Effect of N-methyl deuteration on pharmacokinetics and pharmacodynamics of enzalutamide

Article abstract of DOI:10.1002/jlcr.3516

Enzalutamide, a second-generation antiandrogen, has been developed for the treatment of castration-resistance prostate cancer. We synthesized the deuterated analogues 6 and found that it showed higher drug exposure and thus stronger antitumor potency in preclinical settings. Compound 6 is being developed clinically for the potential to be differentiated from enzalutamide through reduced dosages and a higher safety margin.

Full text of DOI:10.1002/jlcr.3516

Effect of N-methyl deuteration on pharmacokinetics and pharmacodynamics of
Enzalutamide
Xuehai Pang a,b,†, Lingling Peng c,† and Yuanwei Chen a,b,c,d, *
aUniversity of Chinese Academy of Sciences, Chengdu Institute of Organic Chemistry,
Chengdu, 610041, China.
bUniversity of Chinese Academy of Sciences, Beijing, 100049, China.
cLab of YWChen, Cancer Center, West China Hospital, Sichuan University and
Collaborative Innovation Center, Chengdu, 610041, China.
dHinova Pharmaceuticals Inc., Suite 301, Rongyao Building B, #5 South KeYuan Road,
Chengdu 610041, China
ABSTRACT
Enzalutamide, a 2nd generation antiandrogen, has been developed for the treatment of CRPC.
We synthesized the deuterated analogues 6 and found that it showed higher drug exposure
and thus stronger anti-tumor potency in preclinical settings. Compound 6 is being developed
clinically for the potential to be differentiated from enzalutamide through reduced dosages
and a higher safety margin.
Keywords
CRPC; Enzalutamide; Deuterated drug; PK/PD; LNCaP/AR xenograft.
*
Corresponding author. Tel.: +86-18602831330; fax: +86-02885058465; e-mail:
ywchen@scu.edu.cn
† These authors contributed equally.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/jlcr.3516
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Introduction
Prostate cancer (PC) is the most common cancer in males in many developed countries, and
the incidence of prostate cancer has been increasing in the developing world as well.1
Androgens, especially testosterone (T) and dihydrotestosterone (DHT) (Figure 1, 1 and 2),
play
an
important
role
in
the
progression
of
PC
through
the
hypothalamic-pituitary-adrenal/gonadal axis.2-4 The androgen deprivation therapy (ADT),
orchidectomy combined with antiandrogens, has significantly prolonged the survival of PC
patients.5-7 However, progression of the castration-sensitive disease eventually results in
castration-resistance prostate cancer (CRPC) within 1-2 years. The tumor of CRPC regrows
even in the presence of low circulating endogenous ligand (e.g. DHT), and it no longer
responds to classical AR antagonists, such as bicalutamide (Figure 1, 3).8-12 CRPC has been
indicated to associate with a number of potential mechanisms, such as AR
overexpression13-16, AR gene mutations17,18, and androgens synthesized by the PC cells17,19
,
all of which lead to AR signaling reactivation. On the other hand, some classical AR
antagonists, such as 3, can change into agonists in CRPC.20,21
The dependence of CRPC still on AR signaling has led to the development of two novel
therapies such as abiraterone (Figure 1, 4), a potent and selective inhibitor of CYP17A1, and
enzalutamide, a 2nd generation of AR antagonist for the treatment of CRPC. Enzalutamide
(Figure 1, 5) has 5-10 fold higher affinity for AR than bicalutamide and has no AR agonist
activity.22,23 In a randomized and double-blind phase III trial in patients with mCRPC
progressing after docetaxel therapy, enzalutamide significantly prolonged overall survival,
and was also associated with significant benefits in health-related quality of life and in pain
palliation.24,25 However, 5 carried a potential risk of seizures that appears to be
dose-dependent.24,25
Applying the deuterium kinetic isotope effect (DKIE) by replacing hydrogens with
deuteriums has emerged as a validated strategy to improve PK by blocking metabolic
hotspots.26-29 The strategic deployment of deuterium at sites of metabolism where the
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cleavage of C-H is the rate determining step can impede metabolism and redirect metabolic
pathways. This can often increase drug exposure and reduce drug toxicity.29,30 However,
deuterium replacement strategy may be confounded by metabolic switching and other
metabolic pathways in vivo, for example deuterated Propofol and Imatinib.31,32
According to the metabolic profile of 5, CYP2C8 and CYP3A4/5 (the major human
cytochrome P450 isozymes) were responsible for the major metabolism of 5 to N-demethyl
enzalutamide (M2).33 To attenuate the N-demethylation pathway, hydrogen atoms of the
N-methyl moiety were replaced by deuterium. We synthesized the N-methyl deuterated
enzalutamide analogues and found that it has an improved PK profile and a better anti-tumor
activity in animals.
Results and discussion
Chemistry
Enzalutamide 5, and its deuterated version 6 were prepared as shown in Schemes 1. Key
intermediates (12 and 13) were synthesized from commercially available
2-fluoro-4-nitrobenzoic acid (7). Starting from compound 7, 12 and 13 were obtained via
condensation with methylamine hydrochloride or methyl-d3-amine hydrochloride (to 8 or 9),
reduction (to 10 or 11), and a final Strecker reaction with ketone. Preparation of the target
compounds 5 and 6 was finally achieved via cyclization. Aminoacetonitrile compound 12,
aniline 14 and thiophosgene (CSCl2) were cyclized in N,N-Dimethylacetamide (DMAc)
under 60 °C for 14h, followed addition of 2M HCl and ethanol, and refluxed for 2h, giving
target product 5 in 41% yield. Under similar conditions, deuterated thiohydantoin 6 in 38%
yield were produced through cyclization of compound 13 with 14 and CSCl2 in DMAc under
60°C for 14h, and addition of 2M HCl and ethanol for reflux 2h.
In vitro proliferative and AR binding assays
The in vitro anti-proliferation activities were evaluated by using LNCaP/AR cells (prostate
cancer cells overexpressing AR). In order to mimic the CRPC state, the cells were cultured in
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charcoal stripped serum and treated with compounds for 6 days.13 Cell viability was
measured with the Cell Counting Kit-8. As expected, both compound 5 and 6 had similar
anti-proliferation activities (data not shown). Meanwhile, the LNCaP/AR cells were used to
study the affinity of 6 for AR by the competitive inhibition experiment. Both 6 and 5
inhibited the binding of [3H] -R1881 (methyltrienolone) to AR with IC50 values of 0.58 ±
0.56 μM and 0.35 ± 0.24 μM, respectively. Due to the datum from three independent
experiments, the SD (standard deviation) for the cell-based assay was very large.
Although there are measurable differences in physical chemical properties between hydrogen
and deuterium,29,34 they are too small to impact the in vitro biologic activities except
metabolic stability of a deuterated drug. Particularly when only three H atoms of 5 have been
replaced by D, 6 showed very similar inhibitory effects on prostate cancer cells and potency
to bind to AR.
Pharmacokinetics study
PK properties of compounds 6 and 5 were assessed in male ICR mice with a single oral dose
of 10 mg/kg (Figure 2A). As shown in Table 1, compound 6 exhibited an almost 2-fold
increase in t1/2 (24.7 h) compared to 5 (12.8 h); the AUC0-24h was also increased to 150.0
h∙μg/ml (6) from 116.4 h∙μg/ml (5), n=3.
Given the animal-to-animal variability, the deuterated compound 6 and non-deuterated
analog 5 were co-administrated to male Balb/c mice (10/10 mg/kg), SD male rats (5/5 mg/kg),
and male Beagle dogs (4/4 mg/kg) for side-by-side PK studies. All PK parameters in Table
2 were calculated based on the mean concentration vs time curves (Figure 2B, 2C, and 2D).
In mice, compound 6 exhibited increases in t1/2 (12.5 h) and AUC0-24h (124 h∙μg/ml)
compared to 5 (11.0 h and 87.7 h∙μg/ml). In rats, compound 6 had much better PK profiles
than compound 5, with ~1.5-fold increases in t1/2 (18.3 vs 9.0 h) and ~1.5-fold increases in
AUC0-24h (25.4 vs 17.0 h∙μg/ml). In dogs, compound 6 also showed improvements in t1/2 (41.6
h vs 34.5 h) and AUC0-24h (112.0 vs 96.6 h∙μg/ml), compared to compound 5. Overall, the
N-methyl deuteration caused increases in t1/2 and AUC, but had little, if any, effects on Cmax
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in these animals (Table 2). This suggests that at the same drug exposure compound 6 could
have a reduction of Cmax-driven toxicological effects.
In vivo efficacy studies using LNCaP/AR xenograft models of CRPC
SCID mice were castrated and subcutaneously inoculated with LNCaP-AR cells. When the
tumor volumes reached 100-250 mm3, the mice were randomly grouped (8 animals each
group) and p.o. administrated with compound 6 or 5 at the doses of 1, 3, and 10 mg/kg,
respectively, per day for 28 days. As shown in Figure 3A, except for 1 mg/kg of 5, all other
doses of 6 and 5 significantly inhibited the xenograft tumor growth in the SCID mice
(p<0.05, compared with the vehicle group, by Kruskal Wallis Test). The values of tumor
growth inhibition rate were summarized in Table 3. The changes in tumor volumes of the
individual animal and body weight changes were summarized in Figure 3B and Figure 3C,
respectively.
Collectively, it appears that at lower doses the tumor growth inhibition by 6 was more potent
than 5. The tumor suppression effect by 6 at 1 mg/kg was equivalent to that by 5 at 3 mg/kg
(Figure 3A and 3B, Table 3). This could be caused by the improved pharmacokinetic
property of 6 due to the ‘deuteration effect’ (the drug plasma concentrations shown in section
4.4). However, at dosage of 10 mg/kg/ 6 and 5 showed no differentiation in the anti-tumor
activity, suggesting the dose saturation effect.
The drug concentrations in plasma, brain and tumor tissues following 28-day daily dosing
In the in vivo efficacy studies (section 4.3), 24 hours post last dosing, plasma, tumor and
brain tissues were collected for the determination of parental drug and metabolites
concentrations. As shown in Figure 3D, 3E and 3F, compound 6 had ~2-fold higher
concentrations in the plasma, tumor, and brain tissues than compound 5 at the same dosages.
This suggested that the stronger tumor suppression effect by 6 was indeed due to the elevated
drug concentrations of 6 in vivo.
In our previous report, two major metabolites of 5 and 6 have been identified in vivo, oxidative
metabolite N-demethyl enzalutamide (M2, active metabolite) and a carboxylic acid derivative
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(M1) ( Figure 1).31 There were no differences in M1 concentrations of 6 and 5. However, M2
concentrations of compound 6 were only 30-43% of compound 5. These results suggest that
blocking M2 production due to the ‘deuteration effect’ is one of the reasons for the elevated
drug exposure of compound 6. Though M2 could be the active metabolite, the concentrations
of M2 in mice was quite low (Figure 3D, 3E and 3F) and its contribution to the in vivo efficacy
should be limited.
Pharmacokinetic and pharmacodynamic (PK/PD) correlation analysis
In LNCaP/AR xenograft assay, the tumor proliferation rate of compound 6 at day 28 (last
dosing) were graphed against the drug concentrations in the tumor tissues. As shown in
Figure 4, in the dose range of 1, 3, and 10 mg/kg/day, 6’s tumor suppression effect increased
in the dose-dependent manner and was strongly correlated with the drug concentrations in the
tumor tissue. In addition, as show in Figure 5, in the same dose range, the drug concentrations
of 6 in the tumor tissue were strongly correlated with its concentrations in the plasma. These
results suggest that the in vivo efficacy is reflected by the plasma drug concentrations.
Conclusion
The deuteration at N-methyl of 5 (primary metabolic site) had a better PK profile and
stronger in vivo anti-tumor activity due to the elevated drug exposure in preclinical settings.
Compound 6 is being developed clinically for the potential to be a best-in-class AR
antagonist for the treatment of CRPC.
Experimental
Chemistry
Unless specified otherwise, starting materials and reagents were obtained from commercial
sources and were used without further purification. Analytical TLC (thin-layer
chromatography) was performed using 0.2mm silica gel plates (Yantai Jiangyou). And flash
1
column chromatography was performed using silica gel with the indicated solvents. H and
13C NMR spectra were recorded on a Bruker 400 MHz instrument using CDCl3, CD3OD, or
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DMSO-d6 as the solvent and TMS as the internal reference. Chemical shifts (δ) were reported
with parts per million (ppm) relative to the residual solvent peak (for CDCl3, δ (1H) = 7.26
ppm and δ (13C) = 77.16 ppm; for CD3OD, δ (1H) = 3.31 ppm and δ (13C) =49.00 ppm; and
for DMSO-d6, δ (1H) = 2.50 ppm and δ (13C) = 39.52 ppm). Abbreviations used for signal
patterns are as follows: s, singlet; d, doublet; dd, doublet of doublets; ddd, doublet of doublet
of doublets; t, triplet; q, quadruplet; m, multiplet; and br, broad. High-resolution mass
spectrometry (HRMS) was performed with a Bruker micrOTOF-Q II (ESI) instrument.
Liquid chromatography-Mass (LC/MS) were recorded on an Agilent LCMS 1200-6110
(ESI). All of the test compounds had purity >95% as determined by LC/MS.
General procedure for preparation of the intermediates 12 and 13
2-fluoro-N-trideuteriomethyl-4-nitrobenzamide
(9).
To
a
solution
of
2-fluoro-4-nitrobenzoic acid (10.0g, 54.0mmol) and NEt3 (16.4g, 162.2mmol) in DCM
(100ml) was added CDI (10.5g, 64.9mmol) portion-wise under N2 at room temperature. The
reaction mixture was stirred for 1h. To this reaction mixture were added CD3NH2∙HCl (5.7g,
81.1mmol), then the reaction mixture was stirred for 4h. After completion of the reaction
water (100ml) was added. The organic layer was washed with 1M HCl (100ml) and water
(100ml) orderly. The organic layer was dried over anhydrous sodium sulfate, and
concentrated under reduced pressure. The residue was purified by flash column
chromatography on silics gel (ethyl acetate/ petroleum ether=1/2) to afford
1
2-fluoro-N-trideuteriomethyl-4-nitrobenzamide (9) (10.3g, 95%). H NMR (400 MHz,
CDCl3) δ 8.30 (dd, J = 8.6, 7.8 Hz, 1H), 8.13 (dd, J = 8.6, 2.1 Hz, 1H), 8.02 (dd, J = 11.1, 2.1
13
Hz, 1H), 6.79 (br, 1H). C NMR (101 MHz, CDCl3) δ 161.9, 159.7 (d, J =252.3Hz), 150.1
(d, J = 9.4 Hz), 133.4 (d, J = 2.7 Hz), 126.8 (d, J = 12.7 Hz), 119.7 (d, J = 3.6 Hz), 112.1 (d,
J =30.7Hz), 26.5 (m). MS (ESI): m/z 201.7 (M+H+, positive mode).
4-amino-2-fluoro-N-trideuteriomethylbenzamide (11). To a solution of compound 9
(10.0g, 49.8mmol) in EA (100ml) and AcOH (9.2ml, 0.50mol) was added Fe (13.9g,
249.0mmol) portion-wise under N2 at room temperature. The reaction mixture was refluxed
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for 16h. After cooling to room temperature, the reaction mixture was filter, then the filtrate
was concentrated under reduced pressure. The residue was purified by flash column
chromatography
on
silics
gel
(EA/PE=1/1)
to
afford
1
4-amino-2-fluoro-N-trideuteriomethylbenzamide (11) (5.9g, 69%). H NMR (400 MHz,
CDCl3) δ 7.89 (t, J = 8.6 Hz, 1H), 6.61 (d, J = 12.2 Hz, 1H), 6.48 (dd, J = 8.6, 2.2 Hz, 1H),
6.33 (dd, J = 14.4, 2.2 Hz, 1H), 4.15 (br, 2H). 13C NMR (101 MHz, CDCl3) δ 164.4, 162.2 (d,
J =245.7Hz), 151.5 (d, J = 12.8 Hz), 133.3 (d, J = 4.3 Hz), 110.9 (d, J = 1.8 Hz), 110.2 (d, J
= 11.9 Hz), 100.8 (d, J =28.8Hz), 25.8 (m). MS (ESI): m/z 172.2 (M+H+, positive mode).
4-((2-cyanopropan-2-yl)-amino)-2-fluoro-N-trideuteriomethylbenzamide (13). In a sealed
tube with a magnetic stir bar, compound 11 (171mg, 1.0 mmol) was added, followed by
TMSCN (298mg, 3.0mmol), acetone (5ml) and AcOH (5ml). The sealed tube was heated to
80°C and stirred for 16h. After cooling to room temperature, EA (20ml) was added and then
washed with water (20ml). The organic layer was dried over anhydrous sodium sulfate, and
concentrated under reduced pressure. The residue was purified by flash column
chromatography
on
silics
gel
(EA/PE=1/2)
to
afford
4-((2-cyanopropan-2-yl)amino)-2-fluoro-N-trideuteriomethylbenzamide (13)
(217mg,
90%). 1H NMR (400 MHz, CDCl3) δ 7.79 (t, J = 8.7 Hz, 1H), 7.06 (d, J = 10.3 Hz, 1H), 6.70
13
(dd, J = 8.7, 2.3 Hz, 1H), 6.59 (dd, J = 14.7, 2.2 Hz, 1H), 6.41 (br, 1H), 1.72 (s, 6H). C
NMR (101 MHz, CDCl3) δ 164.2, 161.4 (d, J = 246.1Hz), 149.3 (d, J = 12.0 Hz), 132.1 (d, J
= 4.3 Hz), 121.4, 110.8, 110.7 (d, J = 13.2 Hz), 100.7 (d, J = 29.3 Hz), 47.6, 27.5, 25.8 (m).
MS (ESI): m/z 239.3 (M+H+, positive mode).
General procedure for preparation of compounds 5 and 6.
To a solution of compound 12 (235mg, 1.0mmol) and compound 7 (223mg, 1.2mmol) in
DMA (0.5ml) was added thiophosgene (172mg, 1.5mmol) under N2 at 0°C. The reaction
mixture was stirred for 16h at 60°C. To this mixture were added MeOH (2ml) and 2M HCl
(2ml), then the reaction mixture was reflux for 2h. After cooling to room temperature, the
reaction mixture was poured into ice water (10ml) and extracted with EA (10ml). The organic
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layer was dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The
residue was purified by flash column chromatography on silics gel (EA/ PE=1/1) to afford
4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fl
uoro-N-methylbenzamide (5) (190mg, 41%).
4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-
1
2-fluoro-N-methylbenzamide (enzalutaimde, 5). White solid; yield: 41 %; H NMR (400
MHz, DMSO-d6): δ 8.44 (dd, J = 20.7, 5.8 Hz, 2H), 8.30 (s, 1H), 8.09 (d, J = 8.1 Hz, 1H),
7.79 (t, J = 8.0 Hz, 1H), 7.44 (d, J = 10.6 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 2.80 (d, J = 4.2
Hz, 3H), 1.55 (s, 6H). 13C NMR (100 MHz, DMSO-d6): δ 180.04, 174.70, 163.42, 158.90 (d,
J = 251.49Hz), 138.24(d, J = 10.1Hz), 137.88, 136.22, 133.91，131.12(q, J = 33.33Hz),
130.85(d, J = 4.04Hz), 127.95(q, J = 4.04Hz), 126.08 (d, J = 3.03Hz), 125.12 (d, J =
15.15Hz), 122.17(q, J =274.72Hz), 118 (d, J = 24.24Hz), 114.97, 108.71 (d, J =2.02Hz),
66.55, 25.54, 22.89. MS (ESI): m/z 465.2 (M+H+, positive mode). HRMS (ESI): mass
calcd for C21H17F4N4O2S (M+H+, positive mode), 465.1008; found, 465.1028.
4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-
1
2-fluoro-N-trideuteriomethylbenzamide (6). White solid; yield: 39%; H NMR (400 MHz,
DMSO-d6) δ 8.50-8.36 (m, 2H), 8.30 (s, 1H), 8.09 (d, J = 8.1 Hz, 1H), 7.79 (s, 1H), 7.44 (d, J
13
= 10.5 Hz, 1H), 7.34 (d, J = 8.2 Hz, 1H), 1.54 (s, 6H). C NMR (100 MHz, DMSO-d6): δ
180.04, 174.70, 163.42, 158.90 (d, J = 251.5Hz), 138.24(d, J = 10.1Hz), 137.88, 136.22,
133.91, 131.12(q, J = 33.3Hz), 130.85(d, J = 4.0Hz), 127.95(q, J = 4.0Hz), 126.08 (d, J =
3.0Hz), 125.12 (d, J = 15.2Hz), 122.17(q, J =274.7Hz), 118 (d, J = 24.2Hz), 114.97, 108.71
(d, J =2.0Hz), 66.55, 25.54 (m, J = 21.2Hz), 22.89. MS (ESI): m/z 468.2 (M+H+, positive
mode). HRMS (ESI): mass calcd for C21H14D3F4N4O2S (M+H+, positive mode), 468.1197;
found, 468.1191.
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Bioassay
Animals and cells
ICR mice (males, 23-33 g) and SD rats (males, 180-220 g) were purchased from Shanghai
Lingchang biological technology co., LTD, (Shanghai, China). CB17 SCID mice (males, 4-5
weeks old, 18-22 g) were purchased from Beijing HFK Bioscience co., LTD. (Beijing,
China). Beagle dogs (males, 6-9 monthes old, 6-9 kg) were purchased from Beijing Marshall
Biotechnology Co., LTD. (Beijing, China). LNCaP/AR cell lines were obtained from Sichuan
University and Collaborative Innovation Center for Biotherapy (Chengdu, China).
All animals were kept in an animal room with controlled illumination (12/12 h light/dark
cycle), temperature (20-25°C), and humidity (over 40 %). The animals were fasted for 12 h
before dose administration. All PK studies were performed at Shanghai Institute of Materia
Medica (Chinese Academy of Sciences), and all in vitro and in vivo biologic activity studies
performed at Sichuan Kangcheng biological co., LTD. The animal studies were carried out
under protocols approved by the animal protection law of the People's Republic of China
Care and Use Committees and institutional guidelines for the proper, humane use of animals.
All rodent studies were also carried out in accordance with the Guidelines for Care and Use
of Laboratory Animals approved by the Shanghai Institute of Materia Medica, Chinese
Academy of Sciences (Shanghai, China).
Proliferation assays
Trypsinized LNCaP/AR cells were adjusted to a concentration of 60,000 cells per mL in
RPMI 1640 (with 5% CSS), and dispensed in 100 μl aliquots into 96 well plates (Corning).
Cells were cultured for 24 h and then 100 μl of culture medium containing compounds were
added. The cells continued to incubate for 6 days and the cell viability was measured using
the Cell Counting Kit-8 (Dojindo) according to the manufacturer’s instructions. Percent
inhibition of the samples was calculated as follows: %inhibition= [ODvalue of control cells - ODvalue
of sample treated cells]/[ ODvalue of control cells- ODvalue medium without cells]. The IC50 values were calculated
by using the Prism Graph software.
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AR binding assays
The LNCap/AR cells were cultured with RPMI1640 plus 10% FBS at 37°C, 5% CO2. 6×104
cells were added to the 96-well plate (Poly-D pre-coated), and incubated for overnight. The
medium was removed, then, 25μl compound dose (top concentration: 200 μM, three-fold
dilution) was added. Firstly, the cells were incubated for 30 min, then 25μM [3H]-R1881
(final top concentration is 100μM, three-fold dilution, final [3H]-R1881 concentration is
7nM) was added, the cells were incubated for 30 min again. The medium was removed, then
the cells were washed with uptake buffer (10mM HEPEs, pH 7.5, 125mM NaCl, 4.8mM KCl,
1.2mM CaCl2, 1.2mM MgSO4, 1.2mM KH2PO4, 5.6mM D-Glucose), and 100μl lysis buffer
was added. Shaking the plate for 1 hour, the 50μl lysis buffer was transfered to an isotope
plate, 200μl scintillation cocktail was added, and the dates were counted on the MicroBeta.
Single-dosing ICR mice, SD rat, and Beagle dogs PK study
The 5 and 6 were dissolved in 0.5% HPMC at a concentration of 1 mg/ml. A total of nine
male ICR mice were randomly divided into three groups (A group, four rats; B group, four
rats; C group, three rats). The A and B groups were orally given 10 mg/kg 5 and 6,
respectively. The blood sampling time points were pre-dose (0) and 0.5, 1, 2, 4, 6 and 24 h
post-doses. Group C was given a 1:1 formulation of 5 and 6 (10/10 mg/kg) by oral
administration. The blood sampling time points were pre-dose (0) and 0.25, 0.5, 1, 2, 3, 5, 7,
9, 24, 32 and 48 h post-doses. Four male SD rats were given a 1:1 formulation of 5 and 6 (5/5
mg/kg) by oral administration. The blood sampling time points were pre-dose (0) and 0.5, 1,
3, 5, 7, 9, 24, 48, 72, 96 and 120 h post-doses. Three-hundred microliters of blood was
collected from the orbital plexus into tubes after these animals were temporarily anesthetized
by ether inhalation. Three Beagle dogs were given a 1:1 formulation of 5 and 6 (4/4 mg/kg)
by oral administration. The blood sampling time points were pre-dose (0) and 0.5, 1, 2, 3, 4,
6, 8, 12, 24, 48, 96, 114, 192, 240, 288 and 336 h post-doses. One milliliter of blood was
collected from the upper arm cephalic vein into tubes. Immediately after collection, blood
collection tubes were gently inverted at least five times, ensuring complete mixing, and were
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then immediately placed on ice. The blood was centrifuged for 10 min at 3500 rpm and 4°C
to separate the plasma from the red blood cells. After separation, 200μl disposable pipettes
were used to take 100μl aliquots from the top plasma layer into tubes, with each being labeled
by the name of analysis unit. Plasma was stored at −20 °C until removal for LC-MS/MS
analysis for the test compound concentration.
In vivo LNCaP/AR xenograft experiments.
In vivo xenograft experiments to determine anti-tumor response were carried out in CB17
SCID male mice. Mice were orchiectomized under pentobarbital sodium and were given 2-3
days to recover prior to tumor cell injection. LNCaP/AR cells were suspended in 50% RPMI,
50% Matrigel, and 1×107 cells/xenograft were injected in a volume of 100μl. Animals were
observed 3 times/week until tumor growth was apparent. From 21 day post-injection, tumors
were measured weekly, and after 40-60 days post-injection, animals were randomized into
cohorts of equivalent mean (100-250 mm3) and range tumor burden. The 5 and 6 were
administered daily by oral gavage at the dose of 1, 3, and 10 mg/kg/d, respectively. For all
LNCaP/AR xenograft studies 5 and 6 drug stocks were prepared in 18% PEG-400, 1%
Tween-80 and 1% povidone, and were formulated for dosing in 15% Vitamin E-TPGS and
65% of a 0.5% w/v CMC solution in 20mM citrate buffer (pH 4.0).
The blood sample, tumor tissue, and brain were collected 24 hours later after the last
administration, sequentially. The blood samples were added EDTA, and the samples were
stored at -20 °C for LC-MS/MS analysis after being thoroughly vortexed. The tumor tissue
and brain were accurately weighed and taken photos, then were stored at -20 °C for
LC-MS/MS analysis.
Acknowledgments
This work is financially supported by Hinova Pharmaceuticals Inc. and also partially
supported by National Natural Science Foundation of China (No. 81472418). We also thank
Prof. Xiaoyan Chen, and Prof. Zhiwei Gao (Shanghai Institute of Materia Medica, Chinese
Academy of Sciences) for the technical support of Pharmacokinetic experiments.
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Conflict of Interest
The authors have declared that there is no conflict of interest.
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Table 1. Pharmacokinetic parameters orally respective administration of 6 and 5.a
Compound
Dose (mg/kg)
Tmax (h)
5.0
Cmax (μg/ml) AUC0-24h (h∙μg/ml)
t1/2 (h)
5
6
10
10
5.35±0.32
4.13±0.54
116.4±4.2
12.8±0.9
24.7±3.9
4.0
150.0±21.3
aData are presented as the mean of values obtained from three ICR mice.
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Table 2. Pharmacokinetic parameters of PO combination dosing of compounds 5 and 6. a
Dose
Cmax
(μg/ml)
5.48
AUC0-24h
Animal Compound
Tmax (h)
t1/2 (h)
(h∙μg/ml)
(mg/kg)
10
10
5
5.0
5.0
6.0
6.0
2.0
2.0
87.7
124
11.0
12.5
9.0
5
Mouse
5.97
6
0.9
17.0
25.4
96.6
112.0
5
Rat
5
0.87
18.3
34.5
41.6
6
4
4.13
5
Dog
4
4.25
6
aData are presented as the mean of values obtained from three animals.
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Table 3. Tumor weight and inhibitory rate.a
Group
Dose (mg/kg)
n
10
8
Tumor weight (g)
0.199±0.028
Inhibitory rate (%)
0.00
Vehicle
-
10
3
0.049±0.010*
0.094±0.030**
0.149±0.032
75.49
8
50.73
5
6
1
8
25.40
10
3
8
0.039±0.005**
0.062±0.014**
0.082±0.008**
80.57
8
68.72
1
7▲
58.73
aEach value represents the mean ± SEM. ▲ One animal died after grouping; *
P<0.05，** P<0.01.
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Figure 1. Structures of androgens (1 and 2), antiandrogens (3 and 5), inhibitor of CYP17A1 (4), and
metabolites of enzalutamide (M1 and M2).
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Figure 2. The concentration of 5 and 6 in plasma. A, Orally respective administration of 6 and 5 in mice; B,
Orally co-administration of 6 and 5 in mice; C, Orally co-administration of 6 and 5 in rats; D, Orally
coadministration of 6 and 5 in dogs.
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Figure 3. A, The fold change in tumor volume of xenografts with 5 and 6 in 28 days; B, The percentage of
change in individual tumor volume; C, The weight fold change of tumor-burdened mice in 28 days; D, The
concentration of 5, 6, and major metabolites (M1 and M2) in plasma 24 h post last dosing after 28-day
dosing; E, The concentration of 5, 6, M1 and M2 in tumor; F, The concentration of 5, 6, M1 and M2 in brain.
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Figure 4. The tumor suppression effect of 6 correlating with its concentrations in the tumor tissue.
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Figure 5. The concentrations of 6 in the tumor tissue correlating with its concentrations in the plasma.
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Scheme 1. Synthesis of 5 and 6. Reagents and conditions: (a) MeNH2∙HCl or CD3NH2∙HCl, CDI, NEt3, DCM, 3h,
93-95%; (b) Fe, AcOH, EA, reflux, 16h, 69-73%; (c) TMSCN, AcOH, Acetone, sealed tube, 80°C, 16h,
76-90%; (d) CSCl2, DMA, 60°C, 16 h, then 2M HCl, EtOH, reflux, 2h, 38-41%.
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