Developing a novel dual PI3K-mTOR inhibitor from the prodrug of a metabolite

Article abstract of DOI:10.2147/OTT.S142492

This study presents a process of developing a novel PI3K-mTOR inhibitor through the prodrug of a metabolite. The lead compound (compound 1) was identified with similar efficacy as that of NVP-BEZ235 in a tumor xenograft model, but the exposure of compound

Full text of DOI:10.2147/OTT.S142492

OncoTargets and erapy
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O r i g i n a l r e s e a r c h
Developing a novel dual Pi3K–mTOr inhibitor
from the prodrug of a metabolite
This article was published in the following Dove Press journal:
OncoTargets andTherapy
20 October 2017
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Yan Zhou1,2
genyan Zhang2
Feng Wang2
Jin Wang2
Abstract: This study presents a process of developing a novel PI3K–mTOR inhibitor through
the prodrug of a metabolite. The lead compound (compound 1) was identified with similar effi-
cacy as that of NVP-BEZ235 in a tumor xenograft model, but the exposure of compound 1 was
much lower than that of NVP-BEZ235. After reanalysis of the blood sample, a major metabolite
(compound 2) was identified. Compound 2 exerted similar in vitro activity as compound 1,
which indicated that compound 2 was an active metabolite and that the in vivo efficacy in the
animal model came from compound 2 instead of compound 1. However, compound 1 was
metabolized into compound 2 predominantly in the liver microsomes of mouse, but not in the
liver microsomes of rat, dog, or human. In order to translate the efficacy in the animal model
into clinical development or predict the pharmacokinetic/pharmacodynamic parameters in the
clinical study using a preclinical model, we developed the metabolite (compound 2) instead
of compound 1. Due to the low bioavailability of compound 2, its prodrug (compound 3) was
designed and synthesized to improve the solubility. The prodrug was quickly converted to com-
pound 2 through both intravenous and oral administrations. Because the prodrug (compound 3)
did not improve the oral exposure of compound 2, developing compound 3 as an intravenous
drug was considered by our team, and the latest results will be reported in the future.
Keywords: PI3K, mTOR, NVP-BEZ235, prodrug, metabolite, antitumor
Yanwei Ding2
Xinyu li2
chongtie shi2
Jiakui li2
chengkon shih2
song You1
1The school of life science and
Biopharmaceutical, shenyang
Pharmaceutical University,
shenyang, 2Department of Project
Management, Medicinal chemistry,
Pharmacology, Drug Metabolism,
and Pharmacokinetics,Toxicology,
Xuanzhu Pharma, Jinan, china
Introduction
With rapid progress in molecular biology and translational medicine, the focus of
malignant-tumor treatment has moved from conventional chemotherapy to targeted
therapy, especially in cases with multiple alterations, overexpression, and mutations of
genes, such as EGFR, ALK, MEK, BRAF, VEGFR, and MET.1 A great amount of effort
has also been put into exploring the PI3K–Akt–mTOR signaling pathway for cancer
therapy. This pathway not only plays a major role in normal physiological function
but also impacts the progress of growth, proliferation, and metastasis of tumor cells.2
The PI3K family are lipid kinases. Their function is the phosphorylation of phospho-
inositides on 3-OH when it is activated by the upstream receptor tyrosine kinases.3
mTOR is a serine/threonine kinase that belongs to the series of PI3K-related protein
kinases. First identified in yeast in 1994, mTOR is made up of multiple proteins that
drive the process of the cell life cycle. Since PI3K–Akt–mTOR is the downstream
element of growth-factor receptor tyrosine kinases, when the mutation or amplifica-
tion of the key encoding genes in this pathway occurs, it will cause abnormal signal
transcription or translation and lead ultimately to cancer.4,5 It has been reported that
in breast cancer, the most frequent mutation occurs in the PI3K gene, and overall
abnormal activation on the PI3K–Akt–mTOR signaling pathway reaches approximately
70% for these patients.6,7
correspondence: song You
The school of life science and
Biopharmaceutical, shenyang
Pharmaceutical University, 103 Wenhua
road, shenhe District, shenyang,
liaoning 110016, china
Tel +86 24 2398 6436
email yousong206_spu@aliyun.com
chengkon shih
Department of Pharmacology,
Xuanzhu Pharma, 2518 Tianchen street,
Jinan, shandong 250101, china
Tel +531 8098 9255
email sck@xuanzhupharm.com
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Extensive research has been conducted on targeting the inhibitors combined with other pathway inhibitors could
PI3K–Akt–mTOR pathway in oncology therapy, especially have a synergistic effect in treating malignant tumors.14,15
in breast and ovarian cancers.8 Many new chemical entities Indeed, as an example, the combination of PD98059
are being assessed in preclinical or clinical stages. Rapamycin (MEK inhibitor) with NVP-BEZ235 induces cell-cycle
and everolimus, successful mTOR-inhibitor drugs, have arrest and apoptosis more effectively than single inhibitors.
been approved by the US Food and Drug Administration
Furthermore, there are large unmet medical needs for the
(FDA).9 The pioneer dual inhibitor of PI3K–mTOR, NVP- development of new drugs with activity on the PI3K–Akt–
BEZ235, was developed by Novartis and is currently in clini- mTOR pathway. In our earlier work, a series of compounds
cal Phase II trials. Other dual inhibitors, such as apitolisib were designed and synthesized with a similar scaffold
(Genentech), PQR-309 (Universität Basel), and voxtalisib as NVP-BEZ235. After evaluation of the structure–activity
(Exelixis), are also being developed and are in different relationship, absorption, distribution, metabolism, and
clinical phases.10–12 Most dual inhibitors are designed by excretion properties, and in vivo efficacy, a lead compound
modifying the structure of NVP-BEZ235. NVP-BEZ235 (compound 1) was identified and selected for further develop-
structure is shown in Figure 1.
ment.Figure1illustratesthechemicalstructureofcompound1.
It is well established that drug tolerance is one of the key In this paper, we disclose the process of discovering a metabo-
challenges for dual PI3K–mTOR inhibitors, which explains lite of compound 1 and developing it into a novel dual PI3K–
the slow progress of research and development of these inhibi- mTOR inhibitor through the prodrug strategy (Figures 2–4
tors. Currently, only eleven new dual PI3K–mTOR inhibitors and Tables 1–7).
are being evaluated actively in the clinical stage, but none has
Materials and methods
been approved by the FDA. Although significant therapeutic
effects against cancer have been demonstrated, drugs target- chemicals and materials
ing the PI3K–Akt–mTOR pathway, (eg, everolimus) have NVP-BEZ235 was purchased from Selleckchem (S1009).
shown significant toxicity, including stomatitis, noninfectious Compounds 1–3 were synthesized in the laboratory at
pneumonitis, rash, hyperglycemia, and immunosuppression.13 Xuanzhu Pharma, and the synthetic routes of the three com-
Therefore, developing new drug candidates with reduced pounds are shown in Figures 5–7, respectively. In addition,
side effects is necessary. In addition, dual PI3K–mTOR detailed synthetic routes have been described in the patents.16–18
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activity relationship study from nVP-BeZ235; (C) compound 2 was the metabolite of compound 1; (D) compound 3 was a prodrug of compound 2.
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a novel dual Pi3K–mTOr inhibitor
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Notes: compound 1 was incubated with liver microsomes of nude mice, sprague
Dawley rats, beagles, and humans. concentrations of compounds 1 and 2 were
quantified by LC-MS/MS. Compound 1 was metabolized rapidly into compound
2, and compound 2 was the dominant metabolite only in mouse microsomes.
However, the metabolic profiling of compound 1 in microsomes of rats, dogs, and
humans showed low levels of compound 2 in the products.
Figure 2 In vivo efficacy of compound 1 and NVP-BEZ235.
Notes: Dosed orally in tumor-bearing immunocompromised mice at approximately
15–60 mg/kg and 30 mg/kg, respectively. Pc-3 cells were injected subcutaneously
into nude mice (n=8 per group), and the observation groups were treated orally once
daily for 14 days. Tumor volumes were measured twice weekly; antitumor activity
is expressed as treatment/control (T/c) and tumor-growth inhibition (Tgi). The
dose–effect relationship showed perfect linearity. Furthermore, Tgi increased from
15 mg/kg to 60 mg/kg for compound 1, while compound 1 and nVP-BeZ235 showed
broadly similar effects, both at 30 mg/kg.
Abbreviations: lc, liquid chromatography; Ms, mass spectrometry.
DMEM for U87MG were purchased from GE Healthcare.
Ham’s F-12K medium for PC-3/A549 was purchased from
Thermo Fisher Scientific, and FBS was purchased from GE
Healthcare. All the liver microsomes were purchased from
the Research Institute for Liver Diseases. BALB/c nude
mice were obtained from Huafukang Biological Technology,
and Sprague Dawley rats were purchased from Vital River
Laboratories.
All three compounds were characterized by nuclear magnetic
resonance (NMR) and mass spectrometry (MS) and their
structures further qualified. Other materials – β-cyclodextrin,
HEPES, Tween 20, penicillin, streptomycin, MgCl2, and
β-NADPH – were all of analytical grade.
PI3K and mTOR enzymes were purchased from Thermo
Fisher Scientific (A11815) and EMD Millipore (14-770),
respectively. A Kinase-Glo Plus luminescent kinase-assay
kit was purchased from Promega (V3771). U87MG, BT474,
A549, SKOV-3, PC-3, HCT116, and 786-0 cell lines were
purchased from ATCC. RPMI- 1640 medium for 786-0/
BT474, McCoy’s 5A medium for HCT116/SKOV-3, and
ethics statement
All animal experiments were performed according to the
Guide for the Care and Use of Laboratory Animals. The
protocol was approved by the institutional animal care and
use committee of Xuanzhu Pharma. All experiments were
carried out in strict compliance with the regulations. Animals
were monitored daily for physical appearance and mobility
and were killed when they met specific criteria or showed
signs of distress. Animals were acclimatized for more than
3 days before experiments and were housed under controlled
temperature and humidity with a 12/12-hour light/dark cycle
with ad libitum feeding in an Association for Assessment and
Accreditation of Laboratory Animal Care-certified animal
facility. All procedures in the pharmacokinetic (PK) experi-
ments in rats and mice were performed under isoflurane anes-
thesia, and all efforts were made to minimize suffering.
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Figure 3 Plasma-concentration data of compounds 1 and 2 following a 60 mg/kg
oral dose of compound 1 in BalB/c nude mice.
in vitro Pi3K- and mTOr-inhibition
assays
To evaluate the inhibitory activities of the compounds
on PI3Ks and mTOR, in vitro kinase assays were per-
formed at ChemPartner. Compounds were serially diluted
Notes: nude mice were dosed orally with compound 1 at 60 mg/kg (n=9 per
group). Blood was collected from different animals and prepared by centrifugation.
concentrations of compounds 1 and 2 in plasma were determined by lc-Ms/Ms.
as compound 2 was a dominant metabolite in mice, its aUc was about 50-fold higher
than compound 1, and the half-life of compound 2 was longer than compound 1.
Abbreviations: lc, liquid chromatography; Ms, mass spectrometry; aUc, area
under the curve.
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Table 1 PK/PD exploration in BalB/c nude-mouse xenograft model of Pc3 cell line (n=8)
Body weight (g)
Tumor
volume
(mm3)
T/C (%)
TGI (%)
AUC0–24
in plasma
(ng⋅h/mL)
Concentration
in tumor
tissue (ng/g)c,d
Before
treatmentd
After
treatmentd
control
23.1±0.5
23.1±0.5
23.2±0.4
23.2±0.4
23.2±0.5
22.8±0.5
22.4±0.4
21.5±0.4
19.4±0.3
20.8±0.3
1,026±68
643±40
412±38
185±21
331±16
–
–
–
–
compound 1 (15 mg/kg)
compound 1 (30 mg/kg)
compound 1 (60 mg/kg)
nVP-BeZ235 (30 mg/kg)
62.7
40.3
17.9
32.4
37.3a
59.7b
82.1b
67.6b
c
30
5.49±1.86
5.33±1.97
15.3±4.29
310±61.8
d
111
328
4,779
Notes: aP,0.05 (vs vehicle control using one-way anOVa); bP,0.001 (vs vehicle control using one-way anOVa); at 4 hours after the last dose; data presented as
mean ± standard deviation. Pc-3 cells were injected subcutaneously into nude mice (n=8 per group), and observation groups were treated orally once daily for 14 days.
Tumor volumes were measured twice weekly, and antitumor activity is expressed as T/c (%) and Tgi (%). The dose–effect relationship showed perfect linearity, Tgi
increased from 37.3% to 82.1% as doses ascended from 15 mg/kg to 60 mg/kg for compound 1 (compared with vehicle control, P,0.05), while compound 1 and nVP-BeZ235
showed broadly similar effect, both at 30 mg/kg.
Abbreviations: T/c, treated/control; Tgi, tumor growth inhibition; aUc, area under the curve; anOVa, analysis of variance; PK/PD, pharmacokinetic/pharmaco-
dynamic.
(ten concentrations) and prepared as source plate and assay supplemented with 10% FBS, 100 U/mL penicillin, and
plate. Kinase (4×) and (2×) substrate solutions were also 100 μg/mL streptomycin at 37°C under a 5% CO2 atmo-
prepared, and the assay plate was subsequently incubated sphere. Cells that were 80% confluent were used in the
at room temperature for 1 hour. After kinase detection, data assay. The suspension of freshly trypsinized cultured cells
were collected on an EnVision (Caliper reading). Lance signal was spun at 1,000 rpm for 4 minutes and subsequently
(665 nm) values were copied and subsequently converted to resuspended in fresh medium supplemented with 10%
percentage-inhibition values:
FBS. After adjustment of cell density, cells were seeded
in 96-well plates. After 24 hours, test-compound solutions
of different concentrations were prepared with culture
medium, and 100 μL of each compound solution was
transferred to each well according to the plate maps. Cells
were treated for 72 hours at 37°C under a 5% CO2 atmo-
sphere. After the medium had been removed, 150 μL of
XTT reagent was added to each well. The plate was then
incubated at 37°C for 120 minutes and the testing wells
read at a wavelength of 450 nm. All tests were performed
in duplicate.
Lance signal − Min
Max − Min
Percentage inhibition =
× 100
where min indicates the Lance signal of no enzyme control
and max indicates the Lance signal of the dimethyl sul-
foxide control. Compounds 1 and 2 were tested for their
potency against class I PI3K, mTOR, and a panel of rep-
resentative kinases using Kinase-Glo assays for PI3Kα,
Lance Ultra kinase assay for mTOR, and Caliper for other
kinases, with ATP concentrations in Km (Michaelis–Menten
constant).
In vivo efficacy studies in tumor xenograft
model
Tumor-cell growth-inhibition assays
In vitro compound potency was determined using dif-
ferent tumor-cell lines and methods that have been
described in the patents.16,17 Cells were cultured in medium
Tumor-growth-inhibition studies were performed in
BALB/c nude mice. Initial body weight of mice on arrival
was 18–22 g. Briefly, PC3 cells were grown in RPMI
1650 medium supplemented with 10% FBS, 100 U/mL
Table 2 Pharmacokinetic parameters of compound 1 and its metabolite compound 2 after oral dosing of compound 1 in nude mice
(60 mg/kg)
Animal
t
(hours)
tmax (hours)
Cmax (ng/mL)
AUC0–24 (ng⋅h/mL)
½
compound 1a
BalB/c nude mice
BalB/c nude mice
nab
10.1
0.5
0.5
77.6
448
61.3
3,404
b
compound 2a
a
Notes: Dosing compound 1 and detecting compound 1 and compound 2. nude mice were dosed orally with compound 1 at 60 mg/kg (n=9 per group); not available.
Blood was collected from different animals and prepared by centrifugation. The concentration of compounds 1 and 2 in plasma was determined by lc-Ms/Ms. The aUc of
compound 2 was about 50-fold higher than compound 1, and the half-life (t ) of compound 2 was much longer than compound 1.
½
Abbreviations: Cmax, maximum concentration; tmax, time to Cmax; aUc, area under the curve; lc, liquid chromatography; Ms, mass spectrometry; na, not available.
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a novel dual Pi3K–mTOr inhibitor
Table 3 in vitro enzyme and cellular activity of compounds 1
and 2 and nVP-BeZ235 (ic50, nM)
Table 5 Pharmacokinetics of compound 2 in rats with different
preformulations
Assay
NVP-BEZ235
Compound 1
Compound 2
Dose
Cmax
tmax
AUC0–t
F (%)
(mg/kg) (ng/mL) (hours) (ng⋅h/mL)
enzyme
Pi3Kα
mTOr
cell line
U87Mg
BT474
a549
sKOV-3
Pc-3
hcT116
786-0
33
8.5
16
7.8
2.5
Formulation 1a
Formulation 2b
Formulation 3c
Formulation 4d
5
5
4
4
50.8±17.5 4
34.5±11.7 4
449±55.6
281±73.6
1,568±58.3 8.45±0.32
2,549±352 14±1.93
2.02±0.28
1.33±0.34
0.85
129±11
6
6
3.7
10.7
11.8
6.7
8.3
50
104.8
127.3
265.9
98.22
103.6
217.9
264.5
29.48
33.66
63.16
51.17
33.73
100.2
65.65
205±32.6
Notes: a0.5% Mc (n=3); b0.5% Mc + 0.5% sDs (n=3); csolid-dispersion technology
(n=3); d30% DMF + 50% Peg400 + 20% (0.9% saline) (n=3). compound 2 was dosed
orally in rats at 4 or 5 mg/kg (n=3 per group). Blood was collected and the
concentrationofcompound2determinedbylc-Ms/Ms.Pharmacokineticparameters
were calculated by Winnonlin software with a noncompartmental model. The
bioavailability of compound 2 was particularly poor in routine formulations. Data
presented as mean ± standard deviation.
6.5
Abbreviations:Cmax, maximumconcentration;tmax, timetoCmax;aUc, areaunderthe
curve; Mc, methylcellulose; sDs, sodium dodecyl sulfate; DMF, dimethylformamide;
Peg, polyethylene glycol; lc, liquid chromatography; Ms, mass spectrometry.
Notes: inhibitory activity of compounds 1 and 2 and nVP-BeZ235 on Pi3Kα and
mTOr was assessed and cellular assay performed. compound 2 showed similar
inhibitory activity against Pi3K and mTOr to compound 1 and nVP-BeZ235. it also
exerted good inhibitory activity on the proliferation of several tumor-cell lines, eg,
U87Mg, sKOV-3, and Pc-3.
Plasma and tumor-tissue samples were collected for
PK analysis. Fresh tumor tissue obtained from nude mice
was collected and placed in cold 0.9% NaCl solution.
Samples were homogenized under standardized conditions
(tumor:buffer [v:v] 1:4). After homogenization, samples were
centrifuged at 22,000 g for 5 minutes at 4°C. The supernatant
was transferred into a clean microcentrifuge tube and kept
in ice-cold conditions until assayed.
penicillin, and 100 μg/mL streptomycin at 37°C under
5% CO2 atmosphere. Resuspended PC3 cells (5×106 cells
per animal) were injected subcutaneously into the right
flank in 90 mice. When the average tumor volume reached
100 mm3, animals were randomly assigned to four groups
(n=8 each). Animals were treated with compound 1 (15,
30, and 60 mg/kg) and vehicle (β-cyclodextrin) orally
once daily for 14 days. Tumor volume and body weight
were measured twice a week. The treatment:control (T/C)
ratio (tumor-volume ratio [%]) and tumor-growth inhibi-
tion (TGI; %) were calculated, and data are presented as
mean ± SEM. The experiment was terminated when the
mean tumor volume of the vehicle groups reached 1,500 mm3.
in vivo pharmacokinetics in animals
Compound 1 was dosed orally (60 mg/kg) with a for-
mulation of 10% β-cyclodextrin in BALB/c nude (n=9
male animals/group). Subsequently, 200 μL whole blood
was collected at 0.083, 1, and 6 hours for animals 1–3,
0.25, 2, and 8 hours for animals 4–6, and 0.5, 4, 24
hours for animals 7–9.
Table 4 liver-microsome stability of compound 1 in mouse, rat,
dog, and human species at 1 μM
Table 6 Plasma concentration of compounds 3 and 2 at the
same time points after dosing compound 3 in rats (intravenous
injection, 2 mg/kg)
Time point,
minutes
0
5
10
(nM)
20
(nM)
30
(nM)
60
(nM)
(nM)
(nM)
nude mouse
compound 1
compound 2
rat
compound 1
compound 2
Dog
compound 1
compound 2
human
compound 1
compound 2
Time (hours)
Compound 3 (ng/mL)
Compound 2 (ng/mL)
804
0
30.85
13.15
13.27
34.7
24.55
77.65
26.15
114.5
18.95
180.5
0.083
0.25
0.5
1
719±226
47.5±5.86
BQl
2,383.3±255.4
2,230±212.8
1,996.7±160.7
1,733.3±184.8
1,193.3±141.5
685.7±98
387.7±95.5
196.3±51.6
8.4±4.2
776
0
527
0
356
0
198
0
118
0
33
2.5
BQl
2
BQl
4
BQl
764
0
662
0
515
0
327
0
257
0
86
0
6
BQl
8
BQl
847
0
598
0
530
0
256
0
142
0
29
5
24
BQl
Notes: compound 3 was dosed as intravenous bolus in rats at 2 mg/kg (n=3 per
group), and blood was collected and prepared by centrifugation. The concentration
of compounds 2 and 3 was separately quantified by LC-MS/MS. After dosing,
compound 3 disappeared, while the concentration of compound 2 decreased slowly
with time. Data presented as mean ± standard deviation.
Notes: compound 1 was incubated with liver microsomes of the nude mice, sprague
Dawley rats, beagles, and humans. concentration of compounds 1 and 2 were
quantified by LC-MS/MS. Compound 1 was metabolized rapidly into compound 2,
and compound 2 was the dominant metabolite only in mouse microsomes. however,
the metabolic profiling of compound 1 in rat, dog, and human microsomes showed
small amounts of compound 2 in the products.
Abbreviations: BQL, below quantifiable limit; LC, liquid chromatography; MS,
mass spectrometry.
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Table 7 Pharmacokinetics in rats of compound 2 after iV and PO dosing of compound 3
IV
PO
Cl (L/h/kg) Vss (L/kg) AUC0–t (ng⋅h/mL)
t
(hours) Cmax (ng/mL) AUC0–t (ng⋅h/mL)
t
(hours) F (%)
½
½
compound 2a 0.23±0.03
0.85±0.05
8,687±1,209
3.24±0.22
143.3±25.9
1,695±1,033
9.04±4.33
12.2±9.4
Notes: aDosing compound 3 (dosage conversion calculated as compound 2) and detecting compound 2; PO at 4 mg/kg, iV at 2 mg/kg (n=3). compound 3 was dosed in rats
via iV (2 mg/kg) and PO (4 mg/kg) routes, with three animals per group; blood was collected and prepared by centrifugation. The concentration of compounds 2 and 3 was
separately quantified by LC-MS/MS. Pharmacokinetic parameters were calculated by WinNonlin software with a noncompartmental model. The bioavailability of compound 2
was only 12.2%, which was too low to be administered as an oral drug via the strategy of the prodrug. Data presented as mean ± standard deviation.
Abbreviations: iV, intravenous; PO, per os (oral); cl, clearance; Vss, volume of distribution at steady state; aUc, area under the curve; t , half-life; Cmax, maximum
½
concentration; lc, liquid chromatography; Ms, mass spectrometry.
Compound 2 in different formulations was administered points and prepared by centrifugation. Blood was centrifuged
orally to male Sprague Dawley rats (four groups, three male at 4,500 rpm for 5 minutes at 4°C, and plasma was subse-
animals/group). Formulations were: formulation 1, 5 mg/kg quently harvested and stored at -20°C until further analysis.
of compound 2 in 0.5% methylcellulose; formulation 2, Finally, animals were killed. Compound concentrations in the
5 mg/kg of compound 2 in 0.5% methylcellulose +0.5% plasma samples were determined by liquid chromatography
sodium dodecyl sulfate; formulation 3, 4 mg/kg of com- (LC)–MS/MS. PK parameters were calculated by WinNonlin
pound 2 (solid-dispersion technology); and formulation 4, software with a noncompartmental model.
30% dimethylformamide +50% polyethylene glycol
in vitro metabolic stability assay with
multiple-species liver microsomes
400 +20% saline. A total of 200 μL whole blood was col-
lected at 0.167, 0.5, 1, 2, 4, 6, 8, and 24 hours.
The PK parameters of compound 2 via dosing with In vitro metabolic stabilities of compound 1 in different
compound 3 were studied in male Sprague Dawley rats species were tested by incubating the compound with liver
(two groups, three male animals/group). Compound 3 was microsomes of nude mice, Sprague Dawley rats, beagles,
intravenously dosed at 2 mg/kg with a formulation of 30% and humans. The incubated system was composed of PBS
dimethylformamide and 70% sterile water. Subsequently, (100 mM), MgCl2 (20 mM), liver microsomes (20 mg
200 μL whole blood was collected at 0.083, 0.25, 0.5, 1, 2, protein/mL), compound 1 (1 μM), and β-NADPH (10 mM).
4, 6, 8, and 24 hours. Compound 3 was also orally dosed with Incubation of compound 1 and liver microsomes in the
the same formulation at 4 mg/kg, and blood-collection time absence of NADPH served as a negative control. Incubation
points were 0.167, 0.5, 1, 2, 4, 6, 8, and 24 hours.
was terminated by acetonitrile at different incubation times
All the aforementioned blood samples were collected (0, 5, 10, 20, 30, and 60 minutes). Subsequently, samples
into K2-EDTA-coated (2.5%) tubes at the designated time were centrifuged for 5 minutes at 12,000 rpm to precipitate
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a novel dual Pi3K–mTOr inhibitor
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the proteins. Supernatants were analyzed using optimized was operated at a temperature of 500°C, and the ion-spray
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compound 2 were monitored at the same time.
451.1/411.2, 451.1/368.1, 561.2/441.2, and 470.4/443.1 for
compounds 1, 2, 3, and NVP-BEZ235, respectively. The
major compound-specific parameter was collision energy at
Procedure of lc-Ms/Ms
All biological samples were analyzed by LC-MS/MS using a 40, 52, 42, and 48 eV, and the declustering potentials were
Shimadzu high-performance liquid-chromatography system 100, 101, 97, and 100 V for compounds 1, 2, 3, and NVP-
and MS (API 4000 equipped with an electrospray ioniza- BEZ235, respectively.
tion probe in positive-ion mode). The column used was a
statistical analysis
Waters XBridge C18 (2.1×50 mm, 5 μm). The binary-gradient
method consists of mobile phases A (0.1% formic acid in
water) and B (0.1% formic acid in acetonitrile) with a flow
rate of 0.50 mL/min. The gradient used was B held at 95%
for 1 minute, then decreased linearly to 10% in 4.5 minutes.
After B had been held at 10% for another 0.5 minutes, it was
brought back to 95% in 0.5 minutes, followed by a 1-minute
re-equilibration. The total cycle duration was 7.5 minutes.
All results are presented as mean ± SD. Differences between
treated groups and the vehicle-control group were analyzed
using one-way analysis of variance. The level of significance
was established at P,0.05.
Results
Discovery of the major metabolite of
The injection volume was 3 μL. MS with electrospray ioniza- compound 1
tion was used to quantify compounds by multiple-reaction PK/PD study of compound 1
monitoring. Curtain, nebulizing, auxiliary, and collision During preclinical development of the lead compound, ie,
gases were all nitrogen at settings of 30, 60, 65, and 6 psi, compound 1, the PK/PD study was conducted in a BALB/c
respectively. The LC-MS/MS turbo ion-spray interface nude mouse xenograft model with the PC3 cell line. Mice
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were dosed with compound 1 at 15, 30, and 60 mg/kg, was subsequently identified. After enrichment and purifi-
together with NVP-BEZ235 at 30 mg/kg. Compared with cation of the major metabolite from the plasma samples,
the vehicle group, the TGI of compound 1 increased from the chemical structure was confirmed by nuclear magnetic
37.3% to 82.1% as doses ascended from 15 mg/kg to 60 resonance (Figure 1) and named compound 2.
mg/kg; a dose–effect relationship of compound 1 was
To study fully the in vivo metabolite profile of com-
clearly observed. In the same experiment, NVP-BEZ235 pound 1, a separate PK study was conducted in BALB/c nude
also showed a similar effect, as illustrated in Figure 2. At mice by oral dosing at 60 mg/kg. The concentration of com-
30 mg/kg, the TGI of NVP-BEZ235 was 67.6% vs 59.7% pound 1 and its major metabolite (compound 2) in the plasma
for compound 1.
samples was analyzed and quantified using authentic com-
Combined with early research data, compound 1 and pounds 1 and 2 as reference standards. The PK parameters of
NVP-BEZ235 had comparable in vitro potency against PI3K compounds 1 and 2 are listed in Table 2, and the PK profile
andmTOR.However,withsimilarinvivoefficacyat30mg/kg, is shown in Figure 3. As can be seen in Table 2, the area
drugconcentrationsinplasmaandtumortissueofcompound 1 under the curve (AUC) of compound 2 was 3,404 ng⋅h/mL,
and NVP-BEZ235 differed largely, as shown in Table 1. which was approximately 50 times higher than compound 1.
At 30 mg/kg, concentrations of compound 1 in plasma and The maximum plasma concentration of compound 2
tumor tissue were 111 ng⋅h/mL and 5.33±1.97 ng/g, respec- was 448 ng/mL, approximately six times higher than
tively. Even at 60 mg/kg with an inhibition rate of 82.1%, compound 1.
concentrations of compound 1 in plasma and tumor tissue
were 328 ng⋅h/mL and 15.3±4.29 ng/g, respectively. Concen- characterization of compound 2
trations of both doses were much lower than NVP-BEZ235 enzyme and cellular inhibition assays
in plasma (4,779 ng⋅h/mL) and tumor tissue (310±61.8 Since the in vivo pharmacodynamic (PD) effect could not
ng/g). The discordance in exposures of the two compounds be explained by exposure to compound 1 and since in vivo
indicated the possibility of the contribution of another factor exposure to its major metabolite, compound 2, was much
to efficacy besides compound 1.
higher than the parent compound, enzyme activities against
PI3Kα and mTOR and antiproliferation of different cell
lines of compound 2 were tested. The results are presented
in Table 3. The IC50 of compound 2 against PI3Kα was
Identification and quantification of the major
metabolite
Compound 1 blood samples from the efficacy study in nude 7.8 nM, which was equivalent to compound 1 and lower
mice were reanalyzed by LC-MS/MS, and a major metabolite than NVP-BEZ235. The IC50 of compound 2 against mTOR
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a novel dual Pi3K–mTOr inhibitor
ratio of compound 3 transformed to compound 2
In an attempt to improve the solubility of compound 2, a
phosphate ester prodrug (compound 3) was synthesized. The
structure is shown in Figure 1. The PK study of compound 3
was performed in rats. Both the prodrug and the parent
drug were monitored simultaneously. As shown in Table 6,
after intravenous bolus dosing, plasma concentrations of
compound 3 and compound 2 were 719±226 ng/mL and
2,383.3±255.4 ng/mL, respectively, at the first time point
(0.083 hours), and the ratio of compound 2 to compound 3 was
3.3:1. At the second time point (0.25 hours), the concentration
of compound 2 was 2,230±212.8 ng/mL, which was much
higher than that of compound 3, and the ratio of compound 2
to compound 3 was 46.9:1. At 0.5 hours, compound 3 totally
disappeared, while the concentration of compound 2
decreased slowly with time. It was concluded that com-
pound 3 can be converted to compound 2 quickly and com-
pletely in vivo. After oral administration of compound 3 in
rats, however, it was undetectable, and only compound 2 was
detected in rat plasma (data not shown).
was 2.5 nM, which was slightly higher than NVP-BEZ235,
but much lower than compound 1. Overall, the enzymatic
potency of compound 2 against PI3Kα/mTOR was better
than compound 1 and equivalent to NVP-BEZ235. Com-
parison of the antiproliferative effect on different carcinoma
cell lines among three compounds showed compound 2
had strong inhibition on selected tumor-cell lines, with IC50
values equal or less than 100 nM, which was more potent
than compound 1. However, the IC50 values of NVP-BEZ235
on these cell lines were lower than compound 2 (Table 3).
ratio of compound 1 transformed to compound 2
Compound 1 was incubated with mouse, rat, dog, and
human liver microsomes. At six time points, concentrations
of compound 1 and compound 2 were analyzed. Results
are summarized in Table 4 and data at 60 minutes plotted
in Figure 4.
As incubation time increased, the concentration of
compound 1 in mouse liver microsomes decreased, while
the concentration of compound 2 increased. At 60 minutes,
the concentration of compound 2 was nearly ten times
higher than compound 1 (180.5 nM vs 18.95 nM). The
in vitro metabolic profiles of compound 1 in mouse liver
microsomes corresponded to those obtained from in vivo
xenograft models. The data indicated that in the mouse
liver microsomes, compound 1 metabolized rapidly to the
dominant metabolite – compound 2.
rat PK study of compound 2 via dosing compound 3
The PK parameters of compound 2 after dosing compound 3
in rats are summarized in Table 7. The clearance of com-
pound 2 in rats was 0.23±0.03 L/h/kg, the AUC0–t of intra-
venous and oral routes was 8,687±1,209 ng⋅h/mL and
1,695±1,033 ng⋅h/mL, respectively, and the bioavailability
of compound 2 in rats was 12.2%±9.4%, which was the same
as compound 2 with formulation 4 (Table 5).
However, in the rat, dog, and human liver microsomes,
no compound 2 was detected up to 30 minutes. Furthermore,
only 2.5 nM and 5 nM of compound 2 in the liver microsomes
of rats and humans were detected, respectively, at the
60-minute time point. It was concluded that compound 1
was metabolized into compound 2 dominantly only in mice
and not in rats, dogs, or humans.
Discussion
During the process of profiling a clinical lead candidate, it
was found that the drug concentration of compound 1 was
not correlated with its PD effect in the nude-mouse xenograft
model. Compound 1 had equivalent in vitro activity on
PI3Kα and mTOR to NVP-BEZ235. However, at a similar
tumor-inhibition rate in the nude-mouse xenograft model,
drug exposure of compound 1 was approximately 2.3% of
NVP-BEZ235. The question arises as to why the in vivo effi-
cacy in the xenograft model did not align with the exposure
to compound 1. We speculated that there was some “sub-
stance” contributing the PD effect to compound 1, which was
not detected at the time.
Oral bioavailability of compound 2 in rats
Compound 2 was dosed orally in rats using four different
formulations with doses of 5 mg/kg or 4 mg/kg. As shown in
Table 5, with simple suspensions A and B, the bioavailability of
compound 2 was very low: ~1%–2%. To improve the bioavail-
ability of compound 2, amorphous solid-dispersion formulation
and solvent-solubilized solution were developed and tested.
Bioavailability increased to 8.45% and 14%, respectively, but
was still insufficient to develop the oral dosage form later.
In order to answer this question, we reanalyzed plasma
samples of the nude-mouse xenograft model, and a major
metabolite (compound 2) was identified. In subsequent
studies, compound 2 was characterized, synthesized, and
quantified in the plasma samples of nude mice. These results
Prodrug strategy of compound 2
To improve further bioavailability, compound 3, the prodrug
of compound 2, was synthesized and characterized.
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were reconfirmed by another independent PK study in mice. A permeability study across the Caco-2 monolayer
After dosing compound 1, the AUC of compound 2 was showed that compound 2 had low permeability (PappAB
50 times higher than that of its parent, compound 1, and the #2.5×10-6 cm/s) and high efflux ratio of 22.7. Compound 2
PK/PD results revealed a good correlation with compound 2 can be classified as a Biopharmaceutic Classification System-
instead of compound 1.
In the subsequent experiment, the enzyme and cellular would be the reasons for the low bioavailability.
inhibitory activities of compound 2 were tested. Compound 2 Prodrug development is a common strategy to improve
Class Four compound, and poor solubility and permeability
had better in vitro activity than compound 1. Combined with the solubility of an active ingredient.19,20 Compound 3, a
the fact that the exposure of compound 2 was much higher phosphate ester of compound 2, was synthesized. The solu-
than compound 1 in nude mice, it can be concluded that the bility of the prodrug in pH 7 buffer was improved 5,000-fold
in vivo antitumor activities observed in the mouse xenograft compared to compound 2. Furthermore, PK studies through
model were due to compound 2 rather than compound 1.
intravenous and oral routes indicated that compound 3 con-
One thing to note is that compound 1 was metabolized verted into compound 2 immediately and completely, which
into compound 2 dominantly only in mice, but not in rats, reduced the complexity in developing this prodrug.
dogs, or humans. Since compound 2 is responsible for the
However, the PK study of compound 3 showed that
antitumor efficacy in the mouse xenograft model, but com- systemic exposure of compound 2 was not improved and
pound 1 is not metabolized into compound 2 in humans, the calculated bioavailability of compound 2 in rats was
the question arises: How can the efficacy observed in the about 12.2%±9.4%. Therefore, oral administration of the
xenograft models be translated into clinical practice? Would prodrug compound 3 may not be a valid option. Phosphate
compound 1 have enough clinical response if it were not ester is hydrolyzed by alkaline phosphatases,21,22 which are
metabolized into compound 2 in humans?
present in all tissues throughout the body and particularly
Inconsiderationofthemetabolicdifferenceofcompound1 enriched in the liver, bile duct, intestinal mucosa, kidney,
between mice and other species and the unavailability of a rat bone, and placenta. A phosphate ester prodrug introduced
xenograft model, further development of compound 1 was sus- intravenously is metabolized mainly by the enzymes in
pended. Moreover, due to the highly encouraging in vitro and the blood and liver. When orally dosed, compound 3 was
in vivo data of compound2, we decided to profile compound2, metabolized to compound 2 by the alkaline phosphatases in
the metabolite of compound 1, as a clinical lead candidate.
intestinal mucosa first. Due to poor permeability and high
Solid oral dosage is a preferred route of administra- efflux of compound 2, this explains why the prodrug did not
tion for medications, and thus the oral bioavailability of improve the exposure of compound 2 by oral administration.
compound 2 is a key parameter in its drug-like properties. Based on these results, further development of compound 3
Rat PK studies of compound 2 were conducted, and indicated will focus on the parenteral pathway.
thatitsbioavailabilitywasmuchlowerthan20%inratsandthat
In summary, the process of discovering and develop-
it was not a good candidate to be developed into an oral drug. ing a dual PI3K–mTOR inhibitor was not straightforward.
For oral drugs, bioavailability is impacted by absorp- Compound 2, a metabolite of compound 1, was discovered
tion and metabolism. As shown in Table 7, the clearance of firstly in plasma samples due to a lack of PK/PD correlation
compound 2 was 0.23±0.03 L/h/kg in rats, and the hepatic of compound 1 in the mouse xenograft model. Although
extraction ratio was only 5.4% based on the hepatic blood compound 1 was identified as the clinical lead candidate, its
flow of 70 mL/min/kg in rats. Such a low hepatic extraction candidacy was suspended because of greatly different meta-
ratio value indicated that the poor bioavailability of com- bolic profiles across species. On the basis of better inhibitory
pound 2 was not due to fast metabolism.
activities in enzymatic and cellular assays, compound 2, the
Solubility and permeability are the two major factors that active metabolite of compound 1, was explored further for
influence drug absorption. Therefore, the physicochemical possibilities of developing it as a clinical lead candidate.
properties of compound 2 were tested. The solubility of However, the poor solubility and permeability of compound 2
compound 2 in pH 7 buffer was less than 1 μg/mL, indicating made this difficult to pursue further. Fortunately, the phos-
compound 2 would not dissolve well in the gastrointestinal phate ester prodrug, compound 3, resolved the solubility
tract, or would precipitate out with increasing pH values issue. Although the low bioavailability of compound 3 is
when moving down the gastrointestinal tract. Therefore, problematic for its oral dosing, it has adequate solubility
the solubility may be the reason for the poor bioavailability. for intravenous formulation. Therefore, the development of
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a novel dual Pi3K–mTOr inhibitor
10. Netland IA, Førde HE, Sleire L, et al. Dactolisib (NVP-BEZ235) toxicity
in murine brain tumour models. BMC Cancer. 2016;16:657.
11. Wen PY, Omuro A, Ahluwalia MS, et al. Phase I dose-escalation study
of the PI3K/mTOR inhibitor voxtalisib (SAR245409, XL765) plus
temozolomide with or without radiotherapy in patients with high-grade
glioma. Neuro Oncol. 2015;17(9):1275–1283.
12. Dolly SO, Wagner AJ, Bendell JC, et al. Phase I study of apitolisib
(GDC-0980), dual phosphatidylinositol-3-kinase and mammalian target
of rapamycin kinase inhibitor, in patients with advanced solid tumors.
Clin Cancer Res. 2016;22(12):2874–2884.
a parenteral dosage form via the prodrug strategy became
attractive, especially for immuno-oncology therapy.23,24 The
combination of PD-1/PD-L1 antibody with traditional small
molecules has been confirmed to be beneficial to patients.25
It is thus desirable to develop an intravenous medication
of a novel dual PI3K–mTOR inhibitor for cancer therapy.
As such, further work will be published in the near future.
13. Chia S, Gandhi S, Joy AA, et al. Novel agents and associated toxicities
of inhibitors of the PI3K/Akt/mTOR pathway for the treatment of breast
cancer. Curr Oncol. 2015;22(1):33–48.
14. Wong MH, Xue A, Baxter RC, Pavlakis N, Smith RC. Upstream and
downstream co-inhibition of mitogen-activated protein kinase and PI3K/
Akt/mTOR pathways in pancreatic ductal adenocarcinoma. Neoplasia.
2016;18(7):425–435.
Acknowledgments
The authors sincerely acknowledge the previous work from
Dr Jianjun Xue, Dr Peng Peng, Dr Yan Zhang, Dr Chutian
Shu, and Dr Frank Wu.
15. Simioni C, Ultimo S, Martelli AM, et al. Synergistic effects of selective
inhibitors targeting the PI3K/AKT/mTOR pathway or NUP214-ABL1
fusion protein in human acute lymphoblastic leukemia. Oncotarget.
2016;7(48):79842–79853.
16. Wu F, Shu CT, inventors; Xuanzhu Pharma, assignee. PI3K and/or
mTOR inhibitor. World Intellectual Property Organization patent
WO2014040373. 2013 Sep 12.
17. Wu F, Zhang Y, inventors; Xuanzhu Pharma, assignee. Three-ring PI3K
and/or mTOR inhibitor. World Intellectual Property Organization patent
WO2013071698. 2012 Nov 16.
Disclosure
Yan Zhou, Genyan Zhang, Feng Wang, Jin Wang, Yanwei
Ding, Xinyu Li, Chongtie Shi, Jiakui Li and Chengkon Shih
all work for Xuanzhu Pharma, Jinan, China. The authors
report no other conflicts of interest in this work.
18. Wu F, Xue JJ, Li L, inventors; Xuanzhu Pharma, assignee. PI3K and/or
mTOR inhibitor prodrug. World Intellectual Property Organization
patent WO2014154026. 2014 Mar 28.
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