Novel alkynylphosphonate analogue of calcitriol with potent antiproliferative effects in cancer cells and lack of calcemic activity

Article abstract of DOI:10.1021/ml200034w

Here, we describe the design and synthesis of diethyl [(5Z,7E)-(1S,3R)-1,3- dihydroxy-9,10-secochola-5,7,10(19)-trien-23-in-24-yl] phosphonate (compound 10), which combines the low calcemic properties of phosphonates with the decreased metabolic inactivat

Full text of DOI:10.1021/ml200034w

LETTER
pubs.acs.org/acsmedchemlett
Novel Alkynylphosphonate Analogue of Calcitriol with Potent
Antiproliferative Effects in Cancer Cells and Lack of Calcemic Activity
Dꢀebora G. Salomꢀon,† Silvina M. Grioli,† Maximiliano Buschiazzo,† Evangelina Mascarꢀo,‡ Cristian Vitale,‡
Gabriel Radivoy,‡ Manuel Perez,§ Yagamare Fall,§ Enrique A. Mesri,|| Alejandro C. Curino,† and
María M. Facchinetti*,†
†Laboratorio de Biología Bꢀasica del Cꢀancer, Instituto de Investigaciones Bioquímicas Bahía Blanca (INIBIBB), 8000, Bahía Blanca,
Argentina
‡Departamento de Química, Instituto de Química del Sur (Inquisur-Conicet), Universidad Nacional del Sur, 8000 Bahía Blanca,
Argentina
§Departamento de Química Orgꢀanica, Facultad de Química, Universidad de Vigo, 36200, Espa~na
Microbiology and Immunology & Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami,
Florida, 33136, United States
S Supporting Information
b
ABSTRACT: Here, we describe the design and synthesis of diethyl [(5Z,7E)-(1S,3R)-
1,3-dihydroxy-9,10-secochola-5,7,10(19)-trien-23-in-24-yl] phosphonate (compound
10), which combines the low calcemic properties of phosphonates with the decreased
metabolic inactivation due to the presence of a triple bond in C-24 and studied its in vitro
effects on several cancer cell lines and its in vivo effects on blood calcium levels. We
demonstrate that this compound is a potent antiproliferative vitamin D analogue,
showing lack of calcemic effects in vivo.
KEYWORDS: 1R,25-(OH)2 vitamin D3, alkynylphosphonate analogues, cancer, hyper-
calcemia, cellular proliferation
ihydroxyvitamin D3 (calcitriol) has been
1R,25-Dshown to exert control over a multitude of
biological processes related to calcium and phosphorus home-
ostasis, cell proliferation, differentiation, and apoptosis.1ꢀ3 The
potent growth inhibitory effect, combined with the presence of
the vitamin D receptor (VDR) in a wide variety of cells, makes
calcitriol an ideal compound to treat hyperproliferative disorders
such as cancer. However, major side effects such as hypercalce-
mia have severely hampered its therapeutic application. One way
to overcome this problem is to design structural analogues of
calcitriol with the same or even increased antiproliferative and
pro-differentiating activities and with reduced undesired effects
on calcium and bone metabolism. Several of these analogues have
been synthesized and tested in various cell lines and animal
models, in some cases with promising results.3ꢀ5 Nevertheless,
considerable variation in the antitumoral response to analogues
has been observed among different types of cells and different
type of tumors. The differential response of tumor cells to these
analogues might be explained by the different level of interaction
of VDR with coactivators upon analogue binding,3 differences in
binding to VDR or to vitamin D binding protein (DBP), or
differences in drug metabolism.4,6 Although the molecular path-
ways involved in the antitumor effects of calcitriol and analogues
are not clear, substantial preclinical data support the hypothesis
that vitamin D compounds may play an important role in cancer
therapy and prevention. The preclinical data were rapidly
followed by clinical trials in humans, and although many trials
with vitamin D analogues have been conducted in cancer
patients, the results have sometimes been disappointing.5 For
example, although initial phase II data suggested some beneficial
effects of vitamin D analogue EB1089, a large trial in patients with
hepatocellular carcinoma was negative.7 Most studies have
administered vitamin D analogues orally on a continuous daily
dosing schedule, and hypercalcemia or hypercaliuria was en-
countered, thus limiting dose escalation. These concerns about
induction of hypercalcemia by the analogues and the desire for
more potent agents have prompted the development of less
calcemic vitamin D analogues. These have to achieve high
concentrations in cancer cells to be biologically active, this being
greatly controlled by their cellular catabolism initiated by C-24
hydroxylation. The design of new analogues employs structural
modifications on the side chain or A ring to prevent the
inactivating hydroxylation, oxidation, and epimerization, which
are characteristic of calcitriol catabolism. In this regard,
Received: February 4, 2011
Accepted: April 11, 2011
Published: April 11, 2011
r
2011 American Chemical Society
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|

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Scheme 1. Synthesis of Alkynylphosphonate Vitamin D3 Analogue 10a
a Reagents and conditions: (a) TESCl, Im., DMAP, CH2Cl2, 0 °C, (97%). (b) DIBAL-H, CH2Cl2, ꢀ10 °C. (c) CBr4, PPh3, CH2Cl2, 0 °C to room
temperature. (d) (i) LDA, THF, ꢀ78 °C; (ii) ClPO(OEt)2, ꢀ78 °C (50% three steps from 2). (e) HF, CH3CN, room temperature, (92%). (f) PDC,
PPTS, CH2Cl2, room temperature, (97%). (g) (i) Compound 8, n-BuLi, THF, ꢀ78 °C; (ii) compound 7, ꢀ78 °C (60%). (h) HF, CH3CN, room
temperature, (84%).
Table 1. Half Maximal Inhibitory Concentration (IC50) of
the Cell Lines That Responded to Analogue Treatmenta
IC50
cell line
HN12
compound 10
calcitriol
22.3
113
553
SVEC vGPCR
T98G
0.52
36.1
1.68
4.78
LM05e
0.03
1.30
T47D
a Values are the means of at minimum three experiments and are given in
the nanomolar range.
Figure 1. Timeꢀcourse response analysis of compound 10 on cellular
survival of human head and neck squamous cell carcinoma HN12 cell line.
Cells were exposed to the indicated concentrations (nM) of compound 10
(C10) over a total time of 72 h. The experiment was repeated twice.
P values from Bonferroni post-test of ANOVA analysis are shown.
analogues from the corresponding alkynylphosphonate precursor
5. The synthesis of such analogues is currently under way in our
laboratory with a view to their biological evaluation.
phosphonate analogues have been shown to display a certain
degree of dissociation between the vitamin D activity in vitro and
undesired hypercalcemia in vivo.8 The first reference to vitamin
D analogues possessing phosphorus atoms in the side chain can
be found in the work of Dauben et al.9 Since then, few of such
derivatives have been described until Steinmeyer worked on
vitamin D phosphonate hybrids.8
We have now designed a convergent route for the synthesis of a
novel vitamin D analogue 10 bearing an alkynylphosphonate
moiety. This analogue combines the low calcemic properties of
phosphonates8 with the decreased metabolic inactivation due to the
presence of a triple bond in C-24.10 The synthetic approach is also
useful for the preparation of structurally related phosphonate
Our synthetic approach involves the construction of the vitamin
D triene system employing the convergent WittigꢀHorner cou-
pling between the ketone 7 and the phosphine oxide 8. The
synthesis of the key precursor 7 takes advantage of the readily
available Inhoffen diol to afford the nitrile 1 (Scheme 1).11,12
Protection of the hydroxyl group in 1 as a triethylsilyl ether
under standard conditions gave compound 2.13 This was treated
with diisobutylaluminium hydride in dichloromethane and
yielded the aldehyde 3, which was submitted as a crude to the
ylide prepared from CBr4 and PPh3 using CoreyꢀFuchs condi-
tions to give a dibromoalkene 4.14,15 Treatment of 4 with an
excess of lithium diisopropyl amide in THF led to the corresponding
acetylenic anion, which was trapped with diethyl chlorophosphate
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Figure 2. Doseꢀresponse effects of compound 10 (C10) on cellular survival and its comparison with calcitriol. (A) Human head and neck squamous
cell carcinoma HN12 (p = 0.0499), (B) human glioma T98G (p = 0.0014), (C) Kaposi sarcoma SVEC vGPCR (p e 0.0001), (E) human mammary
adenocarcinoma T47D (p e 0.0001), and (F) murine mammary adenocarcinoma LM05e (p e 0.0001) responded to compound 10 treatment with
reduced survival, whereas (D) the nonmalignant cell line SVEC, (G) human colorectal carcinoma HCT116, and (H) murine mammary adenocarcinoma
LM3 were not responsive to analogue treatment. Cells were exposed to the indicated doses of vehicle (isopropanol), compound 10, or calcitriol over a
total time of 72 h. Cellular proliferation was expressed as percentage of the vehicle. The experiments were repeated at least three times for each cell line.
P values from Bonferroni post-test of one-way ANOVA analysis for comparisons of reduction in cellular survival with compound 10 are shown.
to afford the alkynylphophonate 5.16 This was deprotected
by treatment with HF in acetonitrile to give alcohol 6, which
afforded ketone 7 after further oxidation with pyridinium
dichromate.
The WittigꢀHorner reaction of 7 with the anion derived from
the phosphine oxide 8 yielded the protected analogue 9. Sub-
sequent removal of the silyl protecting groups afforded the
desired analogue 10.11,12 Following this eight-step synthetic
sequence, 10 was achieved in 22% overall yield from 1.
The synthesized vitamin analogue (compound 10 hereafter)
was tested for its antiproliferative effects on several human and
murine tumor cell lines. In preliminary testing, we performed
a timeꢀcourse response experiment (0ꢀ72 h) in the human
squamous cell carcinoma cell line HN12 with different
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Figure 3. VDR silencing does not alter the antiproliferative effects exerted by compound 10 on the glioma cell line T98G. (A) Cellular count following
transfection of cells with a plasmid silencing VDR (shRNA VDR) or a scrambled shRNA (CTRL shRNA). Cells were treated for 48 h after transfection
with compound 10 (C10, 10 nM) or vehicle, and cell count was performed 72 h following treatments. The experiment was repeated three times and was
done in triplicate. Shown is the mean ( SE. (B) Silencing of VDR with shRNA plasmid (p FIV-H1-Puro shRNA VDR) in T98G cells: CTRL plasmid
(lane 1) or 2 (lane 2) and 4 μg (lane 3) of VDR shRNA plasmid were transfected using lipoafectamine reagent, and protein lysates were performed
48 h later.
concentrations of compound 10 (0.1ꢀ100 nM) and observed
a significant decrease in cell number at 72 h (Figure 1) starting
at 1 nM.
limitations in its metabolization through 24-hydroxylation due to
the presence of a triple bond between the carbons 23 and 24, so
its metabolic transformation might be reduced.10 This is relevant
in cells showing important activity of CYP24A1 such as astro-
cytes,20 prostate cells,21 and also in colon, ovary, lung tumors, 22
and glioma.23 Importantly, in human glioma cell lines, the natural
hormone either does not exert antiproliferative activity or it
increases proliferation,23,24 whereas compound 10 potently in-
hibits cellular survival, as shown in this report. The potential
differences in the metabolic degradation between calcitriol and
compound 10 might account for the differences observed in the
antiproliferative response; therefore, compound 10 might be
useful for the treatment of human gliomas. Preliminary experi-
ments showed that VDR is not necessary for the antiproliferative
effects observed in the glioma cell line following compound 10
treatment (Figure 3), thus suggesting the involvement, at least in
part, of nongenomic effects elicited by this analogue. This is in
accordance with previously published results showing no upre-
gulation of VDR by calcitriol in several glioblastoma cell lines and
the presence of low levels of the receptor mRNA in human
biopsies of these tumors.23 Our results demonstrating compound
10 antiproliferative effects on breast cancer cell lines that are
hormone-responsive are also supported by previous observations
showing that the sensitivity to calcitriol is higher in those
mammary cancer cell lines that express estrogen receptors.25
Because of its significant in vitro antiproliferative activity in
some tumor cells, compound 10 was evaluated for hypercalcemic
effects in vivo. Previous pharmacokinetic studies performed in
normal mice indicated that calcitriol at 0.125 μg/mouse (appro-
ximately 5 μg/kg body weight) results in a Cmax >10. 0 ng/mL
and AUC > 40.0 ng h/mL,26 which exceeds the concentration
needed for calcitriol antitumor activity in vitro. Therefore, we
chose doses of 5 and 20 μg/kg that have antitumor effects in vivo.
Mice were divided into three groups (n = 5/group) and given a
daily intraperitoneal injection of calcitriol, compound 10, or
vehicle at 5 or 20 μg/kg body weight for 5 days. Blood was
collected prior to dose administration, then at 24, 48, 72, and 96 h
post-treatment. Plasma calcium levels were measured by reading
the absorbance of metallochromic indicator Arsenazo III. Inter-
estingly, compound 10 showed no calcemic activity as observed
in Figure 4. Instead, calcitriol was effective at causing an increase
in plasma calcium, as expected. Moreover, mice that were treated
with calcitriol died after 3 days, whereas mice treated with
We subsequently performed 72 h doseꢀresponse analyses for
all of the cell lines, comparing the effects of compound 10 with
those elicited by the natural hormone calcitriol. Table 1 contains
the IC50 of cell lines that responded to compound 10 treatments.
As shown in Figure 2, we observed a significant decrease in cell
counting after treatment with compound 10 in human squamous
cell carcinoma HN12 (Figure 2A), human glioma T98G
(Figure 2B), and Kaposi sarcoma SVEC vGPCR (Figure 2C)
cell lines. Furthermore, in the human glioma T98G and in the
human squamous cell carcinoma HN12 cell lines, the antiproli-
ferative effects exerted by compound 10 were greater than those
elicited by calcitriol. Importantly, although calcitriol was more
potent than compound 10 in inhibiting the growth of Kaposi
sarcoma cells, the nonmalignant parental cell line, SVEC, did not
respond to compound 10 (Figure 2D). The human T47D
(Figure 2E) and the murine LM05e (Figure 2F) hormone-
sensitive breast adenocarcinoma cell lines also responded to
compound 10 with decreased survival. In contrast, the human
colorectal carcinoma HCT116 (Figure 2G) and the murine
hormone-insensitive breast adenocarcinoma LM3 (Figure 2H)
cell lines did not show reduced survival following compound 10
treatment. Longer treatment periods or higher concentrations of
compound 10 did not show significant antiproliferative effects.
It is known that cancer cell lines display a range of sensitivities
to the antiproliferative effects of calcitriol and its derivatives,
although the reason for this is largely unknown and could result
from defects in any of the components in the VDR signaling
pathway including VDR and 24-hidroxylase (CYP24A1). Calci-
triol action is limited by its catabolism, occurring mainly by the
CYP24A1 resulting in 1R,24,25-(OH)3-D3, a metabolite with
substantially lower affinity for the VDR. Although this enzyme is
located primarily in liver, many studies have demonstrated that it
can also be expressed by many tissues.17 The augmented expres-
sion of CYP24A1 has been shown to be detrimental to calcitriol
antiproliferative effects. For example, in prostate cancer cell lines,
it has been demonstrated that enzyme expression was inversely
correlated to the antiproliferative effects displayed by the cells.18
In addition, antagonists of the CYP24A1, such as azoles, have
been shown to potentiate the antitumor effects of calcitriol
in vitro and in vivo.19 In this regard, compound 10 presents
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LETTER
Dr. Tokino T. from the Sapporo Medical University (Japan)
for providing pFIV-H1-Puro shRNA VDR plasmids.
’ REFERENCES
(1) Bouillon, R.; Okamura, W. H.; Norman, A. W. Structure-func-
tion relationships in the vitamin D endocrine system. Endocr. Rev. 1995,
16, 200–257.
(2) Jones, G.; Strugnell, S. A.; DeLuca, H. F. Current understanding
of the molecular actions of vitamin D. Physiol. Rev. 1998, 78, 1193–1231.
(3) Eelen, G.; Verlinden, L.; De Clercq, P.; Vandewalle, M.; Bouillon,
R.; Verstuyf, A. Vitamin D analogs and coactivators. Anticancer Res. 2006,
26, 2717–2721.
(4) Beer, T. M.; Myrthue, A. Calcitriol in cancer treatment: from the
lab to the clinic. Mol. Cancer Ther. 2004, 3, 373–381.
(5) Trump, D.; Muindi, J.; Fakih, M.; Yu, W.; Johnson, C. Vitamin D
compounds: Clinical development as cancer therapy and prevention
agents. Anticancer Res. 2006, 26, 2551–2556.
(6) Brown, A. J.; Slatopolsky, E. Vitamin D analogs: Therapeutic applica-
tions and mechanisms for selectivity. Mol. Aspects Med. 2008, 29, 433–452.
(7) Dalhoff, K.; Dancey, J.; Astrup, L.; Skovsgaard, T.; Hamberg,
K. J.; Lofts, F. J.; Rosmorduc, O.; Erlinger, S.; Bach Hansen, J.; Steward,
W. P.; Skov, T.; Burcharth, F.; Evans, T. R. A phase II study of the
vitamin D analogue Seocalcitol in patients with inoperable hepatocel-
lular carcinoma. Br. J. Cancer 2003, 89, 252–257.
(8) Steinmeyer, A.; Schwarz, K.; Haberey, M.; Langer, G.; Wiesinger,
G. Synthesis and biological activities of a new series of secosteroids:
vitamin D phosphonate hybrids. Steroids 2001, 66, 257–266.
(9) Dauben, W. G.; Ollman, R.; Funhoff, A. S.; Neidlen, R. The
synthesis of 25-oxo-25 phosphavitamin D3. Tetrahedron Lett. 1991,
32, 4643–4646.
(10) Uskokovic, M. R.; Norman, A. W.; Maanschand, P. S.; Studzinski,
G. P.; Campbell, M. J.; Koeffler, H. P.; Takeuchi, A.; Siu-Caldera, M. L.;
Rao, D. S.; Reddy, G. S. Highly active analogs of 1alpha,25-dihydroxyvi-
tamin D(3) that resist metabolism through C-24 oxidation and C-3
epimerization pathways. Steroids 2001, 66, 463–471.
(11) Sardina, F. J.; Mouri~no, A.; Castedo, L. Studies on the synthesis
of side-chain hydroxylated metabolites of vitamin D. 2. Stereocontrolled
synthesis of 25-hydroxyvitamin D2. J. Org. Chem. 1986, 51, 1264–1269.
(12) Toh, J. T.; Okamura, W. H. Studies on a convergent route to
side-chain analogues of vitamin D: 25-hydroxy-23-oxavitamin D3. J. Org.
Chem. 1983, 48, 1414–1417.
(13) Fall, Y.; Torneiro, M.; Castedo, L.; Mourin~o, A. An efficient
stereoselective synthesis of 1R, 24(R)-dihydroxyvitamin D3 by the
dienyne route. Tetrahedron 1997, 53, 4703–4714.
(14) Corey, E. J.; Fuchs, P. L. A synthetic method for formylfethy-
nyl conversion (RCHOfRCtCH or RCtCR0). Tetrahedron Lett.
1972, 13, 3769–3772.
(15) Wang, L.; Shen, W. The stille reaction of 1,1-Dibromo-1-
alkenes: preparation of trisubstituted alkenes and internal alkynes.
J. Org. Chem. 1999, 64, 8873–8879.
Figure 4. Plasma calcium levels in mice in response to daily intraper-
itoneal injections of vehicle, calcitriol, or compound 10 during a period
of 5 days. Animals were injected with 5 μg/kg body weight of compound
10, calcitriol, or vehicle (isopropanol), and plasma calcium was mea-
sured before the injection (basal levels, 0 h) and at 24, 48, 72, and 96 h.
Values for calcitriol at 96 h are missing because animals died following
3 days of treatment due to hypercalcemia. Values are means ( SEs from
five animals in each group. The experiment was repeated two times.
compound 10 remained alive and healthy during the entire
examination period. The 20 μg/kg dose gave similar results for
compound 10 (not shown). Visual observation of the internal
organs of the animals such as liver, duodenum, lungs, and kidneys
showed no macroscopic morphological alterations after the
treatment with compound 10. Thus, this compound appears to
be well tolerated even at high doses.
Altogether, these results suggest that compound 10 exerts
considerable antiproliferative activity at nonhypercalcemic do-
sages and may have therapeutic potential for the treatment of
different hyperproliferative disorders.
’ ASSOCIATED CONTENT
Supporting Information. Synthetic procedures, 1H, 13
C
S
b
and 31P NMR spectral data of compounds 2ꢀ10, HRMS data for
compounds 5, 9ꢀ10 and HPLC data for alkynylphosphonate
analogue 10, and experimental procedures for biological assays.
This material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
(16) Gil, J. M.; Sung, J. W.; Park, C. P.; Oh, D. Y. One-pot synthesis
of 1-alkynylphosphonates. Synth. Commun. 1997, 27, 3171–3174.
(17) Trump, D. L.; Deeb, K. K.; Johnson, C. S. Vitamin D:
Considerations in the continued development as an agent for cancer
prevention and therapy. Cancer J. 2010, 16, 1–9.
(18) Moreno, J.; Krishnan, A. V.; Feldman, D. Molecular mechan-
isms mediating the anti-proliferative effects of vitamin D in prostate
cancer. J. Steroid Biochem. Mol. Biol. 2005, 97, 31–36.
(19) Muindi, J. R.; Yu, W. D.; Ma, Y.; Engler, K. L.; Kong, R. X.;
Trump, D. L.; Johnson, C. S. CYP24A1 inhibition enhances the
antitumor activity of calcitriol. Endocrinology 2010, 151, 4301–4312.
(20) Garcion, E.; Wion-Barbot, N.; Montero-Menei, C. N.; Berger,
F.; Wion, D. New clues about vitamin D functions in the nervous system.
Trends Endocrinol. Metab. 2002, 13, 100–105.
(21) Skowronski, R. J.; Peehl, D. M.; Feldman, D. Vitamin D and
prostate cancer: 1,25 dihydroxyvitamin D3 receptors and actions in
human prostate cancer cell lines. Endocrinology 1993, 132, 1952–1960.
Corresponding Author
*Tel: 54-291-4861700. Fax: 54-291-4861200. E-mail: facchinm@
criba.edu.ar.
Funding Sources
This work was supported by grants and fellowships awarded by
the National Council of Scientific and Technical Research
(CONICET), the National Agency for Scientific and Techno-
logical Promotion (ANPCyT), and the Universidad Nacional del
Sur (SGCyT-UNS), Argentina.
’ ACKNOWLEDGMENT
We thank the spectral service provided by the CACTI,
University of Vigo, Dr. Dodero V. I. from Inquisur (Bahía Blanca,
Argentina) for her kind assistance on HPLC analysis, and
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(22) Anderson, M. G.; Nakane, M.; Ruan, X.; Kroeger, P. E.;
Wu-Wong, J. R. Expression of VDR and CYP24A1 mRNA in human
tumors. Cancer Chemother. Pharmacol. 2006, 57, 234–240.
(23) Diesel, B.; Radermacher, J.; Bureik, M.; Bernhardt, R.; Seifert,
M.; Reichrath, J.; Fischer, U.; Meese, E. Vitamin D3 metabolism in
human glioblastoma multiforme: Functionality of CYP27B1 splice
variants, metabolism of Calcidiol, and effect of Calcitriol. Clin. Cancer
Res. 2005, 11, 5370–5380.
(24) Reichrath, S.; M€uller, C. S.; Gleissner, B.; Pfreundschuh, M.;
Vogt, T.; Reichrath, J. Notch- and vitamin D signaling in 1,25(OH)-
(2)D(3)-resistant glioblastoma multiforme (GBM) cell lines. J. Steroid
Biochem. Mol. Biol. 2010, 121, 420–424.
(25) Colston, K. W.; Hansen, M. Mechanisms implicated in the
growth regulatory effects of vitamin D in breast cancer. Endocr.-Relat.
Cancer 2001, 9, 45–59.
(26) Muindi, J. R.; Modzelewski, R. A.; Peng, Y.; Trump, D. L.;
Johnson, C. S. Pharmacokinetics of 1alpha,25-dihydroxyvitamin D3 in
normal mice after systemic exposure to effective and safe antitumor
doses. Oncology 2004, 66, 62–66.
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