Interactions of Artefenomel (OZ439) with Milk during Digestion

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Interactions of artefenomel (OZ-439) with milk during digestion: insights into digestion-driven solubilization and polymorphic transformations Malinda Salim, Jamal Khan, Gisela Ramirez, Andrew J. Clulow, Adrian Hawley, Hanu Ramachandruni, and Ben J. Boyd Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00541 • Publication Date (Web): 22 Jun 2018 Downloaded from http://pubs.acs.org on June 24, 2018

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Molecular Pharmaceutics

Interactions of artefenomel (OZ439) with milk during digestion: insights into digestion-driven solubilization and polymorphic transformations Malinda Salim,1 Jamal Khan,1 Gisela Ramirez,1 Andrew J. Clulow,1 Adrian Hawley,2 Hanu Ramachandruni3, Ben J. Boyd1,4* 1

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences,

Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC 3052, Australia 2

SAXS/WAXS beamline, Australian Synchrotron, ANSTO, 800 Blackburn Rd, Clayton, VIC 3169, Australia 3

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Medicines for Malaria Venture, 20, Route de Pré-Bois, 1215 Geneva 15, Switzerland

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC 3052, Australia

KEYWORDS: milk, lipid-based formulation, drug solubilization, polymorphism, X-ray scattering, in vitro digestion, antimalarial, artefenomel, weakly basic drug, precipitation

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ABSTRACT: Milk has been used as a vehicle for the delivery of antimalarial drugs during clinical trials to test for a food effect and artefenomel (OZ439) showed enhanced oral bioavailability with milk. However, the nature of the interaction between milk and OZ439 in the gastrointestinal tract remains poorly understood. To understand the role of milk digestion on the solubilization of OZ439 and polymorphism, we conducted real-time monitoring of crystalline drug in suspension during in vitro intestinal lipolysis of milk containing OZ439 using synchrotron X-ray scattering. OZ439 formed an unstable solid state intermediate free base form (OZ439-FB form 1) at intestinal pH and was partially solubilized by milk fat globules prior to lipolysis. Dissolution of the free base form 1 and re-crystallization of OZ439 in a more stable polymorphic form (OZ439-FB form 2) occurred during in vitro lipolysis in milk. Simply stirring the milk/drug suspension in the absence of lipase or addition of lipase to OZ439 in a lipid-free buffer did not induce this polymorphic transformation. The formation of OZ439-FB form 2 was therefore accelerated by the solubilization of OZ439-FB form 1 during the digestion of milk. Our findings confirmed that although crystalline precipitates of OZ439-FB form 2 could still be detected after in vitro digestion, milk-based lipid formulations provided a significant reduction in crystalline OZ439 compared to lipid-free formulations, which we attribute to the formation of colloidal structures. Milk may therefore be particularly suited as a form of lipid-based formulation (LBF) for co-administration with OZ439, from which both an enhancement in OZ439 oral bioavailability and the delivery of essential nutrients should result.

INTRODUCTION Malaria is a mosquito-borne parasitic disease that poses a global concern; about half a million children under the age of 5 die each year.1 The emergence of parasite resistance to the current artemisinin-based therapy led to the need for new antimalarial drugs.2,

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OZ439 is a synthetic


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trioxolane drug (Figure 1) that is highly potent against arteminisin-resistant strains of Plasmodium falciparum, and can potentially be used as a single-dose cure in combination with a partner drug.4-6 OZ439 is an amphiphilic poorly water-soluble drug7 that can self-assemble into colloidal liquid crystal structures in aqueous solution.8 Although the mesylate salt of OZ439 has an apparent high solubility in water, the free base forms of the drug are poorly soluble in fluids representative of those in the gastrointestinal (GI) tract. Consequently, solubilization of drug in the aqueous GI environment is anticipated to be a limitation to absorption through the intestinal membrane.9, 10 Lipid-based formulations (LBFs) such as triglycerides, mixed glycerides, and self-micro-, or nano-emulsifying drug delivery systems (SMEDDS/SNEDDS), have been widely used to improve the oral bioavailability of poorly soluble lipophilic drugs by enhancing drug solubilization in the GI tract, often due to enhanced solubility in the fatty acids and monoglycerides generated upon digestion of the lipid components.11-13 The digestion process is commonly assessed by in vitro lipolysis experiments followed by separation of the colloidal phases14,

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, and more recently has been adapted to the study of the fate of the drug during

digestion.16 Milk can be considered in some respects as the ultimate lipid formulation – it serves a range of functions, including supporting the solubilization and delivery of poorly soluble vitamins and nutrients in the critical early stages of life. Milk fat is composed of 98% triglycerides emulsified as fat globules,17 and the underlying concept for milk to serve as a lipid formulation to improve the oral bioavailability of poorly water soluble drugs is therefore similar to other digestible lipidbased formulation systems.16, 18, 19 The potential use of milk and milk components as formulation excipients targeted for pediatric populations has been reported in the literature,20-24 although the


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impact of milk digestion and consequent lipid digestion products on drug solubilization has not been studied. The dispersion of antimalarial active pharmaceutical ingredients (APIs) in milk-based LBFs could therefore serve as suitable means for oral drug delivery with pediatric acceptability. Milk has been used to co-administer OZ439 in laboratory studies and clinical trials to enhance drug absorption.6, 25, 26 An increased level of OZ439 in blood was observed when full cream cow’s milk was co-administered with the drug when compared with fasted volunteers.25,

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suggested the capability of milk fat globules (MFG) and or the products of milk digestion to maintain and present OZ439 in a solubilized or absorbable form to enable intestinal absorption, although the mechanistic processes remain unknown. Hence, this study aims to provide the physical-chemical understanding of the role of milk as a LBF for delivery of OZ439. Drug crystallinity during dispersion of drug in milk and during digestion of the formulation in vitro was monitored in real-time using synchrotron small angle X-ray scattering. High performance liquid chromatography (HPLC) was used in tandem to quantify the phase distribution of drug during the dispersion and digestion of milk containing OZ439.

Figure 1. The chemical structure of OZ439-free base4 MATERIALS AND METHODS Materials. OZ439 was provided as the mesylate salt form by the Medicines for Malaria Venture. Full fat bovine milk (3.8 wt% fat) was either supplied by the Australian Synchrotron or purchased from Coles supermarkets (Brunswick or Mt Waverly, Victoria, Australia). Trizma


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maleate (reagent grade), trifluoroacetic acid (≥99% purity), 4-bromophenylboronic acid (4BPBA, >95% purity), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, Missouri). Calcium chloride dihydrate (>99% purity) and sodium hydroxide pellets (min 97% purity) were purchased from Ajax Finechem (Seven Hills, New South Wales, Australia). Hydrochloric acid (aqueous solution) was purchased from LabServ (Ireland). Sodium chloride (>99% purity) was purchased from Chem Supply (Gillman, South Australia, Australia). Sodium azide (≥99% purity) was purchased from Fluka (Sigma-Aldrich, St. Louis, Missouri), acetonitrile (liquid chromatography grade) was purchased from Merck (Darmstadt, Germany), and phospholipid (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) was purchased from Sapphire Bioscience (New South Wales, Australia). USP grade pancreatin extract was purchased from Southern Biologicals (Nunawading, Victoria, Australia). Unless otherwise stated all chemicals were used without further purification and water was sourced from a Millipore Milli-Q purification system. Preparation of OZ439-HCl and OZ439-FB powders. The HCl-salt of OZ439 was prepared by dissolving 99 mg of OZ439-mesylate in 2.5 mL of water, followed by the addition of 0.25 mL of 1 M HCl solution. Samples were vortexed until thoroughly mixed and passed through a vacuum filter unit with 0.45 µm nylon membrane. OZ439-FB (form 1) was prepared by addition of 1 M NaOH solution to a solution of OZ439-mesylate (99 mg) in water (2.5 mL) to raise the pH to approximately 8.0. The sample was vortexed until thoroughly mixed and vacuum filtered as described above. The resultant powders were analyzed using SAXS within 1 hour of preparation. Preparation of OZ439 in milk formulations and their in vitro lipolysis. OZ439-mesylate salt (99.0 mg, equivalent to 82.2 mg OZ439-FB) was added to 2.75 mL water containing 0.25


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mL of 1 M HCl solution. The drug sample was vortexed until thoroughly mixed prior to the addition of 17.5 mL of milk. This dosage was selected based on a clinically effective dose of 800 mg of OZ439 free base in 200 mL of fluid, i.e. 4 mg OZ439-FB/mL.28, 29 The resulting drug/milk mixture that contained 123.6 mg OZ439-FB equivalents/gram of milk fat (or 4 mg OZ439FB/mL) was incubated in a thermostatted (37 °C) glass vessel under constant magnetic stirring. The pH of each sample was adjusted to 6.500±0.005 prior to each measurement. For dispersion only measurements the samples were only stirred in the glass vessel, whilst for digestion measurements pancreatin lipase suspension (2.25 mL, 1,000 TBU/mL of digest) was remotely injected into the vessel to initiate lipolysis. The pH of the sample in the digestion vessel was maintained at 6.5 during digestion using either 2 M or 1 M NaOH solution titrated in by a pH stat control module (Metrohm AG, Herisau, Switzerland). 2 M NaOH was used to minimize the effects of titrant dilution during SAXS and HPLC runs, while 1 M NaOH was used during titration and back-titration experiments to determine the extent of lipolysis, as described forthwith. Tris-maleate buffer (50 mM pH 6.5) was used for the digestions and was prepared by dissolving 11.86 g of Trizma-maleate, 0.74 g of CaCl2.2H2O (5 mM), and 8.77 g of NaCl (150 mM) in water to a total volume of 1000 mL. Calcium chloride was added to remove the free fatty acids from the lipolysis medium that could inhibit lipolysis and to more closely resemble in vivo conditions where the free fatty acids are absorbed.14 The amount of titrated (ionized) fatty acids during the digestion of milk and OZ439/milk suspensions was calculated from the volume of NaOH required to maintain the pH of the samples at 6.5, with background correction from a digestion without lipid present. To determine the total amount of fatty acids released (ionized and unionized), back-titrations were performed after 1 hour of lipolysis that increased the pH to 9.0. The extent of digestion was calculated


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based on Equation 1; and the theoretical amount of fatty acids (FA) released (2 mmol) was estimated using the total FA content in bovine milk reported in the literature,30 assuming that 2 moles of free FA were liberated from 1 mole of triglyceride. Extent of digestion %=

Ionised FA mol+ Unionised FA mol × 100 (%) Theoretical free FA in milk mol (1)

Isolation of drug precipitates for characterization of the solid state structures. Precipitates containing OZ439 were isolated from digested milk samples to identify the solid-state forms of the OZ439. 15 mL of sample was collected from the digestion vessel after 1 hour in vitro lipolysis of 247.2 mg OZ439-FB equivalent/gram of milk fat and centrifuged for 15 min at 7,378 × g (25 °C). The upper layers of the centrifuged samples that contained both the lipid phase and the aqueous supernatant phase were discarded and the pellets were rinsed by adding 10 mL of water prior to re-centrifugation under the same conditions as above. The aqueous layer was again removed, and the pellets were analyzed using SAXS. Synchrotron small angle X-ray scattering: flow through digestion measurements. The small and wide angle X-ray scattering (SAXS/WAXS) beamline at the Australian Synchrotron (ANSTO, Clayton, Victoria) was used to monitor real-time liquid crystalline structure formation in milk, the solid state of OZ439 (inferred from the presence of crystalline drug peaks), and polymorphic transformations of OZ439. The instrumental setup of the digestion apparatus interfaced to the SAXS has been described previously.16 The digestion medium containing the formulation was re-adjusted to pH 6.5 and aspirated from the digestion vessel (thermostatted to 37 °C) through a fixed quartz capillary mounted in the X-ray beam using a peristaltic pump operating at approximately 10 mL/min. An X-ray beam with a wavelength λ of 0.954 Å (13 keV) was used throughout this study. The liquid crystalline structures in milk were detected using a


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sample-to-detector distance of approximately 1.6 m (covering a q range of 0.01 < q < 0.4 Å-1), while the solid crystalline drugs were detected with a sample-to-detector distance of approximately 0.6 m (0.04 < q < 2.00 Å-1). q is the length of the scattering vector defined by (4π/λ) sin (2θ/2), where 2θ is the scattering angle. The 2D SAXS patterns were recorded using a Pilatus 1M detector with a 5 s acquisition time and a delay of 15 s between measurements (one measurement every 20 s). The raw data were reduced to 1D scattering functions I(q) by radial integration using the in-house-developed software ScatterBrain. Synchrotron small angle X-ray scattering: static capillary measurements. SAXS patterns of the OZ439-mesylate, the OZ439-HCl, and the OZ439 free base powders were collected by loading the powder samples into 1.5 mm outer-diameter glass microcapillaries placed in the Xray beam. The sample-to-detector distance was approximately 0.6 m to give a q range of (0.04 < q < 2.00). The same setup was used to analyze the precipitates of OZ439 after 1 h digestion in milk and tris buffer. Distribution and quantification of OZ439 in the digested milk phases. Quantification of the amount of drug solubilized after dispersion and digestion of OZ439 in milk and tris-maleate buffer (i.e. dynamic solubilization) were carried out using HPLC. Digestion experiments were carried out using methods described in the in vitro lipolysis section. To quantify the amount of drug dissolved during the digestion of milk and the amount of undissolved drug remaining in the system, aliquots (300 µL) were taken at various time points throughout the digestion experiments (0, 2, 5, 10, 30 and 60 min). These aliquots were pipetted into 2 mL glass vials containing 30 µL of lipase inhibitor solution (0.05 M 4-BPBA in methanol).31 Subsequently, an aliquot (200 µL) was then transferred from each glass vial to a polycarbonate centrifuge tube (7 × 20 mm, Beckman Coulter) and centrifuged for 40 min at 329,177 × g using an Optima MAX-TL ultra-


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centrifuge (Beckman Coulter, Indiana, U.S). After the digested samples were centrifuged, the resultant samples consisted of three phases (except in the case of buffer-only samples). The three phases were the upper lipid layer and the middle aqueous layer that consist of digested milk products and colloidal phases, as well as the pellet phase, which contained precipitated drug. The aqueous colloidal phase was first removed with a 1 mL syringe and 25-gauge needle and transferred to a separate 2 mL glass vial. The remaining lipid layer that settled at the top of the centrifuge tube was then also transferred to a separate 2 mL glass vial. A 25-gauge needle was used for this purpose and the lipid layer was collected into the vial. The needle was subsequently rinsed with DMSO, and 800 µL of DMSO was added to the separated aqueous colloidal phase and lipid layer phase. These samples were then diluted in 5:95 v/v mobile phase B: mobile phase A (mobile phase A was water with 0.1% TFA; and mobile phase B was acetonitrile with 0.085% TFA) before the drug was quantified using HPLC. The pellet phase was also dissolved separately in DMSO and diluted in mobile phase before analysis by HPLC. The HPLC system included a Shimadzu CBM-20A system controller, an LC-20AD solvent delivery module, an SIL-20A auto sampler and a CTO-20A column oven set at 35 °C, coupled to an SPD-20A UV-detector (Shimadzu Corporation, Kyoto, Japan). A reverse-phase C18 column was used (4.6 × 75 mm2, 3.5 µm; Waters Symmetry®, MA, USA) and the UV detector was set to 260 nm for the detection of OZ439 using a binary gradient of mobile phase A and B. The buffer gradient consisted of 5-95% B for 6 min, 95-5% B for 0.2 min and 5% B for 5.8 min at 0.5 mL/min flowrate. The injection volume was 50 µL. A standard curve was prepared from a stock solution of OZ439 prepared in DMSO at a concentration of 5 mg/mL. This stock solution was diluted in a mixture of DMSO and digested milk, which represented the sample matrix (1:4 v/v of digested milk to DMSO), to provide standards at different concentrations ranging from 1 to 40


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µg/mL. Diazepam was used as an internal standard in all the HPLC measurements. The retention time of OZ439 is 7 min, while that for diazepam is 5 min. The limit of detection for OZ439 is 0.05 µg/mL and the limit of quantitation is 0.10 µg/mL, with the respective signal-to-noise ratio of 5 and 10.

RESULTS AND DISCUSSION Effects of OZ439 on the extent of digestion and lipid self-assembled structures in milk. Our group has previously shown that liquid crystalline nanostructures were formed during the digestion of bovine milk due to the self-assembly of amphiphilic digestion products that consist of fatty acids, monoglycerides, and diglycerides.32, 33 As shown in the X-ray scattering pattern of milk in Figure 2(a) of reference32, an inverse bicontinuous cubic phase Im3m, an inverse hexagonal phase H2, and a lamellar phase Lα coexist upon complete digestion of full cream milk. However, these structures were altered when OZ439 was present in the digestion system at different concentrations [Figure 2(a)]. Major changes in the self-assembled structures in milk occurred at > 50 mg OZ439-mesylate, i.e. 62.4 mg OZ439-FB equivalent/gram of milk fat. Above this concentration, the Im3m phase did not form during the 1 hour digestion period. Only the Lα phase related to the formation of the calcium soaps of fatty acid digestion products, and an H2 phase with the prominent (100) peak at q = 0.12 Å−1 were seen. The time-dependent colloidal structure formation during the digestion of milk in the presence of OZ439 is shown in Figure 2(b). A shift of the scattering peaks of the H2 phase towards higher q values with increasing concentration of drug is illustrated in Figure 2(c), signifying a contraction of the lattice spacing, i.e. closer packing of adjacent cylindrical aqueous channels between lipid domains.


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The finding that OZ439 interferes with the self-assembly of the lipids during the digestion of milk was anticipated. The presence of drug molecules in liquid crystalline systems does not necessarily impact on the self-assembly of lipids,34, 35 however, in the case of OZ439 which is amphiphilic, it is expected to participate in altering the global curvature of the amphiphilic digestion components, in this case driving the self-assembly towards a less swollen inverse hexagonal phase. This drive towards more negative overall curvature indicates that OZ439 is acting as a more hydrophobic component than the overall influence on packing of the lipid digestion components alone. The total amount of liberated fatty acids after 60 minutes digestion of milk and milk containing 99 mg OZ439 (2.35 ± 0.13 vs. 2.29 ± 0.20 mM respectively) were similar, indicating that although the surface-active OZ439 molecules altered the lipid packing on digestion, the overall digestibility of milk was not significantly affected by the presence of OZ439. Changes in the structures were therefore not attributable to the different extents of digestion. The influence of loading of amphiphilic drug on the release of fatty acids from LBFs has been reported previously with variable findings.36-38 For example, Williams et al. reported no significant effects of varying the concentration of danazol on the digestibility of medium and long chain lipids,38 while Thomas et al. observed a decrease in the titrated fatty acids as a function of simvastatin concentration during digestion in medium chain-SNEDDS but not long chain-SNEDDS.37 Meanwhile, contrary to Williams et al.,38 studies by Arnold et al. suggested that the presence of a variety of amphiphilic drugs (including danazol) in LBFs reduced the extent of lipolysis of medium chain triglycerides.36 Whilst the basis of the discrepancies between these observations were not initially clear due to the complexity of the involved mechanisms, it was generally recognized that poorly water soluble drugs may change the surface compositions of the lipid


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droplets available for digestion.36, 37 This could arise from interactions between the drugs and the lipid droplets, surface localization or partial solubilization of the drugs.36, 37 In addition to the effects of drugs on the removal of lipolytic products from the oil/water interface through changes in surface characteristics, interactions between the drugs and the lipid digestion products such as ion-pairing may also occur, which could potentially affect the formation of liquid crystalline structures as was seen in the case of OZ439 in milk.


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Figure 2. (a) X-ray scattering patterns of milk after 60 minutes digestion in the absence of OZ439-mesylate salt (0 mg OZ439-mes)32, and with increasing concentrations of OZ439mesylate salt from 25 mg to 50 mg and 99 mg. The corresponding amount of OZ439-free base per gram of milk fat was 31.2, 62.4, and 123.5 mg/g. ‘A1’, ‘A2’, and ‘A3’ represented the first, second, and third peak of an Im3m phase with the reciprocal spacing ratios of 1: √2 ∶ √4. The three peaks of the H2 phase with the reciprocal spacing ratios of 1: √3 ∶ √4 are annotated as ‘B1’, ‘B2’, and ‘B3’ respectively. The Lα phase was denoted as ‘C’. (b) X-ray scattering plot of the time-dependent structure formation during the digestion of milk in the presence of OZ439. (c) Effect of increasing amount of OZ439 on the lattice parameters of the H2 phase and the Lα phase present at the end of digestion. Solid-state properties of OZ439 prior to exposure to milk. Transit of a drug through the GI tract following oral administration may result in dissolution, salt formation and/or solid state transformations of the drug due to variations in the intraluminal pH across the different segments.39 The pH-induced changes in the solid-state properties of OZ439 have been previously investigated,8 and the findings are summarized in Figure 3. Briefly, the mesylate salt of OZ439 has a high apparent solubility in pure water due to the formation of micelles and other structures, however transformation of OZ439 to the insoluble HCl salt occurred after addition of HCl solution at pH 1.2 to simulate stomach conditions, with the formation of white crystalline solids. This transformation also occurred in the current study up to this point in the preparation of the formulation prior to exposure to milk. In our previous work, when the pH was increased 6.5 (representative of duodenal pH), conversion of the HCl salt to a free base form of OZ439 occurred. The solid powder of the free base initially formed (OZ439-FB form 1) then underwent


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a solid-solid polymorphic transformation to the more thermodynamically stable form (OZ439-FB form 2) after storage at room temperature and upon heating.

Figure 3. Proposed solid state transformations of OZ439 starting from the mesylate salt due to changes in storage and formulation conditions, the gastrointestinal pH conditions and intestinal lipolysis. Solid state disposition of OZ439 in milk and buffer pre- and post-digestion. Milk fats are dispersed as globules that are composed principally of triglycerides, which are anticipated to be capable of solubilizing the anti-malarial drugs. Solubilization of poorly water-soluble drugs and the ability of LBFs to maintain drug solubility in solution during GI transit have been reported as key to improved oral bioavailability.15, 40 Solubilization of drugs in LBF during in vitro digestion has recently been used to predict the performance of the lipid excipients following oral administration by assessing the fraction of precipitated and dissolved drugs.14, 15, 41-43 Samples were removed at time intervals and the lipolysis reactions were inhibited prior to ultracentrifugation of the inhibited digests. This typically led to separation into three layers – an upper lipid layer containing the digestion products and associated drugs, the aqueous supernatant and a lower pellet layer containing undissolved drug. The distribution of the drugs in the separated phases were then analyzed and quantified using HPLC. However, recent studies have highlighted the importance of in situ monitoring of drug solubilization using a synchrotron small


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angle X-ray scattering 16, 44-46 and Raman spectroscopy47 to account for the dynamic processes of the lipid digestion. These techniques therefore provide critical information regarding the solubilization of OZ439 in the rapidly digesting milk, analogous to studies on colloidal phase transitions.32, 33 In addition, elucidation of the solution-mediated44, 46, 48 or digestion-mediated49, 50 polymorphic transformations of drugs that typically occurred within short time frames could also be achieved. Figures 4(a) and 4(b) summarize the SAXS profiles of milk in the high-q range during the course of 25 minutes dispersion (stirring drug in the milk) and digestion in the presence of OZ439. The proportional changes in the amount of OZ439-FB form 1 and form 2 detected were determined by measuring the area of the characteristic peaks at q = 1.31 (OZ439-FB form 1) and 1.27 Å-1 (OZ439-FB form 2) shown in Figure 4(c). Formation of OZ439-FB form 1 was observed immediately after the addition of milk to OZ439-HCl solution and pH adjustment to 6.5. With continuous stirring at 37 °C for 25 minutes at pH 6.5 no further changes in diffraction from the drug were evident. Interestingly, a decrease in the peak area of OZ439-FB form 1 was seen upon dispersion in milk, which could be related to the solubilization into the undigested milk fat globules [Figure 4(c)]. Injection of pancreatic lipase to initiate the digestion of milk containing OZ439 resulted in a decrease in the area of the characteristic peak in OZ439-FB form 1, which disappeared completely after approximately 15 min digestion. Concurrent with the disappearance of the characteristic peaks of OZ439-FB1, new peaks appeared at q values of 1.27 and 1.35 Å-1 approximately 3 min into the digestion. The positions of these new peaks point to the formation of the thermodynamically stable form of the free base (OZ439-FB form 2) as was discussed previously.

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. A progressive decrease in the peak area of OZ439-FB form 2 was subsequently


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observed from about 8 min of digestion until the conclusion of the measurement. This decrease was not due to dilution from the addition of the NaOH titrant (titrated volume of NaOH was about 3% of the total sample volume after 60 minutes digestion), but was attributed to gradual but incomplete solubilization of the crystalline FB form 2 into the digested milk products. Redissolution of crystalline drug precipitates (collected after digestion) has also been reported by Stillhart et al.45 and Sassene et al.51 but more research is needed to understand the influences of self-assembled colloidal structures formed by the lipid digestion products on the dissolution of the crystalline drugs. Simply stirring OZ439 in milk without digestion for an extended period of time did not induce the formation of OZ439-FB form 2 [Figure 4(a)], indicating that digestion provided the required conditions for the polymorphic transformation from OZ439-FB1 to OZ439-FB2. This transformation was not influenced by the initial form of the drug (both the HCl and the mesylate salts of the OZ439 converted to the FB form 2 upon digestion), similar to observations made by Porter et al. with halofantrine.50 Clinical exposure of these drugs would therefore be largely determined by the final form of the drugs post-digestion, and their dissolution into the digests.45


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Figure 4. X-ray scattering patterns of OZ439 during (a) dispersion at 37 °C in milk (pH 6.5), and (b) during in vitro digestion in milk between q = 1.2 and 1.40 Å-1. The crystalline Bragg peaks (of OZ439-FB 1) remained throughout the dispersion in panel (a), while new peaks belonging to OZ439-FB 2 appeared during digestion in panel (b). The asterisks point to the characteristic prominent peaks for OZ439-FB form 1 and form 2 of which the corresponding area under the peaks during dispersion and digestion were shown in panel (c) and (d) respectively. (e) X-ray scattering patterns of OZ439-FB form 2 powder (OZ439-FB 2, black trace), and the pellets collected after 1 hour digestion of OZ439 in milk (red trace). The background intensity of an empty capillary has been subtracted from the scattering intensities of the samples.


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Precipitation of OZ439 by crystallization of the FB form 2, which was not completely solubilized during digestion, was confirmed by the solid-state measurements of the pellet after 60 min of lipid digestion. It has been previously suggested that weakly basic drugs can readily form amorphous non-crystalline precipitates during digestion in LBFs, depending on the pKa values of the drugs and the pH of the digestion medium.31,

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Ionized drugs have been shown to

precipitate in the amorphous form due to charge interactions with liberated fatty acids, while neutral drugs are generally precipitated as crystalline solids.52 OZ439 has a pKa value of approximately 6.5−6.7 (extracted from the literature7 and from Scifinder based on ACD/Labs Software V11.02), and would therefore exist both in the unionized and ionized states (in equal concentrations) under the digestion conditions at pH 6.5. As a consequence it was anticipated that OZ439 would be precipitated in a mixture of crystalline and amorphous solids, but solidstate measurements of the precipitated OZ439 revealed a predominantly crystalline solid [Figure 4(d)]. However, the slight increase in the background intensities observed between q values of 1.3 and 1.7 Å-1 in the collected pellets compared to as-prepared OZ439-FB form 2 powder may indicate presence of some amorphous precipitates. Precipitation of predominantly crystalline solids with some degree of amorphous-salt after lipolysis of cinnarizine at pH similar to its pKa has also been previously reported.31 Finally, a ‘sham’ digestion of OZ439 in tris buffer medium was carried out as a control to determine the behavior of drug in the absence of digesting milk fat lipids. No decrease in the peak area of OZ439-FB form 1 was observed after pancreatin addition indicating no solubilization occurred (see Figure S1 in the Supporting Information). No polymorphic transformations were also observed. Schematic representation of the dynamic processes that


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occur during the digestion of milk containing OZ439 and the control experiment where drug was dispersed in tris buffer and the solid state interrogated after addition of lipase (Figure 5).

Figure 5. Schematic representation of the time-course formation of liquid crystalline structures and the corresponding solid state form of OZ439 during digestion in buffer and milk.

Quantification of the dynamic solubilization of OZ439 in milk and buffer during in vitro digestion using HPLC. The phase distribution of OZ439 into the colloidal lipid phases during the course of digestion in milk and lipid-free buffer was quantified using HPLC after ultracentrifugation. The process of ultra-centrifugation of the digested milk sample resulted in the formation of three phases (i.e. the upper lipid layer, the aqueous layer, and the pellet layer), while only two phases were seen in tris buffer due to the absence of lipids. Separation of the digested milk-only formulation (no OZ439) resulted in only an upper lipid layer enriched in lipid digestion products and a middle aqueous layer, with no observable precipitates that were more dense than the aqueous supernatant.32


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Figure 6(a) shows that OZ439 was largely present in an undissolved state during the initial dispersion prior to the addition of lipase at the 0 min time point. As the digestion of milk was initiated, OZ439 was solubilized into the digested milk lipids, which was evident from the increase of OZ439 content in the lipid and aqueous layers, and the reduction in the amount of OZ439 in the pellet phase. These observations were supported by the visual observations of the separated phases after ultra-centrifugation in Figure S2 in the Supporting Information. The solubilization of OZ439 in the lipid and aqueous phases were quantified in mg per gram of milk fat [Figure 6(d)].The amount of OZ439 that resided in the separated phases remained relatively constant after about 10 minutes of digestion, which coincided with the formation of the final liquid crystalline structure (the H2 phase) of the digesting milk containing OZ439, which occurred at approximately 9 min into each digestion.


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Figure 6. Partitioning of OZ439 in the pellet, aqueous, and the lipid layers after ultracentrifugation of OZ439 in (a) milk and (b) tris buffer, before (0 min) and after digestions for 2, 5, 10, 30, and 60 min. The digestion reactions were terminated using 0.05 M 4-BPBA in methanol.31 The extent of digestion of milk in the presence of 99 mg OZ439-mesylate was also shown in panel (a). (c) Comparison between the effects of inhibitor on the partitioning of OZ439 in the digested milk phases at the 60 minute time-point (validating its use for the shorter time points to suspend lipolysis during ultracentrifugation) (d) Solubilized OZ439 in the lipid and aqueous layers during 60 min digestion in mg per gram of milk fat. The theoretical maximum value for the amount of OZ439 that can be solubilized per g of milk fat was presented as the dashed line. Data are mean ± s.d, n=3 separate digestions. In contrast to the phase distribution during digestion of milk, there was little to no solubilization of OZ439 when tris buffer alone was used as the digestion medium [Figure 6(b)]. OZ439 remained in the pellet phase at all sampling points throughout the 60-minute lipid-free digestion. The formation of digestion products during lipid hydrolysis therefore was essential to the solubilization of OZ439 in milk, where the drug may be partitioned into the various colloidal


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structures. Previous studies have shown that the aqueous supernatant layer of the digested milk medium consisted of mainly unilamellar vesicles with multilamellar fragments, while the H2 phase was predominantly located in the upper lipid layer.32 It was therefore speculated that OZ439 was preferentially localized within the H2 phase instead of the vesicular phase, which may also be possibly linked to the greater oil-water surface area provided by the H2 phase for the amphiphilic OZ439 molecules. Although inhibition of the lipase activity can be achieved using 4-BPBA to halt the digestion of triglycerides in order to obtain time-dependent drug solubilization profiles, it was not known if the presence of 4-BPBA (prepared in methanol) would alter the distributions of OZ439. To investigate this, comparisons were made between the inhibited and non-inhibited samples collected at the 60 minute end point. It was assumed that little or no further lipolysis could occur following the 40 minutes ultra-centrifugation at 37 °C, as the digestion had reached completion [Figure 6(a)]. As was shown in Figure 6(c), no significant differences in the distributions of OZ439 between samples with and without added 4-BPBA were observed. This observation may reflect the minimal interference of methanol on the partitioning of OZ439 in the digested milk phases.

CONCLUSIONS Assessing the solubilization behavior of drugs during digestion is important to understand their fate after oral administration. In this study, in situ monitoring of diffraction peaks and thereby solubilization of OZ439 in milk as the formulation vehicle (inferred through the loss of Bragg peak intensity) during the course of digestion was investigated using synchrotron X-ray scattering. The following key conclusions can be drawn from the observations. OZ439


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transforms from the mesylate salt form to the hydrochloride salt form at the low pH of the gastric phase, which then changes to the free base form when the pH of the environment increases to 6.5 mimicking the small intestine. This free base form (FB form 1) is an unstable intermediate and can undergo polymorphic transformation to FB form 2 during digestion but not during dispersion in milk. Digestion of OZ439 in lipid-free formulation (buffer) also does not cause the polymorphic transformation, suggesting that the formation of OZ439 free base form 2 in digesting milk is triggered by solubilization of the form 1. This highlights the potential complexity in the solid-state behavior of poorly water soluble drugs, and that consideration of the solid state of drug and the colloidal state of the lipids at the end point of digestion may be critical to understanding consequent patterns of absorption. In addition, the presence of fat in milk and the process of digestion are essential for the solubilization of OZ439 under intestinal conditions based on the in vitro findings. Inclusion of a digestion step to the drug solubilization experiments in milk-based oral formulations is therefore strongly recommended. The influence of fat content on the solubilization of OZ439 also warrants further investigation and by introducing milk powders, modulation of the ratios of milk fat to the solubilization of a fixed dose of OZ439 may become possible.

ASSOCIATED CONTENT Supporting Information Additional figures show the X-ray scattering patterns of OZ439 during dispersion and digestion in tris buffer and the changes in the area under the peak of OZ439-FB form 1 during digestion; and visual observations of the separated phases in the non-digested (0 min) and digested (2-60


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min) OZ439-containing milk samples after ultra-centrifugation.

AUTHOR INFORMATION Corresponding Author * Postal Address: Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, VIC 3052, Australia Telephone: +61 3 99039112; Fax: +61 3 99039583 Email: [email protected] Funding Sources Bill & Melinda Gates Foundation (Investment ID OPP1160404). Australian Research Council (DP160102906) Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was funded by the Bill and Melinda Gates Foundation grant number OPP1160404 in collaboration with the Medicines for Malaria Venture (MMV). Funding is also acknowledged from the Australian Research Council under the Discovery Projects scheme DP160102906. The SAXS experiments for this work were conducted on the SAXS/WAXS beamline of the Australian Synchrotron, part of ANSTO. The authors thank Dr Niya Bowers, Dr Michael Mitchell and Dr Drazen Ostovic for technical and historical discussions around co-administration of OZ439 with milk. ABBREVIATIONS


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FB, free base; LBF, lipid-based formulation; GI, gastrointestinal; SMEDDS, self-microemulsifying drug delivery systems; SNEDDS, self-nano-emulsifying drug delivery systems; API, active pharmaceutical ingredient; MFG, milk fat globules; HPLC, high performance liquid chromatography; SAXS, small angle X-ray scattering; 4-BPBA, 4-bromophenylboronic acid; FA, fatty acid. REFERENCES 1. WHO World Malaria Report; 2016. 2. WHO Status report on artemisinin and ACT resistance (April 2017); 2017. 3. WHO A framework for malaria elimination; 2017. 4. Charman, S. A.; Arbe-Barnes, S.; Bathurst, I. C.; Brun, R.; Campbell, M.; Charman, W. N.; Chiu, F. C. K.; Chollet, J.; Craft, J. C.; Creek, D. J.; Dong, Y.; Matile, H.; Maurer, M.; Morizzi, J.; Nguyen, T.; Papastogiannidis, P.; Scheurer, C.; Shackleford, D. M.; Sriraghavan, K.; Stingelin, L.; Tang, Y.; Urwyler, H.; Wang, X.; White, K. L.; Wittlin, S.; Zhou, L.; Vennerstrom, J. L. Synthetic ozonide drug candidate OZ439 offers new hope for a single-dose cure of uncomplicated malaria. P. Natl. A. Sci. 2011, 108, (11), 4400-4405. 5. Moehrle, J. J.; Duparc, S.; Siethoff, C.; van Giersbergen, P. L. M.; Craft, J. C.; ArbeBarnes, S.; Charman, S. A.; Gutierrez, M.; Wittlin, S.; Vennerstrom, J. L. First-in-man safety and pharmacokinetics of synthetic ozonide OZ439 demonstrates an improved exposure profile relative to other peroxide antimalarials. Brit. J. Clin. Pharmaco. 2013, 75, (2), 535-548. 6. Phyo, A. P.; Jittamala, P.; Nosten, F. H.; Pukrittayakamee, S.; Imwong, M.; White, N. J.; Duparc, S.; Macintyre, F.; Baker, M.; Möhrle, J. J. Antimalarial activity of artefenomel (OZ439), a novel synthetic antimalarial endoperoxide, in patients with Plasmodium falciparum and Plasmodium vivax malaria: an open-label phase 2 trial. Lancet Infect. Dis. 2016, 16, (1), 6169. 7. Dong, Y.; Wang, X.; Kamaraj, S.; Bulbule, V. J.; Chiu, F. C. K.; Chollet, J.; Dhanasekaran, M.; Hein, C. D.; Papastogiannidis, P.; Morizzi, J.; Shackleford, D. M.; Barker, H.; Ryan, E.; Scheurer, C.; Tang, Y.; Zhao, Q.; Zhou, L.; White, K. L.; Urwyler, H.; Charman, W. N.; Matile, H.; Wittlin, S.; Charman, S. A.; Vennerstrom, J. L. Structure–Activity Relationship of the Antimalarial Ozonide Artefenomel (OZ439). J. Med. Chem. 2017, 60, (7), 2654-2668. 8. Clulow, A. J.; Salim, M.; Hawley, A.; Gilbert, E. P.; Boyd, B. J. The curious case of the OZ439 mesylate salt – an amphiphilic antimalarial drug with diverse solution and solid state structures. Mol. Pharm. 2018, 15, (5), 2027-2035. 9. Amidon, G. L.; Lennernas, H.; Shah, V. P.; Crison, J. R. A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm. Res. 1995, 12, (3), 413-20. 10. Hörter, D.; Dressman, J. B. Influence of physicochemical properties on dissolution of drugs in the gastrointestinal tract. Adv. Drug. Deliver. Rev. 1997, 25, (1), 3-14.


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Graphical Table of Contents


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Interactions of Artefenomel (OZ439) with Milk during Digestion

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