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IJP 14431 1–13 International Journal of Pharmaceutics xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

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Novel semisolid SNEDDS based on PEG-30-dipolyhydroxystearate: Development and characterization

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Tamer H. Hassan, Hendrik Metz, Karsten Mäder *

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Department of Pharmaceutics and Biopharmaceutics, Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, D-06120 Halle (Saale), Germany

A R T I C L E I N F O

A B S T R A C T

Article history: Received 9 August 2014 Received in revised form 27 October 2014 Accepted 28 October 2014 Available online xxx

The aim of the current study is to explore the potential of PEG-30-dipolyhydroxystearate (Cithrol1 DPHS) and Soluplus1 as ingredients in novel semisolid self-nanoemulsifying drug delivery systems (SNEDDS). Semisolid SNEDDS consisting of Cithrol1 DPHS, Capmul1 MCM and Kolliphor1 HS 15 were successfully prepared. The formulations were comprehensively characterized by photon correlation spectroscopy (PCS), differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), electron spin resonance (ESR) and proton nuclear magnetic resonance (1H NMR). All formulations were semisolid at room temperature and melted at body temperature. The hydrodynamic diameter of the dispersions was less than 25 nm. The ratio Cithrol1 DPHS:Capmul1 MCM was found to be critical for the dispersibility and the stability of the formed nanoemulsion. ã 2014 Published by Elsevier B.V.

Keywords: Selfemulsifying SNEDDS PEG-30-dipolyhydroxystearate Cithrol1 DPHS Kolliphor1 HS 15 Capmul1 MCM Soluplus1 Tempolbenzoate ESR

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1. Introduction

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Poorly water-soluble drugs represent circa 40–75% of currently marketed drugs and drugs under investigation (Di et al., 2012; Lipinski et al., 1997). The enhancement of their solubilisation is a prerequisite for their bioavailability. Otherwise, they may fail to reach the market despite their pharmacological activity. Several approaches have been established to improve the water solubility and the bioavailability of poorly water-soluble drugs (Williams

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Abbreviations: %T, percentage transmittance; 1H NMR, proton nuclear magnetic resonance; aN, hyperfine coupling constant; D2O, deuterium oxide; dB, decibel; dDMSO, deuterated dimethyl sulfoxide; DLS, dynamic light scattering; DPHS, Cithrol1 DPHS; DSC, differential scanning calorimetry; EPR, electron paramagnetic resonance; ESR, electron spin resonance; GMS, Cithrol1GMS 40; HLB, hydrophilic– lipophilic balance; HPLC, high performance liquid chromatography; HS15, Kolliphor1 HS 15; LFCS, lipid formulation classification system; MCM, Capmul1 MCM; mT, millitesla; O/W, oil in water; PCS, photon correlation spectroscopy; PDI, polydispersity index; PEG, polyethylene glycol; PXRD, powder X-ray diffraction; R1, rotation state 1; R2, rotation state 2; rpm, revolutions per minute; SD, standard derivation; SEDDS, self-emulsifying drug delivery systems; SMEDDS, self-microemulsifying drug delivery systems; SNEDDS, self-nanoemulsifying drug delivery systems; TB, tempolbenzoate; USP, United States Pharmacopeia; W/O, water in oil; W/O/W, water in oil in water; t c, rotation correlation time. * Corresponding author. Tel.: +49 345 55 2516; fax: +49 345 55 27029. E-mail address: [email protected] (K. Mäder).

et al., 2013). Among them, the use of lipid formulations has generated considerable interest and finally therapeutically and commercial success (Hauss et al., 1998; O'Driscoll, 2002). Lipid formulations present and maintain the drug in the solubilised form, in which absorption takes place (Humberstone and Charman, 1997). Furthermore, they can enhance the bioavailability by different mechanisms depending on their type and amounts (Porter et al., 2007; Rahman et al., 2013; Song et al., 2013) such as:

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a) Prolongation of the gastric emptying time. b) Stimulation of bile secretion and interaction with mixed

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micelles. Reduction of the first pass metabolism via stimulation of intestinal lymphatic transport for highly lipophilic drugs (log P more than 5) and reduction of the enterocyte based metabolism. Modulation of intestinal efflux transporters such as P-glycoprotein. Permeation enhancement. Generation and maintenance of a metastable supersaturable drug state.

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Lipids can be classified according to their chemical structures, origin, solubility in organic solvents or biochemical interactions

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c)

d) e) f)

http://dx.doi.org/10.1016/j.ijpharm.2014.10.063 0378-5173/ ã 2014 Published by Elsevier B.V.

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(Akoh and Min, 2008; Fahy et al., 2011, 2005, 2009; Hauss, 2007). A pioneer in the field of lipid based systems was Small, who introduced in 1968 a lipid classification system based upon lipid– water interactions in bulk water and the behavior of lipids at the air –water interface (Small, 1968). Later, a lipid formulation classification system (LFCS) has been published by Pouton (2000, 2006). He arranged lipid-based formulations into four classes based upon their composition and the expected impact of dilution and digestion on the biofate of the lipid carrier and the drug. Class I formulations are the most lipophilic. They have the disadvantage that digestion is required to facilitate their dispersion. The digestion products might have different drug solubilisation capacity and the solubilisation is strongly dependent from gall bladder activity. This might introduce a variability and food dependency. On the opposite side, Class IV formulations are oilfree and rather polar systems based on surfactants and co-solvents. In general, they are less sensitive toward digestion but more sensitive toward dilution. Self-emulsifying drug delivery systems are composed of two or more ingredients, which provide the self-emulsifying properties: more hydrophilic amphiphiles, more lipophilic amphiphiles and sometimes co-solvents or a precipitation inhibitor. Upon mild agitation and dilution in the gastrointestinal fluids, these systems transform into oil in water (O/W) emulsions (SEDDS), microemulsions (SMEDDS) or nanoemulsions (SNEDDS). Self-emulsification increases the bioavailability by the circumvention of drug crystal dissolution, which is often insufficient and highly variable for poorly soluble drugs. However, SEDDS show in many cases low drug loads and sensitivity to dilution. An increase of the lipophilic components leads in many cases to higher drug loads. However, the self-emulsifying properties are decreased. Therefore, a balanced composition is necessary to ensure high drug loads and self-emulsification. SEDDS should not only be able to solubilize the drug, but also be able to maintain drug solubilization throughout the gastrointestinal tract (Dokania and Joshi, 2014). Therefore, co-solvents (e.g., ethanol, propyleneglycol) should be avoided due to the risk of drug precipitation upon dilution in gastrointestinal fluids and the negative impact on capsule shelf-life stability. Furthermore, a precipitation inhibitor (e.g., HPMC) could be added to generate and maintain a metastable supersaturated drug state throughout the gastrointestinal tract (supersaturable SEDDS) (Date et al., 2010; Kohli et al., 2010; Patel et al., 2010; Rahman et al., 2011, 2013; Song et al., 2013). SEDDS are typically formulated as a liquid dosage form and are usually contained in soft gelatine capsules. A good example of a very successful formulation is Neoral1/Optoral1 (Novartis). It is composed of a mixture of mono-, di- and triglycerides as lipophilic amphiphiles, Cremophore1 RH 40 as a hydrophilic amphiphile, propylene glycol and ethanol as co-solvents and tocopherol as an antioxidant (Strickley, 2004). It forms spontaneously a transparent dispersion with particle sizes below 100 nm upon dilution with aqueous media (Vonderscher and Meinzer, 1994). Nevertheless, disadvantages of soft gelatine capsules include the requirement of specialized manufacturing equipment requirements the high potential of stability issues due to incompatibility problems between the filling and the capsule shell (Balakrishnan et al., 2009 Yi et al., 2008). The incorporation of self-emulsifying mixture into a solid or a semisolid dosage form is an alternative approach (Abdalla and Mäder, 2007). It combines the benefits of liquid SNEDDS with those of solid dosage forms. Nevertheless, it is very challenging because self-emulsifying properties are more difficult to achieve with solid materials. However, the potential advantages of solid self-emulsifying dosage forms have motivated several authors (Agarwal et al., 2012; Beg et al., 2013; Breitkreitz et al., 2013; Kanuganti et al., 2012; Li et al., 2013; Onoue et al., 2012;

Salve, 2011; Shah and Serajuddin, 2012; Shanmugam et al., 2011; Singh et al., 2014, 2013; Tang et al., 2008). Several approaches were developed to produce solid SNEDDS (Mallikarjun and Rajesh Babu, 2011). One approach is the solidification of selfemulsifying liquids by encapsulation or adsorption. The alternative is the use of amphiphiles with strong solid character. This approach provides a simple way of production and scale up. As semisolid amphiphiles, Cithrol1 DPHS (DPHS) and Kolliphor1 HS 15 (HS15) were selected. As solid amphiphiles, Soluplus1 and Cithrol1 GMS 40 (GMS) were selected. Capmul1 MCM was tested as a co-emulsifier. Cithrol1 DPHS (DPHS), formerly Arlacel1 P135, (PEG-30dipolyhydroxystearate) is a polymeric W/O emulsifier with a HLB value of 5.5. DPHS is an ABA block copolymer. The middle block (B) is the hydrophilic poly(ethylene oxide) while the two outer blocks (A) are poly(12-hydroxystearic acid) (Plaza et al., 1999). DPHS offer some advantages that enable it to be a good candidate for SNEDDS. However, to best of our knowledge, its potential use as a lipophilic excipient in SNEDDS was not yet explored. The proposed advantages include:

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a) DPHS is semisolid at room temperature. Semisolid formulations

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have higher viscosity than the liquid ones. Therefore, they usually afford high drug stability and better handling (Singh et al., 2014). b) is a saturated long chain lipid. Saturated lipids are more resistant to oxidation offering longer shelf-life stability than the unsaturated ones, while long chain lipids are known to enhance the lymphatic uptake of the poorly water-soluble drugs. Therefore, the accompanied drug may avoid the first pass metabolism (Singh et al., 2014). Furthermore, the digestion products of long chain lipids are able to enhance the solubilization capacity of the SNEDDS, when combined to the bile mixed micelles (Porter et al., 2007). c) The polymeric nature of the DPHS enables the manufacture of very stable emulsions at low concentrations in the presence of additives that are incompatible with traditional W/O emulsifier systems (Croda GmbH, 2009). DPHS has been used to prepare stable semisolid multiple W/O/W emulsions (Dai et al., 2006; Ferrari and da Rocha, 2011; Kanouni et al., 2002; Vasiljevic et al., 2006, 2005, 2009). d) DPHS is a non-ionic, high molecular weight amphiphile. Therefore, it offers low oral toxicity. A published report from the European Food Safety Authority in 2004 demonstrates its safety based on the safety of its starting substances (PEG and 12hydroxystearic acid) and its average molecular weight (>5000 D). Furthermore, the Committee for Veterinary Medicinal Products has demonstrated that PEG stearates with 8– 40 ethylene oxide units are considered of low oral toxicity. The acute oral median lethal dose (rats) of DPHS is estimated to be more than 2000 mg/kg. (Committee for Veterinary Medicinal Products, 2003; Croda GmbH, 2010; European Food Safety Authority, 2004).

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Soluplus1 is a relatively new excipient, which was developed for melt extrusion. It consists of polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Nagy et al., 2012). It has a strong solid character and amphiphilic properties. Soluplus1 was investigated as a precipitation inhibitor in a supersaturating SEDDS (Song et al., 2013). Kolliphor1 HS 15 (HS15), formerly known as Solutol1 HS 15, is a non-ionic amphiphile that has proved its potential usefulness for drug delivery and possesses a low toxicity (Coon et al., 1991; Kraus et al., 1990). HS15 consists of a mixture of circa 70% PEG mono- and di-esters of 12-hydroxystearic acid and circa 30% PEG (BASF AG, 2012; Strickley, 2004). The HLB is about 15 and the critical micelle concentration lies between

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0.005 and 0.02% (BASF AG, 2012). Capmul1 MCM (MCM) is a medium chain mono- and di-glyceride. It is widely used as a lipophilic excipient or co-surfactant in SEDDS with HLB value of 4.7. Cithrol1 GMS 40 is a long chain monoglyceride with an HLB value of about 3. It was therefore the aim of the current study to explore the potential of PEG-30-dipolyhydroxystearate and Soluplus1 as ingredients in novel semisolid/solid SNEDDS. The prepared SNEDDS should conform to the following criteria: a) Semisolid or solid consistency at room temperature. b) pH-independent self-emulsification upon mild agitation at

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C. c) High lipid mobility after dilution at physiological condition. d) Less susceptibility to oxidation due to absence of excipients with unsaturated bounds. e) Avoidance of co-solvents. The preparation of the semisolid SNEDDS is not trivial. The excipient selection is usually based on their solid properties, melting points, toxicity, drug solubility and HLB values (Rahman et al., 2013). However, the performance and dispersibility of the different combinations are difficult to predict. Therefore, phase diagrams are always made to help understanding the phase behavior of the combinations and to estimate the nanodispersion region. After ingestion, the SNEDDS will be subjected to gastric and intestinal fluids with different pH values and ions respectively. Semisolid formulations may suffer from poor in vivo performance due to the presence of the high melting point lipids (Singh et al., 2014). Furthermore, during melting and dispersion of the semisolid lipid formulations in physiological conditions, several phases might occur based on the temperature as well as the volume, pH and ionic strength of the dispersion media. These phases (ranging from crystals, gel, and lyotropic liquid crystals to micelles)- have different impact on the in vivo performance (dispersibility, digestibility, absorption of the lipids and the accompanied poorly water-soluble drug). For example, we observed in previous studies that sucrose ester nanodispersions are pH and ion-sensitive and might precipitate under physiological relevant conditions (Ullrich, 2008). Therefore, it is important to ensure sufficient lipid mobility and to avoid lipid crystallization and precipitation. Therefore it is very important to study the robustness of the semisolid formulation to dilution and to evaluate the mobility of the lipid formulation after dilution, especially at body temperature. Accordingly, the dispersibility and the droplet size distribution were evaluated in pH 1.2 and 6.8 as well as double distilled water to anticipate the effect of different pH-values and ions on the self-emulsifying properties. In addition, proton nuclear magnetic resonance (1H NMR) was used to assess the molecular mobility of the dispersed SNEDDS components. Photon correlation spectroscopy (PCS) was used to characterize the droplet size distribution after dilution. Differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD) were used to evaluate the physical state of SNEDDS. ESR has shown its usefulness for the characterisation of lipid nanodispersions in previous studies. For example, ESR has been used in the area of solid lipid nanoparticles, which quite frequently do not crystallise and remain for long periods in the state of supercooled melts. It has been shown by ESR that crystallisation dramatically decreases the drug load capacity of the lipid nanosdispersions and induces a translocation of incorporated compounds from the desired lipophilic environment into more polar microenviroments of the interphase and into the aqueous phase (Jores et al., 2003). It is obvious, that the characteristics of the microenvironment affect drug stability and release processes. Using the poorly water-soluble spin probe (Tempolbenzoate) as a

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drug model, electron spin resonance (ESR) was also used in the current study to characterize the micropolarity and microviscosity and to quantify the distribution of the probe between different microenvironments.

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2. Materials and methods

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2.1. Materials

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Cithrol1 DPHS and Cithrol1 GMS 40 were kindly provided by Croda GmbH, Nettetal, Germany. Kolliphor1 HS 15 and Soluplus1 were generous gifts from BASF AG, Ludwigshafen, Germany. Capmul1 MCM was kindly provided by Abitec Corporation, Janesville, Wisconsin, USA. Deuterium oxide, deuterated DMSO and Tempolbenzoate were purchased from Sigma–Aldrich, Steinheim, Germany. All other materials were of analytical grade and were used as received.

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2.2. Preliminary screening experiments

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DPHS, MCM and Cithrol1 GMS (GMS) were selected as lipophilic amphiphiles. As hydrophilic amphiphiles, HS15 and Soluplus1 were screened. Several combinations (Table S1 of the Supplementary data) were melted together and mixed. The mixtures were cooled down to room temperature and allowed to equilibrate for 24 h at 23
C prior to analysis. One percent dispersion of each formulation (m/V) was prepared in 10 ml of double distilled water, 0.1 N HCl or phosphate buffer pH 6.8 USP. The dispersions were incubated in a preheated (37  1
C) end over end mixer rotating at 10 rpm. The self-emulsification spontaneity and the appearance of the final dispersion were monitored visually at the specified time intervals (10 min, 30 min, 1 h, 2 h, 3 h, 4 h and 24 h).

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2.3. Pseudo-ternary phase diagram

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HS15 and DPHS were screened in different ratios (M1–M4). MCM was tested as a co-emulsifier. Two ratios of DPHS:MCM were extensively tested: 1:1 (M5–M15) and 2:1 (M16–M26). Furthermore, different ratios of DPHS:MCM (1:2.5, 1:3 and 1:4) were tested at HS15 content of 40–60% (M27–M35). In addition, at constant DPHS content of 20%, different ratios of MCM:HS15 (M36–M38) were tested. The exact composition of DPHS, MCM and HS15 combinations is summarized in Table S2 of the Supplementary data. The combinations were prepared and dispersed as described in Section 2.2. Furthermore, selected dispersions were investigated with a Zeiss Axiolab microscope (Carl Zeiss, Oberkochen, Germany).

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2.4. Effect of dilution

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One gram of the samples were heated to 37  1
C in an orbital mixing chilling/heating dry bath (Torrey Pines Scientific Inc., California, USA) operated at 600 rpm. The samples were then diluted with 0.1 N HCl to yield a 90% m/V lipid dispersion. The diluent was added in 10% (m/V) incremental steps every 15 min. The appearance of the final emulsion was monitored visually.

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2.5. Droplet size distribution

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Based on the results of the pseudo-ternary phase diagram, three formulations were selected for further characterization (Table 1). Formulations were analysed for droplet size distribution by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, U.K.) with a back scattering angle of 173
. Samples were dispersed as described at Section 2.2 and

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T.H. Hassan et al. / International Journal of Pharmaceutics xxx (2014) xxx–xxx Table 1 Composition of the PEG-30-dipolyhydroxystearate formulations (m%).

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Formulation code

Cithrol1 DPHS (%)

Capmul1 MCM (%)

Kolliphor1 HS 15 (%)

F1 F2 F3

20 26.67 30

20 13.33 10

60 60 60

measured at 37
C after an equilibration time of 20 min. Each sample was measured 3 times with 12–17 runs over 10 seconds. The measurement position was fixed in the middle of the cuvette. Z-average diameters and polydispersity indices (PDI) were determined by the cumulant analysis software of the instrument (Zetasizer Software 6.32, Malvern Instruments, Worcestershire, U. K.). The viscosity of the sample was evaluated at 37
C using Ubbelohde-Viscometer type 1 (Schott Geräte, Hofheim, Germany) using the following equation:

All 1H NMR spectra were recorded by a Gemini 2000 spectrometer (Varian, Les Ullis, France) operated at 400 MHz at 37
C. The analysis of the relative signal area and peak width at half amplitude was performed using MestReNova 6.0.2–5475 software (Mestrelabs Research S.L., Santiago de Compostela, Spain). Expected relative signal area calculations were based on the molar fraction of the individual excipient in the dispersion and the relative areas obtained from the dissolved DPHS and MCM in d-DMSO and micellar dispersion HS15 in D2O.

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h ¼ K  ðt  FÞ  r

2.10. Electron spin resonance (ESR)

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The nitroxide spin probe Tempolbenzoate (TB) was used as a model for poorly water-soluble drugs in a concentration of 2 mM/ kg. Several SNEDDS-dispersions; 1, 5, 10 and 20% (m/V) in phosphate buffer pH 6.8 USP were prepared. ESR spectra were recorded by means of a 9.3–9.55 GHz X-band spectrometer equipped with a nitrogen gas flow temperature regulation (MiniScope MS200, Magnettech, Berlin, Germany). The following parameters were used for emulsions: sweep 4.95 mT, sweep time 1200 s, modulation amplitude 0.1 mT and microwave attenuation 10 dB. The following parameters were used for excipients and anhydrous formulations: sweep 6.97 mT, sweep time 60 s, modulation amplitude 0.06 mT and microwave attenuation 10 dB. The obtained spectra were then fitted for either one isotropic rotation state (excipients) or two isotropic rotation states (anhydrous formulations and emulsions). Fitting of the EPR spectra was performed using EPRSIM Nitroxide spectra simulation software V. 4.99 (Biophysical laboratory EPR center, Jožef Stefan Institute, Ljubljana, Slovenia).

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3. Results and discussion

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3.1. Preliminary experiments

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Soluplus1 represents one of the recently developed amphiphiles that has strong solid character and is scarcely used in the development of SEDDS. Soluplus1 was screened as hydrophilic amphiphile in the proposed self-emulsifying formulations (P1– P9). The results are summarized in Table S1 of the Supplementary data. Formulations of Soluplus1 with GMS (P1–P2) had a solid consistency and could be moulded into a tablet-like dosage form. However, a very slow dispersion rate was observed. The produced mass did not disperse over 24 h, which is too long for a meaningful oral administration. GMS is a solid long chain lipid with low polarity and high melting temperature (60
C). Therefore, its combination with Soluplus1 has lower polarity and strong solid character. This could be the cause of the very slow dispersibility. Increasing Soluplus1 content (P2) slightly promoted the 24 h dispersibility. One approach to enhance the formulation dispersibility is to reduce the melting point of the formulation. Therefore, Soluplus1 was partially substituted with HS15 (P3). HS15 has a semisolid consistency, lower melting point and nearly the same HLB value of Soluplus1. The produced formulation retained the solid characteristics. However, compared to a published semisolid formulation consisting of GMS and HS15 (Abdalla and Mäder, 2007), they showed a slow dispersibility. Another approach is to

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where h is the sample viscosity, K is a device constant, F is Hagenbach correction, t is the time to pass through the two marks (average, n = 5) and r is the density of the sample. The density of the samples was determined by Mohr–Westphal balance (Wissenschaftlicher Apparate-bau Johannes Hammer, Leipzig, Germany). The sample density was found to be constant for all samples (0.997 g/cm3). The measured viscosity for F1 was 0.7622 mPa s, F2 was 0.7722 mPa s and F3 was 0.7821 mPa s.

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2.6. Percentage transmittance

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One percent dispersions (m/V) were prepared as described in Section 2.2. The optical clarity expressed as percentage transmittance (%T) was measured spectrophotometrically at 650 nm using a Spekol 1200 spectrometer (Analytik Jena GmbH, Jena, Germany). Blank dispersion media were used as References.

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2.7. Differential scanning calorimetry (DSC)

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DSC investigations were performed using a nitrogen purged Netzsch DSC 200 (Netzsch-Gerätebau GmbH, Selb, Germany). All samples (approx. 15 mg) were accurately weighed in aluminum pans and hermetically sealed to avoid water evaporation. DSC thermograms were generated by cooling the samples to 20
C. The samples were then equilibrated for 3 min and the heating thermograms at a rate of 5 K/min up to 200
C were recorded. Furthermore, the second and the third heating and cooling thermograms of F2 were recorded.

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2.8. Powder X-ray diffraction (PXRD)

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Samples were measured using a powder X-ray diffractometer (STADI MP, Stoe, Darmstadt, Germany) equipped with a curved Ge (1 11) monochromator (CoKa radiation, l = 0.178896 nm) operated at 40 KV and 30 mA and an image plate detector at room temperature. The samples were scanned from 5
(2u) to 123
(2u) with a step size of 0.03
(2u ) and a count time of 1000 s per step.

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2.9. Proton nuclear magnetic resonance (1NMR)

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Formulations were dispersed in deuterium oxide (D2O) (5% m/ V) using an end over end mixer at 37  1
C. Excipients were dissolved in d-DMSO. HS15 was also dispersed in D2O (3% m/V) to investigate the chemical shift corresponding to the micellar form.

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increase the polarity of the formulation. Accordingly, the content of the hydrophilic excipients were increased on the favor of the lipophilic one (P4–P5). The formulations have kept the solid character to certain extent. Nevertheless, the dispersibility did not improved, especially with higher Soluplus1 content. GMS (HLB ca. 3) was substituted with DPHS (HLB ca. 5.5) (P6) to increase the formulation polarity and reduce the melting point. A non-dispersible mass with an oily surface was obtained. Further reduction in the melting point and modulation in the formulation polarity (P7) resulted in a milky, incomplete dispersion. Substitution of the solid long chain monoglyceride (GMS) with a liquid medium chain monoglyceride (MCM) (P8) resulted in complete milky dispersions in all media. Formulation with lower content of Soluplus1 (P9) showed pH dependent dispersibility. Nanodispersions were produced in both water and 0.1 N HCl media, whereas milky dispersions were produced in phosphate buffer pH 6.8. However, the formulation has a semisolid consistency. In order to prepare formulations that melt at body temperature, DPHS was selected as a lipophilic amphiphile and HS15 as a hydrophilic one (P10). Both excipients are semisolid at room temperature. The formulation had a semisolid consistency. However, it was non-dispersible. Incorporation of a monoglyceride in the formulation was found to be necessary for the selfemulsifying process (P11–P24). The monoglyceride may act as a cosurfactant and promote the dispersibility of the formulations. Two monoglycerides were evaluated: GMS (solid, long chain) and MCM (liquid, middle chain). DPHS-GMS formulations (P11–P21) were semisolid. The formulations became softer with the increase of the HS15 content relative to the lipophilic excipients. All formulations provided a turbid, milky and pH independent dispersions within 1–3 h. The dispersion rate and turbidity was a function of both HS15 content and DPHS-GMS ratio. Faster dispersion rate and lower turbidity were observed with higher content of HS15 and

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DPHS:GMS ratio close to 1. However, even with relatively high HS15 content (P21, 80%), still a reasonable amount of the droplet size in the micro range. Therefore, the solid monoglyceride was substituted with a liquid one (P22–P24). Compared to the long chain monoglycerides, the middle chain ones has a higher flexibility at the interfacial film, which is a very important requirement for the production of the nanoemulsion (Dokania and Joshi, 2014). All formulations were semisolid at room temperature and were capable of producing nanodispersion in water. However, a pH dependent dispersibility were observed with varying the ratio of DPHS:MCM. Based upon the aforementioned results of the preliminary studies, combinations between DPHS, MCM and HS15 could be able to produce pH-independent nanodispersions. Therefore, these combinations were selected for further studies. Soluplus1 combinations produced milky dispersions and in some cases exhibited a pH dependent dispersibility or even very slow dispersibility, which is out of our scope of work.

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3.2. Pseudo-ternary phase diagrams

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Pseudo-ternary phase diagrams are constructed to identify nanoemulsion regions and accordingly the suitable ratios between hydrophobic and hydrophilic amphiphiles. Many studies in the literature describe pseudo ternary phase diagrams at ambient temperature. However, the mobility and dispersion characteristics of lipid molecules are very sensitive to temperature (Koynova and Tenchov, 2007). It was the aim to prepare SNEDDS that are semisolid or solid at room temperature and have high mobility and kinetic stability at body temperature. Therefore, all experiments were performed at body temperature. In order to identify and optimize the nanoemulsifying range, 38 different combinations of DPHS, MCM and HS15 (Table S2 of the

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Fig. 1. Physical state of different combinations of Kolliphor1 HS 15, Cithrol1 DPHS and Capmul1 MCM after equilibration at 23
C (a) and pseudo-ternary phase diagrams of Q5 their 1% (m/V) dispersions in (b) double distilled water, (c) 0.1 N HCl and (d) phosphate buffer pH 6.8 at 37
C. For the physical state, the green zone represents semisolid state. The blue zone represents liquid state without any phase separation. The red zone represents liquid state with phase separation. For the pseudo-ternary phase diagram, the green zone represents the nanoemulsion region. The blue zone represents the milky dispersions. The red zone represents no dispersion or a dispersion with very large droplet size. The value of 100 in the phase diagram represents 1% (m/V) dispersion of the corresponding excipient. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Effect of dilution on combinations prepared from Kolliphor1 HS 15 and Cithrol1 DPHS: Capmul1 MCM (a) 1:1 and (b) 2:1 using 0.1 N HCl as a diluent. The green zone represents the one phase or nanoemulsion. The blue zone represents emulsions with large droplet size. The red zone represents 2 phases. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479

Supplementary data) were prepared. The physical state of these combinations is represented in Fig. 1a. All DPHSHS15 combinations (M1–M4) were semisolid at room temperature. Combinations of HS15 and 1:1 DPHS:MCM (M5–M15) were liquid at lower HS15 amounts. After storage at room temperature for several months, phase separation was observed. At least 40% m/ m of HS15 is required to develop a semisolid formulation with 1:1 DPHS:MCM. Increased DPHS to MCM ratios (M16–M35) resulted in semisolid formulations, regardless of the HS15 amounts. At 20% DPHS, the variation of HS15:MCM ratio (M36–M38) resulted in liquid formulations that did not exhibit phase separation. Fig. 1b–d illustrates the pseudo-ternary phase diagrams of 1% m/V dispersions obtained in different media. The phase diagrams are divided into 3 distinct regions: (a) region in which phase separation was observed or dispersions with rather large droplet sizes were obtained, (b) region with a milky dispersion and (c) isotropic nanoemulsion region. Examples of the optical microscopic images of selected dispersions in the three mentioned regions are shown in Fig. S1 of the Supplementary data. Although both DPHS and HS15 has an amphiphilic properties, their combinations (M1–M4) were not found to be self-emulsifying over 24 h. The presence of MCM was essential for the selfemulsification even in a small concentration. At lower HS15 concentration (below 60 %), the process of self-emulsification was sensitive to the ionic strength and pH of the media. The selfemulsification process was better in water and 0.1 N HCl compared to pH 6.8 buffer. However, above 60% of HS15, this sensitivity was less pronounced. Therefore, at least 60 % of HS15 is required to obtain a transparent nanoemulsion in almost all media.

Lower amounts of MCM resulted in a slower kinetics of selfemulsification (minutes to hours) while higher amounts resulted in pH (or ionic strength) dependent dispersibility as well as the formation of more coarse and instable dispersions. Substitution of MCM (liquid) with Capmul1 MCM C10 (solid) or Cithrol GMS 40 (solid) did not alter the overall consistency, but did result in dispersions with increased turbidity. Formulations with a DPHS: MCM ratio of 2:1 was optimal in terms of the emulsification rate and has a low sensitivity to pH and ionic strength and formulation stability. Increasing the DPHS:MCM ratio to 4:1 or even 5:1 gave SNEDDS with slower emulsification rates (in terms of hours). The increase in the viscosity of the more lipophilic part and its semisolid properties accompanied with the increase of DPHS ratio relative to MCM can be attributed to this effect.

480

3.3. Effect of dilution

494

After oral administration, the gastrointestinal fluids will dilute the SNEDDS. The degree of dilution and the local pH might be highly variable in vivo. Therefore, it is important to mirror the in vivo situation by in vitro tests. Fig. 2 demonstrates the effect of dilution on the appearance of the corresponding emulsion. Although the proposed dilution test is not a good simulation to the physiological dilution, it helps us to understand what is happening to the formulation during the dilution and which composition gives a stable dispersion upon dilution. Dilution by up to 20% 0.1 N HCl yields single phase mixtures. The presence of DPHS supports the emulsification of a considerable high amount of water into the oil phase. Upon further dilution, the emulsion is inversed and an O/W emulsion is formed at higher

495

Table 2 Average droplet size diameter of the Kolliphor1 HS 15 micelles and the nanoemulsion produced by 1% (m/V) dispersion in different media measured by dynamic light scattering at 37
C (mean, n = 3). Formulation/excipient

Double distilled water

0.1 N HCl

Phosphate buffer pH 6.8

Z-average (nm)

PDI

Z-average (nm)

PDI

Z-average (nm)

PDI

Kolliphor1 HS 15 F1 F1 (filtered)b

12.5 25.1 27.0

0.033 0.16 60.145

12.7 75.3 69.5

0.021 0.277 0.250

11.1 NAa 41.3

0.022 NAa 0.238

F2 F2 (filtered)b F2 (cent.)c

18.2 16.6 17.8

0.245 0.091 0.150

21.5 18.3 19.1

0.299 0.086 0.090

21.7 19.3 21.2

0.272 0.107 0.114

F3 F3 (filtered)b

19.9 17.2

0.334 0.179

21.7 17.4

0.334 0.121

20.5 16.9

0.310 0.070

a b c

NA is not available. Sample was too polydisperse. As a result, data quality was too poor for cumulant analysis. 0.22 mm filter. 12,045 g/5 min.

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HS15 concentrations. The stability of the formed nanoemulsions was found to be dependent on the HS15 level and DPHS:MCM ratio. At lower HS15 content, the formed emulsions can easily separate into two phases. Higher DPHS:MCM ratio (2:1) are more hydrophobic and were not able to accommodate more water at lower contents of HS. Therefore, phase separation was observed. At moderate level of HS15 (40–50%), a stable dispersion with large droplet was formed during dilution.

516

3.4. Selection of formulations for further investigation

517

Based on the aforementioned results, three formulations (Table 1) were selected for further investigations. The HS15 content was kept constant at 60% while the lipophilic part of the formulations was kept at 40% with different DPHS:MCM ratios. All formulations are semisolid at room temperature (Fig. S2a of the Supplementary data) and provided a fine dispersion in the three tested media within few minutes (Fig. S2b–d of the Supplementary data). An exception was noticed in the case of the 1% (m/V) F1 formulation in phosphate buffer pH 6.8. It produced a milky dispersion at 37
C (Fig. S2d left of the Supplementary data). A clear dispersion with low turbidity was obtained after this dispersion was cooled to room temperature.

509 510 511 512 513 514

518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546

3.5. Droplet size distribution One percent dispersions (m/V) of formulations and HS15 in different media were incubated in an end over end mixer at 37  1
C and the formed nanoemulsion was evaluated for the droplet size distribution (Table 2). HS15 micelles showed a monomodal distribution with an average hydrodynamic diameter of 11–13 nm. Incorporation of more lipophilic excipients (DPHS and MCM) increased the hydrodynamic diameter. F1 dispersions showed a high degree of sensitivity toward the ionic strength and pH of the dispersion media. It gave a milky dispersion in the buffer media, which was not suitable for PCS measurements due to the high degree of polydispersity. F2 and F3 formulations showed monomodal volume distributions in all media with an average hydrodynamic diameter of less than 25 nm. The effect of the ionic strength and pH on the particle size distribution of F2 and F3 nanoemulsions was minimal. Moreover, filtered and centrifuged nanoemulsions of F2 did not show a prominent change in the droplet size volume distribution (Fig. S3 of the Supplementary data).

Fig. 3. Percentage transmittance of 1% m/V dispersions of formulations in different media at 650 nm (mean  SD, n = 3).

7

Fig. 4. Heating DSC thermograms of excipients, different excipient combinations and formulations.

3.6. Percentage transmittance

547

In order to evaluate the optical clarity quantitatively, UV–visible spectrophotometer was used to measure the transmitted light at 650 nm. Fig. 3 demonstrates the values of the percentage transmittance of 1% (m/V) dispersions of formulations in different media. All dispersions were transparent (%T > 94%) with the exception of the formulation F1 in phosphate buffer pH 6.8.

548

3.7. Differential scanning calorimetry (DSC)

554

To examine the thermal behavior and possible excipient interactions DSC measurements were performed on the single excipients, excipient combinations and formulations (Fig. 4). The melting endotherm of DPHS was above the body temperature. HS15, being a mixture of two main components (free PEG and PEG esters), showed two melting endotherms. MCM showed a very broad melting endotherm, which is typical for lipids mixtures. The DSC study indicated an interaction between the more lipophilic components of the formulations (DPHS and MCM) with a shift of the DPHS melting endotherm toward the MCM melting range. MCM is probably capable of dissolving DPHS in a ratio of 1:1 and 1:2 and to a certain extent in a ratio of 1:3. This points toward the importance of MCM in the formulations and explains the slower dispersibility observed with decreasing MCM content. Furthermore, the enhancement of the mobility of the

555

Fig. 5. PXRD pattern of F2.

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mixture compared to DPHS alone at body temperature is expected. A similar pattern was observed between MCM and HS15. On the other hand, the interaction between HS15 and DPHS was not pronounced. All formulations showed a melting endotherm above room temperature and below 37
C with a very slight positive shift in the melting endotherm with the increase of the DPHS content in the formulation. Accordingly, all formulations are expected to be highly mobile at body temperature. F2 showed a peak melting endotherm at about 31.5
C. The second heating cycle showed a very broad melting endotherm and a shifting of the peak melting temperature to about 27.2
C and crystallization exotherm at about 12.1
C (Fig. S4 of the Supplementary data). The third heating cycle was overlaying the second heating one.

584

3.8. Powder X-ray diffraction (PXRD)

571 572 573 574 575 576 577 578 579 580 581 582

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PXRD was used to evaluate the solid state of the SNEDDS (F2). PXRD pattern of F2 is shown in Fig. 5. Based on the chemical composition, X-ray signals from lipid structures and from PEGunits can be expected to occur. The diffraction pattern of F2 revealed the presence of broad signals between 15
–30
, which reflect the short spacings. The signals can be used to evaluate the cross sectional packaging of the lipid hydrocarbon and PEG chains. Short spacing is independent on the chain length and can be used to characterize the polymorphism of lipids (D'Souza et al., 1990;

deMan, 1992). Two stronger signals were observed at 4.6 Å and 3.8 Å. Overall, the signals indicate a partial amorphous and partial crystalline system with a superposition of different spacings. Due to the chemical composition of the excipients, the formation of crystals with very well defined spacings and small line widths is not expected.

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3.9. Proton nuclear magnetic resonance (1NMR)

600

1 H NMR spectroscopy measurements can provide good information about the structure and mobility of the lipid in the nanodispersed systems (Jores et al., 2003). The mobility of nanodispersed systems are essential for its in vivo performance. After ingestion of lipids, different phases with different degree of mobilities were developed. The 1H NMR signals of mobile lipids are characterized by narrow lines with high amplitudes. On the other hand, signals of less mobile lipids are characterized by broader lines and lower amplitudes. Immobile lipids cannot be detected by standard 1H NMR spectroscopy because of their very short relaxation times (Jores et al., 2003; Schubert et al., 2006). To facilitate the identification of the signals corresponding to each excipient in the nanoemulsion, 1H NMR spectra of all excipients were recorded at 37
C in d-DMSO (Fig. 6). 1H NMR spectra of 5% (m/V) SNEDDS dispersions in D2O at 37
C as well as micellar dispersion of 3% (m/V) HS15 at 37
C in D2O are shown in Fig. 7.

601

Fig. 6. 1H NMR spectra of excipient dissolved in d-DMSO at 37
C. All signals are signed corresponding to the positions of the protons in the chemical formula above.

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Fig. 7. 1H NMR spectra of 5% (m/V) dispersions of formulations in D2O at 37
C compared to the micellar spectra of Kolliphor1 HS 15 in D2O. 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 Q2 652 653 654 655 656 657 658 659

MCM is a medium chain complex lipid mixture of mono-, diand triglycerides of caprylic acid (C8) and capric acid (C10) as well as free glycerol (Chen et al., 2009). The assignment of the 1H NMR signals to MCM was performed in agreement with the literature (Knothe, 2006; Lie Ken Jie and Lam, 1995). The multiplet (e) with a shift between 4.0 and 4.2 ppm corresponding to the esterified glycerol protons can be used to identify MCM in emulsified formulations. No interference of these signals with signals from the other excipients was observed. The MCM multiplet (e) was also detected in the three emulsified formulations (Fig. 7 bottom center). The observed decrease in the signal amplitudes for (e) from F1 to F3 is caused by the decrease of the MCM content in the formulations. DPHS is an ABA block copolymer. The middle block (B) is a PEG chain while the two outer blocks (A) are poly(12-hydroxystearic acid) (Plaza et al., 1999). HS15 consists of a mixture of circa 70% PEG mono- and di-esters of 12-hydroxystearic acid and circa 30% PEG (BASF AG, 2012; Strickley, 2004). Due to the ownership of the same building blocks, DPHS and HS15 proton NMR signals have nearly the same chemical shifts. However, the relative intensity of the signals and some line widths differ according to the different ratios of the building blocks and their different mobility. In d-DMSO solution of DPHS, the PEG signal was broader due to fact that PEG is localized in the central B block of the polymeric emulsifier (Fig. 6). Due to the covalent linkage at both sides, its flexibility is restricted compared to free PEG and PEG linked only at one end. Furthermore, all DPHS signals are very broad and -in contrast to HS15- no real baseline could be observed in the region between 0.5 and 1.5 ppm. The PEG chains of HS15 are free either at one (the other linked to hydroxystearic acid) or even at both ends (free PEG content ca. 30%). Therefore, compared to the DPHS, a higher mobility is expected. Indeed, the PEG specific proton NMR signals of HS15 solutions in d-DMSO have narrow line widths and high signal amplitudes with a good signal resolution (Fig.7). The values of the chemical shifts of HS15 micelles (Fig.7) in our study are in good agreement with literature data (Momot and Kuchel, 2003). All signals of the aqueous micellar dispersion (Fig.7) were shifted downfield compared to the HS15 signals in d-DMSO solutions (Fig. 6). The change of the solvent and the proximity of the amphiphilic chains to each other in the micellar structure may be responsible for such effect. In the aqueous dispersions shown in Fig. 7, we did not observe two separated signals for the same

protons which could occur due to their localisation in different environments (e.g., molecular dispersed in water and associated in micelles or SNEDDS droplets). One explanation for this observation would be the dominance of one species. Another reason could be a fast exchange between both species. Micelles are not static entities. They are constantly reorganizing themselves by rapid reversible exchange between the surfactant molecules localised in the micelles and molecular dissolved surfactants. Under the used experimental setup, we assume that this exchange is faster than the NMR-timescale. As a result, both signals are averaged and not recognisable as two different species. The terminal methylene group of the free PEG (G0 ) can serve as an identification of HS15 in the emulsified formulations (Fig. 7 bottom right). Because of the constant free PEG fraction in the formulations, the signal area remained unchanged. However, it was more difficult to assign a signal for DPHS. Due to its higher molecular weight (circa 5000 Dalton) compared to HS15 (ca. 854 Da) and its relatively lower content, the DPHS fraction in the dispersion was too low to be compared to HS15. The (D0 ) signal corresponding to C12 in the di-ester is stronger in DPHS compared to HS15. An increase in the (D0 ) signal area in the emulsified formulations was observed with the slight increase in DPHS mole fraction (Fig. 7 bottom left). 1 H NMR spectra of the SNEDDS formulations showed four distinct signals; (a) the terminal methyl signal at 0.92 ppm, (b) the fatty acid chain signal at 1.33 ppm, (c) the PEG signal at 3.70 ppm and (d) the HOD signal at 4.66 ppm. In order to assess the mobility of the hydrophilic and lipophilic part of the formulations, a

Table 3 Comparison between expected and measured relative signal areas of the emulsified formulations. —CH2—/—CH3

F1 F2F3 F2F3

PEG/fatty acid chain (—CH3 + —CH2—)

Expected*

Measured

(%)

Expecteda

Measured

(%)

6.2:1 6.8:1 7.1:1

6.5:1 7.0:1 7.4:1

104.2 103.4 104.0

1.80:1 1.77:1 1.76:1

1.59:1 1.44:1 1.38:1

88.3 81.1 78.1

* Calculations are based on the relative signal areas obtained from dissolved Cithrol1 DPHS and Capmul1MCM in d-DMSO and micellar dispersion of Kolliphor1 HS 15 in D2O. a Calculations are based on the relative signal areas obtained from dissolved Cithrol1 DPHS in d-DMSO and micellar dispersion of Kolliphor1 HS 15 in D2O.

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Table 4 Line width at half amplitude of the main signals of emulsified formulations and micellar Kolliphor1 HS 15. Line width at half amplitude of the respective signal (Hz)

Kolliphor1 HS 15 F1 F2 F3

688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721

Terminal —CH3(0.92 ppm)

Chain —CH2—(1.33 ppm)

PEG(3.70 ppm)

13.6 13.6 14.0 14.4

16.0 18.0 17.6 18.0

4.4 7.6 6.8 6.4

comparison between expected and measured relative signal areas was constructed in Table 3. Furthermore, the spectra were assessed in terms of line width at half amplitude of the respective signal (Table 4) (Jores, 2004; Noack, 2012). The line widths indicate an overall high degree of mobility. Due to its position, the terminal lipid methyl signal is the most sensitive signal to the mobility of the hydrophobic part of the emulsified formulations. A small increase in line width was observed with the increase in DPHS content in the hydrophobic part of the formulation (Table 4). This reflects a small decrease in mobility of the hydrophobic part with the increase of the local viscosity caused by an increased DPHS content. However, the restriction in the mobility was not pronounced compared to micellar HS15. The line width of the —CH2— groups of the fatty acid chain signal did not considerably change within the formulations (Table 4). Nevertheless, the signals were broader compared to micellar HS15 signals due to the incorporation of the hydrophobic part within the micelles. The measured —CH2/—CH2— ratios were nicely in agreement with the calculated ratios (Table 3), indicating that all excipients were in a mobile status. The PEG signal was used to evaluate the mobility of the hydrophilic part of the emulsified formulations (Fig. 7). The relative value of the PEG/—CH2— + —CH3 ratio of the NMR signals of micellar HS15 was about 84% of that of the dissolved HS15. This result can be attributed to the increased broadness of the micellar signals due to the decreased mobility of PEG at the interface. All dispersed formulations showed a broader PEG signal compared to the micellar HS15. Surprisingly, the observed PEG signal line width decreased with the increase of DPHS ratio. This could be attributed to a partial replacement of HS15 by DPHS at the interface. The replaced HS15 would have a higher water affinity and show a narrow line width in the molecular dispersed state. The results indicate that only a part (78–88%) of the PEG groups was detectable by NMR under our experimental conditions. The missing part is

most likely localised at the water–lipid interface with a restricted mobility.

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3.10. Electron spin resonance (ESR)

724

ESR, also known as electron paramagnetic resonance (EPR), is a beneficial non-invasive tool to detect and characterise free radicals and other paramagnetic compounds. It is very useful to explore the microenvironment inside drug delivery systems with respect to micropolarity, mobility, microviscosity as well as pH. Most of the drug delivery systems are diamagnetic and EPR silent. Consequently, incorporation of spin probes is essential. In many cases, nitroxides are used, which show in solution 3 ESR lines due to impact of the 14N- nuclear spin (I = 1). A wide range of spin probes with different physicochemical properties is commercially available (Kempe et al., 2010). The spin probe Tempolbenzoate (TB) was used as a model compound with submillimolar solubility in water, a log P of 2.46 and HLB of 6.7 (Abdalla and Mäder, 2009; Kuentz and Cavegn, 2010). The hyperfine coupling constant (aN) can serve as an indication of the polarity of the microenvironment. The value of aN increases with polarity (Fig. S5 of the Supplementary data). The hyperfine coupling between the unpaired electron and the nuclear spin is anisotropic. Because of the non-spherical structure of the TB molecule, the spectrum can be described as a weighted average of two rotation states, a slower and a faster one. Microviscosity affects the tumbling speed expressed as the rotation correlation time (t c). In a low viscosity microenvironment, the anisotropy is nearly averaged due to the rapid movement of the spin probe and a nearly isotropic hyperfine splitting is obtained. Under such conditions, the ESR spectra of nitroxides show three similar lines with similar small line widths and signal amplitudes. An increase of the microviscosity results in an incomplete averaging of the anisotropy. Consequently, broader peaks are obtained. The broadening

725

Fig. 8. ESR spectra of Tempol benzoate in different excipient and combinations at 37
C (a) and the values of the hyperfine coupling constant (aN) and rotation correlation time (t c) of Tempol benzoate obtained after data fitting (b). Squares symbolize t c while bars symbolize aN. Error bars are obtained from the fitting process and denote the accordance of the simulated states to the original data.

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effect is different for each line and most pronounced for the third peak (Mäder, 2006). ESR spectra of TB loaded excipients at 37
C and the results of their spectral fitting are shown in Fig. 8. MCM showed the highest polarity and lowest viscosity. On the other hand, DPHS showed the lowest polarity and the highest viscosity. A decrease in polarity accompanied with an increase in viscosity was noticed with the increase of DPHS ratio in the lipophilic mixtures. The ESR spectra of different MCM–DPHS combinations are shown in Fig. S6 of the Supplementary data. The ESR spectra were normalized to the same first line amplitude. Accordingly, the effect of viscosity can be observed on the peak height of the third peak of the normalized spectra. The spectra indicate a slight increase of microviscosity with increasing DPHS content. The results of the ESR fitting of DPHS formulations and their dispersions are shown in Fig. 9. The data can be fitted into two rotation states: R1 and R2 (Fig. S7 of the Supplementary data). The superposition of different rotation states is seen for both waterfree SNEDDS and 5% and 10% SNEDDS dispersions. Compared to R2, R1 has a lower hyperfine coupling constant (indicating a lower polarity) and a higher rotation correlation time (t c) (indicating a higher viscosity). In water-free SNEDDS, increasing the temperature from 25
C to 37
C leads to a decrease of the R1–R2 differences in the hyperfine splittings (Fig. 9a) und to a decrease of the rotation correlation times (Fig. 9c and d). The data indicate that different

11

nanocompartments exist at lower temperatures and that the temperature increase causes structural changes which transform the SNEDDS toward a more isotropic, less structured low viscous liquid. For SNEDDS dilutions, the hyperfine splittings are comparable to water-free SNEDDS despite a water content of 90% or 95% (Fig. 9b vs. a). The aN value of R1 decreases with increasing DPHS content (from F1 to F3), indicating the formation of a more lipophilic compartment. In contrast, the aN value of the more polar rotation state R2 does not change from F1 to F3 (Fig. 9b). R2 accounts for about 70% of the overall signal EPR signal intensity in the SNEDDS dispersions (Fig. 9e and f), but only to about 45% (Fig. 9c) and 30% (Fig. 9d) for undiluted SNEDDS at 25
C and 37
C, respectively. The SNEDDS composition (F1, F2 or F3) does not affect the rotation correlation time (t c) of the R2 rotation state (Fig. 9d–f), which is around 0.3 ns at 37
C both for undiluted and dilutes SNEDDS. In contrast, the R1 rotation state changes from F1 over F2 to F3, especially in SNEDDS dispersions (from 0.7 to 1.1 ns). It also depends on the dilution degree, especially for the more hydrophilic SNEDDS dispersions F1 and F2 (Fig. 9e and f). An overlay of the normalized ESR spectra of emulsified F2 spectra in phosphate buffer pH 6.8 is shown in Fig. 10. Samples were measured at higher modulation and longer time to increase the signal-to-noise ratio. ESR spectra of the one percent dispersions were difficult to fit because of the low signal-to-noise ratio. However, the 1% dispersion spectra clearly show a

Fig. 9. ESR fitting data of Tempol benzoate in formulations and emulsions. (a) The hyperfine coupling constant (aN) in formulations at 25
C and 37
C. (b) aN in 10% and 5% emulsions at 37
C. (c) The rotation correlation time (t c) and the proportion of each fitted states in the formulation at 25
C and (d) at 37
C. (e) t c and the proportion of each fitted states in the formulation in 10% and (f) 5% emulsion 37
C. Squares symbolize t c while bars symbolize aN. Error bars are obtained from the fitting process and denote the accordance of the simulated states to the original data.

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Fig. 10. ESR spectra of emulsified Tempolbenzoate loaded F2 in phosphate buffer pH 6.8. The spectra are normalized to the same amplitude of the first peak. The extra signal marked with (*) is a background signal of the resonator.

804

811

considerable localisation of the spin probe in a polar environment, indicated by the line shape and shift of the third peak. It can be concluded, that small molecules with a similar characteristics as TB (log P between 2 and 3; aqueous solubility submillimolar) will be mainly associated with the SNEDDS up to a dilution of 5% and will translocate into water with further dilution (or relocate in vivo into other sides such as proteins and lipid tissue). Furthermore, no evidence of TB precipitation was detected upon dilution.

812

4. Conclusion

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The feasibility of using the polymeric emulsifier PEG-30dipolyhydroxystearate as a lipophilic excipient for the production of SNEDDS was for the first time explored. Based on phase diagrams and dilution assay, semisolid SNEDDS with good selfemulsifying properties was successfully prepared. The formulations and the produced nanodispersions were comprehensively characterized by PCS, DSC, PXRD, ESR and 1H NMR. Presence of MCM is essential for the self-emulsifying process as it contributes to the flexibility of the interfacial layer. Higher MCM content results in a pH and ionic strength dependent dispersibility, while lower MCM content results in a slower dispersibility. The ratio DPHS:MCM 2:1 is ideal in term of the particle size, dispersibility and stability of the formed nanoemulsion. F2 and F3 show mono modal volume distributions in all tested media with an average hydrodynamic diameter less than 25 nm. All excipients can be detected by standard 1H NMR in their dispersions, reflecting a high molecular mobility, which is essential for the in vivo performance of the SNEDDS. Among the excipients, DPHS showed the lowest polarity and the highest viscosity. TB was mainly associated with the SNEDDS up to a dilution of 5% and translocated into water with further dilution. We believe that our study adds to the better understanding of the performance of the SNEDDS. Further investigations are carried out to study the digestibility of the proposed excipients and formulations as well as its impact on the biofate of the accompanied poorly water-soluble drug.

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Acknowledgements

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We would like to thank the Egyptian Ministry of Higher Education and Scientific Research and the Deutscher Akademischer Austauschdienst (DAAD) for the financial support as a PhD scholarship to Tamer H. Hassan, Assistant Lecturer, Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Suez

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Canal University, Ismailia, Egypt. Prof. Peter Imming is appreciatively acknowledged for his support in the 1H NMR section. Furthermore, we would like to thank Mrs. Kerstin Schwarz for the DSC measurements, Dr. Dieter Ströhl for the 1H NMR measurements and Dr. Christoph Wagner for the PXRD measurements. Abitec Corporation, BASF AG, and Croda GmbH are gratefully acknowledged for the excipient donation.

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Appendix A. Supplementary data

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijpharm. 2014.10.063.

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