Whey proteins as stabilizers in amorphous solid dispersions

Accepted Manuscript Whey proteins as stabilizers in amorphous solid dispersions

Jaya Mishra, Adam Bohr, Thomas Rades, Holger Grohganz, Korbinian Löbmann PII: DOI: Reference:

S0928-0987(18)30531-1 https://doi.org/10.1016/j.ejps.2018.12.002 PHASCI 4774

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

9 July 2018 16 November 2018 3 December 2018

Please cite this article as: Jaya Mishra, Adam Bohr, Thomas Rades, Holger Grohganz, Korbinian Löbmann , Whey proteins as stabilizers in amorphous solid dispersions. Phasci (2018), https://doi.org/10.1016/j.ejps.2018.12.002

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ACCEPTED MANUSCRIPT Whey proteins as stabilizers in amorphous solid dispersions Jaya Mishraa, Adam Bohra, Thomas Radesa,b, Holger Grohganza and Korbinian Löbmanna(*) a

Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100

Copenhagen, Denmark Department of Pharmacy, Faculty of Science and Engineering, Åbo Akademi University,

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20521 Turku, Finland

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*Correspondence: [email protected]; Tel.: + 45 35 32 25 41

Abstract

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Graphical Abstract

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Whey proteins are extensively used as nutritional supplements but have so far not been investigated as co-formers for amorphous solid dispersions (ASD) to enhance the solubility and dissolution rate of poorly water soluble drugs. In this study, whey protein isolate (WPI) and whey protein hydrolysate (WPH) were each mixed with three poorly water soluble drugs (indomethacin: IND, carvedilol: CAR and furosemide: FUR) and prepared as ASDs at 50% (w/w) drug loading using vibrational ball milling. Subsequently, solid state characteristics, dissolution rate and physical stability of the obtained samples were analyzed. All ASDs showed

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ACCEPTED MANUSCRIPT a significant increase in their glass transition temperatures, as well as faster dissolution rates and higher apparent solubilities compared to both the respective pure crystalline and amorphous drugs. The saturation solubility of the drugs was increased in the presence of the whey proteins, and the investigated ASDs showed supersaturation by attaining higher drug concentrations compared to the respective saturation solubilities. Upon storage, ASDs

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containing IND were found to be physically stable for at least 27 months, whereas, ASDs

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containing CAR or FUR were stable for about 8 months and 17 months, respectively. This was

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a tremendous increase in physical stability compared to the pure amorphous drugs which recrystallized within less than one week. Overall, WPI and WPH proved to be promising co-

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formers and amorphous stabilizers in ASD formulations.

Keywords: amorphous, solid dispersions, dissolution, whey proteins, poorly water-soluble

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drugs

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ACCEPTED MANUSCRIPT 1. Introduction Amorphous formulations are a very promising approach to increase the solubility and dissolution rate of poorly water-soluble drugs compared to drug delivery systems containing the drug in a crystalline form (Hancock and Parks, 2000). However, pure amorphous drugs are usually physically too unstable and tend to crystallize quickly back into a poorly water-soluble

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crystalline form of the drug (Laitinen et al., 2013). Thus, research efforts are directed towards

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finding excipients that can stabilize the drug in its amorphous form. In the past, various studies

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have been conducted to find appropriate stabilization approaches such as amorphous solid dispersions (ASDs) using polymers (Paudel et al., 2013) and co-amorphous (CoA)

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formulations using low molecular weight co-formers such as amino acids (AAs) (Dengale et

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al., 2016).

An ASD is defined as a dispersion of the drug in an amorphous polymer matrix at the molecular

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level (also known as a glass solution) (Huang and Dai, 2014), i.e. the drug is dissolved in the

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polymer. Several polymers, such as the water soluble polymer polyvinylpyrrolidone (PVP), have been extensively used as co-formers in ASDs. Since the drug is dissolved in the polymer, in theory a polymeric ASD is thermodynamically stable in case the drug loading is below the

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saturation solubility of the drug in a given (amorphous) polymer (He and Ho, 2015; Knopp et

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al., 2015). In this regard, one of the major disadvantages of polymeric ASDs is that drug solubility in the polymeric carriers is often rather limited, i.e. usually below 35% (w/w) (He and Ho, 2015; Knopp et al., 2015). Hence, in order to achieve stable ASDs, often large amounts of polymers are required to incorporate the drug dose, resulting in large bulk volumes of the final dosage form (Vasconcelos et al., 2007).

Abbreviations- aDS: apparent degree of supersaturation, ASD: amorphous solid dispersion, BM: ball-milling, CAR: carvedilol, CoA: co-amorphous, FUR: furosemide, IDR: intrinsic dissolution rate, IND: indomethacin, WPI: whey protein isolate, WPH: whey protein hydrolysate 3

ACCEPTED MANUSCRIPT CoA formulations are defined as amorphous multi-component systems consisting solely of low molecular weight compounds, where the components are usually mixed together at fixed molar ratios (most often at a 1:1 molar ratio (Löbmann et al., 2013b; Mishra et al., 2018)). In a CoA system, the drugs are frequently stabilized in the amorphous form by strong molecular interactions between the drug and the co-former (Chieng et al., 2009; Löbmann et al., 2013c).

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Especially AAs have been shown in the past to be excellent co-formers in CoA formulations

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(Löbmann et al., 2013a). Because of the low molecular weight of the excipients, CoA

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formulations usually require less stabilizing excipient and therefore, have been introduced as an alternative to polymer based ASD, allowing higher drug loadings. However, because the

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thermodynamically most stable forms for both, drug and excipient, are their respective crystalline counterparts, CoA systems are thermodynamically unstable and potentially may not

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offer sufficient amorphous stability (Newman et al., 2018). Recently, Pas et al. used proteins, i.e. polymers made of AAs, for ASD formulations (Pas et

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al., 2018). They examined gelatin as an alternative co-former for ASDs for twelve poorly

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water-soluble drugs at various drug loadings (5% to 40% (w/w)), prepared by freeze-drying, and found that it was possible to form either fully amorphous or partially amorphous systems,

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depending on the drug to gelatin ratio (w/w). It was found that at lower drug loadings (less than

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20%-30% (w/w)), most of the drugs could be prepared in a fully amorphous form, and these ASDs in general also had a significantly higher dissolution rate compared to the crystalline drug. However, these drug loading levels are comparable to those achievable with common polymeric ASDs (see above). Furthermore, the authors did not investigate the physical stability of these gelatin based ASDs. The purpose of the current study was to further explore the feasibility of proteins in the preparation and stabilization of ASDs. Herein, we investigate the use of whey proteins as an ASD co-former. Whey proteins are obtained from the dairy industry, and as shown in Table 1

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ACCEPTED MANUSCRIPT consist mainly of β-lactoglobulin, α-lactalbumin, bovine serum albumin (BSA), immunoglobulin G (IgG) and lactoferrin (de Wit, 1998; Eigel et al., 1984). Whey proteins are further processed and usually sold either as whey protein isolate (WPI) or whey protein hydrolysate (WPH). The exact composition of both WPI and WPH may vary depending upon the manufacturer. However, the WPI contains a high protein content (over 90% w/w) and less

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than 1% lactose and fat, while WPH contains a comparatively lower amount of protein with up

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to 10% of lactose and 8% fat (Qi and Onwulata, 2011; Bylund, 1995; EFSA Panel on Dietetic

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Products, 2010; Kilara and Vaghela, 2018; Mollea et al., 2013). The European Food Safety Authority (Regulation (EC) No 258/97 and Regulation (EC) No 1924/2006) defines whey

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proteins as novel food ingredient, which is completely safe for consumption (EFSA Panel on Dietetic Products, 2010). Furthermore mentioned is the application of whey proteins in the

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European Union as an ingredient in infant and follow-on formulae, meal replacement, maintenance of lean body and muscle mass, dietary foods for special medical purposes and

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reduction of body fat, physical performance (tissue repair, muscle recovery), and food

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supplements (EFSA Panel on Dietetic Products, 2010). The United States Food and Drug Administration (US-FDA) has also determined that native whey proteins in food are generally

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recognized as safe (GRAS) (under FDA 21 CFR 184.1979, FDA 21 CFR 170.30(b), FDA 21

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CFR 170.36(62 FR 18938)).

This study uses ball-milling (BM) to produce ASDs of poorly water-soluble drugs in combination with WPI and WPH as co-formers and stabilizing excipients. Three poorly watersoluble model drugs i.e. indomethacin (IND), carvedilol (CAR) and furosemide (FUR) were studied (Figure 1). The thermal properties, apparent solubility, dissolution performance and physical stability profile of the prepared ASDs were investigated.

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Figure 1. Chemical structure of (a) indomethacin, (b) carvedilol and (c) furosemide.

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Table 1: General composition of unprocessed whey protein mixture, commercial whey protein isolate (WPI) and commercial whey protein hydrolysate (WPH). Whey protein Weight % in Composition % Composition % of of WPI (Morr and WPH (Hernándezcomposition unprocessed whey protein mixture (Qi Ha, 2009) Ledesma et al., and Onwulata, 2011) 2005) β-lactoglobulin ~50-65 ~67.6-74.8 ~60 α- lactalbumin ~20 ~8.3-17.5 ~20 Bovine serum ~6 ~7.2-10.9 NR* albumin Immunoglobulin G ~10-14 ~5.9-7.5 NR* (IgG) Lactoferrin ~3 NR* NR* ** Lactose ~0.8-10 <1 ~10** ** Fat ~0.2-8 <1 ~8** *NR: Not reported, **(Bylund, 1995; EFSA Panel on Dietetic Products, 2010; Kilara and Vaghela, 2018; Mollea et al., 2013)

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ACCEPTED MANUSCRIPT 2. Materials and methods 2.1 Materials Indomethacin (IND, γ-IND, melting point (Tm): 160.5 °C) was purchased from Fagron (Copenhagen, Denmark), carvedilol (CAR, Form-II, Tm: 115 °C) from Cipla Ltd. (Mumbai,

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India) and furosemide (FUR, Form-I, Tm: 206 °C) from Sigma–Aldrich (St. Louis, MO, USA). Whey protein isolate (WPI) (commercial name: +ZERO WPI 90) and whey protein hydrolysate

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(WPH) (commercial name: WPI HD) were purchased from LSP Sporternährung (Bonn,

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Germany). All materials were used as received.

2.2 Methods Ball milling

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2.2.1

A total mass of 700 mg (1:1 weight ratios of drug-whey protein mixtures) was placed inside

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25 ml milling jars with two 12 mm stainless steel balls and continuous milling was performed

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at 30 Hz using a vibrational BM (MixerMill MM400, Retsch GmbH & Co., Haan, Germany), placed in a cold room (4 °C). In order to prepare the amorphous form of the pure drugs, the

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respective crystalline drugs were ball-milled for 60 min (IND, CAR) and 90 min (FUR), respectively. For preparation of ASDs, the IND-whey protein mixtures were ball-milled for 25

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min, whereas mixtures of CAR and FUR with whey proteins were ball-milled for 60 min, respectively.

2.2.2

X-Ray powder diffractometry (XRPD)

XRPD was performed in triplicate using a X´Pert PANanalytical PRO X-ray diffractometer (PANanalytical, Almelo, The Netherlands) with CuKα radition: 1.54187 Å, current: 40 mA and acceleration voltage: 45 kV. Each sample was scanned in reflectance mode between 2° and 7

ACCEPTED MANUSCRIPT 35° 2θ, at a scan rate of 0.067° 2θ/s and step size of 0.026°. The collected data was analysed using X´Pert PANanalytical Collector (PANanalytical, Almelo, The Netherlands) software.

2.2.3

Modulated differential scanning calorimetry (mDSC)

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Thermal analysis was performed using a Discovery DSC (TA Instruments, New Castle, DE, USA). Calibration of the equipment was carried out with indium. Each sample weighing

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approx. 6-8 mg was placed in an aluminium Tzero pan and sealed with an aluminium hermetic

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lid that had a pierced top. The samples were heated to 110 °C and kept isothermal for 10 min

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to remove any moisture in the samples, and subsequently cooled to -10 °C. The samples were then heated to 30 °C above the melting points of the respective drugs using a heating rate of

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2 °C/min with an underlying modulation amplitude of 0.2120 °C and a period of 40 s. A constant nitrogen flow rate of 50 mL/min was applied during each measurement. Using Trios

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software (TA Instruments, New Castle, DE, USA), the glass transition temperature (Tg,

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midpoint) was determined from the second heating run of each sample. All mDSC experiments were conducted in triplicate, and Tg values are reported as mean ± standard deviation.

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The theoretical Tgs of the drug-WPI and drug-WPH mixtures were calculated using the Fox

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equation (Lodge and McLeish, 2000) which is given by: 1 𝑇𝑔(𝑖,𝑖𝑖)

=

𝑤𝑖 𝑤𝑖𝑖 + 𝑇𝑔(𝑖) 𝑇𝑔(𝑖𝑖)

where Tg(i,ii) is the theoretical Tg of a fully amorphous drug-WPI or drug-WPH mixture, Tg(i) and Tg(ii) are the Tgs of the drug and co-former (WPI or WPH), respectively, and wi and wii are their weight fractions.

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Intrinsic dissolution

The intrinsic dissolution rate (IDR) of pure crystalline and amorphous drugs along with the ASDs of drug-WPI and drug-WPH samples was determined from powder compacts obtained with a hydraulic press (PerkinElmer, Hydraulische Presse Model IXB-102-9, Ueberlingen, Germany). Compacts of 150 mg were directly compressed at a pressure of 124.9 MPa for 45 s

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into stainless steel cylinders that served as intrinsic dissolution sample holders. The compacts

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had a flat surface with a surface area of 0.7854 cm2 and were placed in 900 ml of 0.1M

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phosphate buffer (pH 7.2, 37°C) dissolution medium at a rotation speed of 100 rpm. At predetermined time points (1, 3, 5, 10, 15 and 20 min), 5 ml aliquots were withdrawn and

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immediately replaced with dissolution medium of 37 °C. The obtained samples were then analyzed to obtain drug concentrations in mg/ml using an UV Evolution 300 spectrophotometer

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(Thermo Scientific, Cambridge, UK) at 320 nm, 302 nm and 295 nm for IND, CAR and FUR, respectively, since whey proteins have no absorption at these wavelengths (Wetlaufer, 1962).

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The drug release was then calculated as release per accessible surface area (mg/cm2), i.e. by

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multiplying the drug concentration with 900 ml and dividing it by the surface area occupied by the drug on the powder compact. It was assumed that the surface area of each powder compact

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(0.7854 cm2) exposed to the medium remained constant during the entire experiment.

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Since the drug only makes up a certain fraction in the ASD compacts next to the WPI or WPH, it was assumed that it also occupies a smaller relative area of the entire surface area of the compact. Hence, the available surface for the drug was calculated. Initially, the volume occupied (by both drug and whey proteins) was calculated by dividing mass (mg) of each compound within the compact by its true density. These volumes were then used to calculate the drug percentage within the compact. For ASDs the volume percentage was assumed to be similar to the surface area percentage of the drug within the compact and the drug release was eventually calculated in mg/cm2. A detailed explanation of the IDR determination can be found

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ACCEPTED MANUSCRIPT in the supplementary materials and has been previously reported (Löbmann et al., 2013a). The IDR was calculated using linear regression as the slope in the linear equation, where the accumulated released drug per surface area was plotted as a function of time, and is reported as the slope (± standard deviation) of the dissolution profile. Table 2 summarizes the mass and true density values used in calculation of the IDR. All intrinsic dissolution experiments were

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conducted in triplicate.

Powder dissolution

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2.2.5

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Table 2: Density, molar mass and pKa of all three drugs and whey proteins used in this study. Samples Density (g/cm3) Molar mass pKa (g/mol) Indomethacin 1.379 (Löbmann et al., 2011) 357.79 4.5 Carvedilol 1.26 (Planinšek et al., 2011) 406.47 7.8 Furosemide 1.594 (Babu et al., 2010) 330.74 3.6 Whey protein isolate 1.088 (Carvalho-Silva et al., 2013) ~15000 -Whey protein hydrolysate 1.093 (Carvalho-Silva et al., 2013) ~15000 --

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Non-sink powder dissolution was performed in a custom-made downscaled dissolution set-up (Klein and Shah, 2008), combined with an Erweka GmbH, DT70 dissolution tester

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(Heusenstamm, Germany). Samples containing a total of 300 mg IND, 100 mg CAR and 200 mg FUR, respectively, were added to a dissolution medium of 100 ml 0.1M phosphate buffer

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(pH 7.2), in 250 ml dissolution vessels, with rotating mini paddles (50 rpm) at a temperature of 37 °C. Samples of 5 ml were collected at 1, 3, 5, 10, 20, 30, 40, 60, 90, 120, 180, 240, 360 and 1440 min, and subsequently filtered using a 0.22 µm latex free syringe filter (Qmax, Frisinette APS, Knebal, Denmark) and replaced with 5 ml dissolution medium (pre heated at 37 °C). From the filtrate, the first 2 ml of each sample were discarded to minimize drug losses due to adsorption to the filter membrane. From the remaining filtrate, the drug concentration

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ACCEPTED MANUSCRIPT in buffer was measured in triplicate using UV spectroscopy (see 2.2.4). All powder dissolution experiments were conducted in triplicate.

2.2.6

Equilibrium solubility of drugs in the presence of whey proteins

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Solubility of IND, CAR and FUR was determined in the presence of WPI and WPH using the shake-flask method. Excess crystalline drug was added to the buffer containing 3 mg/ml (for

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IND), 1mg/ml (for CAR) and 2mg/ml (for FUR) of pre-dissolved whey proteins, i.e. a

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concentration of whey proteins equivalent to the amount added in the powder dissolution

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experiments. These mixtures were then continuously rotated at 10 rpm for 72 hours using a mechanical rotor from Heto Lab equipment (Birkerod, Denmark). The samples were filtered

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and measured in triplicate using UV spectroscopy (see 2.2.4).

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Physical stability

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2.2.7

All samples were stored in a desiccator over silica gel (3.2% relative humidity) at room temperature and a physical stability study was performed for all pure amorphous drugs and the

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ASDs. Each sample was analyzed by XRPD at day 0, 2, 4, 8 and once a month thereafter.

3. Results and discussion 3.1.Solid state characterization

XRPD was used to confirm whether an amorphous product was obtained from ball milling. Both WPI and WPH were amorphous starting materials, whereas IND and CAR were successfully converted into their respective amorphous forms by milling them individually for 60 min, whilst FUR became amorphous by milling it alone for 90 min (data not shown). These

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ACCEPTED MANUSCRIPT milling times are in accordance to values reported in literature (Mishra et al., 2018a; Mishra et al., 2018b; Wu et al., 2018). As seen in Figure 2, the appearance of an amorphous halo upon milling each drug (crystalline starting material) with either WPI or WPH, suggests a complete amorphization of the drug in the presence of the proteins. For IND, the mixtures with WPI and WPH remained partly crystalline after 15 min of milling (Figure S1) but amorphized

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completely after 25 min of milling (Figure 2), whereas for both CAR and FUR the respective

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ASDs were formed after 45 min and 60 min of milling. The reduced milling time of

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amorphization for IND-whey protein mixtures suggests that the IND interacts faster or stronger with whey proteins which induce and facilitate fast amorphization when compared to whey

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protein mixtures with CAR and FUR.

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Thermal analysis using mDSC revealed a single Tg event in the thermograms of all ASDs, indicating the formation of a single phase homogeneous amorphous system, where the drug

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molecule and whey proteins are mixed at the molecular level (Van Drooge et al., 2006). Table

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3 summarizes all experimentally obtained Tg values. In general, the ASDs of drug-WPI samples showed higher Tg values compared to the ASDs of drug-WPH samples, which correlates well with the higher Tg of WPI compared with WPH. The IND-WPI samples showed the highest Tg

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of 132.1 °C followed by FUR-WPI (127.9 °C), FUR-WPH (126.1 °C), CAR-WPI (115.3 °C),

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IND-WPH (113.1 °C) and CAR-WPH samples (98.9 °C). These experimental Tg values of the respective drug-whey protein ASDs were found to be higher than the theoretical Tg values calculated with the Fox equation for the respective systems (see table 3, Figure S2). In general, when using the Fox equation to calculate a theoretical Tg of a mixture, one assumes that there are either no or limited specific interactions between the two components in the blend (Genina et al., 2018). Hence, the positive deviation of the experimental Tgs compared to the theoretical Tgs suggests that the drug and whey proteins in the ASDs are forming strong molecular interactions with each other in the mixture (Lu and Weiss, 1992).

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Figure 2. XRPD diffractograms of ball-milled amorphous solid dispersions (ASD) of (a) drugwhey protein isolate (WPI) and (b) drug-whey protein hydrolysate (WPH). The drugs are indomethacin (IND), carvedilol (CAR) and furosemide (FUR).

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Table 3: Experimental and theoretical glass transition temperatures (Tg) as well as intrinsic dissolution rates of all samples (± standard deviations). Experimental Tg Theoretical Tg Intrinsic dissolution (°C) (°C) rate (mg cm-2 min-1) WPI 228.5 ± 1.4 --WPH 155.4 ± 2.9 --C IND --0.079 ± 0.009 A IND 36.7 ± 1.7 -0.140 ± 0.019 ASD IND-WPI 132.1 ± 2.4 111.5 0.836 ± 0.037 ASD IND-WPH 113.1 ± 1.6 97.2 0.363 ± 0.016 C CAR --0.036 ± 0.004 A CAR 34.2 ± 0.9 -0.066 ± 0.007 ASD CAR-WPI 115.3 ± 1.4 109.1 0.326 ± 0.015 ASD CAR-WPH 98.9 ± 0.4 85.3 0.188 ± 0.003 C FUR --0.215 ± 0.008 A FUR 44.2 ± 0.5 -0.240 ± 0.012 ASD FUR-WPI 127.9 ± 1.4 117.5 0.658 ± 0.039 ASD FUR-WPH 126.1 ± 1.8 91.8 0.473 ± 0.025 *WPI: whey protein isolate, WPH: whey protein hydrolysate, C: crystalline, A: amorphous, IND: indomethacin, ASD: amorphous solid dispersion, CAR: carvedilol, FUR: furosemide.

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Samples

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3.2 Intrinsic dissolution testing ASDs are known to enhance the dissolution rate of poorly water-soluble drugs (Chiou and Riegelman, 1970). Thus, the intrinsic dissolution rate (IDR) for all the ASDs of drug-WPI and

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drug-WPH samples was compared with the crystalline and amorphous forms of the respective drug. Figure 3 shows the IDR of the pure crystalline and amorphous drugs together with the

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different ASD formulations prepared with IND, CAR and FUR. The IDR of the individual

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amorphous IND (0.14 mg cm-2 min-1) was found to be 1.8-fold higher than the IDR of

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crystalline IND (0.079 mg cm-2 min-1). In comparison, a substantially greater increase in the IDR was observed for the ASDs of IND-WPI and IND-WPH mixtures. In case of IND-WPI

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(0.836 mg cm-2 min-1) there was a 10.6-fold and 6-fold increase in dissolution rate when compared to crystalline and amorphous IND, respectively. For IND-WPH samples (0.363 mg

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cm-2 min-1) there was a 4.6-fold increase compared to the IDR of crystalline IND and a 2.6-

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fold increase compared to amorphous IND. The IDR of IND-WPI samples was 2.3-fold higher than for IND-WPH mixtures, see table 3. This is possibly due to the surface properties of the

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proteins used in WPI and WPH. The proteins in WPI are natural and intact whereas those in WPH have been digested (hydrolyzed) and consist of smaller fragments with higher exposure

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of the hydrophobic fractions (Zhou et al., 2014). The intact proteins (WPI) are likely to be more hydrophilic and surface active (Donz et al., 2014; Nishanthi et al., 2017), where the hydrophobic segments are mostly inside the protein core whereas the surface of the proteins are mostly consisting of hydrophilic AAs. A similar finding has also been reported for coamorphous drug-AA mixtures, where a faster dissolution was observed for highly soluble and hydrophilic AA compared to less soluble and more hydrophobic AAs (Jensen et al., 2014).

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Figure 3. Intrinsic dissolution of (a) indomethacin (IND), (b) carvedilol (CAR) and (c) furosemide (FUR) in the respective pure crystalline (C) and amorphous (A) forms, as well as from their amorphous solid dispersions (ASD) with whey protein isolate (WPI) and whey protein hydrolysate (WPH).

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Figure 3b and 3c show that the IDRs of the individual amorphous CAR (0.066 mg cm-2 min-1) and FUR (0.24 mg cm-2 min-1) were 1.8 and 1.1-fold higher than the IDR of crystalline CAR (0.036 mg cm-2 min-1) and FUR (0.215 mg cm-2 min-1), respectively. For the ASDs with CAR and FUR, overall, similar findings as with IND were achieved. There was a significant increase

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in the IDR for the ASDs of drug-WPI and drug-WPH mixtures. The IDR of the CAR-WPI

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(0.326 mg cm-2 min-1) and CAR-WPH samples (0.188 mg cm-2 min-1) showed a 9.1-fold and 5.2-fold increase compared to crystalline CAR, respectively, and a 4.9-fold and 2.8-fold

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increase compared to the individual amorphous CAR. Similarly, FUR-WPI (0.658 mg cm-2

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min-1) and FUR-WPH mixtures (0.473 mg cm-2 min-1) showed a 3.1-fold and 2.2-fold increase in dissolution rate when compared to the crystalline FUR, and a 2.7-fold and 2-fold increase

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compared to the individual amorphous FUR.

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3.3 Powder dissolution testing

Powder dissolution was performed to investigate the possibility for supersaturation from the

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ASD formulations. Figure 4 shows the powder dissolution profiles of all three drugs and their

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respective amorphous formulations. In general, similar findings as from the IDR study were found for all three drugs, i.e. the WPI based ASDs showed the fastest drug release, followed by the WPH based ASDs and the pure amorphous drugs. As expected the crystalline drugs had the lowest dissolution rates. Furthermore, during the powder dissolution experiments, the pure amorphous drugs (except pure amorphous FUR) as well as the drug-whey protein ASDs reached higher drug concentrations with respect to the crystalline drugs, suggesting apparent drug supersaturation from these formulations. It should be noted that the crystalline reference drugs did not undergo a milling process; hence, a particle sizing effect may have additionally

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ACCEPTED MANUSCRIPT contributed to the faster dissolution of the amorphous samples, however, not to the obtained apparent degree of supersaturation (aDS). The degree of supersaturation has been defined as the ratio between the activity of the dissolved species in the supersaturated solution and the activity of the species in a saturated solution (Mullin and Söhnel, 1977). However as previously explained by Blaabjerg et al., the thermodynamic activity is often difficult to obtain especially

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when the drug be complexed or is dispersed in micelles or a colloidal phase (Blaabjerg et al.,

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2018). Hence, the concentration of the drug is used instead of the activity to calculate an

𝐶𝑠𝑢𝑝𝑒𝑟𝑠𝑎𝑡 𝐶𝑒𝑞𝑢𝑖𝑙𝑖𝑏𝑟𝑖𝑢𝑚

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𝑎𝐷𝑆 =

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apparent degree of supersaturation (aDS) and can be calculated by:

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Where Csupersat is the concentration of the drug in a supersaturated solution and Cequilibrium is the concentration of the drug in a saturated crystalline solution.

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As seen in Figure 4, the equilibrium solubilities of crystalline IND, CAR and FUR in the

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dissolution medium (pH 7.2) were found to be ~700 µg/ml, ~40 µg/ml and ~100 µg/ml, respectively. Compared to the equilibrium solubility of crystalline IND, it was found that

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amorphous IND reached an apparent degree of supersaturation (aDS) of 1.5. This is due to an enhanced apparent solubility and dissolution rate of an amorphous drug itself. The drug from

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the IND-WPI and IND-WPH mixtures reached a higher aDS of 3.6 and 2.8, respectively. This further increase in the aDS of the drug from the ASDs is most likely due to the presence of the carriers (here: whey proteins), which can lead to even higher aDS, compared to the free amorphous drug, due to an increase of the apparent solubility of drug in presence of the carriers, as has also been seen for other polymeric carriers (Lainé et al., 2016). Similarly, amorphous CAR, CAR-WPI and CAR-WPH samples reached an aDS of 1.8, 3.5 and 2.8, respectively, compared to the equilibrium solubility of crystalline CAR. FUR-WPI and FUR-WPH mixtures

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ACCEPTED MANUSCRIPT also had 9 and 7 times, respectively, higher aDS compared to the equilibrium solubility crystalline FUR. As mentioned above, amorphous FUR did not show any significant increase in the maximum drug concentration when compared to crystalline FUR.

A drug is in a thermodynamically unstable state when supersaturated in solution, hence it

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possesses the risk for precipitation towards the equilibrium solubility of the drug (Six et al., 2004). This is seen for example in the dissolution profile of pure amorphous indomethacin,

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which initially supersaturates followed by a slow precipitation and decrease in the dissolved

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drug fraction towards the equilibrium solubility of the crystalline drug over the period of

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around 24h (Figure 4a).

Even though reaching a higher aDS compared to the pure amorphous drugs, both WPI and

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WPH were able to maintain the higher aDS obtained from the whey protein ASDs. One possible explanation for this may be that the whey proteins increase the solubility of the drug

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in the dissolution medium. Therefore, saturation solubility studies of the drugs in the presence

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of the whey proteins where conducted and compared to the saturation solubilities of the drug in pure dissolution medium. Indeed, the solubility of the three drugs was increased in presence

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of whey proteins when compared to their solubilities in buffer alone. The saturation solubility of IND was found to be almost 1.7 times higher in presence of WPI (~1150 µg/ml), and 1.5

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times higher in presence of WPH (~1020 µg/ml). Similar findings were observed for CAR and FUR. The solubility of crystalline CAR increased 2 times and 1.7 times in presence of WPI (~85 µg/ml) and WPH (~78 µg/ml), respectively. The solubilities of crystalline FUR increased 1.5 times in presence of WPI (~150 µg/ml) and 1.2 times in presence of WPH (~120 µg/ml). From the dotted and dashed lines in Figure 4, it can be seen that the whey protein ASDs reached higher drug concentrations than the determined equilibrium solubilities in the presence of the whey proteins, hence suggesting true supersaturation. Even though the whey protein ASDs

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ACCEPTED MANUSCRIPT supersaturate, no precipitation is observed and supersaturation is maintained throughout the time frame of the experiment (24 h). Hence, another possible explanation for the achieved supersaturation from these formulations may be that the whey proteins act as precipitation inhibitors and hence, prevent the recrystallization of the drug from the supersaturated solution.

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Overall, the drug-WPI samples showed the fastest dissolution and reached the highest aDS, followed by the drug-WPH samples, pure amorphous drug and crystalline drug. Furthermore,

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the pure presence of WPI and WPH in the dissolution medium increased the solubility of the

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drug. Nevertheless, the drugs supersaturared form drug-whey protein ASDs and

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supersaturation was maintained within the time frame of the dissolution experiments (24 h).

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Figure 4. Powder dissolution testing of (a) indomethacin (IND), (b) carvedilol (CAR) and (c) furosemide (FUR) in crystalline (C) and amorphous (A) forms of the drugs, as well as from their amorphous solid dispersions (ASD) with whey protein isolate (WPI) and whey protein hydrolysate (WPH). The dotted (···) and dashed (---) line in each case corresponds to the saturation solubility of the crystalline drug in the presence of WPI and WPH, respectively.

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ACCEPTED MANUSCRIPT 3.4 Physical stability

During the physical stability study, the individual amorphous drugs IND, CAR and FUR were found to recrystallize in less than one week. Recrystallization peaks were observed within 8 days in the XRPD diffractograms for all 3 drugs (Figure S3). In contrast, the ASDs of IND-

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WPI and IND-WPH mixtures were found to be stable for more than 27 months (Figure 5a). Figure 5b shows that ASD of CAR-WPI and CAR-WPH were stable for 8 months and CAR

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recrystallization peaks were observed at month 9. The FUR-WPI and FUR-WPH samples were

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stable up to 17 months and FUR started to recrystallize from these ASDs at month 18 (Figure

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respective starting material (Figure 5d).

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5c). Furthermore, both CAR and FUR crystallized back into the crystalline form of the

Figure 5. XRPD diffractograms of amorphous solid dispersions (ASD) of (a) indomethacin (IND)-whey protein isolate (WPI) and IND-whey protein hydrolysate (WPH) stored for 27 months; (b) carvedilol (CAR)-WPI and CAR-WPH stored for 9 months; (c) furosemide

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ACCEPTED MANUSCRIPT (FUR)-WPI and FUR-WPH stored for 18 months; as well as (d) crystalline (C) CAR and FUR for comparison.

4. Conclusion This study highlighted that WPI and WPH are promising new excipients for the formation of

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ASDs with poorly water-soluble drug compounds at weight ratios of 1:1. These new ASD

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formulations have shown higher Tg, faster dissolution rates and higher apparent solubilities when compared to the individual crystalline or amorphous drugs. Amongst the whey protein

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ASDs, the drug-WPI ASDs in general had a higher Tg and performed slightly better in the

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dissolution studies compared to the drug-WPH ASDs. Furthermore, the drug-WPI and drugWPH samples also had significantly higher physical stability compared to their individual

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amorphous drug.

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Acknowledgments: Jaya Mishra would like to thank the Lundbeck Foundation (R180-2014-

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3606) for the financial support.

Conflicts of Interest: The Lundbeck Foundation had no role in the design of the study; in the

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collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. The authors declare no conflict of interest.

References Babu, N.J., Cherukuvada, S., Thakuria, R., Nangia, A., 2010. Conformational and synthon polymorphism in furosemide (Lasix). Cryst. Growth Des. 10, 1979–1989. Blaabjerg, L.I., Lindenberg, E., Löbmann, K., Grohganz, H., Rades, T., 2018. Is there a correlation between the glass forming ability of a drug and its supersaturation propensity? Int. J. Pharm. 538, 243–249. Bylund, G., 1995. Whey processing. In Tetra Pak Dairy Processing Handbook, Tetra Pak

22

ACCEPTED MANUSCRIPT

PT E

D

MA

NU

SC

RI

PT

Processing Systems AB. Lund, Sweden, 331–351. Carvalho-Silva, L.B. de, Vissotto, F.Z., Amaya-Farfan, J., 2013. Physico-Chemical Properties of Milk Whey Protein Agglomerates for Use in Oral Nutritional Therapy. Food Nutr. Sci. 4, 69–78. Chegini, G., HamidiSepehr, A., Dizaji, M.F., Mirnezami, S.V., 2014. Study of physical and chemical properties of spray drying whey powder, Int. J. Recycl. Org. Waste Agric. 3, 62. Chieng, N., Aaltonen, J., Saville, D., Rades, T., 2009. Physical characterization and stability of amorphous indomethacin and ranitidine hydrochloride binary systems prepared by mechanical activation. Eur. J. Pharm. Biopharm. 71, 47–54. Chiou, W.L., Riegelman, S., 1970. Oral Absorption of Griseofulvin in Dogs: Increased Absorption via Solid Dispersion - in Polyethylene Glycol 6000. J. Pharm. Sci. 59, 937– 942. de Wit, J.N., 1998. Nutritional and Functional Characteristics of Whey Proteins in Food Products. J. Dairy Sci. 81, 597–608. Dengale, S.J., Grohganz, H., Rades, T., Löbmann, K., 2016. Recent advances in co-amorphous drug formulations. Adv. Drug Deliv. Rev. 100, 116–125. Donz, E., Boiron, P., Courthaudon, J.L., 2014. Characterization of industrial dried whey emulsions at different stages of spray-drying. J. Food Eng. 126, 190–197. EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), 2010. Scientific Opinion on the substantiation of health claims related to whey protein and increase in satiety leading to a reduction in energy intake (ID 425), contribution to the maintenance or achievement of a normal body weight (ID 1683), growth and maintenance of muscle mass (ID 418, 419, 423, 426, 427, 429, 4307), increase in lean body mass during energy restriction and resistance training (ID 421), reduction of body fat mass during energy restriction and resistance training (ID 420, 421), increase in muscle strength (ID 422, 429), increase in endurance capacity during the subsequent exercise bout after strenuous exercise (ID 428), skeletal muscle tissue repair (ID 428) and faster recovery from muscle fatigue after exercise (ID 423, 428, 431), pursuant to Article 13(1) of Regulation (EC) No 1924/2006. EFSA J. 8(10):1818, 28 pp. Eigel, W.N., Butler, J.E., Ernstrom, C.A., Farrell, H.M., Harwalkar, V.R., Jenness, R., Whitney, R.M., 1984. Nomenclature of Proteins of Cow’s Milk: Fifth Revision. J. Dairy Sci. 67, 1599–1631.

CE

Genina, N., Hadi, B., Löbmann, K., 2018. Hot melt extrusion (HME) as solvent-free technique for a continuous manufacturing of drug-loaded mesoporous silica. J. of Phar. Sci. 107, 149-155.

AC

Hancock, B.C., Parks, M., 2000. What Is the True Solubility Advantage for Amorphous Pharmaceuticals? Pharaceutical Res. 17, 397–404. He, Y., Ho, C., 2015. Amorphous Solid Dispersions: Utilization and Challenges in Drug Discovery and Development. J. Pharm. Sci. 104, 3237–3258. Hernández-Ledesma, B., Dávalos, A., Bartolomé, B., Amigo, L., 2005. Preparation of Antioxidant Enzymatic Hydrolysates from α-Lactalbumin and β-Lactoglobulin. Identification of Active Peptides by HPLC-MS/MS. J of Agri. and Food Chem. 53, 588– 593. Huang, Y., Dai, W.-G., 2014. Fundamental aspects of solid dispersion technology for poorly soluble drugs. Acta Pharm. Sin. B. 4, 18–25. Jensen, K.T., Löbmann, K., Rades, T., Grohganz, H., 2014. Improving co-amorphous drug formulations by the addition of the highly water soluble amino acid, Proline. Pharmaceutics. 6, 416–435. Kilara, A., Vaghela, M.N., 2018. Whey proteins, Proteins in Food Processing (Second Edition). Edited by Yada, R.Y. Cambridge, Woodhead Publishing Ltd. 93–126. Klein, S., Shah, V.P., 2008. A standardized mini paddle apparatus as an alternative to the

23

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

standard paddle. AAPS PharmSciTech. 9, 1179–84. Knopp, M.M., Tajber, L., Tian, Y., Olesen, N.E., Jones, D.S., Kozyra, A., Löbmann, K., Paluch, K., Brennan, C.M., Holm, R., Healy, A.M., Andrews, G.P., Rades, T., 2015. Comparative Study of Different Methods for the Prediction of Drug-Polymer Solubility. Mol. Pharm. 12, 3408–3419. Lainéa, A.-L., Pricea, D., Davis, J., Roberts, D., Hudson, R., Back, K., Bungaya, P., Flanagana, N., 2016. Enhanced oral delivery of celecoxib via the development of a supersaturable amorphous formulation utilising mesoporous silica and co-loaded HPMCAS. Int. J. of. Pharm. 512, 118-125. Laitinen, R., Löbmann, K., Grohganz, H., Strachan, C., Rades, T., 2014. Amino acids as Coamorphous excipients for simvastatin and glibenclamide: Physical properties and stability. Mol. Pharm. 11, 2381–2389. Laitinen, R., Löbmann, K., Strachan, C.J., Grohganz, H., Rades, T., 2013. Emerging trends in the stabilization of amorphous drugs. Int. J. Pharm. 453, 65–79. Lodge, T.P., McLeish, T.C.B., 2000. Self-concentrations and effective glass transition temperatures in polymer blends. Macromolecules. 33, 5278-5284. Löbmann, K., Grohganz, H., Laitinen, R., Strachan, C., Rades, T., 2013a. Amino acids as coamorphous stabilizers for poorly water soluble drugs - Part 1: Preparation, stability and dissolution enhancement. Eur. J. Pharm. Biopharm. 85, 873–881. Löbmann, K., Laitinen, R., Grohganz, H., Gordon, K.C., Strachan, C., Rades, T., 2011. Coamorphous drug systems: Enhanced physical stability and dissolution rate of indomethacin and naproxen. Mol. Pharm. 8, 1919–1928. Löbmann, K., Laitinen, R., Grohganz, H., Strachan, C., Rades, T., Gordon, K.C., 2013b. A theoretical and spectroscopic study of co-amorphous naproxen and indomethacin. Int. J. Pharm. 453, 80–87. Löbmann, K., Laitinen, R., Strachan, C., Rades, T., Grohganz, H., 2013c. Amino acids as coamorphous stabilizers for poorly water-soluble drugs - Part 2: Molecular interactions. Eur. J. Pharm. Biopharm. 85, 882–888. Lu, X., Weiss, R.A., 1992. Relationship between the glass transition temperature and the interaction parameter of miscible binary polymer blends. Macromolecules 25, 3242–3246. Mishra, J., Rades, T., Löbmann, K., Grohganz, H., 2018a. Influence of solvent composition on the performance of spray-dried co-amorphous formulations. Pharmaceutics. 10, 47. Mishra, J., Löbmann, K., Grohganz, H., Rades, T., 2018b. Influence of preparation technique on co-amorphization of carvedilol with acidic amino acids. Int. J. Pharm. 552, 407-413. Mullin, J.W., Söhnel, O., 1977. Expressions of supersaturation in crystallization studies. Chem. Eng. Sci. 32, 683-686. Mollea, C., Marmo, L., Bosco, F., 2013. Valorisation of Cheese Whey, a By-Product from the Dairy Industry. Food Ind. 549–588. Morr, C. V., Ha, E. Y. W., 2009. Whey protein concentrates and isolates: Processing and functional properties. Critical Reviews in Food Sci. and Nutrition. 33, 431-476. Newman, A., Reutzel-Edens, S.M., Zografi, G., 2018. Coamorphous Active Pharmaceutical Ingredient–Small Molecule Mixtures: Considerations in the Choice of Coformers for Enhancing Dissolution and Oral Bioavailability. J. Pharm. Sci. 107, 5–17. Nishanthi, M., Vasiljevic, T., Chandrapala, J., 2017. Properties of whey proteins obtained from different whey streams. Int. Dairy J. 66, 76–83. Pas, T., Vergauwen, B., Van den Mooter, G., 2018. Exploring the feasibility of the use of biopolymers as a carrier in the formulation of amorphous solid dispersions – Part I: Gelatin. Int. J. Pharm. 535, 47–58. Paudel, A., Worku, Z.A., Meeus, J., et al, 2013. Manufacturing of solid dispersions of poorly water soluble drugs by spray drying: Formulation and process considerations. Int. J.

24

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

NU

SC

RI

PT

Pharm. 453, 253–284. Planinšek, O., Kovačič, B., Vrečer, F., 2011. Carvedilol dissolution improvement by preparation of solid dispersions with porous silica. Int. J. Pharm. 406, 41–48. Qi, P.X., Onwulata, C.I., 2011. Physical properties, molecular structures, and protein quality of texturized whey protein isolate: Effect of extrusion moisture content, J. Dairy Sci. 94, 2231–2244. Six, K., Verreck, G., Peeters, J., Brewster, M., Mooter, G. Van den, 2004. Increased Physical Stability and Improved Dissolution Properties of Itraconazole, a Class II Drug, by Solid Dispersions that Combine Fast‐ and Slow‐Dissolving Polymers. J. Pharm. Sci. 93, 124– 131. Van Drooge, D.J., Hinrichs, W.L.J., Visser, M.R., Frijlink, H.W., 2006. Characterization of the molecular distribution of drugs in glassy solid dispersions at the nano-meter scale, using differential scanning calorimetry and gravimetric water vapour sorption techniques. Int. J. Pharm. 310, 220–229. Vasconcelos, T., Sarmento, B., Costa, P., 2007. Solid dispersions as strategy to improve oral bioavailability of poor water soluble drugs. Drug Discov. Today 12, 1068–1075. Wetlaufer, D., 1962. Ultraviolet spectra of proteins and amino acids. Adv Prot Chem 17, 303– 390. Wu, W., Löbmann, K., Rades, T., Grohganz, H., 2018. On the role of salt formation and structural similarity of co-formers in co- amorphous drug delivery systems. Int. J. of Pharm. 535, 86-94. Zhou, P., Liu, D., Chen, X., Chen, Y., Labuza, T.P., 2014. Stability of whey protein hydrolysate powders: effects of relative humidity and temperature. Food Chem. 150, 457–62.

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