Nanostructured Drugs Embedded into a Polymeric Matrix: Vinpocetine

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Nanostructured Drugs embedded into a Polymeric Matrix: Vinpocetine/ PVP Hybrids investigated by the Debye Function Analysis Dritan Hasa, Carlotta Giacobbe, Beatrice Perissutti, Dario Voinovich, Mario Grassi, Antonio Cervellino, Norberto Masciocchi, and Antonietta Guagliardi Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00124 • Publication Date (Web): 18 Jul 2016 Downloaded from http://pubs.acs.org on July 20, 2016

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Molecular Pharmaceutics

Nanostructured Drugs embedded into a Polymeric Matrix: Vinpocetine/PVP Hybrids investigated by the Debye Function Analysis

Dritan Hasa,a,b Carlotta Giacobbe,c,§ Beatrice Perissutti,b Dario Voinovich,b,* Mario Grassi,d Antonio Cervellino,e Norberto Masciocchic,* and Antonietta Guagliardif

a

Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW, Cambridge, UK

b

Department of Chemical and Pharmaceutical Sciences, University of Trieste, 34127 Trieste, Italy

c

Department of Sciences and High Technology and To.Sca.Lab, University of Insubria, 22100 Como, Italy d

Department of Engineering and Architecture, University of Trieste, 34127 Trieste, Italy

e

Swiss Light Source, Paul Scherrer Institute, CH-5232 Villigen, Switzerland

f

Istituto di Cristallografia and To.Sca.Lab, Consiglio Nazionale delle Ricerche, 22100 Como, Italy

§

Present address: European Synchrotron Radiation Facility, 71 Avenue des Martyrs, 38000 Grenoble, France

* E-mail: [email protected]; Phone: (+39)-031-2386613; Fax: (+39)-031-2386659

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For the Table of Contents use only:

Using the Debye Function Analysis on Wide-Angle Synchrotron X-ray Total Scattering data of pharmaceutical solid dispersions, the enigmatic difference between in vitro and in vivo solubility tests performed on nanosized Vinpocetine / polymeric matrix was reconciled.


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ABSTRACT

Microcrystalline Vinpocetine, co-ground with crosslinked polyvinylpyrrolidone, affords hybrids containing nanosized drug nanocrystals, the size and size distributions of which depend on milling times and drug-to-polymer weight ratios. Using an innovative approach to microstructural characterization, we analyzed Wide-Angle X-ray Total Scattering data by the Debye Function Analysis and demonstrated the possibility to characterize pharmaceutical solid dispersions obtaining a reliable quantitative view of the physico-chemical status of the drug dispersed in an amorphous carrier. The microstructural properties derived therefrom have been successfully employed in reconciling the enigmatic difference in behaviour between in vitro and in vivo solubility tests performed on nanosized Vinpocetine embedded in a polymeric matrix.

KEYWORDS: Mechanical Activation, Solid Dispersion, Total Scattering, Debye Scattering Equation, Vinpocetine.



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INTRODUCTION The oral delivery of drugs represents the most natural way to reach the systemic circulation.1 However, at present more than 70% of existing drugs are poorly soluble in the gastrointestinal fluid, 2 being placed in the second group of the Biopharmaceutical Classification System (BCS).3 The low bioavailability is hence the main reason of discarding the majority of new drug candidates. 4 , 5 To overcome these problems, Class II drugs are often appropriately engineered to improve their solubility and dissolution properties by optimized structural or morphological modification. 6 As reviewed by Laitinen and coworkers, 7 there exist several emerging alternative methods to amorphous polymer dispersions for stabilizing the amorphous or nanocrystalline forms of drugs. Indeed, as things currently stand, despite apparent solubility issues with many existing and new APIs, solid dispersions are actually widely formulated and marketed. 8 In this context, mechanical activation (MA), i.e. the use of the mechanical energy to induce microstructural changes, represents a straightforward, green and innovative approach that has been successfully introduced as a pharmaceutical processing technique.9 During the MA process, the mechanical energy (usually produced in ball mills) is transferred to the loaded crystalline substance favoring the formation of nanocrystalline, defective and/or amorphous phases through an interplay of several factors that remain subject of numerous investigations.9,10,11 Typically, nanocrystals show higher solubility with respect to the original microcrystalline phase thanks to the enormous specific surface area (increased by several orders of magnitude with respect to the bulk) and, in the droplet-like approximation (Kelvin’s law), also to the reduced curvature radius.12 However, nanocrystals and amorphous materials are metastable, and are likely subjected to re-crystallization. Therefore, MA of drugs is usually performed in presence of a stabilizing agent (carrier), giving rise to solid dispersions,13 maintaining the sought nanocrystalline and/or amorphous aggregation states.


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The structural, morphological and quantitative

phase analyses of less-than-ideal

polycrystalline species, alone or in complex mixtures, typically requires a combination of analytical methods, ranging from microscopic (Transmission or Scanning Electron Microscopy − TEM and SEM, respectively) investigations, to spectroscopic, calorimetric or chromatographic experiments, and, ultimately, to (conventional) X-ray powder diffraction (XRPD) techniques, 14 typically providing volume- or mass-based average sizes, with no information about size distribution. When the sizes of crystal domain are small enough, XRPD (though powerful in many cases) and standard crystallographic methods additionally neglect the diffuse scattering below and between Bragg peaks and, therefore, can only afford limited, and approximated, answers. However, it was recently proven that, within a physically sound and robust approach, Wide Angle X-ray Total Scattering (WAXTS) methods could be successfully used to determine all this complementary information without resorting to multiple (often destructive) experiments and deriving all the sought structural and microstructural properties from the same set of scattering data.15 This innovative technique, which is based on the (reciprocal space) Debye Function Analysis (DFA) performed on accurately measured and suitably corrected synchrotron X-ray total scattering data, has been used for a number of inorganic systems (photocatalytically active titania nanoparticles16 and silica-titania nanocomposites, 17 bioinspired polyphasic calcium phosphates mimicking the initial steps of nanoapatite growth in bone, 18 and superparamagnetic core-shell iron oxide nanoparticles19). However, although DFA has been applied to different types of materials, we are unaware of its use in the field of pharmaceuticals. Suffice here to say that the only reports in field of molecular organic compounds appear to be the “total-diffraction” semi-quantitative estimation of the hydration level within freeze-dried (amorphous) sucrose and (crystalline + amorphous) raffinose samples,20 and a few total scattering studies based on the real-space



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Pair-Distribution Function technique, 21 including the very recent attempt of polymorph quantification. 22 Pushed by some inconsistencies between in-vivo pharmacokinetic results and the extensive physico-chemical characterization recently performed by some of us23 on Vinpocetine (VIN, a nootropic drug used for the treatment of cognitive disorders and related symptoms24 ) coground within a pharmacologically inert carrier (crosslinked polyvinylpyrrolidone, PVP-Cl), we report here the complete WAXTS characterization of a series of Vinpocetine / PVP-Cl hybrids, mechanically activated at different milling times. 25 We further discuss the capabilities of the DFA method, and the tremendous potentiality it bears, in the organic as well as in other fields such as nanocomposites, when nanocrystalline powders are intimately mixed within an amorphous matrix. The obtained results, in terms of average sizes of VIN nanocrystals and of their distributions, and their modification upon prolonged milling, are subsequently correlated with functional properties (dissolution rate), the differences between

in vitro and in vivo tests representing a still unsolved conundrum,23 that the innovative physico-chemical DFA-based characterization approach used in this work has eventually elucidated.

MATERIALS AND METHODS Materials. Microcrystalline Vinpocetine (purity of 99.8%) was a kind gift from Linnea SA (Riazzino-Locarno, CH). Micronized crospovidone (PVP-Cl) was purchased from BASF (Ludwigshafen, Germany). All other analytical-grade chemicals and HPLC grade solvents were provided by Carlo Erba (Milan, Italy) and used without further purification.

Preparation of VIN/PVP-Cl solid dispersions. The mechanochemical experiments performed in this study were in accordance to the experimental conditions already reported in


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ref. 23. A total of nine solid dispersions, in 1:4 or 1:7 VIN-to-PVP-Cl weight ratios were prepared by ball milling using a planetary mill (Fritsch P5, Pulverisette, Milan, Italy). A mean amount of 5 g of powder mixture containing VIN and PVP-Cl in 1:4 or 1:7 weight ratio was transferred to an agate cylindrical grinding chamber (internal height Hv = 2.6 cm, internal radius Rv = 3.2 cm, internal volume = 27.5 cm3) and co-ground using agate balls having a 2.2 cm diameter as grinding media. Different milling times were adopted: 60, 120, 180, 240, 300 and 360 min for the solid dispersions in a 1:4 VIN-to-PVP-Cl weight ratio, and 60, 120, 180 min for the solid dispersions in a 1:7 VIN-to-PVP-Cl weight ratio). Additionally, a few samples of pure PVP-Cl were also subjected to the same treatment, to be used as reference standards. The mechanochemical experiments were stopped for 5 min every 30 min of grinding to avoid excessive temperature increase: at the end of each milling experiment, the temperature increase inside the grinding bowls (measured by using thermocouples) did not exceed 40°C.

Dissolution tests. The dissolution experiment for each VIN/PVP-Cl solid dispersion was performed using 150 ml of 0.2 M KH2PO4 / 0.2 M NaOH (pH 7.4) buffer at 37 °C, as VIN solubility is pH-dependent and particularly low in intestinal buffer. 23 Accordingly, at time zero, a suitable amount of solid dispersion to give 5 mg of VIN (non-sink conditions), was added to the phosphate buffer. Each test lasted 15 min, and the uniformity conditions were constantly ensured by an impeller at a stirring rate of 200 rpm. Subsequently, 100 µl of solution were taken after 3 min of analysis, filtered through a syringe filter (RC 0.45 µm, Phenomenex, Castel Maggiore, Bologna), and immediately added to 600 µl of acetonitrile. The solution was then sonicated for 10 min and stored at −25°C. The amount of VIN solubilized in such samples was quantified by a previously described High Pressure Liquid Chromatography (HPLC) method. 23



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Analytical Characterization. The VIN content in solid dispersions and in the samples obtained during the dissolution experiments was measured using a previously published HPLC method.21 The HPLC samples were prepared as following: a suitable amount of powdered sample was accurately weighted and transferred to a 100 ml volumetric flask with acetonitrile. The solution was then sonicated for 10 min and subsequently filtered through a syringe filter (RC 0.45 µm, Phenomenex, Castel Maggiore, Bologna). Finally an appropriate amount of the filtered solution was further diluted with acetonitrile and assayed by a validated HPLC with mass spectrometry detection method. 23

X-ray Total Scattering Analysis: Data collection strategy and Data Pre-processing. Synchrotron WAXTS patterns were acquired for all the co-ground VIN/PVP-Cl samples and for two samples of pure PVP-Cl (the pristine sample and that ground at 180 min) at the X04SA Material Science beamline26 of the Swiss Light Source of the Paul Scherrer Institut (located in Villigen, CH), with a beam energy of 16 keV (λ = 0.775332(2) Å after calibration with a NIST Silicon powder standard - SRM 640d). Each sample was filled in a 0.5 mm borosilicate glass capillary spun at a rate of 1 Hz about their axis. Data were collected at 100 K using a cold nitrogen stream, to minimize sample damage by the beam. 16 different runs lasting 10 s each, and measured at different detector positions (Mythen II, 27 covering, with 24 single modules, a large 120° angular range) to cover intermodule gaps, were performed for each sample (V1-16 patterns), accompanied by further measurements for the evaluation of empty capillary (C1-16 patterns) and air scattering (A1-16 patterns) contributions. After merging the pertinent data, full patterns V, C and A with high counting statistics became accessible within about 15 min (each), thanks to the availability of the single-photon-counting silicon microstrip detector.27 The high angular resolution of the instrumental set-up ensured negligible instrumental contribution to the peak broadening and allowed accurate crystal sizes to be estimated. 8 ACS Paragon Plus Environment

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Using the experimentally accessible beam attenuation factor µt = ln(I0/I) (obtained by separately measuring the intensity of incident, I0, and sample/capillary attenuated, I radiation), “pure” sample contributions to the scattering were obtained by sagaciously combining the V,

C and A patterns and computing sample/capillary weighted angle-dependent absorption corrections and pattern subtractions28 prior to the Debye Scattering Equation29 (DSE) analysis (as shown in the Supporting Information file). Such data reduction process was carried on through a complex, locally developed, X-ray tracing procedure. Figure 1 collectively reports the reduced WAXTS pattern, in two different angular ranges.

Figure 1. Reduced WAXTS pattern for the PVP-Cl polymer (bottom) and the nine co-ground VIN/PVP-Cl mixtures (λ = 0.775332 Å). Left: full range (up to 120° 2θ). Right: the lowangle region (3

Nanostructured Drugs Embedded into a Polymeric Matrix: Vinpocetine

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