Study of the Preparation of Amorphous Itraconazole Formulations

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Study of the preparation of amorphous Itraconazole formulations María P. Fernández-Ronco, Matteo Salvalaglio, Johannes Kluge, and Marco Mazzotti Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/cg501892j • Publication Date (Web): 13 Apr 2015 Downloaded from http://pubs.acs.org on April 20, 2015

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Crystal Growth & Design

Study of the preparation of amorphous Itraconazole formulations Mar´ıa P. Fernández-Ronco,∗,†,¶ Matteo Salvalaglio,†,‡ Johannes Kluge,†,§ and Marco Mazzotti† ETH Zurich, Institute of Process Engineering, Sonneggstrasse 3, 8092 Zurich, Switzerland, and Facolt` a di Informatica, Istituto di Scienze Computazionali Universit` a della Svizzera Italiana, 6900 Lugano, Switzerland E-mail: [email protected] Phone: +41 44 632 22 45. Fax: +41 44 632 11 41

Abstract The formulation of poorly water-soluble Active Pharmaceutical Ingredients (APIs) has been widely studied in the last decades to overcome the limited bioavailability imposed by these pharmaceutical ingredients. In this work, supercritical fluid extraction of emulsions has been applied using two different types of excipients, i.e. polyvinyl alcohol (PVA) and glycyrrhizic acid (GA) to address the precipitation of itraconazole (ITZ) as an amorphous solid. The delivery profiles of the in-vitro dissolution tests of the ITZ-GA particles confirmed the advantage of the manufactured amorphous material in terms of apparent solubility in comparison to the raw itraconazole. Furthermore, the stability of an amorphous glassy product and its potential to recrystallize ∗

To whom correspondence should be addressed ETH Zurich–Institute of Process Engineering ‡ ETH Zurich–Istituto di Scienze Computazionali ¶ Current address: EMPA, Laboratory for Advanced Fibers, Lerchenfeldstrasse 5, 9014 St. Gallen, Switzerland. E-mail: [email protected] § Current address: Novartis Pharma AG, 4056 Basel, Switzerland. †

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in the presence of water have been experimentally determined using both excipients and interpreted applying molecular dynamics (MD) simulations. Results manifest that recrystallization of the amorphous ITZ particles starts at their surface, corroborating that the better adsorption of GA into the surface of the particles inhibits the crystallization and provides long term stability products. This study shows the importance of the selection of the excipient in the preparation of amorphous formulations of API’s, as well as their effect on the stability to recrystallize over storage or dosing in the presence of water.


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Introduction

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The formulation of poorly water-soluble active pharmaceutical ingredients (APIs) exhibiting

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low and erratic bioavailability represents a major challenge in pharmaceutical development.

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In fact, about 30% of approved drugs, as compared to 40 - 70% of new chemical entities

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(NCEs) currently under development, belong to this category.

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Classical formulation development has worked out a number of strategies to overcome the

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issue of limited bioavailability, including the reduction of API particle size, the modification

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of API solid form, i.e. the formation of polymorphs as well as of salts and co-crystals, and

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the association of the API with a well-soluble carrier (e.g. the formation of drug-polymer

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solid dispersions, cyclodextrin inclusion complexes and self-emulsifying systems). Some of

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these approaches involve a change of the physical state of the drug, namely from the crys-

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talline to a high-energy amorphous state, which promises an enhancement of bioavailability

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by increasing both the apparent solubility and the rate of dissolution of the API 1 . However,

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the main drawback of amorphous formulations, as solid dosage forms and/or as suspen-

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sions, lies in their inherent physical metastability and the associated risk of uncontrolled

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API recrystallization during their preparation and storage as well as during dissolution/re-

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suspension 2,3 . This means that such formulations may reach the market only if the time scale

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of recrystallization is large enough that sufficient stability of the pharmaceutical product can

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be guaranteed 4 . In many cases, this is only feasible if stabilizing excipients are added and

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adequate storage conditions are defined.

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The formation of amorphous particles by supercritical CO2 (scCO2 ) is a common and

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widely used practice in the pharmaceutical literature. Several are the publications dealing

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with the use of precipitation by compressed antisolvent (PCA) technique, where the scCO2

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acts as an antisolvent agent of the API, generating high supersaturations which usually lead

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to the precipitation of solid amorphous particles 5,6 . Morevover, the formation of particles

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through emulsions, combined with the use of supercritical CO2 as solvent extracting agent, is

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well-established in the literature, particularly in the formulation of amorphous solids such as 3 ACS Paragon Plus Environment


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polymeric materials and drug-polymer composites 7,8 . This Supercritical Fluid Extraction of

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Emulsions (SFEE) process should be applicable also in the formulation of sparingly soluble

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APIs, aiming at an aqueous suspension of submicron and amorphous API particles that is

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both highly supersaturated and finely dispersed, so as to maximize bioavailability. However,

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since also the recrystallization of API from supersaturated amorphous suspensions has been

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observed 9,10 , adequate stabilization of such suspensions remains a challenge. Recent studies

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have also shown the role that the particle’s surface plays in this context. For example, in

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the recrystallization of phenanthrene from emulsions 10 , it has been observed that this pro-

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cess is triggered by surface contact of amorphous particles with already existing crystals.

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In another study concerning the recrystallization of celecoxib from amorphous particles 11 ,

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it was concluded that crystallization of celecoxib mostly originates on the particle surface

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exposed to the aqueous medium, where it can be successfully inhibited by irreversibly ad-

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sorbing ammonium glycyrrhizinate molecules from the surrounding solution. Ammonium

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glycyrrhizinate, the excipient used in that paper, is also investigated in the present study.

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With an extremely low aqueous solubility of 98%, Fig 1a), was purchased from TCI Chemicals (Tokio, Japan). Both excip-

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ients glycyrrhizic acid ammonium salt (GA) and polyvinyl alcohol Mowiol 4-88 (PVA), whose

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molecular structures can be compared in Fig. 1b and c, as well as HPLC grade acetonitrile

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(ACN), triethylamine (TEA), and hydrochloric acid (37%), were supplied by Sigma Aldrich

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(Buchs, Switzerland). Sodium chloride for simulated gastric fluid and dichloromethane

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(DCM), employed in emulsion preparation, were obtained from Merck (Darmstadt, Ger-

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many). CO2 of 99.9 % purity was supplied from PanGas (Dagmersellen, Switzerland).

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Preparation of glassy itraconazole

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Glassy itraconazole was prepared by melt quenching as follows: 1 g of crystalline itraconazole

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is melted by heating up above the melting temperature, i.e. 166 ◦C, in a Petri dish followed

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by a quick cooling of the molten liquid by pouring liquid N2 over it. Afterwards, the glassy

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drug is scratched and ground by mortar and pestle to produce a fine glassy ITZ powder whose

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lack of crystallinity was confirmed by XRD and DSC measurements. The final product was

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then stored in a desiccator at room temperature until further use.



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Preparation of itraconazole emulsions

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An oil in water emulsion (o/w) was prepared by pouring at room temperature the organic

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phase, consisting of a 5 wt% itraconazole in dichloromethane solution, into the water phase,

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which contains 1.2 wt% excipient, i.e. GA or PVA. The final mixture, containing both

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phases at a weight ratio of 1:4, respectively, was then mixed by ultrasonication using a

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BransonSonifier 450 (Skan AG, Basel, Switzerland). Sonication was applied at maximum

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power output while cooling the emulsion on ice and stirring with a magnetic stir bar at 500

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rpm. For 200 mL of emulsion, a sonication time of 2 minutes was applied in 4 cycles of 30

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seconds each; intermediate breaks allowed for sufficient cooling of the emulsion

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Supercritical Fluid Extraction of Emulsions (SFEE)

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Directly after the preparation of the emulsion, the organic solvent was extracted from the

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emulsion by SFEE, a process where supercritical CO2 acts as an extracting solvent agent of

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the solvent contained in the emulsion droplets, i.e. DCM in this case, so as to produce solid

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particles of the API. A scheme of the experimental setup used for SFEE experiments and a

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more detailed description of the process is provided in a previous work 8 . Briefly, both CO2

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and emulsion are delivered from their storage vessels at continuous flowrates of 80 g/min

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and 2 mL/min, respectively. The two streams are mixed in a coaxial nozzle located at the

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entrance of a high pressure vessel, i.e. the emulsion stream is finely dispersed by the much

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larger stream of scCO2 , thus creating conditions where DCM is extracted selectively and

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efficiently from the droplets of the organic phase. Throughout the experiment, the vessel

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is kept at constant pressure and temperature, i.e. 80 bar and 45 ◦C, by means of a back-

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pressure regulator and a thermostat, respectively. While the stream of supercritical CO2

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leaves the reactor together with the organic solvent through an outlet located at the top of

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the vessel, a solvent free aqueous suspension of itraconazole particles is recovered through a

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separate outlet at the bottom of the reactor. The product suspension is then stored at room

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temperature for specific time intervals, tlag , prior to its post-processing. 6 ACS Paragon Plus Environment

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Post-processing of SFEE product suspensions

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For sample characterization, solid particles were recovered from the aqueous suspension

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and separated from the free surfactant remaining in solution by means of centrifugation

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and freeze-drying. After centrifugation at 20,000 rpm for 15 min (Avanti J-20, Beckman,

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USA) the supernatant was discarded. Sedimented particles were re-suspended in 2 mL of

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cold deionized water and frozen immediately by liquid N2 . Subsequently, freeze-drying was

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carried out in a FlexiDry equipment (FTS Systems, USA) for 24 hours, and the collected

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dried particles, containing less than 50 ppm of DCM, were stored at room temperature in a

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desiccator for further characterization.

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Precipitation by compressed antisolvent (PCA)

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The precipitation of itraconazole by PCA was carried out mixing a stream of CO2 , which is

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drawn from a dip tube cylinder and precooled in a pressure module (PM101, NWA GmbH,

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Lörrach, Germany) before being delivered to the reactor by a piston pump (PP200, Thar

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Design Inc., Pittsburgh, USA), and the DCM solution containing the ITZ, which is delivered

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by an HPLC pump (PU2080 plus, Jasco, Easton, USA). Both the CO2 and the solution feed

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stream are mixed by a two substance nozzle (Schlick, Untersiemau, Germany) at the inlet

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of the reactor (Premex, Lengnau, Switzerland). The streams are brought to the operating

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temperature by a thermostat (CC230, Huber, Offenburg, Germany). The solid particles

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precipitated are retained within the reactor by a filter bag made of Teflon tissue, and two

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sintered metal filters are placed at the outlet of the reactor in order to remove all particles

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from the off-gas stream. A more in detail description of the process and of the experimental

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setup employed can be found elsewhere 8,14 .



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Characterization of Itraconazole content

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For particles, a known amount of the freeze-dried powder was dissolved in DMSO, and the

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solution was filtered using a PTFE 0.22 µm syringe filter and loaded into HPLC vials. The

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itraconazole content of the resulting solution was determined by high-performance liquid

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chromatography on an Agilent 1100 (Agilent Technologies, USA) with a DAD detector.

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Itraconazole was detected at 263 nm using as eluent acetonitrile:water (70:30 v/v) + 0.05%

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TEA with a flow rate of 1 mL. At these conditions, and using a GROMSIL 100 ODS-2 FE

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column (125 × 4.6 mm), the peak of ITZ eluted at 3.2 min. Using the same chromatographic

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method and static phase, the ITZ concentration of the aqueous samples collected from in-

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vitro tests was also determined.

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SEM, DSC and XRD analysis

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Scanning electron microscopy (SEM) images of samples, previously sputter-coated with 5 nm

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platinum, were carried out in a Zeiss Gemini 1530 FEG scanning electron microscopy to

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analyse their morphology. Differential scanning calorimetry (DSC) measurements were con-

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ducted by using a Mettler Toledo DSC 822e. Indium was used for calibration. Accurately

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weighed samples (5 mg to 10 mg) were placed in hermetically sealed aluminium crucibles and

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scanned from 20 ◦C to 220 ◦C at a rate of 5 ◦C/min under nitrogen purge. To determine the

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degree of crystallinity of different products, X-ray diffraction (XRD) patterns were obtained

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on a Bruker AXS D8 Advance diffractometer with Cu Kα radiation (λ = 1.5406 ) and a

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voltage and current applied of 40 kV and 30 mA, respectively. The crystallinity of samples

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was calculated by minimizing the square error of the linear regression of the XRD patterns

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in the 2θ range of 6◦ to 60◦ , namely raw ITZ (100 % crystalline), excipient, and glassy ITZ

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(100% amorphous), whose summation constitute the pattern of the SFEE product.


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In-vitro drug dissolution test

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In-vitro itraconazole dissolution data was obtained by means of a dialysis method 15 as fol-

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lows: 5 mg of the corresponding ITZ product were mixed with 10 mL of simulated gastric

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fluid without pepsin 16 and placed inside a semipermeable regenerated cellulose dialysis tube

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(length 10 cm, ID = 4.5 cm, CO = 14,000), which was closed at both ends with a clamp to

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form a tight bag and to avoid any contamination. Then, the bag was immersed in 250 mL

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of the dissolution medium, i.e. simulated gastric fluid, and kept at 37 ◦C in a shaking water

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bath at a speed of 60 rpm. Aliquots of 5 mL were withdrawn from the dissolution medium at

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specific time intervals and replaced by the same volume of fresh simulated gastric fluid. After

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filtration with a 0.22 µm PTFE filter, the ITZ content of the samples was directly analysed

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by HPLC according to the method previously described. Experiments were replicated twice

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and the average values reported.

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Molecular Dynamics simulations

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For the investigation of the surface recrystallization in water, a small amorphous particle

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of ITZ was simulated using classical molecular dynamics (MD). The initial structure was

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prepared using the open source software packmol 17 in order to randomly arrange 230 ITZ

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molecules in a spherical aggregate characterized by a radius of approximately 4 nm. Such a

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structure has been solvated in a box of TIP3P 18 water molecules resulting in a symulation box

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composed of 72,618 atoms. The system was then relaxed with a 2,000 cycles minimization.

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Temperature and pressure were then equilibrated with a 1 ns simulated annealing and a

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subsequent 5 ns NPT calculation, respectively. A NVT production run of 300 ns was then

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performed in order to characterize the dynamic evolution of the ensemble of ITZ molecules.

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In order to investigate the excipient interaction with the ITZ surface, a small slab composed

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of 40 ITZ molecules has been prepared and solvated with TIP3P water molecules following

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the same protocol used in the preparation of the particle model previously described. The

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simulation boxes resulted approximately in 10,000 atoms per simulation. As a potential 9 ACS Paragon Plus Environment


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energy function, the Generalized Amber Force Field (GAFF) 19,20 was chosen. The ITZ

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and GA structures were obtained from their crystal structure and optimized with Density

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Functional Theory (DFT) calculations performed at the B3LYP/6-31G(d,p) level 21 . The

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structure of the PVA oligomer has been constructed by replicating the monomer structure

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optimized within a small chain of three monomer units. The partial atomic charges of ITZ,

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GA and PVA were assigned using the restrained electrostatic potential formalism 22 based

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on the electrostatic potential computed with DFT at the B3LYP/6-31G(d,p) level. All DFT

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calculations were carried out using Gaussian 09 23 . In the MD simulations, we applied three-

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dimensional periodic boundary conditions. Long range electrostatics were treated with the

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Particle Mesh Ewald (PME) approach and a non-bonded cut-off of 1.0 nm was used 24 . The

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shake algorithm 25 was used to constrain the length of hydrogen bonds, thus enabling a 2 fs

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time step. The MD simulations were performed with the AMBER 12 suite of programs 26 .

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Minimization was performed with the sander MD engine, while the equilibration and the

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production runs were performed using pmemd engine on a GPU workstation 24 .

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Results and Discussion

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A first set of experiments addressed the feasibility and reproducibility of SFEE-based process

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as means to manufacture suspensions of amorphous itraconazole particles using two different

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excipients at otherwise identical operating conditions. The expected enhanced solubility

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of the manufactured amorphous particles in comparison to the crystalline raw API was

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confirmed by conducting in-vitro dissolution tests. In a second part of the study, the effect

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of the excipients in the stabilization of aqueous suspensions of amorphous pure glassy ITZ

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was evaluated experimentally and rationalized by means of molecular dynamics simulations.


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Supercritical processing for Itraconazole amorphization

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To investigate the potential of different supercritical CO2 based technologies to produce

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amorphous itraconazole products, three different experiments were carried out (Table 1).

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In Run 1, the precipitation of raw itraconazole as a solid powder from a dichloromethane

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solution was evaluated by compressed antisolvent (PCA) technique using experimental con-

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ditions based on earlier studies 27 . Runs 2 and 3 addressed the use of supercritical CO2

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as solvent extracting agent of an emulsion containing the low-water soluble itraconazole by

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SFEE technique. This process leads to an aqueous suspension of formulated particles, i.e.

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ITZ-excipient, that might be post-processed to obtain a solid product. Both Runs 2 and 3

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were conducted at identical operating conditions, while the excipient used as surfactant in

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the emulsion preparation changed from PVA in Run 2 to GA in Run 3.

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Figures 2 and 3 show representative SEM images of the solid powders recovered from all

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experimental runs. As it can be observed in Figure 2, the product collected after Run 1 con-

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sists of rather big crystalline particles, whereas Runs 2 and 3 yield both ITZ particles much

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smaller in size than those of Run 1, varying from flake like morphologies when PVA was

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used as excipient to spherical microparticles using GA. Besides, the degree of crystallinity of

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these products (fig. 4) shows that contrary to what happened using PCA (crystallinity ca.

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100%), the products recovered from SFEE processing were amorphous or semi-crystalline

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depending on the excipient.

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It is important to notice that the fundamental difference between PCA and SFEE is the

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precipitation mechanism. Whereas in PCA the precipitation of the particles starts in a ho-

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mogeneous solution, therefore the size of particles depends on the supersaturation during

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mixing and on the nucleation and growth kinetics, in SFEE technique the nucleation and

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growth are confined within the droplets of the emulsion. As a result, the particle size of the

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final product is typically smaller than that obtained by PCA 7 , as it was confirmed in our

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experiments. Moreover, PCA is certainly the technique that generates higher supersatura-

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tion compared to SFEE, therefore, the precipitation of amorphous ITZ particles would be 11 ACS Paragon Plus Environment


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expected at the conditions described for Run 1, i.e. 100 bar and 40 ◦C, which characterize

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a system above the critical point of the binary CO2 -DCM mixture at this temperature (81

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bar) 28 . However, it seems obvious at the light of our results for Run 1 that not only high

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supersaturation is required to produce amorphous ITZ particles, but also a good stabiliza-

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tion of these amorphous particles, once formed, is an important requirement that has to be

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taken into account.

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The results from Runs 2 and 3 seem to indicate that the use of excipients might promote

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this stabilization, but its selection is a key parameter for a successful effect. For example,

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considering the products recovered at different tlag , i.e. the time lag between the collection

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of the suspension product after the SFEE processing and its post-processing, this time can

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offer a way to compare the stability of the manufactured suspension over time, hence the

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stabilization effect of the excipient. Figure 4 shows that the product precipitated in Run 3

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using GA as excipient is amorphous and stable over time. Besides, it contains 19wt.% of

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GA according to the HPLC measurements. On the contrary, the collected ITZ-PVA semi-

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crystalline particles, which contain 3wt.% of PVA, increased their degree of crystallinity

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from 63% to 75% in 20 hours, thus indicating a recrystallization effect during this time, and

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therefore the lack of stability of such ITZ formulation.

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Analysing in detail the morphology of the ITZ-PVA particles for a tlag of 0 hours, some

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spheres associated to amorphous state are still visible (see insert in fig. 3d) which exhibit a

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rougher surface than that of the spheres obtained using GA as excipient. Coupling this ob-

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servation with the appearance of some rougher particles at high tlag when employing GA (see

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insert in fig. 3c) and with the conclusions reported by other authors who observed crystal-

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lization triggered at the surface of the particles 10,11 , both facts suggest that recrystallization

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of the amorphous material starts indeed at the surface. This preliminary hypothesis about

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the recrystallization mechanism in an amorphous particle will be analysed later in detail.


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In-vitro dissolution tests

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In order to evaluate whether the apparent solubility of the amorphous ITZ-GA product man-

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ufactured by SFEE is higher than that of the crystalline raw API, in-vitro drug dissolution

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tests were conducted using both solid materials.

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It is expected that the apparent solubility of ITZ-GA experiences a significant increase

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due to the combined effect of the reduction in particle size, amorphization and association

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of the API with the excipient. In fact, Sun et al. 29 reported an increase of five-fold in the

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percentage of itraconazole dissolved by reducing the average particle size of the tested product

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from 5.5 µm to 300 nm. Barret et al. 6 analysed the effect of Polyethylene glycol (PEG) as

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excipient to assist the precipitation of itraconazole, concluding that for itraconazole-PEG

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(50:50) particles, the percentage of itraconazole dissolved in the dissolution test was 2.7 times

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higher after 100 minutes; and the amorphous state of APIs is well-known to significantly

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promote an increase in the apparent solubility of the drug 30 . In particular for pure ITZ, the

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supersaturation at 37 ◦C has been theoretically calculated and established between 95 and

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125 31 .

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Figure 5 shows the evolution in the delivery of ITZ-GA in comparison to raw ITZ, where

262

bars indicate the deviation of two experimental measurements and straight lines guide for

263

the eyes. Results highlight the higher apparent solubility of ITZ-GA product that reaches

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concentrations of 6.42 ppm after 1,600 minutes in comparison to 0.66 ppm of the crystalline

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ITZ over the same delivery period. Due to the lack of experimental data points in the interval

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between 200 - 1,000 minutes, the kinetic evaluation of the global curve is not possible.

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Nevertheless, the initial linear part of the curves, i.e. from zero to ca. 60 minutes, can

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be used to evaluate the release of the API to the medium as a function of the solubility

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of the API 32 . Consequently, the gradient in the API concentration represents the driving

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force in the mass transfer process in which the external mass-transfer coefficient (kext ) is the

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most characteristic parameter. Calculations for both amorphous ITZ-GA and crystalline

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raw ITZ revealed that the kext does not differ significantly, namely 0.245 h−1 and 0.312 h−1 13 ACS Paragon Plus Environment


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respectively, indicating that the dissolution rate of the API is rather equal for both products

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even though the apparent solubility differs almost ten times. This increase in the apparent

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solubility highlights in fact the enhancement in the bioavailability of the amorphous ITZ-GA

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product, which moreover remains stable without crystallizing from solution over the delivery

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period.

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Stability and recrystallization of amorphous glassy ITZ

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As well-known, amorphous solids are by definition thermodynamically metastable, i.e. they

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require an extra effort to keep their stability and therefore to exploit the promised enhanced

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solubility of the API’s formulated in that way. To determine how recrystallization happens

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in an amorphous itraconazole particle, glassy ITZ prepared as described in the experimental

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section, which has been shown to be stable for more than six months (data not shown), was

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redispersed in deionised water containing different amounts and type of excipients, namely

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GA and PVA, for 96 hours. After this time, the suspension was filtered and the recovered

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solids analysed by XRD. During the whole duration of the experiment, temperature was

287

kept constant at 25 ◦C and the stirring speed was set at 500 rpm.

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Figure 6 presents all XRD patterns of the products obtained using as excipients both GA

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(fig. 6a) and PVA (fig. 6b). From these results, it is evident that whereas with no excipient

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recrystallization of glassy ITZ occurs, the selection of the right excipient and its addition into

291

the suspension, even at low concentrations, allows inhibiting the recrystallization. In fact,

292

comparing the patterns obtained using both excipients, and as expected from our previous

293

results, PVA is not capable to avoid the recrystallization of the glassy ITZ, even at a 1:1

294

ratio of glassy ITZ to excipient. On the other hand, ratios of 1:0.05 are sufficient to prevent

295

the recrystallization of the amorphous solid when using GA as excipient. In the latter case,

296

(see fig. 6a) the increase in the amount of excipient up to ratios 1:1 yields a second halo

297

in the XRD pattern, which falls within the 2θ angle range representative of pure GA. This

298

might be explained by the excess of GA that does not adsorb on the surface of the glassy 14 ACS Paragon Plus Environment

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299


ITZ and therefore remains free in solution.

300

In order to further validate the experimental observations and to gain further insight

301

into the recrystallization mechanisms, we performed molecular dynamics (MD) simulations

302

with two aims: providing a deeper understanding of the surface-initiated recrystallization

303

of amorphous glassy ITZ particles, and developing a rationale for the differences observed

304

experimentally in the stabilization effects of GA and PVA.

305

Surface recrystallization To address the first issue, we have performed a MD simulation

306

of a small amorphous particle with full atomistic detail (fig. 7a).

307

The experiments show that the recrystallization of amorphous ITZ nanoparticles at ambient

308

pressure occurs over rather long timescales, rendering a direct investigation of the crystal-

309

lization mechanism in the nanoparticle through MD practically unattainable. We observe

310

in fact, that in the amorphous phase at ambient temperature, the ITZ molecules tend to

311

form an entangled structure in which both diffusion and configurational rearrangements are

312

extremely slow, as typically observed in glassy systems 33,34 . Nevertheless, MD simulations

313

provide a molecular level description of the dynamics of ITZ molecules in the amorphous

314

state. In particular we can follow the dynamics of individual ITZ molecules within the

315

amorphous phase, thus computing their Mean Square Displacement as an index of their

316

propensity to move. The Einstein equation establishes a relationship between the diffusion

317

coefficient (D) and the Mean Square Displacement (MSD), namely D = MSD/(6t), that

318

holds rigorously in the long time limit 35 .

319

From our simulations, we observe that the diffusion coefficient is not constant within the

320

amorphous particle. In fact, it shows a rather marked dependence on the position within

321

the particle. As shown in Fig. 7c, molecules on the surface, characterized by a high solvent

322

exposure (right hand side of the figure), exhibit larger values of the diffusion coefficient

323

compared to molecules that are buried within the particle (low solvent exposure, left hand

324

side of the figure). ITZ molecules located on the surface of the particle have D values up to



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325

one order of magnitude larger than those in the bulk. This observation is in agreement with

326

previous results reported by Zhu et al., where they observed surface diffusion faster than

327

bulk diffusion for indomethacin 36 .

328

Since recrystallization, i.e. the transition from amorphous state to crystalline state, im-

329

plies a reorganization of the ITZ molecules towards a higher level of order, molecular mobility

330

is a prerequisite for such a reorganization to occur. As a consequence, the key molecules that

331

trigger recrystallization are those that have the highest mobility, i.e. those on the surface of

332

the amorphous particles, as to the MD simulations have shown. Under the hypothesis that

333

there is a direct relationship between mobility, i.e. D, and recrystallization rate, we estimate

334

recrystallization rates at least one order of magnitude larger on the surface than in the bulk

335

of the particle. These results support the experimental observation of a recrystallization

336

process triggered at the particle surface. Adding an excipient that provides a coating for

337

the particle and avoids the direct exposure of ITZ molecules at the particle/solvent interface

338

results in a key factor towards obtaining a substantial slowing-down of the recrystallization

339

process.

340

ITZ-excipient interactions In order to investigate the interactions of the GA and PVA

341

excipients with the amorphous ITZ surface, we have simulated the adsorption of the excipient

342

on the surface of an amorphous ITZ particle, as well as its behaviour when dispersed in

343

aqueous solution. The model of the ITZ surface has been prepared by relaxing 40 ITZ

344

molecules randomly packed in a planar slab geometry. A single GA molecule and a PVA

345

oligomer composed of 20 monomeric units have been prepared in solution and allowed to

346

spontaneously adsorb on the surface, thus adopting a stable interacting configuration. The

347

adsorbed configurations of both GA and PVA excipients have been simulated for 200 ns. In

348

Fig. 8 we report two sample structures exhibiting the additive adsorbed on the surface of the

349

ITZ particle. The amorphous ITZ surface favors non polar interactions with the excipients.

350

The GA molecule has a very well defined hydrophilic head and hydrophobic tail. As shown


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351

in Fig. 8, an isolated GA molecule in solution interacts with water molecules in a localized

352

region that can be identified as the polar, hydrophilic head. A large hydrophobic tail can

353

also be identified as the molecular region that does not significantly interact with water

354

molecules. The PVA oligomer exhibits instead a different interacting pattern within the

355

water environment. The hydroxyl groups are by construction distributed along the whole

356

polymer chain, and thus polar/apolar domains cannot be clearly identified. This difference

357

in the distribution of the polar moieties is reflected in the adsorbed configuration.

358

In its adsorbed configuration, GA has the apolar tail strongly interacting with the surface,

359

while the polar head fluctuates, while interacting with the water molecules in solution. PVA

360

exhibits larger flexibility instead: while its adsorbed configuration is maintained for hundreds

361

of nanoseconds, it is subject to fluctuations associated with the formation and breakage of

362

polar interactions with the solvent. These characteristics of the interaction between the

363

excipient and the ITZ surface determine a tighter binding of the GA on the surface, that

364

likely limits the surface-initiated recrystallization of ITZ amorphous particles. While PVA

365

also binds to the amorphous ITZ surface, it is characterized by much larger fluctuations both

366

in its structure and in the formation of hydrophobic interactions with the surface. These

367

observations seem to be in agreement with the recrystallization experiments and provide

368

further evidence to support the hypothesis that the recrystallization of the ITZ particles

369

is more effectively inhibited by excipient molecules that can establish stable interactions

370

with the particle surface as previous authors have suggested 3,37 , thus hindering the surface

371

reorganization that is considered to be the initial step towards ITZ recrystallization.

372

Conclusions

373

SFEE has been demonstrated as a feasible technique to manufacture totally amorphous

374

formulations of itraconazole as a function of the excipient used, whose correct selection allows

375

stable amorphous products that can therefore provide the promised enhanced solubility and



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376

bioavailability. In fact, the in-vitro delivery tests have concluded that in the case of the stable

377

amorphous ITZ-GA product manufactured by SFEE, the apparent solubility is almost ten

378

times higher than the corresponding for crystalline ITZ.

379

Molecular dynamics simulations have proved that recrystallization of the amorphous ITZ

380

particle starts at its surface in the presence of water, highlighting that the addition of suitable

381

excipients, such as GA, allows an adequate adsorption of the excipient onto the ITZ particle

382

and hinders its recrystallization.

383

The work presented here not only shows the importance in the selection of the type of ex-

384

cipient to formulate an amorphous product of a low water-soluble API, but also it highlights

385

the relevance of evaluating the stabilizing effect of the excipients over the recrystallization

386

of the pure amorphous product when it is redispersed in water, such as for formulating a

387

pharmaceutical suspension or during dissolution. Indeed, the selection of the excipient plays

388

a crucial role in the recrystallization of the glassy itraconazole, which seems to start at the

389

surface of the particle, and must be carried out based on the molecular interactions between

390

the API and the excipient. Actually, a preliminary evaluation of the interactions between

391

excipients and amorphous material might allow to gain further insight into the affinity of

392

the excipient towards the particle surface, and hence, into its capability to inhibit recrystal-

393

lization in the presence of water.

394

Acknowledgements

395

The authors thank the group of Prof. Michele Parrinello for the scientific discussions and

396

the computational resources. The corresponding author thanks Junta de Comunidades de

397

Castilla-La Mancha (Spain) for the postdoctoral grant co-financed by the European Union

398

within the framework of 2007-2013 ERDF Operational Programme.


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400

401


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(2) Matteucci, M.; Paguio, J.; Miller, M.; Williams III, R.; Johnston, K. Highly Supersat-

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urated Solutions from Dissolution of Amorphous Itraconazole Microparticles at pH 6.8.

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Mol. Pharm. 2009, 6, 375–385.

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(3) Alonzo, D.; Zhang, G.; Zhou, D.; Gao, Y.; Taylor, L. Understanding the behaviour of

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amorphous pharmaceutical systems during dissolution. Pharm. Res. 2010, 27, 608–618.

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(4) Six, K.; Verreck, G.; Peeters, J.; Augustijns, P.; Kinget, R. Int. J. Pharm. 2001, 213,

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163–173. (5) Miller, D. A.; McConville, J. T.; Yang, W.; Williams III, R. O.; McGinity, J. W. J. Pharm. Sci. 2007, 96, 361–376.

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(6) Barrett, A. M.; Dehghani, F.; Foster, N. R. Pharmaceut. Res. 2008, 25, 1274–89.

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(7) Shekunov, B. Y.; Chattopadhyay, P.; Seitzinger, J.; Huff, R. Pharmaceut. Res. 2006,

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23, 196–204. (8) Kluge, J.; Fusaro, F.; Muhrer, G.; Thakur, R.; Mazzotti, M. J. Supercrit. Fluids 2009, 48, 176–182. (9) Kluge, J.; Fusaro, F.; Mazzotti, M.; Muhrer, G. J. Supercrit. Fluids 2009, 50, 336–343.

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(10) Kluge, J.; Joss, L.; Viereck, S.; Mazzotti, M. Chem. Eng. Sci. 2012, 77, 249–258.

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(11) Margulis-Goshen, K.; Weitman, M.; Major, D. A. N. T.; Magdassi, S. J. Pharm. Sci.

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2011, 100, 4390–4400. (12) Six, K.; Leuner, C.; Dressman, J.; Verreck, G.; Peeters, J.; Blaton, N.; Augustijns, P.; Kinget, R.; Mooter, G. V. D. J. Therm. Anal. Calorim. 2002, 68, 591–601. 19 ACS Paragon Plus Environment


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(13) Six, K.; Berghmans, H.; Leuner, C.; Dressman, J.; Werde, K. V.; Mullens, J.;

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Benoist, L.; Thimon, M.; Meublat, L.; Verreck, G.; Peeters, J.; Brewster, M.; Mooter, G.

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V. D. Pharmaceut. Res. 2003, 20, 1047–1054.

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(14) Fernández-Ronco, M. P.; Kluge, J.; Krieg, J.; Rodr´ıguez-Rojo, S.; Andreatta, B.; Luginbuehl, R.; Mazzotti, M.; Sague, J. J. Supercrit. Fluids 2014, 95, 204–213.

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(15) Abdel-Mottaleb, M. M.; Lamprecht, A. Drug Dev. Ind. Pharm. 2011, 37, 178–84.

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(16) The United States Pharmacopea, U. United States PharmacopeaConvention, Inc.,

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Rockville, MD. 2005, (17) Mart´ınez, L.; Andrade, R.; Birgin, E. G.; Mart´ınez, J. M. J. Comput. Chem. 2009, 30, 2157–2164. (18) Jorgensen, W. L.; Chandrasekhar, J.; Madure, J. D.; Impery, R. W.; Klein, M. L. J. Chem. Phys. 1983, 926–935. (19) Wang, J.; Wolf, R.; Caldwell, J.; Kollman, P.; Case, D. J. Comput. Chem. 2004, 25, 1157–1174. (20) Cornell, W.; Cieplak, P.; Bayly, C.; Gould, I.; Merz, K.; Ferguson, D.; Spellmeyer, D.; Fox, T.; Caldwell, J.; Kollman, P. J. Am. Chem. Soc. 1995, 117, 5179–5197. (21) Stephens, P.; Devlin, F.; Chabalowski, C.; Frisch, M. J. Phys. Chem. 1994, 98, 11623– 11627.

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(22) Bayly, C.; Cieplak, P.; Cornell, W.; Kollman, P. J. Phys. Chem. 1993, 97, 10269–10280.

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(23) Frisch, M. J. et al. Gaussian 09 Revision A.1. Gaussian Inc. Wallingford CT 2009.

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(24) Salomon-Ferrer, R.; Gotz, A. W.; Poole, D.; Le Grand, S.; Walker, R. C. J. Chem.

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Theory Comput. 2013, 9, 3878–3888. (25) Ryckaert, J.-P.; Ciccotti, G.; Berendsen, H. J. Comput. Phys. 1977, 23, 327–341. 20 ACS Paragon Plus Environment

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(26) Case, D. A. et al. AMBER 12. 2012; http://ambermd.org/.

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(27) Fusaro, F.; Kluge, J.; Mazzotti, M.; Muhrer, G. J. Supercrit. Fluids 2009, 49, 79–92.

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(28) Gonzalez, A. V.; Tufeu, R.; Subra, P.; Inge, L.; Nord, P.; Jean, A.; Cle, B. J. Chem.

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Eng. Data 2002, 492–495.

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(29) Sun, W.; Mao, S.; Shi, Y.; Chiu Li, L.; Fang, L. Nanonization of Itraconazole by High

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Pressure Homogenization: Stabilizer Optimization and Effect of Particle Size on Oral

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Absorption. J. Pharm. Sci. 2011, 100, 3365–3373.

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(30) Yang, W.; Johnston, K.; Williams III, R. Comparison of bioavailability of amorphous

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versus crystalline itraconazole nanoparticles via pulmonary administration in rats. Eur.

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J. Pharm. Biopharm. 2010, 75, 33–41.

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(31) Matteucci, M.; Miller, M.; Williams III, R.; Johnston, K. Highly Supersaturated Solu-

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tions of Amorphous Drugs Approaching Predictions from Configurational Thermody-

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namic Properties. J. Phys. Chem. B 2008, 112, 16675–16681.

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(32) Cabezas, L. I.; Gracia, I.; Lucas, A. D.; Rodr´ıguez, J. F. Ind. Eng. Chem. Res. 2014, 40, 15374–15382. (33) Xiang, T.-X.; Anderson, B. Molecular dynamics simulation of amorphous indomethacin. Mol. Pharm. 2013, 10, 102–114.

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(34) Gupta, J.; Nunes, C.; Jonnalagadda, S. A molecular dynamics approach for predicting

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the glass transition temperature and plasticization effect in amorphous pharmaceuticals.

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Mol. Pharm. 2013, 10, 4136–4145.

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(35) Frenkel, D.; Smit, B. Understanding Molecular Simulation, 2nd ed.; Academic Press, Inc.: Orlando, FL, USA, 2001. (36) Zhu, L.; Brian, C.; Swallen, S.; Straus, P.; Ediger, M.; Yu, L. Surface Self-Diffusion of an Organic Glass. Phys. Rev. Lett. 2011, 106, 256103(1)–256103(4). 21 ACS Paragon Plus Environment


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(37) Rumondor, A.; Taylor, L. Effect of Polymer Hygroscopicity on the Phase Behaviour

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of Amorphous solid Dispersions in the Presence of Moisture. Mol. Pharm. 2010, 7,

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477–490.


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472

List of Tables

473

Table 1. Experimental conditions for PCA and SFEE runs. Solution flow refers to the flow

474

of the oil-in-water emulsion in the SFEE experiments.

475



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Page 24 of 35

Table 1: Experimental conditions for PCA and SFEE runs. Solution flow refers to the flow of the oil-in-water emulsion in the SFEE experiments.

Run #

Technique

P (bar)

T (◦C)

ITZ in oil phase (wt%)

1∗ 2∗ 3∗

PCA SFEE SFEE

100 80 80

40 45 45

1 5 5

∗

Excipient in water phase (wt%)

CO2 Flow (g/min)

Solution flow (mL/min)

– PVA (1.2) GA (1.2)

80 80 80

0.67 2 2

All the experiments represent the average of two replica runs.


Page 25 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


476

List of Figures

477

Figure 1:

478

Molecular structures of (a) itraconazole API; (b) glycyrrhizic acid (GA) excipient; (c) polyvinyl

479

alcohol (PVA) excipient.

480

Figure 2:

481

SEM images of (a) pure itraconazole as received from supplier, and (b) itraconazole product

482

obtained from PCA experiment (run 1). Scale bars indicate 100 µm.

483

Figure 3:

484

SEM images of the post-processed products collected from SFEE experiments at tlag of 0, 5

485

and 20 hours. a), b), and c) refer to the products manufactured using GA as excipient; and

486

d), e) and f) refer to those using PVA. White arrows underline the recrystallization at the

487

surface of the particles.

488

Figure 4:

489

Effect of the type of excipient in the evolution of SFEE product crystallinity as a function

490

of tlag . Bars indicate the experimental error of two replica experiments.

491

Figure 5:

492

Delivery profiles obtained from in-vitro tests of raw crystalline ITZ and the post-processed

493

SFEE product obtained using GA as excipient. Bars indicate the standard deviation.

494

Figure 6:

495

XRD patterns of the products collected after 96 hours of redispersing glassy ITZ in deionised

496

water. Percentages indicate the weight of excipient in the solid mixture. a) Glycyrrhizic acid;

497

b) Polyvinyl alcohol.

498

Figure 7:

499

MD simulations of an amorphous ITZ nanoparticle. The three parts of the figure provide

500

different information on the same simulation. Individual ITZ molecules are always associated

501

to one specific color; atoms are displayed as spheres when the focus is on the surface and

502

points connected by bonds when we look at the interior structure. This allows for a visual25 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

503

isation of the molecules within the nanoparticle as well as on its surface. a) Surface of the

504

nanoparticle. b) Section of the nanoparticle highlighting its amorphous internal structures.

505

c) Diffusion coefficient plotted against the average solvent exposure. Each point in the plot

506

represents a single ITZ molecule. Negative numbers in the solvent exposure represent the

507

ITZ molecules that do not enter in contact with the particle/solution interface.

508

Figure 8:

509

MD simulations of surfactants in solution (a,b) and interacting with the ITZ surface (c,d).

510

The water molecules within 0.3 nm of polar moieties of either GA or PVA molecules are ex-

511

plicitly represented, together with their VdW surface reported as a red transparent surface.

512

The ITZ surface in contact with the GA or the PVA molecules is represented in green. GA

513

both in solution (a) and adsorbed on the surface (c), interacts with water molecules in a

514

localised domain acting as a polar head. The PVA oligomer instead establishes polar interac-

515

tions with the solvent all along its chain both in solution (b) and adsorbed on the surface (d).

516


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(a)

(b)

(c)

Figure 1: Molecular structures of (a) itraconazole API; (b) glycyrrhizic acid (GA) excipient; (c) polyvinyl alcohol (PVA) excipient.



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Page 28 of 35

(b)

(a)

Figure 2: SEM images of (a) pure itraconazole as received from supplier, and (b) itraconazole product obtained from PCA experiment (run 1). Scale bars indicate 100 µm.


ACS Paragon Plus Environment

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2 mm

tlag = 0 h

e

b

3 mm

1 mm

tlag = 5 h

tlag = 5 h

f

c

5 mm

1 mm

tlag = 20 h

tlag = 20 h

Figure 3: SEM images of the post-processed products collected from SFEE experiments at tlag of 0, 5 and 20 hours. a), b), and c) refer to the products manufactured using GA as excipient; and d), e) and f) refer to those using PVA. White arrows underline the recrystallization at the surface of the particles.

d

1 mm

tlag = 0 h

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a

Page 29 of 35 Crystal Growth & Design


1 0 0 9 0 8 0

IT Z -P V A

7 0

C r y s ta llin ity ( % )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 35

6 0 5 0 4 0 3 0 2 0 1 0

IT Z -G A 0 0

5

1 0

1 5

2 0

T im e ( h ) Figure 4: Effect of the type of excipient in the evolution of SFEE product crystallinity as a function of tlag . Bars indicate the experimental error of two replica experiments.


Page 31 of 35

1 0

IT Z -G A

1

IT Z (p p m )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


R a w

IT Z

0 .1

0 .0 1 0

5 0

1 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

1 6 0 0

1 8 0 0

T im e ( m in )

Figure 5: Delivery profiles obtained from in-vitro tests of raw crystalline ITZ and the postprocessed SFEE product obtained using GA as excipient. Bars indicate the standard deviation.


ACS Paragon Plus Environment

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Intensity (AU)

10

15

20

25

35

40

2 (degrees)

30

45

50

65

5

10

15

20

25

30

35

40

45

50

2 (degrees)

55

60

ITZ

ITZ

60

Glassy

Glassy

55

PVA

0%

5%

GA

0%

5%

Figure 6: XRD patterns of the products collected after 96 hours of redispersing glassy ITZ in deionised water. Percentages indicate the weight of excipient in the solid mixture. a) Glycyrrhizic acid; b) Polyvinyl alcohol.

5

19 %

19 %

9%

33 %

33 %

9%

50 %

(b) 50 %

Intensity (AU)

65

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(a)

Crystal Growth & Design Page 32 of 35

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Figure 7: MD simulations of an amorphous ITZ nanoparticle. The three parts of the figure provide different information on the same simulation. Individual ITZ molecules are always associated to one specific color; atoms are displayed as spheres when the focus is on the surface and points connected by bonds when we look at the interior structure. This allows for a visualisation of the molecules within the nanoparticle as well as on its surface. a) Surface of the nanoparticle. b) Section of the nanoparticle highlighting its amorphous internal structures. c) Diffusion coefficient plotted against the average solvent exposure. Each point in the plot represents a single ITZ molecule. Negative numbers in the solvent exposure represent the ITZ molecules that do not enter in contact with the particle/solution interface.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 8: MD simulations of surfactants in solution (a,b) and interacting with the ITZ surface (c,d). The water molecules within 0.3 nm of polar moieties of either GA or PVA molecules are explicitly represented, together with their VdW surface reported as a red transparent surface. The ITZ surface in contact with the GA or the PVA molecules is represented in green. GA both in solution (a) and adsorbed on the surface (c), interacts with water molecules in a localised domain acting as a polar head. The PVA oligomer instead establishes polar interactions with the solvent all along its chain both in solution (b) and adsorbed on the surface (d).


Page 34 of 35

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Study of the preparation of amorphous Itraconazole formulations Mar´ıa P. Fernández-Ronco,∗,†,¶ Matteo Salvalaglio,†,‡ Johannes Kluge,†,§ and Marco Mazzotti† ETH Zurich, Institute of Process Engineering, Sonneggstrasse 3, 8092 Zurich, Switzerland, and Facolt` a di Informatica, Istituto di Scienze Computazionali Universit` a della Svizzera Italiana, 6900 Lugano, Switzerland E-mail: [email protected] Phone: +41 44 632 22 45. Fax: +41 44 632 11 41

AMORPHOUS ITRACONAZOLE Redispersed in water

+ Glycyrrhizic acid

+ Polyvinyl alcohol STABLE

NOT STABLE

5% 0% GA Glassy ITZ 5

50 % 33 % 19 % 9% 5% 0%

Intensity (AU)

50 % 33 % 19 % 9%

Intensity (AU)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PVA Glassy ITZ 5

10 15 20 25 30 35 40 45 50 55 60 65

2θ(degrees)

10 15 20 25 30 35 40 45 50 55 60 65

2θ(degrees)

Synopsis – Schematic representation of the effect of excipients in the stability to recrystallize of amorphous pure glassy itraconazole when redispersing it in water.

∗

To whom correspondence should be addressed ETH Zurich–Institute of Process Engineering ‡ ETH Zurich–Istituto di Scienze Computazionali ¶ Current address: EMPA, Laboratory for Advanced Fibers, Lerchenfeldstrasse 5, 9014 St. Gallen, Switzerland. E-mail: [email protected] § Current address: Novartis Pharma AG, 4056 Basel, Switzerland. †


Study of the Preparation of Amorphous Itraconazole Formulations

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