2-Methoxyoestradiol (2-MeOE2, Figure 1
Figure 1
Structures: compound 1, 2-MeOE2; compound 2, 2-MeOE2bisMATE; compound 3, 2-MeOE2-3S;
compound 4, EMATE; compound 5, 2-MeOE1; compound 6, 2-MeOE2-3MATE; compound 7, 2-MeOE2-17MATE.
, 1), a metabolite of oestradiol, is generated in vivo by catechol-O-methyl transferase,
an enzyme which is expressed in a plethora of mammalian tissues including the liver,
kidney, brain and red blood cells (Mannisto and Kaakkola, 1999). 2-Methoxyoestradiol
has been shown to inhibit the growth of ER+ and ER− breast cancer cell lines and HeLa
cells (Seegers et al, 1989; Lottering et al, 1992; Raobaikady et al, 2003). It does
not stimulate uterotropic growth, and has a low binding affinity for the oestrogen
receptor (Ball and Knuppen, 1980; Merriam et al, 1980), and therefore it must act
via an oestrogen receptor-independent mechanism (LaVallee et al, 2002). It also inhibits
the in vivo growth of xenografts derived from human MDA-MB-435 melanoma cells, Meth
A sarcomas, B16 melanomas and the multiple myeloma cell line KAS-6/1 in immunodeficient
mice (Fotsis et al, 1994; Klauber et al, 1997; Dingli et al, 2002). However, comparatively
high oral or intraperitoneal doses of 75 and 150 mg kg−1 day−1, respectively, of 2-MeOE2
were necessary to reduce the growth of melanoma or myeloma tumours (Klauber et al,
1997; Dingli et al, 2002). The relatively high doses of 2-MeOE2 required to inhibit
tumour growth in vivo in the mouse may be a corollary of poor gastrointestinal absorption
and/or rapid metabolic deactivation of the agent, although this remains to be experimentally
determined.
In addition to inhibiting the proliferation of cancer cells, 2-MeOE2 also possesses
antiangiogenic properties (Fotsis et al, 1994). Inhibition of human umbilical vein
endothelial cell (HUVEC) growth is used as a predictive assay of a drug's antiangiogenic
potential. The sulphamoylated product of 2-MeOE2, 2-methoxyoestradiol-bis-sulphamate
(2-MeOE2bisMATE, Figure 1, 2), was 60-fold more potent in an in vitro HUVEC growth-inhibition
assay (Newman et al, 2004). 2-Methoxyoestradiol-bis-sulphamate has also been shown
to inhibit neo-vascularisation in vivo in the mouse Matrigel plug model of angiogenesis
(Chander et al, 2003).
2-MeOE2bisMATE is a derivative of oestrone-3-O-sulphamate (EMATE, Figure 1, 4) which
was originally developed as a steroid sulphatase (STS) inhibitor (Purohit et al, 1998).
Unexpectedly, EMATE proved to be a potent oestrogen on oral application in rats (Elger
et al, 1995). The observed oestrogenicity of oestrogen sulphamates, such as EMATE,
is thought to be a consequence of their sequestration into red blood cells (RBCs)
and slow release of the oestrogen moiety into plasma (Elger et al, 1998). It is conceivable
that, similarly, the sulphamoylation of 2-MeOE2, and the subsequent uptake of the
ester into erythrocytes, may reduce the first pass metabolism of the agent and thus
enhance its oral bioavailability. To investigate this postulate we compared the pharmacokinetics
of 2-MeOE2bisMATE and 2-MeOE2 following administration of a single oral or intravenous
dose in rats.
In addition, as part of the pharmacokinetic study, we also examined the metabolism
of these two compounds in vivo. As sulphamoylated steroids can act as STS inhibitors,
the ability of 2-MeOE2bisMATE to inhibit the in vivo activity of this enzyme was also
examined (Reed et al, 1996). To compare the abilities of 2-MeOE2 and 2-MeOE2bisMATE
to inhibit tumour growth in vivo, their effects on the growth of xenografts derived
from MDA-MB-435 (ER−) human melanoma cells were also examined. This cell line was
chosen for in vivo studies, as it had previously been used to test the efficacy of
2-MeOE2 (Klauber et al, 1997). Since the present study was initiated, it has been
revealed that the MDA-MB-435 cell line is not derived from a breast carcinoma, but
probably originates from a melanoma (Ellison et al, 2002). However, as this cell line
is highly metastatic and ER−, it remains a good model for testing agents which should
be active against a wide range of hormone-independent cancers.
MATERIALS AND METHODS
Chemicals and reagents
The following reagents were purchased from the suppliers listed: high-performance
liquid chromatography (HPLC) grade methanol, diethyl ether (Fisher Scientific UK Limited,
Loughborough, Leicestershire, UK); Halothane Astra Zeneca, Cheshire, UK); propylene
glycol, ammonium sulphate, sodium azide, tetrahydrofuran (THF) (Sigma-Aldrich Comp.
Ltd, Poole, Dorset, UK); 17α-epitestosterone (Steraloids, Newport, RI, USA); [6,7-3H]
oestrone sulphate (43-50 Ci mmol−1, Perkin-Elmer, Boston, MA, USA). 2-methoxyoestrone
(2-MeOE1, Figure 1, 5), 2-MeOE2, 2-MeOE2bisMATE, 2-MeOE2-3S (Figure 1, 3), 2-methoxyoestradiol-3-sulphamate
(2-MeOE2-3MATE, Figure 1, 6) and 2-methoxyoestradiol-17-sulphamate (2-MeOE2-17MATE,
Figure 1, 7) were synthesised from oestrone. All new compounds exhibited spectroscopic
and analytical data in accordance with their structure. Full details of their synthesis
will be reported elsewhere.
In vivo pharmacokinetic and metabolism studies
Female Wistar (155–165 g) rats were purchased from Charles River UK Ltd (Margate,
Kent, UK) and housed in a dedicated animal facility. Rats received RM1 rodent maintenance
diet (SDS, Kent, UK), water ad libitum, and were maintained in positive pressure isolators
under a 12 h light–dark cycle. These experiments were carried out under conditions
that complied with institutional requirements. Rats received 2-MeOE2 or 2-MeOE2bisMATE
(10 mg kg−1, oral or intravenous), with control animals receiving vehicle only (propylene
glycol: THF, 9 : 1 v v−1). There were two reasons for selecting 10 mg kg−1 for both
oral and intravenous dosing. Firstly, a single dose of EMATE administered by these
routes has been shown to inhibit rat liver sulphatase by at least 99% (Purohit et
al, 1995). Secondly, administration of this dose was found to elicit sufficiently
high levels of the agents for their detection in plasma. Rats were subjected to terminal
anaesthesia (Halothane) and blood removed by cardiac puncture at 5, 15 and 30 min
and 1, 3, 8 and 24 h after intravenous administration, and 15 and 30 min and 1, 3,
8, 24 and 48h following oral administration of 2-MeOE2 or 2-MeOE2bisMATE. Plasma was
prepared from whole blood by centrifugation (2800 g, 4°C, 15 min). Plasma (0.5 ml)
was extracted with diethyl ether (4 ml) and frozen in a methanol : solid carbon dioxide
mixture. 17α-Epitestosterone (28 μg ml−1) was used as an internal standard after purification
by HPLC. The organic phase was decanted to a fresh tube and evaporated to dryness
under a stream of air at room temperature. The extraction efficiencies for 2-MeOE2
and 2-MeOE2bisMATE from plasma were 71±5 and 75±4% (n=6), respectively. The residues
were stored at −20°C until analysis by HPLC.
HPLC analysis
2-MeOE2bisMATE was separated from its putative metabolites, 2-MeOE2, 2-MeOE2-3MATE
and 2-MeOE2-17MATE, using a modified version of a reversed-phase HPLC method described
previously (Hildago Aragones et al, 1996). An Agilent 1100 (Cheshire, UK) autosampler,
photodiode array detector and solvent delivery system were used. The agents were separated
from endogenous plasma components by an isocratic mobile phase consisting of 58% methanol
in 0.02 M ammonium sulphate. Sodium azide (1 mM) was added to the mobile phase in
order to decrease microbial growth. Extracted samples were reconstituted in mobile
phase and aliquots of 100 μl were injected on to a C3-phenyl column (250 × 5 mm, 5 μm)
purchased from Phenomenex (Cheshire, UK). 2-Methoxyoestradiol-bis-sulphamate, 2-MeOE2
and their metabolites were analysed with a photodiode array detector with detection
at 285 nm. The method was validated by spiking plasma with 2-MeOE2bisMATE (960 ng ml−1).
The inter-day and intra-day coefficients of variation were 8.2% (n=6) and 3.8% (n=6),
respectively. Quantification was achieved by spiking plasma with 2-MeOE2bisMATE and
the internal standard 17α-epitestosterone, extraction with diethyl ether and subsequent
analysis by HPLC. Plasma calibration curves were found to be linear from 40 to 9000 ng ml−1.
The limits of detection (LODs) and quantification of 2-MeOE2bisMATE in plasma were
11 and 40 ng ml−1, respectively.
Pharmacokinetic analysis
Pharmacokinetic parameters were calculated using WinNonlin software (Pharsight Corporation,
Mountview, CA, USA). The area under the curve (AUC) was calculated using the linear
trapezoidal method, with extrapolation of the terminal phase to infinity. Other parameters
calculated were: distribution and elimination rate constants (α and β); total body
clearance (Cl)=dose/AUC; volume of distribution (Vd)=Cl/β; distribution half-life
(t
1/2
α)=0.693/α; elimination half-life (t
1/2
β)=0.693/β; bioavailability (% F=(AUCoral/AUC
i.v
) × 100).
Liver oestrone sulphatase activity
Livers were obtained from animals administered with 2-MeOE2 or 2-MeOE2bisMATE either
orally or intravenously, to assess their effects on steroid sulphatase activity. Steroid
sulphatase activity was measured as described previously (Purohit et al, 1995).
In vivo inhibition of tumour growth
To compare the anticancer effects of 2-MeOE2 and 2-MeOE2bisMATE, xenografts derived
from MDA-MB-435 (ER−) human melanoma cells were transplanted into female nude mice
with eight mice per group. These studies were carried out by Anti-Cancer Inc. (San
Diego, CA, USA). Treatment was initiated when tumour volumes reached 100–200 mm3.
Drugs were dissolved in a minimum volume of THF, diluted with propylene glycol and
administered at 20 mg kg−1, oral, daily for 28 days. The length (l) and width (w)
of tumours was measured at weekly intervals, from which the tumour volumes were calculated
using the formula (l × w
2
/2). Monitoring of tumour volumes continued for a further 28-day period after the
end of drug administration. The body masses of control and treated animals were also
measured at weekly intervals as an indicator of any toxicity that might be associated
with the use of these drugs.
Statistics
Student's t-test was used to assess the significance of differences in tumour volumes
between control and treated animals.
RESULTS
HPLC analysis
A reproducible, robust and sensitive analytical method was developed for the detection
of 2-MeOE2 and 2-MeOE2bisMATE in plasma. The method facilitated separation of these
agents from the putative metabolites 2-MeOE1, 2-MeOE2-17MATE and 2-MeOE2-3MATE, the
internal standard, 17α-epitestosterone, and endogenous plasma components.
Pharmacokinetics of 2-MeOE2 and 2-MeOE2bisMATE
In order to compare the pharmacokinetics of 2-MeOE2 and 2-MeOE2bisMATE, rats were
administered with either a single intravenous or oral dose of the agents. Using the
HPLC analytical method developed, it was possible to detect both compounds in plasma
after intravenous administration (Figure 2B, C
Figure 2
HPLCs of diethyl ether plasma extracts. Plasma was prepared from whole blood following
administration of intravenous (A) propylene glycol, (B) 2-MeOE2, (C) 2-MeOE2bisMATE
or oral, (D) propylene glycol, (E) 2-MeOE2 and (F) 2-MeOE2bisMATE. The blood was removed
from animals 0.25 and 3 h after intravenous and p.o. dosing, respectively. The identities
of 2-MeOE2, 2-MeOE2bisMATE and 17α-epitestosterone (internal standard) as being peaks
1, 2 and 3, respectively, were corroborated by co-elution with authentic standards
and liquid chromatography-mass spectrometry. For details of extraction and HPLC analysis,
see ‘Materials and Methods’. The peak eluting at 3.9 min was an endogenous plasma
component.
), but only 2-MeOE2bisMATE was detected after oral administration (Figure 2E, F).
The identities of these agents were corroborated by electrospray ionisation liquid
chromatography-mass spectrometry in the selected ion mode (results not shown). When
2-MeOE2 was given to rats as an intravenous bolus, the agent was found to be rapidly
removed from the plasma (Figure 3A
Figure 3
Concentrations of 2-MeOE2 and 2-MeOE2bisMATE in rat plasma following a single intravenous
(A) or oral (B) bolus of drug (10 mg kg−1) in THF/propylene glycol. The values shown
are the means ±s.e.m. (n=3). 2-Methoxyoestradiol was below the LOD (11 ng ml−1) in
plasma after oral administration. Where no error bars are shown, the coefficient of
variation is <10%.
) and was below the LOD (11 ng ml−1) 1 h after administration of the dose. Consequently,
it was not possible to determine the pharmacokinetic parameters for 2-MeOE2. In contrast,
when 2-MeOE2bisMATE was administered as an intravenous bolus, the parent compound
was quantifiable in plasma 8 h after administration of the agent (1.52±0.10 μg ml−1),
although it was not detectable by 24 h. The decrease in plasma concentration of 2-MeOE2bisMATE
followed a biexponential pattern with an initial distribution half-life (t1/2
α) of 0.22±0.10 h and a terminal half-life (t
1/2
β) of 8.50±1.16 h. The area under the plasma concentration–time curve (AUC) was 513.99±153.88 h μg ml−1.
Pharmacokinetic parameters after oral administration were calculated using an extravascular
noncompartmental model. Maximum levels of 2-MeOE2bisMATE in plasma (C
max=3.90±0.25 μg ml−1) were reached 3 h after oral administration of the agent (Figure
3B and Table 1
Table 1
Summary of 2-MeOE2bisMATE pharmacokinetic data after bolus intravenous or oral administration
PK parameters
Intravenous
Oral
Cmax (μg ml−1)
6.55±0.92
3.90±0.25
t
1/2
α (h)
0.22±0.10
—
t
1/2
β (h)
8.50±1.16
6.51±1.09
AUC (h μg ml −1)
513.99±153.88
388.84±28.84
Vd (l)
0.28±0.09
0.35±0.02
Cl (l h−1)
0.05±0.01
0.036±0.001
% F (bioavailability)
—
85.19±16.09
Values shown are mean±s.e.m. (n=3).
) with 0.53±0.04 μg ml−1 still detectable after 24 h. A terminal plasma half-life
of 6.51±1.09 h with an AUC of 388.84±28.84 h μg ml−1 was achieved. The bioavailability
for 2-MeOE2bisMATE based on AUC after intravenous or oral administration was 85.19±16.09%.
The putative metabolites of 2-MeOE2bisMATE, 2-MeOE2-17MATE, 2-MeOE2-3MATE and 2-MeOE2
were not above the LOD in plasma.
Inhibition of liver sulphatase
Hepatic sulphatase activity was inhibited by 99.5±0.5% compared with the activity
measured in control animals, following administration of a single oral or intravenous
dose of 2-MeOE2bisMATE. This level of inhibition of sulphatase activity was maintained
for 48 h following administration of 2-MeOE2bisMATE. 2-Methoxyoestradiol did not affect
rat liver sulphatase activity (data not shown).
In vivo inhibition of tumour growth
To compare the effects of 2-MeOE2 and 2-MeOE2bisMATE on in vivo tumour growth in nude
mice that had been inoculated with xenografts derived from MDA-MB-435 human melanoma
cells, animals were treated with drugs (20 mg kg−1, oral) daily for 28 days (Figure
4
Figure 4
In vivo effect of 2-MeOE2 or 2-MeOE2bisMATE on the growth of MDA-MB-435 (ER−) xenografts
in nude mice. Vehicle (THF/propylene glycol) or compounds were administered daily
(20 mg kg−1, oral) for 28 days. Tumour volumes were monitored at weekly intervals
for the duration of drug administration and for a further 4-week period after the
end of drug administration (means±s.e.m., n=8). Results are expressed as percentage
change in tumour volumes detected at weekly intervals. At this dose, 2-MeOE2 had no
significant effect on tumour growth during the treatment or post-dosing periods. For
2-MeOE2bisMATE-treated animals, tumour volumes were significantly smaller than those
in control animals by week 2 (P<0.05) and remained significantly smaller (P<0.01)
for the rest of the study period.
). At this dose, 2-MeOE2 had no significant effect on tumour growth during the treatment
or post-dosing periods. In contrast, by week 2 of dosing, the tumour volumes of mice
receiving 2-MeOE2bisMATE were significantly smaller (P<0.05) than those of the control
group. Tumour volumes for mice treated with 2-MeOE2bisMATE then remained significantly
smaller (P<0.01) for the remaining period of the study. At 4 weeks, at the end of
the dosing period, the mean tumour volumes in mice receiving 2-MeOE2bisMATE were 14%
of that of tumour volume in the control animals. This difference (86%) in tumour volumes
between the two groups was maintained for a further 4 weeks after the cessation of
drug administration. No significant differences in the change in weights between the
control and treated animals were detected during the treatment or post-dosing periods
(Figure 5
Figure 5
Changes in weights of control animals and those administered 2-MeOE2 or 2-MeOE2bisMATE.
Animals were weighed at weekly intervals and the results are expressed as the percentage
change compared with their pre-dosing weights (means±s.e.m., n=8).
).
DISCUSSION
The sulphamoylated oestrogen derivative 2-MeOE2bisMATE has previously been shown to
be a more potent inhibitor of cancer cell proliferation than its parent compound,
2-MeOE2 (Raobaikady et al, 2003). Furthermore, in in vitro models of angiogenesis,
2-MeOE2bisMATE proved to be considerably more potent as an inhibitor of angiogenesis
than 2-MeOE2 (Newman et al, 2004). The main finding to emerge from the present in
vivo study goes some way to offer an explanation for the enhanced potency of 2-MeOE2bisMATE.
In this study, the plasma concentrations of both drugs were determined after oral
or intravenous dosing, together with an examination of the duration for which the
compounds and putative metabolites were detectable in plasma. After intravenous administration
of 2-MeOE2, the compound was rapidly cleared from the plasma, suggesting metabolic
removal. 2-Methoxyoestradiol eluded detection in plasma after its oral administration
and, by 1 h after intravenous dosing, levels were below the LOD for the assay. In
contrast, significant concentrations of 2-MeOE2bisMATE were detectable in plasma after
its oral or intravenous administration with 450 ng ml−1 still being detectable at
24 h after oral dosing. Recently, a daily oral dose of 1000 mg 2-MeOE2 was given to
24 patients with advanced metastatic breast cancer (Sledge et al, 2002; Lakhani et
al, 2003). It was shown that 2-MeOE2 was metabolised to 2-MeOE1 and 2-MeOE2/2-MeOE1
glucuronide and sulphate conjugates. Demethylated 2-MeOE1 (2-OHE1) has also been measured
in the urine of humans administered with [3H]2-MeOE2, although this metabolite could
not be detected in plasma (Longcope et al, 1980).
After intravenous administration of 2-MeOE2, its half-life in plasma was approximately
14 min. Pharmacokinetic studies in rats indicated that 2-MeOE2bisMATE was detectable
in plasma for up to at least 24 h after oral administration. The calculated bioavailability
for 2MeOE2bisMATE from the AUC was 85.2%. A possible explanation for this finding
may be that, initially, there is sequestration of 2-MeOE2bisMATE into blood components,
possibly RBCs (unpublished data), or binding to plasma proteins, resulting in high
AUC orally. This may, in part, account for the slow clearance rates and the high volume
of distribution achieved by both intravenous and oral administration. Nevertheless,
it is apparent that 2-MeOE2bisMATE has a higher level of bioavailability after oral
administration, compared with that of the non-sulphamoylated 2-MeOE2.
Further evidence of the enhanced potency of 2-MeOE2bisMATE compared with that of 2-MeOE2
was obtained from the in vivo study carried out in nude mice bearing xenografts of
MDA-MB-435 tumours. 2-Methoxyoestradiol had little effect on the growth of these tumours.
In contrast, for animals receiving 2-MeOE2bisMATE, tumour growth was almost completely
inhibited for the 4-week treatment period, and its growth-inhibiting effects were
maintained for 4 weeks after the cessation of dosing. It is evident from the present
study that 2-MeOE2bisMATE can inhibit in vivo tumour growth. Further studies will
be required to ascertain whether it is acting by inhibition of angiogenesis or induction
of apoptosis or, as most likely, a combination of both mechanisms. No decreases in
body weights were detected in animals receiving either 2-MeOE2 or 2-MeOE2bisMATE,
indicating that, at the dose tested, these compounds have little or no toxicity.
From these studies, it is evident that 2-MeOE2bisMATE is resistant to metabolism in
vivo, and this finding goes some way to account for its enhanced potency compared
with 2-MeOE2. In vivo, the C3 phenolic group and 17β hydroxyl group of 2-MeOE2 are
likely to be subjected to rapid conjugation or oxidation, thus reducing the potency
of this compound as an anticancer and antiangiogenic drug. Several derivatives of
2-MeOE2 have been developed, such as 2-methoxymethyloestradiol and a series of 2-methoxytetrane
oestradiols, which are more potent inhibitors of in vitro cancer cell proliferation
than 2-MeOE2 itself (Bruggemeier et al, 2001; Tinley et al, 2003). However, as these
compounds retain the C3/17β configuration of 2-MeOE2, it is unlikely that they will
prove to be more potent in vivo and will undergo the same route of rapid inactivation.
EMATE was previously found to have a half-life of 4.5 h in plasma (Hildago Aragones
et al, 1996), and this compares with the much shorter half-life for unconjugated oestrogen
of 20–30 min (Ruder et al, 1972). Like EMATE, 2-MeOE2bisMATE is a potent steroid sulphatase
inhibitor and, in the present study, liver sulphatase was rapidly inactivated after
its oral or intravenous administration. Almost complete inhibition of steroid sulphatase
activity (>99%) was achieved within 5 and 15 min of intravenous and oral dosing, respectively.
Inhibition of steroid sulphatase activity will prevent removal of the sulphamoyl group
at the C3 position, thus greatly prolonging the plasma, and presumably tissue, concentrations
of the active sulphamoylated drug. The relatively high plasma concentrations of 2-MeOE2bisMATE
(up to 5 μ
M) detected are within the range that has been shown to inhibit cancer cell and HUVEC
proliferation in vitro (Newman et al, 2004). Thus, sulphamoylation of 2-MeOE2 yields
a drug that is resistant to in vivo metabolism. In vivo, its ability to inhibit STS
appears to block its de-sulphamoylation, ensuring that the biologically active drug
is available to exert its effects for a prolonged period of time. The findings from
in vitro studies that 2-MeOE2bisMATE is more potent than 2-MeOE2, together with the
results of the in vivo studies showing no evidence of conversion to 2-MeOE2, indicate
that it is not acting as a pro-drug for the formation of 2-MeOE2.
There is currently considerable interest in exploring the mechanisms by which 2-MeOE2
and its sulphamoylated derivates act to inhibit cancer cell growth and angiogenesis.
2-Methoxyoestradiol is an endogenous oestrogen metabolite, and it has been suggested
that it may be the body's natural anticancer metabolite (Zhu and Conney, 1998). This
contention is supported by a significant body of research carried out by Bradlow et
al (1998), who have convincingly demonstrated that production of 2-MeOE2 is increased
in women at low risk of breast cancer. 2-Methoxyoestradiol and its sulphamoylated
derivatives are thought to bind to the colchicine-binding site of tubulin, where they
act to alter microtubule dynamics, leading to activation of the intrinsic apoptotic
pathway via BCL-2 phosphorylation (D'Amato et al, 1994; MacCarthy-Morrogh et al, 2000).
In addition, it is possible that, like 2-MeOE2, the sulphamoylated derivatives will
also act to induce apoptosis via activation of the extrinsic apoptotic pathway, which
involves upregulation of death receptors, such as DR5 (LaVallee et al, 2003).
In vitro, 2-MeOE2 has been shown to inhibit the proliferation of a wide range of cancer
cells including those derived from the breast, prostate, ovary, pancreas, lung and
multiple myelomas. At relatively high doses (75–150 mg kg−1), its efficacy in mouse
models against melanomas, Meth A sarcomas and multiple myelomas has been demonstrated
(Klauber et al, 1997; Dingli et al, 2002). As shown in the present in vivo efficacy
study at a dose of 20 mg kg−1, while 2-MeOE2 was ineffective, 2-MeOE2bisMATE almost
completely blocked tumour growth. The identification of 2-MeOE2bisMATE as a potent
anticancer, antiangiogenic drug that is orally available, yet resistant to metabolism,
should make it a good candidate for development for cancer therapy.