Towards improved solubility of poorly water-soluble drugs: cryogenic co-grinding of piroxicam with carrier polymers.

http://informahealthcare.com/ddi ISSN: 0363-9045 (print), 1520-5762 (electronic) Drug Dev Ind Pharm, Early Online: 1–11 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2015.1054400

RESEARCH ARTICLE

Towards improved solubility of poorly water-soluble drugs: cryogenic co-grinding of piroxicam with carrier polymers

Drug Dev Ind Pharm Downloaded from informahealthcare.com by Nyu Medical Center on 06/16/15 For personal use only.

Anna Penkina1, Kristian Semjonov1, Maija Hakola2, Sirpa Vuorinen2, Timo Repo2, Jouko Yliruusi3, Jaan Aruva¨li4, Karin Kogermann1, Peep Veski1, and Jyrki Heina¨ma¨ki1 1

Department of Pharmacy, Faculty of Medicine, University of Tartu, Tartu, Estonia, 2Department of Chemistry, Faculty of Science, University of Helsinki, Laboratory of Inorganic Chemistry, Helsinki, Finland, 3Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland, and 4Institute of Ecology and Earth Sciences, University of Tartu, Tartu, Estonia Abstract

Keywords

Amorphous solid dispersions (SDs) open up exciting opportunities in formulating poorly watersoluble active pharmaceutical ingredients (APIs). In the present study, novel catalytic pretreated softwood cellulose (CPSC) and polyvinylpyrrolidone (PVP) were investigated as carrier polymers for preparing and stabilizing cryogenic co-ground SDs of poorly water-soluble piroxicam (PRX). CPSC was isolated from pine wood (Pinus sylvestris). Raman and Fourier transform infrared (FTIR) spectroscopy, X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) were used for characterizing the solid-state changes and drug–polymer interactions. Highresolution scanning electron microscope (SEM) was used to analyze the particle size and surface morphology of starting materials and final cryogenic co-ground SDs. In addition, the molecular aspects of drug–polymer interactions and stabilization mechanisms are presented. The results showed that the carrier polymer influenced both the degree of amorphization of PRX and stabilization against crystallization. The cryogenic co-ground SDs prepared from PVP showed an enhanced dissolution rate of PRX, while the corresponding SDs prepared from CPSC exhibited a clear sustained release behavior. In conclusion, cryogenic co-grinding provides a versatile method for preparing amorphous SDs of poorly water-soluble APIs. The solid-state stability and dissolution behavior of such co-ground SDs are to a great extent dependent on the carrier polymer used.

Amorphous solid dispersion, dissolution, polyvinylpyrrolidone, pretreated softwood cellulose, solid-state stability

Introduction Enhancing solubility and bioavailability of poorly water-soluble active pharmaceutical ingredients (APIs) is a great challenge in pharmaceutical research. In recent years, there has been an increasing interest in using amorphous solid dispersions (SDs) for obtaining a good physical stability of the API, as well as an enhanced solubility and bioavailability1–4. In these systems, amorphous drug is formulated together with a polymer to produce a single-phase amorphous mixture of API and polymer. These formulations are proven to be essential for an improved long-term physical and chemical stability, as well as enhanced bioavailability2. The amorphous SDs of poorly water-soluble APIs can be obtained with a number of techniques, such as mechanical activation (ball milling), cryogenic co-grinding and quench cooling of a melt5–9. Cryogenic co-grinding is a promising

Address for correspondence: Anna Penkina, Department of Pharmacy, Faculty of Medicine, University of Tartu, Nooruse 1, 50411 Tartu, Estonia. Tel: +372 737 5285. Fax: +372 7375289. E-mail: [email protected]

History Received 27 January 2015 Revised 15 April 2015 Accepted 20 May 2015 Published online 11 June 2015

technique for both size reduction and obtaining amorphous form of APIs6,10–12. The milling process is a combination of strain field formation and relaxation, wherein the first phenomena leading to the crystal crushing. With more energy applied to the system the defects on the crystalline surface lead to the complete amorphization of the system6. Ball-milling at extremely low temperature prevents the occurrences of thermal damage and undesirable chemical reactions between phases6. In addition, the solubility of API is expected to be improved via amorphization. However, physical and chemical stabilities remain still challenging in cryogenic co-grinding13, and consequently, the inclusion of a stabilizing carrier polymer or polymer mixture is essential. Piroxicam (PRX) is a poorly water-soluble, non-steroidal anti-inflammatory drug, which belongs to Class II (high permeability, low solubility) in the Biopharmaceutics Classification System. For highly permeable and low soluble drugs, the limiting factor for oral absorption is the dissolution rate of drug and formulation. Amorphous state of PRX has been obtained by mechanical activation, cryogenic grinding, electrospinning and quench cooling of a melt7,11,14. Recently, Kogermann et al. reported that the amorphous state of PRX can be obtained by ball-milling at low temperature but the major


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limitation is that amorphous PRX is very unstable15. Further investigations on co-milling of PRX with several polymers in different ratios revealed that this approach enables to stabilize the amorphous state of PRX16. A new catalytic oxidation and acid precipitation pretreatment method for the isolation of cellulose and lignin from lignocellulosic biomass was recently introduced by Hakola and coworkers17. The present technology produces cellulose with characteristic material properties, avoids the formation of toxic compounds and offers an environmentally benign concept for the cellulose production17,18. Catalytic pretreated softwood cellulose (CPSC) with special material properties may have uses as a stabilizing carrier polymer in the SDs of poorly water-soluble APIs (for more detailed information about the material properties of CPSC see Penkina et al.18). Polyvinylpyrrolidone (PVP) is an amorphous polymer, which has been reported to be an effective carrier polymer to inhibit drug recrystallization when used in amorphous SDs19,20. PVP has been shown to be applicable in preparing and stabilizing amorphous API in the SDs obtained with different techniques including cryogenic co-grinding11,12,16,21. The aim of the present study was to investigate CPSC and PVP as carrier polymers in cryogenic co-grinding (amorphization) of poorly water-soluble PRX. Special attention was paid on the effects of carrier polymers on the physical solid-state stability of cryogenic co-ground SDs of PRX and polymer. In addition, the in vitro dissolution of the present cryogenic co-ground SDs was studied.

Materials and methods Materials Piroxicam anhydrous form I (PRXAH I) was purchased from Letco Medical, Inc., Decatur, AL. Isolation of CPSC from pine soft wood (Pinus sylvestris) was carried out according to the method described by Hakola et al. with a slight modification related to the pre-extraction of birch chips. PVP (Kollidon 25, BASF, Ludwigshafen, Germany) was used as a reference carrier polymer for cryogenic co-grinding. Methods Preparation of physical and cryogenic co-ground solid dispersions Binary physical mixtures (PMs) of PRXAH I and polymers (CPSC or PVP) were prepared manually by gently mixing with spatula. For preparing cryogenic co-ground SDs, CPSC was first pre-milled separately for 30 min with a laboratory-scale Retsch MM 400 Mixer Mill (Retsch GmbH, Haan, Germany) to reduce the particle size and to obtain more uniform particle size distribution close to that of PRX. Cryogenic co-grinding of PRXAH I with a carrier polymer (CPSC, PVP) was performed with the same laboratory-scale Retsch MM 400 Mixer Mill. The API–polymer ratio used in all cryogenic co-ground SDs was 1:3. The samples (1 g of PRXAH I and 3 g of polymer) were placed in a 25-ml volume stainless steel milling jar containing one 12-mm diameter stainless steel ball, and milled at 28 Hz frequency for 180 min. Milling jars were wrapped with parafilm and immersed into liquid nitrogen for 2 min prior to milling. This procedure was repeated every 15 min to provide a low temperature during milling. X-ray powder diffraction (XRPD) was used to determine the solid-state changes during cryogenic co-grinding (loss of crystallinity). Samples were taken during milling after specified time-periods: 30, 60, 90, 120, 150 and 180 min. Tests were performed at least in triplicate.

Drug Dev Ind Pharm, Early Online: 1–11

Physical stability of cryogenic co-ground solid dispersions Immediately after preparation, the cryogenic co-ground SDs were placed in desiccators over sodium silicate (silica gel) at room temperature (25 ± 2 C). The effect of short-term aging on the physical stability of samples was investigated by storing the samples at ambient room temperature and humidity conditions (25 ± 2 C/23% RH). The samples were analyzed over time at 24 h, 48 h, 72 h, 96 h, 1 week and 4 weeks. Physicochemical characterization of cryogenic co-ground solid dispersions Fourier transform infrared spectroscopy (FTIR) FTIR spectra of samples were collected with IRPrestige-21 Spectrophotometer (Schimadzu Corp., Kyoto, Japan) and Specac Golden Gate Single Reflection ATR crystal (Specac Ltd., Orpington, UK). Operating range was 4000–600 cm1. Differential scanning calorimetry (DSC) DSC thermograms were obtained using DSC 4000 (Perkin Elmer Ltd., Shelton, CT). Samples of 2.8–3.3 mg were placed in aluminium pans with pinholes in a lid and analyzed under dry nitrogen flow. Heating rate was 10 C/min and the temperature range from 30 to 208.5 C. The DSC system was calibrated for temperature and enthalpy using indium as a standard. Raman spectroscopy Data were collected using Raman spectroscopy (B&W TEK Inc., Newark, NJ) equipped with thermoelectrically cooled CCD detector (2048 64), two-fiber coaxial optic probes and a 300mW diode laser source operating at 785 nm. Spectra were recorded from 900 to 1700 cm1 with an integration time of 4 s. At an average three spectra per sample were collected. X-ray powder diffraction (XRPD) The XRPD patterns of samples were collected using Bruker D8 ˚ , operAdvance diffractometer using Cu radiation l ¼ 1.5418 A ated at 40 kV and 40 mA. Data were collected in Theta–Theta geometry in the range of 5 –30 2, with the step size of 0.01 2 using LynxEye positive sensitive detector. Diffraction patterns were analyzed in Topas software using (1) full pattern Rietveld modeling of PRX and cellulose structures, and (2) peak deconvolution using Pearson VII functions. Calculation of crystallite sizes Changes in the crystallite size of materials induced by cryogenic grinding was quantified by using full pattern Rietveld modeling and measuring the full width at half maximum of diagnostic deconvulated peaks. Average crystallite size was calculated by Scherrer equation (Equation (1)): FWHM ¼

k cos

ð1Þ

where Scherrer constant k is 0.89, which varies depending on the ˚ ), shape of the crystal, l is the wavelength of radiation (1.5418 A is the width of the peak broadening at half the maximum intensity (in radians), is a Bragg angle. Scanning electron microscopy (SEM) Diameter and surface morphology of starting materials and cryogenic co-ground SDs were investigated with a high-resolution SEM (Zeiss EVO MA 15, Germany). Pure materials were

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sputter coated with gold in argon atmosphere on carbon tape and examined with vacuum under high pressure. Cryogenic co-ground SDs were analyzed in the same conditions but without coating. Contact angle measurement The wettability of starting materials and PMs was determined at ambient room temperature with a contact angle measurement system (CAM200, ver. 4.1) using a Young–Laplace method. For measuring, one standard drop of Milli-Q water was placed on the tablet surface. The tablets were prepared with an instrumented Korsch EK-0 eccentric tableting machine (Erweka Apparatebau, Germany) equipped with 9-mm flat-faced punches. The contact angle value is an average of three measurements.


Dissolution test The dissolution behavior of PMs and corresponding cryogenic coground SDs was investigated in an automated dissolution system (Termostat-Sotax AT7, Sotax AG, Switzerland) using USP paddle method. The samples (n ¼ 5–6) were analyzed at regular intervals over 8 h using an UV–VIS spectrophotometer (Ultrospec III, Biochorm Ltd., UK) at 354 nm. The standard solution was prepared by dissolving 0.04 g of PRXAH I in 20 ml of methanol. The dissolution media was 900 ml of distilled water at 37.0 ± 0.5 C. The paddle rotation speed was set at 100 rpm. All samples were tested immediately after the preparation. Three separate parallel dissolution tests were performed for each formulation at the same dissolution conditions.

Results and discussion Physical material properties PRXAH I powder consisted of white prisms with a particle size ranging from 5 to 35 mm (Figure 1A). Pre-milled CPSC exhibited

Towards improved solubility of poorly water-soluble drugs

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the particles with relatively round but irregular shape with a particle size ranging from 10 to 100 mm (Figure 1B). PVP showed almost round granular particles in the range of 10–100 mm and with a particle size close to that observed with CPSC (Figure 1C). Figure 2 illustrates the XRPD patterns of PRXAH I, pre-milled CPSC and PVP. The XRPD pattern of PRXAH I was in good agreement with the calculated theoretical patterns obtained from Cambridge Structural Database and with previously published data15,22. According to our previous study, a non-milled raw cellulose (CPSC) consists of both crystalline and amorphous regions18. The XRPD pattern of the pre-milled CPSC (Figure 2) revealed that vibrational ball milling for 30 min resulted in XRPD amorphous cellulose. Characteristic Raman and FTIR spectra of PRXAH I and PVP were in good agreement with previously published spectra of PRX AH I15,23–26 and PVP27,28. Due to an exceptionally high fluorescence, there were no specific Raman spectral peaks visually detectable in the CPSC samples. For interpreting the FTIR spectrum of CPSC, readers are referred to our previously published paper18. Solid-state changes of piroxicam during cryogenic co-grinding The XRPD analyses were performed at regular intervals in order to monitor the effects of carrier polymers on the physical solidstate changes of PRX (loss of crystallinity) during cryogenic cogrinding. The carrier polymers influenced both the degree of amorphization and stabilization of amorphous PRX during and after cryogenic co-grinding (Figure 3). With pre-milled CPSC, cryogenic co-grinding for 90 min resulted in significant reduction of peak intensities in the region of 20 –25 2, thus indicating the formation of amorphous PRX. However, a sign of very lowintensity characteristic crystalline peak of PRXAH I at 8.6 2

Figure 1. Scanning electron micrographs (SEMs) of (A) piroxicam anhydrous form I, PRXAH I; (B) catalytically pretreated softwood cellulose, CPSC (pre-milled); (C) polyvinylpyrrolidone, PVP. Magnification 1000.


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Figure 2. XRPD patterns of piroxicam anhydrous form I (PRXAH I), catalytically pretreated softwood cellulose (CPSC) (pre-milled) and polyvinylpyrrolidone (PVP). The characteristic reflections of PRXAH I are indicated with asterisks (*) and monitored in the subsequent cryogenic co-grinding experiments.

Figure 3. The effects of cryogenic co-grinding on the solid state of piroxicam anhydrous form I (PRXAH I). The carrier polymers studied were (A) premilled catalytic pretreated softwood cellulose (CPSC) and (B) polyvinylpyrrolidone (PVP). X-ray powder diffraction (XRPD) patterns from down to top: Physical mixture (PM) of PRXAH I and a carrier polymer (1:3 w/w), and the corresponding cryogenic co-ground solid dispersions (SDs) sampled at 30, 60, 90, 120, 150 and 180 min during a cryogenic co-grinding process (n ¼ 3).


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Figure 4. Scanning electron micrographs (SEMs) of cryogenic co-ground solid dispersions (SDs) of piroxicam anhydrous form I (PRXAH I) and carrier polymers. Key: (A) PRXAH I and non-milled catalytic pretreated softwood cellulose (CPSC) (1:3 w/w), (B) PRXAH I and pre-milled CPSC (1:3 w/w) and (C) PRXAH I and polyvinylpyrrolidone (PVP) (1:3 w/w). Magnifications 1000 (A, C) and 100 (B).

could be observed in XRPD pattern presumably indicating still some degree of crystallinity (Figure 3A). With PVP, cryogenic co-grinding resulted in XRPD pattern representing an amorphous halo of PRX already after 90 min suggesting faster amorphization of API compared to the corresponding co-ground SDs obtained with pre-milled CPSC (Figure 3B). When non-milled CPSC was used for preparing cryogenic coground SDs, only partially XRPD amorphous PRX was obtained after 180 min of cryogenic co-grinding (data not shown). This could be explained by the fact that the reduced particle size of CPSC increased the specific surface area of the material, hence maximizing the number of contact points and the amount of possible hydrogen bonding sites available for interaction with PRX. The observed difference in amorphization of PRX with premilled and non-milled CPSC can be due to increased amorphous part of CPSC after 30 min grinding and the reduced particle size of pre-milled CPSC. In recent studies, a correlation between the miscibility of drug/polymer and development of amorphous SDs have been reported29,30. Increase in the XRPD amorphous part of CPSC induced the miscibility and formation of a single amorphous phase of the binary mixture. The difference can also be explained by the effect of water content on the physical transformations during milling. CPSC contains residual water of approximately 4.5% (w/w), and pre-milling removes some of the residual water due to the temperature increase in the ball-milling jars on the course of a continuous applied mechanical stress. Consequently, the recrystallization facilitating effect of residual water will be partially lost during milling compared to the corresponding cryogenic mixtures containing non-milled CPSC. To sum up, the XRPD results suggest that the cryogenic co-grinding of PRX with CPSC and PVP for 180 min produces amorphous PRX (with minor crystallinity) and completely amorphous PRX, respectively. Since amorphous PRX is very

unstable, it was of our interest to further characterize the prepared cryogenic co-ground SDs and to investigate their physical solidstate stability over time. Solid-state properties and stability of cryogenic co-ground solid dispersions Particle size, shape and morphology The SEMs on the cryogenic co-ground SDs of PRX with carrier polymers (after co-grinding for 180 min) are shown in Figure 4. The cryogenic co-ground SDs of PRX with non-milled CPSC exhibited particles with an irregular shape, rough surface (partially covered with PRX) and particle size ranging from 5 to 50 mm (Figure 4A). Some larger agglomerates with a particle size up to 200 mm were also found. The cryogenic co-grinding of PRX with pre-milled CPSC resulted in large particle size variation (10–400 mm and the formation of agglomerates as shown in Figure 4B). It is evident that the formation of agglomerates was due to the small particle size CPSC and hygroscopicity of cellulose as a material31. The cryogenic coground SDs with PVP showed round particles with a size range of 1–30 mm (Figure 4C). Physical solid-state stability As shown previously, both carrier polymers (CPSC and PVP) reduced the crystallinity of a poorly water-soluble PRX, and the co-ground SDs of PRX and a carrier polymer were in XRPD amorphous state or amorphous with some degree of crystallinity immediately after preparation (Figure 3). The effects of a shortterm aging on the physical solid-state stability of cryogenic co-ground SDs are presented in Figure 5. When pre-milled CPSC was used as a carrier polymer, amorphization of PRX was


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Figure 5. The effects of short-term aging on the solid state of cryogenic co-ground solid dispersions (SDs) of piroxicam anhydrous form I (PRXAH I) and carrier polymer: (A) pre-milled catalytic pretreated softwood cellulose (CPSC) and (B) polyvinylpyrrolidone (PVP). X-ray powder diffraction (XRPD) patterns from down to top: Cryogenic co-ground SD of PRXAH I and carrier polymer (1:3 w/w) immediately after preparation, and after aging of 24 h and 1 week at controlled room temperature and humidity (25 C/23% RH) (n ¼ 3).

only temporary. As seen in Figure 5(A), the PMs of PRX and premilled CPSC prepared by cryogenic co-grinding for 180 min, showed numerous distinct crystalline reflections already after a 24-h aging at a controlled temperature and humidity conditions (25 C/RH 23%). Fast recrystallization occurred during the first 24 h of storage, since at later time-points (1 week versus 4 weeks) samples showed no differences (data not shown). Characteristic reflections of PRX appeared at a diffraction angle of 2 at 8.6 , 11.7 , 14.5 , 17.7 , 21.8 and 27.4 , being comparable to that shown in the diffractogram of pure PRXAH I (Figure 2). The intensity of reflections was the same as found with the corresponding PM prior to cryogenic co-grinding. It is evident that CPSC acts as a physical matrix, where PRX is located during cryogenic co-grinding at a particle level between cellulose microfibers. Even though, cellulose repeating unit cellobiose has eight free hydroxyl groups, which makes it possible to form strong intermolecular bonds, this unit structure did not significantly influence the interaction between PRX and CPSC.

Controversially, the XRPD patterns for the corresponding cryogenic co-ground SDs of PRX and PVP showed XRPD amorphous structure and physical stability against crystallization up to one week (Figure 5B). The molecule of PRX contains O–H group and one N–H functional group, which are able to form hydrogen bonding with a carbonyl group of PVP10, thus protecting the corresponding SDs against external factors (e.g. moisture), which can induce the recrystallization process. This molecular interaction between the API and polymer is likely to improve the stability of amorphous state in the system. Nevertheless, numerous low-intensity characteristic reflections of PRX at a diffraction angle of 2 at 8.6 and 17.7 can be detected in the diffraction patterns obtained with a 1-week aged cryogenic co-ground SD of PRX and PVP (Figure 5B). It is well known that residual water could act as a plasticizer32,33, decreasing the glass transition temperature (Tg) and inducing molecular mobility and promoting amorphous PRX recrystallization. The presence of residual absorbed water in


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CPSC structure (4.5% w/w) obviously induces the recrystallization of amorphous PRX in cryogenic co-ground SDs. The possible mechanism may be the disruption of H-bonding interactions (between –OH group of CPSC and the amide –NH group or pyridine N atom in PRXAH I) and bridging between the carrier polymer and PRX structural units. Additionally, for preventing crystallization the polymer should be molecularly miscible with the drug34,35. The presence of residual water may drive amorphous SD toward immiscibility followed by phase separation. The solid-state changes of amorphous PRX in cryogenic co-ground SDs with PVP were different. The first signs of recrystallization in the XRPD diffractograms were observed after a 1 week of storage at controlled room temperature and humidity (25 C/23% RH) (Figure 5B). Sekikawa et al. reported that molecular interaction between the drug and PVP inhibits crystal nucleus growth36. The present results suggest that pre-milled CPSC used as a carrier polymer in cryogenic co-grinding can stabilize the amorphous state of PRX only temporary, while PVP provides a longer-term stabilization through molecular interaction with PRX. The XRPD amorphous form of PRX was not obtained with cryogenic grinding of pure crystalline PRXAH I for 180 min without using neither CPSC or PVP. The Scherrer equation showed crystallinity reduction from 188.6 nm (before mechanical activation) to 29.0 nm (at the end of the process). Stability measurement showed that amorphous PRX crystallized back to the more stable form with increased crystal size to 47.6 nm within 24 h. As pointed out in previous paragraph, the XRPD amorphous PRX was obtained after 180 min of cryogenic co-grinding with PVP at low temperature. The crystallinity of cryogenic co-ground SDs of PRX and non-milled CPSC revealed crystal size variation from 1.9 to 2.7 nm. The cryogenic co-ground SD of PRX and premilled CPSC elucidated crystal size reduction from 5.5 to 1.4 nm. Average crystallite size of CPSC for whole XRPD pattern was 3.1 nm, and after 180 min of cryogenic grinding of CPSC, the crystallite sizes were reduced to 1.2 nm. Interestingly, calculations using single peak reflection (002) imparted crystallite size variation 4.0–4.8 nm and revealed no correlation between grinding time and particle size, albeit amorphous phase of CPSC increased with cryogenic grinding time. In overview, PVP showed good solid-state stabilizing properties in cryogenic co-ground SDs preventing amorphous PRX recrystallization for up to 1 week when only the first signs of crystallinity were observed (Figure 5B). On the contrary, the CPSC samples were completely crystalline (Figure 5A). A strong hypothesis is made that the improved solid-state stabilization effectiveness of PVP over CPSC is due to the differences in interaction mechanisms between PRX and PVP as compared to those between PRX and CPSC. Intermolecular interactions between piroxicam with carrier polymer Possible intermolecular interactions between PRX and carrier polymers in the solid state were further studied by FTIR and Raman spectroscopy. The results obtained with FTIR spectroscopy (Figure 6) were consistent with the XRPD results. According to the literature, the amorphization of PRXAH I with polymers can be predicted by the disappearance of –NH stretching vibration band of PRXAH I27,37. In the present study this characteristic peak was at 3327 cm1. The FTIR spectra of the respective PMs (PRXAH I and polymer) showed a distinct PRXAH I peak at 3327 cm1, thus indicating the absence of intermolecular interactions between PRX and polymers (Figure 6). With the cryogenic co-ground SDs, the disappearance of the peak suggests that intermolecular hydrogen bonding between


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PRX and CPSC was formed either by the amide (–NH) group or protonated pyridine N atom of PRX and free –OH groups of CPSC (Figure 6A). However, the amorphous PRX was unstable in the cryogenic co-ground SDs since the peak at 3327 cm1 was observed again after 24 h of storage, thus indicating the recrystallization of PRX. Most likely these intermolecular interactions were not strong enough for the stabilization of amorphous SDs and CPSC molecules preferably link with each other. FTIR spectra of the cryogenic co-ground SDs prepared with PRX and PVP showed no characteristic peak of PRXAH I indicating the hydrogen bond formation between NH group of PRX molecule and the 4N– or C ¼ O group of PVP, which matched nicely with the reported results by Tantishaiyakul et al.38. Thus, the absence of a peak at 3327 cm1 also after one week of storage indicated the improved amorphous solid-state stabilizing properties of cryogenic co-ground SDs of PRX and PVP (Figure 6B). Due to a high autofluorescence of CPSC, the analysis of both PMs and cryogenic co-ground SDs by Raman spectroscopy was very challenging. The absence of characteristic peaks of pure CPSC (because of a high fluorescence) resulted in significant peak broadening and intensity decrease in the Raman spectra of PMs and cryogenic co-ground SDs (data not shown). The Raman spectra obtained with the cryogenic co-ground SDs of PRX and PVP showed some peak shifts and decrease in intensity compared to the respective PMs spectra (Figure 7). A sharp peak of PRXAH I at 1331 cm1 changed to a broader peak. In the literature, this peak has been suggested to be related to a CH3 deformation pyridinic N1626. A number of other changes were also observed, such as decrease in intensity, peak broadening, and gradual peak shifts of PRXAH I from 1519 to 1523 cm1. Moreover, several peaks started to grow or appeared at 930, 1390 and 1461 cm1 after cryogenic co-grinding of PRX and PVP for 180 min. The present changes in Raman spectra suggest that hydrogen bonding mediated intermolecular interactions between PRX and PVP during cryogenic co-grinding. Raman spectroscopy results verified the FTIR results and showed improved stability of amorphous PRX within cryogenic co-ground SD mixtures with PVP during short-term storage. Thermal properties DSC analyses were performed to study the thermodynamic properties of cryogenic co-ground SDs and to distinguish between the amorphous and microcrystalline states (the degree of crystallinity) based on the determination of Tg and melting temperature (Tm) (Figure 8). The cryogenic co-ground SDs prepared with PRX and CPSC showed a characteristic melting endotherm (198.0 C) for PRXAH I on DSC thermogram, thus indicating that the amorphization and stabilizing effect of CPSC as a carrier polymer was only temporary. This is also consistent with the XRPD, FTIR and Raman spectroscopy data showing recrystallization. The cryogenic co-ground SDs prepared with PRXAH I and PVP exhibited a broad dehydration endotherm (62–81 C) obviously due to the removal of the absorbed water from the sample, which has been also reported by Shakhtshneider et al.11. The specific melting point for PRXAH I was not observed suggesting complete amorphization (not microcrystallization) of the API (Figure 8). Dissolution in vitro The effects of cryogenic co-grinding and carrier polymer on the dissolution behavior of poorly water-soluble PRX were studied in purified water. The in vitro dissolution of cryogenic co-ground SDs was compared to that obtained with pure API and the corresponding PMs (Figure 9). Amorphous state or almost amorphous (vague residual crystallinity) of PRX in the cryogenic


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Figure 6. FTIR spectra of physical mixture (PM) (1:3) and cryogenic co-ground solid dispersions (SDs) (1:3) of piroxicam anhydrous form I (PRXAH I) and carrier polymer: (A) pre-milled catalytic pretreated softwood cellulose (CPSC) and (B) polyvinylpyrrolidone (PVP). The cryogenic co-ground SDs were analyzed immediately after preparation, at 24 h and at one week of storage at controlled room temperature and humidity (25 C/23% RH) (n ¼ 3).

co-ground SDs was verified by means of XRPD immediately after preparation and before dissolution tests. According to the literature, the solution-mediated transformations (recrystallization) may occur in amorphous SD while being in contact with dissolution media32,39,40. With PMs, the presence of carrier polymer (CPSC or PVP) increased the dissolution rate of PRX compared to the dissolution rate of pure API (Figure 9). Interestingly, the dissolution behavior of PRXAH I from the PMs did not depend on the carrier polymer used. The enhanced dissolution of PRXAH I from the PMs was obviously due to the surface covering of polymer particles with more fine API particles, thereby increasing the effective surface area that is in contact with a dissolution medium. According to Brouwers et al., hydrophilic polymers can enhance the surface area of a powder mixture available for dissolution, and thus wettability41. Moreover, the carrier polymers can inhibit the transformation of PRXAH I to a less-soluble PRX monohydrate (PRXMH) during dissolution testing, and consequently, increase the dissolution rate of PRX. Recently, the solid-state

transformation of PRXAH I to PRXMH during the dissolution test was verified (quantified) by means of Raman spectroscopy42. Furthermore, the dissolution-mediated solid-state changes of PRXAH I were monitored in the presence of surfactant (sodium lauryl sulphate, SLS), and it was found that SLS can also delay the conversion of PRXAH I to PRXMH42. Since hydrophilic polymers can enhance the wettability of the particles in powder mixtures41, the contact angle of the present carrier polymers and PMs (both compressed into tablets) was measured in order to investigate the wettability of the mixtures (Table A1 shown as an Appendix). All samples tested showed good wettability (590 ). In case of pure materials, pre-milled CPSC exhibited the lowest contact angle of 28.0 , and the highest contact angle value was obtained for pure PVP (53.9 ). The contact angle values for the corresponding 1:3 (w/w) PMs were 31.2 and 49.5 , respectively. Both PMs showed good wettability, thus enhancing the dissolution rate of PRXAH I compared to that obtained with pure PRXAH I. The present results are in agreement with the previous studies showing faster dissolution


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Figure 7. Raman spectra of physical mixture (PM) (1:3) and cryogenic co-ground solid dispersion (SD) (1:3) of piroxicam anhydrous form I (PRXAH I) and polyvinylpyrrolidone (PVP). The cryogenic co-ground SD was analyzed immediately after preparation, at 24 h and at one week of storage at controlled room temperature and humidity (25 C/23% RH) (n ¼ 3).

Figure 8. Differential scanning calorimetry (DSC) thermograms of pure materials and cryogenic co-ground solid dispersions (SDs) of piroxicam anhydrous form I (PRXAH I) and carrier polymers. Key: (A) PRXAH I, (B) pre-milled catalytic pretreated softwood cellulose (CPSC), (C) polyvinylpyrrolidone (PVP), (D) cryogenic co-ground SD (1:3) of PRXAH I and pre-milled CPSC and (E) cryogenic co-ground SD (1:3) of PRXAH I and PVP. The cryogenic co-ground SDs were analyzed immediately after preparation (n ¼ 3).

rate of PRX in powder mixtures (PVP K-30) compared to that obtained with pure PRX, and this was suggested to be related to the improved wetting of PRX by lowering surface tension38. The cryogenic co-ground SDs prepared from PVP enhanced the dissolution rate of PRX, while the use of CPSC as a carrier polymer resulted in the sustained release behavior of PRX (Figure 9). The complete release of the PRX from the SDs prepared with CPSC was accomplished within 8 h (data not shown). With the cryogenic co-ground SDs of PRX and PVP, the complete dissolution of PRX in purified water was achieved within 10 min. Kogermann et al. reported that PVP can form the diffusion layer around drug particles during dissolution, thus making the dissolution medium more readily penetrating into particles and dissolving API16.

As seen in Figure 9, the dissolution rate (and the total amount of PRX dissolved) was significantly decreased when CPSC was used as a carrier polymer in cryogenic co-ground SDs. This sustained-release dissolution behavior may be explained by the recrystallization of amorphous PRX after preparation of cryogenic co-ground SDs followed by hydrate formation during dissolution (some vague XRPD crystalline reflections were still observed after preparation of cryogenic co-ground SDs). Obviously, the slow drug release was also attributed to the presence of agglomerates/clumps (confirmed by SEM) in the cryogenic co-ground SDs of PRX and pre-milled CPSC, and subsequent phase separation. Even though CPSC is a hydrophobic polymer, it contains a relatively high amount of residual water (4.5% w/w). By pre-milling the amount of water in the polymer


10

A. Penkina et al.


Figure 9. Dissolution profiles (n ¼ 5–6) of physical mixtures (PMs) and cryogenic co-ground solid dispersions (SDs) of piroxicam anhydrous form I (PRXAH I) and carrier polymer. Key: () PRXAH I; (D) PM (1:3) of PRXAH I and pre-milled catalytic pretreated softwood cellulose (CPSC); (œ) PM (1:3) of PRXAH I and polyvinylpyrrolidone (PVP); (m) cryogenic co-ground SD (1:3) of PRXAH I and pre-milled CPSC and (g) cryogenic co-ground SD (1:3) of PRXAH I and PVP (n ¼ 3).

can be reduced, but it seems that still the presence of water evidently induces the separation of cryogenic co-ground SD into amorphous drug-rich (PRX) and polymer-rich (CPSC) domains followed by recrystallization of amorphous PRX. According to the literature, the disruption of drug–polymer interactions in amorphous SDs can be induced by absorbed water, and thereby the formation of drug–drug interactions leading to phase separation is evident34.

Acknowledgements

Conclusions

The authors report no declarations of interest. This research was supported by the European Social Fund’s Doctoral Studies and Internationalisation Programme DoRa and by NordForsk. This work is part of the ETF grant project no. ETF7980 and IUT-34-18 project. The Estonian Ministry of Education and Research is acknowledged for their financial support.

The solid-state stability and dissolution of cryogenic co-ground SDs of API are greatly dependent on the carrier polymer used. With a novel cellulosic polymer (CPSC), the induction of cryogenic co-ground amorphous state and subsequent stabilization of PRX against crystallization are only limited and temporary. CPSC seems to act as a physical matrix, where PRX is located at a particle level between cellulose microfibers. The results suggest that the presence of residual water in CPSC affects the solid-state stability and dissolution of PRX in cryogenic coground SDs. Therefore, the moisture content of cellulose-based carrier polymer(s) is crucial to be controlled in formulating such SDs for poorly water-soluble APIs. PVP is an efficient solid-state stabilizing carrier polymer for amorphous PRX obtained by cryogenic co-grinding. The cryogenic co-ground SDs prepared from PVP enhance the dissolution rate of PRX, while the corresponding co-ground SDs prepared from CPSC result in sustained release behavior. Therefore, cryogenic co-grinding provides a versatile method for preparing both immediate-release and modified-release co-ground SDs for poorly watersoluble APIs.

Mr. K. Kirsimaë (Institute of Ecology and Earth Sciences, University of Tartu) is gratefully acknowledged for performing the crystallite size calculations. Mr. L. Joosu (Institute of Ecology and Earth Sciences, University of Tartu) is gratefully acknowledged for performing SEM experiments.

Declaration of interest

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Appendix

Table A1. Contact angles of pure materials and PMs pressed into the tablets. Sample PRXAH I PVP CPSC (pre-milled) CPSC (non-milled) PRX:PVP 1:3 PM PRX:CPSC 1:3 (pre-milled)

11

Contact angle () 61.7 53.9 28.0 31.3 49.5 31.2

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