Cavitron complex, a prototypic parenteral drug formulation

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/ Cavitron complex, a prototypic parenteral drug formulation

MARK

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: Anthra[2,3-b]furan 2-Hydroxypropyl-β-cyclodextrin Drug formulation Anticancer activity Acute toxicity

To improve the water solubility of the anticancer drug candidate LCTA-2034 (A1), we investigated the formation of complexes of this anthrax[2,3-b]furan congener with the solubilizing 2-hydroxypropyl derivative of β-cyclodextrin HP-βCD (Cavitron®). The interaction of A1 with HP-βCD resulted in the inclusion complex A1/HPβCD in 1:1 stoichiometry. The A1/HP-βCD complex was used to develop a prototype of a lyophilised drug formulation with enhanced (> 10-fold) aqueous solubility than A1 and a long-term stability. The use of HP-βCD 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 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, valrubicin, 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 anthraquinone and identified the prospective chemotypes (Cogoi et al., 2015; Shchekotikhin et al., 2014; Tikhomirov et al., 2015). Recently, a

⁎

highly potent anthra[2,3-b]furan LCTA-2034 (A1, Fig. 1) has been 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 acceptable aqueous media under physiological conditions. This obstacle substantiates the necessity to obtain a soluble and stable drug formulation 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 oligoglucosides 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: [email protected] (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.

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

H.M. Treshalina et al.

O

OH

O

N

O

CH3

2.3. Preparation of Anthrafuran/Cavitron complex (a lyophilized drug formulation A1/HP-βCD)

NH2 *MsOH

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 stirred for 10 min. After cooling the bacterial contamination and mechanical 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 20 ml. After checking A1 concentration by HPLC (the required concentration 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 allowed 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 pressure (0.01 mbar). The vials were then sealed with sterile rubber stoppers and rolled with aluminium caps. Each vial contained an amount of A1/HP-βCD equivalent to 20 ± 1 mg of A1 (pharmacologically active component).

O

OH 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-cyclodextrin complexes for an increased solubility of A1 and to develop a prototype for the parenteral drug formulation. We investigated the interaction 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 improved solubility and excellent stability. Finally, comparison of cytotoxicity, acute toxicity and in vivo anticancer potency of A1 and A1/HPβCD clearly demonstrated the advantages of the new formulation.

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 respective 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. 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 methanesulfonate dyhydrate (A1) was synthesized following the previously reported method (Shchekotikhin et al., 2016). The (2-hydroxypropyl)-β-cyclodextrin (Сavitron® W7 HP5 Pharma, Ashland Inc.) was generously donated by Ashland Specialty Ingredients. All other solvents, chemicals and reagents were purchased from Sigma-Aldrich (unless specified otherwise) and used without purification.

2.5. Determination of saturating concentrations Compound A1 (20 mg) or the lyophilised A1/HP-βCD drug formulation (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 supernatant 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 highperformance liquid chromatography (HPLC) analysis.

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:

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 stability 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 measurements 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

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 calculated 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

H.M. Treshalina et al.

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 transplantation 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.

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% relative 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%).

2.11. Drugs for animal treatment Solutions of A1 substance or lyophilized A1/HP-βCD drug formulation 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 animals. 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 formulation 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 maintained 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).

2.8. 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 formulation were dissolved in 10% aqueous DMSO as 10 mM stock solutions followed by serial dilutions in water immediately before experiments. 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).

2.12. 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 peritoneal leukaemia canceromatosis: increasing of lymphatic nodules and ascites liquid in peritoneal cavity.

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 LitchfieldWilcoxon probit method for the standard calculating LD50 value (Litchfield and Wilcoxon, 1949). 2.10. In vivo tumor models

3. Results and discussion

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, daunorubicin, taxanes, vinca alkaloids, and other bulky chemotherapeutic agents due to drug efflux associated with over-expression of transmembrane 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 sensitive 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,

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) 633

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

H.M. Treshalina et al.

Absorption of A1 with the addition of HP-βCD did not change significantly. 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 characterizes 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 effective 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 A1/HP-βCD complex. Although the stoichiometry of complexation was defined as 1:1, the amount of cyclodextrin in pharmaceutical formulations not necessarily reflects this ratio. Normally the concentrations of cyclodextrin bigger than a stoichiometric (that depends on complexation efficiency) are needed to achieve drug solubilisation (Rao and Stella, 2003; Brewster and Loftsson, 2007; Loftsson et al., 2007). The molar ratio of A1 and HP-βCD for drug formulation was calculated considering the complexation efficiency:

Fig. 2. The fluorescence intensity of A1 and fluorescence polarization increase with increasing concentrations of HP-βCD.

1000

free 1 mM

800

2 mM

600

4 mM 6 mM

400

10 mM

200 0

450

550 650 Wavelength, nm

750

A1: HP − βCD = 1: (1 + EC So) = 1: 1.4 where EC = 1.0 mmol/l (the effective concentration of binding); S0 = 2.4 mmol/l (the solubility of free A1, see Table 2). Optimization of the drug formulation showed that the molar ratio can be slightly reduced to 1:1.3. Freeze-drying of the aqueous A1/HPβCD mixture in this ratio led to the formation of uniform red-colored cakes that quickly formed a homogeneous solution after the addition of water or 5% ISG at room temperature. Next, the saturating concentrations of A1 for the drug substance and A1/HP-βCD formulation were compared to determinate whether the addition of HP-βCD increases water solubility of A1. As shown in Table 2, HP-βCD significantly increased the solubility of A1. The saturating concentration of A1/HP-βCD was 19 mg/ml vs 1.2 mg/ml for A1 substance. This confirms that holding the hydrophobic chromophore of A1 in the cavity of HP-βCD can efficiently improve water solubility. The inclusion of A1 into HP-βCD was reversible as determined by HPLC and mass-spectrometry data. Indeed, A1/HP-βCD was detected as free A1 by HPLC analysis: the retention time (tr) of the main peak in chromatograms of the reconstituted lyophilized A1/HPβCD at different eluting conditions (pH 2.5 and 7.4) corresponded to tr of the main peak of the reference A1 (Figs. S1–4, Supplementary material). Moreover, free A1 and HP-βCD in a reconstituted lyophilized A1/HP-βCD formulation were also detected by mass-spectrometry (Fig. S8, Supplementary material). Thus, the developed freeze-dried A1/HPβCD composition demonstrates a convenient prototypic formulation for the parenteral application of A1. Fluorescence intensity, au

Fluorescence intensity, au

measurements were carried out for free A1, HP-βCD and their stoichiometric mixture in aqueous solutions at equal concentrations (4 mmol/l, Fig. 4A). The analysis of size distribution showed a monodisperse nature of samples. The solutions of free A1 and HP-βCD had mean hydrodynamic size values 0.72 and 0.83 nm with the polydispersity index (PDI) 0.11 and 0.17, respectively. The equimolar mixture A1/HP-βCD also showed unimodal dispersion with the hydrodynamic size 1.12 nm and PDI = 0.27. Accordingly, the interaction of A1 with HP-βCD at the molar ratio 1:1 led to an increased hydrodynamic volume (compared with free A1 and HP-βCD) and to the formation of particles with a size corresponding to the inclusion complexes (Connors, 1997; Lucio et al., 2017). The increase of HP-βCD concentrations (> 4 mmol/l) led to appearance of another size of scattering objects in the solution. At the molar ratio 1:2.5 the second distribution appeared around 68 nm and this signal increased with the concentration of HP-βCD (Fig. 4B). Similar bimodal distribution of scattering objects can be attributed to self-aggregation of cyclodextrins and drug-cyclodextrin complexes (Lucio et al., 2017). Formation of nanoaggregates in solutions with higher molar ratios of HP-βCD may be important in further increase of A1 solubility. An approximation of the fluorescence and polarization changes by the equation 1 / (1 + [HP-βCD] / EC) was used to determine the effective concentration of binding of A1 to HP-βCD. The approximation is shown as continuous curves in Fig. 2. The fluorescence changes reflect ECf = 1.2 mmol/l, and for changes in the fluorescence polarization, ECp = 1.0 mmol/l. The fluorescence lifetime of A1 was measured in the presence and absence of HP-βCD. Fig. 3B demonstrates that a higher concentration of HP-βCD was accompanied by an increase of the fluorescence lifetime. The increased fluorescence intensity is associated with an increase in average lifetime and hence the quantum yield of fluorescence.

A

1000

IRF free

800

1 mM

600

2 mM 4 mM

400

6 mM 10 mM

200 0

58

63

68 Time, ns

73

78

B

Fig. 3. The fluorescence spectra (A) and fluorescence decay curves (B) of A1 upon binding to HP-βCD at different concentrations.

634

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

H.M. Treshalina et al.

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).

98,3

HP-βCD, mM

Fluorescence lifetime, ns

χ2

Θ, ps

0 10

3.1 3.8

1.05 1.00

179 ± 5 671 ± 3

Anthrafuran A1 content, %

Table 1 Fluorescence lifetime and rotational correlation time of free A1 and in complex with HPβCD.

Table 2 Solubility of A1/HP-βCD and A1 in distilled water and parameters of in vitro/in vivo toxicity. Agent

A1 saturating concentrationa,b, mg/ml

IC50a,c HCT116, μmol

LD50d on F1♀, mg/kg

MTDe on F1♀, mg/kg

A1 A1/HPβCD

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

98,2 60OC 22OC

98,1 98,0 0

2

4

6

8

Time, months

10

12

Fig. 5. Average anthrafuran A1 content with SD (n = 4) of the lyophilized A1/HP-βCD drug formulation stored at 22 °C and 60 °C.

a

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. e MTD (maximum tolerated dose; LD10), dose causing death of 10% animals after i.p. administration.

does not affect the antiproliferative potency of A1. The lack of significant differences in the activity of free A1 and A1/HP-βCD drug formulation also indicates that the efficacy of the developed drug formulation is associated with the released A1. In contrast to the results in cell culture, evaluation of the toxicity in vivo revealed differences between the lyophilized A1/HP-βCD formulation and A1. For A1/HP-βCD, the LD50 value 68.3 ± 6.2 mg/kg was notably higher than that for A1 (LD50 = 52.5 ± 5.4 mg/kg, Table 2). Thus, although the inclusion of the active ingredient in cyclodextrin does not influence for the growth rate of tumor cells in culture, the complexation markedly reduced the acute toxicity. This fact opens up an opportunity for widening the therapeutic window and escalation of doses of A1.

Finally, physical and chemical stability of the A1/HP-βCD prototype were screened by long-term storage and accelerated aging. The lyophilized A1/HP-βCD drug formulation was chemically stable, with ~ 99% (from an initial quantity of the agent) of A1 content remaining after 12 months at 22 °C and at 60 °C for 2 months (Fig. 5). During the long-term storage a small increase of some degradation products was observed, most of which were components B and C (Figs. S1, 2, 5, 6, Supplementary material). The same unidentified impurities were found in the samples of A1 substance. Most likely they are anthrafuran related 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 samples. We therefore evaluated the A1/HP-βCD formulation for antitumor activity and toxicology.

3.4. Antitumor activity The specific activity of A1/HP-βCD was evaluated in in vivo murine tumor models. First, we compared the therapeutic efficacy of A1/HPβCD and A1 in the P388 model under control of tolerance. The results 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 a significant level of ILS = 140% (p < 0.05). There was no statistical deviation among the compared drugs (p > 0.05). Autopsy results demonstrated an absence of increasing lymphatic nodules or ascite accumulation in all mice. The treatments with each substance were tolerated equally well without any side effects or death from toxicity. Since A1 potently inhibited the proliferation of MDR cells

3.3. Cytotoxicity in cell culture and acute in vivo toxicity of Anthrafuran/ Cavitron drug formulation Complexation of the active ingredient may result in changes to its specific activity or toxicity, so we compared the cytotoxicity and acute toxicity of A1 and the A1/HP-βCD complex. The antiproliferative effect of A1 and lyophilized A1/HP-βCD was investigated against human colon cancer cell line HCT116 (Table 2). Values of IC50 were similar for A1 and A1/HP-βCD indicating that the complexation with HP-βCD 635

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

H.M. Treshalina et al.

prototype of lyophilised A1/HP-βCD formulation with improved solubility and long-term stability was developed and evaluated. Measurements of the saturating concentration showed that the A1/HPβCD complexation enhanced the aqueous solubility >10-fold compared to A1. The formation of the inclusion complex is reversible as determined by detection of free A1 in the solution of the reconstituted lyophilized A1/HP-βCD formulation by HPLC and MS. Similar antiproliferative activity of A1 and A1/HP-βCD also confirmed the release of A1 from the A1/HP-βCD complex at physiological conditions. The in vivo study demonstrated that HP-βCD clearly decreased the acute toxicity of A1, probably via changes in distribution, bioavailability, and pharmacokinetics of the active ingredient. Notably, LD50 for the A1/HP-βCD drug formulation was 30% higher than for A1 (52.5 ± 5.4 and 68.3 ± 6.3 mg/kg, respectively). Thus, the application of β-CD derivatives can be useful for improvement of solubility of A1 as well as optimization toxicology, safety and tolerance. Finally, the A1/HP-βCD drug formulation showed a potent antitumor activity in the murine tumor models. The A1/HP-βCD complex and A1 in equal doses (5 × 30 mg/kg) increased life spans up to 140% of mice with i.p. transplanted P388 leukaemia. Furthermore, the A1/ HP-βCD drug formulation demonstrated significant and reliable antitumor efficacy on the Р388/ADR resistant tumor model and B16/F10 melanoma. Further studies on animal models are underway to verify optimal protocols for application of the A1/HP-βCD formulation. Additional studies are needed to fully characterize the toxicology, biodistribution, and pharmacokinetics of the A1/HP-βCD formulation for further in-depth preclinical evaluation of A1.

Table 3 Antitumor efficacy of A1 and A1/HP-βCD in the Р388 model. Group

Single dosea

Antitumor efficacy ILS, %

Control (5% glucose) A1 A1/HP-βCD

Number of mice with symptoms of peritoneal canceromatosisb, Ascites

Lymphatic nodules

0.5 ml

–

5/5

5/5

30 mg/kg

143c 142c

0/6 0/5

0/6 0/6

a

Daily i.p. for 5 days; On the day of death from leukaemia; Significant deviation from control (p < 0.05) without significant deviation between the treated groups. b c

Table 4 Efficacy of A1/HP-βCD on MDR tumor model Р388/ADR. Group

Single dose

Antitumor efficacy ILS, %

Control (5% glucose) A1/HP-βCD ADR

0.5 m

–

b

b

40 mg/kg 7.5 mg/kgc

36 1

c

Number of mice with symptoms of peritoneal canceromatosisa, Ascites

Lymphatic nodules

5/5

5/5

3/6

3/6 5/5

Acknowledgements

a

On the day of death from leukaemia; b Daily for 5 days; c Significant deviation were p < 0.05 against control or single dose of ADR, needed for to maintain resistance.

This study was supported by the Ministry of Industry and Trade of the Russian Federation (state contract 12411.1008799.13.007). The 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 assays, HPLC and MS analysis, and to Olga Tyurina (Ashland Specialty Ingredients) for providing HP-βCD (Cavitron®).

Table 5 Efficacy of the A1/HP-βCD against i.p. B16/F10 melanoma. Group

Single dosea

ALSb, days

ILS, %

Control (5% glucose) A1/HP-βCD

0.5 ml 30 mg/kg

17.8 [17.6 ÷ 18.0] 26.6 [25.9 ÷ 27.3]

– 50c

a b c

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ejps.2017.09.025.

Daily for 5 days; Average of life span; 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 melanoma (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/HPβCD significantly increased the lifespan up to the level of ILS = 50% (p < 0.001) with good tolerance.

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 anticancer 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.

4. Conclusion We developed a drug formulation for a poorly water soluble anticancer drug candidate, the anthrax[2,3-b]furan derivative A1. To improve 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 636

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

H.M. Treshalina et al.

Nourbakhsh, M., Jaafari, M.R., Lage, H., Abnous, K., Mosaffa, F., Badiee, A., Behravan, J., 2015. Nanolipoparticles-mediated MDR1 siRNA delivery reduces doxorubicin resistance 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]indole5,10-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: optimization 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 pyramyd. 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 inclusion 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 derivatives 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.

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 investigations. 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 ATPdependent 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 inclusion 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 vorinostatloaded 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