In Vivo Formation of Cubic Phase in Situ after Oral Administration of

Nov 15, 2015 - Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade,. Parkville ...
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In Vivo Formation of Cubic Phase in Situ after Oral Administration of Cubic Phase Precursor Formulation Provides Long Duration Gastric Retention and Absorption for Poorly Water-Soluble Drugs Anna C. Pham,†,‡ Linda Hong,†,‡,∥ Oliver Montagnat,† Cameron J. Nowell,§ Tri-Hung Nguyen,† and Ben J. Boyd*,†,∥ †

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia § Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia ∥ ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia S Supporting Information *

ABSTRACT: Lipid-based liquid crystalline systems based on the combination of digestible and nondigestible lipids have been proposed as potential sustained release delivery systems for oral delivery of poorly water-soluble drugs. The potential for cubic phase liquid crystal formation to induce dramatically extended gastric retention in vivo has been shown previously to strongly influence the resulting pharmacokinetics of incorporated drug. In vitro studies showing the in situ formation of cubic phase from a disordered precursor comprising a mixture of digestible and nondigestible lipids under enzymatic digestion have also recently been reported. Combining both concepts, here we show the potential for such systems to form in vivo, increasing gastric retention, and providing a sustained release effect for a model poorly water-soluble drug cinnarizine. A mixture of phytantriol and tributyrin at an 85:15 mass ratio, shown previously to form cubic phase under the influence of digestion, induced a similar pharmacokinetic profile to that in the absence of tributyrin, but completely different from tributyrin alone. The gastric retention of the formulation, assessed using micro-X-ray CT imaging, was also consistent with the pharmacokinetic behavior, where phytantriol alone and with 15% tributyrin was greater than that of tributyrin in the absence of phytantriol. Thus, the concept of precursor lipid systems that form cubic phase in situ during digestion in vivo has been demonstrated and opens new opportunities for sustained release of poorly watersoluble drugs. KEYWORDS: cubic phase, pharmacokinetics, oral drug delivery, gastric retention, micro-X-ray CT imaging

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INTRODUCTION

number of different types of in vitro digestion experiments; initially the formation of cubic phases was inferred from microscopy experiments on exposure of triglyceride to lipase,15 and digestion of triolein has been shown to form cubic phases at certain pH values,16,17 while the formation of cubic phases during the digestion of milk has also been recently shown.18 The potential of nondigestible lipids to form cubic phases under gastrointestinal conditions has also been shown;19 however, to our knowledge the formation of cubic phases has not yet been shown in vivo.

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Cubic phases and liquid crystalline materials in general are receiving increasing interest for their application in drug delivery,2−4 as the internal nanostructure of aqueous channels of the materials (Figure 1) can be used to control the release of drugs.5−8 Cubic phases are formed by a range of lipids in excess water, including glyceryl monooleate (GMO)9 and phytantriol (PHY).10 Cubic phases can be prepared as bulk materials, or dispersed as particles which retain the internal liquid crystalline structure of the bulk material, known as cubosomes.11−14 There are opportunities to utilize structured cubic phases in drug delivery, functional foods, and nutrition; however, an understanding of their formation and behavior after oral ingestion is crucial but lacking. The formation of cubic phases under the conditions of digestion has been reported in a © XXXX American Chemical Society

Received: October 15, 2015 Revised: November 12, 2015 Accepted: November 15, 2015

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DOI: 10.1021/acs.molpharmaceut.5b00784 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 1. Chemical structures of lyotropic lipids, glyceryl monooleate (GMO) and phytantriol (PHY), and the triglyceride, tributyrin (TB). When hydrated, GMO and PHY can self-assemble into the cubic Pn3m phase, and when PHY is mixed with TB and water in the right proportions, an inverse micellar emulsion can result, which upon digestion forms the “parent” cubic phase formed by PHY + water alone. The cubic Pn3m structure was reproduced from Seddon et al.22 by permission of the PCCP Owner Societies.

(such as phytantriol and selachyl alcohol27) with digestible lipids (such as triglycerides). In vitro lipolysis studies showed the transformation of the precursor emulsion into cubosomes as a result of enzymatic attack on the triglycerides. It is apparent that the process could be used to prepare cubosomes in a manufacturing context, but could also be used in vivo to generate liquid crystalline structures in the gastrointestinal tract after oral administration as a precursor formulation. Consequently, as a first step toward evaluating the in vivo utility of the enzymatic approach to forming liquid crystalline structures, the pharmacokinetics of an incorporated highly permeable, poorly water-soluble drug, cinnarizine (CZ), were determined after oral administration when formulated in tributyrin alone (TB) and phytantriol containing tributyrin at the critical ratio of 85% phytantriol:15% tributyrin (w/w, PHY +TB) known (in vitro at least25,26) to transform to a liquid crystalline phase on digestion. This was compared with the pharmacokinetic profile and parameters obtained previously for phytantriol alone (PHY). Thus, the hypothesis was that the unstructured PHY+TB formulation would perform similarly to PHY alone after oral administration due to cubic phase formation, exhibiting prolonged gastric retention and a long duration of drug absorption, while the TB-only formulation would behave similarly to the oleic acid formulation in previous studies, with more “typical” rapid gastric emptying and a shorter duration of absorption of CZ. The gastric retention of all three formulations was also compared using micro-X-ray CT-imaging techniques by incorporation of gold nanoparticles (GNP) to relate the gastric retention of the formulations to the drug absorption kinetics.

In an oral drug delivery context, the digestibility of the lipid has been shown to contribute greatly to the pharmacokinetic outcome for the drug. Despite forming cubic phase in aqueous environments, GMO is susceptible to hydrolytic degradation by lipases present in the gastrointestinal tract due to the ester linkage in its chemical structure (Figure 1). In contrast, lipids such as PHY, which also a forms cubic phase but does not have an ester linkage between the hydrophobic tail and hydrophilic headgroup, are not subject to breakdown by lipases. Consequently, for poorly water-soluble compounds, the PHYbased cubic phase provides sustained gastric retention for both the lipid and drug and a steady absorption profile in vivo,20 whereas the equivalent GMO formulation does not. The general concept was confirmed by administration in a different liquid crystal forming nondigestible lipid formulation based on selachyl alcohol,21 which also showed the long duration effect, while a simple oleic acid emulsion formulation, which does not form a liquid crystal in gastrointestinal fluids, did not provide the gastric retention needed for the long duration of absorption.19 Interestingly, predispersion of the PHY formulation to form drug-loaded cubosomes did not significantly increase the rate of gastric emptying, again leading to the long duration of absorption seen with the bulk phase, whereas the administration of GMO cubosomes resulted in rapid emptying.20 Thus, in situ formation of liquid crystalline systems is linked directly to beneficial gastric retention and increased exposure. Although PHY has not yet been used in oral commercial formulations, there has been increasing evidence which suggests that PHY can be utilized as an excipient as it provides sustained absorption effects20 not seen with current excipients. The bulk cubic phase is not ideal for oral administration due to the high viscosity making for difficult handling and administration,23 while cubosome dispersions are difficult to form and generally lack the stability for a viable formulation with sufficient shelf life.24 To address these issues, a new approach to preparing cubosomes has been demonstrated where a stable, readily formed emulsion is transformed by lipase enzymes to form cubosomes in situ.25,26 The concept relies on forming an emulsion from a mixture of nondigestible lipids

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MATERIALS Phytantriol (3,7,11,15-tetramethylhexadecane-1,2,3-triol) was purchased from DSM Nutritional Products (Singapore), with a minimum purity of 95%. Tributyrin was purchased from TCI Chemicals (Tokyo, Japan). All lipids were used without further purification. Gold(III) chloride (AuCl3), trisodium citrate dihydrate, potassium chloride, cinnarizine (CZ), diazepam, and formic acid (MS grade) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Sodium citrate, disodium hydrogen B

DOI: 10.1021/acs.molpharmaceut.5b00784 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics orthophosphate, and potassium dihydrogen orthophosphate were purchased from APS Ajax Finechem (Auburn, Australia), and sodium chloride was purchased from Chem-Supply, Australia. Hydrochloric acid (AR) and acetonitrile (HPLC analysis grade) were obtained from Merck Millipore (Bayswater, Australia). Lethabarb (325 mg/mL pentobarbitone sodium) was purchased from Abbott Laboratories Pty. Ltd. (NSW, Australia). Milli-Q water used was from a Millipore water purification system (Billerica, MA, USA).

The processed cinnarizine plasma samples were analyzed on a single quadrupole LCMS system (model 2010) with an electrospray ionization (ESI) interface (Shimazu, Kyoto, Japan). The LCMS Solutions software (Shimadzu, Kyoto, Japan) was used for data acquisition and processing. The column used was the Phenomenex Gemini C18 110 Å column (3 μm, 50 mm × 2.00 mm, Phenomenex, CA). The mobile phase A consisted of 95:5 (v/v) water/acetonitrile with 0.2% (w/v) ammonium hydroxide, and mobile phase B consisted of 95:5 (v/v) acetonitrile/water with 0.2% (w/v) ammonium hydroxide). A gradient elution was performed at 50 °C with 10−80% mobile phase B from 0 to 0.3 min, then 80−100% B from 0.3 to 1 min, holding at 100% B from 1 to 10 min, then reducing it from 100 to 10% B from 10 to 10.5 min, and finally 10% B from 10.5 to 14.5 min. A sample volume of 5 μL was used in each run, with halofantrine as the internal standard. The retention time of CZ was 3.8 min and halofantrine was 6.0 min, with a selective ion monitoring (SIM) of m/z 368.80 and 499.80, respectively. Both CZ and halofantrine were detected in positive ion mode. The nebulizing gas flow rate was set at 1.5 L/min, with a heat block temperature of 200 °C and curved desolvation line temperature of 250 °C. The voltage setting of the detector was 1.90 kV, with the probe and curved desolvation line set at 4.5 kV and 25.0 V, respectively. Formulation Preparation for Imaging. Citrate-capped gold nanoparticles (16 nm in diameter) were synthesized according to the previously reported procedures29 based on Turkevich methods.30 The GNP suspension was centrifuged in 1.5 mL microcentrifuge tubes at 12054g to form a GNP pellet. The supernatant containing the dissolved salt was removed, and the GNP pellet was redispersed in Milli-Q water for transfer into lipid formulations. The water was removed from the GNP +lipid mixture via benchtop lyophilization (Virtis AdVantage 2.0 BenchTop Freeze-Dryer/Lyophilizer by SP Scientific (Warminster, PA)) for at least 1 day so that only the bulk lipid containing 5% GNP w/w was dosed to rats. Micro-X-ray CT-Imaging Procedure. Male Sprague− Dawley rats weighing 280−320 g were used for assessing gastric retention for each formulation. Imaging procedures were conducted using methods described by Pham et al.29 Briefly, rats were fasted for at least 12 h prior to dosing, with free access to water. Following anesthesia with isoflurane (5% v/v for induction and 1.5% v/v for maintenance) a predose scan was performed on a nanoCT (Mediso Medical Imaging Systems, Budapest, Hungary). Each X-ray CT scan was isotropic, with a post-reconstruction resolution of 76.9 μm in each dimension. After the initial scan (approximately 9 min) while rats were kept unconscious, lipid formulation (300 mg) containing 5% w/w GNP was dosed to rats via oral gavage. An X-ray CT scan was immediately performed under anesthesia. Rats were then allowed to recover from anesthesia in individual metabolic cages under a heat lamp. Scans were continued at 1, 2, 4, and 8 h after administration. Imaging studies were performed in triplicate. Analysis of Imaging Results. Imaging results were qualitatively and quantitatively analyzed on Imaris by Bitplane (Zurich, Switzerland) using the method previously described in recent studies.29 Briefly, a core was manually inserted through the spine and rib regions to quantitatively calculate the sum of intensity within the gastric region. Contrasts outside of the gastric region (i.e., small intestine) were also excluded by including only contrasts visible two vertebrae above and one vertebra below the last floating ribs (total of 4 vertebrae). The

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METHODS All animal studies were approved by the Monash Institute of Pharmaceutical Sciences Animal Ethics Committee. Preparation of Dosing Formulations for Pharmacokinetic Studies. Cinnarizine was added to molten lipid formulation such that the 300 mg dose of lipid equated to a dose of drug equivalent to 2.1 mg/kg to each rat. The mixture was equilibrated overnight at 37 °C prior to dosing to ensure that the cinnarizine was fully dissolved in the lipid. Both PHY and TB are low in toxicity (LD50 of 5000 mg/kg and 3200 mg/ kg in rats, respectively), therefore the dose was well below toxicity levels. Tributyrin is already used as a food grade flavoring agent. Surgical Procedures for Pharmacokinetic Studies. Male Sprague−Dawley rats (280−320 g) separate from those of the imaging cohort were used for pharmacokinetic studies. Rats were induced under anesthesia via inhalation of isoflurane (5% v/v, maintained at 2.5% v/v for the duration of the surgery). The right carotid artery was isolated and cannulated with a 0.96 × 0.58 mm (o.d. × i.d.) polyethylene tubing to allow serial blood sampling. The rats were then placed in a tether swivel system in individual wire bottom metabolic cages to recover overnight prior to dosing and blood sampling. The rats were then fasted for at least 12 h prior to dosing and 8 h after dosing, with free access to water. The lipid formulations were then dosed via oral gavage (300 mg of lipid), with the dose accurately calculated by weighing the gavage and syringe before and after dosing. Serial blood sampling was performed at 0.5, 1, 2, 3, 4, 6, 8, 10, 24, and 30 h with 250 μL drawn at each time point. To allow patency, cannulas were flushed with 10 IU/mL sodium heparin saline solution after each blood sampling. Blood samples were dispensed in 1.5 mL microcentrifuge tubes containing 10 IU of sodium heparin, which were then centrifuged for 5 min at 6700g. Aliquots of plasma (50 μL) were collected in duplicate and frozen at −20 °C until plasma analysis for CZ concentration. Pharmacokinetic studies with each formulation were performed in triplicate. Analysis of CZ Content in Plasma. Plasma samples were analyzed on a single quadrupole LCMS using the method described by Sahbaz et al.28 Briefly, CZ standards with the range of 10 ng/mL to 2000 ng/mL were prepared by spiking each standard into 50 μL of blank rat plasma in 5 μL of acetonitrile. Halofantrine was used as an internal standard with 5 μg/mL in acetonitrile spiked in 50 μL plasma samples and standards. The spiked samples and standards were then vortexed for 1 min, followed by protein precipitation with saturated ammonium sulfate solution (25 μL), which was then vortexed for 30 s. Acetonitrile (90 μL) was then added, and the samples were vortexed for 1 min before being left at room temperature for 20 min. The samples were then centrifuged at 9600g for 5 min at room temperature before 25 μL aliquots of the supernatant were analyzed. C

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PHY+TB formulations after 10 h. For two rats dosed with the TB formulation, the plasma concentrations fell below limit of quantitation (LOQ) at 30 h. All three formulations showed an improvement in relative bioavailability when compared to CZ suspension formulations (suspension data obtained from Nguyen et al.).19 There were no significant differences between the AUCs for the three lipid formulations (T = 0 to Tlast) despite having very different profiles, however, if the studies were able to be extended beyond 30 h, the PHY and PHY+TB formulations would be expected to get close to or achieve significantly greater AUC compared to TB. Gastric Retention of Lipid Formulations Containing GNP Using Micro-X-ray CT Imaging. In order to assess the gastric retention of each formulation, rats were imaged following oral administration of lipid formulations (300 mg) containing 5% GNP w/w. The imaging process was repeated at 1, 2, 4, and 8 h, and the images were then analyzed and qualitatively processed using Imaris software as shown in Figure 3. The gastric retention of each formulation was quantitatively analyzed using Imaris, with the sum of intensity remaining over time presented in Figure 4. The contrast from the PHY formulation was approximately constant over the 8 h period, while the contrast from the PHY+TB decreased a little over time, with approximately 50% of the contrast still apparent from 1 to 4 h, and 30% of contrast still evident at 8 h after administration. In comparison, the TB formulation was almost completely emptied by the 4 h time point.

approximate spatial location of the stomach was predetermined via dissection of rats with similar weight ranges, separate from the cohort used for the imaging studies.

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RESULTS Pharmacokinetic Results. After the oral administration of CZ in 300 mg of the PHY, PHY+TB, and TB formulations, the mean dose normalized plasma concentrations were obtained over 30 h as shown in Figure 2. The pharmacokinetic

Figure 2. Dose normalized CZ concentrations in plasma after orally administered CZ incorporated in PHY*, PHY+TB, and TB over 30 h. CZ was dissolved in PHY, PHY+TB, or TB and dosed as a lipid solution. The concentration of CZ in each lipid formulation enabled administration at a dose of cinnarizine at 2.1 mg/kg, in a mass dose of 300 mg of lipid. Data are mean ± SEM, n = 3. *Data for PHY reproduced from Nguyen et al.,19 truncated at 31 h.

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DISCUSSION The transformation of a precursor lipid formulation from a stable emulsion to liquid crystalline nanoparticle system has been established in vitro.25,26 While it is not yet possible to directly confirm the formation of liquid crystalline structures in vivo, in this study, the pharmacokinetic profile of cinnarizine administered in the precursor PHYT+TB formulation provided a surrogate indicator, where the previous link to liquid crystal formation and gastric retention had already been established. Previous studies have demonstrated that PHY alone will provide a sustained absorption effect for coformulated cinnarizine as a result of cubic phase formation in situ, so the pharmacokinetic profile was considered as an indicator for in situ formation of liquid crystalline structures. The sustained release profile provides a reduced Cmax, avoiding acute toxicity that may occur in the case of very rapid and extensive absorption,31 and may also provide an overall greater extent of exposure by maintaining elevated plasma levels for much longer than a “typical” lipid formulation, where rapid elimination results in low plasma concentrations and reduced area under the curve. The administration of CZ in the PHY+TB “cubic phase precursor” formulation clearly yielded a profile that closely

parameters (Cmax, Tmax, AUC, and bioavailability relative to a suspension formulation, F%) derived from the pharmacokinetic profiles are presented in Table 1 (data for the suspension were obtained from Nguyen et al.).19 The pharmacokinetic profiles for the PHY and PHY+TB formulations were very similar and almost superimposable with steady absorption occurring over an extended period of time and a Cmax of 55.1 ± 11.4 ng/mL and 59.7 ± 31.9 ng/mL, respectively. There was a difference in the calculated Tmax (23.8 ± 6.0 h and 6 ± 2.3 h respectively), however the atypical flat shape of the profiles meant that this difference is not significant because the Cmax are very similar and the profiles at the respective nominal Tmax are also similar. The shape of both the PHY and PHY+TB profiles did not appear to be influenced by the reintroduction of food at 8 h after administration. In the case of the TB formulation a more typical pharmacokinetic profile was obtained, with a peak in plasma concentration at Tmax 2.3 ± 0.5 h, followed by a rapid decrease in plasma concentration falling below those of the PHY and

Table 1. Mean Pharmacokinetic Parameters Following Oral Administration of CZ Incorporated in PHY, PHY+TB, and TB Formulations formulation

mean Cmax (ng/mL)

mean Tmax (h)

AUC0−last sample (ng/mL·h)

F%a,b

PHYb PHY/TB TBc

55.1 ± 11.4 59.7 ± 31.9 165.8 ± 59.4

23.8 ± 6.0 6 ± 2.3 2.3 ± 0.5

1248.0 ± 224.8 918.8 ± 79.2 919.3 ± 145.0

398.1 293.1 293.0

a

F% is the bioavailability relative to an equivalent aqueous suspension of cinnarizine reported previously. bPHY and suspension data (used to calculate relative bioavailability F%) were obtained from Nguyen et al.19 truncated at 31 h. cTB data was obtained from Pham et al.29 D

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Figure 3. Micro-X-ray CT images of the stomach region of rats administered PHY, PHY+TB, and TB formulations containing GNR. The green contrast represents the contrast from GNR entrained in lipid formulations. Data for PHY and TB formulations were previously reported by Pham et al.29

was less apparent than for PHY alone, and the concentrations in plasma were slightly higher for PHY+TB than for PHY, but were clearly much more similar to PHY than to TB. It is not clear how much TB would be required in the PHY+TB formulation in order for the gastric retention to be compromised; the 15% TB in the formulation in the current study is at the limit of TB content to enable disruption of the cubic phase in the precursor formulation on exposure to aqueous environments, and only a small amount of digestion is required to initiate formation of the cubic phase, triggering the gastric retention effect not apparent for non-liquid crystalline systems. Increasing the proportion of tributyrin in the formulation would require more digestion to take place before formation of the cubic phase is possible, but without a more systematic study of the TB content, it is not possible to anticipate the content where the effect is compromised. Gastric lipase has been shown to be more active against shorter chain triglycerides and may contribute up to 30% of digestion of longer chain lipids, meaning that it is reasonable to expect gastric digestion of tributyrin to be significant.33 In this study, the formulations were administered as bulk lipids, rather than as dispersions. This was considered to be an appropriate starting point for examining the in situ formation of liquid crystalline structures by these systems, as the dispersions introduce a variable in particle size and require the addition of a further colloidal stabilizer. On the other hand, for PHY alone, the behavior of the bulk lipid has been demonstrated to be similar to that of the predispersed cubosomes, indicating that gastric emulsification may occur anyway for the bulk lipid, meaning the predispersion step may not be critical to dictating the in vivo performance. However, in the context of a future product, while the bulk lipid may be suitable for a lipid-filled capsule, the precursor emulsion dispersion presents options for oral liquid formulations and solid lipid-containing dose forms without the colloidal stability issues of cubosomes. Future studies will also examine the propensity for the predispersed precursor emulsions to provide the gastric retention and consequent extended absorption of poorly water-soluble drugs. Applicability of the effect in higher species is also required to be evaluated to ensure translatability into human or veterinary products.

Figure 4. Mean values for sum of intensity in the gastric region remaining over time from X-ray CT imaging for PHY, PHY+TB, and TB formulations. The PHY system exhibited the greatest retention of contrast over time, followed by PHY+TB systems, with rapid emptying of the TB formulation being apparent. Data are mean ± SEM, n = 3.

resembled that of PHY alone, even though it is known that the addition of 15% TB inhibits cubic phase formation, under which conditions it is expected that the pharmacokinetic profile of cinnarizine would resemble that after administration in unstructured digestible or nondigestible lipid systems, i.e., TB, oleic acid,32 and GMO.19,20 In that case a “typical” profile would be expected as illustrated for the TB formulation in Figure 2 in this study. Neither the triglyceride nor the short chain fatty acids generated on digestion of TB form liquid crystalline structures, so this behavior was entirely expected. When combined with TB, in vitro digestion studies indicate that the formation of cubic phase is facilitated by breakdown to butyric acid, which does not disrupt the liquid crystalline structure formation of the residual nondigestible phytantriol. As has been previously seen in studies on oral delivery using lipidic liquid crystalline systems, the gastric retention of the formulation is a strong determinant in the duration of the sustained absorption effect for cinnarizine. The stomach appears to act as a nonsink environment, where release of drug from the formulation is understood to be dictated primarily by partitioning and hence the concentration of drug “free” in gastric fluids. For cinnarizine, this is expected to be low, and consequently the pattern of drug absorption is expected to be primarily dictated by the rate of gastric emptying. The PHY and PHY+TB formulations were emptied slowly resulting in low, but extended, plasma concentrations, while TB was rapidly emptied resulting in high but not sustained plasma concentrations. The retention of PHY+TB

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CONCLUSION In summary, the PHY+TB formulation was comparable to PHY-only in terms of sustained drug absorption for the model poorly water-soluble drug cinnarizine. Inclusion of gold nanoparticles in the formulation provided a convenient means of assessing the disposition of the formulation using E

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Delivery Systems: I. In Vitro Evaluation and Enhanced Oral Bioavailability of the Poorly Water-Soluble Drug Simvastatin. AAPS PharmSciTech 2009, 10 (3), 960−966. (7) Negrini, R.; Mezzenga, R. pH-responsive lyotropic liquid crystals for controlled drug delivery. Langmuir 2011, 27 (9), 5296−5303. (8) Burrows, R.; Collett, J. H.; Attwood, D. The release of drugs from monoglyceride-water liquid crystalline phases. Int. J. Pharm. 1994, 111 (3), 283−293. (9) Landh, T. Phase Behavior in the System Pine Oil Monoglycerides-Poloxamer 407-Water at 20C. J. Phys. Chem. 1994, 98, 8453−8467. (10) Barauskas, J.; Landh, T. Phase Behavior of the Phytantriol/ Water System. Langmuir 2003, 19 (17), 9562−9565. (11) Lindström, M.; Ljusberg-Wahren, H.; Larsson, K.; Borgström, B. Aqueous lipid phases of relevance to intestinal fat digestion and absorption. Lipids 1981, 16 (10), 749−754. (12) Ljusberg-Wahern, H.; Nyberg, L.; Larsson, K. Dispersion of the cubic liquid crystalline phase − structure, preparation, and functionality aspects. Chim. Oggi 1996, 14, 40−43. (13) Siekmann, B.; Bunjes, H.; Koch, M. H. J.; Westesen, K. Preparation and structural investigations of colloidal dispersions prepared from cubic monoglyceride − water phases. Int. J. Pharm. 2002, 244 (1), 33−43. (14) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Cubic Lipid-Water Phase Dispersed into Submicron Particles. Langmuir 1996, 12 (20), 4611−4613. (15) Patton, J. S.; Carey, M. C. Watching Fat Digestion. Science 1979, 204 (4389), 145−148. (16) Salonen, A.; Moitzi, C.; Salentinig, S.; Glatter, O. Material Transfer in Cubosome−Emulsion Mixtures: Effect of Alkane Chain Length. Langmuir 2010, 26 (13), 10670−10676. (17) Barauskas, J.; Anderberg, H.; Svendsen, A.; Nylander, T. Thermomyces lanuginosus lipase-catalyzed hydrolysis of the lipid cubic liquid crystalline nanoparticles. Colloids Surf., B 2015, DOI: 10.1016/j.colsurfb.2015.04.052. (18) Salentinig, S.; Phan, S.; Khan, J.; Hawley, A.; Boyd, B. J. Formation of highly organized nanostructures during the digestion of milk. ACS Nano 2013, 7 (12), 10904−10911. (19) Nguyen, T.-H.; Hanley, T.; Porter, C. J. H.; Larson, I.; Boyd, B. J. Phytantriol and glyceryl monooleate cubic liquid crystalline phases as sustained-release oral drug delivery systems for poorly water-soluble drugs II. In-vivo evaluation. J. Pharm. Pharmacol. 2010, 62 (7), 856− 865. (20) Nguyen, T. H.; Hanley, T.; Porter, C. J. H.; Boyd, B. J. Nanostructured Liquid Crystalline Particles Provide Long Duration Sustained-Release Effect for a Poorly Water Soluble Drug After Oral Administration. J. Controlled Release 2011, 153 (2), 180−186. (21) Nguyen, T. H.; Hanley, T.; Porter, C. J. H.; Boyd, B. J. Nanostructured reverse hexagonal liquid crystals sustain plasma concentrations for a poorly water-soluble drug after oral administration. Drug Delivery Transl. Res. 2011, 1 (6), 429−438. (22) Seddon, J. M.; Robins, J.; Gulik-Krzywicki, T.; Delacroix, H. Inverse micellar phases of phospholipids and glycolipids. Phys. Chem. Chem. Phys. 2000, 2 (20), 4485−4493. (23) Shah, J. C.; Sadhale, Y.; Chilukuri, D. M. Cubic Phase Gels as Drug Delivery Systems. Adv. Drug Delivery Rev. 2001, 47 (2−3), 229− 250. (24) Lakshmi, N. M.; Yalavarthi, P. R.; Vadlamudi, H. C.; Thanniru, J.; Yaga, G.; K, H. Cubosomes as Targeted Drug Delivery Systems - A Biopharmaceutical Approach. Curr. Drug Discovery Technol. 2014, 11, 181−188. (25) Fong, W. K.; Salentinig, S.; Prestidge, C.; Mezzenga, R.; Hawley, A.; Boyd, B. J. Generation of Geometrically Ordered Lipid-Based Liquid-Crystalline Nanoparticles Using Biologically Relevant Enzymatic Processing. Langmuir 2014, 30 (19), 5373−5377. (26) Hong, L.; Salentinig, S.; Hawley, A.; Boyd, B. J. Understanding the Mechanism of Enzyme-Induced Formation of Lyotropic Liquid Crystalline Nanoparticles. Langmuir 2015, 31 (24), 6933−6941.

imaging. X-ray CT showed that although the PHY+TB formulation did not appear to be as extensively retained as the PHY-only formulation, it was more extensively retained than the TB-only formulation. The addition of TB as a digestible additive provided the means to form a stable precursor and could be degraded in vivo to trigger the formation of the liquid crystalline phase that demonstrated the sustained release. By establishing the applicability of this enzymatic approach to generating liquid crystals in vivo, it may now be possible for the commercialization of liquid crystal drug delivery systems, albeit in the form of a precursor emulsion.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.5b00784. Statistical analyses of the pharmacokinetic data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +61399039112. Fax: +61399039583. E-mail: ben. [email protected]. Author Contributions ‡

A.C.P. and L.H. contributed equally to this paper as first authors. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Funding is acknowledged from the Australian Research Council under the Discovery Projects Scheme DP120104032. B.J.B. is a recipient of an Australian Research Council Future Fellowship (FT120100697).

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ABBREVIATIONS USED CZ, cinnarizine; GMO, glyceryl monooleate; GNP, gold nanoparticles; TB, tributyrin; PHY, phytantriol; PHY+TB, 85% phytantriol +15% tributyrin

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REFERENCES

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DOI: 10.1021/acs.molpharmaceut.5b00784 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.molpharmaceut.5b00784 Mol. Pharmaceutics XXXX, XXX, XXX−XXX