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High-Energy Ball Milling as Green Process to Vitrify Tadalafil and Improve Bioavailability Anna Krupa, Marc Descamps, Jean-Francois Willart, Beata Strach, El#bieta Wyska, Renata Jachowicz, and Florence Danède Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00688 • Publication Date (Web): 12 Sep 2016 Downloaded from http://pubs.acs.org on September 15, 2016

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Molecular Pharmaceutics

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High-Energy Ball Milling as Green Process to

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Vitrify Tadalafil and Improve Bioavailability

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Anna Krupa,*,† Marc Descamps,‡ Jean-François Willart,‡ Beata Strach,# Elżbieta Wyska,#

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Renata Jachowicz†, Florence Danède‡

5 †

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Department of Pharmaceutical Technology and Biopharmaceutics, Faculty of Pharmacy, Jagiellonian University Collegium Medicum, 9 Medyczna Street, Cracow, Poland

‡

UMET, Unité Matériaux et Transformations, CNRS, INRA, University of Lille, F 59 000 Lille,

9 10 11

France #

Department of Pharmacokinetics and Physical Pharmacy, Faculty of Pharmacy, Jagiellonian University Collegium Medicum, 9 Medyczna Street, Cracow, Poland

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*Corresponding author: Anna Krupa, Ph.D., Department of Pharmaceutical Technology and

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Biopharmaceutics, Faculty of Pharmacy, Jagiellonian University, Medical College, 9 Medyczna

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Str., 30-688 Cracow, Poland, Tel: +48 12 620 56 08, Fax: +48 12 620 56 19, e-mail:

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[email protected]

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ACS Paragon Plus Environment

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ABSTRACT GRAPHIC

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ABSTRACT

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In this study, the suitability of high-energy ball milling was invesitaged with the aim to vitrify

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tadalafil (TD) and improve its bioavailability. To achieve this goal, pure TD as well as binary

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mixtures composed of the drug and Soluplus® (SL) were co-processed by high-energy ball

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milling. Modulated differential scanning calorimetry (MDSC) and X-ray powder diffraction

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(XRD) demonstrated that after such co-processing, the crystalline form of TD was transformed

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into an amorphous form. The presence of a single glass transition (Tg) for all the co-milled

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formulations indicated that TD was dispersed into SL at the molecular level, forming amorphous

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molecular alloys, regardless of the drug concentration. The high values of Tg determined for

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amorphous formulations, ranging from 70 °C to 147 °C foreshow their high stability during

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storage at room temperature, which was verified by XRD and MDSC studies. The remarkable

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stabilizing effect of SL on the amorphous form of TD in co-milled formulations was confirmed.

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Dissolution tests showed immediate drug release with sustained supersaturation in either

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simulated gastric fluid of pH 1.2 or in phosphate buffer of pH 7.2. The beneficial effect of both

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amorphization and co-amorphization on the bioavailability of TD was found. In comparison to

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aqueous suspension, the relative bioavailability of TD was only 11 % for its crystalline form, and

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53 % for the crystalline physical mixture, whereas the bioavailability of milled amorphous TD

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and the co-milled solid dispersion was 128 % and 289 % respectively. Thus, the results provide

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evidence that not only the presence of polymeric surfactant but also the vitrification of TD is

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necessary to improve bioavailability.

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KEYWORDS: tadalafil, co-milling, vitrification, solid dispersion, dissolution enhancement,

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bioavailability improvement

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ABBREVIATIONS: TD, tadalafil; SL, Soluplus®; PM, physical mixture; SGF, simulated gastric

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fluid; PBS, phosphate buffer solution; C, concentration of drug in dissolution medium; Cs,

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saturation solubility of drug; HME, hot melt extrusion; DSC, differential scanning calorimetry;

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MDSC, temperature modulated differential scanning calorimetry; XRD, X-Ray powder

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diffraction;

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chromatography; Ph.Eur., European Pharmacopoeia; RT, room temperature; SD, standard

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deviation; Tg, glass transition temperature; Tm, melting point; ∆Cp, heat capacity; ∆H, enthalpy;

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Q120, amount of TD dissolved after 120 min; P-gp, P-glycoprotein; PVP, polyvinylpyrrolidone;

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HPMC, hydroxypropyl methylcellulose; HPMCAS, hydroxypropyl methylcellulose acetate

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succinate; Cmax, maximum plasma concentration; tmax, time to reach peak concentration; AUC,

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area under plasma concentration-time curve; AUMC, area under the first moment plasma

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concentration-time curve; AUClast, area under plasma concentration-time curve measured to the

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last concentration; AUC0-∞, area under plasma concentration-time curve measured to infinity; λz,

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terminal elimination rate constant; t0.5λz, terminal half-life; CL/F, oral clearance; FR, relative

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bioavailability, MRT, mean residence time; HaVD, half value duration; cGMP, cyclic guanosine

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monophosphate; PDE-5, phosphodiesterase type-5; PAH, pulmonary arterial hypertension

SEM,

scanning

electron

microscopy;

HPLC,

high-performance

liquid

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1. INTRODUCTION

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The manufacturing and characterization of amorphous forms of drug products with the aim to

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improve solubility and bioavailability is one of the most rapidly developing areas in the

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pharmaceutical technology. It is estimated that more than 40 % of approved drugs and 90 % of

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new active pharmaceutical ingredients under development is poorly soluble in water. Moreover,

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in the 1990s the majority of compounds classified as poorly soluble had the solubility about 100

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µg/mL, whereas nowadays the solubility of new compounds is often far lower than 10 µg/mL.1

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That is the reason why the number of publications dealing with the entry “amorphous” AND

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“drugs” obtained from Scopus database, increased from more than 80 in 1995 to almost 850 in

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2015.

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The properties of amorphous forms of either drugs or excipients, as well as methods of their

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preparation, are investigated in many research articles and reviews.2-7 Nevertheless, the majority

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of them present only a detailed in vitro evaluation of solid-state interactions that may influence

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stability, intrinsic solubility, dissolution or mechanical resistance. The assessement of the

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relationship between the physical solid state of the drug and bioavailability can be difficult to

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establish, especially if the amorphous form is not stable at room temperature (RT) due to low

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glass transition temperature (Tg). Moreover, the bioavailability of amorphous forms is often

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evaluated after their oral administration to experimental animals in the form of aqueous

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suspension. In such a case, the amorphous drug may recrystallize in contact with solvent just

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before, or during oral administration. Despite these strains, there are numerous examples which

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confirm improved bioavailability of amorphous drugs, such as itraconazole,8-9 danazole,10

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sorafenib.11 The opportunity to increase the effectiveness of pharmacotherapy by increasing

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solubility and bioavailability of poorly soluble drugs has led to the development of new

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amorphous drug products. During the last decade, fifteen new drugs in the form of amorphous


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solid dispersions, containing everolimus (Zortress®/Votubia®), ritonavir (Norvir®), vemorafenib

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(Zelboraf®),

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ombitasvir/paritaprevir/ritonavir (Viekira®/Viekirax®) or lumacaftor/ivacaftor (Orkambi®) have

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been introduced on the market.6

posaconazole

(Noxafil®),

including

combination

drugs,

such

as

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Tadalafil (TD) was developed in the late 1990s for the treatment of urinary dysfunctions. It is a

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long-acting inhibitor of phosphodiesterase type-5 (PDE-5), inactivating cyclic guanosine

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monophosphate (cGMP) in vascular smooth muscles. Its main indication concerns the therapy of

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erectile dysfuntions (2.5 - 20 mg). Due to the fact that TD was reported to reduce mean

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pulmonary artery pressure, recently it has also been approved for the polytherapy of a rare

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disease, i.e. pulmonary arterial hypertension (PAH) in a single dose ranging from 10 mg to 40

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mg.12 As a poorly soluble drug (< 10 µg/mL) of high permeability, TD is assigned to BCS class

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II. The bioavailability of BCS class II drugs is dissolution rate limited, since the rate at which

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they dissolve in the gastrointestinal fluids is usually the slowest step. Thus, the limited solubility

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of such drugs can be troublesome.

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As far as TD is concerned, there are three main problems in the development of new solid

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dosage forms of enhanced bioavailability, which are related to the following issues: (i) high

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melting point (Tm = 299 °C); (ii) decomposition after melting; (iii) low solubility in organic

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solvents approved by ICH. So far, the amorphous form of pure TD has been obtained by spray-

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drying, antisolvent precipitation or milling process.13 An important advantage of the amorphous

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TD is a high glass transition temperature (Tg = 147 ⁰C), which can ensure stability during

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storage. However, despite vitrification, the particles of pure amorphous TD remain strongly

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hydrophobic, showing tendency to form agglomerates, floating on the surface of hydrophilic

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solvents. Thus, the amorphization of the pure drug has been found to cause only a slight

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improvement in dissolution.14


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Polyethylene glycol, polyvinyl acetate and polyvinylcaprolactame based graft co-polymer

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(Soluplus®, SL) is an amphiphilic matrix of Tg = 70 °C, designed to prepare glassy solutions of

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poorly soluble drugs by hot-melt extrusion (HME).9-15 Nevertheless, the high melting point of

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some drugs (like TD) requires high processing temperatures, which in general unavoidably

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induce strong decomposition, making it unusable.

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In contrast to HME, the drug amorphisation by co-milling is known to be able to force

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molecular mixing at low temperatures. However, this requires the co-milling to be carried out at

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a temperature significantly lower than the expected Tg of the amorphous mixture.2,3 This latter

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condition seems to be fulfilled in the case of TD and SL since both components have Tg

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noticeably higher than RT (Tg = 70 °C for SL and Tg = 147 °C for TD).2,5 By the way, it has

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been shown recently that the high-energy ball milling process was suitable to prepare glassy

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solutions of TD and SL.14

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The aim of the present study is to: (i) prepare amorphous solid dispersions of TD in SL by

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high-energy ball milling; (ii) examine the stability of these co-processed formulations, and (iii)

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determine dissolution (in vitro) and bioavailability (in vivo) of TD from amorphous systems. We,

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thus, expect to demonstrate the strengths and the weaknesses of co-amorphous formulations over

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crystalline formulations and to understand better the mechanism responsible for the improvement

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of bioavailability.

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The physicochemical properties of co-milled formulations have been determined by X-Ray

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diffraction (XRD) and temperature modulated differential scanning calorimetry (MDSC). The

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influence of the vitrification process on dissolution of TD in solvents simulating either gastric or

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intestinal fluids was also studied, using the paddle apparatus. Then, formulations placed in hard

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gelatin capsules were administrated orally to rats in order to validate the results of in vitro

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experiments. The influence of amorphization, carried out either in the presence of SL or without


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the polymer, on pharmacokinetic parameters was determined on the basis of plasma levels of

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TD.

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2. MATERIALS AND METHODS

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2.1.

Materials

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Tadalafil (TD) was purchased from Polpharma S.A., Poland. Polyethylene glycol, polyvinyl

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acetate and polyvinylcaprolactame graft co-polymer (Soluplus®, SL) was obtained from BASF,

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Poland.

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Hydrochloric acid 37 % was obtained from Merck Millipore, Poland. Sodium chloride,

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potassium dihydrogen phosphate and sodium hydroxide was purchased from Avantor

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Performance Materials Poland S.A. All the reagents used were of the analytical grade.

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Acetonitrile and methanol of HPLC grade were from Merck (Darmstadt, Germany).

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Dichloromethane was purchased from Sigma Aldrich (Steinheim, Germany). Ultrapure

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deionized water (0.1 µS/cm) was prepared, using Hydrolab water purification system (Poland)

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with the 0.2 µm microfiltration capsule.

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2.2.

Methods

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2.2.1. Preparation of binary formulations and co-milling to prepare solid dispersions

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Nine binary physical mixtures (PM) composed of TD and SL, containing 0.1, 0.2, 0.3, 0.4, 0.5,

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0.6, 0.7, 0.8 and 0.9 wt. % of TD were prepared. Pure TD, SL, as well as their binary PM, were

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milled at room temperature, using a high-energy planetary ball mill Pulverisette 7, Fritsch

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(Germany). The zirconium milling jars of 45 mL with seven milling balls of 1 cm in diameter,

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made of the same material, were used. The milling was performed using 1 g of the sample. A


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ball to sample weight ratio was 75:1. The rotation speed of the solar disc was set at 400 rpm. The

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milling was performed for 12 h. The milling periods of 20 min were followed by 10-minute-

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pause periods to avoid overheating of samples.

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2.2.2. Morphological analysis by scanning electron microscopy (SEM)

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The morphology of TD, as well as the binary mixture 0.5TD before and after milling, was

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analyzed by scanning electron microscope Hitachi S-4700 (Japan). The powder was adhered to a

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sample holder by double-sided copper tape. Its surface was coated with carbon, using 208 HR

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carbon sputter coater (Cressington, USA). The images were taken at the magnification of 400 x,

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700 x and 2000 x.

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2.2.3. DSC and MDSC measurements

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Differential Scanning Calorimetry (DSC) and Modulated Differential Scanning Calorimetry

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(MDSC) were used to characterize solid-state properties of TD, SL and their co-milled binary

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mixtures.

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DSC measurements were carried out using DSC 821e (Mettler Toledo, Germany) apparatus,

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equipped with intracooler option (Haake). The samples were hermetically sealed in an aluminum

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crucible and measured in the temperature range from 0 °C to 400 °C at the heating rate of 5 °C

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min-1. The empty pan was used as a reference sample.

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The majority of DSC measurements of co-milled samples were performed by DSC Q200 (TA

180

Instruments, Guyancourt, France) in the modulation mode (MDSC). The temperature and

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enthalpy readings were calibrated, using standard aluminum pans and pure indium at the same

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scan rates as used in all the experiments. The milling process increases specific surface area of

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particles, in consequence, they become hygroscopic. Thus, MDSC measurements were


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performed in open pans (without cover) to facilitate the evaporation of water adsorbed on the

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surface. The sample corresponding to 2 - 5 mg was placed in an aluminum standard pan. During

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the measurement, it was flushed with highly pure nitrogen gas (50 mL/min). The heating rate

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was 50 °C min-1, the amplitude of modulation was 0.663 °C every 50 s ("heat only mode"). The

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scans were performed between 10 °C and 250 °C.

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2.2.4. XRD analysis

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The experiments were carried out with X’Pert PRO MPD diffractometer (Philips,

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Netherlands), equipped with X’Celerator detector. Samples were placed into Lindemann glass

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capillaries of 0.7 mm in diameter (Hilgenberg GmbH, Masfeld, Germany), which were then

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exposed to X-ray radiation (Cu-Kα) with wavelength of 1.540 Å. The scanning rate was 0.02

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°/min in 2Θ range from 5 to 60 °.

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2.2.5. Stability studies

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All the co-milled formulations were placed in Eppendorf test tubes and stored at room

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temperature (RT) for two months and five months. Additionally, milled TD and co-milled

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samples 0.1TD, 0.4TD and 0.6TD were placed in open DSC pans and stored for two months at

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60 °C, using the crystallization system DSC Crystal Breeder (Netherlands). The physicochemical

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properties of these samples were evaluated on the basis of XRD and MDSC measurements as

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described above.

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2.2.6. In vitro dissolution studies

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The dissolution studies were performed using the automated pharmacopoeial paddle

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dissolution apparatus Dissolution Station SR8 Plus (Hanson Research, USA) with autosampling


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device Dissoette II (Hanson Research, USA) at 37 °C ± 0.5 °C. The paddle rotation speed was

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set at 100 rpm. The dissolution tests were carried out for 24 h.

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They were performed in non-sink conditions (C > 0.1 Cs) to mimic in vitro the best, the

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behavior of TD in vivo after oral administration. Two kinds of solvents, corresponding to either

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gastric or intestinal milieu, were used in dissolution studies, i.e. simulated gastric fluid (SGF,

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Ph.Eur.) of pH = 1.2 and phosphate buffer of pH = 7.2 (PBS, Ph.Eur.). 500 mL of each solvent

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was used for the study. A certain number of powdered samples, equivalent of 50 mg of TD, were

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placed into the dissolution tester. At each time point, the samples of 5 mL were withdrawn

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through polyethylene filters (Ø = 10 µm) and then filtered through the syringe filter of 0.45 µm

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(Millex Millipore, USA). The amount of TD dissolved was determined by UV-VIS

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spectrophotometer UV-1800 (Shimadzu, Japan) at 284 nm wavelength. The standard curve was

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linear over the range 1.5-30 µg/mL with correlation coefficients 0.9991 and 0.9983 in SGF and

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PBS respectively. The concentration of TD dissolved over time in µg/mL, as well as standard

221

deviation (SD), were calculated (n = 3).

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ANOVA test was applied to evaluate differences between concentrations of TD dissolved after

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120 min (Q120). If the analysis of variance showed a significant difference between groups,

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Tukey post-hoc test was performed to identify disparate groups. Furthermore, the significance of

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differences between Q120, determined in either SGF or phosphate buffer, was tested with

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Student’s t-test for independent samples. The significance was assumed if p-value was below

227

0.05. The statistical analysis was performed, using Statistica software v.12 (StatSoft, Poland).

228 229

2.2.7. Pharmacokinetic studies


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Male Wistar rats, weighting 250 - 300 g were implanted with catheters (SAI Infusion

231

Technologies, USA) in the jugular vein under ketamine/xylazine anesthesia. The implantation

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was performed at least 2 days prior to the experiment.

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Before the drug administration, the animals were fasted overnight with free access to water.

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The food was given 12 h after the dosing. Formulations containing TD were accurately weighted

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and placed in hard gelatin capsules for rodents (size no 9), using a capsule filling funnel (Torpac,

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USA). The dose of TD in the capsule corresponded to 5 mg per kg of the rat body weight. The

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capsules were administered orally, using a dosing syringe provided by Torpac (n = 4). After

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administration of the capsule, 0.5 mL of water was given via an oral gavage.

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Additionally, TD in the form of the aqueous suspension of crystalline TD in distilled water

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was administered orally to rats (n = 4). Blood samples of 200 µL were collected from the jugular

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vein catheter pre-dose and 1, 2, 4, 6, 8, 10, 12, 16, and 24 h after the drug administration. The

242

samples were collected into propylene tubes containing heparin. The catheters were flushed with

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200 µL of heparinized normal saline after each blood withdrawal to prevent blood clotting and to

244

compensate for the fluid loss. The samples were centrifuged at 12.000 rpm for 5 min and the

245

plasma was stored frozen at – 80 °C until analysis.

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All the procedures used in the present study were approved by the Local Ethics Committee of the Jagiellonian University - permission no 129/2012, granted for 4 years from 2014.

248 249

2.2.8. Bioanalytical method

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The concentrations of TD in rat plasma were measured by the HPLC method. TD was isolated

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from plasma by a simple one-step liquid–liquid extraction with dichloromethane. Prior to the

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extraction, all the samples were alkalized with 0.1 mol/L Na2CO3. The samples were shaken for

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10 min (IKA VXR Vibrax, Germany) and centrifuged at 12.000 rpm for 5 min (ABBOTT


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254

Diagnostic, Germany). The organic layer was transferred into a new test tube and evaporated to

255

dryness at 37 °C under a stream of nitrogen. The residue was dissolved in 50 µL of methanol and

256

20 µL of this solution were injected into the LaChrom Elite HPLC system with an L-2485

257

fluorescence detector (Merck Hitachi, Japan). The excitation and emission wavelengths were set

258

at 281 and 330 nm respectively. LiChrospher® 100RP-18 column (25 cm x 4 mm) of the particle

259

size of 5 µm coupled with LiChroCART guard column (4.0 x 4.0 mm), containing the same

260

packing material as the analytical column (Merck, Germany) was used. The mobile phase

261

consisted of acetonitrile and water mixed at 45:55 (v/v) ratio. It was pumped at a flow rate of 0.7

262

mL/min at 35 °C. Under these conditions, retention times were 7.9 min for TD and 9.7 min for

263

internal standard (IS). No interfering peaks from plasma were observed. The calibration curve

264

was constructed by plotting the peak area ratios of the analyte to IS versus corresponding

265

concentrations of the analyte, and it was linear in the range of tested concentrations. The

266

detection limit was 0.5 ng/mL and the lower limit of quantification was 10 ng/mL, using 100 µL

267

of the plasma sample. The assay was reproducible with low intra- and inter-day variation (CV

268

was less than 10 %).

269 270

2.2.9. Pharmacokinetic and statistical analysis of in vivo data

271

The maximum plasma concentration (Cmax) and the time to reach peak concentration (tmax),

272

following oral dosing, were taken from the concentration-time curves. The area under the curve

273

(AUC) and the area under the first moment curve (AUMC) were calculated by the linear

274

trapezoidal rule from the time of dosing to the last measured concentration (AUClast) or to the

275

infinity (AUC0-∞). The terminal elimination rate constant (λz) was assessed by linear regression.

276

The terminal half-life (t0.5λz) was calculated as ln2/λz. The oral clearance (CL/F) was assessed by


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dividing the dose by the AUC0-∞. The relative bioavailability (FR) was calculated as the

278

geometric mean ratio of AUC0-∞ capsule and AUC0-∞ suspension. The mean residence time (MRT) was

279

derived from AUMC over AUC. Half value duration (HaVD) was obtained by linear

280

interpolation between the respective concentrations above and below 50 % of Cmax.

281

All the pharmacokinetic parameters were calculated by noncompartmental analysis with

282

Phoenix WinNonlin software v. 6.3 (Certara, St. Louis, MO, USA) and expressed as arithmetic

283

mean±SD. The statistical analysis was performed using Statistica software v.10 (StatSoft,

284

Poland). Prior to the analysis, the values for Cmax, AUClast, AUC0-∞, λz, t0.5λz, and MRT were

285

natural log-transformed.

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287


3. RESULTS AND DISCUSSION

288 289

3.1.

Amorphicity and thermal properties of milled formulations

290 291

3.1.1. Pure milled compounds: TD and SL

292

Fig. 1a shows the diffractograms of TD before and after 12 hours’ milling. Before milling, the

293

diffractogram shows Bragg’s peaks characteristic of crystalline materials. Their position is

294

compatible with the expected structure I of TD.16 After 12 h of milling, all Bragg’s peaks

295

disappeared to give way to a wide halo pattern, typical of amorphous materials. These results

296

strongly suggest that the initially crystalline TD was amorphized during the milling process.

297

Fig. 1b shows the heating DSC scans (5 °C/min) of TD before and after milling. Before

298

milling, the thermogram shows an endotherm, corresponding to the melting of the crystal. The

299

temperature and the enthalpy of melting are Tm = 299 °C, and ∆H = 105 J/g respectively, which

300

is in agreement with the values reported in literature.13 At higher temperature, we can also see a

301

broad exotherm, which peaks at about 360 °C, indicating that TD undergoes chemical

302

degradation after melting. It must be noted that because of this thermal degradation and due to

303

the fact that TD strongly sublimates above 250 °C, this DSC experiment was performed using a

304

hermetic DSC pan. After milling, the thermogram reveals some new thermodynamic events: (1)

305

a broad endotherm which ranges from 20 °C to 70°C. It corresponds to the loss of some free

306

water caught by the sample during milling; (2) Cp jump starting at 140 °C, which corresponds to

307

the glass transition of TD; (3) a broad exothermic recrystallization between 150 °C and 200 °C,

308

which starts before the end of glass transition. This recrystallization and glass transition prove

309

that TD underwent a direct crystal to glass transformation upon milling.


15


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Page 16 of 44

310

311 312

Figure 1. XRD patterns on the left hand side and DSC on the right hand side, heating scans

313

(5 °C ·min-1) of: (a-b) TD and (c-d) SL recorded before and after milling. Arrow shows the

314

position of Tg.

315 316

X-ray diffraction patterns of unmilled and milled SL are shown in Fig. 1c. The pattern of the

317

unmilled material is free of Bragg’s peaks which is in agreement with the expected amorphous

318

character of SL. This pattern is unchanged after milling, which points out that the milling process

319

has no impact on amorphicity. Fig. 1d shows DSC scans (5 °C/min) of SL before and after

320

milling. Before milling, the DSC scan only shows the signature of a broad glass transition with

321

midpoint at 70 °C. After milling, the DSC scan shows a broad endotherm, ranging from 15 °C to


16

Page 17 of 44

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322

50 °C, slightly overlapping the Cp jump of glass transition. The endotherm corresponds to the

323

evaporation of some free water, and is not visible on the reversible signal (data not shown),

324

which only shows glass transition.

325 326

3.1.2. Example of co-milled binary mixture containing 80 wt. % of TD

327

The thermal behavior of the binary mixture composed of 80 wt. % of TD and 20 wt. % of SL,

328

(0.8TD) co-milled for 12 h, is representative of other co-milled formulations. Because of that,

329

0.8TD was chosen to explain the thermal properties of co-milled systems.

330

Fig. 2a shows complete amorphisation of 0.8TD upon milling, confirmed by an X-ray

331

diffraction pattern. The DSC signals (total and reversible heat flow) derived from the heating

332

DSC scan (5 °C/min) of the co-milled mixture are shown in Fig. 2b. Prior to the scan, the sample

333

was heated for 30 min at 60 °C in order to remove residual water, caught by micronized

334

particles. Thus, there is no dehydration endotherm around 50 °C, which might overlap the Cp

335

jump. Neither are there signs of glass transition of pure SL, nor of pure TD. However, there is a

336

signature of the unique glass transition (Tg = 130 °C), which is located between Tg of pure SL

337

and pure TD. The co-milled sample, thus, appears to be an amorphous molecular alloy, where

338

TD is homogenously dispersed in the matrix of SL at the molecular level. At higher

339

temperatures, there is also a strong endotherm visible in Fig. 2b with the onset about 162 °C,

340

signalizing the recrystallization of TD. Since TD is molecularly dispersed in the matrix of SL, its

341

recrystallization upon heating requires demixing of the two compounds. Such a phenomenon

342

points out that the molecular alloy 0.8TD is highly supersaturated in TD.

343

The diffractogram of the recrystallized sample is reported in Fig. 2a. It was obtained by

344

heating the solid dispersion composed of 80 wt. % of TD and 20 wt. % of SL at 180 °C for 1 h to

345

induce the recrystallization process of amorphous TD. The positions of Bragg’s peaks in the


17


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Page 18 of 44

346

recrystallized sample are the same as those of the unmilled TD, indicating that polymorphic

347

forms of TD are not formed during recrystallization.

348

349 350 351

Figure 2. (a) XRD patterns recorded at RT. From top to bottom: pure crystalline TD; co-milled

352

solid dispersion 0.8TD, recrystallized after heating for 1 h at 180 °C; 0.8TD immediately after

353

milling;

354

(b) heat flow (solid line) and reversible heat flow curve (dash line) of 0.8TD (5 °C/min),

355

recorded immediately after co-milling.


18

Page 19 of 44

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356


3.1.3. Influence of the composition of co-milled solid dispersions on thermal properties

357

The diffractograms shown in Fig. 3a demonstrate complete amorphization of the crystalline

358

drug upon milling. Fig. 3b presents DSC scans of all the co-milled mixtures. Despite the drug to

359

polymer ratio, there is a single Tg in heat flow curves of all the co-milled mixtures. The position

360

of Tg shifts to higher values as the concentration of TD in the binary mixture increases. Thus, it

361

can be concluded that the co-milling process carried out at RT enables to obtain fully amorphous

362

glassy solutions, in which TD and SL are mixed at the molecular level no matter what the

363

concentration of the drug is. Finally, the high-energy ball co-milling process can also be

364

considered as an effective method to prepare amorphous systems rich in TD.

365 366

Figure 3. (a) XRD patterns recorded at RT and (b) heat flow curves (5 °C/min) of co-milled solid

367

dispersions. From top to bottom: formulations containing a decreasing amount of TD. Arrow

368

shows the position of Tg.


19


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Page 20 of 44

369 370

3.2.

Stability of milled amorphous formulations

371 372

3.2.1. Influence of annealing temperature on the stability of pure amorphous TD

373

In order to determine the influence of temperature on the stability of pure TD milled for 12 h,

374

the samples were stored for 2 months at RT and at 60 °C. MDSC signals (heat flow and

375

reversible heat flow at 5 °C/min) of the annealed amorphous TD are shown in Fig. 4. The

376

signals, corresponding to the non-annealed material are also shown for comparison. The sample

377

analyzed immediately after milling shows the exotherm of recrystallization on the heat flow

378

curve (DSC) and the glass transition on the reversible heat flow curve (MDSC), proving clearly

379

the complete vitrification of TD. After ageing at RT, the DSC curve still shows the exotherm of

380

recrystallization but the enthalpy of this process is slightly lower as compared to that of TD,

381

examined directly after milling. Furthermore, glass transition is still clearly visible on the

382

reversible heat flow, but the heat capacity jump (∆Cp) at Tg is lower than that measured directly

383

after milling. These two points indicate that the majority of the sample remains amorphous, even

384

though presumably, the recrystallization process has already been initiated. After ageing at 60

385

°C, the jump of Cp at Tg is ten times lower than that of TD scanned immediately after milling,

386

and the exotherm of recrystallization is now hardly visible. This indicates that a great part of the

387

milled TD recrystallized during the annealing at 60 °C. The amorphous milled TD, thus, appears

388

to be much less stable as the storage temperature increases.

389


20

Page 21 of 44

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390 391 392

Figure 4. Impact of ageing conditions on thermal properties of pure milled TD:

393

(a) heat flow curves (DSC),

394

(b) reversible heat flow curves (MDSC),

395

recorded just after milling, after two months of storage at RT in closed Eppendorf test tubes, or

396

after two months of storage in open DSC pans at 60 °C (Crystal Breeder). Arrows show the

397

position of Tg.


21


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

398 399

Page 22 of 44

3.2.2. Impact of annealing temperature on the stability of amorphous TD in co-milled solid dispersions

400

The examples of diffractograms recorded for co-milled solid dispersions after ageing for two

401

months in closed plastic containers at RT are shown in Fig. 5. As there is no trace of

402

recrystallization, it can be concluded that regardless of the concentration of TD in the binary

403

mixture, all the samples remain amorphous (Fig. 5). Moreover, prolongation of storage of the

404

solid dispersion 0.1TD from two to five months has no impact on the X-ray pattern, which is that

405

of fully amorphous material.

406

407 408

Figure 5. X-ray diffraction patterns of co-milled solid dispersions after ageing at RT in closed

409

Eppendorf test tubes. From top to bottom: 1.0TD, 0.8TD, 0.6TD and 0.4TD after two months’

410

annealing as well as 0.1TD after five months’ annealing. 0.1TD recorded immediately after

411

milling is shown for comparison.


22

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412

These findings are in agreement with the results of thermal analysis presented in Fig. 6. Either

413

the position of Tg on the reversible heat flow curve, or ∆Cp on the heat flow curve is not

414

changed after annealing at RT, independently of the concentration of TD. Small changes appear;

415

however, on DSC scans after storage at the temperature which is rather close to Tg. An

416

overshoot appears at Tg position, which is related to glass annealing. This confirms that the

417

samples continue to be amorphous, despite storage at a relatively high temperature.

418 419

Overall, these results demonstrate that the co-milling process provides a stable amorphous form of the drug.

420 421 422 423


23


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Page 24 of 44

424 425 426

Figure 6. Influence of ageing conditions on heat flow curves (left hand side) and reversible heat

427

flow curves (right hand side) of co-milled solid dispersions:

428

(a, b) 0.1TD determined after two months of storage at RT in closed Eppendorf test tubes, or in

429

open DSC pans at 60 °C (Crystal Breeder),

430

(c, d) 0.6TD determined after two months of storage at RT in closed Eppendorf test tubes, or in

431

open DSC pans at 60 °C (Crystal Breeder).

432

Arrows show the position of Tg.

433 434 ACS Paragon Plus Environment

24

Page 25 of 44

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435 436 437


3.3.

Dissolution assessment

In general, amorphous solid dispersions are developed with the aim to enhance intestinal absorption by inducing the drug supersaturation in the gastrointestinal tract.17,18

438

The solubility of TD does not depend on pH; however, there are reports that it may have an

439

impact on the properties of SL, and thus, indirectly influence the dissolution rate of TD from

440

solid dispersions.19 That was the reason why dissolution studies were carried out in pH = 1.2

441

(SGF) and pH = 7.2 (PBS). Furthermore, to mimic as close as possible the conditions of

442

dissolution study to the environment of the gastrointestinal tract, the volume of media in these

443

tests was reduced to 500 mL. In consequence, the concentration of SL dissolved ranged from

444

11.1 mg/L to 900 mg/L, which was far above its critical micelle concentration of 7.6 mg/L.15 It

445

has been assumed that such a procedure may allow to check if supersaturation occurs, and

446

whether solubilizing properties of SL are sufficient to prevent recrystallization of the amorphous

447

TD in different pH for 24 h.

448 449

3.3.1. Influence of SL on dissolution of crystalline TD from binary PM

450

Fig. 7 shows concentration-time profile of crystalline physical mixture (PM) 0.1TD

451

determined in SGF or PBS. The concentration of TD dissolved after 120 min (Q120) in SGF is

452

about 16 µg/mL, which is more than six times as high as that of pure crystalline TD (Fig. 7). The

453

solubilizing effect of SL on the dissolution of TD from PM is less pronounced in phosphate

454

buffer of pH = 7.2 (p < 0.0001). After 120 min, only 4.40 µg/mL of TD is dissolved (Fig. 7). It

455

can be related to phosphate ions, which might have an impact on the cloud point of SL.19 Hence,

456

viscosity of gelled polymer can increase and the dissolution rate decreases.

457

Fig. 8c shows the microstructure of binary PM 0.5TD composed of TD and SL in 1:1 wt. %

458

ratio which was submitted to slight manual grinding. Both needle-like particles of TD and


25


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Page 26 of 44

459

spherical in shape SL is still visible. The diameter of TD is similar to the bulk drug presented in

460

Fig. 8a. Likewise, the diameter of SL is about 340 µm, which is in agreement with the

461

specification provided by the manufacturer.15 It means that manual grinding during the

462

preparation of PM did not cause significant changes in the particle size, which might have an

463

impact on dissolution.

464

465 466

Figure 7. In vitro concentration-time profiles determined in: (a) SGF, and (b) phosphate buffer of

467

pH = 7.2. Co-milled solid dispersion 0.1TD (red solid circles); PM 0.1TD (red open symbols);

468

pure amorphous TD (black solid squares) and pure crystalline TD (grey stars).

469


26

Page 27 of 44

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

470


3.3.2. Impact of amorphization and co-amorphization on dissolution of TD

471

The concentration-time profiles of pure amorphous TD determined in SGF of pH = 1.2 or PBS

472

of pH = 7.2 are presented in Fig. 7, whereas Fig. 8 shows morphology of pure TD before and

473

after milling. The dissolution profiles exhibit slight improvement in dissolution after the

474

vitrification process. Analogously to crystalline TD, the influence of pH of the solvent on the

475

dissolution of amorphous TD is not significant (p > 0.05).

476

SEM pictures presented in Fig. 8a-b point out that 12 h of high-energy ball milling causes

477

micronization of the drug. Upon milling, the particle size of TD decreases from about 10 µm to

478

0.5 - 1 µm. However, as shown in Fig. 8b, primary particles form irregular agglomerates of

479

various sizes, i.e. 10 µm or 50 µm, which makes the diameter of primary particles of milled TD

480

difficult to estimate accurately. The agglomeration of micronized particles may be the reason

481

why there is only slight improvement in dissolution stated for amorphous TD as compared to the

482

crystalline drug.

483

As can be seen in Fig. 7, the concentration of amorphous TD dissolved increases in the first

484

120 min (Q120) of the dissolution study, and then the equilibrium seems to be reached,

485

corresponding to 3.48 µg/mL in SGF and 3.30 µg/mL in PBS respectively. There is no decrease

486

in the concentration of amorphous TD, dissolved within 24 h in the conditions of the dissolution

487

study. The samples withdrawn after 24 h show no birefringence (polarized light microscope -

488

Hund Wetzlar H600/12, Germany), indicating that in contact with the solvent, amorphous TD

489

does not recrystallize or the crystals are too small to be observed.

490


27


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Page 28 of 44

491 492 493

Figure 8. SEM pictures of:

494

(a) crystalline TD before milling,

495

(b) TD amorphised by milling,

496

(c) crystalline PM 0.5TD,

497

(d) 0.5TD co-amorphised by milling.

498 499

If medium soluble polymers, such as SL are used as carriers for solid dispersions, the solubility

500

of the drug increases and temporary supersaturation can occur, which can lead to nucleation and

501

then to recrystallization of the amorphous drug.20 This phenomenon was reported for hot-melt

502

extruded formulations, containing SL and fenofibrate,21 oxyglitazar22 or itraconazole.10


28

Page 29 of 44

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503

After co-amorphization of TD with SL in 1:10 wt. % ratio, immediate dissolution is obtained

504

in either SGF or PBS (Fig. 7). Q120 and dissolution rate of solid dispersion 0.1TD increases

505

significantly (p < 0.001) as compared to crystalline PM, crystalline TD and pure amorphous TD

506

(Fig. 7, Tab. 1). Similarly to PM, the kind of the solvent influences Q120 of co-amorphous

507

formulations significantly. The dissolution rate and the concentration of TD dissolved in PBS are

508

lower than in SGF (p < 0.001). Regardless of the solvent used, the concentration of TD dissolved

509

from glassy solution 0.1TD only after 15 min exceeds significantly the level of saturation.

510

Nevertheless, precipitation of the amorphous drug was not observed within 24 h of the

511

dissolution study.

512

Different mechanisms have been described to explain the ‘parachute’ effect related to

513

recrystallization of an amorphous drug in supersaturated solutions, and finally to avert this

514

phenomenon from occuring.23-25 In spite of that, thermodynamic properties and phase behaviour

515

of supersaturated systems become a complex issue, especially if solid dispersion contains

516

surfactants, like SL. On the other hand, the application of selected polymers, namely

517

polyvinylpyrrolidone (PVP), hydroxypropyl methylcellulose (HPMC), and hydroxypropyl

518

methylcellulose acetate succinate (HPMCAS) as crystallization/nucleation inhibitors in

519

supersaturated solutions was also proposed.1, 23-25

520

Friesen et al.24 proved that the presence of colloidal drug/polymer nanostructures of 20 to 100

521

nm as well as nanoaggregates of 70 – 300 nm in diameter, created in aqueous solution of

522

amorphous solid dispersions containing HPMCAS, hindered drug recrystallization for over at

523

least 100 min. As it is within the gap of the residence time for small intestine, supersaturation

524

kept over such a period should be sufficient to enhance absorption of the drug in vivo.

525

Furthermore, it is estimated that even 30 - 50 % of the drug molecules in aqueous polymer

526

solutions can be bound to the water-soluble part of the polymer via hydrogen bonds, which in


29


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Page 30 of 44

527

consequence increases a diffusive barrier in the hydrodynamic boundary layer, and finally

528

influences solubility.24,26

529

The presence of hydrogen bonds between the secondary amine group of TD and vinyl

530

caprolactam and/or the acetate carbonyls of SL as well as between hydroxyl groups of SL and

531

carbonyl groups of TD in co-milled solid dispersions was shown in our previous study.14 Their

532

presence may stabilize the amorphous TD, and despite high supersaturation, dissolution profiles

533

are closer to ‘spring-plateau’ than to ‘spring-parachute’ curves.

534

SEM pictures of the surface of solid dispersion 0.5TD in comparison to crystalline PM 0.5TD

535

are shown in Fig. 8. They indicate that after milling, needle-like particles of TD are no longer

536

visible. Neither are spherical particles of SL visible. After co-milling, the sample of solid

537

dispersion is composed of irregular agglomerates of about 150 - 200 µm in diameter, which are

538

covered with fine particles of less than 2 µm (Fig. 8d). However, agglomerates formed by co-

539

milled formulations are less coherent/electrostatic as compared to pure milled TD. In general, the

540

higher the concentration of SL is, the less coherent co-milled formulations are. Thus, it can be

541

concluded that SL prevents agglomeration of micronized TD, separating micronized particles of

542

the drug. The initial dissolution rate (0-15 min) of amorphous solid dispersion 0.1TD was

543

thirteen or fourteen times as high as the pure amorphous drug in SGF or PBS respectively.

544

Altogether, amphiphilic properties of SL facilitate wetting, and consequently, TD from solid

545

dispersions dissolved far better than the pure, amorphous drug in spite of the presence of

546

agglomerates (Fig. 7, Tab. 1).


30

Page 31 of 44

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


547

Table 1. Influence of formulation on the amount of TD dissolved after 2 h in SGF or PBS estimated on the basis of p-values of

548

ANOVA test followed by post-hoc Tukey’s test. post-hoc Tukey’s test Formulation

549

SGF of pH = 1.2

Phosphate buffer of pH = 7.2

1.0TDunmilled

1.0TDmilled

0.1PM

0.1TD

1.0TDunmilled

1.0TDmilled

0.1PM

0.1TD

1.0TDunmilled

/

n.s.*

p < 0.001

p < 0.001

/

n.s.*

p < 0.05

p < 0.001

1.0TDmilled

n.s.*

/

p < 0.001

p < 0.001

n.s.*

/

p < 0.05

p < 0.001

0.1PM

p < 0.001

p < 0.001

/

p < 0.001

p < 0.05

p < 0.05

/

p < 0.001

0.1TD

p < 0.001

p < 0.001

p < 0.001

/

p < 0.001

p < 0.001

p < 0.001

/

*n.s. – non significant

550 551


31


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

552

3.4.

Page 32 of 44

Bioavailability of tadalafil from amorphous and co-amorphous formulations

553

TD plasma concentration versus time profiles obtained after oral administration of crystalline

554

TD, amorphous milled TD, 0.1TD co-milled solid dispersion, as well as PM at TD dose of 5

555

mg/kg to rats, are shown in Fig. 9. Pharmacokinetic parameters estimated on the basis of these

556

profiles are summarized in Table 2.

557 558

Figure 9. Rat plasma concentrations of TD as a function of time following oral administration of

559

gelatine capsules containing TD (5 mg/kg) in the form of: co-milled glassy solution 0.1TD (red

560

solid circles), milled amorphous TD (black solid squares), unmilled crystalline PM 0.1TD (red

561

open circles), or unmilled crystalline TD (grey stars) (n=4).


32

Page 33 of 44

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

562


Table 2. Pharmacokinetic parameters determined after oral administration of TD (dose of 5 mg/kg, n = 4). Capsule Pharmacokinetic parameter

563

Suspension Crystalline TD

Amorphous TD

of crystalline TD 1.0TDunmilled

PM** 0.1TDunmilled

1.0TDmilled

0.1TDco-milled

Cmax (ng/mL)

410 ± 120

54 ± 21#

240 ± 140*

570 ± 200$

1110 ± 180

tmax (h)

5.5 ± 1.0

7.0 ± 2.0

6.3 ± 4.5

8.00 ± 1.63

4.00 ± 0.01

AUClast (ng h/mL)

3000 ± 1000

410 ± 200#

1800 ± 1100*

4800 ± 2500$

9500 ± 2700

AUC0-∞ (ng h/mL)

4000 ± 1800

450 ± 210#

2100 ± 1100*

4900 ± 2500$

10900 ± 2400

λz (h-1)

0.191 ± 0.053

0.244 ± 0.058

0.210 ± 0.058

0.34 ± 0.11

0.217 ± 0.088

t0.5λz (h)

3.9 ± 1.2

2.98 ± 0.82

3.46 ± 0.81

2.23 ± 0.67

3.8 ± 1.9

CL/F (L/h/kg)

1.44 ± 0.55

12.6 ± 4.1#

3.6 ± 3.4*

1.4 ± 1.2*

0.48 ± 0.12

FR (%)

−

11

53

128

289

MRT (h)

8.8 ± 2.2

9.4 ± 1.6

9.6 ± 2.9

9.7 ± 1.7

8.6 ± 2.2

HaVD (h)

7.9 ± 1.2

6.3 ± 2.3

7.1 ± 2.8

7.8 ± 3.3

9.0 ± 1.2

* p < 0.05; 1.0 TDunmilled vs. PM** 0.1 TDunmilled (one-way ANOVA followed by the Tukey’s post hoc test)


33


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

564

# p < 0.05; 1.0 TDunmilled vs. 1.0 TDmilled (one-way ANOVA followed by the Tukey’s post hoc test)

565

$ p < 0.05; 1.0 TDmilled vs.0.1 TD co-milled (one-way ANOVA followed by the Tukey’s post hoc test)

566

** PM – physical mixture

Page 34 of 44

567 568 569 570 571 572 573 574 575 576 577 578


34

Page 35 of 44

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579

As presented Fig. 9, the absorption of TD from amorphous milled formulations improved in

580

comparison to the crystalline unmilled drug. Pure amorphous formulation of TD reached ten

581

times higher Cmax in comparison to the crystalline drug, i.e. 570 ± 200 ng/mL versus 54 ± 21

582

ng/mL. In turn, when the glassy solution 0.1TD was administered orally to rats, TD Cmax was as

583

high as 1110 ± 180 ng/mL. This value is more than twenty times higher than in the case of pure

584

crystalline TD, twice as high as that of amorphous TD and five times higher in comparison to

585

PM. Furthermore, the value of tmax determined for amorphous TD was similar to that of the

586

crystalline form, i.e. 8.0 ± 1.7 h and 7.0 ± 2.0 h. In contrast to the pure drug, tmax of TD

587

administered orally in the form of solid dispersion, was about 4 h, which was almost twice

588

shorter than that of crystalline TD.

589

Table 2 shows that terminal half-lives, HaVD, and MRT values determined after oral

590

administration of crystalline TD in the form of suspension are not significantly different from

591

those of solid formulations (p > 0.05). The area under the concentration-time curve (AUClast),

592

following the administration of amorphous TD is more than eleven times higher than after dosing

593

of the crystalline drug, i.e. 4800 ng·h/mL and 410 ng·h/mL respectively. The value of this

594

parameter for crystalline PM is 1800 ng·h/mL, whereas the vitrification process increased

595

AUClast of TD to 9500 ng·h/mL, which is more than twenty times higher than that of pure

596

crystalline TD. Similar observations are made for AUC0-∞. The relative bioavailability of TD

597

from milled formulations, determined in relation to suspension, improved significantly in

598

comparison to crystalline drug and PM, such as 128 % for amorphous TD and 289 % for the

599

glassy solution.

600

It is well-known that supersaturation and solubilisation are the two main mechanisms that

601

enable to increase the oral bioavailability of poorly soluble drugs.1 In supersaturated state, the


35


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Page 36 of 44

602

concentration of the free drug increases, whereas in the case of solubilisation, the drug can be

603

entrapped in polymeric micelles, which may finally reduce permeability.17 Thus, in practice, the

604

metastability of supersaturation may limit the benefits of glassy molecular alloys unless it is

605

maintained for a period sufficient for adsorption.23-26

606

In vitro dissolution studies of amorphous forms of TD show sustained supersaturation in the

607

physiological milieu (Fig. 7). Accordingly, the bioavailability of TD administered orally in the

608

form of amorphous and co-amorphous formulations is significantly higher in comparison to

609

crystalline forms. Moreover, despite the fact that amorphous TD was relatively low-soluble in

610

dissolution media (Fig. 9), especially in comparison to PM 0.1TD, the absorption of TD from the

611

gastrointestinal tract was much more effective than that of PM 0.1TD, as reflected by higher

612

Cmax, AUC, and Fr values.

613

The bioavailability improvement of TD from solid dispersion 0.1TD, administered orally to

614

rats, can be related not only to the vitrification of the drug but also to the modulatory effect of the

615

polymer on the function of intestinal P-glycoprotein (P-gp). Interestingly, it was found that TD

616

induces the expression of P-gp more than three times and increases its function twice in LS180

617

cells, which may be the reason for poor bioavailability of the pure crystalline drug.27 In general,

618

P-gp transports drugs from the intestinal cells into the lumen, limiting their absorption.28,29

619

Therefore, oral bioavailability enhancement is possible by the inhibition of P-gp function. Shen

620

et al.28 reported the concentration dependent inhibition of P-gp by polyoxyethylene glycols

621

(PEG) and their molecular derivatives. The graft copolymer used for co-processing in the present

622

study (SL) has backbone consisting of PEG 6000 (13 %) with one or two side chains of vinyl

623

acetate (30 %), randomly copolymerized with vinyl caprolactam (57 %).15 The PEG component

624

predisposes SL for modulation of P-gp. Moreover, there is a report which indicates an inhibitory

625

effect of tadalafil on ABCB1/P-gp.30 Thus, the co-processing of TD with SL, which can act


36

Page 37 of 44

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626

synergistically as P-gp inhibitors, may have a beneficial effect on the intestinal absorption of the

627

drug. Similar findings were described for solid dispersions, containing SL and itraconazole.9 It

628

was shown that tmax determined for amorphous solid dispersions of itraconazole in SL was

629

shorter as compared to amorphous formulations without SL.

630

Jinn et al.31 showed the advantages of P-gp inhibition by SL in the case of doxorubicin. The

631

incorporation of doxorubicin into the polymeric micelles, composed of SL, resulted in the

632

enhancement of both permeability and retention of the drug after oral administration,

633

significantly increasing its antitumor efficacy. SL was found to interact indirectly with P-gp,

634

triggering changes in the fluidity of cell membranes.

635

In the present study, the values of parameters related to the elimination process of TD, such as

636

λz and t0.5λz are similar for all the TD formulations and the suspension and ranged from 0.191 ±

637

0.053 to 0.34 ± 0.11 h-1 and from 2.23 ± 0.67 to 3.8 ± 1.9 h, respectively. The observed

638

differences in oral clearances are the results of significantly different values of AUC0-∞ (Tab. 2).

639

Finally, from the pharmacokinetic point of view, it seems that the co-milled solid dispersion

640

0.1TD revealed the most favourable TD concentration versus time profile, with a fast absorption

641

phase, followed by a slow elimination process, and concentrations measurable up to 24 h post-

642

dose. Overall, the mechanism of this phenomenon seems to be complex since not only the

643

physical form of the drug but also solubilizing properties and P-gp modulation/inhibition of the

644

polymer can have an impact on the bioavailability of TD.


37


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Page 38 of 44

4. CONCLUSIONS In the present study, the opportunity to improve the bioavailability of TD by co-milling the drug with SL was described. The stable amorphous solid dispersions of TD in Soluplus® (polyethylene glycol, polyvinyl acetate and polyvinylcaprolactame graft co-polymer) were obtained by high-energy ball milling process, carried out at room temperature. The highenergy ball milling process was able to provide co-amorphous systems, regardless of the drug to polymer ratio. Moreover, the remarkably higher stability of solid dispersions was stated in comparison to the pure amorphous drug. The results of dissolution studies of all the solid dispersions showed that the coamorphization of TD with SL enabled to obtain a long-term supersaturation, with no precipitation for 24 h. These findings were in line with in vivo studies. The glassy solution 0.1TD showed a fast absorption phase, followed by the slow elimination process with concentrations of TD measurable up to 24 h post-dose. The comparison of pharmacokinetic parameters determined for TD administered orally in the form of amorphous and crystalline formulations showed a considerable increase in bioavailability after vitrification. The presence of SL in the solid dispersion resulted in rapid absorption of TD followed by the slow elimination process. Since neither organic solvents nor high temperature were involved in the described co-processing, high-energy ball milling can be considered to be a ‘green’ approach to enhance the bioavailability of TD, avoiding the risk of degradation at high temperatures. Further studies would be necessary, especially in the frame of scaling-up of this process.

38 ACS Paragon Plus Environment

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AUTHOR INFORMATION *Corresponding author: Anna Krupa, Ph.D., Department of Pharmaceutical Technology and Biopharmaceutics, Faculty of Pharmacy, Jagiellonian University, Medical College, 9 Medyczna str., 30-688 Cracow, Poland, Tel: +48 12 620 56 08, Fax: +48 12 620 56 19, email: [email protected]

ACKNOWLEDGMENTS

The research was performed thanks to Sonata grant no DEC-2012/07/D/NZ7/01673 financed by the National Science Centre in Poland as well as the One-to-One Mentoring Program supported by the Foundation for Polish Science.

.



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