Impact of polymer type on bioperformance and physical stability of hot melt extruded formulations of a poorly water soluble drug

Accepted Manuscript Title: IMPACT OF POLYMER TYPE ON BIOPERFORMANCE AND PHYSICAL STABILITY OF HOT MELT EXTRUDED FORMULATIONS OF A POORLY WATER SOLUBLE DRUG Author: Amitava Mitra Li Li Patrick Marsac Brian Marks Zhen Liu Chad Brown PII: DOI: Reference:

S0378-5173(16)30236-8 http://dx.doi.org/doi:10.1016/j.ijpharm.2016.03.036 IJP 15634

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

23-10-2015 13-3-2016 20-3-2016

Please cite this article as: Mitra, Amitava, Li, Li, Marsac, Patrick, Marks, Brian, Liu, Zhen, Brown, Chad, IMPACT OF POLYMER TYPE ON BIOPERFORMANCE AND PHYSICAL STABILITY OF HOT MELT EXTRUDED FORMULATIONS OF A POORLY WATER SOLUBLE DRUG.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2016.03.036 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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IMPACT OF POLYMER TYPE ON BIOPERFORMANCE AND PHYSICAL STABILITY OF HOT MELT EXTRUDED FORMULATIONS OF A POORLY WATER SOLUBLE DRUG Amitava Mitraa* [email protected], Li Lib, Patrick Marsacc,e, Brian Marksb, Zhen Liuc, Chad Brownd a Biopharmaceutics, Pharmaceutical Sciences and Clinical Supply, Merck & Co. Inc. b Analytical Sciences, Pharmaceutical Sciences and Clinical Supply, Merck & Co. Inc. c Preformulation, Pharmaceutical Sciences and Clinical Supply, Merck & Co. Inc. d Formulation Sciences, Pharmaceutical Sciences and Clinical Supply, Merck & Co. Inc. e Current Affiliation: College of Pharmacy, University of Kentucky * Corresponding Author at: Merck & Co., Inc, West Point, PA-19486, USA; Tel.: +1 215 652 8551; Fax: +1 215 993 1245

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Graphical abstract

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Abstract

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Amorphous solid dispersion formulations have been widely used to enhance bioavailability of poorly

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soluble drugs. In these formulations, polymer is included to physically stabilize the amorphous drug by

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dispersing it in the polymeric carrier and thus forming a solid solution. The polymer can also maintain

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supersaturation and promote speciation during dissolution, thus enabling better absorption as compared to

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crystalline drug substance. In this paper, we report the use of hot melt extrusion (HME) to develop

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amorphous formulations of a poorly soluble compound (FaSSIF solubility = 1 μg/mL). The poor

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solubility of the compound and high dose (300 mg) necessitated the use of amorphous formulation to

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achieve adequate bioperformance. The effect of using three different polymers (HPMCAS-HF,

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HPMCAS-LF and copovidone), on the dissolution, physical stability, and bioperformance of the

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formulations was demonstrated. In this particular case, HPMCAS-HF containing HME provided the

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highest bioavailability and also had better physical stability as compared to extrudates using HPMCAS-

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LF and copovidone. The data demonstrated that the polymer type can have significant impact on the

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formulation bioperformance and physical stability. Thus a thorough understanding of the polymer choice

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is imperative when designing an amorphous solid dispersion formulation, such that the formulation

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provides robust bioperformance and has adequate shelf life.

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Keywords: hot melt extrusion; dissolution; pharmacokinetics; stability; solid dispersion; anti-nucleation

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1. INTRODUCTION

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The use of amorphous solid dispersion (ASD) formulations to enhance bioavailability of poorly soluble

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drugs has been widely published (Serajuddin, 1999; Newman et al. 2012; Paudel et al. 2013; Lang et al.

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2014). The ASDs typically enhance bioavailability due to higher kinetic solubility of the drug substance

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and increased dissolution rate of the formulation, by the virtue of the fact that the drug molecule exists in

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the formulation in a high energy amorphous state. The hot melt extrusion (HME) process has been

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successfully used in pharmaceutical applications to produce ASDs and several of these products have

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been approved by the FDA including Noxafil®, Kaletra™, and Norvir™ (Lang et al. 2014; Crowley et al.

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2007; Repka et al. 2007). Briefly, in the HME process the drug substance and stabilizing polymer are melt

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compounded in an extruder forming a solid solution. Upon exiting the extruder the molten mixture is

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quickly quenched such that the temperature drops below its glass transition temperature thus kinetically

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inhibiting recrystallization. These extrudates are then processed to produce the final tablet product. While

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the HME process has certainly been a great addition in the pharmaceutical scientist’s repertoire to

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formulate poorly soluble drugs, the successful development of a product using HME is determined by

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careful consideration of material properties (drug and polymer), process (temperature, shear) and

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equipment design. For a more detailed description of the HME process and operations, interested readers

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are referred to several in-depth reviews on this topic (Lang et al. 2014; Crowley et al. 2007; Repka et al.

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2007). Most often, unique formulations yield unique physical stability, dissolution performance, and

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ultimately bioperformance.

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particularly complex – each formulation may require unique processing conditions given the differences

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in the material properties and the associated phase diagrams. A balance must be struck between

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processing, physical stability, and measures of in vitro performance without compromising

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bioperformance. Although there are reports which focus on physical stability, dissolution, and other

The number of formulation options makes the production of ASDs

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measures of in-vitro performance (Ilevbare et al. 2013; Sarode et al. 2014), relatively few reports

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highlight the influence of formulation on bioperformance.

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The amorphous nature of the active pharmaceutical ingredient (API) could lead to physical instability in

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the drug product such as conversion to the crystalline state. One common approach to physically stabilize

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the amorphous drug is to disperse the API in a polymeric carrier and thus form a solid solution of the drug

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and the polymer. Another goal of using the polymer matrix is to maintain the supersaturation achieved

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during dissolution over an extended period of time so as to better enable absorption of the solubilized API

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i.e. the higher energy amorphous form of the drug substance transiently increases solubility relative to that

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of the stable crystalline form and the polymer inhibits nucleation and crystal growth and maintains

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supersaturation for an extended time period (Guzman et al. 2007; Brouwers et al. 2009; Augustijns et al.

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2012). The polymer can also promote speciation during dissolution, which also would enhance

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bioperformance of the formulation (Friesen et al. 2008). Several polymers have been reported in literature

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for use in pharmaceutical ASDs, interested readers are referred to the following references (Paudel et al.

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2013; Lang et al. 2014; Konno et al. 2008; Curatolo et al. 2009; Rumondor et al. 2009; Tajarobi et al.

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2011).

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In this paper, we report the development of an HME formulation, in-vitro characterization including

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dissolution and physical stability, as well as preclinical pharmacokinetics (PK) data for Merck compound

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A. In addition, we also report the impact of three different polymers used in the HME formulation-

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Copovidone, HPMCAS-HF, and HPMCAS-LF, on the physical stability and bioperformance. Compound

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A is a low solubility and high permeability (BCS class 2) compound (Table 1) with a fairly high

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efficacious dose projection of approximately 300 mg. This results in a very high dose to volume ratio i.e.

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high dose number (Do = dose/FaSSIF solubility/250 mL) of 1200 indicating significant solubility limited 5

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absorption for this compound (Oh et al. 1993). Hence there was a need to develop an enabled formulation

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such as an ASD so as to transiently increase the concentration in solution and the dissolution rate. Further,

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the data shown in this paper also demonstrates that the choice of polymer can have a significant impact on

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the performance (physical stability and bioavailability) of the ASD formulation. It is the aim of this

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publication to highlight the influence that formulation selection has on bioperformance and physical

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stability of amorphous formulation so as to facilitate improved approaches and methodologies employed

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in the careful balance between process, formulation, and performance.

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2. MATERIALS and METHODS

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2.1 Preparation of Hot Melt Extrusion (HME) formulations of compound A:

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The melting point (Tm) of compound A is approximately 140°C and it is thermally stable up to

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approximately 220°C by thermal gravimetric analysis (TGA), making the compound a prime candidate

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for HME. Thus, formulations of compound A were extrusion compounded at a 20% drug load in a custom

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built co-rotating 7.5 mm twin screw extruder with L/D=15 and 1 cm slit die (MP&R, Hackensack, NJ)

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with three individual polymers- copovidone (Kollidon VA-64™, BASF), hydroxypropyl methyl cellulose

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LF grade (HPMCAS-LF, Shin Etsu), hydroxypropyl methyl cellulose HF grade (HPMCAS-HF, Shin

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Etsu). These three polymers were chosen based on high-throughput screening to assess compatibility of

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the drug and the polymer. This was achieved by film-casting of the drug with several polymers, and

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analysis of the film casts by XRD, DSC and dissolution studies (data not shown). The extruder was

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equipped with all conveying screws and heated to target a product temperature of 145°C to ensure facile

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processing of each polymer. This temperature was above the Tm of compound A thus making this a facile

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compounding process as the drug was completely melted. The screw speed was set at 50 revolutions per

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minute. Approximately 7.5g of feed stock for each formulation was pre-blended in a turbula blender for

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10 minutes prior to extrusion to help ensure compositional homogeneity. A VIBRI (SympaTec, Germany) 6

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vibratory feeder was used to convey the formulation into the extruder. The gap width was set to 8 mm

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and a V-shaped tray was used to convey the material to the feed port on the extruder. The vibration setting

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was set at 35% to provide a feed rate of approximately 1 g/minute. Initial breakthrough of the extruded

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formulation through the slit die (1 mm x 10 mm) was approximately 3.5 minutes after the start of feeding.

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Strands of clear, glassy extrudate were collected on a custom built take-off belt equipped with a dual

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nozzle cold air gun Vortec™ (AiRTX, Cincinnati, OH) to provide rapid quenching. Extrudates of each

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composition (20% compound A and 80% polymer) were milled in a coffee grinder (Krups, Milville, NJ)

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on the fine setting for approximately 30 seconds. The particle size of the extrudates was approximately

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200 μm and 100 μm for the HPMCAS and copovidone based extrudates, respectively. Approximately 300

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mg of extrudate (60 mg potency) were hand-filled into hard gelatin capsules (size 00) for dosing to beagle

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dogs and for biorelevant dissolution testing. An overall yield of approximately 55% for the process was

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achieved. The low yield from this process is primarily because of the small batch size (~7.5g), which

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results in fixed losses such as approximately 2g loss in the extruder due to free volume and approximately

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1g loss during milling. This yield is not representative of large scale HME process.

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2.2 Physical characterization of the extrudates:

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Milled extrudate were tested by X-ray diffraction (XRD) and differential scanning calorimetry (DSC) to

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ensure a single phase, amorphous solid dispersion was formed. XRD was performed on a Philips X’Pert

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with a 1 hour scan over a 2Ө of 2-40 (PANalytical, Westborough, MA). Modulated differential scanning

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calorimetry (DSC) was conducted on a TA instrument Q2000 over a temperature range of 0˚C to 130˚C or

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145˚C (TA Instruments, New Castle, DE). The heating rate was 2˚C /min with a modulation frequency of

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±0.5˚C every 60 seconds. Solid dispersions prepared with HPMCAS-L and HPMCAS-H were placed on

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stability at 40°C/35%RH and 40°C/75%RH in open containers and analyzed after 4 weeks of storage. The

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copovidone systems were stored at 30°C/65%RH and 40°C/35%RH. For the stability studies a 7

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combination of temperature and moisture was used to trigger physical instability in the HME formulation.

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The reason of selecting those temperature and relative humidity combinations was to expedite instability

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kinetics (phase separation and/or drug recrystallization) as well as ranking the physical stability of various

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HME formulations. The differences in temperature and relative humidity conditions selected for

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HPMCAS and copovidone formulations were deliberate, since copovidone is known to be significantly

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hygroscopic so much so that it absorbs enough water at the 40°C/75%RH condition that the entire

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dispersion coalesces into a single liquid mass (data not shown).

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2.3 Dissolution studies of the formulations:

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The dissolution studies were conducted in an USP apparatus II (Vankel VK 7000) using 500 mL fasted

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state simulated intestinal fluid (FaSSIF, pH 6.5) at 37 °C and paddle speed of 100 rpm. Samples were

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manually collected at pre-determined time intervals (15, 30, 60 and 120 minutes). 1 mL of sample was

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ultracentrifuged (Beckman Coulter Optima TLX Ultracentrifuge) at 80,000 rpm for 15 minutes and was

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diluted immediately with 500 uL of 50:50 v/v water: acetonitrile to prevent any further precipitation of

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compound A from the dissolution medium. These samples were analyzed by reverse phase HPLC using a

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mobile phase of 30% 0.1% H3PO4 and 70% acetonitrile at a flow rate of 5 mL/minute, UV detection at

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260 nm and a Merck KGaA Chromolith SpeedROD 18e monolithic column. The compound A retention

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time was 0.4 minutes.

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2.4 Dog study and pharmacokinetic analysis:

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In order to investigate the bioperformance of the HME formulations, pharmacokinetic studies were

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conducted in male beagle dogs at a dose of 5 mg/kg (equivalent to the projected human dose of 300 mg).

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After an overnight fast, the dogs were dosed with one capsule each containing HPMCAS-HF, HPMCAS-

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LF or copovidone based HME formulation or one conventional dry filled capsule (DFC) formulation 8

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containing the crystalline form of compound A, followed by 3.5 mL/kg water rinse. Water was restricted

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for 1 hour post dose. Food was returned at 4 hours after dosing. 1 mL blood sample was drawn from a 21g

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catheter placed in the cephalic vein into EDTA tubes at pre-dose and 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours

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after dosing. The plasma was separated by centrifugation (10 minutes at 2500 rpm) and kept frozen at -

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70ºC until analysis. Concentrations of compound A in dog plasma were quantified by LC-MS/MS

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analysis. All studies were conducted under a protocol approved by the Merck IACUC.

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Area under the curve (AUC0-24hr), observed maximum plasma concentration (Cmax), and time of Cmax

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(Tmax) were calculated using the linear trapezoidal, non-compartmental model in WinNonLin v5.2

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(Certara, Princeton, NJ). Plasma concentration values below LOQ were set at zero for PK calculation

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purposes.

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3. RESULTS

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3.1 Physical characterization:

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The XRD and DSC profiles of the extrudates are shown in Figures 1 and 2, respectively. All three HME

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formulations were found to be x-ray amorphous at the initial time-point, indicating the successful

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formation of amorphous solid dispersions. The modulated DSC profiles also showed a single glass

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transition temperature (Tg) of approximately 73°C, 74°C and 81°C for the HME formulations using

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HPMCAS-HF, HPMCAS-LF and copovidone polymers, respectively. These were well above the Tg of

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the pure drug which resides at about 24°C. As confirmed by the XRPD and DSC results, the ASDs

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produced by HME were x-ray amorphous and displayed no amorphous phase separation for any of these

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formulations at the initial time-point.

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After stressing these HME formulations for four weeks under different conditions clear distinction

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between physical stability of each formulation was observed. The HPMCAS-HF based HME maintained 9

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a single amorphous phase at 40°C/75% relative humidity (RH) open dish condition (Figures 1 and 2).

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The HPMCAS-LF based HME showed subtle signs of crystallization at 40°C/75% RH open dish

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condition but was amorphous at 40°C/35% RH open dish. Peaks at 2θ of 17.78 to 18.40 were used to

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determine drug recrystallization for HPMCAS-LF based HME. For better visualization, a close up of this

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region is shown as inset in Figure 1. The copovidone based HME showed signs of crystallization at

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30°C/65% RH open dish condition and showed signs of amorphous-amorphous phase separation (by

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modulated DSC) at 40°C/35% RH open dish condition. These data highlights the need to consider both

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crystallization and amorphous phase behavior since it is reasonable to consider the amorphous phase

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separation of multi-component amorphous systems as a precursor to crystallization. It should be noted

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that the crystalline anhydrous form of compound A used to make the HME formulations was shown to be

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highly crystalline, non-hygroscopic, physically and chemically stable at room temperature and the

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relevant processing conditions (data not shown).

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3.2 Dissolution data:

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The in vitro dissolution showed that the crystalline compound A DFC formulation only achieved a

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concentration of approximately 1.4 μg/mL at 60 minutes (Figure 3), which was consistent with the

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FaSSIF solubility of the crystalline form (1 μg/mL). In contrast, at 60 minutes the HME formulations

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showed dissolution of approximately 13.3, 4.7 and 5.3 μg/mL for HPMCAS-HF, HPMCAS-LF and

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copovidone, respectively (Figure 3). The dissolution data also clearly demonstrated that HPMCAS-HF

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can achieve and maintain a higher level of free drug concentration than HPMCAS-LF and copovidone

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polymers with highest concentrations achieved of approximately 16.4, 14.7, 7.1 μg/mL at 15 minutes,

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respectively.

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3.3 Pharmacokinetics in dogs:

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Table 2 and figure 4 summarize the mean pharmacokinetic parameters and plasma concentration profiles,

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respectively, after oral administration of compound A formulations in beagle dogs. These data clearly

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demonstrated that the HME formulations provided significantly higher bioavailability as compared to the

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dry filled capsule (DFC) formulation containing crystalline form of compound A. The PK data were

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consistent with the biorelevant dissolution data shown in figure 3. For example in the dissolution study,

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compound A concentration at 60 minutes was 0.3-fold for the DFC, as compared to the copovidone based

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HME formulation (Table 3). Similarly, the relative bioavailability of compound A from the dog PK study

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was 0.2 for DFC as compared to copovidone HME. Of the three HME formulations evaluated in this PK

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study, the copovidone based HME showed the worst bioperformance, with the HPMCAS-HF and

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HPMCAS-LF based formulations showing approximately 2.2 and 1.4-fold higher bioavailability,

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respectively, when compared to the copovidone formulation. These PK data were also in agreement with

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the biorelevant dissolution data, which showed that at 60 minutes compound A concentration was 2.5-fold

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for HPMCAS-HF and 0.9-fold for HPMCAS-LF as compared to copovidone (Table 3).

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4. DISCUSSION

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ASDs are a very valuable formulation tool to enhance bioavailability of poorly soluble drugs and enable

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development of compounds, which otherwise would not achieve adequate exposures in human. However

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due to the high energy state of the amorphous drug, these formulations have an inherent physical stability

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liability (due to crystallization of the amorphous drug or drug-polymer phase separation) during storage

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and/or during dissolution. Hence, there is a lot of interest in identifying and selecting polymers that can

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afford sufficient shelf-life to these formulations (Ilevbare et al. 2012; Ilevbare et al. 2013; Rumondor et al.

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2009) as well as be able to maintain supersaturation (Konno et al. 2008; Curatolo et al. 2009; Sarode et al.

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2014) and promote speciation (Friesen et al. 2008) during dissolution so that greater bioavailability can be 11

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achieved. In addition, mechanistic understanding of the interaction of the polymer with the drug substance

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is also critical in gaining an understanding of the formulation behavior as well as for rational formulation

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design to achieve adequate performance (Curatolo et al. 2009; Ilevbare et al. 2012; Friesen et al. 2008). In

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this paper, we report the comparison of three different polymer types on physical stability and

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bioperformance of HME formulations of an extremely poorly soluble compound. These results highlight

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the importance of robust formulation design to ensure adequate product bioperformance and shelf life.

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The physical stability, dissolution and PK data we have shown here clearly indicates that the polymers

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used have an influence on the performance of the HME formulations, with HPMCAS-HF showing the

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best overall performance in this particular case. It is obvious from the dissolution data that amorphous

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compound A has a strong tendency to crystallize during dissolution (Figure 3). Hence the selection of

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polymer to be used in the HME formulation is of critical importance to sustain supersaturation of the

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metastable amorphous drug, which in turn is a key driver for enhancing bioperformance. The dissolution

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profile is characterized by an initial drug supersaturation, followed by nucleation and/or recrystallization,

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resulting in a decrease in free drug concentration. The dissolution data indicated that the crystallization

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inhibition performance was in the order of HPMCAS-HF > HPMCAS-LF > copovidone. The HPMCAS-

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HF and LF systems were able to achieve an apparent solubility of 16.4 and 15.7 ug/ml at 15 minutes,

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respectively, representing a degree of supersaturation (Ds = solubility of an amorphous drug/solubility of

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crystalline drug) of approximately 15. In contrast, the copovidone based system showed lower degree of

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supersaturation of approximately 7. While all the HME formulations showed indications of initial

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supersaturation followed by precipitation, HPMCAS-HF was able to maintain higher degree of drug

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supersaturation during the entire dissolution process as compared to the other two polymer systems. These

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dissolution data also indicated that in this particular case the extrudate particle size had minimal impact on

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their performance. This was demonstrated by the fact that the copovidone extrudates had smaller particle 12

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size (~100 μm) as compared to the HPMCAS extrudates (~200 μm), however HPMCAS extrudates

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showed better dissolution than copovidone. The dissolution data was also corroborated with PK data in

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dogs. The XRD and DSC data also demonstrated that the HPMCAS based HME had superior physical

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stability than the copovidone HME (Figures 1 and 2). These results are in agreement with previous

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studies that have shown that HPMCAS in general is superior to copovidone at both achieving high levels

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of supersaturation as well as inhibiting the recrystallization of dissolved drugs in solution (Konno et al.

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2008; Yin et al. 2014; Ilevbare et al. 2012; Ilevbare et al. 2013; Sarode et al. 2014), which can

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significantly enhance bioperformance of these formulations. In addition HPMCAS is better at resisting

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moisture-induced crystallization of amorphous solid dispersions during storage, as compared to other

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polymers such as PVP (Friesen et al. 2008; Rumondor et al. 2009). The better antinucleation properties of

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HPMCAS as compared to copovidone are often attributed to its unique physicochemical characteristics

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(Ting et al. 2015; Curatolo et al. 2009; Ilevbare et al. 2012). HPMCAS is a cellulose derivative of two

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ethers - methoxy and hydroxypropoxy as well as two esters - acetate and succinate. The hydrophobic

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acetate groups are thought to facilitate the molecular dispersions of hydrophobic drugs within the

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polymeric matrix through hydrophobic-hydrophobic interactions, and the small amount of unreacted

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hydrophilic hydroxyls allows sufficient hydration of the resulting ASDs in solution. In addition, the

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carboxylic acids from succinates (pKa ~ 5) are ionized at intestinal pH (approximately pH 6-7) and these

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negative charges can stabilize polymer - drug nanostructures that are formed during dissolution, through

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repulsive surface charge, which can be important in preventing crystallization of the dissolved drug

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(Friesen et al. 2008). This amphiphilicity is not present in a hydrophilic polymer such as copovidone.

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Hence it cannot provide sufficient stabilization of supersaturation through hydrophobic interactions or

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repulsive charge (Ting et al. 2015). In addition, copovidone has the lowest glass transition temperature at

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around 105°C and it is the most hygroscopic of the polymers studied, these properties also reduce its anti-

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nucleation abilities. 13

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The dissolution and physical stability data also clearly showed that HPMCAS-HF was further

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differentiated from HPMCAS-LF in its ability to better inhibit recrystallization of compound A, in spite

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of the structural similarity between the two polymers. We believe that enhanced hydrophobicity of

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HPMCAS-HF is responsible for the superior crystallization inhibition of compound A. In fact, Ueda et al.

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(Ueda et al. 2013; Ueda et al. 2014) have shown that HPMCAS-HF inhibited the recrystallization of

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several poorly soluble drugs (e.g. carbamazepine) more strongly than HPMCAS-LF. Solution NMR

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studies showed that the molecular mobility of these drugs were clearly suppressed in the HPMCAS-HF

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solution compared to that in the HPMCAS-LF solution. These studies also revealed strong interaction of

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the drug with the acetate substituent of HPMCAS, however interaction with the succinate substituent was

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quite small. Since HPMCAS-HF is more hydrophobic than HPMCAS-LF due to its higher ratio of acetate

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to succinate, this might be responsible for the stronger hydrophobic interaction between HPMCAS-HF

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and poorly soluble drugs. Similar intermolecular hydrophobic interactions between the drug and

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HPMCAS-HF are most likely responsible for substantially better inhibition of recrystallization of

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compound A during dissolution and better physical stability, as compared to HPMCAS-LF.

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Finally, PK studies in dogs demonstrated that bioperformance of these formulations were in agreement

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with the in-vitro dissolution data. A valid question when using preclinical animal models for formulation

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screening prior to human studies is whether metabolic and/or physiological differences between species

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can lead to different formulation performance. However this will appear unlikely for Compound A.

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Preclinical studies using liver microsome suggested similar primary metabolic route (CYP3A4) in both

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dogs and humans and the compound exhibited low in vivo clearance in dogs (plasma clearance = 3.1

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mL/min/kg). While no intravenous data is available in the clinic to allow for calculation of clearance, the

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predicted clearance in human is also low (< 5 mL/min/kg). These data suggested that there are minimal 14

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metabolic differences for compound A in dogs and human. It should be also noted that given the high

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permeability (BCS 2) of the compound, differences in intestinal permeability between species is also

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unlikely to contribute to differential formulation behavior in the preclinical model and in the clinic. Thus

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it could be assumed that dog is an appropriate species to investigate the formulation bioperformance and

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guide formulation development for clinical evaluation. In addition, dogs are generally considered to be an

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appropriate species for screening formulations for clinical evaluation due to similarities in gastrointestinal

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physiology (Kararli 1995; Hasiwa et al. 2011) and there are numerous literature examples of use of dogs

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to successfully guide formulation development (Miller et al. 2015; Jang & Kang, 2014; Zheng et al.,

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2007). As is seen in table 3, relative bioavailability of the formulations in dog were very close to the

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relative concentrations achieved at 60 minutes in the biorelevant dissolution study. These data showed

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that the ASD formulations achieved significantly higher exposures of compound A as compared to the

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DFC formulation containing crystalline form of the drug substance. These results are similar to several

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previous publications (Lakshman et al. 2008; He et al. 2010; Newman et al. 2012; Lang et al. 2014),

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which have demonstrated the use of HME based ASD formulations to enhance bioavailability of poorly

315

soluble drugs. In addition, clear PK (AUC0-24hr and Cmax) differentiation was observed between the three

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ASD formulations in these dog studies (Table 2), with the HPMCAS-HF based HME showing the highest

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bioavailability. This indicated that the dissolution differences observed between the ASD formulations

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were significant enough to result in differences in the extent and rate of absorption of compound A in

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dogs. The prolonged Tmax observed for HPMCAS-LF and copovidone based systems as compared to

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HPMCAS-HF (4 hours vs. 1 hour) cannot be explained with the current data and might indicate the need

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for further mechanistic understanding of the in-vivo behavior of these formulations. The data presented

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here demonstrate that not only the type of polymer used (e.g. HPMCAS vs. copovidone) but also the

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substituents on HPMCAS (e.g. HPMCAS-HF vs. HPMCAS-LF) can have a profound effect on the

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performance (physical stability and bioavailability) of an ASD formulation. As a result, careful

325

consideration should be given on the choice of polymer used as a matrix in ASD formulations.

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5. CONCLUSION

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The data shown here demonstrate the effect of polymer type (e.g. HPMCAS vs. copovidone) on

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bioperformance and physical stability of a hot melt extruded formulation. It was further shown that the

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substituents on HPMCAS (e.g. HPMCAS-HF vs. HPMCAS-LF) had profound effect on the performance

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of the HME. In this particular case HPMCAS-HF containing HME provided the highest bioavailability in

332

dogs and also had better physical stability as compared to extrudates with HPMCAS-LF and copovidone.

333

These data highlight the influence that polymer selection can have on the performance of amorphous

334

formulations. Thus it is imperative that a thorough understanding of the interaction of polymer with drug

335

substance is gained during formulation design. This will enable rational formulation design to achieve

336

adequate product bioperformance and shelf life.

337 338

ACKNOWLEDGEMENTS

339

The authors would like to thank Kim Manser and Becky Nissley for conducting the dog studies.

340

16

340

REFERENCES

341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384

Augustijns, P., Brewster, M.E. 2012. Supersaturating drug delivery systems: fast is not necessarily good enough. J. Pharm. Sci. 101, 7-9. Brouwers, J., Brewster, M.E., Augustijns, P. 2009. Supersaturating drug delivery systems: the answer to solubility-limited oral bioavailability? J. Pharm. Sci. 98, 2549-2572. Crowley, M.M., Zhang, F., Repka, M.A., Thumma, S., Upadhye, S.B., Battu, S.K., McGinity, J.W., Martin, C. 2007. Pharmaceutical applications of hot-melt extrusion: part I. Drug Dev. Ind. Pharm. 33, 909-926. Curatolo, W., Nightingale, J.A., Herbig, S.M. 2009. Utility of hydroxypropyl methyl cellulose acetate succinate (HPMCAS) for initiation and maintenance of drug supersaturation in the GI milieu. Pharm. Res. 26, 1419-1431. Friesen, D.T., Shanker, R., Crew, M., Smithey, D.T., Curatolo, W.J., Nightingale, J.A. 2008. Hydroxypropyl methylcellulose acetate succinate-based spray-dried dispersions: an overview. Mol. Pharm. 5, 1003-1019. Guzmán, H.R., Tawa, M., Zhang, Z., Ratanabanangkoon, P., Shaw, P., Gardner, C.R., Chen, H., Moreau, J.P., Almarsson, O., Remenar, J.F. 2007. Combined use of crystalline salt forms and precipitation inhibitors to improve oral absorption of celecoxib from solid oral formulations. J. Pharm. Sci. 96, 26862702. Hasiwa, N., Bailey, J., Clausing, P., Daneshian, M., Eileraas, M., Farkas, S., Gyertyán, I., Hubrecht, R., Kobel, W., Krummenacher, G., Leist, M., Lohi, H., Miklósi, A., Ohl, F., Olejniczak, K., Schmitt, G., Sinnett-Smith, P., Smith, D., Wagner, K., Yager, J.D., Zurlo, J., Hartung, T. 2011. Critical evaluation of the use of dogs in biomedical research and testing in Europe. ALTEX. 28, 326-340. He, H., Yang, R., Tang, X. 2010. In vitro and in vivo evaluation of fenofibrate solid dispersion prepared by hot melt extrusion. Drug Dev. Ind. Pharm. 36, 681-687. Ilevbare, G.A., Liu, H., Edgar, K.J., Taylor, L.S. 2012. Understanding polymer properties important for crystal growth inhibition impact of chemically diverse polymers on solution crystal growth of ritonavir. Cryst. Growth Des.12, 3133–3143. Ilevbare, G.A., Liu, H., Edgar, K.J., Taylor, L.S. 2013. Impact of polymers on crystal growth rate of structurally diverse compounds from aqueous solution. Mol. Pharm. 10, 2381-2393. Jang, S.W., Kang, M.J. 2014. Improved oral absorption and chemical stability of everolimus via preparation of solid dispersion using solvent wetting technique. Int J Pharm. 473, 187-193. Kararli, T. 1995. Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals. Biopharm. & Drug Disposition. 16, 351-380.

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Konno, H., Handa, T., Alonzo, D.E., Taylor, L.S. 2008. Effect of polymer type on the dissolution profile of amorphous solid dispersions containing felodipine. Eur. J. Pharm. Biopharm. 70, 493-499. Lakshman, J.P., Cao, Y., Kowalski, J., Serajuddin, A.T. 2008. Application of melt extrusion in the development of a physically and chemically stable high-energy amorphous solid dispersion of a poorly water-soluble drug. Mol. Pharm. 5, 994-1002. Lang, B., McGinity, J.W., Williams, R.O. 3rd. 2014. Hot-melt extrusion-basic principles and pharmaceutical applications. Drug Dev. Ind. Pharm. 40, 1133-1155. Miller, D.A., Keen, J.M., Brough, C., Ellenberger, D.J., Cisneros, M., Williams, R.O. 3rd, McGinity, J.W. 2015. Bioavailability enhancement of a BCS IV compound via an amorphous combination product containing ritonavir. J Pharm Pharmacol. 1-14. Newman, A., Knipp, G., Zografi, G. 2012. Assessing the performance of amorphous solid dispersion. J. Pharm. Sci. 101, 1355-1377. Oh, D.M., Curl, R.L., Amidon, G.L. 1993. Estimating the fraction dose absorbed from suspensions of poorly soluble compounds in humans: a mathematical model. Pharm. Res. 10, 264-270. Paudel, A., Worku, Z.A., Meeus, J., Guns, S., Van den Mooter, G. 2013. Manufacturing of solid dispersions of poorly water soluble drugs by spray drying: formulation and process considerations. Int. J. Pharm. 453, 253-284. Repka, M.A., Battu, S.K., Upadhye, S.B., Thumma, S., Crowley, M.M., Zhang, F., Martin, C., McGinity, J.W. 2007. Pharmaceutical applications of hot-melt extrusion: Part II. Drug Dev Ind Pharm. 33, 10431057. Rumondor, A.C., Stanford, L.A., Taylor, L.S. 2009. Effects of polymer type and storage relative humidity on the kinetics of felodipine crystallization from amorphous solid dispersions. Pharm. Res. 26, 25992606. Sarode, A.L., Wang, P., Obara, S., Worthen, D.R. 2014. Supersaturation, nucleation, and crystal growth during single and biphasic dissolution of amorphous solid dispersions: polymer effects and implications for oral bioavailability enhancement of poorly water soluble drugs. Eur. J. Pharm. Biopharm. 86, 351-360. Serajuddin, A. T. 1999. Solid dispersion of poorly water-soluble drugs: early promises, subsequent problems, and recent breakthroughs. J. Pharm. Sci. 88, 1058-1166. Tajarobi, F., Larsson, A., Matic, H., Abrahmsén-Alami, S. 2011. The influence of crystallization inhibition of HPMC and HPMCAS on model substance dissolution and release in swellable matrix tablets. Eur. J. Pharm. Biopharm. 78, 125-133. Ting, J. M., Navale, T. S., Jones, S. D., Bates, F. S., Reineke, T. M. 2015. Deconstructing HPMCAS: Excipient design to tailor polymer–drug interactions for oral drug delivery. ACS Biomater. Sci. Eng. 1, 978-990. 18

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Ueda, K., Higashi, K., Yamamoto, K., Moribe, K. 2013. Inhibitory effect of hydroxypropyl methylcellulose acetate succinate on drug recrystallization from a supersaturated solution assessed using nuclear magnetic resonance measurements. Mol. Pharm. 10, 3801-3811. Ueda, K., Higashi, K., Yamamoto, K., Moribe, K. 2014. The effect of HPMCAS functional groups on drug crystallization from the supersaturated state and dissolution improvement. Int. J. Pharm. 464, 205213. Yin, L., Hillmyer, M.A. 2014. Preparation and performance of hydroxypropyl methylcellulose esters of substituted succinates for in vitro supersaturation of a crystalline hydrophobic drug. Mol. Pharm. 11, 175185. Zheng, X., Yang, R., Zhang, Y., Wang, Z., Tang, X., Zheng L. 2007. Part II: bioavailability in beagle dogs of nimodipine solid dispersions prepared by hot-melt extrusion. Drug Dev Ind Pharm. 33, 783-789.

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449 450 451

FIGURES CAPTION

452

Figure 1: X-ray diffraction of HME formulations of compound A, at initial time-point and after stressing

453

for 4 weeks.

454

Figure 2: Modulated DSC thermograms of HME formulations of compound A, at initial time-point and

455

after stressing for 4 weeks.

456

Figure 3: Dissolution of compound A formulations in fasted state simulated intestinal fluid (FaSSIF)

457

Figure 4: Mean plasma concentration versus time profiles of compound A in fasted male beagle dogs

458

(n=3, mean ± SE).

459 460

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460 461

TABLE CAPTION

462

Table 1: Physicochemical properties of compound A

463

Table 2: Pharmacokinetic parameters (n=3, mean ± SE) of compound A in fasted male beagle dogs

464

following oral administration of several formulations.

465

Table 3: Comparison of dissolution of compound A formulations to their bioperformance in dogs.

466 467 468

21

468 469

470 471 472 473 474 475 476 477 478 479 480 481 482 483 484

Figure 1:

From bottom to top: HPMCAS-HF initial and after 4 weeks of storage at 40°C/75%RH open, HPMCAS-LF initial and after 4 weeks of storage at 40°C/35%RH open and 40°C/75%RH open, and copovidone initial and after 4 weeks of storage at 40°C/35%RH open and 30°C/65%RH open. All samples are at a drug loading of 20 wt% of compound A in polymer. Inset figure: Close-up of X-ray spectrum image focusing on crystalline peaks of compound A at 17.78 and 18.40 2θ angles (arrows), indicating significant drug crystallization in the copovidone based HME after 4 weeks at 30°C/65%RH open (top), subtle signs of drug crystallization in the HPMCAS-LF based HME formulation after 4 weeks at 40°C/75%RH open (middle) and no drug crystallization in the HPMCAS-HF based HME formulation after 4-week at 40°C/75%RH open (bottom).

22

484

485 486 487 488 489 490 491 492 493 494

Figure 2:

From bottom to top: HPMCAS-HF initial and after storage for 4 weeks at 40°C/75%RH open, HPMCASLF initial and after storage for 4 weeks at 40°C/35%RH open, and copovidone initial and after storage at 4 weeks at 40°C/35%RH open. All samples are at a drug loading of 20 wt% of compound A in polymer.

23

495 496 497

Figure 3:

498 499 500 501 502 503 504 505 506 507

24

507 508

Figure 4:

509 510 511 512 513 514 515 516

25

516 517 518 519

Tables Table 1: Physicochemical properties of compound A

520 Melting point (crystalline anhydrous form II) = 140°C Caco-2 permeability = 14.6 x 10-6 cm/sec Solubility (crystalline anhydrous form II): Simulated Gastric Fluid (SGF, pH 1.2) = 0.001 mg/mL Fasted State Simulated Intestinal Fluid (FaSSIF, pH 6.5) = 0.001 mg/mL Fed State Simulated Intestinal Fluid (FeSSIF, pH 5.0) = 0.002 mg/mL Water = 0.001 mg/mL 521 522 523 524 525 526

26

Table 2: Pharmacokinetic parameters (n=3, mean ± SE) of compound A in fasted male beagle dogs following oral administration of several formulations.

a

Formulation

Dose (mg/kg)

AUC0-24hr (µM*hr)

Dose normalized AUC0-24hr (µM*hr/mg/kg)

Cmax (µM)

Tmax (hr) a

Crystalline API in Dry Filled Capsule (DFC) c

15

4.76 ± 1.12

0.32

0.58 ± 0.11

2.0 (1.0-2.0)

HME (HPMCAS-HF)

5

18.1 ± 2.93

3.62

1.51 ± 0.34

HME (HPMCAS-LF)

5

11.2 ± 2.07

2.24

0.73 ± 0.08

HME (copovidone)

5

8.19 ± 2.91

1.64

0.59 ± 0.26

1.0 (0.5-4.0) 4.0 (1.0-24.0) 4.0 (2.0-6.0)

Tmax is reported as median with range in parenthesis Calculated using dog IV data (AUC0-24hr = 4.3 µM*hr at a dose of 1 mg/kg) c Crystalline jet milled compound A with mean particle size of 4.9 μm was used in the DFC formulation b

27

A Bioav

Table 3: Comparison of dissolution of compound A formulations to their bioperformance in dogs. Dissolution data Formulation

Crystalline API in Dry Filled Capsule (DFC) HME (HPMCAS-HF) HME (HPMCAS-LF) HME (copovidone) a

Relative bioavailability from dog PK study a

Concentration at 60 minutes (ug/mL)

Relative concentration a

1.46

0.3

0.2

13.3 4.7 5.3

2.5 0.9 --

2.2 1.4 --

Relative ratios were calculated with respect to copovidone based HME

28