The influence of amorphization methods on the apparent solubility and dissolution rate of tadalafil

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The influence of amorphization methods on the apparent solubility and dissolution rate of tadalafil

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K. Wlodarski a,⇑, W. Sawicki a, K.J. Paluch b, L. Tajber b, M. Grembecka c, L. Hawelek d, Z. Wojnarowska e, K. Grzybowska e, E. Talik e, M. Paluch e a

Medical University of Gdansk, Department of Physical Chemistry, Hallera 107, 80-416 Gdansk, Poland Trinity College Dublin, School of Pharmacy and Pharmaceutical Sciences, College Green, Dublin 2, Ireland Medical University of Gdansk, Department of Food Science, Hallera 107, 80-416 Gdansk, Poland d Institute of Non Ferrous Metals, Sowinskiego 5, 44-100 Gliwice, Poland e University of Silesia, Institute of Physics, Uniwersytecka 4, 40-007 Katowice, Poland b c

a r t i c l e

i n f o

Article history: Received 19 February 2014 Received in revised form 28 April 2014 Accepted 28 May 2014 Available online xxxx Keywords: Amorphous drugs Tadalafil Apparent solubility Dissolution rate Precipitation from solution

a b s t r a c t This study for the first time investigates the solubility and dissolution rate of amorphous tadalafil (Td) – a poorly water soluble chemical compound which is commonly used for treating the erectile dysfunction. To convert the crystalline form of Td drug to its amorphous counterpart we have employed most of the commercially available amorphization techniques i.e. vitrification, cryogenic grinding, ball milling, spray drying, freeze drying and antisolvent precipitation. Among the mentioned methods only quenched cooling of the molten sample was found to be an inappropriate method of Td amorphization. This is due to the thermal decomposition of Td above 200 °C, as proved by the thermogravimetric analysis (TGA). Disordered character of all examined samples was confirmed using differential scanning calorimetry (DSC) and X-ray powder diffraction (PXRD). In the case of most amorphous powders, the largest 3-fold increase of apparent solubility was observed after 5 min, indicating their fast recrystallization in water. On the other hand, the partially amorphous precipitate of Td and hypromellose enhanced the solubility of Td approximately 14 times, as compared with a crystalline substance, which remained constant for half an hour. Finally, disk intrinsic dissolution rate (DIDR) of amorphous forms of Td was also examined. Ó 2014 Published by Elsevier B.V.

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1. Introduction

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Solubility and dissolution rate are the factors considered to play a key role in the two first phases of the time course of drug distribution (LADME), i.e. liberation and absorption, significantly affecting bioavailability. The increasing number of poorly soluble drug substances in the contemporary pharmaceutical market forces to continuously search for new methods to improve these parameters (Kawabata et al., 2011). Strategies concerning the enhancement of apparent solubility and dissolution rate of drugs at the solid state present an important research area, justified by prevalence and great importance of oral solid dosage forms as means of active pharmaceutical ingredients (APIs) administration. These approaches include micro and nanosizing of API, formation of

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⇑ Corresponding author. Address: Medical University of Gdansk, Faculty of Pharmacy, Department of Physical Chemistry, Hallera 107, 80-416 Gdansk, Poland. Tel./fax: +48 58 349 16 52. E-mail address: [email protected] (K. Wlodarski).

cocrystals and complexes, manufacture of solid dispersions and solutions, formation of various crystal polymorphs and amorphization (Anjana et al., 2013; Saharan et al., 2009). The amorphization process leads to the amorphous form of a drug which, in contrast to the crystalline form, can be characterized by the disordered arrangement of molecules in the solid state. There are numerous methods and physical processes which enable formation of amorphous forms of active pharmaceutical ingredients, e.g. vitrification, grinding, freeze-drying, spray drying and rapid precipitation from a solution. The amorphous form, in comparison to the crystalline form, frequently improves the physical properties, such as solubility and dissolution rate, while maintaining the identical chemical structure and therefore the pharmacological activity of API (Nagapudi and Jona, 2008). This favorable alteration in properties is due to the higher internal energy and lower thermodynamic stability. Reported changes in solubility enhancement varied depending on chemical compounds and in respect to their lowest energy crystalline forms were, for instance, 5-fold, 50-fold and even 250-fold for caffeine, theophylline and

http://dx.doi.org/10.1016/j.ejps.2014.05.026 0928-0987/Ó 2014 Published by Elsevier B.V.

Please cite this article in press as: Wlodarski, K., et al. The influence of amorphization methods on the apparent solubility and dissolution rate of tadalafil. Eur. J. Pharm. Sci. (2014), http://dx.doi.org/10.1016/j.ejps.2014.05.026

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morphine, respectively (Huang and Tong, 2004). Nevertheless, it has been documented that physicochemical properties of amorphous forms can be largely dependent on methods chosen for their production, as well. For instance, a study of two amorphous samples of cefditoren pivoxil obtained using spray drying at different inlet-air temperatures revealed differences in their water vapor desorption and physical stability. This diversity was present despite the same glass transition temperature of amorphous samples (Ohta and Buckton, 2005). Certain attempts to theoretically explain existence of differing amorphous forms have been made so far (Poole et al., 1995). Tadalafil (Td), phosphodiesterase type 5 (PDE5) inhibitor used in the treatment of erectile dysfunction, is a drug substance belonging to class 2 of Biopharmaceutics Classification System (Abdel-Aziz et al., 2011). Therefore, in spite of good permeability, its bioavailability is limited by solubility and dissolution rate. However, there is little data on physicochemical properties of solid Td and inclusion in microporous silica has been the only method used for its amorphization so far (Mehanna et al., 2011). Thus, the objective of this study was to apply several different approaches in order to obtain amorphous tadalafil in a bulk state and further assess the impact of production method on the apparent solubility over time and the dissolution rate of such amorphous powders. The following methods: cryogenic grinding, ball milling, spray drying, freeze-drying and antisolvent precipitation were attempted to produce amorphous Td.

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2. Materials and methods

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2.1. Materials

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Tadalafil (series 20211) was kindly donated by Polpharma S.A., Tween 80 was purchased from Sigma–Aldrich Chemie Company (Germany), Pharmacoat – hypromellose was bought from ShinEtsu Chemical Company (Japan) and acetone was purchased from Corcoran Chemicals (Ireland). Ultrapure water was produced by Millipore Direct-Q 3UV-R water purification system. Sodium lauryl sulfate (SLS) and all other chemicals of analytical grade were purchased from POCH Company (Poland).

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2.2. Methods

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2.2.1. Preparation of amorphous tadalafil using cryogenic grinding Cryogenic grinding of tadalafil was carried out by means of a 6770 SPEX freezer/mill. The total mass of the milled tadalafil was 1 g. The sample was placed in a stainless steel vessel and then immersed in liquid nitrogen. The stainless steel rod present in the vessel was 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. Ten minute grinding intervals were separated by 3 min cool-down periods. The effective grinding times were 120 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.

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2.2.2. Preparation of amorphous tadalafil using ball milling The room temperature ball milling was performed using a Planetary Ball Mill (Retsch, Germany). A zirconium jar (250 ml) was filled with the examined material and 6 zirconia balls (20 mm in diameter). The rotation speed was set to 400 rpm. Three separate tests with the same amount of material (16 g) applying different grinding times were performed. Each milling cycle lasted 15 min and was followed by a 5 min break. The total milling time of tadalafil was 24 h.

2.2.3. Preparation of amorphous tadalafil using spray drying Spray drying of Td 2% w/v solution (acetone/water 9:1, v/v) was performed using a Büchi Mini Spray Dryer B-290 (Büchi, Switzerland). The spray dryer was used in an open system with nitrogen applied as a drying and atomizing gas (open, suction mode). Spray dryer was equipped in standard atomization nozzle with a 1.5-mm cap and 0.7-mm tip. The drying gas pressure was of 6 bar at 4 cm gas flow (rotameter setting), equivalent to 473 norm liters per hour of gas flow in normal conditions (P = 1013.25 mbar and T = 0 °C). The nozzle pressure drop was measured to be 0.41 bar. The pump speed was set to 30% (9–10 ml/min) and the aspirator was operated at 100%. Inlet temperature was set to 160 °C and such setup resulted in outlet temperature of 85 °C. The additional, secondary drying was performed in an incubator with forced air flow (Gallencamp economy incubator with fan; Weiss-Gallencamp, UK) at 80 °C for 24 h.

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2.2.4. Preparation of amorphous tadalafil using freeze-drying 100 mg of Td was dissolved in 50 ml of each of the following solvents or mixture of solvents (given in volume ratios): glacial acetic acid, glacial acetic acid/purified water (90/10), dioxane, dioxane/water (50/50), dioxane/methanol (90/10), dioxane/isopropanol (80/20), acetonitrile/water (45/55), acetonitrile/glacial acetic acid (50/50). The resulting solutions were obtained by stirring in a round-bottomed flask, rapidly frozen in the atmosphere of liquid nitrogen and subsequently freeze-dried for 48 h at 50 °C on the vapor capacitor, using a freeze-dryer (Alpha 1-2 LD, Germany). The sample vessel was attached externally to a manifold of the freeze dryer and the sample was subjected for 24 h to subambient temperatures due to an ongoing sublimation process. After 24 h the sample reached ambient temperature allowing for secondary drying of the residual solvents (Pikal et al., 1990). Secondary drying was continued for additional 24 h.

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2.2.5. Preparation of amorphous tadalafil using antisolvent precipitation Five various samples with composition as outlined in Table 1 were prepared. Briefly, 100 mg of Td was dissolved in the organic solvent (acetonitrile or dioxane), while antisolvent phase composed of Tween 80 or hypromellose (if used) dissolved in water. Samples 1 and 3 contained no excipient added and pure water was used an antisolvent. The organic Td solution was placed on a magnetic stirrer (1000 rpm) and the aqueous excipient solution (or water if no excipient was used) was slowly added. After the addition was complete, the formed suspension was frozen in a liquid nitrogen atmosphere and the dispersion medium was removed by freeze-drying for 48 h at 50 °C, 0.2 mbar.

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2.2.6. Powder X-ray diffraction analysis (PXRD) The X-ray diffraction measurements were carried out on the laboratory Rigaku – Denki D/MAX RAPID II-R diffractometer attached with a rotating anode Ag Ka tube (k = 0.5608 Å), an incident beam (0 0 2) graphite monochromator and an image plate in the Debye–Scherrer geometry. The pixel size was

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Table 1 The composition of samples in the antisolvent precipitation process. Components

Tadalafil (mg) Acetonitrile (ml) Water (ml) Dioxane (ml) Tween 80 (mg) Hypromellose (mg)

Sample 1

2

3

4

5

100.0 15.0 80.0 – – –

100.0 15.0 80.0 – 70.0 70.0

100.0 – 80.0 15.0 – –

100.0 15.0 80.0 – 70.0 –

100.0 15.0 80.0 – – 70.0

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100 lm  100 lm. Crystalline and various forms of Td were placed inside glass capillaries (1.5 mm in diameter). Measurements were performed for the sample filled and empty capillaries and the intensity for the empty capillary was then subtracted. The beam width at the sample was 0.1 mm. The two-dimensional diffraction patterns were converted into the one-dimensional intensity data using suitable software. 2.2.7. Differential scanning calorimetry (DSC) Calorimetric measurements were carried out by a MettlerToledo 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. Aluminum pans (40 ll) with samples were top sealed with one vent hole. Standard DSC measurements were performed at the heating rate of 10 °C/min under nitrogen atmosphere (60 ml/min). 2.2.8. Thermogravimetric analysis (TGA) TG analysis was performed using a Mettler TG 50 module linked to a Mettler MT5 balance (Switzerland). Samples were placed into open aluminum pans (5–12 mg). A heating rate of 10 °C/min was implemented in all measurements. Analysis was carried out in the furnace under nitrogen purge. 2.2.9. Fourier transform infrared spectroscopy (FTIR) IR spectra were recorded using a spectrophotometer FTIR 410 (Jasco, Japan) in a scanning range from 4000 to 400 cm1. 1 mg of a substance and 100 mg of desiccated potassium bromide were pulverized in a mortar and pressed into a disk. 2.2.10. SEM analysis Microstructural observations of the examined samples were conducted using the JEOL-7600F scanning electron microscope equipped with an Oxford Instruments X-ray Energy Dispersive Spectroscope (EDS). 2.2.11. Investigation of the thermodynamic solubility and the apparent solubility over time The intrinsic and apparent solubility studies were carried out as follows: 10 mg of each sample was suspended in 25 ml of purified water preheated to 37 °C in 50 ml conical flasks and shaken (150 rpm) for 24 h in a water bath shaker at 37 ± 0.5 °C. The resulting suspensions were filtered through polyester syringe filters with a pore size 0.45 lm (Chromafil PET-45/25), previously heated to 37 °C, at specified time intervals during the first hour (apparent solubility) and after 24 h. The filtered solutions were quantitatively diluted with acetonitrile (1:4 v/v for precipitates and 1:1 v/v for other samples) to prevent possible precipitation and to ensure that the drug concentrations will fall in the linear range of the calibration curve. Additionally, crystalline, ball milled and freeze-dried (from glacial acetic acid) materials were subjected to an analogous study in 0.1 M HCl and phosphate buffer pH 6.8 (samples collected after 24 h), to investigate the impact of pH effect on Td solubility. The amount of dissolved Td was determined by high performance liquid chromatography (HPLC), as described in the further section. Each investigation was examined three times followed by triple injection and calculation of the average result. 2.2.12. Investigation of disk intrinsic dissolution rate (DIDR) The study of intrinsic dissolution rate was carried out for crystalline, spray dried and ball milled forms of Td with the use of Wood’s apparatus (VanKel Industries, USA), according to the USP 32. For this purpose, 600 mg of powder was poured to the die cavity of the apparatus and subsequently subjected to the pressure of 1 ton for 1 min in a compression apparatus (Specac – Atlas power

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T8, UK). Afterwards, Wood’s apparatus with compressed powder was placed in the dissolution apparatus (Erweka DT 70, Germany). Intrinsic dissolution rate was measured in 900 ml of 1% w/v sodium lauryl sulfate (SLS) aqueous solution, with the rotation speed set to 75 rpm at 37 ± 0.5 °C. Solution samples were collected 5, 10, 20, 30, 50, 70, 90 and 120 min after the start of the study and subjected to analytical assay by HPLC, as described in the section below. After each sampling fresh dissolution medium was added to maintain the volume of 900 ml. DIDR was calculated using the following formula:

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j ¼ V dc dt

1

A1

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where j is dissolution flow (mg cm2 s1), V is the volume of dissolution medium (ml), dc is the increase of the concentration of dissolved drug in the medium (mg/ml), dt is time increment (s) and A is the surface area of the compact (cm2). This investigation was carried out in duplicate for each sample of Td with each replicate based on the averaged result of three HPLC injections.

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2.2.13. High performance liquid chromatography (HPLC) for the quantitative analysis of tadalafil A simple and selective HPLC method was developed for the quantitative evaluation of Td concentration in samples. The analysis was performed using a HPLC Ultimate 3000 system (Dionex, Germering, Germany) consisting of a quaternary pump, an autosampler and a column heater (37 °C) and Nucleodur C-18 column (250  4.6 mm, 5 lm), operating in a reverse phase (RP) system with the mobile phase consisting of acetonitrile and water (40:60 v/v) in the isocratic flow and a flow rate of 1 ml/min. The run time and the retention time of Td were 14 min and 10.3 min, respectively. Td concentrations were assessed using a Corona CAD (ESA, Chelmsford, MA, USA) detector or a UV/DAD detector (Ultimate 3000, Dionex) for samples containing SLS from DIDR tests. The wavelength of 220 nm was chosen on the basis of UV–Vis spectrum of Td in the mobile phase in the range of 180–800 nm (Spectrophotometer UV-1800 Shimadzu, Japan). Nitrogen gas flow rate was regulated automatically and monitored by the CAD device and it was supplied by a nitrogen generator Sirocco-5 (Schmidlin-DBS, Switzerland), regulated at 35 psi. Data processing was carried out with Chromeleon 6.8 software (Dionex).

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2.2.14. Validation procedure of analytical method The HPLC-RP-C18 method with both Corona CAD and UV–Vis detection has been validated on the basis of ICH Q2, Eur. Ph. and USP guidelines for the specificity, limit of detection and quantification, linearity, accuracy and precision. The concentration of Td was determined from peak areas in chromatograms, with regard to linear regression obtained from Td standard solutions. Standard solutions were prepared in the mobile phase from a Td stock solution of 0.05 mg/ml. Each standard solution was prepared twice and analyzed by HPLC three times, what resulted in six injections at one level of concentration. For HPLC-Corona CAD system standard solutions in a concentration range of 0.6–7.0 lg/ml with the following linear relationship were prepared:

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y ¼ 23:315x  1:9899

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R2 ¼ 0:9999

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while for HPLC–UV–Vis system a linear function in the concentration range of 0.2–7.0 lg/ml was plotted:

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y ¼ 3:3274x  0:3157

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R2 ¼ 0:9971

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Table 2 Validation parameters for HPLC-Corona CAD method. Parameter

Results

Selectivity (for concentration of 0.6 lg/ml)

Peak symmetry factor (in the range of 0.8–1.5 required) = 1.15 Retention factor (k) (in the range of 0.5–20 required) = 3.89 Signal to noise ratio (S/N) (>10 required) = 29.15 Absence of interfering substances confirmed LOQ = 10 SD/a = 0.6 lg/ml, where SD is the average of standard deviations of determinations in the lower range of linearity and a is the directional coefficient of the plotted linear function LOD = 3 SD/a = 0.18 lg/ml y = 23.315x  1.9899 Balance of residuals less than 10.0% for all points of the linearity range

Limit of quantification (LOQ)

Limit of detection (LOD) Linearity Accuracy

Concentration Average of 9 injections SD RSD (<5% required) Recovery (95–105% required)

Precision

3 lg/ml 2.94 lg/ml 0.132 4.49% 97.66%

5 lg/ml 4.76 lg/ml 0.203 4.26% 95.2%

7 lg/ml 6.71 lg/ml 0.159 2.36% 95.87%

Repeatability

Repeatability with variable

Intermediate precision

For concentration of 5 lg/ml Average of 18 injections SD RSD (<5% required)

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where y is a peak area, x is a concentration of Td (lg/ml) and R2 is a regression coefficient. Table 2 presents other validation parameters obtained for the HPLC-Corona Cad system. All acceptance criteria for the studied parameters were met which means that the applied analytical method is selective, accurate and precise within the specified linearity range.

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3. Results and discussion

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3.1. Amorphization of tadalafil

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The effectiveness of methods applied in the current work for the conversion of crystalline to amorphous forms of a range of APIs has been confirmed many times in literature (Willart and Descamps, 2008; Chadha et al., 2013). Nevertheless, there is no data describing amorphization of tadalafil (Td) and possible impact of such a solid-state change on its solubility. In our study, an amorphous form of Td was successfully produced utilizing cryogrinding, ball milling, spray drying, freeze-drying and antisolvent precipitation.

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3.1.1. Vitrification of tadalafil Owing to thermal decomposition of Td at the melting point temperature, which is 302–303 (Abdel-Aziz et al., 2011), vitrification, defined as the simplest method of amorphization, could not be applied. In spite of the fact that above 200 °C a change of the Q2 color1 from white to yellow–brown accompanied by release of smoke appeared, the thermogram of the crystalline sample did not reveal any event of degradation (Fig. 1). However, further cooling and reheating of this sample showed a glass transition point significantly different from that determined for other amorphous forms (thermogram not presented), which indicates the influence of degradation products on the thermal properties of Td. TGA showed a 2.5% mass loss when Td was heated up to its melting point. HPLC evidenced that there was 98.4 ± 1.2% intact Td present when the powder was heated up to 200 °C, however only 70.5 ± 1.5% Td remained when the sample was heated up to 302 °C. Since the general solubility equation developed by Yalkowsky and co-workers (Jain and Yalkowsky, 2001) indicates that the 1

For interpretation of color in Fig. 1, the reader is referred to the web version of this article.

4.58 lg/ml 0.164 4.22%

4.81 lg/ml 0.217 4.50%

4.70 lg/ml 0.164 3.49%

Fig. 1. DSC thermograms of: crystalline (a), ball milled (b), precipitated 5 (c) and precipitated 2 (d) tadalafil.

lower the solubility the higher the melting point, the phenomenon of thermal decomposition before melting might appear among poorly soluble compounds belonging to the BCS II class, limiting utilization of vitrification as an amorphization method to such compounds.

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3.1.2. Cryogrinding, ball milling and spray drying of tadalafil On the contrary, cryogrinding, ball milling and spray drying enabled to obtain fully amorphous forms of Td. These results were confirmed by PXRD measurements. As it can be seen in Fig. 2, there are no Bragg peaks in diffractograms obtained for these samples. Such abroad diffraction halo is a characteristic feature of totally amorphous materials. DSC analysis additionally confirmed the amorphous nature of these samples. For example, the DSC curve for the ball milled tadalafil is depicted in Fig. 1. In the thermogram obtained during heating from the glassy state, one can observe the characteristic signature for the glass transition with the midpoint at 147 °C, followed by an exothermic peak with onset at 167 °C, which might have indicated the transformation of existing amorphous form to a more stable and lower energy crystalline form (cold crystalliza-

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Fig. 2. X-ray powder diffraction patterns of crystalline (a), cryoground (b), ball milled (c) and spray dried (d) forms of tadalafil.

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Fig. 3. X-ray powder diffraction patterns of lyophilisate from acetic acid (a), lyophilisate from acetonitrile/water (b), lyophilisate from acetic acid/water (c), precipitate 5 (d), precipitate 2 (e) and crystalline form of tadalafil (f).

tion), and a subsequent endothermic peak of melting point at 302 °C. This melting point of crystallized Td is the same as that of the lowest-energy crystalline form of Td what indicates that amorphous Td does not crystallize to another polymorphic form of the drug (Fig. 1).

3.1.3. Freeze-drying of tadalafil There were a few limitations affecting the preparation of samples using freeze-drying. In addition, there is little data on the selection of lyophilisation parameters such as a type of solvent and a rate of freezing, which enable to produce the amorphous form of API without addition of solid excipients (Tang and Pikal, 2004). Since Td is practically insoluble in water and poorly soluble in methanol and isopropanol, other solvents had to be found. Acetonitrile, dioxane and glacial acetic acid appeared to be appropriate to prepare the desirable concentration of Td solutions. However, very low freezing temperatures of some solvents (methanol 98 °C, isopropanol 89 °C, acetonitrile 45.7 °C, water 0 °C, dioxane 10 °C, glacial acetic acid 16 °C) and their eutectic mixtures in relation to freeze-drying conditions (50 °C on the capacitor, 20 °C in the sample) also had to be taken into consideration. Solvents and their mixtures selected as a compromise between their freezing points and adequate Td solubility have been listed in the section describing the method. None of them had the melting temperature below 20 °C. Fluffy white lyophilisates of Td were obtained from glacial acetic acid, glacial acetic acid/purified water (90/10 v/v) and acetonitrile/water (45/55 v/v). In the case of the rest of the samples, there was only a residue on the walls of the round-bottomed flasks, despite the fact that processes run properly without melting. Diffraction patterns of the three successfully obtained lyophilisates revealed the presence of amorphous forms as evidenced by the absence of Bragg diffraction peaks (Fig. 3). The utilization of different solvents for freeze drying aimed to indicate whether their chemical structures are able to affect the formation of amorphous forms and subsequently result in a change of their solubility. In addition to the confirmation of amorphicity, the infrared spectra obtained for Td lyophilisates showed no differences in a short range molecular arrangement between them as well as between them and other amorphous samples. However, their spectra significantly differed from the spectrum of crystalline Td (Fig. 4). The signal of the amino group of amorphous powders was widened and shifted from the wavelength of 3326– 3299 cm1. Additionally, the double signal of the lactame group

Fig. 4. IR spectra of spray dried, lyophilisate from acetic acid/water, lyophilisate from acetonitrile/water and crystalline form of tadalafil.

at a wavelength of 1665 cm1 was blurred, pointing the appearance and participation of both groups in strong hydrogen bonds. The presence of an additional signal at a wavelength of 3399 cm1 may be due to the amino group which, contrary to the ordered crystalline structure, partly enters into strong hydrogen bonds and partly remains free, resulting in broadening of the signal and the appearance of an additional band (Morzyk-Ociepa et al., 2004). For further analysis amorphous lyophilisates of Td from glacial acetic acid and acetonitrile/water (45/55) were chosen.

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3.1.4. Precipitation of tadalafil In order to obtain amorphous precipitates and simultaneously examine the influence of excipients, five different formulations of Td were prepared using antisolvent precipitation, as presented in Table 1. DSC and PXRD analysis revealed no decrease in the crystallinity of precipitates 1, 3, 4 (the diffraction patterns were identical to that of crystalline Td, not presented), partial amorphization of sample 2 and almost complete amorphization of sample 5, indicating the beneficial impact of hypromellose (Figs. 1 and 3). Residual crystallinity of precipitate 5 was confirmed by the presence of only very faint Bragg peaks on the diffraction pattern.

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Additionally, the presence of the crystallization peak on the DSC thermogram (Fig. 1), which appeared at the same temperature as for other amorphous samples at 167 °C, was the subsequent acknowledgment of Td amorphization. The crystallization event of Td was followed by a melting endotherm with an onset at 250 °C, which is at a lower temperature than that for crystalline Td. This significant depression of melting point was attributed to drug–polymer interactions as reported before (Santos et al., 2008). Appearance of the crystallization peak was a proof that Td was not in a molecular dispersion in hypromellose. In such a hypothetical case, the only thermal effect would come from this polymer. Absence of a visible glass transition event might have been caused by the presence of hypromellose in this composite. In order to find out whether the disappearance of Bragg peaks was not the result of dilution of crystalline Td with the naturally amorphous polymer, a physical mixture of tadalafil and hypromellose in the same weight ratio (10:7) was prepared in the mortar and subsequently analyzed with PXRD. The diffractogram of this sample revealed sharp Bragg peaks characteristic of crystalline Td (Fig. 5) indicating that Td in the precipitate was certainly partially amorphous. Apart from excipients, there are other variables of the precipitation process that may affect the physical properties of the product, i.e. stirring rate, the volume ratio of antisolvent to solvent, drug content, viscosity and temperature (Gao et al., 2008). In the case of amorphous forms, these parameters should be optimized so as to ensure a rapid process of precipitation, preventing the slow growth of ordered crystals. Stirring rate must be sufficient to provide good mixing of miscible solvents, even at the molecular level, causing a desirable state of rapid supersaturation. This state is also the result of relatively large amount of antisolvent (80 ml of water) to solvent (15 ml of organic liquid) and an appropriate amount of drug substance. On the one hand, too small quantity of API slows the precipitation but on the other hand, too high content of a substance may increase the viscosity and as a result slow down the mixing rate. However, the viscosity rise should be particularly considered as a result of polymers addition. Temperature plays an important role when solubility of an active substance is its function. Addition of excipients, discussed below, and freeze-drying protect from Ostwald ripening, which may occur and lead to recrystallization in suspensions (Ghosh et al., 2011). Based on the results of powder crystallographic analysis, precipitate 2 and 5 were selected for further investigation. Equilibrium solubility of precipitate 1 was measured for comparison.

Fig. 5. X-ray powder diffraction patterns of precipitate 5 (a), crystalline form of tadalafil (b) and physical mixture of tadalafil and hypromellose 10:7 (c).

3.2. Solubility and dissolution studies of tadalafil

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3.2.1. Apparent and thermodynamic solubility Apparent solubility of processed forms of Td with respect to thermodynamic solubility of its crystalline form was determined in purified water at 37 °C after 1 h and 24 h shaking. Negligible influence of hydrochloric acid and phosphate buffer on Td solubility was first confirmed (Table 3), what allowed to choose purified water as a reliable and simple solubility medium (Fig. 6, Table 4). Despite the fact that apparent solubility of amorphous forms usually shows the greatest enhancement within the initial period of dissolution, what may be easily explained by further crystallization of suspended particles, our experience indicates that sometimes significant solubility increase may last even after 24 h, indicating existing of a water-stable amorphous form (Lepek et al., 2013). The results of solubility studies indicate a slight improvement of solubility of amorphous forms produced by ball milling, cryogenic grinding, spray and freeze-drying after 24 h in comparison to crystalline Td. Solubility of cryoground form increased approximately twofold, and for other forms the increase was less than twice in comparison to crystalline form. In the case of lyophilisation, no impact of different solvents on the solubility of amorphous samples was seen. What is important, Td concentrations obtained after the first hour remained unchanged for another 23 h for almost all amorphous samples. It means that the dissolution process of Td amorphous forms achieved the long-lasting supersaturated state already after the first hour, pointing to the fast partial crystallization of powder. Determination of concentration in subsequent days could reveal the return to the solubility of crystal, what would be in accordance with the thermodynamic theory of solubility, but it would not have any importance concerning bioavailability of Td. The most significant solubility increase after 24 h of shaking was recorded for Td obtained in the precipitation process. The concentration of the drug substance in water for Td precipitated from the aqueous solution of hypromellose and from the aqueous solution of hypromellose and Tween 80 was 12.32 lg/ml and 10.14 lg/ ml, respectively. Even higher concentrations were obtained for both precipitates after the first hour of experiment, which indicated the existence of a transient supersaturation (Fig. 6). Precipitate 5 was selected for further investigations because of its amorphous nature and a greater improvement in solubility of Td. Precipitate 2, containing Tween 80, was excluded from further investigations as it contained a surfactant, making it impossible to distinguish which factor was the key in solubility increase: the presence of a tenside or the disordered properties of the sample. Moreover, as mentioned above, addition of the surfactant decreased the degree of amorphicity and simultaneously solubility of the sample. The increase in crystallinity could have been caused by solubilizing effect of the surfactant which might have delayed the precipitation process of Td and, as a result, contributed to the formation of more orderly crystals. For a more detailed examination of changes in Td concentrations in water, solubility media for the crystalline, ball milled, spray dried, freeze dried forms and precipitate 5 samples were assayed at additional time points and a relationship of a Td concentration as a function of time was plotted (Fig. 7). The graph indicates an increase of apparent solubility for all amorphous forms of Td, especially within first 10 min. At the 5th min the drug concentration reaches a value of even 9.5 lg/ml for the lyophilisate from glacial acetic acid (a threefold greater than that for crystalline Td). After this time, it suddenly and constantly decreases, possibly through the samples crystallization, to achieve the concentration range of approximately 4–5 lg/ml, which is still better than for crystalline Td and does not change considerably for the next 23 h.

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K. Wlodarski et al. / European Journal of Pharmaceutical Sciences xxx (2014) xxx–xxx Table 3 The impact of liquid media on solubility (lg/ml) (after 24 h) of crystalline and amorphous forms of tadalafil. Form of tadalafil

Purified water

0.1 M HCl

Phosphate buffer pH 6.8

Crystalline Ball milled Lyophilisate (from acetic acid)

2.80 ± 0.24 3.52 ± 0.12 3.60 ± 0.39

2.97 ± 0.18 2.35 ± 0.22 3.07 ± 0.19

2.48 ± 0.23 3.35 ± 0.15 2.88 ± 0.12

Fig. 6. Solubility in water (lg/ml) (after1 h and 24 h) of crystalline and processed forms of tadalafil.

Table 4 Solubility in water (lg/ml) (after1 h and 24 h) of crystalline and processed forms of tadalafil. Form of tadalafil

1 (h)

24 (h)

Crystalline Ball milled Cryoground Spray dried Lyophilisate (acetic acid) Lyophilisate (ACN/water) Precipitate 1 Precipitate 2 Precipitate 5

2.63 ± 0.12 3.58 ± 0,15 5.69 ± 0.42 4.21 ± 0.28 4.32 ± 0.48 4.09 ± 0.51 3.09 ± 0.45 25.83 ± 0.35 30.45 ± 1.44

2.80 ± 0.24 3.52 ± 0,22 5.13 ± 0.32 4.43 ± 0.12 3.60 ± 0.39 3.25 ± 0.38 3.77 ± 0.14 10.14 ± 0.76 12.32 ± 0.69

Fig. 7. Changes of apparent solubility of various forms of tadalafil as a function of time. Blank is a physical mixture with the composition identical to precipitate 5 (tadalafil: hypromellose 10:7 w/w).

Nevertheless, the most significant increase of apparent solubility was obtained for precipitate 5, in accordance with previous results of solubility studies after 24 h. The precipitation with hypromellose enabled to achieve the concentration of Td of approximately 44 lg/ml after 10 min of shaking, which was 14 times higher than for the crystalline form. What is important, the supersaturation state remained at the same level for half an hour. To verify whether hypromellose does not act itself as a solubilizer, the physical mixture of polymer and crystalline Td in the same quantitative ratio as precipitate 5 was subjected to the same investigation (Fig. 7). In this case solubility remained at the level of 3 lg/ ml, which proved that the solubility enhancement of Td in precipitate 5 was the result of powder amorphization. SEM images (Fig. 8) of precipitate 5 revealed a significant reduction of the particles size in comparison to the crystalline Td. However, despite the fact that some articles describe antisolvent precipitation as the method enabling to obtain amorphous nanoparticles, these particles were rather of micrometer size (Pandya and Patel, 2011). In comparison to the crystalline form their shape was irregular, slightly dendritic and less elongated. Since in the case of amorphous forms of Td without the addition of hypromellose the enhancement of apparent solubility was not so substantial, the interaction between polymer and amorphous Td seems to have a great impact, warranting further examinations. While the threefold increase of apparent solubility within first 10 min would not probably have a major impact on bioavailability of Td, the apparent solubility increase from 3 lg/ml to 44 lg/ml for precipitate 5 may be considered as potentially substantial. In contrast to other amorphous forms, the supersaturation state after dissolution of precipitate 5 lasted for half an hour what under in vivo conditions might give a sufficient time to absorb dissolved drug molecules.

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3.2.2. Disk intrinsic dissolution rate study Disk intrinsic dissolution rate (DIDR) study was finally carried out for the crystalline and amorphous (ball milled and spray-dried) forms of Td in order to exclude the effect of the surface area of the powder on dissolution rate. In spite of the fact that this study is usually carried out to compare various polymorphic and amorphous forms of API without excipients, precipitate 5 containing hypromellose was investigated additionally. In addition to the elimination of all the factors exhibiting differentiating effect on the dissolution rate (surface of powder, stirring rate and temperature), except for the internal structure of powder, this study allows to investigate the dissolution kinetics of API in the unsaturated state, as well as observe any physical changes in the drug substance occurring during the process, e.g. crystallization or hydration (Issa and Ferraz, 2011). Because of a very small solubility and dissolution rate of Td, the first study conducted with the use of pure distilled water as a release medium resulted in concentrations (in the order of magnitude of approximately several ng/ml), which could not be detected with the use of HPLC-Corona Cad coupled system. Thus, it was decided to apply the addition of SLS at the concentration of 1% w/v, even though it may have affected the release process and distorted potential differences in release profiles. During the HPLC analysis, SLS was slowly leaving the analytical column and it was

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Fig. 8. SEM pictures of crystalline tadalafil (on the left) and amorphous precipitate 5 of tadalafil and hypromellose (on the right).

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detected by the sensitive but non-specific Corona Cad detector, giving a high background and preventing the quantitative analysis. Therefore, UV–Vis detection at a wavelength of 220 nm was utilized in this case. Figs. 9 and 10 show concentrations and disk intrinsic dissolution rates (DIDR) of the various forms of Td in the function of time, respectively, on compacts made applying a compression force of 1 ton. Unexpectedly, concentration of Td obtained for the crystalline form was higher at all time points as compared to both amorphous forms without hypromellose, which did not show any significant differences between each other. Dissolution flow of the crystalline form is clearly higher within only initial 5 min, while remaining at a relatively similar level to the amorphous forms at subsequent time points. This result is in disagreement to the solubility studies results, indicating a threefold improvement in solubility of spray-dried amorphous form at the 5th min when the substance was in excess of its expected solubility. Crystallization of Td during disk surface tests in water was not a reason for this behavior. The dissolution rate from the amorphous sample in such a hypothetical case should be higher than that for the crystalline form during first minutes of the study, followed by a slow decrease. The addition of SLS to the dissolution medium may have only impacted on the magnitude of Td concentrations, but it should not have changed the relative order of the release profiles, particularly at such low concentrations. The explanation might have been a possible disk crystallization during the process

Fig. 9. Changes in concentration of crystalline, ball milled (subjected to different compression force), spray dried and precipitated 5 tadalafil as a function of time in the DIDR study.

Fig. 10. Dissolution flows of crystalline, ball milled (subjected to different compression force), spray dried and precipitated 5 tadalafil as a function of time in DIDR study.

of compression (Dhumal et al., 2007). Therefore, powder subjected to 1 ton compression for 1 min was subsequently pulverized and investigated with the use of XRD. This analysis confirmed partial crystallization induced by pressure for the ball milled powder, but did not reveal any Bragg peaks for spray dried form (Fig. 11).

Fig. 11. Impact of compression on solid state properties of amorphous spray dried and ball milled forms of tadalafil.

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Theoretically, even complete crystallization should have decreased the dissolution flow to the comparable level as that for the pure crystalline form but this was not the case. Thus, this hypothesis could be rejected. The second reasonable explanation of dissolution results might be the difference in compressibility of powders (Lepek et al., 2013). The utilization of the same compression conditions for the amorphous and crystalline forms, which is crucial in this study, can paradoxically lead to varying degrees of compaction. The compressibility of the amorphous form, resulting from the chaotic arrangement of molecules, can be greater and result in an increased density of a tablet when compared with an equivalent compact made of the crystalline material utilizing the same pressure. This may lead to lower water wettability and subsequently lower dissolution rate, which theoretically should be higher for the amorphous form. In order to investigate this issue, additional compression forces of 2 and 3 tons were used to make compacts of amorphous ball milled powder, which were subsequently analyzed by DIDR. The value of less than 1 ton could not be applied due to the limitation of the press. The obtained curves (Figs. 9 and 10) revealed no significant differences between the dissolution profiles of disks compressed at 2 and 3 tones. Only in the case of powder compressed at 1 ton the dissolution rate was higher at the beginning of the study but then it decreased after first 10 min. As expected, the dissolution rate of Td in precipitate 5 was improved significantly in comparison to the crystalline form (Figs. 9 and 10). This enhancement was approximately a 2-fold throughout the entire study. In spite of the fact that addition of hypromellose decreases the contact area of Td molecules with the release medium, the process of dissolution is improved due to the amorphous nature of the powder, molecular dispersibility of Td in the polymer matrix and better wettability caused by the presence of hypromellose.

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4. Conclusions

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Herein, the amorphous forms of tadalafil drug were obtained for the first time by cryogenic grinding, ball milling, spray drying, freeze-drying and antisolvent precipitation, as confirmed by powder X-ray diffraction analysis (XRD) and differential scanning calorimetry (DSC). On the other hand, due to the thermal decomposition of Td above 200 °C quenched cooling of the molten sample was found to be an inappropriate method of Td amorphization. We have found that all the obtained Td forms, except the precipitate with hypromellose, are characterized by approximately threefold increase of apparent water solubility in the first 10 min, in comparison to the thermodynamically stable crystalline form, and insignificant increase of solubility after 24 h, that indicates the crystallization of the amorphous phase. Consequently, one can state that the applied amorphization methods (without excipients) influence the apparent water solubility of Td insubstantially. Nevertheless, the addition of hypromellose to the antisolvent phase during the precipitation process results in a significant increase of Td concentration (up to 44 lg/ml) after 10 min of shaking, which is 14 times higher than the value determined for the crystalline form. Our studies have shown that this supersaturation state remains constant for half an hour what might be sufficient to improve Td bioavailability. Moreover, even after 24 h the Td concentration does not achieve the level of thermodynamic solubility. On the other hand, disk intrinsic dissolution rate studies (DIDR) of

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two amorphous Td forms, obtained by ball milling and spray drying, did not reveal any improvement in the rate of dissolution.

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Acknowledgments

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The authors K.W., W.S., K.G., Z.W. and M.P. 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 Synthesis and Solid State Pharmaceuticals Centre (SSPC), supported by Science Foundation Ireland under Grant No. 12/RC/2275.

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