Inclusion complex of antiasthmatic compound with 2-hydroxypropyl-β-cyclodextrin: Preparation and physicochemical properties

Accepted Manuscript Inclusion complex of antiasthmatic compound 2-hydroxypropyl-β-cyclodextrin: Preparation physicochemical properties

with and

Marina V. Ol'khovich, Angelica V. Sharapova, German L. Perlovich, Sofia Ya. Skachilova, Nikolai K. Zheltukhin PII: DOI: Reference:

S0167-7322(16)34161-7 doi: 10.1016/j.molliq.2017.04.098 MOLLIQ 7254

To appear in:

Journal of Molecular Liquids

Received date: Revised date: Accepted date:

23 December 2016 14 April 2017 20 April 2017

Please cite this article as: Marina V. Ol'khovich, Angelica V. Sharapova, German L. Perlovich, Sofia Ya. Skachilova, Nikolai K. Zheltukhin , Inclusion complex of antiasthmatic compound with 2-hydroxypropyl-β-cyclodextrin: Preparation and physicochemical properties. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Molliq(2017), doi: 10.1016/ j.molliq.2017.04.098

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ACCEPTED MANUSCRIPT Inclusion complex of antiasthmatic compound with 2-hydroxypropyl-βcyclodextrin: preparation and physicochemical properties Marina V. Ol’khovich1*, Angelica V. Sharapova1, German L. Perlovich1,Sofia Ya. Skachilova2, Nikolai K. Zheltukhin2 1

Institute of Solution Chemistry, Russian Academy of Sciences, 1 Akademicheskaya Street, 153045, Ivanovo, Russia 2

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Russian Scientific Center for the Safety of Bioactive Substances, 142450, Staraya Kupavna, Russia *

E-mail: [email protected]

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Phone: 7(4932)351545

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Fax: 7(4932)336246

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ABSTRACT

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This work aims at obtaining 2-hydroxypropyl-β-cyclodextrin inclusion complexe with an original antiasthmatic compound in buffer solution and solid state with higher solubility and, hence, bioavailability. The compound solubility with different 2-hydroxypropyl-β-cyclodextrin concentrations has been studied by the phase solubility method. The linearity of the phase solubility

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diagrams is classified as an AL type and indicates the formation of inclusion complexes with 1:1 stoichiometry. The complex stoichiometry has been confirmed by the Job’s method. The solubility of the studied compound in buffer has been considerably increased by complexation with 2hydroxypropyl-β-cyclodextrin. Complexation stability constants and thermodynamic parameters have been calculated. The obtained inclusion complexes in solid state have been studied by the differential scanning calorimetry, Fourier transform infrared spectroscopy and X-ray powder diffraction. The kinetic characteristics of dissolution for the supramolecular complex have been obtained.

Keywords: antiasthmatic compound, 2-hydroxypropyl-β-cyclodextrin, inclusion complex, improved solubility, UV-vis, FTIR, XPRD

ACCEPTED MANUSCRIPT 1. Introduction

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Recent years have seen a great increase in the number of studies of 1,3,4–thiadiazole derivatives, many of which have a broad spectrum of pharmaceutical activity, namely antimicrobial [1], anticonvulsant [2], antitumor (anticancer) [3], antidiabetic [4], antiinflammatory [5], antitubercular [6] activity, etc. Resistance to the existing drugs is becoming one of the main problems in the world, which makes development of new biologically active compounds a very important research direction. The authors of [7] have determined that adding a phenyl ring into the 1,3,4–thiadiazole molecule increases the biological activity of the molecule, and compounds in which the phenyl ring contains electron-acceptor groups have a higher antimicrobial activity. The results of experimental studies of the pharmacological activity of the original substance N-(5-ethyl-1,3,4-thiadiazole-2-yl)-4-nitrobenzamide (Fig. 1a) have shown that this compound has significant direct bronchorelaxing activity against all kinds of isolated tracheal smooth muscle contractions induced by histamine and ovalbumin [8]. The broncholythic action discovered in vitro has been confirmed by simulating a bronchial spasm in vivo, which proves its antiasthmatic efficiency. By its bronchoprotective effects this drug is as good as the reference medicines - sodium cromoglicate and budesonide, and by its efficiency index it outperforms isoniazid, an antitubercular drug widely used in medicine. One of the ways to improve biopharmaceutical properties of biologically active compounds is creating drugs based on cyclodextrin inclusion complexes [9 – 12]. Cyclodextrins can form noncovalent associates with hydrophobic organic compounds; and they easily crystallize and have a well-defined chemical composition. The interest in cyclodextrins is caused by their unique ability to form “guest – host” inclusion complexes with substances of different nature. These complexes in many cases allow changing the physicochemical properties and, hence, the pharmacological action of these drugs. The most frequently used drugs of this kind are native cyclodextrins α, β, and γ as they are the only ones with a fixed conformation forming the corresponding macrocycle. The structural unit of the macrocycle is α-D-glucose in pyranose form with a chair conformation. Glucopyranose units of a molecule are interconnected by α-D1,4-glycosidic bonds. Despite the fact that natural cyclodextrins are widely used for studying and developing pharmaceutical compositions, there are some limitations on their application. For example, β-cyclodextrin has poor solubility in water due to its rigid structure caused by the formation of intermolecular hydrogen bonds between the secondary hydroxyl groups [13]. To improve its solubility β-cyclodextrin is chemically modified. 2-hydroxypropyl-β-cyclodextrin (2HP-β-CD) (Fig. 1b) obtained by hydroxyl substitution in positions 2, 3 and 6 with hydroxypropyl-substituents has a better solubility and a lower toxicity [14].

N N H5C2

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O

N H

NO2

a) b) Figure 1. Chemical structure of the studied compound studied (a) and 2HP-β-CD (R=CH3-CH(OH)CH2-) (b).

ACCEPTED MANUSCRIPT Thus, the aim of the present work was to study the complexation of the biologically active compound N-(5-ethyl-1,3,4-thiadiazole-2-yl)-4-nitrobenzamide with 2HP-β-CD in buffer solution and solid state. The complex was characterized by UV-spectroscopy, phase solubility diagram, thermal analysis, X-ray powder diffractometry (XPRD) and Fourier transform infrared spectroscopy (FTIR). 2. Materials and methods 2.1. Materials

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N-(5-ethyl-1,3,4-thiadiazole-2-yl)-4-nitrobenzamide (Fig. 1A) was synthesized by the method described in [8]. The structure of the obtained compound was confirmed by Fourier transform infrared spectroscopy and element analysis: Anal. Calc. for C11H10N4О3S: С 47.43%, H 3.59%, N 20.22%, S 11.51%. Found: С 47.48%, H 3.57%, N 20.31%, S 11.49%. FTIR (KBr, cm-1): ν = 3400 (-NH ), 1680 (-C=C arom.), 1520 (-CO-NH amide), 1632, 1310, 890 (thiadiazole ring). The purity of the synthesized compound after recrystallization was determined by HPLC, an Agilent 1100 series apparatus with a Kinetex C18, 2.6 μm, 3x100 mm (Phenomenex, USA). The mobile phase consisted of a mixture of water-methanol (42/58, v/v). The flow-rate was 0.4 mL/min. The detector was operated at 262 nm. The injection volume was 10 μl. A. 2HP-β-CD with the substitution degree 0.6 was purchased from Sigma-Aldrich and used as received. The detailed information about all the used chemicals is given in Table 1.

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Table 1 Source, CAS numbers and purity of chemicals.

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Source

Initial mass fraction purity, %

278.31

Synthesis

97.5

Recrysta llization

99.3

128446-35-5

~1400

Aldrich

≥ 96

None

-

Potassium dihydrogenphosphate

7778-77-0

136.1

SigmaAldrich

≥ 98.0

None

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Disodium hydrogen phosphate dodecahydrate

7758-79-4

358.1

Merck

≥ 98.5

None

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Potassium bromide

7758-02-3

119.0

SigmaAldrich

≥ 99.0

None

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CAS number

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N-(5-ethyl-1,3,4thiadiazole-2-yl)-4nitrobenzamide

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Compound

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2-hydroxypropyl-βcyclodextrin

Purificat ion method

Final mass fraction purity, %

ACCEPTED MANUSCRIPT Phosphate buffer pH 7.4 (I=0.15 mol/l, 0.067 M) imitating the blood plasma medium was prepared combining KH2PO4 (9.073 g in 1 L H2O) and Na2HPO4·12H2O (23.611 g in 1 L H2O) salts [15]. Bidistilled water used to prepare the buffer solution had electrical conductivity of 2.1 μS cm-1. The compounds were weighed in AND Gemini Analytical Balance GR-202 (Japan). The standard uncertainty of measuring the mass of samples was u(m)=0.01 mg. The solution pH was measured by using the pH meter FG2-Kit (Mettler Toledo, Switzerland) standardized with pH 1.68, 6.86 and 9.22 solutions. 2.2. Phase solubility studies

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Phase solubility diagrams of the compound with 2HP-β-CD in phosphate buffer pH 7.4 were obtained according to Higuchi and Connors [16]. An excessive amount of the drug was added to 10 ml of phosphate buffer or buffer solutions with different 2HP-β-CD concentrations (0.009 - 0.09 mol/kg-1) The suspensions were sealed and shaken at 298.15, 303.15, 308.15 and 313.15 ± 0.05 K for 72 hours in the thermostatically controlled box to ensure equilibrium. The saturation was confirmed by the presence of the undissolved part of the substance. Once the equilibrium was achieved, the saturated solution was taken and centrifuged (Biofuge stratos, Germany) under the temperature control for 5 minutes at a fixed temperature. The solid phase was removed through isothermal filtration by the filter MILLEX®HA 0.20 μm (Ireland). An aliquot of the saturated solution was taken at fixed temperature using the thermostated equipment and then diluted by the solvent. The absorbance was measured spectrophotometrically at room temperature at λmax=270 nm. The presence of CDs did not interfere with the spectrophotometric assay of the drug. The experimental results are reported as an average value of at least three replicated experiments. The calibration procedure was made at room temperature using the solutions with known concentrations of the substance in buffer pH 7.4. The appropriate mass of the above-mentioned drug was exactly weighed and totally dissolved in the desired volume of buffer pH 7.4 (V= 25 ml). The standard curve of the drug, a plot of the measured absorbances against the drug concentration, was a straight line (R > 0.999) in the appropriate concentration range. All the data shown were from the average of three determinations. The apparent stability constants (K) were calculated from the phase solubility diagrams according to the Higuchi–Connors equation, where S0 is the intrinsic solubility of the solute in the absence of CD and slope means the corresponding slope of the phase-solubility diagram data by the equation: slope (1) K S 0 (1  slope) The thermodynamic parameters of complexation, i.e. the standard enthalpy change (∆Н0) and the standard entropy change (∆S0) were calculated by the integral form of the Van’t Hoff’s equation (2): H 0 S 0 ln K    (2) RT R where T is the temperature, R is a gas constant. The Gibbs energy change (∆G0) was calculated by equation 3: ∆G0 = -RTlnK (3) 2.3. Job's plot method The stoichiometry of inclusion was also determined by using a continuous variation technique developed by Job [17]. Equimolar solutions of the compound and 2HP-β-CD with the concentration of 1.35∙10-5 mol·kg-1 were mixed to a standard volume (1 ml : 9 ml; 2 ml : 8 ml; 3 ml : 7 ml and so on) varying the molar ratio but keeping the total concentration of the species

ACCEPTED MANUSCRIPT constant. After stirring the UV–vis spectra were recorded. The absorbance (D) at λmax =270 nm was measured for all the solutions and the difference in absorbance (ΔD) in the presence and in the absence of 2HP-β-CD was plotted as a function of (R = [Drug]/[Drug]+[2HP-β-CD]). 2.4. Preparation of the physical mixture and inclusion complex in solid state

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The physical mixture of the compound studied with 2HP-β-CD (1:1 molar ratio) was prepared by gently mixing the accurately weighed components in a mortar with a spatula. The 1:1 M ratio complex of the studied compound with 2HP-β-CD was prepared by the ball-milling method using a planetary micro mill Pulverisette 7 (Fritsch, GmbH, Idar-Oberstein, Germany) in 12 ml agate grinding jars with ten 5 mm agate balls at a rate of 600 rpm for 60 min. To simplify the process of the compound molecules inclusion in the cyclodextrin cavity, 0.05 ml of methanol was added at grinding. To exclude the influence of the sample preparation technique on the physicochemical properties of the guest molecules, pure compounds were treated by the same grinding procedure, omitting 2HP-β-CD from the preparation. All the samples studied were kept in a desiccator until further analysis.

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2.5. Characterization of inclusion complex

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2.5.1. UV-vis spectrophotometry

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UV-vis spectra of the individual compound and inclusion complex were obtained by Cary 50 spectrophotometer (Varian, Palo Alto, USA) using a 1 cm quartz cell. The scans were registered from 200 to 500 nm. 2.5.2. Differential scanning calorimetry (DSC)

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The melting temperatures of the individual compound and inclusion complex were determined using the differential scanning calorimeter of Perkin Elmer Pyris 1 DSC (Perkin Elmer Analytical Instruments, Norwalk, Connecticut, USA). The DSC procedure was conducted in the atmosphere of drained argon of high purity of 99.990 % at the speed of 20 ml/min by using standard aluminum containers for samples. DSC was calibrated by the indium melting peak onset (Perkin Elmer P/N 0319-0033). Indium melting temperature was Tonset=156.5 ± 0.1°C (n=10). The melting enthalpy equaled 28.48 J·g-1 and corresponded to the recommended value of 28.45 J·g-1. All the DSC experiments were conducted at the heating rate of 10°C·min-1 in the range of 25-300oC. The samples (2-5 mg) dried before the experiment were placed into aluminum crucibles. The weighing accuracy was ± 0.05 mg. An empty sealed crucible was used as a reference. 2.5.3. Fourier transform infrared spectroscopy (FTIR) The FTIR spectra of 2HP-β-CD, the compound under study and the inclusion complex were obtained using an infrared Fourier transform spectrometer, model Vertex 80v (Bruker Optik GmbH, Ettlingen, Germany). The spectral range was 400–4600 cm-1 with 128 scans and a resolution of 2 cm-1. The samples were diluted in KBr powder and pellets were made to perform the measurements. 2.5.4. X-ray powder diffraction (XPRD) The powder X-ray diffraction patterns of the pure compound and inclusion complex were recorded on an X-ray diffractometer D8 Advance (Bruker AXS GmbH, Karlsruhe, Germany) using Mo Ka radiation. The voltage and current applied were 40 kV and 40 mA, respectively.

ACCEPTED MANUSCRIPT The data were collected in the range of 2θ = 5–30 with a step size 0.03o, the scan time for one step was 3. 2.5.5. Dissolution kinetics

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The kinetic curves of dissolution of the individual compound and inclusion complex were determined by the classical shake-flask method. An excess amount of the compound or inclusion complex was placed in 10 ml glass vials which were filled with buffer solution pH 7.4. The obtained suspensions were placed in an air thermostat and shaken during the selected dissolution periods at 25  0.1C. The solid phase was separated by filtration (Rotilabo® syringe filter, PTFE, 0.2 μm). The concentration of the dissolved substances was determined spectrophotometrically.

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3. Results and discussion

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3.1. UV–vis spectroscopy

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The absorption spectra of the compound under study in pure buffer solution and buffer solutions with different 2HP-β-CD concentrations were measured within the range of 200-400 nm and are shown in Figure 2. The UV-spectrum of the individual compound buffer solution contains intensive absorption bands with the maxima at 261 nm and 342 nm. The first one is typical of benzene absorption, while the other is caused by conjugation between benzene and thiadiazole rings. 2HP-β-CD addition to the buffer solution considerably increases the optical density of the compound under study and batochromic effect of the absorption band 261→270 nm, which reveals the noncovalent interaction of the compound with cyclodextrin. Similar changes were also reported by other authors [18].

1,8

4

261nm

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1 - S = 0.13 10 mol/kg -4 2 - S = 0.75 10 mol/kg -4 3 - S = 1.54 10 mol/kg -4 4 - S = 5.26 10 mol/kg

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Figure 2. UV–vis absorption spectra of the studied compound in buffer pH 7.4 at different 2HP-β-CD concentrations: 1 – 0.00, 2 – 0.01, 3 – 0.02, 4 – 0.09 mol/kg.

ACCEPTED MANUSCRIPT 3.2. Phase-solubility studies and stoichiometry by Job’s method

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Experimental solubilities of the compound in pure buffer and buffer with different 2HPβ-CD concentrations in the temperature range 298.15 -313.15 K are presented in Table 2. Table 2 Experimental solubilities S of the compound in buffer pH 7.4 and buffer pH 7.4 with different 2HP-β-CD concentrations (SCD) in a temperature range 298.15 - 313.15 K and pressure p = 0.1 MPa. S·104 (mol∙kg-1) SCD (mol∙kg-1) 298.15 K 303.15 K 308.15 K 313.15 K 0 0.13 ± 0.05 0.21 ± 0.05 0.25 ± 0.11 0.34 ± 0.08 0.0099 0.75 ± 0.05 1.01 ± 0.13 1.24 ± 0.13 1.44 ± 0.11 0.0243 1.54 ± 0.13 2.14 ± 0.18 2.83 ± 0.15 3.36 ± 0.09 0.0387 2.14 ± 0.18 3.24 ± 0.16 3.98 ± 0.12 4.58 ± 0.15 0.0907 5.26 ± 0.16 7.42 ± 0.21 9.20 ± 0.18 10.70 ± 0.22 As Table 2 shows, the compound solubility grows with the cyclodextrin concentration increase. This may be associated with the inclusion complex that is formed when a molecule of the compound under study or its hydrophobic fragment enters the 2HP-β-CD cavity creating a lipophilic medium. The main driving force of complexation is dipole-dipole interaction and release of “high-energy water” molecules from the cyclodextrin cavity into the solvent volume [19, 20]. So, the drug solubility at this temperature is a sum of intrinsic solubility and the inclusion complex solubility. As CD concentration grows, more molecules of the compound enter its cavity, which increases the total solubility of the compound. As the table shows, the compounds solubility in 2HP-β-CD presence increases with the temperature growth, which may be caused by the intrinsic solubility increase with the elevated temperature [21]. To study solubilization, we obtained diagrams of phase solubility of the compound under consideration versus 2HP-β-CD concentration in buffer solution in the temperature range of 298 - 313 К (Figure 3).

-3

1.0x10

-4

8.0x10

T = 298 K T = 303 K T = 308 K T = 313 K

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6.0x10

-4

4.0x10

-4

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0.015

0.030

0.045

0.060

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SCD, mol/kg Figure 3. Phase-solubility diagrams of compound studied in the presence of 2HP-β-CD in buffer pH 7.4 at different temperatures (S – concentration of compound, SCD – concentration 2HP-β-CD).

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It has been determined that at the 2HP-β-CD concentration of 0.09 mol/kg the compound solubility in buffer grows from 1.36∙10-5 to 52.59∙10-5 mol/kg, which represents an almost 39 time solubility increase compared to the compound in pure buffer. The obtained phase solubility diagram is linear and can be classified as an AL type according to Higuchi and Connors [16]. The slope of the solubility diagram is less than one; it is therefore assumed that the solubility increase can be attributed to the formation of the first order complex of the bioactive compound with the cyclodextrin molecule. Stoichiometry of the obtained inclusion complex has been confirmed by the Job’s method [17]. According to the continuous variation method, if a physical parameter directly related to the concentration of the complex can be measured for a set of samples with continuously varying molar fraction of its components. The sample with the maximum complex concentration was the one in which the molar ratio R correspondeds to the complexation stoichiometry. In Figure 4, the maximum absorbance variation for the compound in CD is observed for R = 0.5, which might indicate that the main stoichiometry is 1:1, which agrees with the stoichiometry obtained in the phase-solubility study.

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Figure 4. Job’s curve for inclusion complex of the studied compound with 2HP-β-CD in buffer pH 7.4 at 298 K. The stability constant value is an important factor indicating the possibility of using complexes in pharmaceutics. If the stability constant is low (logK < 2), the inclusion complex is unstable and is quickly destroyed before it fulfills its functions in the human body. If, on the other hand, logK > 4, this hinders the drug release from the cyclodextrin cavity [22, 23]. Based on the data of the obtained phase diagrams, we calculated the stability constants of the inclusion complex of the compound under study with CD at different temperatures by equation 1. The parameters of this equation and stability constants are presented in Table 3. The inclusion complex obtained by us has the optimal stability constant values equal to 2.62 at 298 K, which confirms the application potential of this complex in pharmaceutics. Table 3 The values of the parameters of equation (1) and stability constants (K) of the inclusion complex compound with 2HP-β-CD in buffer pH 7.4 at different temperatures and pressure p = 0.1 MPa.

ACCEPTED MANUSCRIPT Correlation coefficient 298.15 0.9990 1.36  0.67 5.60  0.14 415.4 14.6 303.15 0.9999 2.10  0.18 7.93  0.04 381.5 12.4 308.15 0.9996 2.85  0.72 9.82  0.15 347.8  13.5 313.15 0.9993 3.64  0.11 11.37  0.21 316.3  14.2 The values of the stability constants of the studied inclusion complex decrease as the temperature grows, which is typical of an exothermal process. Such thermal effect for stability constants is characteristic of cyclodextrin systems [24, 25]. The temperature increase evidently leads to a decrease in the drug transition to the soluble form as a cyclodextrin complex, which is caused by host and guest molecules mobility growth. Slope·103

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Т (К)

3.3. Thermodynamic parameters of complexation

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Thermodynamic and structural characteristics of complexation can be effectively used to evaluate cyclodextrin solubilizing and stabilizing action on the change in physicochemical properties of the guest-molecules entering the cyclodextrin cavity. The thermodynamic parameters of inclusion complex formation of the studied compound with 2HP-β-CD were calculated from the logarithmic dependence of stability constant on the reciprocal temperature (Figure 5) by equations (2, 3) and are represented in Table 4.

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Figure 5. Logarithmic dependence of the stability constant on the reciprocal temperature: lnK = (0.34  0.16)+ (1696.8  47.8)/Т (R = 0.9992) for the inclusion complex of the studied compound with 2HP-β-CD.

Table 4 Thermodynamic parameters of inclusion complex 2HP-β-CD with the compound in buffer pH 7.4 at 298 K and pressure p = 0.1 MPa. К ( kg·mol-1) ∆G0 (kJ·mol-1) ∆H0 (kJ·mol-1) ∆S0 (J·mol-1·K-1) 415.4  14.6

-14.94  2.68

-14.11  0.39

2.82  0.15

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Analysis of the obtained data has shown that the compound complexation with 2HP-βCD in buffer solution is characterized by negative Gibbs energy and enthalpy values, which is a reflection of a spontaneous exothermal process. The negative enthalpy changes, as a rule, are connected with strong van-der-Waals interaction and hydrogen bond formation between the host and the guest [26, 27]. The much bigger (in absolute magnitude) enthalpy contribution to the Gibbs energy than the entropy one indicates that the compound introduction into the 2HP-β-CD cavity is an enthalpy-driven process. Complexation, on the one hand, is accompanied by a loss of rotational and translational degree of molecule freedom of the compound and CD and, on the other hand, results in a release of highly ordered solvent molecules that surround the host and guest molecules and thus increase the system entropy. The small scale of the entropy change shows that the contributions of these opposite effects are practically equal. The weakly positive entropy values are explained by the structural destruction of solvation shells of the interacting components and dehydration of the cyclodextrin macrocavity [28]. 3.4. Differential scanning calorimetry

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Differential scanning calorimetry (DSC) is a powerful analytical tool for studying substances in solid state as it can provide detailed data about their physical and energy properties [29, 30]. As such, DSC is widely used to study the interactions between drug compounds and cyclodextrins in solid state [31, 32]. By comparing the thermograms of individual components, their physical mixture and the assumed inclusion compound we can get information about solidstate modifications and interactions between the components and confirm complexation. The thermal curves of the bioactive compound before and after 60 min of grinding, 2HPβ-CD, physical mixture and inclusion complex are shown in Figure 6.

Figure 6. DSC-curve of the studied compound (a); compound after milling 60 min (b); 2-HP-β-CD (c); physical mixtures (d) and inclusion complex (e).

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3.5. Fourier transform infrared spectroscopy

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The thermogram of the pure component (Fig. 6a) has a sharp endothermic peak at 282.2 ± 0.2°С (ΔHm = 37.4 ± 0.5 kJ∙mol-1) corresponding to the compound melting temperature and confirming its crystalline nature. Absence of phase transitions between 25°С and the compound melting temperature shows that this substance does not contain polymorph modifications or hydrated forms. 60 minutes after grinding (Fig. 6b), the compound melting temperature and enthalpy reduced insufficiently (Tm = 281.1°С, ΔHm = 35.6 kJ∙mol-1), which can be connected to a small crystallinity reduction at grinding. According to the amorphous nature of 2HP-β-CD, its DSC curve (Fig. 6c) shows a wide endothermic peak in the region between 53°С and 150°С, which can be connected to the release of crystallized water molecules from the CD cavity [35]. The DSC thermogram of the physical mixture (Fig. 6d) contains two separate peaks corresponding to the melting peaks of the individual components. This proves the heterogeneity of the mixture and absence of chemical interaction between the compound and CD, which agrees with the results obtained earlier for similar mixtures [33, 34]. The inclusion complex thermogram only has a wide cyclodextrin peak (Fig. 6e). Complete disappearance of the melting peak of the pure compound indicates amorphization of the crystalline substance in the presence of an amorphous support and confirms inclusion complex formation.

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FTIR spectroscopy is widely used for studying solid inclusion complexes of drugs with CD. This method allows determining which bands of stretching vibrations of the individual compound and cyclodextrin change in the complexation process. Disappearance, widening, intensity change of the peaks and their shifts indicate complex formation. This fact may result from a decrease in guest molecule stretching vibrations caused by CD introduction into the cavity or by weakened interatomic bonds [36]. The IR spectrum of the pure compound before and after 60 min of grinding, 2HP-β-CD, physical mixture and inclusion complex with cyclodextrin are shown in Figure 7. The IR spectrum of the bioactive compound (Fig. 7a) shows bands of stretching vibrations at 3400 cm-1 (NH, amide), 3002 cm-1 (CH, aromatic ring), 1520 cm-1 (CO-NH, amide), 1680 cm-1 (C=C, aromatic ring), 1632, 1310, 890 cm-1 (thiadiazole ring). In the IR spectrum of the compound after grinding (Fig. 7b), a slight shift and a decrease in the intensity of the valence vibration bands are observed, which is associated with a partial loss of crystallinity of the compound during the milling. The 2HP-β-CD IR-spectrum (Fig. 7c) contains a wide band at 3402 cm-1 that belongs to the H-bond stretching vibrations of O-H hydroxyl groups. The other bands are arranged in the following way: 2930 cm-1 (С-Н) stretching vibrations of С-Н bonds in CH and CH2 groups; 1636 cm-1 (O-H, H bonded) deformation vibrations of О-Н bonds in С-О-Н groups and water molecules; 1369 and 1137 cm-1 (С-Н, deformation vibrations in СН2ОН and СНОН groups); an adsorption peak at 1036 cm-1 (С-О-С). The IR spectrum of the 2HP-β-CD physical mixture with the compound (Fig. 7d) is a combination of the spectra of these two molecules. There are considerable differences in the inclusion complex spectrum compared to that of the physical mixture of the components. Almost all of the peaks of the individual compound are smoothed, which indicates a strong intermolecular interaction between the compound and 2HP-β-CD. Thus, the IR spectrum of the complex (Fig. 7e) has no low-frequency band (1680 cm-1) referring to the С=С bond of the aromatic ring. The wide band of O-H stretching vibrations observed in the 2HP-β-CD spectrum at 3402 cm-1 is also seen on the inclusion complex spectrum. The band at 1636 cm−1 in the 2HPβ-CD spectrum, which belongs to the crystallization water, is not observed in the complex spectra. This indicates that the water molecules inside the 2HP-β-CD cavity have been replaced with the compounds molecules thus confirming the formation of the inclusion complex in the solid state.

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Figure 7. FTIR spectrum of the studied compound (a); compound after milling 60 min (b); 2-HP-β-CD (c); physical mixtures (d) and inclusion complex (e). 3.6. X-ray powder diffractometry (XPRD) XPRD analysis has been lately widely used for studying cyclodextrins and their inclusion complexes in powder or microcrystal forms. Comparison of XPRD-patterns of individual components, their physical mixture and inclusion complex helps to confirm changes in solidstate properties resulting from interactions between the components. New diffraction peaks in the spectrum and the shift of the representative guest molecule peaks as well as the changes in their relative intensity confirm the formation of a new solid phase and complexation [37]. The XPRD patterns of the pure compound before and after 60 min of grinding, physical mixture and inclusion complex are shown in Figure 8.

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Figure 8. XPRD spectrum of the studied compound (a); compound after milling 60 min (b); 2-HP-β-CD (c); physical mixtures (d) and inclusion complex (e).

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The XRPD pattern of the compound (2θ = 6.2, 12.6, 13.2, 17.2, 18.1, 26.3) indicates its crystalline nature (Fig. 8a). After 60 min grinding, the X-ray diffraction pattern of the compound is observed with a slight shift, broadening, and a decrease in the intensity of the characteristic bands, which is due to a partial loss of crystallinity of the compound during grinding (Fig. 8b). In contrast, the 2HP-β-CD diffractogram (Fig. 8c) has broad diffraction peaks at about 2θ = 14.9 and 21.7, which indicates a disorder in the crystal structure, i.e. amorphism. The XRPD pattern of the physical mixture of bioactive compound and 2HP-β-CD (1:1) is almost a superposition of the patterns contributed of these two molecules (Fig. 8d). The dominance of the principal peaks of 2HP-β-CD in the physical mixture is a result of its high percentage in the mixture (84%, w/w). The inclusion complex of the studied compound shows broad diffuse peaks with low intensities and a complete disappearance of the principal diffraction peaks apparent in the XRPD patterns of the studied compound, which confirms the formation of a real inclusion complex consisting of a new solid phase of an amorphous structure (Fig. 8e). 3.7. Dissolution kinetics It is known that dissolution rate in aqueous media is one of the key parameters among other physicochemical properties of biologically active compounds as it is effectively correlated with the drugs oral bioavailability. The dissolution profiles for pure compound and inclusion complex with 2HP-β-CD in buffer pH 7.4 at 25oC are shown in Table 5 and Figure 9.

ACCEPTED MANUSCRIPT Table 5 Experimental concentrations S of the compound and inclusion complex with 2HP-β-CD in buffer pH 7.4 at different time intervals, T=298.15 and pressure p = 0.1 MPa S·105 (mol∙kg-1) compound inclusion studied complex 1.56 3.87 1.98 6.14 2.05 6.17 2.23 6.35 2.38 6.46 2.52 6.71 2.88 7.03 2.91 7.28 2.99 7.50

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Figure 9. Dissolution profiles of the studied compound (a) and inclusion complex with 2HP-βCD (b) in buffer pH 7.4 at 37oC.

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The most significant changes in the inclusion complex solubility are observed in the initial part of the kinetic solubility curves. The concentration of the compound reaches a maximum in the first 18 min of the experiment. The individual compound dissolution rate is 1.1∙10-6 mol∙kg-1∙min-1. As expected, the inclusion complexes dissolve much faster and have greater solute quantity than the pure drug. The dissolution rate for the compound complex equals 3.5∙10-6 mol∙kg-1∙min-1, thus increasing by a factor of 3.2. The rate increase may be attributed to both weaker crystallinity and higher hydrophilicity of the complex compared to the pure substance. Enhanced solubility of the drug in the buffer remains unchanged for at least 6 h, which seems enough to affect the human body. 4. Conclusion The 2HP-β-CD effect on the solubility of the original antiasthmatic compound in buffer solution has been studied by the phase solubility method. It has been found that the presence of 0.09 mol/kg of 2HP-β-CD in the solution improves the compound solubility by 39 times. The

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solubility of the studied compound grows with the increase in 2HP-β-CD concentration, which indicates the formation of inclusion complexes with 1:1 stoichiometry. Based on the obtained experimental data, we have calculated the inclusion complex stability constant and the main thermodynamic parameters of its formation. It has been shown that supramolecular complex formation is an exothermic and enthalpy-determined process. The results of this research have proved the possibility to use 2HP-β-CD to considerably increase the solubility of the studied compound in water systems, which will improve its bioavailability. The inclusion complex in solid state was obtained by grinding the individual component and cyclodextrin with additions of ethanol and characterized by DSC, FTIR spectroscopy and X-ray powder diffraction methods. The dissolution profiles of the pure compound and inclusion complex with 2HP-β-CD in buffer solution were determined.

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Acknowledgments

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This work was supported by the grant of RFBR No. 15-43-03085 r_centre_a. We thank “the Upper Volga Region Centre of Physicochemical Research” for technical assistance.

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ACCEPTED MANUSCRIPT Highlights • Inclusion complex of antiasthmatic compound with 2HP-β-CD was prepared • The solubility of compound complexing with 2HP-β-CD was improved

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• Complex formation was confirmed by UV-vis, FTIR, DSC and XPRD methods.