Molecular Dynamics, Physical Stability and Solubility Advantage from

Article pubs.acs.org/molecularpharmaceutics

Molecular Dynamics, Physical Stability and Solubility Advantage from Amorphous Indapamide Drug Z. Wojnarowska,*,† K. Grzybowska,† L. Hawelek,†,‡ M. Dulski,† R. Wrzalik,† I. Gruszka,† and M. Paluch† †

Institute of Physics, University of Silesia, ul. Uniwersytecka 4, 40-007 Katowice, Poland Institute of Non-Ferrous Metals, ul. Sowinskiego 5, 44-100 Gliwice, Poland

‡

K. Pienkowska and W. Sawicki Department of Physical Chemistry, Medical University of Gdansk, Hallera 107, 80-416, Gdansk, Poland

P. Bujak Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00664 Warszawa, Poland

K. J. Paluch and L. Tajber School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Dublin 2, Ireland

J. Markowski ENT Department, Silesian Medical University, ul. Francuska 20, Katowice, Poland ABSTRACT: This study for the first time investigates physicochemical properties of amorphous indapamide drug (IND), which is a known diuretic agent commonly used in the treatment of hypertension. The solid-state properties of the vitrified, cryomilled and ball-milled IND samples were analyzed using X-ray powder diffraction (XRD), mass spectrometry, nuclear magnetic resonance (NMR), infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and broadband dielectric spectroscopy (BDS). These analytical techniques enabled us (i) to confirm the purity of obtained amorphous samples, (ii) to describe the molecular mobility of IND in the liquid and glassy state, (iii) to determine the parameters describing the liquid-glass transition i.e. Tg and dynamic fragility, (iv) to test the chemical stability of amorphous IND in various temperature conditions and finally (v) to confirm the long-term physical stability of the amorphous samples. These studies were supplemented by density functional theory (DFT) calculations and apparent solubility studies of the amorphous IND in 0.1 M HCl, phosphate buffer (pH = 6.8), and water (25 and 37 °C). KEYWORDS: dielectric spectroscopy, molecular dynamics, glass transition, diuretic agents, amorphous active pharmaceutical ingredients, tautomerization

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INTRODUCTION Indapamide (IND) is a sulfonamide diuretic drug used in the treatment of hypertension. Despite having a slightly different chemical structure than thiazides (e.g., hydrochlorothiazide), its mechanism of action remains similar. It increases the urine volume by increasing the renal excretion of sodium, chlorine, potassium and magnesium ions. Apart from the diuretic action indapamide exerts also spasmolytic effects on blood vessels, consequently reducing the blood pressure.1 However, the commercial form of IND available on the market works very weakly. The most probable reason is its low solubility (75 mg/L) and consequently poor bioavailability.2 Thus, the question arises how to improve the solubility of indapamide? © 2013 American Chemical Society

During the past decade improvement of solubility of poorly water-soluble medicines has become one of the most important aspects of pharmaceutical research. In the literature one can find very few different approaches as to how to realize this task. One of the methods is to modify the active component by means of salification. It means that the pure drug is transferred into nitrate, hydrochloric or other salts which usually reveal Received: Revised: Accepted: Published: 3612

February 28, 2013 August 22, 2013 September 5, 2013 September 5, 2013 dx.doi.org/10.1021/mp400116q | Mol. Pharmaceutics 2013, 10, 3612−3627

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better solubility than the original compound.3,4 Additionally, one can also apply several formulation strategies such as micronization,5 molecular dispersion,6 incorporation of surfactants, comilling with sugars,7 cyclodextrins or PVP and amorphization.8,9 The last method is a relatively simple approach and thus widely employed to enhance the apparent solubility of pharmaceutical materials. As examples one can mention amorphous verapamil hydrochloride,10 glipizide11 or celecoxib.12 It should be noted that apart from affording a higher apparent solubility (and consequently higher bioavailability) an amorphous form can also improve processability of drugs.13 For instance, by tableting an amorphous pharmaceutical as opposed to the crystalline form one can reduce the amount of additives in the tablet and thus simplify the formulation. Unfortunately, preparation of amorphous drugs may be also quite complex. First, an amorphous phase is thermodynamically unstable, which in consequence may lead to crystallization of the drug under storage conditions and subsequent loss of the benefits arising from the amorphous state.14 Second, as a result of higher chemical reactivity, amorphous pharmaceuticals are prone to chemical degradation as well as isomeric transformations during manufacturing and storage. Tautomerization, reversible proton migration in an organic molecule, is of a great importance in pharmaceutical science since it has been shown that preparation of an amorphous pharmaceutical, in which tautomerization reaction takes place, can lead to a state which is chemically different from its crystalline counterpart.15 This phenomenon can have positive as well as negative impacts on the quality of amorphous drugs. On the one hand, one can expect that the presence of tautomerization products may affect the drug solubility, and consequently it may enhance the bioavailability of the active substance. Moreover, the occurrence of two or more tautomers in the sample may reduce its crystallization tendency.34 Also, tautomerization of drugs might lead to serious medical consequences, since one cannot exclude that the biological activity of the given tautomers may be different from that of the parent drug. All these factors are reasons why in recent years one of the main challenges of developing amorphous pharmaceuticals is to understand the molecular interactions in the glassy and supercooled liquid phases and subsequently to predict the physical and chemical stability of amorphous drugs. In this study we investigate the amorphous indapamide drug. Polymorphic and pseudopolymorphic forms of indapamide16 have been identified and characterized, however no reports are available on its amorphous form. Three different preparation techniques, such as quench cooling of the melt, ball milling and cryogrinding, were applied to convert the crystalline indapamide into its amorphous form. To confirm the amorphous nature of the examined materials, X-ray powder diffraction (XRD) and differential scanning calorimetry (DSC) were used. The DSC was also applied to determine the glass transition temperature (Tg) of amorphous IND. The results of FT-IR experimental technique give us information about the chemical stability of indapamide drug. Furthermore, dielectric spectroscopy (BDS), which is often applied to study liquid− glass transitions in various pharmaceuticals, was employed to characterize the molecular dynamics of the analyzed diuretic agent. Experimental results obtained were supplemented by atomic density functional theory (DFT) calculations. Finally, the apparent solubility studies of amorphous IND samples were performed highlighting crystallization tendency of amorphous phase in aqueous media.

Article

EXPERIMENTAL SECTION

Material. The tested indapamide hemihydrate 4-chloro-N(2-methyl-2,3-dihydro-1H-indol-1-yl)-3-sulfamoylbenzamide (C16H16ClN3O3S·(1/2)H2O), MW = 365.83 g/mol with the chemical structure presented in Figure 1, was supplied from

Figure 1. Chemical structure of indapamide.

Quimica Sintetica (Madrid, Spain) as a white crystalline powder and was used without further purification. The powder X-ray diffractogram of crystalline IND (see blue line in Figure 2) was

Figure 2. Thermal analysis of crystalline and amorphous forms of IND. All DSC thermograms were obtained during heating at a rate of 10 K/min. The inset panel shows the glass transition regions of quenched, cryomilled and ball-milled IND samples.

in good agreement with that of the commercial material as published recently by Ghugare et al.16 Indapamide is soluble in acetonitrile, ethyl acetate, glacial acetic acid, methanol, ethanol and other alcoholic solvents. It is very slightly soluble in ether and chloroform, while practically insoluble in water. Amorphous Samples Preparation. Quench Cooling of the Melt. Crystalline IND hemihydrate was placed on a stainless steel plate and heated on a hot plate until a complete melt was achieved (the sample becomes a yellow, transparent film). The molten sample was then quenched by being put onto a cold tile. Ball Milling. The room temperature ball milling was performed using a Planetary Ball Mill (Retsch, Germany). A zirconium jar was filled with the examined material and 6 zirconia balls (20 mm in diameter). The rotation speed was set to 400 rpm. We have performed three separate tests with the same amount of material (6 g) applying different grinding times. Each milling cycle lasted 15 min and was followed by a 5 min break. 3613

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Sealed aluminum crucibles (40 μL) with lids containing five punctures to enable water to volatilize during heating were used for all samples. Standard DSC measurements were performed at a heating rate of 10 K/min under a nitrogen purge (60 mL/min). Moreover, using a stochastic temperature-modulated differential scanning calorimetry (TMDSC) technique implemented by Mettler-Toledo, TOPEM , the dynamic behavior of the glass−liquid transition of the studied materials has been analyzed in the frequency range from 4 mHz to 40 mHz in one single measurement at a heating rate of 0.5 K/min. In the experiment, the temperature amplitude of the pulses of 0.5 K was selected. The calorimetric structural relaxation times τα = 1/2πf were determined from the temperature dependences of the real part of the complex heat capacity cp′(T) obtained at different frequencies in the glass transition region. The glass transition temperature Tg was determined for each frequency as the temperature of the half step height of Cp′(T). Infrared Measurements (FT-IR). Infrared measurements were performed using a Bio-Rad FTS-6000 spectrometer equipped with a KBr beam splitter, a standard source and a DTGS Peltier-cooled detector. The single crystalline and glassy spectra of indapamide have been collected using GladiATR diamond accessory (Pike Technologies) in the range from 400 to 4000 cm−1. The temperature measurements were carried out using a heated plate of the GladiATR diamond accessory (Pike Technologies) equipped with a temperature controller with accuracy ±0.1 K. Time-dependent spectra were recorded every 10 min. All spectra were recorded with a spectral resolution of 2 cm−1. Presented results are an average of 16 scans in order to ensure good quality spectra. Mass Spectrometry. Liquid chromatography−mass spectrometry (LC−MS) was carried out using a Thermo Accela Liquid chromatography coupled to a Thermo LTQ-XLOrbitrap Discovery mass spectrometer as the detector. The column used for chromatographic separation was a Waters Acquity HSS T3 C18, 2.1 mm × 150 mm 1.8 μm at operating at 30 °C. A flow rate of 100 μL/min was used with an injection volume of 4 μL. The mobile phase consisted of two solutions: 0.1% v/v formic acid in HPLC grade water as phase A and 0.1% v/v formic acid in HPLC grade acetonitrile as phase B. The total run time was 10 min, and the following gradient method was used: 20% phase B to 80% phase B over 8.00 min, hold until 7.00 min, then 20% phase B at 7.01 min and equilibrate for 3 min. The LTQ-XL ion trap mass spectrometer was coupled to the Accela LC system via an electrospray ionization (ESI) probe. The capillary temperature was maintained at 310 °C, sheath gas flow rate 60 arbitrary units, auxiliary gas flow rate 5 arbitrary units, sweep gas flow rate 0 arbitrary units, source voltage 3.20 kV, source current 100 μA, capillary voltage 48 V and tube lens 82 V. Indapamide was detected in negative ion mode, and its retention time was 6.6 min. Full FTMS scanning (m/z 80−1000) was used to detect degradation products. The samples as methanolic solutions were also infused directly into the ion chamber at a flow rate of 5 μL/min using a Hamilton syringe. Apparent Solubility Measurements. HPLC Equipment for the Solubility Assay. In this study, a high performance liquid chromatography HPLC system (Shimazu LCsolution Chromatography System, model Prominence LC-20A) was used to quantify the amount of indapamide dissolved. The system was equipped with a UV/vis detector (model SPD-20 A), a quaternary gradient pump (model LC-20 AD) with a paralleltype double plunger, a degasser (model DGU-20 A5) with a

Total milling times of indapamide were 1 h, 2.5 h and 5 h. After milling, indapamide becomes a yellow powder. Cryogenic Grinding. Cryogenic grinding of indapamide was carried out by means of a 6770 SPEX freezer/mill. The total mass of the milled indapamide was 3 g. The sample was placed in a stainless steel vessel and was immersed in liquid nitrogen. The stainless steel rod present in the vessel is vibrated by means of a magnetic coil. Prior to the start of grinding, the sample was subjected to 10 min of precooling. The mill was set to function at an impact frequency of 15 Hz. Five minute grinding intervals were separated by three minute cool-down periods. The effective grinding times were 15, 30, and 45 min. After the cryogenic grinding, the vessel with the ground sample was equilibrated in a vacuum oven at 25 °C, until room temperature was reached. After milling, indapamide becomes a yellow powder. Analytical Techniques. XRD measurements. XRD experiments were performed at ambient temperature on a RigakuDenki D/MAX RAPID II-R diffractometer (Rigaku Corporation, Tokyo, Japan) equipped with a rotating anode Ag KR tube (λ = 0.5608 Å), an incident beam (002) graphite monochromator and an image plate in the Debye−Scherrer geometry. The pixel size was 100 μm × 100 μm. Studied samples were placed inside Lindemann glass capillaries (1.5 mm in diameter). Then, the measurements were performed on sample-filled and empty capillaries, and the background intensity of the empty capillary was subtracted from the sample signal. The beam width at the sample was 0.1 mm. The twodimensional diffraction patterns were converted into onedimensional intensity data using suitable software. Nuclear Magnetic Resonance (NMR). The 1H and 13C NMR spectra were recorded at 400 and 100 MHz, respectively, on a Bruker 400 (Germany). 1 H NMR (400 MHz, DMSO-d6): δ = 1.30 ppm (d, J = 6.1 Hz, 3H, CH3); 2.59 (dd, J = 15.3 Hz, J = 11.1 Hz, 1H, CH2); 3.17 (dd, J = 15.3 Hz, J = 8.1 Hz, 1H, CH2); 3.93−3.99 (m, 1H, CH); 6.50 (d, J = 7.8 Hz, 1H, CH); 6.75−6.78 (m, 1H, CH); 7.02−7.06 (m, 1H, CH); 7.11 (d, J = 7.2 Hz, 1H, CH); 7.74 (s, 2H, NH2); 7.80 (d, J = 8.3 Hz, 1H, CH); 8.12 (dd, J = 8.3 Hz, J = 2.2 Hz, 1H, CH); 8.51 (d, J = 2.2 Hz, 1H, CH); 10.53 (s, 1H, NH). 13 C NMR (100 MHz, DMSO-d6): δ = 18.5 ppm (CH3); 35.4 (CH2); 62.9 (CH); 108.7 (CH), 119.9 (CH); 124.4 (CH); 127.0 (CH); 127.1 (C); 128.3 (CH); 131.6 (C); 131.7 (CH); 132.1 (CH); 133.6 (C); 141.3 (C); 151.4 (C); 163.9 (CO) Broadband Dielectric Spectroscopy (BDS). Ambient pressure dielectric measurements of glassy and supercooled indapamide were performed over a wide frequency range from 10−1 to 106 Hz using a Novo-Control GMBH Alpha dielectric spectrometer. For the isobaric measurements, the sample was placed between two stainless steel electrodes of the capacitor (diameter 20 mm) with a gap of 0.1 mm. The dielectric spectra were collected in a wide temperature range from 133 to 441 K. The temperature was controlled by the Novo-Control Quattro system, with the use of a nitrogen gas cryostat. Temperature stability of the samples was better than 0.1 K. Differential Scanning Calorimetry (DSC). Calorimetric measurements of crystalline and amorphous indapamide were carried out by Mettler-Toledo DSC apparatus equipped with a liquid nitrogen cooling accessory and a HSS8 ceramic sensor (heat flux sensor with 120 thermocouples). Temperature and enthalpy calibrations were performed using indium and zinc standards. 3614

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fluoroethylene membrane, an autosampler (model Sil-20 A HT) with two trays for standard 1.5 mL vials and a column oven (model CTO-1-AS VP). A Microsorb 100-5 C8 (S150 × 4.6, Varian USA) column was used. The column was thermostatted at 313 K. The mobile phase was composed of water/methanol/ acetic acid (55:45:0.1 v/v) and used at flow rate of 1.0 mL/min. 20 μL sample volumes were injected into the column, and elution was monitored at 240 nm. Shimadzu LC solution software (ver. 1.25) data processing software was used to record and integrate the chromatograms. Apparent Solubility Study of IND. To determine the solubility of crystalline and amorphous forms of indapamide in purified water, phosphate buffer pH 6.8 and 0.1 M hydrochloric acid, about 100 mg of each sample was added to 50 mL of each medium. The resulting slurry was strirred for 24 h at 25 °C ± 0.5 °C (I series) and 37 °C ± 0.5 °C (II series). The samples were filtered through a sterile syringe filter (25 mm, PET-polyester, pore size 0.45 μm) from MachereyNagel (Bioanalytic, Poland). Each filtrate was diluted 1:200 v/v (0.25 mL of the filtrate solution was made up to 50 mL in a volumetric flask). In order to prevent precipitation of indapamide from the saturated solutions, all the flasks were heated to 25 or 37 °C. Apparent solubility of the various forms of indapamide was determined by HPLC (n = 9) as described above. The concentration of indapamide was calculated from a rectilinear regression equation obtained from measurements of 10 standard solutions in the concentration range of 0.025−2.0 μg/mL. The calibration curve, y = 72615x − 355.8 (R2 = 0.9999), was determined from the relationship between the peak area of the chromatograms and the concentration of standard solutions. The retention time of indapamide was 6.5 min. Nonsink Dissolution Studies. Nonsink dissolution studies were performed in deionized water at 37 °C. 50 mg of the tested material was placed in a glass vial (diameter, 22 mm; height, 70 mm) containing 25 mL of the medium and stirred (Stuart SD162, U.K.) using a magnetic bar (diameter, 4 mm; length, 12 mm) at a rate of 1300 rpm. The vial was placed in a double walled jacketed beaker connected to a water bath (Haake F3, Germany). Sampling of the liquid medium was carried out using a circulation system composed of an LSMatec peristaltic pump fitted with a 10 μm filter (HMWPE, Porex Technologies, Germany). The filtered solution was passed through a 2 mm flow-through UV quartz cuvette placed in a Shimadzu UV-1700 PharmaSpec spectrophotometer (Japan) set to 279 nm. The results are an average of three measurements, and error bars present standard deviations. For statistical comparisons, one-way analysis of variance (ANOVA) followed by the post hoc Tukey’s test was used. Differences were considered significant at p < 0.05.

theory, relaxed geometry scan by changing proper dihedral angle was performed. Thereafter, structures characterized by the highest value of electronic energy were optimized as transition states at the same level by means of the eigenvector following method. Transition states and minima were subsequently confirmed by performing vibrational analysis. Frequencies were calculated numerically at the same level of theory. Molecules were visualized using Avogadro package.18

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RESULTS AND DISCUSSION Part A: Study of the Amorphous IND Obtained by the Vitrification Method. Various methods for preparation of pharmaceutical systems in their amorphous forms have been reported, such as freeze-drying, spray drying or mechanical milling, however the simplest approach is based on the rapid cooling of the molten sample, typically referred as vitrification. However, this method carries a risk associated with chemical decomposition of the drug during melting of the crystalline material. It should be emphasized that if thermal degradation occurs, the decomposed amorphous material obtained is disqualified as a therapeutic agent. This is the reason why chemical purity of drugs subjected to thermal stress conditions in a manufacturing process should be addressed with particular care. Chemical Stability Analysis. To determine the melting point of IND, differential scanning calorimetry was applied. The DSC curve obtained during heating of the crystalline compound up to 473 K is depicted in Figure 2. An endothermic peak, with an onset at 437 K, indicates melting of the sample. The melting point (Tm) value of IND obtained in this study is in good agreement with that determined recently by Ghugare et al.16 Since the commercial form of the examined compound contains 2.5% water, in the thermogram presented in Figure 2 one can also observe a broad endotherm related to evaporation of water. To produce an amorphous form of the drug, indapamide was molten and then cooled to room temperature. The subsequent heating from the glassy state shows the characteristic signature for the glass transition in the temperature dependence of the heat flow at 377 K. Moreover, there was no thermal effect associated with evaporation of water, indicating that indapamide obtained by vitrification is anhydrous. To confirm the disordered nature of the amorphous form of IND, the XRD technique, being one of the most definitive methods for detecting and quantifying molecular order in a system, was applied. As illustrated in Figure 3, the very broad halo presented as a black line, compared to the sharp peaks typical of the crystalline state (blue line), indicates that the vitrified sample is indeed an amorphous material. It should be noted that the amorphous indapamide obtained by means of vitrification becomes yellow in contrast to the white crystalline form. Such observation might indicate that the examined drug undergoes thermal decomposition upon heating. To verify this supposition FT-IR was employed, which is able to detect chemical changes occurring in a sample. First, we have performed the analysis of crystalline IND as a reference. As illustrated in Figure 4, in the FT-IR spectrum of the crystalline compound one can distinguish several characteristic regions. The first one is located between 3700 and 3300 cm−1. There are two sharp absorption bands associated with the symmetric (3499 cm−1) and asymmetric (3655 cm−1) stretching vibrations for the hydroxy functional group of water molecules observed in the commercial IND sample. The sulfonamide group has strong bands due to its NH2 stretching vibrations at 3431 and

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CALCULATION METHODS All quantum chemistry calculations for indapamide (IND) molecule were performed with the use of density functional theory in the ORCA package.17 In the first step we have performed geometry optimizations of a dozen random IND structures with the use of hybrid B3LYP functional and the 6-31g* basis set. Three the most stable structures were then reoptimized using the same functional and 6-31++g(2d,2p) basis set. The structure presented is in the minimum determined by the position of the sulfonamide (−SO2NH2) group, which is the most flexible part of the molecule. In order to simulate the rotation at the B3LYP/6-31+g(2d,2p) level of 3615

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In the next step, FT-IR spectra were recorded in a wide temperature range, from 293 up to 453 K. As one can see in Figure 4, with increasing temperature, broadening of bands and the loss of spectral resolution are observed. Both of these effects are typical for the melting process. Furthermore, there are a few distinct differences in FT-IR spectra between the crystalline and molten samples. The most significant changes are observed in the 3700−3200 cm−1 region where two well resolved bands assigned to vibrations of water molecules are detected i.e. at 3655 and 3499 cm−1. As illustrated in Figure 4, their intensity decreases with increasing temperature, and finally, close to the melting point, the bands are undetectable. It means that the amount of water in the sample decreases with increasing temperature. To better illustrate this process, the integral intensity of two water bands was calculated and plotted versus temperature (Figure 5). From Figure 5 it can be clearly seen that in fact the whole water evaporates below 373 K. This result is in good agreement with calorimetric measurements presented previously. The other noticeable change in the temperature dependent IR spectra, presented in Figure 4, is associated with the N−H band at 3312 cm−1 of the crystalline sample. As it can be seen, with increasing temperature the band becomes less intense and disappears at 453 K. Simultaneously with rearrangement of the N−H environment, the stretching band of the carbonyl moiety shifts toward higher wavenumbers and a new band at 1458 cm−1 is found to appear. According to the theoretical calculation of IND spectrum, this new band is attributed to the bending C−OH vibrations. All these changes indicate that the increase of temperature induces the proton transfer from the nitrogen to the oxygen atom of the carbonyl group. Consequently, the double bond formerly placed between carbon and oxygen atoms shifts to the carbon−nitrogen position (νCN at 1662 cm−1). It means that indapamide molecule may exist in equilibrium with its two easily interconvertible constitutional isomers (see Figure 1). Taking into account the types of bonds involved in this proton transfer, the transition observed in IND may be called an amide− imidic acid tautomerism. Since the FT-IR spectra recorded showed only the tautomeric transformation and did not reveal any decomposition at Tm, in the next step of our experiments we have annealed the examined sample at 453 K i.e. 12 K above its melting point. The spectra collected after one and two hours of heating are presented in Figure 4. It is clearly seen that there is no difference between the isothermally annealed samples. Taking into account the previous IR results one can expect that the indapamide is a thermally stable compound. The thermal decomposition of the examined material was also excluded by HPLC−MS and NMR measurements performed in this study (a detailed NMR description of this sample is presented in the Experimental Section). The above findings are additionally supported by the thermogravimetric analysis (TGA) presented in a previous study.16 The TGA pattern indicates that the onset of thermal decomposition of IND, related to the onset of the mass loss of IND, begins just above 560 K, well above the melting point temperature. Consequently, one can conclude that it is possible to prepare the pure amorphous IND by means of vitrification. At the same time the change in color of IND drug during melting cannot be associated with chemical decomposition of the sample. It is worth noting that dissolution of indapamide in DMSO or ethanol at room temperature also results in the change of solution color from colorless to yellow. Thus, the peculiar behavior of IND changing color may be an effect of IND transition from crystalline to the liquid phase. It is

Figure 3. X-ray diffraction patterns for various solid-state forms of IND performed at room temperature T = 298 K.

Figure 4. FT-IR spectra collected during annealing of IND from 293 to 453 K. As a reference the spectrum of the starting, crystalline material recorded at room temperature is presented as a bold red line. Dashed lines indicate bands undergoing changes during annealing. The upper panel presents FT-IR spectra of IND heated to 16 K above the melting point for one and two hours, respectively.

3342 cm−1 in the same infrared region. Moreover, a peak at 3312 cm−1 of NH associated with the amide moiety of the indapamide molecule is observed. The second characteristic region in the FT-IR spectrum is observed between 1700 and 1000 cm−1. One of the strongest bands visible in this range is located at 1655 cm−1 and arises from the carbonyl stretching vibration. Other band assignments with their wavenumber positions for crystalline IND phase are summarized in Table 1. It should also be noted that infrared spectroscopy has recently been applied by Ghugare et al. to analyze the crystalline form of indapamide.16 Despite the fact that the authors16 dispersed analyzed diuretic drug in KBr, the published diffuse reflectance infrared Fourier transform spectrum of IND is in good agreement with our results. 3616

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Table 1. FT-IR Band Assignments with Their Wavenumber Positions for Crystalline and Glassy IND Samplesa crystalline IND vibration

a

amorphous IND wavenumber [cm−1]

νasym H2O νsym H2O νasym NH2 νsym NH2 νNH

3655 3499 3431 3342 3312

νCO; νNH νasymCC(benzene); νCH δNH2 βCH; βNH βCH3; γNH2; νasym(SO)2; βCH νasymCC at Cl; γNH2; νasym(SO)2 νNN; βNH; βCH βNH; βCH γNH2; νsymCCN(pyrrole ring) ωNH2; νNS

1655 1611 1563 1460 1323 1297 1281 1242 1067 847

vibration

wavenumber [cm−1]

νOH

3604

νasymNH2 νsymNH2

3387 3277

νCN; νOH

1662

νasymCC(benzene); νCH δNH2 βCH; βCOH

1607 1561 1458

γNH2; νasym(SO)2; βCH

1295

βCOH; βCH; βCH2 γCH2; γNH2; γCH3; γNH2; νsym(SO)2 ωNH2; νNS; βCOH; νNC ωNH2; νNS; βCOH; νCH

1242 1090 841 800

δ, twisting; ω, wagging; γ, rocking; β, bending; ν, stretching; asym, asymmetric; sym, symmetric vibrations.

well as theoretical studies of the tautomerization reaction.21−25 This is due to likely serious consequences of the proton transfer. Especially, isomerization of pharmaceuticals becomes an important problem as various tautomers of one drug may reveal different pharmacological activity. A frequently used antibiotic, erythromycin, exhibits as many as three isomers,26 however only one of them, the ketone form, reveals the antibacterial activity. Moreover, it cannot be excluded that tautomers with no desired therapeutic activity may cause a number of side effects. Therefore, the following question arises: Is the activity of the imidic acid form of IND the same as that of the amide isomer? Unfortunately, to answer this question thorough bioactivity studies are required. However, one cannot exclude that the weak diuretic activity of indapamide drug is due to the tautomeric forms that appear after dissolving the tablet in the gastrointestinal tract. Analysis of Molecular Dynamics in Supercooled and Glassy State of IND. In the literature one can find many examples that demonstrate that studies of the molecular mobility in amorphous pharmaceuticals help to control the behavior of these systems at the macroscopic scale.27−30 The most frequently used experimental methods for monitoring the molecular dynamics of amorphous medicines are dielectric spectroscopy, depolarized light scattering, dynamic mechanical analysis, proton correlation spectroscopy or nuclear magnetic resonance. Here, to provide the essential information about the molecular mobility in the supercooled and glassy states of indapamide, broadband dielectric spectroscopy (BDS) was applied. Additionally, temperature-modulated differential scanning calorimetry (TMDSC) was implemented to analyze the dynamic behavior of IND as well as to determine its glass transition temperature. The imaginary part of complex dielectric permittivity spectra measured at ambient pressure over seven decades of frequency in the supercooled liquid state and the amorphous phase of IND are shown in Figure 7. Panel a of this plot presents dielectric loss spectra recorded above Tg: at temperatures from 385 to 441 K with a uniform increment of 4 K. In this temperature range two dielectric processes can be distinguished: (i) conductivity relaxation at low frequencies which is related to

Figure 5. The temperature dependence of integral intensity for two bands, at 3655 and 3499 cm−1, respectively, recorded during heating of crystalline IND from room temperature to 453 K.

noteworthy that there are several compounds that change their color after melting or dissolution. As an example one can mention indomethacin19 or piroxicam,20 which like indapamide become yellow after amorphization. However, it should be emphasized that in the latter case the tautomerization reaction was found to be responsible for changing the color of sample. Since indapamide also reveals the isomerization tendency, one cannot exclude that the yellow color of the sample is due to the tautomerisation, which occurs during melting. To provide additional information about the tautomerization reaction of amorphous IND, FT-IR spectroscopy was employed once again. The infrared spectrum of vitrified indapamide sample is depicted as a top green line in Figure 6. Since this spectrum practically overlaps that which was recorded for the molten sample i.e. there are bands corresponding to νCN and βC−OH vibrations, while the NH band associated with the amide moiety of indapamide molecule is almost absent, one can conclude that the tautomeric equilibrium achieved at the melting temperature is conveyed into the amorphous material. In recent years, there has been a growing interest in experimental as 3617

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Figure 6. FT-IR spectra of IND: crystalline, obtained by quench-cooling, obtained by cryogenic grinding and prepared by room temperature milling. All spectra were collected at room temperature.

the α-relaxation peak toward lower frequencies is related to a decrease of temperature. This behavior reflects the slowing down of cooperative molecular motions of IND. Below Tg the structural relaxation moves out of the frequency measurement window, and then a secondary relaxation, called the γ-process, becomes apparent (see Figure 7b). Similar to the mentioned α-relaxation, the γ-mode also slows down during cooling, but it is far less sensitive to the change of temperature. To move it over 6 decades in frequency, it was necessary to decrease the temperature by 140 K, from 273 to 133 K. The nature of this secondary mode will be discussed latter. One of the most important parts of the dielectric spectra analysis is to investigate the shape of the structural relaxation process. From the pharmaceutical point of view, this parameter is especially important as it was found to be directly related to the physical stability of the amorphous material. As suggested by Shamblin et al.,31 the stability of various amorphous drugs should increase as the α-peak becomes narrower, i.e. with increasing βKWW parameter. Examples of active substances obeying this rule are e.g. nonivamide,32 telmisartan33 or glibenclamide.34 To verify whether this relationship is valid also in the case of indapamide, we first constructed a so-called “masterplot” in which a number of dielectric curves measured at different temperatures (both above and below Tg) have been superimposed. As the reference one we have used the spectrum recorded at T = 385 K. It can be clearly seen in Figure 8 that the shape of the α-process is practically invariant upon cooling. Thus, in the case of IND, similarly to many other glass-forming liquids, the time−temperature superposition principle is fulfilled. Additionally, it can be seen that the α-relaxation process of IND is asymmetric and broader than the classical Debye response. To parametrize the shape of this mode we have applied the Kohlrausch−Williams−Watts (KWW) function:35,36

Figure 7. Dielectric loss spectra of IND obtained by quench cooling of the melt. Panel a presents dielectric loss above the glass transition temperature, whereas in panel b dielectric loss spectra collected below Tg are presented.

⎡ ⎛ ⎞ βKWW ⎤ t ⎥ ϕ(t ) = exp⎢ −⎜ ⎟ ⎢⎣ ⎝ τα ⎠ ⎥⎦

the translational motion of ions and (ii) the structural relaxation process related to the cooperative rearrangements of indapamide molecules. In contrast to other pharmaceuticals, like glibenclamide or indomethacin, the dc contribution for indapamide is not significant, resulting in the maximum of structural relaxation peak being well resolved despite close proximity to the glass transition region. As illustrated in the upper panel of Figure 7 the shift of

(1)

where t is the time, τσ is the characteristic structural relaxation time and βKWW (0 < βKWW ≤ 1) denotes the stretching parameter, which is related to the width of the relaxation peak. 3618

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This procedure has been applied several times in the past to predict the maximum of the hidden β-process. Using the values of βKWW = 0.72 and the relaxation time determined from the maximum of the α-peak recorded at 385 K, we have calculated τ0 for indapamide. The τ0 was found to be equal to 2.2 × 10−4 s, which corresponds to the frequency f 0 = 714 Hz. As illustrated in Figure 8, the value of f 0 determined lies within the frequency range where the excess wing is observed (green arrow). In order to provide more information about the nature of this excess wing visible in dielectric spectra of indapamide, we have also performed an aging experiment. It is well-known that, except for the high pressure measurements,48,49 this is one of the most frequently used methods to explore more thoroughly the origin of excess wing.50 If upon physical aging the excess wing transforms into a well-separated peak (or shoulder), this provides evidence that the excess wing may be classified as a JG-type relaxation. During the aging experiment the examined diuretic sample was annealed isothermally at 363 K, close to Tg. Both the first and the last dielectric spectra (recorded at the beginning and after 22 h of annealing) are depicted in the inset panel of Figure 8. As one can see, the excess wing becomes more pronounced with time. This experimental result suggests that the observed process may be attributed to the JG-relaxation. The dielectric α-relaxation peak of IND can be also analyzed by means of the Havriliak−Negami function with the dcconductivity term added that fits the experimental data more precisely than the KWW function.51

Figure 8. Superimposed dielectric spectra of IND taken at ambient pressure (p = 0.1 MPa), at seven different temperatures above and below Tg. The solid red line is the KWW function with βKWW = 0.72, while the blue line is a Cole−Davidson fit (α = 1, β = 0.63). The green arrow denotes the position of the primitive relaxation calculated from eq 3 at 385 K. The inset panel presents the spectra collected at 363 K during physical aging of IND .

Since the above equation refers to the time not the frequency domain, to describe the ε″( f) peaks the one-sided Fourier transform of the KWW function has to be applied: ε″(ϖ) = Δε

∫0

∞⎛ ⎜

⎝

−

dΦ ⎟⎞ sin(ϖt ) dt dt ⎠

ε″ (ϖ ) =

(2)

(4)

where ε∞ is the limiting high-frequency permittivity, τHN denotes a characteristic relaxation time; exponents αHN and βHN characterize symmetric and asymmetric broadenings of the dielectric loss curve; Δε is relaxation strength and ω is an angular frequency. A representative fit of eq 4 to the dielectric loss curve recorded above Tg is depicted as a blue line in Figure 8. To parametrize the dielectric loss spectra recorded above Tg, the HN function with αHN =1, which is usually called the Cole−Davidson (CD) function,52 was applied. As one can see, the CD equation describes the α-peak better than the previously used KWW function. However, to obtain the perfect fit in whole measured range, the effect of the unresolved β-mode should be taken into account. Consequently, the superposition of the Cole−Davidson and Cole−Cole function has to be applied. Herein, it should be noted that the Cole−Cole function, i.e. when the parameter βHN is set to one, describes well symmetric and broad relaxation modes. Thus, it can also be successfully used to characterize the secondary γ-relaxation process of IND. In the next step of the dielectric data analysis, based on the CC and CD fit parameters determined previously, the characteristic relaxation times of both modes, α and γ, observed in permittivity spectra of IND were calculated according to eq 5:53

where Δε is the dielectric strength of the α-relaxation. The representative fit of the KWW function to the experimental data, with the βKWW exponent equal to 0.72, is shown as a red line in Figure 8. Since the obtained value of βKWW is quite high, one can suppose that amorphous indapamide should not crystallize. This assumption is supported by experimental data because neither dielectric nor calorimetric measurements reveal crystallization signs during heating of the amorphous IND sample. From the dielectric spectra analysis presented in Figure 8 it is also evident that the KWW function describes the data well only in the vicinity of the α-peak maximum. It can be easily seen that at frequencies about two decades higher than the maximum of the α-process the experimental data systematically deviate from the KWW fit. The origin of such a trend, usually called “excess wing”, has remained a matter of hot debate in the past decade.37−41 Some scientists interpreted this deviation as an inherent part of the α-relaxation42 while others said that the excess wing is a high frequency flank of the secondary relaxation process hidden under the dominant α-peak.43,44 According to the second explanation, for the first time postulated by Ngai45 and experimentally verified by Lunkenheimer46 and other researchers, this unresolved secondary mode originates from some local motions of the entire molecule, and it is believed to be a precursor of α-relaxation. To determine the maximum of this process, usually called the Johari−Goldstein relaxation (JG), the coupling model (CM) approach is generally applied,47 τ0 = (tc)1 − βKWW (τα)βKWW

σdc Δε + ε∞ + ε0 ϖ [1 + (iωτHN)αHN ]βHN

⎡ ⎛ α ·π ⎞⎤−1/ αHN ⎡ ⎛ α ·β ·π ⎞⎤1/ αHN ⎢sin⎜⎜ HN HN ⎟⎟⎥ ⎟⎟⎥ τ = τHN⎢sin⎜⎜ HN ⎢⎣ ⎝ 2 + 2βHN ⎠⎥⎦ ⎢⎣ ⎝ 2 + 2βHN ⎠⎥⎦ (5)

(3)

The experimentally determined variations in log τα and log τγ with temperature are depicted as solid squares and circles, respectively, in the form of a so-called Arrhenius plot shown in Figure 9. As illustrated in this figure the log τα(1/T) dependence can be

where τ0, usually called the primitive relaxation time, denotes the possible relaxation time of the hidden secondary mode while the parameters τα and βKWW characterize the α-relaxation, and tc = 2 ps. 3619

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Table 2. The Parameters of Fitting to the VFT and Arrhenius Equations for Anhydrous and Hydrated Lidocaine HCl Samples sample

vitrified IND

α-Process

reasonably well described over the entire measured range by means of the Vogel−Fulcher−Tammann equation,54−56

(6)

From the fit of the VFT equation to τα(T) dependence one can easy determine the glass transition temperature of IND. Applying the most frequently used definition, which describes Tg as the temperature at which the dielectric relaxation time τα reaches 100 s, the glass transition temperature of IND was found to be equal to 374 K, and this value well agrees with the one found from DSC measurements (TgDSC = 377 K). Additionally, in this paper we have determined the structural relaxation times of indapamide from the temperature dependences of the real part of the complex heat capacity Cp′ measured by the stochastic temperature modulated DSC technique. As illustrated in Figure 9, DSC data are in good agreement with the structural relaxation times determined from the dielectric measurements (Table 2). The most characteristic feature of molecular dynamics in vitrifying liquids is non-Arrhenius dependence of the τα(T) curve. However, the degree of deviation from Arrhenius behavior varies from one compound to another. To assess whether or not this divergence is significant, the “steepness index” or “fragility” defined by Bohmer et al.,57 d log τα d(Tg /T )

T = Tg

374.6 76 ± 2 −16.9 ± 0.2 13.6 ± 0.6 285 ± 2 Arrhenius Dependence −15.05 ± 0.06 −15.45 ± 0.13 52.4 ± 0.3 54 ± 0.6 Arrhenius Dependence −13.33 ± 0.5 32.9 ± 0.2

give m = 76. This value is close to those found for other recently investigated amorphous pharmaceuticals such as verapamil hydrochloride (m = 88),10 telmisartan (m = 87),33 glibenclamide (m = 78)34 or indomethacin (m = 83).19 Thus, indapamide investigated in this work can be classified as an intermediate glassformer drug. Now we consider the molecular mobility in the glassy state of anhydrous indapamide that is reflected in secondary relaxation processes. As one can see in Figure 7, except for the excess wing, identified previously as a hidden β-process, in the dielectric spectra of IND only one secondary relaxation (γ) can be found. Since log τγ is linearly related to the inverse of temperature, it is possible to fit the Arrhenius equation (eq 8) to the experimental data and determine the activation energy barrier of this mode.

Figure 9. Relaxations map of vitrified IND. Temperature dependence of structural and secondary γ-relaxation relaxation times are depicted as solid squares and circles, respectively. Solid lines are VFT and Arrhenius fits to the experimental data. Fitting parameters are presented in Table 2. Additionally, solid triangles are the structural relaxation times determined from TMDSC measurements for anhydrous IND.

m≡

ball-milled IND

VFT Parameters

Tg (τ = 100 s) [K] mP (τ = 100 s) log τ0 D T0 [K] υ-Process log τ∞−υ Ea−υ [kJ/mol] γ-Process log τ∞−γ Ea−γ [kJ/mol]

⎛ DT0 ⎞ τα = τ0 exp⎜ ⎟ ⎝ T − T0 ⎠

cryomilled IND

⎛E ⎞ τγ = τ∞ exp⎜ a ⎟ ⎝ RT ⎠

(8)

The activation energy barrier was found to be equal to 34 kJ/mol. This quite low value of Ea, together with the symmetrical shape of the γ-peak, suggests that the motions of a small part of indapamide molecule are responsible for this secondary relaxation. In order to confirm this supposition we have performed the DFT calculations. Since the sulfonamide group seems to be the most interactive part of the indapamide molecule, we have studied the conformational changes of this moiety. Initially we have checked whether the dipole moment is changing during the conversion or not. It should be emphasized that the variation of the dipole moment is necessary to observe intramolecular motions as a secondary mode in the dielectric spectrum. As illustrated in Figure 10, the rotation of the sulfonamide group of indapamide causes a change in the dipole moment. That is why this movement may be considered as an origin of the γ-relaxation. The value of activation energy of the sulfonamide group rotation obtained in the B3LYP/6-31+ +g(2d,2p) model is equal to 25 kJ/mol, and it is slightly lower than the value of Ea determined experimentally. Theoretical Prediction and Experimental Verification of the Physical Stability of Quenched Indapamide. Understanding of the factors affecting crystallization from the glassy state not only is important from a scientific perspective but also has many practical applications e.g. in the pharmaceutical industry. However, in contrast to the chemical stability determination, where the standard analytical techniques are usually

(7)

is usually calculated. Using this parameter we can classify supercooled liquids into two types: (i) “strong”, if the temperature dependence of structural relaxation times is close to Arrhenius behavior (m ≤ 30), and (ii) “fragile”, if log τα(1/T) deviates significantly from the straight line (m ≥ 100). When m falls within the 30 < m < 100 range, the liquid is classified as intermediate glass-former. Fragility calculations performed for IND 3620

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stable imidic acid forms are dispersed in the amide matrix. In the literature one can find some reports that show that this kind of equilibrium detected in the glassy state may strongly reduce the tendency of such a system to crystallize. For example, glibenclamide60 is a hypoglycemic agent with tautomerization tendency or binary mixtures containing active pharmaceutical substance and various excipients like polymers (PVP, PEG or HPMC)61−63 or sugars (octaacetylmaltose)64 which are characterized by highly physical stability in the amorphous state. The above results indicate that factors influencing the stability of an amorphous system should not be considered separately, because only when studied together can they provide full information about the physical stability of glassy pharmaceuticals. Thus, in the context of molecular dynamics investigations as well as the proton transfer in IND one can suppose that the IND system examined should not reveal the crystallization propensity from the amorphous state. To verify these theoretical predictions, X-ray powder diffraction was used. XRD measurements performed after one year of storage of the sample at room temperature showed no sign of crystallization. Part B: Study of Amorphous Indapamide Samples Obtained by Cryogenic Grinding and Room Temperature Milling. Mechanical milling of crystalline solids is a very popular technique applied in many fields of industry to reduce the particle size. It is also used in the pharmaceutical industry since micronization was proven to enhance the dissolution properties of drugs through an increase in the surface area of micro- or nanoparticulates. As shown in the literature, the mechanical treatment may also lead to amorphization of crystalline materials. For instance, amorphization by milling was observed for piroxicam,20 glibenclamide,34 sucrose or ziprasidone.65 Physicochemical Characterization of Milled IND Samples. Herein, to produce the amorphous form of IND drug we have applied two types of grinding: cryomilling, being operated at very low temperatures using cryogenic media such as liquid nitrogen, and ball-milling that is carried out at room temperature. In the first case the IND sample (3 g) was milled for 15, 30, and 45 min. On the other hand, the ball-milling lasted for 1, 2.5, and 5 h. However, contrary to cryogrinding the amount of milled sample was two times higher (6 g). The diffraction patterns of all micronized materials are presented in Figure 3. Significant broadening and decrease of intensity of diffraction peaks attributed to reduction of sample crystallinity was observed with increasing milling time. After 15 min of cryogrinding and 1 h of ball-milling the degree of IND crystallinity, calculated as a ratio of X-ray diffraction peak areas for the partially crystalline sample to that being 100% crystalline, was found to be equal to 34% and 36%, respectively. However, when the milling time was extended to 30 min and 2.5 h, respectively, indapamide became almost completely amorphous. The degrees of crystallinity were calculated as 5% (cryomilled IND) and 8% (ball-milled IND). A complete crystal−glass conversion was observed after 45 min of cryomilling while at room temperature 5 h of grinding was required to obtain fully amorphous IND. As illustrated in Figure 3, Bragg peaks of these samples, characteristic for materials with a long-range three-dimensional molecular order, were replaced by broad halos identical to that recorded for molten-quenched indapamide. Interestingly, amorphous IND powders obtained using the mechanical treatment were yellow, just like the quenched material. The experimental data presented clearly indicate that cryomilling is much more efficient in preparation of the glassy IND than

Figure 10. Diagram representing changes of the dipole moment and energy during rotation of the sulfonamide group of IND molecule.

applied, there is no certain methodology to predict the physical stability of an amorphous material. Until now crystallization of the glassy sample has been many times correlated to the degree of its molecular mobility, thermodynamic properties (such as heat of fusion, heat capacity, configurational entropy) or the methods of amorphization. One of the parameters, considered as a key factor affecting stability of an amorphous system, is dynamic fragility. This is because the steepness index is related to an average degree of molecular mobility reflected in a structural relaxation near the glass transition. According to the TOP (two order parameter) model proposed by Tanaka,58 near the glass transition region competition between long-range density, being a driving force toward formation of the crystals, and short-range ordering favoring formation of the locally preferred structures is postulated. If the second effect dominates, the system becomes “frustrated”. Consequently, the crystallization is expected to be inhibited. It has been shown that the degree of frustration can be related to fragility, i.e. the lower frustrationthe greater fragilitythe greater tendency to crystallize. Taking into account this assumption, IND with the fragility index equal to 76 should be classified rather as a system with high crystallization tendency. On the other hand, as it was stated previously, we can distinguish only one, well-pronounced secondary relaxation process of IND. It means that the molecular mobility of the examined drug is significantly retarded below the liquid−glass transition, which is observed at a relatively high temperature (377 K). Consequently, at room temperature conditions, i.e. almost 80 K below the Tg, motions of IND molecules are deeply frozen, which prevents crystallization of IND. Moreover, there is a general rule that says that the long-term stability of amorphous drugs can be obtained by storage at a temperature where molecular mobility associated with the structural relaxation time approaches zero, i.e. at T0 in the Vogel−Fulcher−Tamman (VFT) equation. This temperature was found to be close to the so-called Kauzmann temperature,59 where entropy of the supercooled liquid and crystal would be equal. In the case of IND T0 is equal to 285 K. Thus, storage of IND at such temperature conditions should guarantee its physical stability for a typical shelf life. To better understand the physical stability of the examined amorphous diuretic agent we have to consider other factors that may affect its crystallization. Specifically, the possibility of isomerization should be also taken into account. As it was mentioned in the previous part of this paper there are two isomers coexisting in the glassy state of IND. Consequently, the amorphous sample becomes a binary mixture in which less 3621

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by ball-milling. On the other hand, cryomilling was believed not to induce chemical changes in the milled material because liquid nitrogen prevents local overheating of the sample. However, Adrjanowicz et al.68 recently have shown that even cryomilling is able to modify the chemical structure of ground materials. Therefore, one can suppose that both room temperature milling and cryogrinding can induce changes in the structure of indapamide. To characterize the structure of cryomilled and ballmilled materials in detail, FT-IR spectroscopy was exploited. The FT-IR spectra of milled IND are presented in Figure 6 together with the data obtained for the crystalline and vitrified samples. It is clearly visible that the data pertaining to all amorphous materials are almost the same. The only difference in the high wavenumber region 3700−3200 cm−1 was the vibrations of water molecules detected for the milled IND. This is because the milled samples, in contrast to the quenched IND, contain water. On the other hand, similarly to the quenched indapamide, significant loss in intensity of the N−H stretching vibration at 3312 cm−1 was found. Furthermore, new bands attributed to CN and C−OH vibrations also appear in the spectra. These changes in the FT-IR spectra indicate that milling of IND both at room temperature and in cryogenic conditions leads to the conversion of the amide form of drug to its imidic acid isomer. There are contradictory literature reports on the tautomerization reaction of milled materials. On the one hand, there are several examples when the less stable isomer appears after grinding, like in the case of glibenclamide or piroxicam. In the former case tautomerism is observed independently of the grinding conditions while for the second compound the transformation was detected after milling at the liquid nitrogen temperature. On the other hand, these findings are in contrast with the experiments performed with sugars. Descamps et al. have shown that mutarotation of saccharides, being a ring− chain tautomerization, does not occur in amorphous trehalose or lactose samples obtained by milling at liquid nitrogen temperature.68 It means that after grinding there is only one, initial anomeric form. However, it should be stressed that in the case of anhydrous lactose 96% of milled material was still in the α-anomeric form. Thus, one can suppose that after milling 4% of the investigated sample was converted into the β-form. Molecular Mobility of Milled IND Samples. In Figure 11 we present the imaginary parts of dielectric permittivity spectra plotted as a function of frequency during heating of cryomilled and ball-milled IND from 153 K up to 443 K. To show the whole sets of data more clearly, dielectric spectra of each sample were divided into two panels presenting the relaxation dynamics above and below the glass transition temperature. As can be seen in panels c and d of Figure 11, the imaginary part of the complex dielectric permittivity, recorded for both milled materials, reveals a well-resolved secondary relaxation process, called υ-peak, which was not observed in dielectric spectra of the melt-quenched sample. Similar to the γ-relaxation, the υ-process shifts toward higher frequencies on heating, but in contrast to the γ-mode it is more sensitive to temperature changes. This behavior suggests that the activation energy of this υ-process is greater than that for the γ-relaxation. The close inspection of the dielectric loss spectra collected at the same temperature conditions (T = 216 K), for milled and quenchcooled samples, shows that the amplitude of the new dielectric mode is higher than that of the γ-relaxation (see Figure 12). Moreover, both processes, γ and υ, appear in the dielectric spectrum at the same temperature range. This explains why the γ-process becomes almost invisible in the dielectric spectra of

grinding at room temperature. The time needed to fully amorphize crystalline IND by cryomilling was approximately seven times shorter compared to with traditional room temperature milling. This result agrees with the general rule that a decrease of the milling temperature brings similar effects as an increase in milling intensity. Additionally, it is worth pointing out that mechanical amorphization of crystalline IND is much more efficient than milling of other organic compounds reported in the literature. For example, full amorphization of indomethacin19 and furosemide66 was achieved after 60 and 120 min of cryomilling, respectively, while in the case of IND only 45 min was necessary. Moreover, it should be stressed that in our experiments the amount of milled material was three times higher than those for mentioned pharmaceutics. On the other hand, the mechanical treatment of IND at room temperature brings the complete amorphization only after 300 min, while the amorphous state of indomethacin and trehalose was achieved after more than 1200 and 1380 min of traditional ball grinding. Hence, it can be stated that the mechanical treatment of IND at both room and liquid nitrogen temperatures works very effectively. Thus, these methods can be successfully applied in the pharmaceutical industry to produce IND in the amorphous form. In the next step we have performed the DSC analysis of milled indapamide samples. In Figure 2 DSC heating scans of cryomilled and ball-milled IND are illustrated. In both cases, two thermal events were observed within the examined temperature range: the first one associated with water evaporation and the second one ascribed to the glass transition. As seen in the inset panel of Figure 2, a heat flow jump characteristic for a glass transition detected in the thermograms of milled IND occurs exactly at the same temperature as that of the quenched liquid, i.e. 377 K. However, in contrast to the anhydrous vitrified sample, the cryomilled and ball-milled materials contain 1.3% and 2.5% water, respectively. The reduced water content in cryomilled IND is due to the fact that before grinding indapamide hemihydrate has been dried at 363 K for 12 h. On the other hand, amorphous sample obtained by means of room temperature milling contains the same amount of water as the starting hemihydrate. Nevertheless, in both cases the whole amount of water evaporates before the glass transition temperature is achieved. It is interesting that in the DSC thermograms of the tested samples there is no exothermic peak ascribed to so-called “cold crystallization”, which is frequently observed during the slow heating of milled materials. This result suggests high stability of ground IND against crystallization. Indeed, X-ray diffraction measurements performed one year after sample preparation did not reveal any Bragg reflections. It means that there is no difference in terms of the physical stability between amorphous IND samples prepared by milling and vitrification techniques. This result is interesting in the context of recent reports of tri-o-methyl-β-cyclodextrin,66 nucleosides67 or glibenclamide34 which demonstrate the opposite behavior. Thus, one can formulate the following question: What factors af fect the stability of milled IND? As shown in part A of this paper, in the case of the vitrified sample the proton transfer was found to be one of the key factors determining the stability of quenched IND. Accordingly, a further important question to pose is: Does the milling induce the amide−imidic acid transformation of indapamide or not? As shown in the literature, the high level of mechanical energy used during milling may cause significant changes in the structure of the ground material. These may be observed especially when the sample is milled above the liquid to glass transition temperature. Such a scenario was shown for a number of pharmaceuticals such as chloramphenicol, cimetidine or indomethacin, where polymorphic transformations were induced 3622

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Figure 11. Dielectric spectra of IND samples obtained by cryogenic grinding (panels a and c) and room temperature milling (panels b and d). The dielectric data were collected above and below the glass transition temperature.

Figure 12. Comparison of dielectric spectra measured at the same temperature T = 216 K for anhydrous IND obtained by vitrification and hydrated samples prepared using cryomilling and ball milling.

the cryomilled and ball-milled IND samples. The only evidence for the existence of this γ-mode is the poorly noticeable high frequency flank of this process visible in Figure 11d. Therefore, the question arises: What is the molecular origin of the υ-mode? As it was mentioned in the previous part of this paper the only difference between the milled and vitrified IND samples is the water content. While the quench-cooled sample is anhydrous, the cryomilled and ball-milled materials contain small amounts of water. Thus, one can suppose that the υ-process is related to the mobility of water molecules present in milled IND. In order to characterize the υ-modes observed for both water-containing IND materials, the υ-relaxation times have been calculated as the inverse of the frequency of the maximum peak position, i.e. τ = 1/2πf max. As clearly seen in Figure 13 the temperature dependences of log τυ, determined for the amorphous samples obtained by various mechanical treatments, overlay each other. This result is not surprising in the light of the data displayed in Figure 12, where the maximum of υ-modes for both water-

Figure 13. Temperature dependence of α (diamond symbols), υ (solid and crossed circle symbols), γ (open symbols), relaxation times determined for anhydrous IND and both examined hydrated samples. Solid lines indicate VFT and Arrhenius fits to the α-relaxation times and secondary γ-, and υ-relaxation times, respectively.

containing IND samples are exactly the same at 216 K. Since the relaxation times of the υ-process plotted as an inverse of temperature exhibit the Arrhenius behavior from fitting of eq 8 to the experimental data, the activation energy of the υ-mode was determined to be 52.4 ± 0.3 kJ/mol. It is worth noting that this value is in good agreement with the activation energy of water relaxation found for other water mixtures such as e.g. DPG + H2O (43 kJ/mol),69 or hydrated lidocaine HCl (49 kJ/mol).70 Moreover it is also close to the value of Ea for supercooled water examined in some confined water systems (Ea ≈ 44.4 ± 3.7 kJ/mol).71 As it was demonstrated above, the effect of water on the molecular dynamics of IND in the glassy state is significant. 3623

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Table 3. Apparent Solubility of Crystalline and Amorphous Forms of IND Obtained by Vitrification, Ball Milling and Cryomilling Determined after 24 h of the Experiment solubility of indapamide mean value n = 9 amorphous form crystalline form media H2O 0.1 M HCl phosphate buffer pH 6.8

vitrified

ball milled

cryomilled

temp [°C]

mg/L

RSD [%]

mg/L

RSD [%]

mg/L

RSD [%]

mg/L

RSD [%]

25 37 25 37 25 37

81 109 88 106 81 106

2.55 2.40 2.63 2.25 2.92 2.57

96 125 102 140 91 120

2.50 1.90 2.50 2.50 2.10 2.10

107 144 125 149 103 136

2.30 0.70 2.20 1.00 2.70 1.50

100 144 122 151 96 140

2.60 1.38 3.10 1.23 2.30 0.40

Thus, the question is: How does water af fect the structural relaxation process and consequently the values of glass transition temperature and f ragility of the examined systems? In the literature one can find abundance of experimental data showing that water is a common plasticizer and an increase of water content leads to a drop of Tg.68 To verify this statement for IND, we analyzed the dielectric spectra of cryomilled and room temperature milled samples collected above Tg. As illustrated in panels a and b of Figure 11, in the supercooled region of the milled materials the well resolved structural relaxation process is observed. Moreover, in both cases the maximum of the αpeak appears in our frequency window at the same temperature conditions as for the quenched IND sample. Additionally, comparison of the shape and position of the structural relaxation processes collected at the same temperature for all examined materials reveals only one difference between dielectric spectra. Namely, the contribution of the dc conductivity is more pronounced in the case of ball-milled IND than for the melt-quenched and cryomilled samples. Thus, one can expect the same temperature dependences of τα for all examined IND materials. Indeed, as depicted in Figure 13, regardless of the amorphization method, the α-process detected in the dielectric loss spectra of IND exhibits the same pattern of behavior. Consequently, the values of the glass transition temperature and dynamic fragility of IND do not depend on the applied amorphization technique. This result is consistent with the DSC thermograms, which show the characteristic signature for the Tg in the heat flow with an onset at 377 K for each examined IND sample (see inset in Figure 2). Part C: Solubility Advantage from an Amorphous IND Drug. As a final point we would like to present results of apparent solubility measurements of crystalline and all examined amorphous forms of indapamide. The obtained values are collected in Table 3. As a reference, crystalline IND was examined. Crystalline indapamide subjected to nonsink dissolution studies at 37 °C dissolved rapidly to a concentration of 106.8 ± 12.1 mg/L within the first minute of the experiment. Solubility of crystalline indapamide reached a plateau at 109.2 ± 9.6 mg/L within 30 min. PXRD analysis confirmed that the solid residue recovered at the end of the experiment was indapamide hemihydrate, indicating that this crystalline form was physically stable and did not convert in water to other forms of indapamide. Additionally, the solubility of crystalline IND in water was found to be equal to 81 mg/L when the experiment temperature was decreased to 25 °C. These results are in good agreement with the literature data (75 mg/L).72 On the other hand, amorphous indapamide, obtained using vitrification, reached a concentration of 394.6 ± 24.0 mg/L within the first two minutes of the experiment, resulting in over 3-fold

Figure 14. Dynamic solubility studies of crystalline and amorphous (vitrified) indapamide (n = 3).

greater concentration of the drug in comparison to that of the crystalline material (Figure 14). Following the supersaturation peak, concentration of indapamide decreased to 129.4 ± 4.3 mg/L at 10 min of the experiment. The drop in the drug concentration was related to solution mediated crystallization of the amorphous phase to indapamide hemihydrate, however the drug levels in solution remained significantly higher (p < 0.05) than the “steadystate” concentration for crystalline indapamide hemihydrate. From Table 3 it can be seen that irrespective of the amorphization technique, in each medium, the apparent solubility of amorphous IND, determined at 24 h of the experiment, is 13% to 42% greater than that of the crystalline form. The greatest value (151 mg/L i.e. 42% higher than for crystalline IND) was obtained for the cryomilled sample in 0.1 M hydrochloric acid at 37 °C. Additionally, it was found that there was only a slight difference between apparent solubility of the cryomilled and ball-milled samples. The solubility of IND samples was determined with high precision. The values of the relative standard deviation (RSD), calculated on the basis of nine replicates for each sample, were in the range of 0.4−3.1%.

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CONCLUSIONS From our studies on amorphous IND we can draw the following conclusions: 1. Quench-cooling of the melt, cryogenic grinding and room temperature milling can be successfully applied to produce IND in the amorphous state. Especially important for commercial application seems to be cryogenic milling, which was found to be very effective. 3624

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2.

3.

4.

5.

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NMR and HPLC measurements combined with mass spectrometry have shown that all examined amorphous samples were of purity satisfying the strict pharmaceutical requirements. Investigations by means of FT-IR in a wide temperature range have shown that conversion to the glassy state of IND leads to an amide−imidic acid tautomerism, which is a reversible proton transfer between different locations in the molecule. Interestingly, the less stable imidic acid form of IND appears independent of the applied amorphization technique. Using BDS spectroscopy, we have studied the molecular dynamics of vitrified and milled IND in the supercooled liquid and glassy states. In all cases, at a given temperature, the position as well as shape of the structural relaxation process was found to be the same (the only difference between the spectra was the higher contribution of dc conductivity in the case of the ball-milled sample). Thus, there was also no difference between the temperature dependence of structural relaxation times determined for all examined samples. The glass transition temperature calculated on the basis of BDS measurements was found to be equal to 374 K (τα = 100 s) for the vitrified and milled materials, and it is in good agreement with the value determined from calorimetric measurements (TgDSC = 377 K). On the other hand, the molecular dynamics of milled and melt-quench samples below Tg differ from each other significantly. In all cases, the dielectric measurements reveal only one, well-pronounced secondary relaxation process, however its molecular origin is completely different. In the case of the vitrified sample it was labeled as γ-mode. This relaxation process with a low value of the activation energy (34 kJ/mol) was found to be due to rotation of the sulfonamide group. Also, for milled samples the υ-relaxation with Ea = 54 kJ/mol, related to mobility of water molecules, was observed. Additionally, for each examined sample, a slower secondary mode (β), identified as a hidden JG relaxation, which originates from intermolecular interactions, was visible in the dielectric spectra. It has been presented, using BDS, DSC and XRD techniques, that amorphous indapamide prepared by quench-cooling, cryogrinding and ball grinding is a physically stable system in a wide temperature range, above as well as below Tg. We did not notice any signs of crystallization even one year after amorphization (storage conditions: T = 293 K, humidity 10%). Amorphous IND crystallizes in aqueous media to indapamide hemihydrate. The supersaturation concentration of amorphous IND undergoing liquid-mediated crystallization at 37 °C in water was found to be nearly four times greater than the equilibrium solubility of crystalline IND. It was found that, irrespective of the amorphization technique, the apparent solubility of amorphous indapamide was greater by 13−42% than that of the crystalline form. The greatest value (151 mg/ L i.e. 42% greater than for crystalline IND) was obtained for cryomilled sample in 0.1 M hydrochloric acid at 37 °C at 24 h of the apparent solubility studies.

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ACKNOWLEDGMENTS

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REFERENCES

Article

The authors Z.W., K.G., M.P. and W.S. are deeply grateful for the financial support by the National Science Centre within the framework of the Opus3 project (Grant No. DEC-2012/05/B/ 1127NZ3/03233). L.T. and K.J.P. wish to acknowledge funding for this research from Solid State Pharmaceutical Cluster (SSPC), supported by Science Foundation Ireland under Grant No. 07/ 1130SRC/B1158. Z.W. acknowledges the financial assistance from FNP START (2013). M.D. is thankful for support from PL-Grid Infrastructure.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. 3625

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