Discovery and development of galeterone (TOK-001 or VN/124-1) for the treatment of all stages of prostate cancer

Article abstract of DOI:10.1021/jm501239f

In our effort to discover potent and specific inhibitors of 17α-hydroxylase/17,20-lyase (CYP17), the key enzyme which catalyzes the biosynthesis of androgens from progestins, 3β-(hydroxy)-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (Galeterone or TOK-001, formerly called VN/124-1) was identified as a selective development candidate which modulates multiple targets in the androgen receptor (AR) signaling pathway. This drug annotation summarizes the mechanisms of action, scientific rationale, medicinal chemistry, pharmacokinetic properties, and human efficacy data for galeterone, which has successfully completed phase II clinical development in men with castration resistant (advanced) prostate cancer (CRPC). Phase III clinical studies in CRPC patients are scheduled to begin in early 2015.

Full text of DOI:10.1021/jm501239f

Drug Annotation
pubs.acs.org/jmc
Discovery and Development of Galeterone (TOK-001 or VN/124-1)
for the Treatment of All Stages of Prostate Cancer
Vincent C. O. Njar*,†,‡,§ and Angela M. H. Brodie†,§
‡
§
†Department of Pharmacology, Center for Biomolecular Therapeutics, and Marlene Stewart Greenebaum Cancer Center,
University of Maryland School of Medicine, 685 West Baltimore Street, Baltimore, Maryland 21201-1559, United States
ABSTRACT: In our effort to discover potent and specific inhibitors of
17α-hydroxylase/17,20-lyase (CYP17), the key enzyme which catalyzes
the biosynthesis of androgens from progestins, 3β-(hydroxy)-17-(1H-
benzimidazole-1-yl)androsta-5,16-diene (Galeterone or TOK-001, for-
merly called VN/124-1) was identified as a selective development
candidate which modulates multiple targets in the androgen receptor
(AR) signaling pathway. This drug annotation summarizes the
mechanisms of action, scientific rationale, medicinal chemistry,
pharmacokinetic properties, and human efficacy data for galeterone,
which has successfully completed phase II clinical development in men
with castration resistant (advanced) prostate cancer (CRPC). Phase III
clinical studies in CRPC patients are scheduled to begin in early 2015.
States in 2014.5 PC is often diagnosed at a stage when it is
restrained to the prostate gland and its immediate surround-
INTRODUCTION
■
On the basis of the success of aromatase inhibitors (AIs,
ings. At this stage, PC is treated either with prostatectomy
inhibitors of CYP19, the enzyme that catalyzes the conversion
(surgical removal of the prostate gland) or with radiotherapy.
of androgens to estrogens) in the treatment of postmenopausal
women with breast cancer,1 we decided that the same approach
For most men, these procedures are successful in curing the
disease. However, for some men, these procedures are not
curative, and their prostate cancer continues to spread to other
organs of the body. Men whose disease progresses following
surgery or radiation are considered to have advanced prostate
cancer.6
PC growth and progression are stimulated by androgens
acting through the androgen receptor (AR). Androgen levels
are mainly regulated through the hypothalamic−pituitary−
adrenal/gonadal axis (Figure 3). Because androgens, predom-
inantly testosterone (T) and dihydrotestosterone (DHT), are
the primary fuels of advanced prostate cancer growth, the first-
line therapy for advanced prostate cancer typically entails
treatment with a class of drugs known as luteinizing hormone
releasing hormone (LHRH) agonists, which reduce testoster-
one to castrate levels. Another class of marketed hormonal
drugs, known as antiandrogens, block the binding of T and
DHT to the AR.7
Nevertheless, resistance to these therapies usually occurs,
rendering them ineffective over time. Approximately 60% of PC
patients are expected to have tumor recurrence after five years
despite the use of androgen deprivation therapy. This stage of
PC is referred to as castration resistant prostate cancer
(CRPC).7 In CRPC patients, tumor growth occurs even
while circulating androgens remain at castrate levels because
tumor cells develop the capacity for alternative androgen
might be used to target the inhibition of androgen synthesis
involved in prostate cancer development and proliferation. We
were also inspired by the realization that the development of
new aromatase inhibitors might no longer be an attractive
research endeavor because, at the time, four novel aromatase
inhibitors, including 4-hydroxyandrostenedione (formestane),
letrozole, arimidex, and exemestane were already approved for
clinical use.2 This early work resulted in our early publications
in this field of research.3
In this drug annotation, we tell the drug discovery story and
the interesting challenges that were overcome by outlining a
medicinal chemistry case study of galeterone, describing the
disease target(s), mechanism(s) of action, and the scientific
rationale for advancing galeterone to clinical trials in men with
prostate cancer (Figure 1).
STRUCTURE
■
Galeterone, 3β-hydroxy-17-(1H-benzimidazole-1-yl)androsta-
5,16-diene (Figure 2), is a 17-heteroazole steroidal analogue
scheduled to enter phase 3 clinical trial for castration resistant
prostate cancer (CRPC).
DISEASE TARGET
■
Prostate cancer (PC) is the most commonly diagnosed cancer
and the second leading cause of cancer-related death in
American men. It is estimated that approximately 240,000 new
cases of prostate cancer will be diagnosed and that nearly
30,000 prostate cancer-related deaths will occur in the United
Received: August 12, 2014
© XXXX American Chemical Society
A
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Drug Annotation
Figure 1. Important research and commercial/clinical milestones in the discovery and development of galeterone: (UMB = University of Maryland,
Baltimore).
Figure 2. (a) 2-D structure of galeterone. (b) Stereo view of the binding mode of galeterone docked in the active site of human androgen receptor
(hAR).4 Stereo view of the binding mode of galeterone (cap, green) in the active site of hAR. The active site residues in the binding pocket are
shown in stick model with element colors and active site bound water molecule in ball−stick model with element color. Hydrogen bonding
interaction between the hydroxyl group of ligand-Gly711 and Phe764 is shown as white dots, and within the amino acids, the water-amino acid is
also shown as white dashed lines.
biosynthesis. In addition, the cells learn how to thrive in the
presence of very low androgen levels, due in part to AR
overexpression. Since 2010, the US Food and Drug
Administration (FDA) has approved two new antihormonal
agents for the treatment of metastatic CRPC: abiraterone
acetate (2, Zytiga) which is administered with prednisone, and
enzalutamide (3, Xtandi) (Figure 4).8 Each drug disrupts a part
of the AR signaling pathway, the key driver of CRPC. It should
be noted that following oral administration of prodrug, 2, the
release of abiraterone (1) is very rapid.9 Compound 1 inhibits
CYP17 to block androgen production, while 3 competitively
blocks androgen binding to the AR. Compound 3 is a second
generation antiandrogen shown to be more efficacious than
bicalutamide (5, Casodex) (Figure 4), a widely used first
generation antiandrogen.7,10 However, there is a strong need
for drugs with improved efficacy and safety profiles to treat
early stage, hormone sensitive prostate cancer and CRPC. In
this respect, it is worth noting that an analogue of compound 3,
i.e., ARN-509 (6) (Figure 4),11 a potent AR antagonist is
currently undergoing Phase 2 trials in patients with biochemi-
cally relapsed, hormone-sensitive prostate cancer
(NCT01790126). In addition, another potent nonsteroidal
inhibitor of CYP17, TAK-700 (Orteronel) (7) (Figure 4),12
advanced rapidly to Phase 3 trials in patients with metastatic,
castration-resistant prostate cancer (mCRPC).13 Disappoint-
ingly, analysis of the Phase 3 data indicated that 7 plus
B
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Drug Annotation
SCIENTIFIC RATIONALE FOR BRINGING
GALETERONE TO THE CLINIC
■
Because of the modest clinical success of ketoconazole (4)
(Figure 4), an FDA approved antifungal drug and a modest
CYP17 inhibitor),15 we became interested in generating potent
and specific inhibitors of CYP17 with the hope of eliminating
the side effects (attributed to the promiscuous inhibition of
several cytochrome P450 enzymes) of ketoconazole. The
design of our steroidal CYP17 inhibitors was based on the
structures of the natural substrates (progesterone and
pregnenolone) of CYP17 with modifications mainly at the
17,20-side chain (azole heterocycles and other nitrogen
containing moieties) and D-ring; sites at or close to the
positions which may interact with the enzyme’s active site (i.e.,
α-orientations at C-16, C-17, and C-20).3b,16 We envisioned
that these substrate analogues will interact with the steroid
binding site of CYP17 in addition to coordination with the
enzyme’s heme iron resulting in highly specific and tight
binding inhibitors (Figure 5).3b,16,17
Figure 3. Endocrine control of prostatic growth.6 The growth and
development of the normal prostate requires a functioning androgen
signaling pathway, which is regulated by the hypothalamic−pituitary−
adrenal/gonadal axis. Androgens [testosterone (T), androstenedione
(AD), and dehydroepiandrosterone (DHEA)] and other steroids are
synthesized in the testes or adrenal glands and released into the
circulation in response to specific hormonal signals [follicle stimulating
hormone (FSH), gonadotropin releasing hormone (GnRH), luteiniz-
ing hormone (LH), and luteinizing hormone releasing hormone
(LHRH)]. Testosterone is transported by steroid hormone binding
globulin (SHBG) to the prostate, where it is predominantly converted
by 5α-reductase to its more active metabolite, 5α-dihydrotestosterone
(DHT). The adrenals are stimulated to produce AD and DHEA by
adrenocorticotropic hormone, released by the pituitary. Note:
Circulating AD and DHEA are precursors of T which is mostly
converted to DHT. Reprinted with permission from ref 6. Copyright
2011 Elsevier.
Figure 5. Proposed model for the CYP17/inhibitor complex.
prednisone did not meet the primary end point of improved
overall survival (OS) when compared to that of the control
arm.13 Thus, further development of compound 7 has been
terminated.11b,14
Over the course of our studies, several lead CYP17 inhibitors
have been discovered. Critical to this work was the invention of
a novel nucleophilic vinylic “addition−elimination” substitution
reaction of 3β-acetoxy-17-chloro-16-formylandrosta-5,16-diene
Figure 4. Structure of FDA approved antihormonal drugs.
C
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Drug Annotation
(8), to facilitate the synthesis of unique steroidal Δ16-17 azoles
(Figure 6).17b The uniqueness stems from the fact that the
Figure 7. C-20 1,2,4-triazole and azide steroids.
steroidal compounds (17−26) and related analogues contain-
ing a saturated D-ring (27 and 28) (Figure 8).17a
Evaluation of these compounds for the inhibition of human
CYP17 enzyme (Table 1) revealed that whereas the imidazole
and triazole compounds (17, 18 20, and 24−26) were potent
inhibitors of CYP17, the 1H-pyrazole (23) was a modest
inhibitor and that unexpectedly, the 2H-1,2,3-triazole (19) and
the two tetrazole regioisomers (21 and 22) were noninhibitory.
Compounds with the corresponding Δ4-3-one moieties (24−
26) were also potent CYP17 inhibitors. Because these Δ16-17-
azole compounds of Figure 8 are structurally similar, the
striking difference in their CYP17 inhibitory activities may be
due to the differences in their basicity profiles, properties
imposed by the inherent different electronic character of each
of the azole heterocycles. These data also strongly suggested
that the presence of a nitrogen atom at either the 3′ or 4′
position seems important for potent inhibition of the enzyme.
The requirement for a 16,17-double bond also descended from
the observation that compounds 27 and 28 (without Δ16)
exhibited diminished potency compared to that of the
corresponding parent Δ16 compounds, 17 and 18, respectively.
We also determined that bulky groups were not tolerated at the
3β position.17a
In other studies not presented here, we also showed that
compound 18 was a slow-binding inhibitor of CYP17.17a Using
UV−vis difference spectroscopy, we demonstrated that most of
these inhibitors induced a type II difference spectrum,
indicating the coordination of steroidal nitrogen to the heme
iron of the CYP17 enzyme.17
On the basis of CYP17 inhibitory activities, compounds 17,
18, and 20 were selected as lead compounds and were each
subjected to rigorous pharmacological studies in models of
human prostate cancer in vitro and in vivo. In these preclinical
biological studies, we clearly established that the lead
compounds, especially, 18 and 20, possessed several anticancer
properties that target the AR. These include (i) CYP17
inhibition to block androgen (T and DHT) synthesis in vivo,20
(ii) direct competitive binding to the ligand binding domain
(LBD) of AR and concomitant AR antagonism,17a,21 and (iii)
significant growth inhibition of LNCaP human prostate tumor
xenografts.20a,21c In addition, the pharmacokinetics in mice of
these lead compounds was also determined.22
Figure 6. Key nucleophilic vinylic “addition−elimination” substitution
reaction of 3β-acetoxy-17-chloro-16-formylandrosta-5,16-diene (8).
azole moiety is tethered to the steroid scaffold through N1 of
the azole, hitherto unknown C-17 steroidal azoles. As with
most important discoveries, this high-yield and expeditious
reaction was discovered serendipitously,17b where intended
hydrolysis of compound 8 to give the corresponding 3β-
hydroxy-17-chloro-16-formylandrosta-5,16-diene (9), instead
provided 3β-hydroxy-17-methoxy-16-formylandrosta-5,16-
diene (10) in quantitative yield, pointing to the possibility
that the chlorine in compound 8 could be readily displaced by
various nitrogen nucleophiles under appropriate reaction
conditions (Figure 6).
Two other important findings were (i) the discovery that our
lead CYP17 inhibitors were potent antagonists of the AR with
activities that were superior to those of compound 518 and (ii)
that our CYP17 inhibitors were also AR degrading agents
(ARDAs).18b,19 Following many in vivo antitumor efficacy
studies, galeterone was identified as the first example of an
antihormonal agent [an inhibitor of androgen synthesis (CYP17
inhibitor)/antiandrogen/AR degrading agent (ARDA)] that is
significantly more efficacious than castration in the suppression
of androgen-dependent prostate tumor growth.18,19 Because of
its impressive anticancer properties, galeterone was selected as a
clinical candidate.
STRUCTURE−ACTIVITY RELATIONSHIP LEADING
TO GALETERONE: KEY CHALLENGES
■
On the basis of our design strategy described above and
summarized in Figure 5, we set out to synthesize several
steroidal compounds tethered with the 1,2,4-triazole moiety or
azide at steroidal C-20 and in some cases with the introduction
of 17β-hydroxyl or the 16/17 double bond (11−16) (Figure
7). However, the realization that these initial set of compounds
were mediocre CYP17 inhibitors suggested to us the importance
of regio-architecture to the production of potent CYP17
inhibitors. This finding was also unexpected because we had
previously shown that 20-amino and 20,21-aziridinyl pregnene
steroids were potent inhibitors of CYP17.3b
A critical review of our data highlighted the realization that
our best leads were only as potent as castration in suppression
of androgen-dependent prostate tumor growth. This realization
led to the design, synthesis, and evaluations of a new class of
potent CYP17 inhibitors/antiandrogens, with the emergence of
Following our discovery of a general method (vide supra) for
introducing the Δ16-17 azole functionality into ring D (Figure
6),17b we readily synthesized a variety of novel Δ16-17 azole
D
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Drug Annotation
Figure 8. Variety of Δ16-17 azolyl steroids and related compounds (compounds 18−23 have the same steroid scaffold as 17, while compounds 25
and 26 have the same scaffold as 24).
pyrimidine, and pyrazine moieties, the so-called “privileged
sub-structures,”24 in the new molecules. These scaffolds,
especially the benzimidazole scaffold, continue to receive
extensive attention in medicinal chemistry because of their
diverse portfolio of biological activities and because they are
entities of a variety of useful drugs.24
Table 1. Inhibition of Human CYP17 by Azolyl
Compounds
a
b
c
d
e,f
compds
% inhibition
IC50 (nM)
Ki (nM)
17
18
19
20
21
22
23
24
25
26
27
28
60
97
NI
94
NI
NI
40
-
90
8
23.0
1.2
-
-
13
-
1.4
-
CHEMISTRY
■
The synthesis of galeterone is presented in Scheme 1. The key
intermediate in our synthesis of galeterone, 3β-acetoxy-17-
chloro-16-formylandtrosta-5,16-dine (30), was obtained by our
routine procedure as previously described starting from the
commercially available 3β-acetoxyandrost-5-en-17-one (29)
(Scheme 1).17,18 Treatment of 30 with benzimidazole in the
presence of K2CO3 in DMF at approximately 80 °C gave the
desired 3β-acetoxy-17-1H-benzimidazole (31) in near quanti-
tative yield. Compound 31 was smoothly deformylated with
10% palladium on activated charcoal in refluxing benzonitrile to
give compound 32 in 93% yield, from which hydrolysis gave the
required 3β-hydroxy 17-benzimidazole (33) (VN/124-1;
galeterone).
-
-
-
-
55
7
41
1.9
8
-
-
19
219
62
78
-
-
-
-
for comparison
67
38
4
a
b
- = not determined. NI = no inhibition up to 10 μM. Each inhibitor
concentration was 150 nM for the initial screening assays.
c
Concentration of substrate, 17α-hydroxypregnenolone = 10 μM.
d
e
Mean
SDM of at least two experiments. Ki values were
determined as described previously.17a Km for substrate, 17α-
f
It is important to state here that a facile and large scale good
manufacturing practice (GMP) synthesis/production of
galeterone was developed in 2009 by Tokai Pharmaceuticals
Inc. but is yet to appear in the literature. We are also aware that
the commercial synthetic procedure retains the basic tenets of
the original procedure.17,18 The major changes of the GMP
hydroxypregnenolone = 560 nM.
the clinical candidate galeterone (33).18a,23 The stimulus for
preparing this class of C-17 heteroaryl steroids was based on
the desire to incorporate benzimidazole, benzotriazole,
Scheme 1. Synthesis of Galeterone
E
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Drug Annotation
process involve the purification and isolation of the
intermediates and the final product.18a
Following subcutaneous administration (s.c.), the plasma
concentration of galeterone declined exponentially with a mean
half-life of about 44.17 min and an elimination rate constant of
56.5 min−1. Galeterone was cleared at a rate of 1986.14 mL/h/
kg from the systemic circulation and was not detected 6 h after
administration. The calculated noncompartmental pharmaco-
kinetic parameters based on the plasma concentration profile
following subcutaneous administration of galeterone are shown
in Table 2. After s.c. administration of galeterone, the observed
plasma concentration in mice reached peak levels 30.0 min
post-dose. Galeterone is well absorbed from the subcutaneous
site, and the area under the curve for the plasma concentration
versus time profiles after s.c. administration increased
proportionately to dose as the administration dose was changed
from 50 to 100 mg/kg. Furthermore, the elimination half-life,
and mean residence time were relatively constant as the dose of
galeterone was increased from 50 to 100 mg/kg (Table 2).
These results indicate that the pharmacokinetic profile of
galeterone is dose independent.
IN VITRO PHARMACOLOGY
■
Utilizing intact-cells CYP17 expressing E. coli,21a galeterone was
shown to be a potent inhibitor of the enzyme with an IC50
value of 300 nM, being 6-fold less potent than our previously
reported compound 18 (IC50 = 50 nM).23 Under the same
assay conditions, abiraterone had an IC50 value of 800 nM.
However, unlike abiraterone, galeterone was subsequently
found to disrupt androgen signaling through multiple targets.18
The increased efficacy of galeterone in several prostate cancer
models both in vitro and in vivo is believed to arise from its
ability to downregulate the AR as well as competitively block
androgen binding. In competitive binding studies against the
synthetic androgen [3H]R1881, galeterone (EC50 = 845 nM)
was equipotent to bicalutamide (EC50 = 971 nM) in LNCaP
cells but had a slightly higher affinity for the wild-type receptor
in PC3-AR cells (galeterone, EC50 = 405 nM versus casodex
EC50 = 4,300 nM). Transcriptional activation assays utilizing a
luciferase reporter showed galeterone to be a pure AR
antagonist of the wild-type AR and the T877A mutation
found in LNCaP cells.18 In prostate cancer cell lines, galeterone
inhibited the growth of CRPCs (IC50 = 2.9 μM), which had
increased AR expression and were no longer sensitive to
bicalutamide (IC50 = 18 μM).25 In addition, galeterone
demonstrated superior synergy for growth inhibition in
combination with everolimus or gefitinib compared with
bicalutamide.25
Galeterone (0.15 mmol/kg, s.c. twice daily) caused a 93.8%
reduction (P = 0.00065) in the mean final LAPC-4 xenograft
volume compared with that of the controls (Figure 9), and this
IN VIVO PHARMACOLOGY [PHARMACOKINETICS
(PK) ANTI-TUMOR EFFICACY] IN PRECLINICAL
SPECIES
■
The pharmacokinetic properties in male SCID mouse for
galeterone were studied following our previously described
procedure for other CYP17 inhibitors.20a,22 The results are
summarized in Table 2.
Figure 9. Effects of galeterone and compound 18, and castration on
the formation and growth of LAPC4 prostate tumors in male SCID
mice. Three × 107 LAPC-4 cells were injected s.c. into the dorsal flank
of SCID mice. One group of mice was castrated. The other groups of
mice received the vehicle, galeterone (0.15 mmol/kg twice-daily), or
18 (0.15 mmol/kg twice-daily). Daily treatment with galeterone or 18
was initiated 1 day after cell inoculation. Tumors volumes were
measured weekly, and the percentage of change in tumor volume was
determined after 16 weeks of treatment. * indicates significant
difference of galeterone versus the control, castration, and 18 at week
14 (P = 0.00065, 0.05, and 0.0097, respectively). ** indicates
significant difference of galeterone versus castration and 18 at week 16
(P = 0.047 and 0.0047, respectively). ↓−↓↓: period of reduced
administration dose of galeterone. At ↓, the dose was reduced by
78.6%, and after ↓↓, the usual dose was resumed.
Table 2. Pharmacokinetic Parameters for Galeterone (50
and 100 mg/kg) in Mouse after s.c. Administration
a
galeterone
b
parameter
t1/2 (min)
Kel (min−1
50 mg/kg
100 mg/kg
36.6 1.6
44.17 1.15
56.5 0.94
)
68.49 1.26
1813.94 10.94
30.00 0.0
AUC (min·μg/mL)
Tmax (min)
1440.00 60.23
30.00 0.0
Cmax (μg/mL)
MRT (min)
16.82 0.37
65.40 0.60
2098.99 4.11
32.23 0.34
60.46 1.54
3276.39 26.71
Vd (mL/kg)
a
Galeterone was formulated in 100 μL of vehicle (40% β-cyclodextrin
b
in water). Values are expressed as the mean SE, n = 5.
efficacy was significantly more effective than castration or
treatment with compound 18, one of our early lead compounds
with a 6-fold higher CYP17 inhibitory potency than
galeterone.18a At the time of this study, we speculated that
the superior antitumor efficacy of galeterone could in part be
due to its better pharmacokinetics and or pharmacodynamics
properties. Another notable aspect of this study was the
observation, for the first time, of an antihormonal agent (an
inhibitor of androgen synthesis (CYP17 inhibitor)/antiandro-
gen) that was significantly more effective than castration.18a On
On reverse phase HPLC, galeterone [retention time (rt) =
21.6 min] was well resolved from the internal standard (18, rt
=11.5 min), a metabolite (rt =17.3 min), and other endogenous
compounds in mouse plasma. The calibration curves derived
for galeterone were linear and reproducible (data not shown),
the inter- and intra-assay variability was less than 10%, and its
limit of detection was 100 ng/mL. The HPLC assay was
validated and used to monitor galeterone in mice plasma.
F
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the basis of these impressive preclinical data, galeterone and
related compounds were licensed to Tokai Pharmaceuticals
Inc., Cambridge, MA who subsequently conducted advanced
preclinical studies to enable the ongoing clinical development
(vide infra).
that disrupts AR signaling via three distinct mechanisms of
action.
SAFETY PHARMACOLOGY AND
■
PHARMACEUTICAL PROPERTIES
We note that since this initial impressive antitumor study
reported in 2005, three other independent antitumor efficacy
studies of galeterone have been reported by our groups. In the
report by Vasaitis et al.,18b we showed that galeterone (0.13
mmol/kg, s.c., twice daily) was very effective in preventing the
formation of LAPC4 tumors (6.94 versus 2410.28 mm3 in the
control group). Galeterone (0.15 mmol/kg, s.c., twice daily)
plus castration induced regression of LAPC-4 tumor xenografts
by 26.55% and 60.67%, respectively. In the latter study, we
measure AR protein levels within the tumors from control and
treated animals and found that treatment with galeterone
caused marked depletion of AR protein expression. Interest-
ingly, the xenograft data also showed that galeterone was more
efficacious than either casodex or castration. In this report, by
using cyclohexamide to inhibit new protein synthesis and
measuring the rate of AR degradation, we also determined that
galenterone’s down-regulation of the AR level was, in part, due
to increased AR degradation. In another study,26 we also
demonstrated that galeterone plus everolimus acted in concert
to reduce castration-resistant prostate cancer (HP-LNCaP)
tumor growth rates. Finally, in 2011 using an in vivo androgen-
dependent LAPC-4 human prostate cancer xenograft model,19a
we demonstrated for the first time that galeterone was more
efficacious than abiraterone acetate (2) or castration. As
observed previously,18b treatment with galeterone caused
marked depletion of AR protein expression.
Rigorous studies directed by Tokai Pharmaceuticals Inc. were
conducted to establish the pharmaceutical properties and safety
of galeterone. These studies although not available in the
literature were deemed adequate [IND 104,183 for 33 (TOK-
001 or galeterone): Safe to Proceed] and enabled the granting of
Investigation New Drug (IND) status to galeterone by the US
Food and Drug Administration on September 4, 2009.
CLINICAL TRIALS WITH GALETERONE: FIRST IN
HUMAN STUDIES
■
On the basis of the preclinical studies reviewed above and
favorable toxicology and pharmacokinetics, an IND application
for galeterone was approved by the FDA for Phase 1 trials with
an orally administered galeterone, as a powder in capsule
(PIC).
Tokai’s clinical development program for the evaluation of
galeterone in patients with prostate cancer is designated
ARMOR (androgen receptor modulation optimized for
response). The ARMOR1 clinical trial was a 49 patient,
multicenter, open-label, dose-escalation study of galeterone that
utilized PIC formulations for the treatment of chemotherapy
̈
naive nonmetastatic and metastatic CRPC (http://clinicaltrials.
gov/show/NCT00959959). Co-administration of prednisone
was not required, and there were no events of adrenal
mineralocorticoid excess (AME), a clinical syndrome that is
commonly associated with other CYP17 inhibitors, were
observed in the study. Overall, galeterone was well tolerated.
Approximately 90% of treatment emergent adverse events were
of grade 1 or grade 2 in severity and were generally manageable
and reversible. Prostate specific antigen (PSA) reductions were
seen in a majority of patients: 24 (49%) patients had ≥30%
maximum PSA reductions, including 11 patients (22%) with
≥50% maximal PSA reductions. Furthermore, tumor reduc-
tions, another sign of antitumor activity were observed
radiographically. In summary, galeterone was well tolerated,
with all cohorts showing an acceptable safety profile. Indeed,
this was the first proof-of-concept study for galeterone in
patients with CRPC.27 On the basis of these promising Phase 1
data, galeterone received the Fast Track designation from the
FDA for the potential treatment of CRPC.
These preclinical studies enabled us to ascertain the
mechanisms of action of galeterone. Specifically, galeterone
inhibits CYP17, antagonizes AR, and induces degradation of
AR (Figure 10). Indeed, this suggests that this multitarget
approach would reduce the risk of patients developing
resistance to therapy and may translate to an advantage in
progression-free survival and overall survival. The above studies
clearly demonstrate that, galeterone is a first-in-class, multi-
target, oral small molecule for the treatment of prostate cancer
A significant food effect, similar to abiraterone, was found
when galeterone, as a PIC, was dosed in a phase I CRPC trial
and confirmed in a PK study in human volunteers. Following
reformulation of galeterone into a spray dried dispersion
formulation,28 the ARMOR2 clinical trial (∼136 patients,
multicenter study) was initiated as a two-part Phase 2 study to
confirm the recommended Phase 2 dose of reformulated
galeterone (Part 1) and to demonstrate safety and efficacy at
the selected 2550 mg dose for Part 1 in four distinct CRPC
patient cohorts (Part 2) (http://clinicaltrials.gov/show/
NCT01709734). Following the conclusion of Part 1 of the
study, a once-daily galeterone dose of 2550 mg was selected for
Part 2, based on safety, efficacy, and pharmacokinetic data. Data
from this ongoing study, as presented during ASCO 2014
showed that for 51 patients followed for at least 12 weeks
across all doses, 82% achieved a PSA decline of at least 30%
(PSA30), and 75% achieved a PSA decline of at least 50%
Figure 10. Multiple mechanisms of galeterone inhibition along the
androgen axis. Galeterone can inhibit androgen signaling at three
separate points. (1) Inhibition of CYP17 systemically blocks
production of androgens from all tissues. (2) The molecule prevents
binding of androgens to the AR and (3) induces degradation of the
receptor.
G
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Drug Annotation
̈
(PSA50). In metastatic treatment naive CRCP, data from 21
patients showed that 85% achieved a PSA30 and that 77%
achieved a PSA50. Furthermore, initial data in abiraterone
refractory patients showed both biochemical PSA activity and
stable disease after 12 weeks of dosing. Galeterone, at the
recommended dose was safe and well tolerated in 87 patients
with CRPC.29 Approximately 90% of all treatment emergent
adverse events were of grade 1 or 2 in maximum severity and
were generally manageable and reversible. Results from the
ongoing Phase 2 trial will be used to guide the strategy for
pivotal Phase 3 trials planned for initiation in early 2015. In
summary, a total of 107 CRPC patients were dosed at the target
dose of 2550 mg of galeterone. Recruitment for Phase 2 trial
has been closed, and treatment with galeterone therapy is
ongoing for patients still on the study.
change the therapeutic management of CRPC. Galeterone
selectively inhibits CYP17 lyase activity to prevent androgen
synthesis, antagonizes androgen binding to the AR, and causes
degradation of both full-length and splice variant ARs.
Galeterone was discovered in a collaboration between a
medicinal chemist and a pharmacologist/cancer biologist, who
set out some 18 years ago to discover and develop potent
inhibitors of CYP17 inspired by the clinical success of the
aromatase (CYP19) inhibitors in breast cancer patients.1a
Through this collaboration, one of us has over the years
established a competitive and comprehensive laboratory with
expertise not only in medicinal chemistry but also in
oncopharmacology and cancer biology.
The mechanisms of the anticancer effects of galeterone are
not completely understood, but it has been shown to impact
multiple targets that disrupt AR signaling. Its actions selectively
alter the transcription of genes and the function of proteins that
are regulators of proliferation, migration, and the death of
malignant cells. Galeterone was invented by hypothesis-driven
research based on the knowledge that androgens are the key
drivers of prostate cancer cell growth. Its design was also based
on the knowledge of the mechanism of action of CYPs and in
particular CYP17 and with benefit from intuitive medicinal
chemistry knowledge and strategies.3b,16,17,19b Although the
initial approach was to identify small molecules (potential
drugs) with high specificity for inhibition of CYP17, we
followed the scientific data acquired during the course of our
research that enabled the identification of other desirable
antiprostate cancer targets, namely, AR antagonism and AR
degradation. Other potentially clinically relevant targets of
galeterone continue to emerge.33
Galeterone also teaches us another important lesson. The
goal in drug discovery and development is to create a new drug
with an acceptable therapeutic index and favorable medicinal
and drug-like properties. Whereas galeterone has a 6-fold lower
CYP17 inhibitory potency, compared to our most potent
CYP17 inhibitor, 18, the latter compound exhibited only a
modest in vivo antiprostate tumor efficacy in a head-to-head
study with galeterone.18a Thus, although galeterone has a lower
potency for CYP17 inhibition used in the early screens, we
believe that galeterone’s two other mechanisms of action, i.e.,
AR antagonism and AR degradation, led to an overall more
efficacious agent.
Since we first reported that AR degradation contributes to
antitumor efficacy of galeterone,18b several academic and
corporate laboratories have attempted to synthesize small-
molecule androgen receptor degrading agents (ARDAs).
However, it remains to be determined whether a molecule
with specific ARDA activity will be therapeutically more useful
than a multitarget agent, such as galeterone that disrupts AR
signaling, for the treatment of advanced prostate cancer.
FOLLOW-ON PRE-CLINICAL STUDIES WITH
■
GALETERONE
Follow-on preclinical studies can provide important data that
can have profound impact on ongoing clinical drug develop-
ment programs. This strategy represents a “laboratory-to-clinic-
to-laboratory-to-clinic” drug discovery and development pro-
gram. Indeed, this has been the case with some highly
significant follow-on studies with galeterone. These include the
following: (i) On the basis of the findings that patients in the
Phase 1 study with galeterone did not experience events of
AME, a clinical syndrome that is commonly associated with
other CYP17 inhibitors, a comprehensive study was conducted
by Tokai researchers to assess the impact of galeterone and
other clinically used CYP inhibitors on CYP17 (17α-
hydroxylase and 17,20-lyase activities) and global steroido-
genesis.30 Galeterone, unlike the other three inhibitors tested,
was shown to be a selective and potent CYP17 lyase inhibitor
and exhibited minimal evidence of deleterious steroid changes
associated with mineralocorticoid excess (ME).30 These
preclinical data recapitulated Phase 1 clinical experience
where no ME was observed. (ii) To improve drug exposure
and eliminate the significant food effect seen when galeterone
was administered as a PIC, an oral formulation was developed
that had improved oral bioavailability and favorable pharmaco-
kinetics.28 (iii) The unexpected finding that galeterone also
degrades splice variant androgen receptors (AR-3/AR-V7 and
19b
ARv568s
)
which are up-regulated in CRPC and in patients
resistant to abiraterone and enzalutamide31 has impacted the
design of parts of pending Phase 3 clinical trials. (iv) Finally, in
a collaboration between Tokai and researchers at Vancouver
Prostate Centre, Canada, it was recently reported that
galeterone suppressed castration-resistant and enzalutamide-
resistant prostate cancer growth in vitro. Galeterone also
blocked nuclear translocation and decreased AR dependent
genes (PSA, TMPRSS2, and Nkx3.1) expressed in CRPC and
enzalutamide-resistant cell lines.32 Together, these studies
strongly suggest that galeterone may represent the next
generation of antihormonal therapy for patients with multiple
stages of prostate cancer, including CRPC, drug-resistant
prostate cancer, and tumors expressing AR splice variants.
EXPERIMENTAL SECTION
■
Chemistry. General procedures and techniques were identical with
those previously reported.17,18a Infrared spectra were recorded on a
PerkinElmer 1600 FTIR spectrometer using solutions in CHCl3. High-
resolution mass spectra (HRMS) were determined on a 3-T Finnigan
FTMS-2000 FT mass spectrometer, in ESI mode (The Ohio State
University, Department of Chemistry). As a criterion of purity for key
target compounds, we provided high resolution mass spectral data with
HPLC chromatographic data indicating compound homogeneity.
Low-resolution mass spectra (LRMS) were determined on a Finnegan
LCR-MS. Melting points (mp) were determined with a Fischer Johns
melting point apparatus and are uncorrected. Dehydroepiandrosterone
CONCLUSIONS
■
Galeterone is a first-in-class, multitarget, oral small molecule in
development for the treatment of prostate cancer. Galeterone
acts by disrupting AR signaling via three independent points in
the pathway. This unique small molecule has the potential to
H
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Journal of Medicinal Chemistry
Drug Annotation
and dehydroepiandrosterone acetate were purchased from Aldrich,
Milwaukee, WI. 5-Tributylstannylpyrimidine and 2-tributylstannylpyr-
azine were purchased from Frontier Scientific, Inc., Logan, UT. The
purity of all compounds is as previously reported in our original
articles, determined to be at least 95% pure by a combination of
HPLC, NMR, and HRMS.
3β-Acetoxy-17-chloro-16-formylandrosta-5,16-diene (30).
This compound, prepared from 3β-acetoxyandrost-5-en-17-one (29)
as previously described, provided spectral and analytical data as
described.17
and supervised the biological/pharmacological experiments,
reviewed, revised, and finalized the manuscript.
Notes
The authors declare the following competing financial
interest(s): The authors, Vincent C. O. Njar and Angela M.
H. Brodie declare competing financial interest because both of
them are co-inventors of galeterone. Galeterone patents and
technologies thereof owned by the University of Maryland,
Baltimore, have been licensed to Tokai Pharmaceuticals, Inc.
Cambridge, Mass, USA. V.C.O.N. (Lead Inventor) and
A.M.H.B. also consult for Tokai.
3β-Acetoxy-17-(1H-benzimidazol-1-yl)-16-formylandrosta-
5,16-diene (31). A mixture of 3β-Acetoxy-17-chloro-16-formylan-
drosta-5,16-diene (30, 2.5 g, 6.65 mmol), benzimidazole (2.35 g, 19.9
mmol), and K2CO3 (2.76 g, 23.9 mmol) in dry DMF (20 mL) was
stirred at ca. 80 °C under Ar for 1.5 h. After cooling to room
temperature, the reaction mixture was poured onto ice-cold water (250
mL), and the resulting precipitate was filtered, washed with water, and
dried to give a crude dirty white solid (ca. 2.9 g). Purification by FCC
[petroleum ether/EtOAc/Et3N (6:4:0.3)] gave 2.7 g (88.7%) of pure
compound 31: mp 227−230 °C; IR (CHCl3) 3691, 3024, 2951, 2359,
1725, 1670, 1604, 1491, 1452, 1375, 1253, 1032, 897, 852, 818, 700,
ACKNOWLEDGMENTS
■
The preclinical studies reviewed in this drug annotation were
supported over the years by grants from NIH and NCI (R01
CA027440 (Brodie AMH, PI, Njar VCO co-I); R21 CA117991
(Njar VCO, PI)) and also from Tokai Pharmaceuticals Inc.
Follow-on preclinical studies were supported in part by NIH
grant R01 CA 129379 (Njar VCO, PI) and grants from Tokai
Pharmaceuticals Inc. The clinical studies were sponsored by
Tokai Pharmaceuticals Inc. The studies have also been possible
by efforts of dedicated graduate students and postdoctoral
fellows who are coauthors on our original research and review
articles cited in this report. We also thank UMB’s Office of
Research and Development (ORD) for the intellectual property
protection for galeterone and related compounds and also for
securing license agreements with Tokai Pharmaceuticals Inc.
We sincerely thank the agencies and all the persons that made
the invention of galeterone possible. Galeterone could not have
advanced to this stage of clinical development without the
foresight of Tokai Pharmaceuticals Inc. that dedicated all their
resources and efforts to the development of galeterone.
1
657, 618, 576, 565, 550, 529, 511, 476 cm−1; H NMR (300 MHz,
CDCl3) δ 1.07 (s, 6H, 18- and 19-CH3), 2.04 (s, 3H, 3β-OCH3), 4.60
(m, 1H, 3α-H), 5.43 (br s, 1H, 6-H), 7.35 (br. s, 2H, aromatic-Hs),
7.85 (s, 1H, aromatic-H), 7.98 (s, 1H, aromatic-H), 7.98 (s, 1H, 21-H)
and 9.59 (s, 1H, 16-CHO). HRMS calcd 481.2462 (C29H34O3N2.Na+),
found 481.2454.
3β-Acetoxy-17-(1H-benzimidazol-1-yl)androsta-5,16-diene
(32). A solution of 3β-acetoxy-17-(1H-benzimidazol-1-yl)-16-formy-
landrosta-5,16-diene (31, 2.04 g, 4.45 mmol) in dry benzonitrile (10
mL) was refluxed in the presence of 10% palladium on activated
charcoal (1.02 g, i.e., 50% weight of 29) for 5 h. After cooling to room
temperature, the catalyst was removed by filtration through a Celite
pad. The filtrate was evaporated, and the residue was purified by FCC
[petroleum ether/EtOAc/Et3N (7.5:3:0.5)] to give 1.41 g (73.8%) of
pure compound 32: mp 159−160 °C; IR (CHCl3) 3687, 2947, 2854,
2358, 2340, 1725, 1633, 1609, 1557, 1489, 1454, 1373, 1291, 1253,
1195, 1136, 1031, 985, 910, 839, 735, 665, 590, 544, 533, 513, 502,
488 cm−1; 1H NMR (300 MHz, CDCl3) δ 1.02 (s, 3H, 18-CH3), 1.07
(s, 3H, 19-CH3), 2.04 (s, 3H, 3β-OCH3), 4.62 (m, 1H, 3α-H), 5.43
(br s, 1H, 6-H), 5.98 (s, 1H, 16-H), 7.30 (m, 2H, aromatic-Hs), 7.49
(s, 1H, aromatic-H), 7.81 (s, 1H, aromatic-H), and 7.95 (s, 1H, 21-H).
HRMS calcd 453.2512 (C28H34O2N2.Na+), found 453.2511.
3β-Hydroxy-17-(1H-benzimidazol-1-yl)androsta-5,16-diene
(33, Galeterone). The acetate 30 (1.3 g 3.02 mmol) was dissolved in
methanol (20 mL) under an inert Ar atmosphere and the resulting
solution treated with 10% methanolic KOH (8 mL). The mixture was
stirred at room temperature for 1.5 h and then concentrated under
reduced pressure at approximately 40 °C to a volume of 10 mL. This
solution was poured into ice water (300 mL), and the resulting white
precipitate was filtered, washed with water, and dried. Crystallization
from EtOAc/MeOH gave 33 (1.10 g, 94%), mp 189−190 °C; IR
(CHCl3) 2934, 2339, 1609, 1490, 1453, 1291, 1040, 837, 808, 705,
663, 608, 578, 550, 517 cm−1; 1H NMR (300 MHz, CDCl3) δ 1.02 (s,
3H, 18-CH3), 1.07 (s, 3H, 19-CH3), 3.55 (m, 1H, 3α-H), 5.41 (br s,
1H, 6-H), 5.99 (s, 1H, 16-H), 7.30 (m, 2H, aromatic-Hs), 7.54 (s, 1H,
aromatic-H), 7.80 (s, 1H, aromatic-H), and 7.96 (s, 1H, 21-H). HRMS
calcd 411.2407 (C26H32ON2.Na+), found 411.2396.
ABBREVIATIONS USED
■
AD, androstenedione; AI, aromatase inhibitors; ME, miner-
alocorticoid excess; AME, adrenal mineralocorticoid excess;
ARDAs, androgen receptor degrading agents; ARMOR,
androgen receptor modulation optimized for response; AR,
androgen receptor; CRPC, castration resistant prostate cancer;
CYP17, cytochrome P450 17α-hydroxylase, C17,20-lyase;
CYP19, cytochrome P45 aromatase; Gal, galeterone; DHEA,
dehydroepiandrosterone; DHT, 5α-dihydrotestosterone; FDA,
Food and Drug Administration; FCC, flash column chromatog-
raphy; FSH, follicle stimulating hormone; GnRH, gonadotropin
releasing hormone; LH, luteinizing hormone; LHRH, luteiniz-
ing hormone releasing hormone; NCI, National Cancer
Institute; NIH, National Institutes of Health; PC, prostate
cancer; mCRPC, metastatic castration resistant prostate cancer;
PIC, powder in capsule; PK, pharmacokinetics; SHBG, steroid
hormone binding globulin; T, testosterone
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AUTHOR INFORMATION
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Corresponding Author
*Department of Pharmacology, University of Maryland School
of Medicine, 685 West Baltimore Street, HSF1, Suite 580E,
Baltimore, MD 21201, USA. Tel: (410) 706 6364. Fax: (410)
706 0032. E-mail: vnjar@som.umaryland.edu.
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V.C.O.N. designed and synthesized the novel compounds and
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NOTE ADDED IN PROOF
■
During the review of this manuscript, Tokai Pharmaceuticals
announced that the company remains on track to initiate the
K
DOI: 10.1021/jm501239f
J. Med. Chem. XXXX, XXX, XXX−XXX