Impact of Drug-rich Colloids of Itraconazole and HPMCAS on

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Impact of Drug-rich Colloids of Itraconazole and HPMCAS on Membrane Flux In Vitro and Oral Bioavailability in Rats Aaron M. Stewart, Michael E. Grass, Timothy J. Brodeur, Aaron K. Goodwin, Michael M. Morgen, Dwayne T. Friesen, and David T. Vodak Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00338 • Publication Date (Web): 07 Jun 2017 Downloaded from http://pubs.acs.org on June 13, 2017

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Molecular Pharmaceutics

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Impact of Drug-rich Colloids of Itraconazole and HPMCAS on Membrane Flux In

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Vitro and Oral Bioavailability in Rats

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Aaron M. Stewart, Michael E. Grass*, Timothy J. Brodeur, Aaron K. Goodwin, Michael M. Morgen,

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Dwayne T. Friesen, David T. Vodak

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Global Research and Development, Pharmaceutical Science, Capsugel, Bend, Oregon 97701, USA

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*Corresponding Author

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Postal address: 64550 Research Rd, Bend, Oregon, USA, 97701

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Phone: (541)-706-8268

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Fax: (541)-382-2713

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Email: [email protected]

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ACS Paragon Plus Environment


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Abstract Graphic

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Abstract

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Improving the oral absorption of compounds with low aqueous solubility is a common challenge that often requires an enabling technology. Frequently, oral absorption can be improved by formulating the compound as an amorphous solid dispersion (ASD). Upon dissolution, an ASD can reach a higher concentration of unbound drug than the crystalline form, and often generates a large number of submicron, rapidly-dissolving drug-rich colloids. These drug-rich colloids have the potential to decrease the diffusional resistance across the unstirred water layer of the intestinal tract (UWL) by acting as rapidlydiffusing shuttles for unbound drug. In a prior study utilizing a membrane flux assay, we demonstrated that for itraconazole, increasing the concentration of drug-rich colloids increased membrane flux in vitro. In this study, we evaluate spray-dried amorphous solid dispersions (SDDs) of itraconazole with hydroxypropyl methylcellulose acetate succinate (HPMCAS) to study the impact of varying concentrations of drug-rich colloids on the oral absorption of itraconazole in rats, and to quantify their impact on in vitro flux as a function of bile salt concentration. When Sporanox® and itraconazole/HPMCAS SDDs were dosed in rats, the maximum absorption rate for each formulation rankordered with membrane flux in vitro. The relative maximum absorption rate in vivo correlated well with the in vitro flux measured in 2% SIF (26.8 mM bile acid concentration), a representative bile acid concentration for rats. In vitro it was found that as the bile salt concentration increases, the importance of colloids for improving UWL permeability is diminished. We demonstrate that drug-containing micelles and colloids both contribute to aqueous boundary layer diffusion in proportion to their diffusion coefficient and drug loading. These data suggest that for compounds with very low aqueous solubility and high epithelial permeability, designing amorphous formulations that produce colloids on dissolution may be a viable approach to improve oral bioavailability.

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Keywords: flux, dissolution, amorphous solid dispersion, spray-dried dispersion, membrane, diffusion, itraconazole, HPMCAS, bioavailability, colloids

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Introduction

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An often critical requirement for successful oral drug delivery is that a compound dissolve rapidly to its

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saturated solubility in gastrointestinal fluid in order to maximize the amount of drug that can be absorbed

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through the epithelium. Recent studies have shown that as many as 90% of new compounds are classified

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as BCS class II or IV (low solubility with either high or low permeability)1. Amorphous solid dispersions

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(ASD) are utilized to improve the bioavailability of low solubility compounds by creating supersaturated

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drug concentrations upon dissolution2,3,4,5,6,7,8,9. ASDs are metastable, typically requiring incorporation

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into a matrix with a high glass transition temperature. An ideal matrix leads to rapid dissolution and also

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prevents crystallization over a relevant timeframe2,10,11,12,13. It has been shown that some ASDs rapidly

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disintegrate into drug-rich colloidal structures stabilized by an amphiphilic excipient, usually a polymer or

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surfactant2,14. The colloids may improve ASD performance because they have a very fast dissolution rate

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and may diffuse into the unstirred water layer (UWL)2,14,15,16,17.

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For some lipophilic low solubility compounds, diffusion across the UWL is much slower than absorption

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through the epithelial membrane of the intestinal tract18,19. In this case, increasing UWL permeability can

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be accomplished by maximizing the total drug available to diffuse across the UWL, which can be in the

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form of unbound drug, micelle-bound drug, inclusion complexes, and nano-sized drug-rich colloids2,16,20.

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If a compound is limited by absorption through the epithelium, rapidly-diffusing species are less critical

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as the neutral unbound drug concentration is the largest contributor to absorption21,22. In both cases, drug-

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rich colloids can improve dissolution rate by providing a high surface area and rapidly-dissolving source

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for unbound drug as it is absorbed.

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In vitro flux studies are commonly used for studying the transport of drug in various forms (unbound,

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micelle-bound, inclusion complexes, colloids) in an effort to establish an in vivo relevant evaluation of

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formulation performance4,6,21,23,24,25,26,27. Specific to this study, we use an in vitro flux assay to investigate

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the mechanism by which drug-rich colloids formed from itraconazole ASDs improve in vitro and in vivo


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performance28. While there are several single phase dissolution methods available that are able to

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quantify the concentration of drug-rich colloids29,30, they are unable to establish their significance with

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respect to absorption. Performing dissolution-flux experiments in vitro can provide valuable insight into

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not only the concentration of drug-rich colloids upon dissolution, but also the impact they may have with

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respect to mass transport, and potentially oral absorption. Furthermore, a dissolution-flux assay can also

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be used to study the mechanism in which drug-rich colloidal species contribute to flux and can help

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develop a formulation strategy during formulation screening.

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In this work, we have investigated amorphous formulations of itraconazole (ITZ) with hydroxypropyl

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methylcellulose acetate succinate (HPMCAS) both in vitro and in vivo because these formulations are

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capable of forming a high and tunable concentration of drug-rich colloids. All ASDs of ITZ increase the

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concentration of unbound drug relative to crystalline ITZ. These formulations are compared to

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Sporanox®.

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Sporanox® was used as a control formulation in this study because it does not form any drug-rich

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colloids in vitro. Sporanox®, a commercial formulation of ITZ, is a spray layered amorphous dispersion

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of ITZ and HPMC on inert sugar cores. The plasma AUC of 50 mg of amorphous ITZ (Sporanox®) is 5 –

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30 fold higher than 50 mg of crystalline ITZ in dogs31. ITZ is also known to exhibit a positive food effect

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in humans, likely due to increased bile salt secretion in the fed state32,33. Thus, absorption of ITZ is likely

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limited by UWL diffusion due to its lipophilicity (clogD 5.7 at pH 6.5)32 and very low aqueous solubility

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(< 10 ng mL-1 for crystalline ITZ, ca. 0.1 µg mL-1 for amorphous ITZ at pH 6.5).

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In a previous study using an in vitro membrane flux assay28, we demonstrated the integral role of micelle-

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bound and colloidal drug species when flux is limited by diffusion across the aqueous boundary layer

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(ABL). In this study, we further investigate the effect of drug-rich colloid concentration on ITZ

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performance by: (a) quantifying the contribution from these species to the measured flux as a function of

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micelle-bound drug concentration in vitro, (b) confirming the mechanism in which they are improving



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measured flux by employing a pseudo-steady-state mass transport model, and (c) determining their impact

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on oral bioavailability in rats.

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Materials and Methods

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ITZ (>98% purity) and sodium lauryl sulfate were purchased from Spectrum Chemical MFG Corp (New

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Brunswick, New Jersey). Sporanox® was purchased from Drug World (Drug World Pharmacies, New

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City, NY). Hydroxypropyl methylcellulose acetate succinate (Affinisol™) L-HP and H-HP (high

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productivity/low viscosity) grades were a generous gift from DOW (DOW Chemical Company, Midland,

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MI), and HPMCAS-HF grade was purchased from Shin-Etsu (Shin-Etsu Chemical Co., Ltd., Tokyo,

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Japan). Methanol, acetonitrile, and tetrahydrofuran were purchased from Honeywell (Morris Plains, NJ).

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The aqueous medium used for all flux experiments was 67 mM pH 6.5 phosphate buffered saline (PBS)

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containing various levels of SIF bile salts (Biorelevant.com, London, United Kingdom). Gastrointestinal

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tract (GIT-0) lipid was purchased from Pion Inc. (Billerica, MA). The chemical structure and

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physicochemical properties of ITZ are shown in Figure 1 and Table 1.

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Figure 1.

Chemical structure of ITZ

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Table 1.

ITZ physicochemical properties and experimentally determined solubility values in

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various media at pH 6.5 measured via ultracentrifuge assay (method described below) Solubility, cu,m= cu + cm (µg mL-1)

Form Amorphous Crystalline

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MW (g/mol) 705.6

Tg or Tm (°C) 59 (Tg)34 35

167 (Tm)

Basic pKa 3.732

logD 5.732

PBS, 0% SIF 0.10a 7 nm (size of micelle-bound drug) in the supernatant post-

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ultracentrifugation (data not shown). For each sample, 50 µL of supernatant was diluted into 250 µL (6x

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dilution) of 9:1 methanol:water (v/v) and the concentration was determined on an Agilent HP 1100 HPLC

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system using the same standard calibration and isocratic HPLC method described above for crystalline

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and amorphous ITZ solubility measurements. All samples were analyzed in duplicate.

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DLS Measurements

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Dynamic light scattering (DLS) was used to measure the size of drug-rich colloids in the donor solution.

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Samples were collected from the donor vessel in 250 µL aliquots (same aliquot used for measuring donor

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concentration) and centrifuged at 15,800 x g on a Thermo Scientific Legend Micro 21 centrifuge to pellet

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bulk non-colloidal drug (>400 nm). 100 µL of supernatant was added to an Eppendorf UVette cuvette

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(Eppendorf, Hamburg, Germany) with a 10 mm pathlength, 2 mm slit. Samples were analyzed on a

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Malvern Nano-Zetasizer (Nano-ZSP) (Malvern Instruments, Westborough, MA) at 5 minutes and 120

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minutes post-dosing. The instrument was set to backscatter mode at an angle of 173° and samples were

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equilibrated to 37°C prior to analysis.

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Flux Measurements

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A vertical orientation membrane flux cell28,37 was used to measure flux vs. time of ITZ formulations

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dosed at 1,000 µg mL-1. An Accurel PP 1E (55% porous, 100 µm thickness) membrane impregnated with

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50 µL of Pion GIT-0 lipid solution consisting of 20% w/w phospholipid dissolved in dodecane (Pion Inc.,

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Billerica, MA) is used to separate the donor and receiver vessels. The donor vessel contained 6.5 mL of

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dissolution medium, all consisting of 67 mM PBS at pH 6.5 with either 0, 0.5, or 2% w/w of SIF bile salts

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(Biorelevant.com, London, United Kingdom). When added to aqueous buffer at biorelevant pH, SIF bile

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salts (4:1 ratio of sodium taurocholate to phosphatidylcholine)38 form mixed micelles in solution capable

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of solubilizing drug, the amount of which will depend on how much SIF powder is added to the

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dissolution medium and the micelle partition coefficient of the drug (discussed in following sections). The



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receiver vessel contained 10 mL of 67 mM PBS at pH 6.5 with 2% (wt/wt) sodium lauryl sulfate (SLS).

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Similar to SIF, SLS forms micelles in solution capable of solubilizing drug to an extent sufficient in

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providing a large sink condition for ITZ in the receiver compartment (Table 1). The surface area of the

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membrane was 4.90 cm2, making the ratio of membrane surface area to donor volume 0.75-0.98 cm-1 for

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these experiments (this ratio changes over time as volume is removed from the donor compartment for

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assay and size measurements). The temperature for all experiments was maintained at 37°C by

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circulating water through a heating block mounted to a Pion µDiss™ profiler. UV probes (10 mm path

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length) connected to a Pion Rainbow™ UV spectrometer system were used to determine the drug

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concentration in the receiver vessel by monitoring UV spectra vs. time. Receiver concentration vs. time

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plots were generated, and flux was calculated by taking the slope of receiver concentration vs. time from

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30-50 minutes and dividing by the ratio of membrane surface area to the volume of donor medium (0.75

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cm-1). A time range for flux measurements of 30-50 minutes was deemed sufficient for the system to

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reach steady-state while still capturing the initial flux as drug starts to enter the receiver compartment.

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Samples were analyzed in duplicate.

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In Vivo Study Protocol

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The in vivo study was performed by Covance Laboratories (Greenfield, IN) similar to a previously

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reported protocol for dosing ITZ formulations in rats39. All experimental procedures were in accordance

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with Covance Standard Operating Procedures (SOPs). Experiments were conducted in fasted male

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Sprague-Dawley rats (250-325g), 6 subjects per cohort. Animals were fasted overnight through

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approximately 4 hours post-dose, and water was provided ad libitum.

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The formulations dosed in vivo were Sporanox®, 25% ITZ:HPMCAS-H HP, 25% ITZ:HPMCAS-L HP,

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and 25% ITZ:HPMCAS-L HP + HPMCAS-H (suspended with SDD at a ratio of 2:1 HPMCAS-

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L:HPMCAS-H). All formulations were administered by oral gavage at a dose of 50 mg kg-1 (4 mL kg-1).

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Individual doses were adjusted to 50 mg kg-1 based on body weight recorded on the day of administration.

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For the ITZ SDD formulations, powder was suspended in a dosing vehicle (0.5% Methocel™ A4M in


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water) by mortar and pestle at 12.5 mg mL-1 (50 mg mL-1 total solids) ITZ concentration and 4 mL kg-1

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was administered through a 15 gauge, 100 mm oral gavage. For Sporanox®, individual beads were pre-

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weighed into the gavage tip. The formulation was then dosed by flushing through 2 mL kg-1 of dosing

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vehicle, followed by another 2 mL kg-1 rinse to ensure all of the beads were cleared from the gavage.

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Blood samples (approximately 0.3 mL) were collected from the jugular vein via syringe and needle and

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transferred into tubes containing K3 EDTA from each animal pre-dose and at approximately 0.5, 1, 2, 3,

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4, 6, 8, 10, 24, 28, 32, and 48 hours post-dose. Blood was maintained at approximately 5°C prior to

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centrifugation to obtain plasma. Resulting samples were harvested within 40 minutes of the start of

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centrifugation. Plasma were placed into individually labeled 96-well plates and stored at ≤-60°C prior to

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sample analysis via LC-MS.

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Pharmacokinetic Analysis of ITZ In Vivo Data

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All pharmacokinetic analyses were conducted using custom scripts in Python, specifically leveraging

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Anaconda Software Distribution.40 Specifically, the following scientific libraries were used in this

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analysis: 1) Numpy—core library for performing high-level mathematical operations on multi-

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dimensional arrays and matrices; 2) Pandas—data analysis library developed for manipulating labeled and

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relational data structures; 3) SciPy—scientific and engineering library developed for performing

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integration, interpolation, signal processing and statistical analysis; 4) Plotly—data visualization library

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featuring interactive plotting and graphing.

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Primary pharmacokinetic parameters were determined through standard non-compartmental analysis of

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the experimental plasma concentration vs. time data for each subject. Peak plasma concentration, Cmax, is

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the maximum plasma concentration measured for a given subject, and Tmax is the corresponding time at

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which Cmax is observed. Area under the curve, AUC, for each subject was calculated using a linear

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trapezoidal method from the time of dosing, T0, to the last measured time point, Tlast. AUC extrapolated to



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infinity (AUCinf) was not determined for this analysis. The results from this PK analysis are summarized in

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Table 2 in the results section of this manuscript.

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A numerical deconvolution of the experimental plasma concentration vs. time data was used to

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approximate the input function of ITZ into the central compartment. The central compartment is defined

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here as the hypothetical space into which the drug initially distributes following absorption, typically

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representing the freely circulating blood volume. As discussed below, the academic literature suggests

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single compartment pharmacokinetics for ITZ, therefore no peripheral compartments were considered in

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these calculations. In this approach, a series of discrete drug impulses with subsequent impulse-response

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disposition curves were superimposed to obtain a best fit of the observed plasma concentration vs. time

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data for each subject. Total absorption into the central compartment was approximated as the cumulative

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summation of these sequential drug impulses.

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The impulse-response function for ITZ (concentration profile following bolus IV dose) in rats was

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approximated as a single-compartment model using Equation 1:

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1)

ܿ௧ = ‫ି ݁ ∗ ܣ‬௞೐೗ ௧

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Where ‫ ܣ‬is the theoretically expected plasma concentration for 1 milligram of ITZ instantaneously

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entering the central compartment and ݇௘௟ , the elimination rate constant, is the natural log divided by the

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half-life of elimination. ‫ ܣ‬in this case can be experimentally determined through in vivo data (particularly

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intravenous dosing) by dividing the dose by the volume of distribution. Using the mean values for

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volume of distribution and half-life of elimination obtained from literature41,42,43,44, ‫ ܣ‬and ݇௘௟ were

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calculated to be 677 ng mL-1 and 0.106 hr-1, respectively.

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Figure 2 shows an example subject deconvolution with: a) observed plasma data and resulting

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superimposed best-fit disposition curve; b) sequence of drug impulses (input function); c) cumulative

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summation of the drug impulses.


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Figure 2. Example Deconvolution Results for a Single Subject: a) plasma profile, b) input function, c) cumulative input (absorption).

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The maximum rate of absorption for each subject was estimated as the maximum impulse value obtained

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through deconvolution divided by the respective time-step used in the deconvolution. In this case, the

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time-step for all deconvolutions was 29 minutes. For IVIVC, the mean of these maximum values was

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calculated for all subjects for a given formulation treatment. For verification of rank-order absorption,

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these mean maximal values were also shown to correlate to maximal absorption rates obtained through

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more traditional absorption estimations such as the Wagner-Nelson method45. The deconvolution



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methodology described above was employed to generate a plot of absorption rate vs. time shown in

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Figure 9 in the discussion.

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Results

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Characterization of ITZ Amorphous Spray-dried Dispersions (SDDs)

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X-ray Powder Diffraction (XRPD)

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XRPD was used to characterize the ITZ SDDs manufactured for the dissolution-flux study. Figure 3

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shows diffractograms of the ITZ SDDs in comparison to crystalline ITZ. Both SDDs manufactured show

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no evidence of diffraction peaks, indicative that the SDDs are amorphous.

282 X-ray diffractograms of crystalline ITZ in comparison to 25% ITZ/H and 25% ITZ/L.

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Figure 3.

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Modulated Differential Scanning Calorimetry (mDSC)

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Modulated differential scanning calorimetry was performed to confirm that the ITZ SDDs were an

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amorphous single phase, represented by a single glass transition temperature (Tg). Figure 4 shows the

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thermograms for both SDD samples, of which both samples exhibited a single Tg of 95 ± 1°C, indicative

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of a single amorphous phase.


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Figure 4.

mDSC thermograms of 25% ITZ/H and 25% ITZ/L HP. Both reversing (solid) and

nonreversing (dashed) heat flow are plotted. Exothermic heat flow is down.

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Drug Species Notation and Description

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In a previous in vitro membrane flux study, amorphous ITZ flux was determined to be limited by

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diffusion across the aqueous boundary layer when tested at pH 6.5 (ITZ >98% neutral) and dosed at 1,000

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µg mL-1. When flux is ABL-limited, increasing the unbound, micelle-bound, or drug-rich colloid

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concentration can lead to an increase in the measured flux28. In the following sections, we will treat ITZ

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as ABL-limited, in which the largest resistance to flux is diffusion across the ABL, and the membrane and

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dissolution rate resistances can be neglected. We will refer to certain concentration terms that are

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considered relevant for calculating ABL-limited flux at steady state. The concentration terms used are

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unbound, micelle-bound, drug-rich colloids, and composites thereof. Below are definitions of each drug

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concentration term:

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ܿ௨

Bulk concentration of unbound drug in the donor compartment (µg mL-1)

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ܿ௠

Bulk concentration of micelle-bound drug in the donor compartment (µg mL-1)

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ܿ௖

Bulk concentration of undissolved drug in colloids in the donor compartment (µg mL-1)



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ܿ௨,௠

Bulk concentration of unbound drug plus micelle-bound drug in the donor compartment,

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measured by ultracentrifuge assay at 300,000 x g for 8 minutes. If no micelles are present, ܿ௨,௠ =

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ܿ௨ (µg mL-1)

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ܿ௨,௠,௖ Bulk concentration of the sum of unbound drug, micelle-bound drug, and drug-rich colloids in the

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donor compartment (“apparent concentration”), measured by microcentrifuge assay at 15,800 x g

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for 1 minute. If no drug colloids are present, ܿ௨,௠,௖ = ܿ௨,௠ . If no micelles or drug colloids are

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present, ܿ௨,௠,௖ = ܿ௨ (µg mL-1)

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Dissolution-Flux Studies of ITZ Formulations

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For all formulations tested, ITZ quickly dissolves to and sustains its “amorphous solubility” such that

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ܿ௨,௠ is the same in each medium at each measured time point >4 minutes (Table 3). Any differences in

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the donor concentration between formulations or time points represents variations in the amount of drug

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in colloids. These colloids consist of undissolved amorphous ITZ, likely associated with polymer as nano-

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sized particles, small enough in size (ca. 150-400 nm) to have a significant diffusion rate across the ABL.

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In all experiments, Sporanox® is used as a control formulation because it does not form measurable

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amounts of these colloids upon dissolution in vitro (i.e. ܿ௨,௠,௖ = ܿ௨,௠ ) over the timescale studied.

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The concentration vs. time profiles for amorphous ITZ formulations dosed to each donor solution (Figure

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5 left) show the apparent drug concentration (ܿ௨,௠,௖ ) vs. time. In PBS alone, the concentration ܿ௨,௠ = ܿ௨ =

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0.1 µg mL-1. In 0.5% and 2% SIF ܿ௨ = 0.1 µg mL-1, but the addition of drug solubilizing micelles

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increases ܿ௨,௠ to 6 µg mL-1 and 20 µg mL-1 in 0.5% and 2% SIF, respectively. The apparent concentration

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(ܿ௨,௠,௖ ) is much greater for the 25% ITZ/HPMCAS formulations compared to Sporanox® due to the

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presence of a significant amount of drug-rich colloids (Figure 5 left). The maximum apparent

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concentration of ITZ reached is ca. 600 µg mL-1 from 25% ITZ/L and 25% ITZ/L+H and ca. 200 µg mL-1

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for 25% ITZ/H in all media regardless of the presence of drug-solubilizing micelles (SIF). The apparent


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concentration of drug-rich colloids, however, decreases over time during the experiment for 25% ITZ/L.

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The decrease in apparent concentration suggests that the amount of drug in colloids that are small enough

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or low enough density to remain suspended after centrifugation, decreases. The mechanism (colloid

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aggregation, growth in size, densification) is outside the scope of this paper. The presence of pre-

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dissolved HPMCAS-H limits the decrease in apparent concentration from this SDD. The colloids that

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form from the 25% ITZ/H SDD (ca. 200 µg mL-1) remain stable throughout the test.

335

The particle size of colloids in the supernatant after microcentrifugation was determined at the first and

336

last time points of each experiment for all of the formulations (Figure 6 and Figure S2). The 25% ITZ/L

337

SDD forms colloids that are initially ca. 150 – 200 nm in diameter, depending on the medium. At the final

338

time point, however, the average diameter increases to ca. 300 – 350 nm for those colloids in the

339

supernatant. In contrast, the colloids formed from 25% ITZ/L+H are initially larger (ca. 200 nm

340

independent of medium), but do not increase in size as much as 25% ITZ/L in the absence of HPMCAS-

341

H. For 25% ITZ/H, the colloid size is the smallest at the start and end of the dissolution test across all

342

formulations. Further characterization of the colloids by nanoparticle tracking analysis and scanning

343

electron microscopy is shown in the supporting information (Figure S3 and Figure S4).

344

The receiver concentration vs. time profiles for each formulation in PBS (no SIF), 0.5%, and 2% SIF at

345

pH 6.5 are shown in Figure 5 (right) and summarized in Table 3. In all media, the initial steady-state

346

flux increases in the order Sporanox® < 25% ITZ/H < 25% ITZ/L+H < 25% ITZ/L. In PBS, the flux

347

measured from 25% ITZ/L is 10-times greater than for Sporanox®. In contrast, the increase in flux is

348

only about 2-fold in media containing 2% SIF. Interestingly, flux of 25% ITZ/L + H was slightly lower

349

than for 25% ITZ/L alone even though the apparent concentration (ܿ௨,௠,௖ ) is sustained for a longer period

350

of time in the donor solution. These results is discussed in more detail in the following sections.

351

Similarly, the flux is much more sensitive to micelle concentration for formulations that generate fewer

352

drug-rich colloids. The flux of ITZ from Sporanox® increases 30-fold between PBS (no SIF) and 2% SIF.

353

For 25% ITZ/L, however, the increase is only 3.5-fold.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

354

355


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356 357

Figure 5. Donor concentration (ܿ௨,௠,௖ ) vs. time (left) and receiver concentration vs. time (right) at a dose

358

concentration of 1000 µg mL-1 ITZ added to the donor vessel: Sporanox® (black ♦), 25% ITZ/H

359

(red ●), 25% ITZ/L (blue ■), 25% ITZ/L+H (green ▲). Donor media: a) no SIF b) 0.5% SIF c)

360

2% SIF, all in 67 mM PBS at pH 6.5. Receiver medium was 2% SLS in 67 mM PBS, pH 6.5 for

361

all experiments. ܿ௨,௠ is constant across all formulations in each medium and at each measured

362

time point >4 minutes throughout the testing period.

363 364 365

Figure 6.

Diameter of drug-rich colloids in the donor compartment separated from larger

precipitate by microcentrifugation (1 minute at 15,800 x g) at 5 minutes and 120 minutes



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

366

determined by dynamic light scattering (DLS): 25% ITZ/H (left), 25% ITZ/L (middle), 25%

367

ITZ/L+H (right). Raw size distribution data are shown in the supporting information.

368

In Vivo Study in Male Sprague-Dawley Rats

369

To determine whether the increase in in vitro flux provided by ITZ SDDs relative to that of Sporanox®

370

would translate to improved absorption in vivo, a proof of concept study was performed in male Sprague-

371

Dawley rats to investigate the impact drug-rich colloids may have on oral bioavailability. Plasma

372

concentration vs. time profiles for all ITZ formulations are shown in Figure 7 with tabulated PK results

373

in Table 2. All SDD formulations show higher and more rapid exposure during the first four hours

374

compared to Sporanox®. The 25% ITZ/L SDD and the same SDD co-dosed with HPMCAS-H reached

375

plasma concentrations of ca. 1000 ng mL-1, compared to ca. 800 ng mL-1 for 25% ITZ/H SDD and

376

Sporanox®. The mean AUC values for 25% ITZ/L and 25% ITZ/L+H were ca. 50% higher than for

377

Sporanox®, while the AUC value for 25% ITZ/H was only slightly higher (ca. 10%). For many subjects

378

across formulations, there was a leveling out of the plasma concentration between ca. 2 – 6 hours, then a

379

subsequent rise up to ca. 10 hours. The cause of this unusual profile is not understood, but may be

380

attributed to the high dose (50 mg kg-1) and dosing volume affecting transit times through the

381

gastrointestinal tract for some subjects.

382


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383


Figure 7.

Plasma concentration vs. time profiles for ITZ formulations dosed at 50 mg kg-1 in

384

Sprague-Dawley rats. Sporanox® (black ♦), 25% ITZ/H (red ●), 25% ITZ/L (blue ■), 25%

385

ITZ/L+H (green ▲). Data are mean ± standard deviation (n=6).

386

Table 2.

Pharmacokinetic parameters for ITZ formulations dosed at 50 mg kg-1 in Sprague-

Dawley rats.

387

p-Valuesa Formulation Sporanox® 25% ITZ/H 25% ITZ/L 25% ITZ/L+H

388

a

AUC (ng h mL-1) 14940 ± 3816 16577 ± 2355 21264 ± 6725 21947 ± 5345

Cmax (ng mL-1) 842 ± 130 933 ± 140 1058 ± 205 1118 ± 199

Tmax (hr) 8.0 ± 1.3 6.7 ± 2.4 6.3 ± 2.3 7.2 ± 2.7

rabs Max (µg min-1) 7.7 ± 3.1 11.9 ± 2.2 17.1 ± 3.3 17.4 ± 5.9

AUC N/A 0.30 0.05 0.03

Cmax N/A 0.25 0.09 0.03

rabs Max N/A 0.08 0.002 0.02

Higher than control formulation, Sporanox®

389

390

Discussion

391

The Formation and Effect of Drug-rich Colloids of ITZ on Flux In Vitro

392

HPMCAS can help prevent many drugs from crystallizing out of a supersaturated solution2,10,11,14,46,47.

393

Depending on the interaction of a drug with HPMCAS, HPMCAS based amorphous dispersions have also

394

shown the ability to form drug-rich amphiphilic colloidal structures upon dissolution. These drug-rich

395

polymer colloids are amorphous, and as unbound drug is absorbed through the intestinal epithelium they

396

can rapidly dissolve to replenish absorbed drug, thereby maintaining the concentration of unbound drug at

397

or near the amorphous solubility2,14,21. The substitution ratio of acetyl/succinoyl on the HPMC backbone

398

dictates the hydrophilicity, resulting dissolution rate, and solution properties of HPMCAS. The

399

HPMCAS-L grade polymer (ca. 7 wt% acetyl, 16 wt% succinoyl) contains a higher percentage of

400

hydrophilic succinoyl groups, resulting in a faster dissolution rate when it becomes ionized at pH 6.5

401

compared to the HPMCAS-H grade polymer (ca. 12 wt% acetyl, 6 wt% succinoyl) (Figure S8). The rapid

402

dissolution rate of the polymer, along with a strong interaction between ITZ and hydrated HPMCAS,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

403

helps drive the formation of drug-rich colloids upon hydration of the SDD particle14. This results in more

404

colloids observed for 25% ITZ/L compared to 25% ITZ/H immediately after dosing. This general

405

mechanism is driven by the dissolution rate of the ionized polymer and drug/polymer interaction (Figure

406

S7). In general, the formation of colloids is independent of bile salt micelle concentration as demonstrated

407

in Figure 5.

408

A high concentration of drug-rich colloids greatly increases in vitro flux when the number of micellar

409

“shuttles” is limited, as is the case for PBS at pH 6.5 without any added SIF (ܿ௨,௠ 4 minutes (ܿ௨,௠ ).

525

While there was a measurable impact observed from drug-rich colloids, it is transient, and only

526

differentiates formulations through the first two hours after administration. This may be attributed to



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

527

biological considerations such as pH, bile acid concentration, and transit times in different sections of the

528

gastrointestinal tract of a rat, all of which can play a role in affecting the concentrations of all rapidly-

529

diffusing drug-containing species in the intestinal fluid, and resulting absorption rate. Additionally, the in

530

vitro data shows that drug-rich colloids of ITZ grow in size over time, such that they may become a less

531

reliable source for unbound drug as residence time in the intestinal medium increases, though this

532

apparent phenomenon did not affect the measured flux in vitro. Conversely, drug-rich colloids eventually

533

form even from Sporanox® under some circumstances after several hours (data not shown).

534 535 536

Figure 9.

Deconvoluted absorption rate of ITZ formulations in Sprague-Dawley rats through 4

hours in units of µg min-1.

537

Rats do not have a gall bladder, and continuously secrete bile acids from the liver regardless of whether

538

fed or fasted51,52. Bile acid concentrations in rats are reported anywhere from 10 mM to 100 mM (0.75-

539

7.5% SIF using the ratio of sodium taurocholate to phosphatidylcholine of 4:1 from the FaSSIF V1

540

composition)38 depending on the section of the intestinal tract and methodology used for

541

measurements53,54,55. Figure 10 compares the measured in vitro flux at 0.5% and 2% SIF to the max

542

absorption rate observed in vivo relative to Sporanox®. The max absorption rate observed in vivo more

543

closely follows the in vitro flux measured in 2% SIF, measuring approximately a two-fold increase in

544

max absorption rate.


Page 28 of 35

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545

At 0.5% SIF, the concentration of sodium taurocholate is ca. 6.7 mM, much lower than the reported bile

546

acid concentrations in rats. As a result, the relative contribution of drug-rich colloids to flux at 0.5% SIF

547

in vitro may be an over-prediction of what would be expected in a rat. At 2% SIF in vitro, the maximum

548

measured flux enhancement relative to Sporanox® is approximately two-fold, similar to that observed in

549

vivo. 2% SIF consists of ca. 26.8 mM sodium taurocholate, within the range of bile acid reported in rats; a

550

potential reason for the closer prediction of in vivo performance. It is apparent that the in vitro flux data

551

was able to predict the relative impact of drug-rich colloids on in vivo absorption rates by measuring flux

552

in a range of media at different bile salt concentrations. As the in vitro data shows, the effect of drug-rich

553

colloids on flux is greatest when the relative concentrations of unbound drug and micelle-bound drug are

554

low. Therefore, the impact of colloid-forming formulations of very low solubility compounds may be

555

particularly pronounced in individuals with low bile excretion or in bile-cannulated animals.

556 557

Figure 10.

Relative flux measured in vitro vs. relative max absorption rate observed in vivo for 0.5%

558

SIF (left) and 2% SIF (right): Sporanox® (black ♦), 25% ITZ/H (red ●), 25% ITZ/L (blue ■),

559

25% ITZ/L+H (green ▲). All values are relative to Sporanox®.

560



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

561

Generalization of the Impact of Drug-rich Colloids on Aqueous Boundary Layer Diffusion of Low

562

Solubility Compounds

563

The impact of drug-rich colloids on the flux of drug in vitro and in vivo is demonstrated in this work for

564

the specific case of ITZ. In order to generalize the phenomenon of colloid diffusion, we can use

565

Equations 2 – 6 to solve for the concentration of drug in colloids required to increase flux 2-fold as a

566

function of the unbound drug concentration and the micelle partition coefficient as:

567

8)

ܿ௖ (double flux) = ವವೠ೎ ∙ ቀ1 + ܸ௠ ∙ ‫ܭ‬௠ ∙ ವವ೘ೠ ቁ ∙ ܿ௨

568

For a concentration of micelles that corresponds to 6.7 mM NaTC (0.5% SIF) and assuming a constant

569

size of micelles (7 nm) and colloids (200 nm), the concentration of colloids needed to double flux as a

570

function of ܿ௨ and ‫ܭ‬௠ is shown in Figure 11. Several low solubility amorphous compounds are also

571

included for reference. The amorphous solubility of ITZ was determined in this study. The micelle

572

partition coefficient (‫ܭ‬௠ ) of ITZ and fenofibrate and the amorphous solubility of fenofibrate were

573

measured (data not shown). The micelle partition coefficient of all other compounds was estimated from

574

clogP and molecular weight. The amorphous solubility of tolnaftate and sorafenib were determined by

575

Almeida e Sousa et al36. The amorphous solubility of clotrimazole, loratadine, and felodipine were

576

determined by Ilevbare and Taylor56. The concentration of drug in colloids required to double flux is

577

lowest at low ܿ௨ and low ‫ܭ‬௠ .

578

In order for this mode of action to be viable for improving bioavailability, the concentration of colloids

579

required to double flux should not be excessive (ca. >500 µg mL-1). Thus, this is likely a viable

580

mechanism only when the amorphous solubility in aqueous buffer (no micelles) is < 3 µg mL-1. For

581

compounds with higher solubility, colloids may still play an important role in increasing dissolution rate,

582

but are unlikely to significantly impact apparent permeability.


Page 30 of 35

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583 584

Figure 11.

The concentration of colloids required to double flux as a function of the amorphous

585

unbound drug concentration (ܿ௨ ). Black lines shown at different values of the micelle partition

586

coefficient (‫ܭ‬௠ ) according to Equation 7.

587

Conclusions

588

This study provides experimental evidence for, and the mechanism by which drug-rich colloids increase

589

flux of ITZ in a membrane flux assay. The absolute contribution from drug-rich colloids to flux via ABL

590

diffusion in vitro remains more or less constant irrespective of unbound and micelle-bound drug

591

concentration. Conversely, the relative contribution of drug-rich colloids to the overall effective diffusion

592

of all rapidly-diffusing drug species is largest when unbound and micelle-bound drug concentrations are

593

low. We have shown the ability to utilize a membrane flux assay to predict the impact of drug-rich

594

colloids on in vivo performance in rats. Measuring the relative contribution of drug-rich colloids to flux in

595

vitro as a function of bile concentration may allow a formulator to estimate relative performance of

596

colloid-forming formulations in vivo across different animal species. For compounds with very low

597

aqueous solubility and high epithelial permeability, designing formulations to produce a large amount of



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

598

sub-micron drug-rich colloids can improve formulation performance in vitro, and may be a viable

599

approach for improving oral bioavailability.

600

Acknowledgements

601

The authors would like to thank many colleagues at Capsugel for their support and ideas. In particularly,

602

we would like to thank Dr. Keith Hutchison for his continued support.

603

Supporting Information

604

DLS size distribution curves and tabulated data for drug-rich ITZ colloids, nanoparticle tracking analysis

605

and SEM images of colloids, experimental data for flux at different SA/V ratios, the impact of

606

acetate/succinate ratio on colloid formation, acetyl/succinoyl substitution map for different HPMCAS

607

grades. Also included is a link (html) to the complete description of pharmacokinetic methodology and

608

analyses used herein.

609

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