CO-AMORPHOUS FORM OF A SUBSTANCE AND A PROTEIN

Abstract

The present invention relates to co-amorphous form of a substance and a protein. The present invention also relates to pharmaceutical, cosmetic or veterinary compositions comprising the co-amorphous form as well as to methods for preparing and using the co-amorphous form.

Claims

1. A co-amorphous form of a therapeutically active substance and a protein, wherein the protein is selected from whey protein isolate, beta-lactoglobulin, and or mixtures thereof; and wherein said co-amorphous form is a mixture of said therapeutically active substance and said protein at the molecular level in a single homogenous amorphous phase and, wherein the therapeutically active substance is a member of Biopharmaceutics Classification System (BCS) class II or IV.

2-25. (canceled)

26. The co-amorphous form according to claim 1 further comprising at least one pharmaceutically, cosmetically or veterinary acceptable excipient.

27. The co-amorphous form according to claim 1, wherein the co-amorphous form has a stability of at least 5 weeks or more when stored in a desiccator over silica gel at 0% relative humidity and room temperature of 18-25 C. and analyzed by XRPD.

28. The co-amorphous form according to claim 27, wherein the co-amorphous form has a stability of 8 weeks or more.

29. The co-amorphous form according to claim 27, wherein the co-amorphous form has a stability of 15 weeks or more.

30. The co-amorphous form according to claim 1, wherein an increase in intrinsic dissolution rate of the co-amorphous form is at least 2-fold higher than the dissolution rate of the therapeutically active substance in crystalline form.

31. The co-amorphous form according to claim 30, wherein the increase in intrinsic dissolution rate of the co-amorphous form is at least 5-fold higher than the dissolution rate of the therapeutically active substance in crystalline form.

32. The co-amorphous form according to claim 1, wherein the therapeutically active substance is a member of BCS class II.

33. The co-amorphous form according to claim 1, wherein the therapeutically active substance is a member of BCS class IV.

34. The co-amorphous form according to claim 1, wherein the protein is beta-lactoglobulin.

35. The co-amorphous form according to claim 1, wherein the protein is whey protein isolate.

36. The co-amorphous form according to claim 1, wherein the co-amorphous form comprises from 25 to 75% w/w of the therapeutically active substance and from 25 to 75% w/w of the protein.

Description

LEGENDS TO FIGURES

[0116] FIG. 1

[0117] XRPD diffractograms of co-amorphous forms of indomethacin (IND), carvedilol (CAR), paracetamol (PAR) and furosemide (FUR) with either whey protein isolate (WPI) or whey protein hydrolysate (WPH). All co-amorphous forms were obtained by spray drying. Panel (i): A=IND-WPI, B=CAR-WPI, C=PAR-WPI, D=FUR-WPI. Panel (ii): A=IND-WPH, B=CAR-WPH, C=PAR-WPH, D=FUR-WPH.

[0118] FIG. 2

[0119] Intrinsic dissolution rate of crystalline indomethacin (C IND), amorphous indomethacin (A IND), co-amorphous indomethacin-whey protein isolate obtained by ball milling (BM IND-WPI), co-amorphous indomethacin-whey protein hydrolysate obtained by ball milling (BM IND-WPH), co-amorphous indomethacin-whey protein isolate obtained by spray drying (SD IND-WPI), and co-amorphous indomethacin-whey protein hydrolysate obtained by spray drying (SD IND-WPH).

[0120] FIG. 3

[0121] Intrinsic dissolution rate of (a) crystalline carvedilol (C CAR), amorphous carvedilol (A CAR), co-amorphous carvedilol-whey protein isolate obtained by ball milling (BM CAR-WPI), co-amorphous carvedilol-whey protein hydrolysate obtained by ball milling (BM CAR-WPH), co-amorphous carvedilol-whey protein isolate obtained by spray drying (SD CAR-WPI), and co-amorphous carvedilol-whey protein hydrolysate obtained by spray drying (SD CAR-WPH); (b) crystalline paracetamol (C PAR), amorphous paracetamol (A PAR), co-amorphous paracetamol-whey protein isolate obtained by ball milling (BM PAR-WPI), co-amorphous paracetamol-whey protein hydrolysate obtained by ball milling (BM PAR-WPH), co-amorphous paracetamol-whey protein isolate obtained by spray drying (SD PAR-WPI), and co-amorphous paracetamol-whey protein hydrolysate obtained by spray drying (SD PAR-WPH); (c) crystalline furosemide (C FUR), amorphous furosemide (A FUR), co-amorphous furosemide-whey protein isolate obtained by ball milling (BM FUR-WPI), co-amorphous furosemide-whey protein hydrolysate obtained by ball milling (BM FUR-WPH), co-amorphous furosemide-whey protein isolate obtained by spray drying (SD FUR-WPI), and co-amorphous furosemide-whey protein hydrolysate obtained by spray drying (SD FUR-WPH)

[0122] FIG. 4

[0123] Intrinsic dissolution rate of (a) crystalline indomethacin (C IND), amorphous indomethacin (A IND), co-amorphous indomethacin-whey protein isolate obtained by spray drying (SD IND-WPI), co-amorphous indomethacin with whey protein isolate (digested with trypsin) obtained by spray drying (SD IND-WPI ENZ T), co-amorphous indomethacin with whey protein isolate (digested with trypsin followed by pepsin) obtained by spray drying (SD IND-WPI ENZ T+P), co-amorphous indomethacin with whey protein isolate (digested with pepsin) obtained by spray drying (SD IND-WPI ENZ P), co-amorphous indomethacin with whey protein isolate (digested with pepsin followed by trypsin) obtained by spray drying (SD IND-WPI ENZ P+T); (b) crystalline indomethacin (C IND), amorphous indomethacin (A IND), co-amorphous indomethacin-whey protein isolate obtained by spray drying (SD IND-WPI), co-amorphous indomethacin-bovine serum albumin obtained by spray drying (SD IND-BSA), co-amorphous indomethacin-alpha-lactalbumin obtained by spray drying (SD IND-a lactalbumin), and co-amorphous indomethacin-beta-lactoglobulin obtained by spray drying (SD IND-b lactoglobulin).

[0124] FIG. 5

[0125] Powder dissolution studies of crystalline indomethacin (C IND), amorphous indomethacin (A IND), physical mixture of indomethacin and whey protein isolate (PM IND-WPI), co-amorphous form of indomethacin and whey protein isolate obtained by ball milling (BM IND-WPI), and co-amorphous form of indomethacin and whey protein isolate obtained by spray drying (SD IND-WPI).

[0126] FIG. 6

[0127] Stability of co-amorphous forms of whey protein isolate (WPI) with indomethacin (IND), carvedilol (CAR), paracetamol (PAR) and furosemide (FUR), respectively. The 5 months stability data was measured for WPI mixtures with IND, CAR and FUR and the 1 month stability was measured for PAR-WPI, assessed using x-ray powder diffraction (XRPD). All co-amorphous mixtures were obtained by spray drying. A=PAR-WPI, B=FUR-WPI, C=CAR-WPI, D=IND-WPI. Stability studies were further carried out each month until the drug substance started to recrystallize and the data is shown in Table 3.

[0128] FIG. 7

[0129] Absolute bioavailability of crystalline furosemide (Crystalline FUR), amorphous furosemide (Amorphous FUR), co-amorphous furosemide-polyvinylpyrrolidone (25:75 w/w) obtained by spray drying (SD FUR-PVP (75:25)), physical mixture (50:50 w/w) of furosemide-whey protein isolate and (PM FUR-WPI (50:50)), co-amorphous furosemide-whey protein isolate (25:75 w/w) obtained by spray drying (SD FUR-WPI (25:75)), co-amorphous furosemide-whey protein isolate (50:50 w/w) obtained by spray drying (SD FUR-WPI (50:50)), and co-amorphous furosemide-whey protein isolate (75:25 w/w) obtained by spray drying (SD FUR-WPI (75:25)). Bioavailability was assessed following oral administration to rats. Polyvinylpyrrolidone was included in the experiment because it is the most commonly used excipient for making solid dispersions, which is the main competing technology for amorphization in terms of optimizing solubility and/or stability of drug substances with poor solubility and/or stability properties. The ratios of WPI and FUR were varied by changing the content of WPI while the content of FUR was kept constant.

[0130] FIG. 8

[0131] Maximum concentration in the bloodstream (Cmax) of crystalline furosemide (Crystalline FUR), amorphous furosemide (Amorphous FUR), co-amorphous furosemide-polyvinylpyrrolidone (25:75 w/w) obtained by spray drying (SD FUR-PVP (75:25)), physical mixture (50:50 w/w) of furosemide-whey protein isolate and (PM FUR-WPI (50:50)), co-amorphous furosemide-whey protein isolate (25:75 w/w) obtained by spray drying (SD FUR-WPI (25:75)), co-amorphous furosemide-whey protein isolate (50:50 w/w) obtained by spray drying (SD FUR-WPI (50:50)), and co-amorphous furosemide-whey protein isolate (75:25 w/w) obtained by spray drying (SD FUR-WPI (75:25)). Cmax was assessed following oral administration to rats. Polyvinylpyrrolidone was included in the experiment because it is the most commonly used excipient for making solid dispersions, which is the main competing technology for amorphization in terms of optimizing solubility and/or stability of drug substances with poor solubility and/or stability properties. The ratios of WPI and FUR were varied by changing the content of WPI while the content of FUR was kept constant.

[0132] FIG. 9

[0133] Intrinsic dissolution rate of crystalline furosemide (Crystalline FUR), amorphous furosemide (Amorphous FUR), co-amorphous furosemide-polyvinylpyrrolidone (25:75 w/w) obtained by spray drying (SD FUR-PVP (75:25)), physical mixture (50:50 w/w) of furosemide-whey protein isolate and (PM FUR-WPI (50:50)), co-amorphous furosemide-whey protein isolate (25:75 w/w) obtained by spray drying (SD FUR-WPI (25:75)), co-amorphous furosemide-whey protein isolate (50:50 w/w) obtained by spray drying (SD FUR-WPI (50:50)), and co-amorphous furosemide-whey protein isolate (75:25 w/w) obtained by spray drying (SD FUR-WPI (75:25)). Polyvinylpyrrolidone was included in the experiment because it is the most commonly used excipient for making solid dispersions, which is the main competing technology for amorphization in terms of optimizing solubility and/or stability of drug substances with poor solubility and/or stability properties. The ratios of WPI and FUR are varied by changing the content of WPI while the content of FUR is kept constant.

[0134] FIG. 10

[0135] XRPD diffractograms of co-amorphous forms of indomethacin (IND) with various proteins. All co-amorphous forms were obtained by spray drying. A: SD IND-Soy, B: SD IND-Rice, C: SD IND-Egg, D: SD IND-Gelatin, E: SD IND-Collagen, F: SD IND-Myoglobin, G: SD IND-Lysozyme and H: SD IND-Casein.

[0136] FIGS. 11A and 11B

[0137] Intrinsic dissolution rate of (i) SD IND-Gelatin, SD IND-Egg, SD IND-Soy, C IND, A IND; and (ii) SD IND-Myoglobin, SD IND-Lysozyme, SD IND-Collagen, SD IND-Casein, C IND, AIND.

[0138] FIG. 12

[0139] XRPD diffractograms of A: SD IND-Ovalbumin, B: SD CEL-WPI, C: SD CEL-Myoglobin, D: SD CEL-Lysozyme, E: SD CEL-Casein, F: SD CEL-Collagen.

[0140] FIGS. 13A-13C

[0141] Intrinsic dissolution rate of FIG. 13A SD IND-Myoglobin, SD IND-Lysozyme, SD IND-Collagen, SD IND-Casein, SD IND-WPI; FIG. 13B SD CEL-Myoglobin, SD CEL-Lysozyme, SD CEL-Collagen, SD CEL-Casein, SD CEL-WPI; and FIG. 13C SD CAR-Myoglobin, SD CAR-Lysozyme, SD CAR-Collagen, SD CAR-Casein, SD CAR-WPI.

[0142] FIGS. 14A-14B

[0143] Intrinsic dissolution rate of FIG. 14A SD IND-EGG, SD IND-RICE, SD IND-SOY, SD IND-WPI, SD IND-Gelatin; and FIG. 14B SD IND-BSA, SD IND-Ovalbumin, SD IND-Casein, SD IND-WPI.

[0144] FIGS. 15A-15C

[0145] Intrinsic dissolution rate (IDR) of FIG. 15A SD IND-Myoglobin, SD IND-Lysozyme, SD IND-Collagen, SD IND-Casein, SD IND-WPI; FIG. 15B SD CEL-Myoglobin, SD CEL-Lysozyme, SD CEL-Collagen, SD CEL-Casein, SD CEL-WPI; and FIG. 15C SD CAR-Myoglobin, SD CAR-Lysozyme, SD CAR-Collagen, SD CAR-Casein, SD CAR-WPI; Where the IDRs are plotted as a function of isoionic points (pl) of the proteins.

[0146] FIGS. 16A-16B

[0147] Intrinsic dissolution rate (IDR) of SD IND-BSA, SD IND-Ovalbumin, SD IND-Casein, SD IND-WPI; where FIG. 16A the IDRs are plotted as a function of molecular weight (Mw) of the proteins; and FIG. 16B the IDRs are depicted as a function of isoionic points (pl) of the proteins.

[0148] FIG. 17

[0149] Intrinsic dissolution rate (IDR) of SD IND-EGG, SD IND-RICE, SD IND-SOY, SD IND-WPI, SD IND-Gelatin; where the IDRs are depicted as a function of isoionic points (pl) of the proteins.

[0150] FIG. 18

[0151] XRPD diffractograms of A: SD CAR-Myoglobin, B: SD CAR-Lysozyme, C: SD CAR-Collagen, D: SD CAR-Casein.

[0152] FIG. 19

[0153] XRPD diffractograms of SD IND-WPI and SD IND-WPH, 20 months after preparation of respective co-amorphous formulations.

ABBREVIATIONS

[0154] BM, ball milling [0155] CAR, carvedilol [0156] CEL, celecoxib [0157] FUR, furosemide [0158] IDR, intrinsic dissolution rate [0159] IND, indomethacin [0160] pl, isoionic point [0161] LC-MS, liquid chromatography-mass spectometry [0162] mDSC, modulated differential scanning calorimetry [0163] Mw, molecular weight [0164] PAR, paracetamol [0165] PM, physical mixture [0166] PVP, polyvinylpyrrolidone [0167] SD, spray drying [0168] TGA, thermogravimetric analysis [0169] UV Vis, ultra-violet spectrophotometry [0170] XRPD, x-ray powder diffraction [0171] WPH, whey protein hydrolysate [0172] WPI, whey protein isolate

Experimentals

[0173] Materials

[0174] Indomethacin (IND) was purchased from Hawkins, Inc. (Minneapolis, MN, USA). Carvedilol (CAR) from Cipla Ltd. (Mumbai, India), paracetamol (PAR) from Fagmn (Copenhagen, Denmark) and Furosemide (FUR) from Sigma-Aldrich (St. Louis, MO, USA). All these powders were of reagent grade and used as received. Whey protein isolate (WPI), whey protein hydrolysate (WPH), rice protein isolate, soy protein isolate and egg protein isolate were purchased from LSP Sporternahrung (Bonn, Germany, www.lsp-sports.de). Polyvinylpyrrolidone (PVP, Kollidon 25), alpha-lactalbumin and beta-lactoglobulin from bovine milk were received from Sigma-Aldrich (Schnelldorf, Germany). Bovine serum albumin (BSA), celecoxib (CEL), ovalbumin, collagen, gelatin, myoglobin, lysozyme, casein and, pepsin from porcine gastric mucosa and trypsin from bovine pancreas were obtained from Sigma-Aldrich (Brondby, Denmark). All materials were of reagent grade and used as received.

Methods

[0175] Spray Drying:

[0176] Physical mixtures (powders mixed together in container with spatula) of IND, CAR, PAR, CEL and FUR with either WPI or WPH were prepared at a 1:1 weight ratios. The mixtures were then dissolved in 250 ml of milliQ water (18.2 MO, 23.8 C.) freshly prepared by MilliQ water system from LabWater (Los Angeles, CA, USA). The concentration of drug substance-WPI/WPH in each respective solution was 4 mg/ml. Spray drying was performed using a Buchi B-290 spray-dryer (Buchi Labortechnik AG, Flawil, Switzerland) equipped with a dehumidifier (Buchi B296). The spray drying conditions were as follows: inlet temperature: 120 C.; outlet temperature: approx. 62 C.; atomizing air flow rate: 667 I/h; drying air flow (nitrogen): 40 m3/h and feed flow rate: 9 ml/min. To further study the dissolution behaviour of the co-amorphous forms, IND was spray dried together with the main components of WPI (alpha-lactalbumin, beta-lactoglobulin and bovine serum albumin (BSA), respectively), and with WPI subjected to enzymatic digestion (trypsin, pepsin, trypsin followed by pepsin, and pepsin followed by trypsin, respectively). Enzymatic digestions was performed overnight using 1 mg enzyme for every 100 mg WPI. Pepsin digestion was performed at pH 8 and trypsin digestion was performed at pH 3. To analyze the dissolution behaviour of a drug substance with proteins of different properties, IND was spray dried with rice protein isolate, egg protein isolate, soy protein isolate, ovalbumin, collagen, gelatin, myoglobin, lysozyme and casein, and CEL and CAR were spray dried with WPI, myoglobin, lysozyme, collagen and casein.

Ball Milling:

[0177] To compare the co-amorphous forms obtained with spray drying to the co-amorphous forms obtained by ball milling, physical mixtures of IND, CAR, PAR, and FUR with either WPI or WPH were subjected to vibrational ball milling using a MixerMill MM400 (Retsch GmbH & Co., Haan, Germany) in a cold room (4 C.). The co-amorphous forms obtained by ball milling were produced by placing a total mass of 700 mg of 1:1 weight ratios (drug substance-WPI/WPH) in 25 ml milling jars with two 12 mm stainless steel balls. Milling was performed at 30 Hz for up to 30 min in case of IND and CAR and up to 60 min in case of FUR and PAR.

X-Ray Powder Diffraction (XRPD) for Measurement of Solid State Form:

[0178] The molecular interactions of the drug substance-WPI/WPH mixtures were investigated by XRPD using an X'Pert PANanalytical PRO X-ray diffractometer (PANanalytical, Almelo, The Netherlands) with Cuk radition: 1.54187 , current: 40 mA and acceleration voltage: 45 kV. Each of the co-amorphous forms obtained either by spray drying or ball milling were scanned (scan rate of 0.067 2/s and step size of 0.026) with reflectance mode between 2 and 350 2. The collected data was analysed using X'Pert PANanalytical Collector software (PANanalytical, Almelo, The Netherlands).

Thermogravimetric Analysis for Measurement of Residual Moisture:

[0179] Thermogravimetric analysis was performed on a TGA Discovery instrument (TA Instruments, New Castle, USA). Samples of 10 mg were placed in a platinum pan and sealed with a lid and heated from 25 to 300 QC at 10 QC/min. Resulting weight-temperature diagrams were analyzed using Trios software (TA Instruments, New Castle, USA) to calculate the weight loss between 25 and 150 QC.

Modulated Differential Scanning Calorimetry (mDSC) for Measurement of Tg and Tm:

[0180] Thermal analysis was performed using a Discovery DSC instrument (TA Instruments, New Castle, USA). Each sample weighing approximately 6-8 mg was placed in an aluminium pan and sealed with lids. Calibration of the equipment was carried out with indium and the samples were then subjected to an amplitude of 0.2120 C. for a period of 40 s. A heating rate of 2 C./min was employed with measurement ranging from 20 C. to 180 C. A constant nitrogen flow rate of 50 mL/min was applied during each measurement. Glass transition temperature (Tg) was found by analyzing the data collected using Trios software (TA Instruments, New Castle, USA), observing the half height of the midpoint of onset and end temperature of the samples.

Intrinsic Dissolution Rate:

[0181] The intrinsic dissolution rate (IDR) was determined from powder compacts obtained with a hydraulic press (PerkinElmer, Hydraulische Presse Model IXB-102-9, Ueberlingen, Germany). Ball-milled powders of pure drug and spray dried powders of drug-protein mixtures were compressed into tablets. Tablets of 150 mg were directly compressed into stainless steel cylinders that served as intrinsic dissolution sample holders at a pressure of 124.9 MPa for 45 secs. Compression of tablets resulted in a flat surface of surface area 0.7854 cm.sup.2 at one end of the cylinder. These cylinders were then placed in 900 ml of 0.1 M phosphate buffer (pH 7.2, 37 C.) dissolution medium and stirred using a magnetic bar at a rotation speed of 50 rpm. At predetermined time points (1, 5, 10, 15, 20, 25 and 30 min, some IDRs were only determined up to 20 min), 5 ml aliquots were withdrawn and immediately replaced with dissolution buffer. The obtained samples were then analyzed using UV spectrophotometer (see below). All dissolution experiments were conducted in triplicate.

Ultra-Violet Spectrophotometry (UV Vis):

[0182] The concentration of each drug in the buffer was measured by an Evolution 300 UV spectrophotometer (Thermo Scientific, Cambridge, UK) at 320 nm, 272 nm, 270 nm, 265 nm and 285 nm for IND, CAR, PAR, CEL and FUR, respectively.

Powder Dissolution (USPII Apparatus):

[0183] Powder dissolution was performed in USP type II apparatus. 200 mg of crystalline IND, amorphous IND, SD IND-WPI and physical mixture (PM) IND-WPI were added in triplicate to 50 ml phosphate buffer of pH 7.2 (sodium phosphate dibasic heptahydrate and sodium phosphate monobasic anhydrous) as the dissolution medium. The dissolution paddles were rotated at 50 rpm for 1 hour taking samples out at 1, 3, 5, 7, 10, 15, 20, 25, 30, 35, 40, 50, 60 and 120 mins. Each sample of 5 ml was taken out and replaced by dissolution medium. To separate powders from medium, the samples were filtered through a 0.45 pm syringe filter (Qmax, Frisinette ApS) and the first 2 ml was discarded to minimize losses due to adsorption. The samples were examined using UV Vis to analyze the drug concentration.

Stability:

[0184] All samples were stored in a desiccator over silica gel (0% relative humidity) at room temperature and a physical stability study was performed for all SD samples. Each sample was analyzed by XRPD at day 0 and subsequently once every month thereafter.

In Vivo Pharmacokinetics Studies:

[0185] The study was carried out under the study protocol approved by the Danish Animal Experiments Inspectorate (approval no. 2014-15-0201-00031). The purpose of this study was to study the performance of (i) crystalline FUR compared to (ii) amorphous FUR; (iii) physical mixture of FUR-WPI (50% FUR, 50% WPI); (iv) SD PVP-WPI (75% PVP, 25% FUR); (v) SD FUR-WPI (75% FUR, 25% WPI); (vi) SD FUR-WPI (50% FUR, 50% WPI); and (vii) SD FUR-WPI (25% FUR, 75% WPI). All ratios are in weight %. SD, XRPD, DSC and intrinsic dissolution studies were all carried using the same conditions mentioned in section 1.2.

[0186] Male Sprague Dawley Rats of 7 weeks weighing 250-348 g (Charles River, Denmark) were used for the experiments. Animals were allowed free access to water and food and were housed under controlled environmental conditions (constant temperature and humidity with a 12 h dark-light cycle). All animals were fasted for approximately 12 hours prior to being administered the drug. The rats were randomly assigned into 8 groups (each consisting of 6-8 rats) including a group receiving FUR intravenously at 1.5 mg/rat (approximately 5 mg/kg) in saline, injected in the tail vein. The remaining 7 groups were administered orally using a gavage of size 2.5 mm tablet thickness. Each tablet was a dose of 4.5 mg FUR per rat, which equals to approximately 15 mg/kg. Blood samples (0.2 ml) were collected from the tail vein after 0.25, 0.5, 1, 2, 4 and 24 hour by puncturing the tail. These blood samples were collected and stored in EDTA coated tubes and until plasma was harvested by centrifugation at 3600 g (12 min, 4 C.) and transfer into microtubes. Plasma samples were stored at 80 C. until used for further analysis. Food was given to rats after approximately 8 h after drug administration. Water was freely available for rats during the entire duration of the experiment.

Quantitative Analysis of Plasma Samples by Liquid Chromatography-Mass Spectrometry (LC-MS):

[0187] The furosemide content in plasma samples was assessed by adding 300 I of acetonitrile to 30 I of plasma to precipitate the proteins. An internal standard consisting of 30 I fenofibric acid (FA) was also added to each sample. These final mixtures were then centrifuged for 10 mins at 8000 rpm (room temperature). After centrifugation, the supernatants were carefully transferred to LC-MS plates and LC-MS was performed using an Agilent technologies 1200 system with a 6140 Quadrupole detector. Chromatographic separations were carried out using an Agilent Zorbax XDB-C18 column (2.150 mm, 3.5 pm). The samples were eluted with a flow rate of 0.5 ml/min in a gradient mixture of 0.04% glacial acetic acid in miiliQ water (solvent A) and acetonitrile (solvent B). Each gradient program was: 0-8 min, 15% solvent B; 8-10 min, from 15% to 80% solvent B; 10-11 min, 80% solvent B; 11-11.10 min, from 80% to 15% solvent B; 11.10-14 min, 15% solvent B. The autosampler temperature was kept at 8 C. and volume of each injection sample was set to 5 L. The LC-MS method was carried out in presence of nitrogen to assist nebulisation.

Pharmacokinetic Analysis:

[0188] The area under the curve (AUC) of the plasma concentration with respect to time was determined by linear log trapezoidal method from time t=0 min to t=1440 min (last plasma concentration). AUC was used to calculate the absolute bioavailability (Fa):

[00001]$\mathrm{Absolute}{F}_a=100.Math.\frac{AU{C}_{P.O.}}{AU{C}_{I.V.}}.Math.\frac{{\mathrm{Dose}}_{I.V.}}{{\mathrm{Dose}}_{P.O.}}$

Where P.O. stands for per oral delivery and I.V. is for intravenous dosage. The maximum FUR plasma concentration C.sub.max was also determined.

EXAMPLES

Example 1: x-Ray Powder Diffraction of Spray Dried Drug Substance-WPI/WPH

[0189] XRPD was used to analyze the amorphous (halo structure in the XRPDno Bragg peaks in the diffractograms) or crystalline phases (distinct peaks in the diffractograms) for all samples. FIG. 1 shows the appearance of the amorphous halo in each cases proving a success in amorphization of all drug-WPI/WPH mixtures.

[0190] FIG. 10 shows the appearance of the amorphous halo in experiments where IND with rice protein isolate, soy protein isolate, egg protein isolate, collagen, gelatin, myoglobin, lysozyme and casein, respectively, were in the co-amorphous from. The figure clearly demonstrates successful amorphization in all cases. Further to this, FIG. 12 shows halo structures of IND with ovalbumin and CEL with myoglobin, lysozyme, casein, collagen and WPI, confirming the formation of co-amorphous formulations. The halo structures of CAR with myoglobin, lysozyme, casein, collagen in FIG. 18 also confirm the co-amorphous formulation.

Example 2: Thermal Analysis of Spray Dried Drug Substance-Protein Mixtures

[0191] TGA confirmed that the residual moisture content in all amorphous drugs and the SD drug substance-protein mixtures was 3.2-8.3%. See table 1a and 1b for the detailed results.

TABLE-US-00001 TABLE 1a TGA data for all amorphous drug substance and co-amorphous forms of drug substance-WP! (including co-amorphous forms of IND with components of WPI and co-amorphous forms of IND-WPI digested with enzymes) and drug substance- WPH mixtures obtained by spray drying. TGA Powders (residual moisture content in %) In vitro samples AIND 3.2 0.4 SD IND-WPI 3.2 0.7 SD IND-WPH 3.5 0.3 SD IND--Lactalbumin 3.2 0.2 SD IND--Lactoglobulin 3.6 0.6 SD IND-BSA 3.7 1.1 SD IND-WPI (ENZ T) 4.3 0.3 SD IND-WPI (ENZ T + P) 4.2 0.4 SD IND-WPI (ENZ P) 3.9 0.5 SD IND-WPI (ENZ P + T) 3.9 0.4 ACAR 3.1 0.3 SD CAR-WPI 3.7 0.2 SD CAR-WPH 3.9 0.4 SD PAR-WPI 4.0 0.7 SD PAR-WPH 4.1 0.6 A FUR 3.6 0.3 SD FUR-WPI 4.5 0.3 SD FUR-WPH 4.2 0.5 In vivo samples SD FUR-PVP (25:75) 4.4 0.8 SD FUR-WPI (25:75) 4.3 0.6 SD FUR-WPI (50:50) 4.5 0.3 SD FUR-WPI (75:25) 4.1 0.2

TABLE-US-00002 TABLE 1b TGA data for all co-amorphous forms of IND, CEL and CAR with respective proteins, obtained by spray drying. TGA Powders (residual moisture content in %) SD IND-Soy 8.1 0.3 SD IND-Rice 8.3 0.3 SD IND-Egg 8.1 0.5 SD IND-Gelatin 1.1 0.4 SD IND-Collagen 4.9 0.6 SD IND-Myoglobin 6.4 0.5 SD IND-Lysozyme 1.2 0.3 SD IND-Casein 0.9 0.2 SD IND-Ovalbumin 3.7 0.2 SD CEL-WPI 2.8 0.7 SD CEL-Casein 2.5 0.4 SD CEL-Collagen 2.3 1.2 SD CEL-Lysozyme 3.1 0.8 SD CEL-Myoglobin 3.4 0.7 SD CAR-Casein 3.1 0.3 SD CAR-Collagen 2.4 0.5 SD CAR-Lysozyme 3.6 0.3 SD CAR-Myoglobin 3.1 0.9

[0192] Each SD drug substance-protein mixture showed a single Tg (Glass transition temperature), which points that a single phase co-amorphous system has been achieved. All the co-amorphous mixtures showed increase in values Tg compared to the amorphous drug itself showing a better miscibility within the mixtures.

Example 3: Intrinsic Dissolution Rate of Different Forms of IND

[0193] As depicted in FIG. 2, the intrinsic dissolution rate (IDR) of the amorphous ball milled IND (0.1333 mg cm-2 min-1) is 1.7 fold higher than the IDR of crystalline IND (0.0787 mg cm-2 min-1). In comparison, a substantially greater increase in the IDR was observed for the co-amorphous IND-WPI and IND-WPH mixtures. In case of spray dried IND-WPI (1.494 mg cm-2 min-1) there is a 19 fold increase in dissolution rate when compared to crystalline IND and 11 fold increase compared to ball milled amorphous IND. For spray dried IND-WPH (1.3066 mg cm-2 min-1) there is 4 fold increase from crystalline IND and about 2 fold increase from ball milled amorphous IND. There is also 1 fold increase in dissolution of spray dried IND-WPI when compared to spray dried IND-WPH. See Table 2a for the relevant line equations and intrinsic dissolution rates. Further, Table 2b presents additional line equations and intrinsic dissolution rates for co-amorphous mixtures.

TABLE-US-00003 TABLE 2a Line equations of intrinsic dissolution testing of crystalline (C) and amorphous (A) drug substances along with spray dried (SD) and ball milled (BM) co-amorphous drug substance with whey protein isolate (WPI) and whey protein hydrolysate (WPH), respectively. Sample y (mg cm.sup.2 min.sup.1) Sample y (mg cm.sup.2 min.sup.1) CIND y = 0.0787x + 4.006 CCAR y = 0.0117x + 2.8082 AIND y = 0.1333x + 4.5538 ACAR y = 0.0214x + 2.973 SD IND-WPI y = 1.494x + 3.3209 SD CAR-WPI y = 0.1948x + 4.7973 SD IND-WPH y = 1.3066x + 0.1726 SD CAR-WPH y = 0.0794x + 3.4102 BM IND-WPI y = 1.187x + 2.4124 BM CAR-WPI y = 0.0266x + 3.0153 BM IND-WPH y = 0.3019x + 4.2494 BM CAR-WPH y = 0.0307x + 2.979 WPI y = 3.8317x + 3.8477 C PAR y = 0.1632x + 2.3462 WPH y = 3.2347x + 42.311 APAR y = 0.201x + 4.5947 SD IND- lactalbumin y = 1.1065x + 5.1568 SD PAR-WPI y = 0.5433x + 3.273 SD IND- lactoglobulin y = 0.3468x + 5.311 SD PAR-WPH y = 0.4664x + 2.4197 SD IND-BSA y = 0.2861x + 5.4 BM PAR-WPI y = 0.3386x + 3.0738 SD IND-WPI (ENZ T) y = 0.2512x + 4.4218 BM PAR-WPH y = 0.2865x + 3.4489 SD IND-WPI (ENZ T + P) y = 0.2271x + 7.1504 C FUR y = 0.1024x + 2.3685 SD IND-WPI (ENZ P) y = 0.1131x + 4.6117 AFUR y = 0.1548x + 2.4064 SD IND-WPI (ENZ P + T) y = 1.1917x + 7.4698 SD FUR-WPI y = 0.4115x + 5.0747 SD FUR-WPH y = 0.2359x + 4.3598 BM FUR-WPI y = 0.2279x + 2.1756 BM FUR-WPH y = 0.1972x + 2.234

TABLE-US-00004 TABLE 2b Line equations for intrinsic dissolution testing performed on all co- amorphous IND-protein, CEL-protein and CAR-protein mixtures. All mixtures were prepared by spray drying (SD). Sample y (mg cm.sup.2 min.sup.1) SD IND-Gelatin y = 0.6263x + 3.5063 SD IND-Egg y = 0.3502x + 2.2251 SD IND-Rice y = 0.1952x + 2.5039 SD IND-Soy y = 0.1801x + 2.6814 SD IND-Lysozyme y = 1.0676x + 4.2697 SD IND-Myoglobin y = 0.9113x + 2.4847 SD IND-Collagen y = 0.2924x + 2.5942 SD IND-Casein y = 0.2224x + 2.2982 SD IND-Ovalbumin y = 0.2733x + 2.8722 SD CEL-WPI y = 0.9972x + 4.7968 SD CEL-Casein y = 0.9714x + 2.9813 SD CEL-Collagen y = 0.6629x + 1.8036 SD CEL-Lysozyme y = 0.2019x + 1.0872 SD CEL-Myoglobin y = 0.2628x + 1.8702 SD CAR-Casein y = 0.1925x + 4.0547 SD CAR-Collagen y = 0.1694x + 3.5951 SD CAR-Lysozyme y = 0.0737x + 3.0801 SD CAR-Myoglobin y = 0.092x + 3.3139

[0194] FIGS. 11A and 11B show the intrinsic dissolution rate (IDR) for the co-amorphous forms of IND with various proteins, where the co-amorphous forms are prepared by spray-drying. Spray dried IND-WPI (1.494 mg cm.sup.2 min.sup.1) has a 19 fold increase in dissolution rate when compared to crystalline IND and an 11 fold increase compared to ball milled amorphous IND. For spray dried IND-WPH (1.3066 mg cm.sup.2 min.sup.1) there is 4 fold increase from crystalline IND and about 2 fold increase from ball milled amorphous IND. There is also a 1 fold increase in the dissolution rate of spray dried IND-WPI when compared to spray dried IND-WPH. The dissolution rate of SD IND-OVALBUMIN, SD IND-GELATIN, SD IND LYSOZYME, SD IND-MYOGLOBIN, SD IND-COLLAGEN and SD IND-CASEIN are 3.5, 7.9, 13.5, 11.6, 3.7, 2.8 fold higher than C IND and 2, 4.7, 8, 6.8, 2.2, 1.7 fold higher than A IND, respectively. On the other hand SD IND-EGG, SD IND-RICE and SD IND-SOY are 4.5, 2.5, 2.3 fold higher than C IND and 2.6, 1.5, 1.4 fold higher than A IND.

[0195] Proteins were paired with the acidic drug IND (pKa: 4.5), neutral drug CEL (pKa: 11.1) and basic drug CAR (pKa: 7.8) based on their isoionic points (pl), pH value at which a zwitterion molecule has an equal number of positive and negative charges, and subsequently the intrinsic dissolution rate (IDR) was determined (FIG. 13A, 13B, 13C). The pl's of lysozyme, myoglobin, collagen and casein are 10.7, 7.4, 5.8 and 4.6, respectively. The pl of WPI is 5, since WPI is a mixture of a-lactalbumin, 13-lactoglobulin and BSA, which have pl's of 5.0, 5.2 and 5.2, respectively. For co-amorphous mixtures with IND, SD IND-LYSOZYME (1.0676 mg cm.sup.2 min.sup.1) had the highest IDR followed by SD IND-MYOGLOBIN (0.9113 mg cm.sup.2 min.sup.1), SD IND-COLLAGEN (0.2924 mg cm.sup.2 min.sup.1) and SD IND-CASEIN (0.2224 mg cm.sup.2 min.sup.1). This shows that at pH 7.2 the negatively charged IND achieves a higher IDR when paired with a co-former protein with high pl, indicating that electrostatic attraction between the negatively charged IND and the protein with net positive charge has a positive influence on the dissolution rate.

[0196] A similar pattern was found in case of CAR, which is positively charged at pH 7.2 and showed higher IDR with decreasing pl of proteins used to form the co-amorphous mixtures. SD CAR-CASEIN (0.1925 mg cm.sup.2 min.sup.1), SD CAR-COLLAGEN (0.1694 mg cm.sup.2 min.sup.1) and SD CAR-WPI (0.1948 mg cm.sup.2 min.sup.1) showed higher IDR compared to CAR mixed with proteins with a net positive charge, lysozyme and myoglobin. SD CAR-CASEIN was 2.6 fold higher than SD CAR-LYSOZYME (0.0737 mg cm.sup.2 min.sup.1) and 2.1 fold higher than SD CAR-MYOGLOBIN (0.092 mg cm.sup.2 min.sup.1), whereas SD CAR-COLLAGEN was 2.3 and 1.8 fold higher than SD CAR-LYSOZYME and SD CAR-MYOGLOBIN, respectively. This suggests that electrostatic attraction between the drug molecule and co-former protein has a positive influence on the resulting IDR compared with electrostatic repulsion. Interestingly, CEL, which is neutral at pH 7.2 also showed higher IDR when combined with proteins with a net negative charge as co-former. SD CEL-CASEIN (0.9714 mg cm.sup.2 min.sup.1) showed the highest IDR followed by SD CEL-COLLAGEN (0.6629 mg cm.sup.2 min.sup.1), SD CEL-MYOGLOBIN (0.2628 mg cm.sup.2 min.sup.1) and SD CEL-LYSOZYME (0.2019 mg cm.sup.2 min.sup.1). This may be due to neutral charge at pH 7.2 and other additional properties of CEL.

[0197] In all cases, when using WPI as a co-former the IDR was found to be higher than that observed with other proteins, irrespective of the pl and independent of the nature of the drug (acidic, basic or neutral). For further visualization of the correlation between the IDR of drugs with different charge and proteins with different net charge the IDR of the different drug-protein combinations were plotted against the pl of the proteins (FIGS. 15A-15C). There is a good correlation between the pl of the proteins and the resulting IDR of the co-amorphous mixtures except for WPI when used as a co-former with IND. This may be explained by the composition and properties of WPI. As mentioned, WPI consists of a mixture of multiple proteins, which could result in higher heterogeneity of the resulting co-amorphous mixtures compared with a co-amorphous mixture consisting of a single protein and drug. This could have a positive effect on the dissolution rate of the drug. Further, it is believed that certain properties of the proteins of WPI make them especially suitable for forming stable interactions with drug molecules, which result in enhanced dissolution rate of the drug.

[0198] FIG. 14A shows co-amorphous forms of IND spray dried with WPI and with other proteins that represent mixtures of several proteins together. Egg protein isolate (EGG) is a mixture consisting mainly of ovalbumin, ovomucoid, ovomucin and lysozyme, whereas, rice protein isolates (RICE) consist of glutenin, globulin, albumin and prolamin. On the other hand, soy protein isolates (SOY) are mixture of globular proteins, conglycinin and glycinin, and gelatin is essentially denatured and hydrolyzed collagen. The IDR of SD IND-EGG was found to be 1.9 fold higher than that of SD IND-SOY and 1.8 fold higher than that of SD IND-RICE. Furthermore, SD IND-gelatin showed a 1.8 fold higher intrinsic dissolution than SD IND-EGG. From all IND-protein mixtures, again SD IND-WPI showed the highest intrinsic dissolution, which was 2.4 fold higher than SD IND-gelatin. Overall, SD IND-WPI had the highest dissolution rate followed by SD IND-gelatin, SD IND-EGG,

[0199] SD IND-RICE and SD IND-SOY. FIG. 17 shows that the isoionic points of these proteins (pl) did not have a direct co-relation with the IDR observed, most likely due to the proteins being a combination of several other proteins. SD IND-WPI, however, had the highest dissolution rate. Each of the proteins comprising WPI indicated a relatively high IDR, especially a-lactalbumin, when used alone (Table 2a) with the drug molecule and resulted in an even higher IDR when mixed as WPI. SD IND-gelatin also indicated a high dissolution rate compared with many other proteins but but both SD IND-WPI and SD IND-WPH resulted in a higher dissolution rate.

[0200] FIG. 14B shows SD IND-protein co-amorphous mixtures where the co-former proteins were selected based on their molecular weight (Mw), BSA having the highest Mw (=66500), followed by ovalbumin (=45000), casein (23000) and WPI (15000). These four proteins, BSA, ovalbumin, casein and WPI also have similar pl's of 5.2, 4.8, 4.6 and 5, respectively. Interestingly it was found that the dissolution rate of SD IND-BSA was around 1.05 fold higher than that of SD IND-OVALBUMIN and 1.3 fold higher than SD IND-CASEIN, although they were relatively similar. This may suggest that the Mw of the proteins has a slight influence on the resulting dissolution rate of the co-amorphous mixture, possibly due due to high diversity in interaction formed with high Mw proteins.

[0201] FIG. 16A also illustrates this trend. Here, SD IND-WPI was again an outlier, resulting in higher IDR irrespective of its lower Mw.

Example 3: Intrinsic Dissolution Rate of Different Forms of CAR, PAR and FUR

[0202] FIG. 3 depicts the IDR of different forms of CAR (FIG. 3A), PAR (FIG. 3B), and FUR (FIG. 3C). See Table 2 for the relevant line equations.

[0203] FIG. 3 demonstrates that the IDR of ball milled amorphous CAR (0.0214 mg cm.sup.2 min.sup.1), PAR (0.201 mg cm.sup.2 min.sup.1) and FUR (0.514 mg cm.sup.2 min.sup.1) is 1.8, 1.2, and 1.5 fold higher than the IDR of crystalline CAR (0.0117 mg cm.sup.2 min.sup.1), PAR (0.1632 mg cm.sup.2 min.sup.1) and FUR (0.1024 mg cm.sup.1 min.sup.1) respectively.

[0204] Moreover, there is a great increase in the IDR of for the co-amorphous drug substance-WPI/WPH mixtures. The IDR of the spray dried (SD) CAR-WPI and SD CAR-WPH (0.194 mg cm.sup.2 min.sup.1 and 0.0794 mg cm.sup.2 min.sup.1, respectively) shows nearly a 17 (WPI) and a 7 (WPH) fold increase compared to crystalline CAR and a 9 (WPI) and 3.7 (WPH) fold increase compared to ball milled amorphous CAR.

[0205] In case of SD PAR-WPI and SD PAR-WPH (0.5433 mg cm.sup.1 min.sup.1 and 0.4664 mg cm.sup.1 min.sup.1) there is a 3.3 (WPI) and 2.8 (WPH) fold increase in dissolution rate when compared to crystalline PAR and 2.7 (WPI) and 2.3 (WPH) fold increase compared to the individual ball milled amorphous PAR.

[0206] For SD FUR-WPI and SD FUR-WPH (0.4115 mg cm.sup.2 min.sup.1 and 0.2359 mg cm.sup.1 min.sup.1) a 4 (WPI), 2.3 (WPH) fold increase was observed in dissolution rate when compared to crystalline FUR and 2.6 (WPI), 1.5 (WPH) fold increase compared to the individual amorphous (BM) FUR. It can be concluded from FIGS. 2 and 3 that spray dried drug-WPI mixtures has the highest dissolution rate when compared to its crystalline or amorphous counterparts.

Example 4: Intrinsic Dissolution Rate of Amorphous IND with Different WP/Components

[0207] FIG. 4 shows that the IDR of WPI (3.8317 mg cm.sup.2 min.sup.1) is 3.4, 11 and 13.4 folds higher than its components: a-lactalbumin, 13-lactoglobulin and BSA, respectively. SD IND-WPI is 6 fold higher than SD IND-WPI with trypsin, 13.2 fold than SD IND-WPI with pepsin and 6.6 fold more than SD IND-WPI with trypsin+pepsin (trypsin added first). SD IND-WPI is 1.25 fold higher SD IND-WPI with pepsin+trypsin (pepsin added first). Consequently, it can be concluded that the intact native form of WPI provides with the highest dissolution rate when compared to co-amorphous forms digested with enzymes.

Example 5: Powder Dissolution Studies

[0208] As seen in FIG. 5, we can see that co-amorphous SD IND-WPI shows higher dissolution rate compared to BM IND-WPI. Amorphous IND itself shows more dissolution than the PM IND-WPI. The solubility of IND in its amorphous state is more than double the value crystalline IND. This is due to the solubility of a compound in the amorphous form is higher than in the more stable crystalline form because the Gibbs free energy is higher. This increase in dissolution rate from the amorphous drug alone is due to the increase in molecular interaction upon co-amorphisation.

Example 6: Stability Studies

[0209] FIG. 6 depicts the physical stability of co-amorphous forms of WPI and WPH with IND, CAR, FUR, and PAR, respectively. Amorphous IND, CAR, FUR and PAR were found to be stable for less than a week shown by recrystallization from XRPD. In contrast, the co-amorphous spray dried drug substance-protein mixtures were found to be stable for several months. SD IND-WPI and SD IND-WPH were found to be stable for more than 20 months (FIG. 19) whereas most other SD IND-protein co-amorphous forms such as for example SD IND-gelatin, SD IND-BSA and SD IND-collagen (see Table 3 below for detailed stability study) were only stable for 2-3 months. Co-amorphous formulations of WPI and WPH with the drugs CAR and FUR were also stable up to 8 and 18 months, respectively. The co-amorphous form of SD CEL-WPI was also stable for more than 8 months. Hence, WPI and WPH were the proteins and co-formers for co-amorphous mixtures with the best stabilizing properties for all of the investigated drugs. It was also more stable than a solid dispersions prepared using PVP (a commonly used co-former for amorphous formulations) and drug, even at a higher drug concentration (drug loading).

[0210] This indicates that WPI and WPH are not only performing superiorly compared with other proteins and protein mixtures with regards to dissolution when combined with poorly soluble drugs to form co-amorphous mixtures or solid dispersions. They are also performing superiorly compared with other proteins and protein mixtures with regards to physical stability with several fold increase in stability observed for WPI and WPH.

TABLE-US-00005 TABLE 3 Stability data showing the number of months SD drug-protein mixtures remained co-amorphous. XRPD was used to conclude this data. On recrystallization, mixtures showed the peaks of respective drugs in diffractograms. The stability study was stopped only for the samples which showed crystalline peaks. Number of months at Number of months which recrystallization of at which drug was SD formulations drug was observed still amorphous IND-WPI 20 IND-WPH 20 SD IND-lactalbumin 3 2 SD IND- lactalbumin 3 2 IND-BSA 3 2 IND-GELATIN 2 1 IND-EGG 3 2 IND-WPI (ENZ P) 2 1 IND-WPI (ENZ T + P) 2 1 IND-WPI (ENZ P) 2 1 IND-WPI (ENZ P + T) 2 1 IND-RICE 2 1 IND-SOY 2 1 IND-LYSOZYME 3 2 IND-MYOGLOBIN 3 2 IND-COLLAGEN 2 1 IND-CASEIN 2 1 CAR-WPI 8 7 CAR-WPH 8 7 PAR-WPI 2 1 PAR-WPH 2 1 FUR-WPI 18 17 FUR-WPH 18 17 PVP 75%-FUR 25% 3 2 WPI 75%-FUR 25% 18 17 WPI 50%-FUR 50% 16 15 WPI 25%-FUR 75% 5 4 IND-OVALBUMIN 6 5 CEL-CASEIN 6 5 CEL-COLLAGEN 6 5 CEL-LYSOZYME 4 3 CEL-MYGLOBIN 4 3 CEL-WPI 8 CAR-CASEIN 1 CAR-COLLAGEN 1 CAR-LYSOZYME 1 CAR-MYGLOBIN 1

Example 7: In Vivo Studies

[0211] FIG. 7 depicts the bioavailability of co-amorphous (spray dried) forms of FUR and WPI following oral administration to rats. The SD WPI:FUR (75% WPI, 25% FUR) showed the highest bioavailability (11.4%) followed closely by SD WPI:FUR (50% WPI, 50% FUR) (11.3%) and SD WPI:FUR (25% WPI, 75% FUR) (10.6%). This indicates that the bioavailability increases with increasing WPI content. The bioavailability of SD WPI:FUR samples was significantly higher than that of SD PVP:FUR (6.3%) and the physical mixture. Crystalline FUR showed the lowest bioavailability (4.7%) as expected and was followed by amorphous FUR (5.1%), both of which were significantly lower than the SD WPI:FUR samples. The ratios of WPI and FUR were varied by changing the content of WPI while the content of FUR is kept constant.

[0212] FIG. 8 depicts the maximum concentration (Cmax) following oral administration to rats. The pattern of Cmax values was in line with the bioavailability results. An increase in the amount of WPI in the co-amorphous resulted in increased Cmax levels.

Example 8: Intrinsic Dissolution Rate of Compounds Used for In Vivo Experiments

[0213] FIG. 9 depicts the IDR of the compositions used for the in vivo experiments. The IDR of the SD WPI-FUR (75% WPI, 25% FUR) was found to have the highest dissolution rate. It was 5.67 fold higher than the crystalline FUR and 3.7 fold higher than the amorphous FUR. It was followed by SD WPI-FUR (50% WPI, 50% FUR) which was 4 fold more than crystalline and 2.6 fold more than amorphous FUR. Interestingly, it was found that traditionally used SD PVP/FUR (75% PVP, 25% FUR) was only 1 fold higher than the SD WPI-FUR (25% WPI, 75% FUR). Amorphous FUR was 0.69 fold higher than PM WPI-FUR (50% WPI, 50% FUR) See table 4 for the relevant line equations.

TABLE-US-00006 TABLE 4 Line equations for intrinsic dissolution testing for all mixtures used in in vivo studies. Sample y (mg cm.sup.1 min.sup.1) C FUR y = 0.1024x + 2.3685 A FUR y = 0.1548x + 2.4064 PM FUR-WPI (50% PUR, 50% WPI) y = 0.1065x + 4.6238 SD PVP 75%-FUR 25% y = 0.1213x + 4.9021 SD WPI 75%-FUR 25% y = 0.5811x + 3.1377 SD WPI 50%-FUR 50% y = 0.4114x + 5.0757 SD WPI 25%-FUR 75% y = 0.119x + 3.7316 C: crystalline, A: amorphous, PM: physical mixture, SD: spray dried.

Overall Conclusion:

[0214] From the above examples, we can conclude that various proteins, notably WPI, are promising new excipients for the co-amorphization of crystalline drug substance. Co-amorphous forms of the drugs IND, CAR, FUR, PAR and CEL show remarkably higher dissolution rate compared to the crystalline or mono-amorphous forms of the drug substances, but most notably also to other competing technologies that are developed to improve dissolution rate and solubility of poorly soluble drug substances. Improved bioavailability and PK-profile was also observed for all formulations with WPI, compared with mono-amorphous drug substances and solid dispersions (with PVP) and physical mixtures. Furthermore, the co-amorphous drug substance-WPI forms also showed an increased physical stability compared to their mono-amorphous counterparts.