European Journal of Pharmaceutical Sciences 109 (2017) 631–637

Contents lists available at ScienceDirect

European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier.com/locate/ejps

Development and pharmaceutical evaluation of the anticancer Anthrafuran/ MARK

Cavitron complex, a prototypic parenteral drug formulation
Helen M. Treshalinaa, Vladimir I. Romanenkoa, Dmitry N. Kaluzhnyb, Michael I. Treshalinc,
Aleksey A. Nikitind,e, Alexander S. Tikhomirovc,f, Andrey E. Shchekotikhinc,f,⁎
a
Federal State Budgetary Scientific Institution “N.N. Blokhin Russian Cancer Research Center” of the Ministry of Health of the Russian Federation, 24 Kashirskoye Shosse,
Moscow 115478, Russia
b
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street, Moscow 119991, Russia
c
Gause Institute of New Antibiotics, 11 B. Pirogovskaya Street, Moscow 119021, Russia
d
National University of Science and Technology “MISIS”, 4 Leninsky prospect, Moscow, 119991, Russia
e
Lomonosov Moscow State University, 1–3 Leninskiye Gory, Moscow GSP-1, 119991, Russia
f
Mendeleyev University of Chemical Technology, 9 Miusskaya Square, Moscow 125190, Russia

A R T I C L E I N F O A B S T R A C T

Keywords: To improve the water solubility of the anticancer drug candidate LCTA-2034 (A1), we investigated the formation
Anthra[2,3-b]furan of complexes of this anthrax[2,3-b]furan congener with the solubilizing 2-hydroxypropyl derivative of β-cy-
2-Hydroxypropyl-β-cyclodextrin clodextrin HP-βCD (Cavitron®). The interaction of A1 with HP-βCD resulted in the inclusion complex A1/HP-
Drug formulation βCD in 1:1 stoichiometry. The A1/HP-βCD complex was used to develop a prototype of a lyophilised drug
Anticancer activity
formulation with enhanced (> 10-fold) aqueous solubility than A1 and a long-term stability. The use of HP-βCD
Acute toxicity
decreased the acute toxicity of A1 by > 30%. The A1/HP-βCD drug formulation as well as A1 in equal doses
(5 × 30 mg/kg) to increase the lifespan by up to 140% for mice with i.p. transplanted P388 leukaemia.
Furthermore, the A1/HP-βCD formulation demonstrated a significant and reliable antitumor efficacy in a Р388/
ADR drug resistant leukaemia and B16/F10 melanoma, proving a perspective of investigations of toxicology,
biodistribution and pharmacokinetics.

1. Introduction highly potent anthra[2,3-b]furan LCTA-2034 (A1, Fig. 1) has been

Cancer remains one of the main causes of mortality worldwide, and
the number of detected incidents increased in recent decades (Torre
et al., 2016; Fortin 2013). The therapeutic efficacy is often limited due
to the development of multidrug resistance (MDR) in tumor cells (Shtil,
2002; Nussinov et al., 2017; Kachalaki et al., 2016; Bugde et al., 2017).
Hence, agents that can circumvent MDR phenotypes are considered
promising for pre-clinical evaluation and clinical development
(Gangwar, et al., 2016; Zha et al., 2017; Yong et al., 2017; Rathore
et al., 2017; Genova et al. 2017).
Derivatives of anthraquinone (doxorubicin, farmorubicin, valru-
bicin, mitoxantrone, etc.) have demonstrated a high antitumor activity.
The anthraquinone scaffold is widely used in medicinal chemistry for
the search of new anticancer drug candidates (Soldi et al., 2015;
Nicolaou et al., 2016; Chen et al., 2016; Ali et al., 2016). Our group has
synthesized and evaluated a series of heterocyclic derivatives of an-
thraquinone and identified the prospective chemotypes (Cogoi et al.,
2015; Shchekotikhin et al., 2014; Tikhomirov et al., 2015). Recently, a
discovered as a result of structural optimization of hit compounds
(Shchekotikhin et al., 2016). The derivative A1 has demonstrated the
effects on multiple intracellular targets (Topoisomerase (Top) 1, Top 2
and protein kinases). At low concentrations A1 triggered apoptotic cell
death in tumor cell lines including the sublines with different MDR
mechanisms. Moreover, A1 showed an outstanding antitumor activity
in a model of murine P388 leukaemia, increasing the animal lifespan up
to 262% at tolerable doses (Shchekotikhin et al., 2016).
Despite these good properties A1 is poorly soluble in distilled water
(~ 1.0 mg/ml at room temperature) and in pharmacologically accep-
table aqueous media under physiological conditions. This obstacle
substantiates the necessity to obtain a soluble and stable drug for-
mulation for parenteral use.
Cyclodextrins and their derivatives, particularly hydroxyalkylated
cyclodextrins, are applied in pharmaceutics as solubilizing agents
(Meinguet et al., 2015; Yankovsky et al., 2016; Thiry et al., 2017;
Mohamed et al., 2017; Vossen et al., 2017). These natural cyclic oli-
goglucosides have an inner cavity that can enclose a wide range of

⁎
Corresponding author at: Gause Institute of New Antibiotics, 11 B. Pirogovskaya Street, Moscow 119021, Russia.
E-mail address: instna@sovintel.ru (A.E. Shchekotikhin).

http://dx.doi.org/10.1016/j.ejps.2017.09.025
Received 22 May 2017; Received in revised form 24 August 2017; Accepted 15 September 2017
Available online 18 September 2017
0928-0987/ © 2017 Elsevier B.V. All rights reserved.
H.M. Treshalina et al.
O 2.3. Preparation of Anthrafuran/Cavitron complex (a lyophilized drug
O OH N formulation A1/HP-βCD)
NH2
*MsOH
CH3
O stirred for 10 min. After cooling the bacterial contamination and me-
O OH
- 20 ml. After checking A1 concentration by HPLC (the required con-
LCTA 2034 (A1)
Fig. 1. Structure of anthrafuran LCTA-2034 (A1).
compounds. The inclusion can significantly improve the solubility and
stability of hydrophobic ingredients of pharmaceutical formulations
(Connors, 1997; Loftsson et al., 2007; Loftsson and Brewster, 2012).
The aim of this study is to evaluate the applicability of β-cyclo-
dextrin complexes for an increased solubility of A1 and to develop a
prototype for the parenteral drug formulation. We investigated the in-
teraction of A1 with (2-hydroxypropyl)-β-cyclodextrin (Сavitron® W7
HP5, HP-βCD) in aqueous solutions and the stoichiometry of
Anthrafuran/Cavitron (A1/HP-βCD) inclusion complexes. We prepared
a prototype of a water-soluble lyophilized composition with an im-
proved solubility and excellent stability. Finally, comparison of cyto-
toxicity, acute toxicity and in vivo anticancer potency of A1 and A1/HP-
βCD clearly demonstrated the advantages of the new formulation.
2. Experimental section
2.1. Materials
An amorphous substance of (S)-3-(3-aminopyrrolidine-1‑carbonyl)-
4,11-dihydroxy-2-methylanthra[2,3-b]furan-5,10-dione methanesulfo-
nate dyhydrate (A1) was synthesized following the previously reported
method (Shchekotikhin et al., 2016). The (2-hydroxypropyl)-β-cyclo-
dextrin (Сavitron® W7 HP5 Pharma, Ashland Inc.) was generously do-
nated by Ashland Specialty Ingredients. All other solvents, chemicals
and reagents were purchased from Sigma-Aldrich (unless specified
otherwise) and used without purification.
2.2. Evaluation of Anthrafuran/Cavitron complex formation
A solution of (2-hydroxypropyl)-β-cyclodextrin (HP-βCD; 200 mM)
was prepared by dissolving HP-βCD in distilled water. A solution of A1
(50 mmol/l in 20 mmol/l Na acetate buffer, pH 5.0, 23 °C) was titrated
with the HP-βCD solution. Fluorescence spectra were obtained with a
Varian Cary Eclipse spectrofluorimeter (USA). The fluorescence at
540 nm was registered with excitation at 450 nm. Spectral width of the
slit was 5 nm. The fluorescence polarization (P) was calculated using
the equations:
P = (Ivv –G × Ivh ) (Ivv + G × Ivh ); G = Ihh Ihv
where Ivv and Ivh are the intensities of the vertical and horizontal
fluorescence components when excited vertically with polarized light,
Ihv and Ihh are the intensities when excited horizontally (Lakowicz,
2006).
The kinetics of the fluorescence decay was measured on an
EasyLife™ V taumeter (OBB Corp) with excitation by a pulsed LED with
spectral maximum 435 nm and IRF (Fig. 3). The lifetime (τ) was cal-
culated by the single-exponential decay law with EasyLife™ software.
The rotational relaxation time (Θ) was calculated as:
Θ = τ (1 P0 −1 3) (1 P − 1 P0 ),
where P0 = 0.5 is the limiting polarization.
632
European Journal of Pharmaceutical Sciences 109 (2017) 631–637
A mixture of A1 (200 mg), (2-hydroxypropyl)-β-cyclodextrin
(Сavitron® W7 HP5 Pharma, 800 mg), sodium citrate (10 mg) and
distilled water (15.0 ml) in a sterile flask was heated to 95–98 °C and
chanical impurities were removed by filtering the solution under
aseptic conditions through a 0.22 μm filter (Os050, GE Osmonics,
Lenntech BV). The resulting solution was diluted with sterile water to
centration is 10.0 ± 0.5 mg/ml), the solution was aliquoted (2 ml) in
sterile glass vials. The vials were closed with sterile tampons and al-
lowed for 12 h at − 70 °C. The vials with the frozen solution were put
into a freeze-drying machine (Alpha 1–2 LDplus, Martin Christ
Gefriertrocknungsanlagen) and lyophilised for 24 h at a reduced pres-
sure (0.01 mbar). The vials were then sealed with sterile rubber stop-
pers and rolled with aluminium caps. Each vial contained an amount of
A1/HP-βCD equivalent to 20 ± 1 mg of A1 (pharmacologically active
component).
2.4. Dynamic light scattering (DLS) measurements
These measurements were carried out using the Malvern Zetasizer
Nano ZS analyzer (Malvern Instruments Ltd., Malvern) at a wavelength
of 633 nm with a solid-state He–Ne laser at a scattering angle of 173° at
23 °C. Solutions of A1, HP-βCD (4 mmol/l) and their mixture in molar
ratios 1:1 and 1:2.5 were prepared by dissolving of samples in the re-
spective volumes of Na acetate buffer (20 mmol/l, pH 5.0) at room
temperature. For DLS analysis, each sample was filtered through a
0.2 μm membrane (Macherey-Nagel) and immediately investigated.
The size distribution of scattering objects was calculated with Zetasizer
Nano 4.2 software using an algorithm based upon the Mie theory which
transforms time-varying intensities to particle diameters.
2.5. Determination of saturating concentrations
Compound A1 (20 mg) or the lyophilised A1/HP-βCD drug for-
mulation (100 mg) were added to double-distilled water (1 ml) in a
sealed vial and incubated in a water bath at 22 °C for 1 h. The super-
natant was filtered through Millex-HV Durapore® PVDF filter
(0.45 μm), the first 3 drops were discarded, and the resulting solution
(0.25 ml) was diluted with double-distilled water to 50 ml for high-
performance liquid chromatography (HPLC) analysis.
2.6. HPLC
The concentration of A1 in the aqueous solutions for preparation of
the lyophilized drug formulation, as well as in the solubility and sta-
bility tests was quantified by a validated HPLC method using a
Shimadzu Class-VP V6.12SP1 system. A GraceSmart® RP 18 analytical
column with 5 μm particles (250 × 6 mm) was used with a mobile
phase composed of a mixture of 0.01 M H3PO4 and acetonitrile (pH 2.7,
System A). The column temperature was kept constant at 22 °C. The
sample (20 μl) was injected and eluted with the gradient of acetonitrile
(from 20 to 60%) in the mobile phase. The chromatographic run time
was set to 30 min, the flow rate was set at 1 ml/min, the detection
wavelength was 260 nm, and the retention time of A1 (tR) was close to
17 min (see representative HPLC tracks in Supplementary material,
Figs. S1, 2, 5, 6). Quantification of A1 was based on peak area mea-
surements using the interim reference standard Anthrafuran A1. The
purity of A1 was calculated by dividing the area of the A1 peak by total
peak area (100%). An alternative HPLC method with a mobile phase
composed of 0.2% ammonium formiate and acetonitrile (pH 7.4,
System B) was used for an additional analysis of quality of A1 substance
and A1/HP-βCD drug formulation. The retention time of A1 (tR) under
H.M. Treshalina et al.
the same HPLC condition as for System A, was close to 21 min
(Supplementary material, Figs. S3, 4).
2.7. Stability studies
Samples of the lyophilized A1/HP-βCD drug formulation in capped
glass vials were stored in a climate cabinet at 22 ± 1 °C, 60 ± 5% re-
lative humidity (RH) and at 60 ± 1 °C, 60 ± 5% RH (Jouan EU170).
Each vial contained an amount equivalent to 20 mg of A1 substance.
Samples stored at long-term storage conditions (22 °C) were tested at 0,
1, 3, 6 and 12 months. Samples stored at accelerated conditions (60 °C)
were also analysed at 0, 1 and 2 months. The studied parameters were
physical stability (by visual inspection) and chemical stability using the
HPLC method described in Section 2.6. Physical instability was defined
as changes in color or quality of a freeze-dried cake or by the visible
precipitation after reconstitution of samples. None of the above signs of
physical instability were observed in any stored samples. The purity of
A1/HP-βCD drug formulation was determined by HPLC (UV detection,
260 nm) and calculated by dividing the area of the A1 peak by total
peak area (100%).
2.8. Cytotoxicity
The HCT116 colon carcinoma cell line (ATCC) was cultured in
Dulbecco modified Eagle's medium supplemented with 5% fetal calf
serum (HyClone, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin,
and 100 μg/ml streptomycin. Cells in logarithmic phase of growth were
used in the experiments. The substance A1 and A1/HP-βCD drug for-
mulation were dissolved in 10% aqueous DMSO as 10 mM stock solu-
tions followed by serial dilutions in water immediately before experi-
ments. The assays were performed in 96-well microtiter plates. To each
well 5 × 104 tumor cells and a given concentration of the tested
compound were added. Cells were allowed to proliferate for 72 h at
37 °C in a humidified CO2-controlled atmosphere. At the end of the
incubation period, cells were counted in a Coulter counter. The IC50
was defined as the concentration of the compound that inhibited cell
proliferation by 50%. Cell viability was evaluated by an MTT test
(Shchekotikhin et al., 2016).
2.9. Toxicity in vivo
The acute toxicity of A1 and A1/HP-βCD was evaluated in
F1[DBA2 × C57Bl6j] female mice 20–24 g (bred at the Andreevka
branch of Scientific Center of Biomedical Technologies, Russia). Each
cohort consisted of 6 mice. A1 and A1/HP-βCD (0.2% in 5% glucose)
and administered i.p. at single doses (20–90 mg/kg). Lethal doses were
evaluated using a “StatPlus-2006” software, based on Litchfield-
Wilcoxon probit method for the standard calculating LD50 value
(Litchfield and Wilcoxon, 1949).
2.10. In vivo tumor models
For the study were chosen the following transplantable tumor
strains growing on the regular mice intraperitoleally (i.p.) P388 or
P388/ADR lympholeukaemia, which is primarily resistant to
Adriamycin (ADR) and cross-resistant to other anthracyclines, dau-
norubicin, taxanes, vinca alkaloids, and other bulky chemotherapeutic
agents due to drug efflux associated with over-expression of trans-
membrane transporter P-gp in the tumor cells (Donenko et al., 1993;
Gupta et al., 1991; Borst et al., 2000; Gottesman et al., 2002; Szakács
et al., 2006). Moreover was used i.p. transpnanted murine B16/F10
melanoma (original cell culture №CRL-6475™ ATCC®) with more sen-
sitive to i.p. chemotherapy then its subcutaneous strain (Goldin et al.,
1980).
Choosing tumor strains were obtained from the Tumor Strains
Collection of the «N.N. Blokhin Russian Cancer Research Center» (TSC,
633
European Journal of Pharmaceutical Sciences 109 (2017) 631–637
Moscow). To obtain the inoculating material each of strain tumor
models was transplanted twice on the donor mice of DBA2 or C57Bl6j
accordingly. Then suspension of 1 × 106 leukaemia cells or 50 mg/
0.2 ml of melanoma tissues with the culture medium 199 were prepared
and implanted into each of mice. All the mice for the tumor trans-
plantation were obtained from branch of Center of Biomedical
Technologies “Stolbovaya” (Russia) and contained in the Animal
Department of the “N.N. Blokhin Russian Cancer Research Center”. For
experimental evaluation there were used 2–10 passages of tumor in
mice.
2.11. Drugs for animal treatment
Solutions of A1 substance or lyophilized A1/HP-βCD drug for-
mulation contained 2 mg/ml of the active ingredient A1. The substance
of A1 in the isotonic solution glucose (ISG) was pre-warmed to 50–60°С,
then cooled down to room temperature and injected i.p. into the ani-
mals. The solution of A1/HP-βCD was prepared by adding 10 ml ISC
per one vial (contained 20 mg of A1) of the lyophilized drug formula-
tion and shook 2–3 min for complete dissolution of the composition.
Control mice were injected i.p. with ISC. Treatment was performed
daily for 5 days beginning on day 2 after tumor inoculation. The daily
dose of the drugs was 30 mg/kg or 40 mg/kg; total doses were 150 mg/
kg or 200 mg/kg, respectively. These dosages were chosen as equally
effective according to screening of a wide range of doses and similar
schedule (Shchekotikhin et al., 2016). MDR in P388/ADR was main-
tained with 7.5 mg/kg single dose of Doxorubicin (Lance-Pharm Ltd.,
Russia) (Goldenberg et al., 1986; Deffie et al., 1988; De Jong et al.,
1995; Nourbakhsh et al., 2015).
2.12. Evaluation of the antitumor activity
For calculation of the standard criteria of the antitumor activity was
used the standard criteria as increasing of life span ILS ≥ 25% for the
mice with P388 (parental sensitive), and average of life span (ALS,
days) for P388/ADR or B16/F10. Effectiveness of A1/HP-βCD on the
above-mentioned criteria was used for the definition of significant and
reliable antitumor efficacy. Adequate groups of the mice for the both
tumor model were used for the efficacy control. Statistical analysis of
obtained data was made with Fisher method through statistically
Program Excel 2013 with Fisher t-test; the reliable differences were
calculated for p < 0.05. Calculated experimental results are presented
in corresponding illustrations. Tolerance of the therapy was evaluation
based on the mice condition and behaviour. At the final stage of the
experiment, all the remaining animals were euthanized under the
general anaesthesia using an ether overdose, in accordance with the
European Convention for the Protection of Vertebrate Animals Used for
Experimental and Other Scientific Purposes (Newcomer, 2012). By the
autopsy of the dead mice were revealed the local symptoms of perito-
neal leukaemia canceromatosis: increasing of lymphatic nodules and
ascites liquid in peritoneal cavity.
3. Results and discussion
3.1. Evaluation of Anthrafuran/Cavitron complex formation
The interaction of A1 with HP-βCD in an aqueous solution was
evaluated by fluorescence intensity and fluorescence polarization. The
fluorescence and polarization of A1 synchronously increased with the
concentration of HP-βCD (Fig. 2). This suggests that both parameters
reflect the process of the complexation. The shape of the fluorescence
spectra did not change, indicating that the fluorophore in the same form
are free in the solution and are present in the complex (Fig. 3A).
The increased fluorescence and polarization indicate an increase in
the hydrodynamic volume of the complex compared to free compound.
To confirm this observation dynamic light scattering (DLS)
H.M. Treshalina et al. European Journal of Pharmaceutical Sciences 109 (2017) 631–637

Absorption of A1 with the addition of HP-βCD did not change sig-

nificantly. The rotational correlation time of A1 and its complex with
HP-βCD (Table 1) was calculated with the lifetime of fluorescence and
polarization. The interaction of A1 with HP-βCD resulted in an increase
in the rotational correlation time by 3.7-fold, which corresponds to the
ratio of molecular masses of free molecule and the complex and char-
acterizes the formation of a complex between one molecule of A1
(Mr = 503) and one molecule of HP-βCD (Mr ~ 1579).

3.2. Preparation and evaluation of Anthrafuran/Cavitron drug formulation

Interaction of A1 with HP-βCD in aqueous solutions and the effec-

tive concentration of binding strongly suggested the inclusion of A1 in
the HP-βCD cavity (Connors, 1997; Loftsson and Brewster, 2012;
Mallick et al., 2014). The parameters of complexation of A1 were used
to develop a prototype of lyophilized drug formulation based on the
Fig. 2. The fluorescence intensity of A1 and fluorescence polarization increase with in- A1/HP-βCD complex. Although the stoichiometry of complexation was
creasing concentrations of HP-βCD. defined as 1:1, the amount of cyclodextrin in pharmaceutical for-
mulations not necessarily reflects this ratio. Normally the concentra-
measurements were carried out for free A1, HP-βCD and their stoi- tions of cyclodextrin bigger than a stoichiometric (that depends on
chiometric mixture in aqueous solutions at equal concentrations complexation efficiency) are needed to achieve drug solubilisation (Rao
(4 mmol/l, Fig. 4A). The analysis of size distribution showed a mono- and Stella, 2003; Brewster and Loftsson, 2007; Loftsson et al., 2007).
disperse nature of samples. The solutions of free A1 and HP-βCD had The molar ratio of A1 and HP-βCD for drug formulation was calculated
mean hydrodynamic size values 0.72 and 0.83 nm with the poly- considering the complexation efficiency:
dispersity index (PDI) 0.11 and 0.17, respectively. The equimolar A1: HP − βCD = 1: (1 + EC So) = 1: 1.4
mixture A1/HP-βCD also showed unimodal dispersion with the hy-
drodynamic size 1.12 nm and PDI = 0.27. Accordingly, the interaction where EC = 1.0 mmol/l (the effective concentration of binding);
of A1 with HP-βCD at the molar ratio 1:1 led to an increased hydro- S0 = 2.4 mmol/l (the solubility of free A1, see Table 2).
dynamic volume (compared with free A1 and HP-βCD) and to the Optimization of the drug formulation showed that the molar ratio
formation of particles with a size corresponding to the inclusion com- can be slightly reduced to 1:1.3. Freeze-drying of the aqueous A1/HP-
plexes (Connors, 1997; Lucio et al., 2017). The increase of HP-βCD βCD mixture in this ratio led to the formation of uniform red-colored
concentrations (> 4 mmol/l) led to appearance of another size of cakes that quickly formed a homogeneous solution after the addition of
scattering objects in the solution. At the molar ratio 1:2.5 the second water or 5% ISG at room temperature.
distribution appeared around 68 nm and this signal increased with the Next, the saturating concentrations of A1 for the drug substance and
concentration of HP-βCD (Fig. 4B). Similar bimodal distribution of A1/HP-βCD formulation were compared to determinate whether the
scattering objects can be attributed to self-aggregation of cyclodextrins addition of HP-βCD increases water solubility of A1. As shown in
and drug-cyclodextrin complexes (Lucio et al., 2017). Formation of Table 2, HP-βCD significantly increased the solubility of A1. The sa-
nanoaggregates in solutions with higher molar ratios of HP-βCD may be turating concentration of A1/HP-βCD was 19 mg/ml vs 1.2 mg/ml for
important in further increase of A1 solubility. A1 substance. This confirms that holding the hydrophobic chromo-
An approximation of the fluorescence and polarization changes by phore of A1 in the cavity of HP-βCD can efficiently improve water
the equation 1 / (1 + [HP-βCD] / EC) was used to determine the ef- solubility. The inclusion of A1 into HP-βCD was reversible as de-
fective concentration of binding of A1 to HP-βCD. The approximation is termined by HPLC and mass-spectrometry data. Indeed, A1/HP-βCD
shown as continuous curves in Fig. 2. The fluorescence changes reflect was detected as free A1 by HPLC analysis: the retention time (tr) of the
ECf = 1.2 mmol/l, and for changes in the fluorescence polarization, main peak in chromatograms of the reconstituted lyophilized A1/HP-
ECp = 1.0 mmol/l. βCD at different eluting conditions (pH 2.5 and 7.4) corresponded to tr
The fluorescence lifetime of A1 was measured in the presence and of the main peak of the reference A1 (Figs. S1–4, Supplementary ma-
absence of HP-βCD. Fig. 3B demonstrates that a higher concentration of terial). Moreover, free A1 and HP-βCD in a reconstituted lyophilized
HP-βCD was accompanied by an increase of the fluorescence lifetime. A1/HP-βCD formulation were also detected by mass-spectrometry (Fig.
The increased fluorescence intensity is associated with an increase in S8, Supplementary material). Thus, the developed freeze-dried A1/HP-
average lifetime and hence the quantum yield of fluorescence. βCD composition demonstrates a convenient prototypic formulation for
the parenteral application of A1.

1000 1000
free IRF
Fluorescence intensity, au
Fluorescence intensity, au

800 1 mM 800 free

2 mM 1 mM
600 600 2 mM
4 mM
4 mM
400 6 mM 400
6 mM
10 mM
200 200 10 mM

0 0
450 550 650 750 58 63 68 73 78
Wavelength, nm A Time, ns B
Fig. 3. The fluorescence spectra (A) and fluorescence decay curves (B) of A1 upon binding to HP-βCD at different concentrations.

634
H.M. Treshalina et al. European Journal of Pharmaceutical Sciences 109 (2017) 631–637

Fig. 4. DLS measurements of size distribution in acetate buffer pH 5.0, 23 °C: A1, HP-βCD and A1/HP-βCD at 4 mmol/l (A); 4 mmol/l A1 in the presence of 10 mmol/l A1/HP-βCD (B).

Table 1 98,3
Fluorescence lifetime and rotational correlation time of free A1 and in complex with HP-

Anthrafuran A1 content, %
βCD.

HP-βCD, mM Fluorescence lifetime, ns χ2 Θ, ps

98,2
0 3.1 1.05 179 ± 5
10 3.8 1.00 671 ± 3 60OC
22OC
Table 2 98,1
Solubility of A1/HP-βCD and A1 in distilled water and parameters of in vitro/in vivo
toxicity.

Agent A1 saturating IC50a,c LD50d on F1♀, MTDe on F1♀,

concentrationa,b, mg/ml HCT116, mg/kg mg/kg 98,0
μmol

A1
A1/HP-
1.2 ± 0.1
19.1 ± 0.3
6.4 ± 0.6
6.2 ± 0.7
52.5 ± 5.4
68.3 ± 6.2
39.4 ± 1.9
47.6 ± 2.3
0 2 4 6 8 10 12
βC- Time, months
D
Fig. 5. Average anthrafuran A1 content with SD (n = 4) of the lyophilized A1/HP-βCD
a drug formulation stored at 22 °C and 60 °C.
Values are mean ± SD (n = 3).
b
T = 22°С.
c
IC50, concentration of A1 that inhibited cell proliferation by 50%.
d
LD50, dose causing death of 50% animals after i.p. administration. does not affect the antiproliferative potency of A1. The lack of sig-
e
MTD (maximum tolerated dose; LD10), dose causing death of 10% animals after i.p. nificant differences in the activity of free A1 and A1/HP-βCD drug
administration. formulation also indicates that the efficacy of the developed drug for-
mulation is associated with the released A1.
Finally, physical and chemical stability of the A1/HP-βCD proto- In contrast to the results in cell culture, evaluation of the toxicity in
type were screened by long-term storage and accelerated aging. The vivo revealed differences between the lyophilized A1/HP-βCD for-
lyophilized A1/HP-βCD drug formulation was chemically stable, with mulation and A1. For A1/HP-βCD, the LD50 value 68.3 ± 6.2 mg/kg
~ 99% (from an initial quantity of the agent) of A1 content remaining was notably higher than that for A1 (LD50 = 52.5 ± 5.4 mg/kg,
after 12 months at 22 °C and at 60 °C for 2 months (Fig. 5). During the Table 2). Thus, although the inclusion of the active ingredient in cy-
long-term storage a small increase of some degradation products was clodextrin does not influence for the growth rate of tumor cells in
observed, most of which were components B and C (Figs. S1, 2, 5, 6, culture, the complexation markedly reduced the acute toxicity. This fact
Supplementary material). The same unidentified impurities were found opens up an opportunity for widening the therapeutic window and
in the samples of A1 substance. Most likely they are anthrafuran related escalation of doses of A1.
compounds since they have similar UV spectra (Fig. S7, Supplementary
material). No changes in color, quality of a freeze-dried cake and clarity
after reconstitution of the composition were observed in any of tested 3.4. Antitumor activity
samples. We therefore evaluated the A1/HP-βCD formulation for an-
titumor activity and toxicology. The specific activity of A1/HP-βCD was evaluated in in vivo murine
tumor models. First, we compared the therapeutic efficacy of A1/HP-
3.3. Cytotoxicity in cell culture and acute in vivo toxicity of Anthrafuran/ βCD and A1 in the P388 model under control of tolerance. The results
Cavitron drug formulation are present in Table 3. With daily doses of 30 mg/kg (total dose of
150 mg/kg), both A1 and A1/HP-βCD had similar antitumor activity at
Complexation of the active ingredient may result in changes to its a significant level of ILS = 140% (p < 0.05). There was no statistical
specific activity or toxicity, so we compared the cytotoxicity and acute deviation among the compared drugs (p > 0.05). Autopsy results de-
toxicity of A1 and the A1/HP-βCD complex. The antiproliferative effect monstrated an absence of increasing lymphatic nodules or ascite ac-
of A1 and lyophilized A1/HP-βCD was investigated against human cumulation in all mice. The treatments with each substance were tol-
colon cancer cell line HCT116 (Table 2). Values of IC50 were similar for erated equally well without any side effects or death from toxicity.
A1 and A1/HP-βCD indicating that the complexation with HP-βCD Since A1 potently inhibited the proliferation of MDR cells

635
H.M. Treshalina et al. European Journal of Pharmaceutical Sciences 109 (2017) 631–637

Table 3 prototype of lyophilised A1/HP-βCD formulation with improved solu-

Antitumor efficacy of A1 and A1/HP-βCD in the Р388 model. bility and long-term stability was developed and evaluated.
Measurements of the saturating concentration showed that the A1/HP-
Group Single Antitumor efficacy
dosea βCD complexation enhanced the aqueous solubility >10-fold com-
ILS, % Number of mice with symptoms of pared to A1. The formation of the inclusion complex is reversible as
peritoneal canceromatosisb, determined by detection of free A1 in the solution of the reconstituted
lyophilized A1/HP-βCD formulation by HPLC and MS. Similar anti-
Ascites Lymphatic nodules
proliferative activity of A1 and A1/HP-βCD also confirmed the release
Control (5% 0.5 ml – 5/5 5/5 of A1 from the A1/HP-βCD complex at physiological conditions.
glucose) The in vivo study demonstrated that HP-βCD clearly decreased the
A1 30 mg/kg 143c 0/6 0/6 acute toxicity of A1, probably via changes in distribution, bioavail-
A1/HP-βCD 142c 0/5 0/6
ability, and pharmacokinetics of the active ingredient. Notably, LD50 for
a
Daily i.p. for 5 days; the A1/HP-βCD drug formulation was 30% higher than for A1
b
On the day of death from leukaemia; (52.5 ± 5.4 and 68.3 ± 6.3 mg/kg, respectively). Thus, the application
c
Significant deviation from control (p < 0.05) without significant deviation between of β-CD derivatives can be useful for improvement of solubility of A1 as
the treated groups. well as optimization toxicology, safety and tolerance.
Finally, the A1/HP-βCD drug formulation showed a potent anti-
Table 4 tumor activity in the murine tumor models. The A1/HP-βCD complex
Efficacy of A1/HP-βCD on MDR tumor model Р388/ADR.
and A1 in equal doses (5 × 30 mg/kg) increased life spans up to 140%
Group Single dose Antitumor efficacy of mice with i.p. transplanted P388 leukaemia. Furthermore, the A1/
HP-βCD drug formulation demonstrated significant and reliable anti-
ILS, % Number of mice with symptoms of tumor efficacy on the Р388/ADR resistant tumor model and B16/F10
peritoneal canceromatosisa,
melanoma. Further studies on animal models are underway to verify
Ascites Lymphatic nodules optimal protocols for application of the A1/HP-βCD formulation.
Additional studies are needed to fully characterize the toxicology,
Control (5% 0.5 m b
– 5/5 5/5 biodistribution, and pharmacokinetics of the A1/HP-βCD formulation
glucose)
b c
for further in-depth preclinical evaluation of A1.
A1/HP-βCD 40 mg/kg 36 3/6 3/6
ADR 7.5 mg/kgc 1 5/5
Acknowledgements
a
On the day of death from leukaemia;
b
Daily for 5 days; This study was supported by the Ministry of Industry and Trade of
c
Significant deviation were p < 0.05 against control or single dose of ADR, needed for
the Russian Federation (state contract 12411.1008799.13.007). The
to maintain resistance.
authors are thankful to Dr. L.G. Dezhenkova and N.M. Malyutina and
Dr. A.M. Korolev (Gause Institute of New Antibiotics, Moscow) for MTT
Table 5
Efficacy of the A1/HP-βCD against i.p. B16/F10 melanoma.
assays, HPLC and MS analysis, and to Olga Tyurina (Ashland Specialty
Ingredients) for providing HP-βCD (Cavitron®).
Group Single dosea ALSb, days ILS, %
Appendix A. Supplementary data
Control (5% glucose) 0.5 ml 17.8 [17.6 ÷ 18.0] –
A1/HP-βCD 30 mg/kg 26.6 [25.9 ÷ 27.3] 50c
Supplementary data to this article can be found online at http://dx.
a
Daily for 5 days; doi.org/10.1016/j.ejps.2017.09.025.
b
Average of life span;
c
Significant difference compared with control, p < 0.001. References

(Shchekotikhin et al., 2016), we analysed the anticancer activity of A1/
HP-βCD in vivo on the Р388/ADR MDR transplants (Donenko et al.,
1993). For treatment of this resistant to chemotherapy tumor model,
the single dose of A1/HP-βCD was escalated to 40 mg/kg for 5 days
(total dose of 200 mg/kg). The results are presented in Table 4. Only
A1/HP-βCD with i.p. administration revealed a significant ILS of 36%
(p < 0.05) without symptoms of peritoneal canceromatosis in all mice,
but not ADR.
The investigation of the antitumor activity on i.p. B16/F10 mela-
noma (Table 5) demonstrated a significant ALS = 26.6 [25.9–27.3]
days after A1/HP-βCD therapy with doses of 30 mg/kg (total 150 mg/
kg) against ALS = 17.8 [17.6–18.0] days in the control group. A1/HP-
βCD significantly increased the lifespan up to the level of ILS = 50%
(p < 0.001) with good tolerance.
4. Conclusion
We developed a drug formulation for a poorly water soluble antic-
ancer drug candidate, the anthrax[2,3-b]furan derivative A1. To im-
prove its solubility the interaction of A1 with HP-βCD (Cavitron®) in
aqueous solutions was investigated. The solubilizing agent formed an
inclusion complex with A1 in 1:1 stoichiometry. Based on these data, a
Ali, A.A.A., Lee, Y.R., Chen, T.C., Chen, C.L., Lee, C.C., Shiau, C.Y., Chiang, C.H., Huang,
H.S., 2016. Novel anthra[1,2-c][1,2,5]thiadiazole-6,11-diones as promising antic-
ancer lead compounds: biological evaluation, characterization & molecular targets
determination. PLoS One 11 (4), e0154278.
Borst, P., Evers, R., Kool, M., Wijnholds, J., 2000. A family of drug transporters: the
multidrug resistance-associated proteins. J. Natl. Cancer Inst. 92, 1295–1302.
Brewster, M.E., Loftsson, T., 2007. Cyclodextrins as pharmaceutical solubilizers. Adv.
Drug Deliv. Rev. 59, 645–666.
Bugde, P., Biswas, R., Merien, F., Lu, J., Liu, D.-X., Chen, M., Zhou, S., Li, Y., 2017. The
therapeutic potential of targeting ABC transporters to combat multi-drug resistance.
Expert Opin. Ther. Targets 21 (5), 511–530.
Chen, W.L., Wang, Z.H., Feng, T.T., Li, D.D., Wang, C.H., Xu, X.L., Zhang, X.J., You, Q.D.,
Guo, X.K., 2016. Discovery, design and synthesis of 6H-anthra[1,9-cd]isoxazol-6-one
scaffold as G9a inhibitor through a combination of shape-based virtual screening and
structure-based molecular modification. Bioorg. Med. Chem. 24, 6102–6108.
Cogoi, S., Zorget, S., Shchekotikhin, A.E., Xodo, L.E., 2015. Potent apoptotic response
induced by chloroacetamidine anthrathiophenediones in bladder cancer cells. J. Med.
Chem. 58, 5476–5485.
Connors, K.A., 1997. The stability of cyclodextrin complexes in solution. Chem. Rev. 97,
1325–1357.
De Jong, G., Gelmon, K., Bally, M., Goldie, J., Mayer, L., 1995. Modulation of doxorubicin
resistance in P388/ADR cells by Ro44-5912, a tiapamil derivative. Anticancer Res.
15, 911–916.
Deffie, A.M., Alam, T., Seneviratne, C., Beenken, S.W., Batra, J.K., Shea, T.C., Henner,
W.D., Goldenberg, G.J., 1988. Multifactorial resistance to adriamycin: relationship of
DNA repair, glutathione transferase activity, drug efflux, and P-glycoprotein in
cloned cell lines of adriamycin-sensitive and -resistant P388 leukemia. Cancer Res.
48, 3595–3602.

636
H.M. Treshalina et al.
Donenko, F.V., Sitdikova, S.M., Kabieva, A.O., 1993. The cross resistance to cytostatics of
leukemia P388 cells with induced resistance to doxorubicin. Bull. Exp. Biol. Med. 116
(9), 309–311.
Fortin, S., 2013. Advances in the development of hybrid anticancer drugs. Expert Opin.
Drug Discovery 8, 1029–1047.
Gangwar, S., Vite, G., Pitsinos, M.N., 2016. Streamlined total synthesis of uncialamycin
and its application to the synthesis of designed analogues for biological investiga-
tions. J. Am. Chem. Soc. 138 (26), 8235–8246.
Genova, C., Rijavec, E., Biello, F., Rossi, G., Barletta, G., Dal Bello, M.G., Vanni, I., Coco,
S., Alama, A., Grossi, F., 2017. New systemic strategies for overcoming resistance to
targeted therapies in non-small cell lung cancer. Expert. Opin. Pharmacother. 18 (1),
19–33.
Goldenberg, G.J., Wang, H., Blair, G.W., 1986. Resistance to adriamycin: relationship of
cytotoxicity to drug uptake and DNA single- and double-strand breakage in cloned
cell lines of adriamycin-sensitive and -resistant P388 leukemia. Cancer Res. 46,
2978–2983.
Goldin, A., Kline, I., Sof'ina, Z., Syrkin, A. (Eds.), 1980. Methods of selecting antitumor
drugs in the United States and Soviet Union, chapter in experimental evaluation of
antitumor drugs in USA and USSR and clinical correlation. NCI Monogr. 55, 25–50.
Gottesman, M.M., Fojo, T., Bates, S.E., 2002. Multidrug resistance in cancer: role of ATP-
dependent transporters. Nat. Rev. Cancer 2, 48–58.
Gupta, S., Kim, J., Gollapudi, S., 1991. Reversal of daunorubicin resistance in P388/ADR
cells by itraconazole. Itraconazole and multidrug resistance. J. Clin. Invest. 87,
1467–1469.
Kachalaki, S., Ebrahimi, M., Khosroshahi, L.M., Mohammadinejad, S., Baradaran, B.,
2016. Cancer chemoresistance; biochemical and molecular aspects: a brief overview.
Eur. J. Pharm. Sci. 89 (30), 20–30.
Lakowicz, J.R., 2006. Principles of Fluorescence Spectroscopy, 3rd ed. 954 Springer US.
Litchfield Jr., J.T., Wilcoxon, F., 1949. A simplified method of evaluating dose-effect
experiments. J. Pharmacol. Exp. Ther. 96, 99–113.
Loftsson, T., Brewster, M.E., 2012. Cyclodextrins as functional excipients: methods to
enhance complexation efficiency. J. Pharm. Sci. 101, 10–12.
Loftsson, T., Hreinsdottir, D., Masson, M., 2007. The complexation efficiency. J. Incl.
Phenom. Macrocycl. Chem. 57, 545–552.
Lucio, D., Irache, J.M., Font, M., Martínez-Ohárriz, M.C., 2017. Nanoaggregation of in-
clusion complexes of glibenclamide with cyclodextrins. Int. J. Pharm. 519, 263–271.
Mallick, A., Majumdar, T., Haldar, B., Roy, U.K., 2014. Binding interaction of a newly
developed bisindole drug molecule with α-cyclodextrin: face to face shielding of
indole hoops. RSC Adv. 4, 38206–38212.
Meinguet, C., Masereel, B., Wouters, J., 2015. Preparation and characterization of a new
harmine-based antiproliferative compound in complex with cyclodextrin: increasing
solubility while maintaining biological activity. Eur. J. Pharm. Sci. 77, 135–140.
Mohamed, E.A., Hashim, I.I.A., Yusif, R.M., Suddek, G.M., Shaaban, A.A.A., Badria,
F.A.E., 2017. Enhanced in vitro cytotoxicity and anti-tumor activity of vorinostat-
loaded pluronic micelles with prolonged release and reduced hepatic and renal
toxicities. Eur. J. Pharm. Sci. 96, 232–242.
Newcomer, C.E., 2012. The evolution and adoption of standards used by AAALAC. J. Am.
Assoc. Lab. Anim. Sci. 51 (3), 293–297.
Nicolaou, K.C., Wang, Y., Lu, M., Mandal, D., Pattanayak, M.R., Yu, R., Shah, A.A., Chen,
J.S., Zhang, H., Crawford, J.J., Pasunoori, L., Poudel, Y.B., Chowdari, N.S., Pan, C.,
Nazeer, A., Gangwar, S., Vite, G., Pitsinos, M.N., 2016. Streamlined total synthesis of
uncialamycin and its application to the synthesis of designed analogues for biological
investigations. J. Am. Chem. Soc. 138 (26), 8235–8246.
637
European Journal of Pharmaceutical Sciences 109 (2017) 631–637
Nourbakhsh, M., Jaafari, M.R., Lage, H., Abnous, K., Mosaffa, F., Badiee, A., Behravan, J.,
2015. Nanolipoparticles-mediated MDR1 siRNA delivery reduces doxorubicin re-
sistance in breast cancer cells and silences MDR1 expression in xenograft model of
human breast cancer. Iran J. Basic Med. Sci. 18, 385–392.
Nussinov, R., Tsai, C.-J., Jang, H., 2017. A new view of pathway-driven drug resistance in
tumor proliferation. Trends Pharmacol. Sci. 38 (5), 427–437.
Rao, V.M., Stella, V.J., 2003. When can cyclodextrins be considered for solubilizing
purposes? J. Pharm. Sci. 92, 927–932.
Rathore, R., McCallum, J.E., Varghese, E., Florea, A.M., Büsselberg, D., 2017. Overcoming
chemotherapy drug resistance by targeting inhibitors of apoptosis proteins (IAPs).
Apoptosis. http://dx.doi.org/10.1007/s10495-017-1375-1.
Shchekotikhin, A.E., Glazunova, V.A., Dezhenkova, L.G., Luzikov, Yu.N., Buyanov, V.N.,
Treshalina, H.M., Lesnaya, N.A., Romanenko, V.I., Kalyuzhny, D.N., Balzarini, J.,
Agama, K., Pommier, Y., Shtil, A.A., Preobrazhenskaya, M.N., 2014. Synthesis and
evaluation of new antitumor 3-aminomethyl-4,11-dihydroxynaphtho[2,3-f]indole-
5,10-diones. Eur. J. Med. Chem. 86, 797–805.
Shchekotikhin, A.E., Dezhenkova, L.G., Tsvetkov, V.B., Luzikov, Y.N., Volodina, Y.L.,
Tatarskiy, V.V., Kalinina, A.A., Treshalin, M.I., Treshalina, H.M., Romanenko, V.I.,
Kaluzhny, D.N., Kubbutat, M., Schols, D., Pommier, D., Shtil, A.A., Preobrazhenskaya,
M.N., 2016. Discovery of antitumor anthra[2,3-b]furan-3-carboxamides: optimiza-
tion of synthesis and evaluation of antitumor properties. Eur. J. Med. Chem. 112,
114–129.
Shtil, A.A., 2002. Multifactorial drug resistance: P-glycoprotein on the apex of the pyr-
amyd. J. Hematother. Stem Cell Res. 11, 437–439.
Soldi, R., Horrigan, S.K., Cholody, M.W., Padia, J., Sorna, V., Bearss, J., Gilcrease, G.,
Bhalla, K., Verma, A., Vankayalapati, H., Sharma, S., 2015. J. Med. Chem. 58,
5862–5864.
Szakács, G., Paterson, J.K., Ludwig, J.A., Booth-Genthe, C., Gottesman, M.M., 2006.
Targeting multidrug resistance in cancer. Nat. Rev. Drug Discov. 5, 219–234.
Thiry, J., Krier, F., Ratwatte, S., Thomassin, J.-M., Jerome, C., Evrard, B., 2017. Hot-melt
extrusion as a continuous manufacturing process to form ternary cyclodextrin in-
clusion complexes. Eur. J. Pharm. Sci. 96, 590–597.
Tikhomirov, A.S., Shchekotikhin, A.E., Lee, Y.H., Chen, Y.A., Yeh, C.A., Tatarskiy, V.V.,
Dezhenkova, L.G., Glazunova, V.A., Balzarini, J., Shtil, A.A., Preobrazhenskaya, M.N.,
Chueh, P.J., 2015. Synthesis and characterization of 4,11-diaminoanthra[2,3-b]
furan-5,10-diones: tumor cell apoptosis through tNOX-modulated NAD+/NADH
ratio and SIRT1. J. Med. Chem. 58, 9522–9534.
Torre, L.A., Siegel, R.L., Ward, E.M., Jemal, A., 2016. Global cancer incidence and
mortality rates and trends - an update. Cancer Epidem. Biomar. 25 (1), 16–27.
Vossen, A.C., Velde, I., Smeets, O.S.N.M., Postma, D.J., Eckhardt, M., Vermes, A., Koch,
B.C.P., Vulto, A.G., Hanff, L.M., 2017. Formulating a poorly water soluble drug into
an oral solution suitable for paediatric patients; lorazepam as a model drug. Eur. J.
Pharm. Sci. http://dx.doi.org/10.1016/j.ejps.2017.01.025.
Yankovsky, I., Bastien, E., Yakovets, Y., Khludeyev, I., Lassalle, H.-P., Grafe, S.,
Bezdetnaya, L., Zorin, V., 2016. Inclusion complexation with β-cyclodextrin deriva-
tives alters photodynamic activity and biodistribution of meta-tetra(hydroxyphenyl)
chlorine. Eur. J. Pharm. Sci. 95, 172–182.
Yong, L.D., Ting-Ren, L., Hong, Z., Jian, D., Bin, X., Shu, Y.W., 2017. Anticancer drug
development, getting out from bottleneck. Int. J. Mol. Biol. 2 (1), 2–10.
Zha, G.F., Qin, H.L., Youssif, B.G.M., Amjad, M.W., Raja, M.A.G., Abdelazeem, A.H.,
Bukhari, S.N.A., 2017. Discovery of potential anticancer multi-targeted ligustrazine
based cyclohexanone and oxime analogs overcoming the cancer multidrug resistance.
Eur. J. Med. Chem. 135, 34–48.