74
`108
`36
`50
`nd
`
`103
`l
`
`4162
`802
`
`{Id
`nd
`
`rid
`90
`873
`+
`nd
`nd
`22
`nd
`nd
`+
`nd
`120
`
`30
`S1
`rid
`94
`nd
`30
`66
`S4
`39
`7'1
`29
`nd
`
`58
`+
`
`S6
`49
`
`4
`nd
`
`nd
`96
`374
`+
`nd
`nd
`22
`83
`83
`4
`-+
`162
`
`1593
`3030
`152
`1834
`I
`10
`I33
`81
`133
`9
`26
`nd
`
`55
`1
`
`4408
`2011
`
`+
`-1-
`
`4
`5372
`45.600
`+
`+
`-1-
`40
`2617
`2617
`+
`+
`161
`
`the backdoor pathway of dihydrotestosterone biosynthesis. One
`possible explanation is that abiraterone can influence backdoor
`pathway by elevating CYP17A1 expression and induce progesterone
`accumulation
`. The progesterone accumulation can compete
`against abiraterone for binding to CYP17A1 leading to induction of
`androgen metabolites involved in backdoor pathway
`. Otter-
`onel seems to be more specific for inhibiting the 17.20 lyase activity
`in the classical androgen biosynthesis pathway and might not in-
`fluence the androgen metabolism via backdoor pathway. Addi-
`tionally, we found that orteronel and abiraterone treatment also
`lead to an increase in other androgen metabolites like etiochola-
`nolone and 11B-hydroxy-etiocholanolone. For the first time the
`data here show the effect of CYP17A1 inhibitors on the human
`
`steroid metabolome especially on androgen metabolites, in adrenal
`H295R cells. These data indicate that orteronel, galeterone and
`abiraterone can influence or alter different steroid metabolites in
`human adrenal cells apart from blocking the CYP17A1 activity.
`Based on our results. we can conclude that orteronel is a more
`specific inhibitor for CYP17A1 17,20 lyase activity compared to other
`inhibitors tested here. Orteronel had no effect on other tested en-
`zymes that are involved in steroid hormone biosynthesis. Hence, it
`can be considered as a treatment option for hyperandrogenic dis-
`orders like polycystic ovary syndrome. Using abiraterone for
`lowering androgen production in hyperanclrogenic disorders may
`create additional undesired complications in patients because of its
`inhibitory effect on CYP21A2 activities. For the first
`time we
`showed the effect of CYP17A1 inhibitors abiraterone, orteronel and
`galeterone on steroid metabolome in human adrenal H295R cells.
`Our data provide evidence for the significantly altered steroids
`
`metabolites by CYP17A1 inhibitors. The steroid metabolome model
`may be used as an efficient diagnostic tool for further studies on
`urine or serum samples of hyperandrogenic patients treated with
`CYP17A1 inhibitors. Our observations that abiraterone is also a very
`potent inhibitor of CYP21A2, suggests that abiraterone should not
`be used to treat hyperandrogenic disorders when inhibition of
`CYP21 A2 is an undesirable side effect. Orteronel may be tried for a
`more specific inhibition of CYP17A1 activities without affecting
`other steroid metabolizing enzymes.
`
`Acknowledgments
`
`This work was supported by grants to AVP from the Swiss Na-
`tional Science Foundation (31003A-134926). We thank Dr. Nasser
`Dhayat from the Department of Nephrology, Hypertension and
`Clinical Pharmacology. University Hospital of Bern. Bern,
`Switzerland for help with the steroid pathway figure.
`
`Appendix A. Supplementary data
`
`Supplementary data related to this article can be found at
`
`Transparency document
`
`Transparency document related to this article can be found
`online at
`
`

`

`1010
`
`References
`
`5.5. Udhcme er al. /Biochemical and Biophysical Research Cammunicatiuns 477 (2016) 1005-1010
`
`[1]
`
`[2]
`
`[3]
`
`[4]
`
`[5]
`
`I7]
`
`[3]
`
`I9]
`
`I10]
`
`[11]
`
`[12]
`
`[13]
`
`[14]
`
`I15]
`
`I15]
`
`I17]
`
`I13]
`
`I19]
`
`[201
`
`[21]
`
`[22]
`
`B. Montgomery. M.A. Eisenberger. M.B. Rettig, F. Chu, R. Pili, j.j_ Stephenson.
`NJ. Vogelzang. A.J.
`l(oletsky_ LT. Nordquist, W.j. Eclenfield, K. Marniouk,
`l(.]. Ferrante. M.E. Taplin, Androgen receptor modulation optimized for
`response (ARMOR) phase ] and II studies: galetercme for the treatment of
`castration-resistant prostate Cancer, Clin. Cancer Res. 22 (2016) 13564363,
`.
`‘
`Publvled
`PMID:
`26527750.
`
`[23]
`
`[24]
`
`125]
`
`'
`
`I25]
`
`I27]
`
`I28]
`
`[29]
`
`[30]
`
`[31]
`
`I32]
`
`I33]
`
`I34]
`
`I35]
`
`[35]
`
`