Chemico-Biological Interactions 240 (2015) 146e152

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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Preclinical drug metabolism and pharmacokinetics of salinomycin, a

potential candidate for targeting human cancer stem cells
Kahkashan Resham a, Prinesh N. Patel b, Dinesh Thummuri a, Lalita Guntuku a,
Vanya Shah c, Ramesh B. Bambal c, V.G.M. Naidu a, *
a
Department of Pharmacology, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad, 500037, Telangana, India
b
Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad, 500037, Telangana,
India
c
Department of Pharmacokinetics and Metabolism, Daiichi Sankyo India Pharma Pvt Ltd, Village Sarhaul, Sector 18, UdyogVihar Industrial Area, Gurgaon,
122015, Haryana, India

a r t i c l e i n f o a b s t r a c t

Article history: There has been a search for new anticancer agents to treat cancer resistance throughout the globe.
Received 11 January 2015 Salinomycin (SAL), a broad spectrum antibiotic and a coccidiostat has been found to counter tumour
Received in revised form resistance and kill cancer stem cells with better efficacy than the existing chemotherapeutic agents;
9 July 2015
paclitaxel and doxorubicin. This refocused its importance for treatment of human cancers. In this study,
Accepted 10 August 2015
we studied the in vitro drug metabolism and pharmacokinetic parameters of SAL. SAL undergoes rapid
Available online 14 August 2015
metabolism in liver microsomes and has a high intrinsic clearance. SAL metabolism is mainly mediated
by CYP enzymes; CYP3A4 the major enzyme metabolising SAL. The percent plasma protein binding of
Keywords:
Salinomycin
SAL in human was significantly lower as compared to mouse and rat plasma. CYP inhibition was carried
Drug metabolism out by chemical inhibition and recombinant enzyme studies. SAL was found to be a moderate inhibitor of
Pharmacokinetics CYP2D6 as well as CYP3A4. As CYP3A4 was the major enzyme responsible for metabolism of SAL, in vivo
CYP pharmacokinetic study in rats was done to check the effect of concomitant administration of Ketoco-
In vitro metabolism nazole (KTC) on SAL pharmacokinetics. KTC, being a selective CYP3A4 inhibitor increased the systemic
In vivo pharmacokinetics exposure of SAL significantly to 7-fold in AUC0ea and 3-fold increase in Cmax of SAL in rats with
concomitant KTC administration.
© 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Salinomycin (SAL) has been used for more than 30 years as an
effective anti-coccidial drug in poultry and is also fed to ruminants
and pigs to improve nutrient absorption and feed efficiency. It is a
751 Da monocarboxylic polyether ionophore isolated from Strep-
tomyces albus, constituting a large pentacyclic molecule with a
unique tricyclic spiroketal ring system and an unsaturated six-
membered ring. It is a lipophilic, anionic and weakly acidic com-
pound with the molecular formula C42H70O11 [1e3]. SAL has been
shown to target Cancer Stem Cells (CSCs) in different types of hu-
man cancers, including gastric cancer [4], lung [5], osteosarcoma
* Corresponding author. Department of Pharmacology, National Institute of
Pharmaceutical Education and Research (NIPER), IDPL R & D Centre, Balanagar,
Hyderabad, Telangana, 500037, India.
E-mail addresses: vgmnaidu@niperhyd.ac.in, vgmnaidu@gmail.com
(V.G.M. Naidu).
[6], colorectal cancer [3], squamous cell carcinoma (SCC) [7], and
prostate CSCs [8] mainly by inhibiting wnt signalling pathway
[9e13]. SAL is able to enhance the cytotoxic effects of conventional
anti cancer drugs such as doxorubicin, gemcitabine, etoposide,
paclitaxel, docetaxel, vinblastine, and trastuzumab, envisioning a
central role for SAL-based combination therapies in the future
treatment of cancer [10,14e17].
One important caveat for developing SAL as a clinical treatment
for cancer is the paucity of preclinical metabolism and pharmaco-
kinetic data. Determination of drug metabolism and pharmacoki-
netic (DMPK) parameters and effect of these parameters on the
pharmacological effects of SAL will help researchers to design the
appropriate dose and also predict the interaction with other drugs
in the body. Since no scientific reports have been published as yet
regarding the DMPK parameters of this drug, it becomes a pre
requisite to perform these studies to minimize its toxicity as well as
have a complete understanding of this drug's behaviour in thera-
peutic use. In the present study, in vitro metabolism of SAL was

http://dx.doi.org/10.1016/j.cbi.2015.08.007
0009-2797/© 2015 Elsevier Ireland Ltd. All rights reserved.
 K. Resham et al. / Chemico-Biological Interactions 240 (2015) 146e152
studied in liver microsomes and S9 fractions for metabolic stability
and identification of CYP enzymes involved in the metabolism of
SAL along with in vitro plasma protein binding affinity studies.
2. Materials and methods
Salinomycin (SAL) was received as a gift sample from Bauji
GuangPvt. Ltd. (China). Potassium phosphate buffer (0.1 M, pH 7.4),
Fluorogenic substrates 7-benzyloxy-trifluromethyl coumarin (BFC),
3-cyano-7-ethoxycoumarin (CEC), di-benzyl fluorescein (DBF), 7-
methoxy 4-trifluromethyl coumarin (MFC), 3-[ 2-(N,N-diethyl-N-
methyl-amino) ethyl]-7-methoxy-4 methyl coumarin (AMMC),
microsomes (human, rat and mouse liver); cofactors, G6PDH,
control protein, potassium phosphate buffer (0.5 M, pH 7.4) were
obtained from BD Gentest Corp. (Woburn, MA), NADP salt, G6PD
salt and G6PDH enzyme were purchased from Sisco research lab-
oratories Pvt. Ltd. (Mumbai).
2.1. Methodology
2.1.1. In-vitro studies
2.1.1.1. Metabolic stability assay in liver microsomes (human, rat and
mouse) and liver S9 fraction (human, rat and mouse). SAL (10 mM)
was incubated with a mixture comprised of microsomes or liver S9
fraction (supernatant fraction obtained from liver homogenate by
centrifuging at 9000g for 20 min in a suitable medium). Micro-
somes and liver S9 fractions equivalent to protein concentration of
0.5 mg/mL were added to phosphate buffer 0.1 M (pH 7.4), NADPH
regenerating system (NRS) which contains 10 mM Magnesium
chloride, 2.5 mM NADP salt, 25 mM G6PD salt, 0.5 units/mL G6PDH
enzyme in a total volume of 1 mL). The control, standard/test
compounds were added to the incubation mixture and further
incubated for 10 min at 37
C. Aliquots of the incubation solution
were withdrawn at different time points after the 10 min incuba-
tion time and the reaction was stopped using quenching solution
(acetone: ethanol: acetic acid: 1 mM niflumic acid (79:20:0.9:0.1) at
0, 6, 12, 15, 24 and 30 min. In order to confirm the metabolism of
SAL by CYP enzymes, SAL was incubated with microsomes in
presence 1-amino benzotriazole (1-ABT) which is a non specific
CYP inhibitor at concentrations of 50 mM, 100 mM, 200 mM and
400 mM. The concentration of parent compound in all the reaction
mixtures was determined by LCMS and calculated in terms of area
ratio with internal standard at each sampling point. In vitro rate
constant (k) and intrinsic clearance were calculated using the
following formula:
Clearance ðmL=min=g liverÞ
¼ KðminÞ  ½ðincubation volumeðmLÞ=microsomal or
S9 protein in incubationðmgÞ  ½microsomal or
S9 proteinðmgÞ=liver weightðgÞ:
Measured CLIN VITRO,INT values were extrapolated to the intact
liver using a scaling factor LS9 (120.7 mg/g liver) and Lmicrosomal
(52.5 mg/g liver) for S9 and microsomal fractions respectively
[18,19].
2.1.1.2. Identification of CYP enzymes involved in the metabolism of
SAL
A. Chemical inhibition study: The experiment with known chem-
ical inhibitors of CYP isoforms were performed at similar SAL
concentration (10 mM) as in liver microsomal incubations. All
the inhibitors were co-incubated with the substrate (SAL) at
37
C.
147
B. Recombinant enzyme study: Incubations with human recom-
binant P450s were also performed using the same conditions
described herein for human liver microsomes, except that the
mixture contained 20 pmol CYP (CYP3A4, CYP2C9, CYP2C19
supersomes) or 50 pmol CYP (CYP1A2, CYP2D6 supersomes)
with SAL at concentration same as in liver microsomal in-
cubations. The concentration of parent compound (SAL) was
determined in terms of area ratio with internal standard for each
sampling point considering concentration at zero min to be
100% determined by LCMS analysis.
2.1.1.3. Plasma protein binding assay. Warfarin and SAL 10 mM
stock solutions were prepared in DMSO. 5 mM of Warfarin and SAL
were incubated in plasma for 10 min at 37
C (final concentration of
DMSO 0.1%). Suitable quantity of the above mixture was taken in
ultracentrifuge tubes and centrifuged at 41,000 rpm for 4.30 h at
4
C. After centrifugation, the middle layer plasma supernatants
were collected and drug concentration was estimated by LCMS
Waters UPLC attached to triple quadrupole mass detector from
ABSCIEX. A calibration curve of standard as well as test drug was
prepared to estimate the unknown concentration in the unbound
fraction of plasma.
 
Cuf
Percent of drug bound to plasma ¼ 100  1 
Cp
Where,Cuf ¼ concentration of standard/test compound in
ultra-filtrate.
Cp ¼ initial concentration of standard/test comp in plasma.
2.1.1.4. Fluorescent based CYP inhibition assay. Fluorogenic CYP in-
hibition studies were conducted at 37
C in 96-well flat bottomed
black polystyrene plates. Enzyme mixture containing recombinant
CYP, control protein, substrate, potassium phosphate buffer (0.1 M,
pH 7.4) and MilliQ water was prepared; pre incubated for 10 min at
37
C. SAL (10 mM) in DMSO (0.1%) was combined with enzyme-
substrate solution and the reaction was initiated by addition of
NRS. A positive control experiment, i.e., with standard inhibitor was
performed simultaneously. The reaction was stopped by addition of
stop solution (acetone: ethanol: acetic acid: 1 mM niflumic acid
(79:20:0.9:0.1)). The fluorescence was detected as per the emission
wavelengths of various substrates by fluorimetric plate reader
(BioTek, USA). The percentage inhibition of test compound and
positive control in comparison to no inhibitor control was deter-
mined using the following equation in Excel fit.
Percent inhibition ¼ 100
average blank corrected fluorescence of test compound
 100
average blank corrected fluorescence of no inhibitor
2.1.1.5. CYP inhibition assay for SAL in HLM using probe substrates.
The inhibitory effects of SAL on CYP activity was examined by using
10 mM phenytoin (CYP1A2), 1 mM diclofenac (CYP2C9), 5 mM S-
mephenytoin (CYP2C19), 0.3 mM dextromethorphan (CYP2D6),
1 mM nifedipine (CYP3A4) as CYP probe substrate (which is
metabolized to a measurable product) using human liver micro-
somes (HLM). SAL was co-incubated with these CYP marker sub-
strates from 0.1 mM to 100 mM with HLM before the reaction was
148 K. Resham et al. / Chemico-Biological Interactions 240 (2015) 146e152

initiated with NRS. Percent inhibition of SAL on CYP enzymes were processed by protein precipitation technique using acetonitrile
calculated based on the % of substrate markers remained and (ACN) and then spiking 1 mM niflumic acid as internal standard
further IC50 value for the SAL for CYP inhibition was calculated by after the addition of ACN and filtered for the detection of plasma
plotting graph between % enzyme activity (100% metabolite con- concentration of SAL in samples by LCMS.
version as 0% enzyme inhibition) with log concentration of SAL.
2.1.2.3. Pharmacokinetic calculations. Pharmacokinetic parameters
2.1.1.6. Metabolism based inhibition (MBI). The assay was designed were calculated by non-compartmental methods using the soft-
to run duplicates of a single reaction along with one MBI positive ware WinNonlin Professional version 6.0.
reference (50 mM ethinylestradiol), and one MBI negative reference
(100 nM KTC), and a vehicle control (reaction mixture without an 2.1.2.4. Statistical analysis. The data obtained in the study was
inhibitor) in a 96-well flat-bottom micro plate heated at 37
C. The expressed as the mean of two values in triplicates (n ¼ 3) plus or
pre-incubation solution have 0.5 mg/mL HLMs in 0.1 M sodium minus standard deviation. The intergroup variations was measured
phosphate buffer, and the incubation solution have 0.1 M sodium by one way or two way analysis of variance (ANOVA) using the
phosphate buffer and 50 mM nifedipine. Pre-incubation solution of Graph Pad Prism, version 6.0. Results with *P values < 0.05 were
volume 160 mL solutions were transferred to the wells of assay plate considered to be significant.
and test compound solutions or control solutions were added to
pre-incubation solutions. The pre-incubation reactions were initi-
3. Results
ated by the addition of NRS containing 25 mM NADPþ, 250 mM G6P,
20 units/mL G6PDH, 100 mM MgCl2. 6H2O, and 0.1 M sodium
3.1. In-vitro studies
phosphate buffer. For the 0-min pre-incubation, 20 mL of pre-
incubation mixture was immediately transferred into 180 mL of
3.1.1. Metabolic stability in liver microsomes and liver S9 fraction
incubation solution and then incubated for 10 min. For the 30 min
Determination of in vitro metabolic stability and prediction of
pre-incubation, each pre-incubation mixture was diluted 10-fold
intrinsic clearance plays crucial role in understanding the meta-
with the incubation solution after 30 min. At the end of the incu-
bolic pathway of the drug in in-vivo. As represented in Fig. 1, there
bation reactions, the reaction was stopped by addition of quenching
was significant change in the clearance with NRS as compared to
solution containing niflumic acid as an internal standard. The so-
control group (without NRS) in all three species with insignificant
lutions were filtered and analysed by LCMS.
interspecies difference in the intrinsic clearance. From the data
Percent enzymatic activity remaining obtained for the present study, it can be inferred that SAL un-
dergoes rapid metabolism in liver microsomes as compared to liver
% activity remaining ð30 minÞ S9 fraction and thus has a high intrinsic clearance in human liver
¼  100
% activity remaining ð0 minÞ microsomes (6.5 mL/min/g liver). As shown in Fig. 2, the intrinsic
clearance of SAL in S9 fraction (2.8 mL/min/g liver in mouse and
Where, >80% remaining ¼ MBI negative 1.33 mL/min/g liver in rat S9) was relatively lower as compared to
microsomes in mouse and rat (8.76 mL/min/g liver and 7.23 mL/
<80% remaining ¼ MBI positive min/g liver and respectively). Whereas difference was not signifi-
cant in human S9 (2.73 mL/min/g liver) and microsomal
2.1.2. In-vivo studies
Male Sprague Dawley (SD) rats (200e220 g, 6e7 weeks old) were
allowed to acclimatize for one week. The animals were housed in
polycarbonate cages of dimension 42 cm  27 cm  20 cm with
stainless steel grid tops and solid bottoms. Each cage was supplied
with a polycarbonate water bottle. A 12 h lightedark cycle was
maintained in the room during the acclimatization period. The rats
were fed with standard pelleted diet (Golden Feeds, Mehrauli, New
Delhi). The diet was available ad libitum. The study was conducted in
accordance with the approval of Animal Ethical Committee Guide-
lines (Protocol Number. DS-2014/024).

2.1.2.1. The effect of CYP3A4 on SAL metabolism in rats.

Animals were divided into two groups (n ¼ 3 in each group). One
group of animals received only SAL at an oral dose of 10 mg/kg and
the other group received 20 mg/kg oral dose of KTC, 20 min prior to
SAL at dose of 10 mg/kg by oral route. SAL was formulated at
2.5 mg/mL solution whereas KTC was formulated at 4 mg/mL
concentration solution. Post dosing, blood samples (~0.1 mL) were
collected from each animal through retro orbital plexus into 1.5 mL
micro centrifuge tubes containing sodium citrate (20% w/v) as
anticoagulant at 0.25, 0.5, 1, 2, 4, 8, 24 h. Plasma (10 mL) was har-
vested from blood samples by centrifuging the samples at
10,000 rpm for 5 min at 4
C and stored at 20
C until analysis.

Fig. 1. Effect of NRS on the calculated intrinsic clearance of SAL in human S9 fraction,
2.1.2.2. LC/MS/MS analysis. For the quantitative analysis of SAL in mouse S9 fraction, rat S9 fraction. Values are expressed as mean ± SD (n ¼ 3). (Two
plasma samples, a calibration curve of SAL in the concentration way ANOVA was made with Bonferroni's Multiple Comparison Test). **P < 0.01 sig-
range of 0.0089e150 mg/mL was prepared. Plasma samples were nificant vs Intrinsic clearance without NRS.
 K. Resham et al. / Chemico-Biological Interactions 240 (2015) 146e152
Fig. 2. Comparison of intrinsic clearance of SAL between microsomes and S9 fraction
of human, mouse and rat (in presence of NRS). Values are expressed as mean ± SD
(n ¼ 3). **P < 0.01 significant vs S9 fractions. Two way ANOVA was made with Bon-
ferroni's Multiple Comparison Test.
incubations (6.5 mL/min/g liver), besides, it showed moderate
clearance pattern in S9 fraction. The decrease in clearance in S9
fraction is because the metabolism of SAL is chiefly CYP mediated
and CYP activity in S9 is almost five fold lower as compared to
microsomes. Similarly, in presence of 400 mM 1-ABT, the intrinsic
clearance of SAL was decreased significantly as shown in Fig. 3,
which suggests that SAL metabolism is mainly mediated by CYP
enzymes since NRS is essential for the activation of these enzymes.
In the present study the phenotypic identification of the metabo-
lites and the enzymes responsible for the metabolite formation was
not investigated. Liver S9 fraction contains cytosolic enzymes such
as esterase, aldehyde and alcohol oxidases and dehydrogenases, but
the contribution of these enzymes for the metabolism of SAL was
not evaluated since the purpose of the study was to estimate overall
in-vitro intrinsic clearance in microsomes and liver S9 fraction
regardless of the enzyme involved.
Fig. 3. Effect of increasing concentrations of 1-ABT on the intrinsic clearance of SAL.
Values are expressed as mean ± SD (n ¼ 3). **P < 0.01, ****P < 0.0001 vs control group
in all three species. Two way ANOVA was made with Bonferroni's Multiple Comparison
Test.
149
Fig. 4. Plasma protein binding profile of SAL. Values are expressed as mean ± SD
(n ¼ 4).
3.1.2. Plasma protein binding assay
SAL being an acidic drug of high molecular weight molecule
binds with high affinity to plasma proteins. The percent binding
was found to be highest in mice with 97% as compared to 92% in
human and 95% in rat plasma respectively as shown in Fig. 4. This
might be a probable cause of its low clearance in vivo as demon-
strated in WT mice [12].
3.1.3. CYP inhibition assay
SAL was found to be a strong inhibitor of CYP3A4 as compared to
CYP2C9 and CYP2C19 as determined in fluorescent assay (Fig. 5)
Fig. 5. Fluorescent probe based CYP inhibition assays for SAL. Values are expressed as
mean ± SD (n ¼ 3). Salinomycin inhibits CYP3A4 significantly ***P < 0.001 vs. CYP 1A2,
2C9, 2C19, & 2D6. One way ANOVA was made with Bonferroni's Multiple Comparison
Test.
150 K. Resham et al. / Chemico-Biological Interactions 240 (2015) 146e152
Fig. 6. Direct CYP inhibition assay by SAL using human liver microsomes (LCMS
detection). Values are expressed as mean ± SD (n ¼ 3). SAL inhibits CYP2D6, CYP3A4
and CYP2C9 significantly ****P < 0.0001 and **P < 0.01 respectively versus CYP1A2 and
2C19. One way ANOVA was made with Bonferroni's Multiple Comparison Test.
whereas it was found to be a moderate inhibitor of CYP2D6 and
CYP3A4 in direct inhibition in HLM assay (Fig. 6). Since results of
CYP inhibition study in HLM is FDA approved and thus more reli-
able, it can be inferred that SAL acts as a moderate inhibitor of
CYP2D6 as well as CYP3A4. As SAL is found to inhibit approximately
50% of CYP3A4 and is metabolized chiefly by the same, experiment
to find out the IC50 of SAL was conducted by using nifedipine as
probe marker substrate in HLM. The IC50 value for SAL on CYP3A4
was found to be 17.44 ± 2.12 mM (Fig. 7). As SAL is found to show
synergistic anti-cancer effects with conventional anti-cancer drugs
like paclitaxel and doxorubicin in various studies [5], it may be
possible that it decreases the metabolism of these drugs reversibly
since these are CYP3A4 substrates. The actual mechanism of this
synergism however still remains to be elucidated by further PKePD
interaction studies.
3.1.4. Metabolism based inhibition assay for CYP3A4
To find whether the inhibition is reversible or irreversible in
nature based upon the binding of SAL with the enzyme, metabolism
Fig. 7. CYP3A4 enzyme inhibition study using probe marker substrate. HLM was
incubated with nifedipine (probe marker substrate for CYP3A4) along with different
concentrations of SAL in presence of NRS. The % of enzyme activity was calculated
based on the amount of metabolite converted by the enzyme. IC50 of SAL on CYP3A4
enzyme inhibition calculated based on the conversion of probe substrate (nifedipine)
to its metabolite analysed by LCMS method.
based assay of SAL was performed. SAL was pre-incubated in a pre-
incubation solution containing 10x of the HLM in presence of NRS
for 0 and 30 min. Incubation at zero time incubation was used as a
control. After pre-incubation (0 or 30 min) an aliquot of a pre-
incubation solution was diluted 10x in an incubation solution
which contained buffer, CYP3A4 substrate nifedipine and NRS.
Percent remaining activity in 30 min pre-incubation was compared
with the 0 min pre-incubation to estimate the MBI potential as
described in experimental section. When the percent activity
remaining in 30 min pre-incubation versus 0 min pre-incubation
was >80% was judged as MBI negative. According to this criteria
the percent remaining activity for SAL was 88.1% and therefore
judged as MBI negative and thus having lesser probability of
drugedrug interactions in in-vivo by irreversible inhibition.
3.1.5. Identification of CYP450 enzymes involved in the metabolism
of SAL
In the chemical inhibition study conducted in vitro where indi-
vidual CYP isoform inhibitors were co-incubated with SAL, it was
observed that KTC being a CYP3A4 inhibitor, inhibits SAL meta-
bolism to the maximum extent as compared to other inhibitors
(Fig. 8).
Also, in-vitro experiment using specific recombinant CYP en-
zymes to determine SAL metabolism has shown minor involvement
of CYP1A2 (30%) and CYP2C9 (33%). whereas. 60% of the SAL was
metabolised with CYP3A4 enzyme as shown in Fig. 9. Further
immunoinhibition and correlation studies are required to confirm
the involvement of the CYP isoforms along with CYP3A4.
3.2. In-vivo studies
3.2.1. Specificity, sensitivity and calibration curve range
Blank rat plasma from different rats were extracted and spiked
only with internal standard as a single blank. No endogenous peaks
were found to interfere with the drug. The lower limit of quanti-
fication (LLOQ) of detection was 0.0089 mg/ml. The calibration
Fig. 8. In vitro chemical inhibition studies to identify the enzymes involved in SAL
metabolism. Individual CYP isoform inhibitors (KTC for CYP3A4, quinidine for CYP2D6,
tranylcypromine for CYP2C19, sulfaphenazole for CYP2C9, a-naphthoflavone for
CYP1A2) were co-incubated with SAL. Values are expressed as mean ± SD (n ¼ 3). KTC
was found to inhibit the metabolism of SAL significantly versus all other inhibitors
(****P < 0.001). Two way ANOVA was made with Bonferroni's Multiple Comparison
Test.
 K. Resham et al. / Chemico-Biological Interactions 240 (2015) 146e152 151

Fig. 10. Plasma timeeconcentration profile of SAL after oral administration to SD rats
at a dose of 10 mg/kg with and without co-administration of KTC (20 mg/kg). Values
are expressed as mean ± SD (n ¼ 3 rats).

In the present study, in order to know the DMPK profile of SAL,

firstly, its metabolic stability was estimated in vitro using human,
Fig. 9. In vitro recombinant enzyme study to identify the enzymes involved in SAL rat and mouse liver microsomes (HLM, RLM and MLM) and S9
metabolism. The incubation mixture contained 20 pmol CYP(CYP3A4, CYP2C9,
fraction since these are stable and robust in vitro models to study
CYP2C19 supersomes) or 50 pmol CYP(CYP1A2, CYP2D6 supersomes) with SAL at
10 mM. Values are expressed as mean ± SD (n ¼ 3). Recombinant CYP3A4 was found to the metabolism of any novel drug. In microsomes, the assay was
metabolize SAL to greatest extent (***P < 0.001). One way ANOVA was made with performed both in presence and absence of NRS to determine the
Bonferroni's Multiple Comparison Test. involvement of CYP enzymes in SAL metabolism. The results ob-
tained showed significant differences in the metabolic stability of
curve was linear ranged from 0.0089 to 150 mg/ml. The coefficient the drug in presence and absence of NRS, suggesting that SAL
of linear regression (r2) was found to be 0.997. The method was metabolism is mainly mediated via Phase 1 pathway, chiefly
validated as per FDA guidelines for bioanalytical method validation. involving CYP enzymes, as NRS is an essential cofactor for the ac-
Following oral dose in both groups of animals, the absorption of tivity of these enzymes in Phase 1 metabolism. In order to confirm
SAL was found to be very small in rats, which is reflected by low the involvement of CYP enzymes for the metabolism of SAL, the
exposure (AUC0e∞ 0.04 mg h/mL) as depicted in Table 1. In contrast, metabolic stability assay was performed using 1-ABT, a non-specific
the animals which received KTC before SAL dosing, showed an inhibitor of CYP enzymes. 1-ABT increased the metabolic stability of
AUC0ea of 1.39 mg h/mL which is 7-fold higher compared to SAL SAL, or in other words, decreased its intrinsic clearance significantly
alone treated rats as shown in Fig. 10. Since KTC is a CYP3A isoform by blocking the activity of all isoforms of CYP in microsomes.
inhibitor (human CYP3A4/5 and Rat CYP3A1/2), we can predict that The assay was also done using liver S9 fraction to determine if
SAL metabolism is mainly mediated through CYP3A isoforms in in- the drug undergoes any phase 2 metabolisms. The metabolic sta-
vivo. bility of SAL was found to be higher in liver S9 (low intrinsic
clearance) as compared to microsomes, suggesting that there was
more involvement of CYPs. The reason why SAL metabolism
4. Discussion
decreased in liver S9 was due to lower CYP activity in liver S9 as
compared to microsomes. These results suggest that CYPs are
Significant advances have been made recently in the discovery
important metabolizing enzymes for SAL. In order to know the CYP
of novel drugs that target CSCs, and the future clinical use of these
isoforms involved in SAL metabolism, two approaches such as
novel agents may represent a powerful strategy for eradicating
chemical inhibition study and metabolism using recombinant
CSCs in cancer patients, contributing to the cure of cancer. SAL
CYP450 enzymes were performed and CYP3A4 was found to be the
seems to have even extended capabilities of eliminating cancer
major enzyme subfamily involved in SAL metabolism.
because it has been demonstrated to effectively target regular
DMPK parameters namely plasma protein binding and CYP in-
cancer cells, multidrug and apoptosis-resistant cancer cells, and
hibition studies were also studied for SAL. SAL was found to have
CSCs.
moderate CYP3A4 inhibitory potential (53%) which was reversible
in nature (as determined by the negative MBI potential of the drug)
Table 1
with an IC50 value of 17.44 ± 2.12 mM (Mean ± SD) indicating that
Pharmacokinetic parameters of salinomycin at 10 mg/kg oral dose (with or without
concomitant administration of 20 mg/kg oral dose of ketoconazole) in male SD rats SAL is much less potent than KTC (IC50 ¼ 0.07 mM) in inhibiting
(n ¼ 3). CYP3A4 activity. SAL was found to be extensively bound to human,
rat and mouse plasma proteins (>90%) as determined in vitro by
Parameters Salinomycin 10 mg/kg p.o Salinomycin 10 mg/kg p.o
(with ketoconazole) (without ketoconazole) ultracentrifugation method.
The involvement of CYP3A4 in SAL metabolism was further
Mean ±SD Mean ±SD
confirmed by dosing of SAL (10 mg/kg, oral dose) to male SD rats
Tmax (h) 4.33** 1.29 2.08 0.68 with and without concomitant administration of KTC (20 mg/kg,
T1/2 (h) 8.15*** 0.76 3.46 1.02
Cmax (mg/mL) 0.12** 0.04 0.04 0.01
oral dose). KTC, being an inhibitor of CYP3A1/2 in rats which are
AUC0et (mg.h/mL) 0.60** 0.27 0.16 0.03 equivalents to human CYP3A4 [20,21]increased the systemic
AUC0e∞ (mg.h/mL) 1.39*** 0.38 0.22 0.06 exposure of SAL significantly, i.e., there was a 7-fold increase in
CL/V (mL/min/kg) 205.89*** 1.45 864.74 2.12 AUC0ef and a 3-fold increase in Cmax in animals with KTC and
V/F (L/kg) 101.67* 1.34 262.71 1.92
salinomycin (Table 1). These results signify the major involvement
152 K. Resham et al. / Chemico-Biological Interactions 240 (2015) 146e152
of CYP3A isoforms in metabolism of SAL in vivo. In future, this data
may prove to be useful for therapeutic use of SAL in terms of dosage
adjustment, if given concomitantly with or without any CYP3A4
inhibitor.
Considering the low plasma concentrations of SAL in in vivo
condition (Cmax ¼ 0.04 mg/mL), and that SAL is extensively bound to
plasma proteins, the inhibitory effects of SAL on CYP3A4 substrates
probably would be minimal. Nevertheless, the in vivo effects of SAL
on pharmacokinetics of CYP3A4 marker substrates remain to be
elucidated. In our study we observed low bioavailability SLN which
is similar to that of results observed in mice [3].
5. Conclusion
SAL, being a promising candidate, its preclinical DMPK data
would be an important asset to exploit its huge therapeutic po-
tential for treatment of cancer. Our results indicate that SAL
metabolism is CYP mediated and being a high molecular weight
acidic compound, has a high affinity towards plasma proteins. In
CYP inhibition study, SAL is found to be a moderate inhibitor of
CYP2D6 and CYP3A4 with negative MBI potential, suggesting pos-
sibility of low drugedrug interactions (DDIs) risk in human. So far,
there have been no reports suggesting the involvement of CYP3A4
in the metabolism of SAL, first time we report CYP3A4 plays a major
role in SAL metabolism as determined by the in-vitro and in-vivo
data thereby showing a good correlation between both the results.
The high variability in the plasma profile of SAL in rats could be due
to its low intestinal absorption. Further studies such as perme-
ability, urine and faecal studies and biliary excretion studies are
needed to explicate the ADME (absorption, distribution, meta-
bolism excretion) properties of SAL.
Acknowledgements
K.R is thankful to Daiichi Sankyo India Pharma Pvt Ltd, for
providing the necessary facilities to carry out this work and NIPER-
Hyderabad for motivation and assistance during the course of
dissertation work. Authors also thankful to Department of Phar-
maceuticals (DOP), Ministry of chemicals & fertilisers for providing
financial support for pursuing masters and PhD degrees.
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.cbi.2015.08.007.
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