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A Comparison across Three Hybrid Lipid-Based Drug Delivery Systems for Improving the Oral Absorption of the Poorly Water-Soluble Weak Base Cinnarizine Paul Joyce, Rokhsana Yasmin, Achal Bhatt, Ben J. Boyd, Anna Pham, and Clive A. Prestidge Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00676 • Publication Date (Web): 26 Sep 2017 Downloaded from http://pubs.acs.org on September 27, 2017

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Molecular Pharmaceutics

A Comparison across Three Hybrid Lipid-Based Drug Delivery Systems for Improving the Oral Absorption of the Poorly Water-Soluble Weak Base Cinnarizine Paul Joyce,a,b Rokhsana Yasmin,a,b Achal Bhatt,a,b Ben J. Boyd,c,d Anna Phamc and Clive A. Prestidgea,b* a

School of Pharmacy and Medical Sciences, University of South Australia, City East Campus, Adelaide, South Australia 5000, Australia b

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, University of South Australia, Mawson Lakes Campus, Mawson Lakes 5095, Australia

c

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, Parkville, Victoria 3052, Australia

d

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville Campus, Parkville, Victoria 3052, Australia

*Corresponding Author. Telephone: +61 8 8302 3569. Fax: +61 8 8302 3683. Email: [email protected]

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Abstract

Three state-of-the-art drug delivery vehicles engineered by nanostructuring lipid colloids within solid particle matrices were fabricated for the oral delivery of the poorly watersoluble, weak base, cinnarizine (CIN). The lipid and solid phase of each formulation was varied to systematically analyse the impact of key material characteristics, such as nanostructure and surface chemistry, on the in vitro and in vivo fate of CIN. The three systems formulated were: silica-stabilised lipid cubosomes (SSLC), silica-solid lipid hybrid (SSLH) and polymer-lipid hybrid (PLH) particles. Significant biopharmaceutical advantages were presented for CIN when solubilised in the polymer (poly(lactic-co-glycolic) acid; PLGA) and lipid phase of PLH particles compared to the lipid phases of SSLC and SSLH particles. In vitro dissolution in simulated intestinal conditions highlighted reduced precipitation of CIN when administered within PLH particles, given by a 4-5 fold improvement in the extent of CIN dissolution compared to the other delivery vehicles. Furthermore, CIN solubilisation was enhanced 1.5-fold and 6-fold under simulated fasted state lipid digestion conditions when formulated with PLH particles compared to SSLH and SSLC particles, respectively. In vivo pharmacokinetics correlated well with in vitro solubilisation data, whereby oral CIN bioavailability in rats, when encapsulated in the corresponding formulations, increased from SSLC ˂ SSLH ˂ PLH. The pharmacokinetic data obtained throughout this study indicated a synergistic effect between PLGA nanoparticles and lipid droplets in preventing CIN precipitation and thus, enhancing oral absorption. This synergy can be harnessed to efficiently deliver challenging poorly water-soluble, weak bases through oral administration.

Keywords: lipid-based formulations, particle-lipid conjugates, poorly water-soluble drugs, weak bases, cinnarizine, pharmacokinetics. 2 ACS Paragon Plus Environment

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Introduction The oral bioavailability of many poorly water-soluble weak bases, such as cinnarizine (CIN), is limited due to their pH-dependent aqueous solubility and subsequent precipitation in the gastrointestinal tract (GIT).1,

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That is, a > 25-fold discrepancy exists between the

solubility of these compounds in the acidic gastric environment and in the neutral conditions of the small intestine, when under fasted state conditions.3 This leads to supersaturation and precipitation under fasted state gastric emptying, which drastically restricts oral absorption.4 Consequently, several formulation strategies have been employed to improve the equilibrium solubility of weak bases in the GIT and optimise dissolution kinetics for improved oral absorption. Lipid-based drug delivery vehicles have been identified as one of the most promising approaches due to their ability to improve the oral bioavailability of a range of poorly water-soluble drugs.5 These vehicles include self-emulsifying lipid systems, solid lipid nanoparticles, liquid crystalline lipid dispersions, lipophilic ionic liquids and others. These formulations act by forming a solubilisation reservoir in the GIT, resulting from the digestion of the lipid excipients. Digestion products combine with endogenous bile salts and phospholipids to generate mixed micelles and other colloidal structures that maintain the lipophilic drug in a dissolved state until absorption.6 Lipid-based formulations also delay gastric emptying and prolong residence time of the drug within the GIT, which increases the time for absorption into the bloodstream.7 A novel solid-state lipid-based formulation with a demonstrated ability to improve the therapeutic potential of several poorly water-soluble compounds is that of silica-lipid hybrid (SLH) particles.8-10 SLH particles were engineered by spray drying emulsion droplets stabilised by porous silica particles to create a highly organised three-dimensional composite structure. This structure is fundamental to enhancing the performance of poorly water-soluble drugs, as it facilitates rapid and complete digestion of the lipid droplets,11, 12 which leads to 3 ACS Paragon Plus Environment


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optimal drug dissolution.13 Furthermore, small changes in carrier nanostructure can help finely tune the release and solubilisation of drugs encapsulated within the SLH particles.14 In vivo and clinical assessments have highlighted the ability for SLH particles to improve the oral bioavailiability through this mechanism for a wide range of challenging drugs, including celecoxib,15 ibuprofen,16 indomethacin17 and several others. However, recent studies have indicated that formulating poorly soluble weak bases with conventional SLH particles and other simple lipid formulations results in low efficacy and requires a high lipid:drug ratio to provide a satisfactory enhancement in oral bioavailability.18, 19

This is due to the drug existing in a metastable supersaturated state when dispersed in the

GIT. That is, the concentration of the drug when released into solution is above the thermodynamic equilibrium solubility and is subsequently in a higher energy state than the stable form of the drug. Thus, for lipid-based systems to successfully overcome equilibrium solubility limitations of weak bases, they must be combined with excipients that prolong supersaturation and slow the rate of drug precipitation. Polymeric precipitator inhibitors (PPIs), such as cellulose derivatives and poloxamers, have been shown to stabilize metastable supersaturated systems by preventing the recrystallization of lipophilic compounds.20 Rao et al.19 recently combined the synergistic effects of PPIs with lipids for the first time by synthesizing Pluronic-functionalised silica-lipid hybrid (Plu-SLH) particles. This formulation successfully outperformed conventional SLH particles and lipid suspensions during in vitro and in vivo assessments for the oral absorption of CIN. The presence of Pluronic molecules within the system facilitated an increase in the hydrophobicity of the microenvironment and thus reduced the risk of drug precipitation. Consequently, conventional SLH particles may act as a template, or a starting point, for the synthesis of novel particle-lipid hybrid composites, whereby modifications to the structure and composition of these hybrid particles can trigger

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improved delivery performance of poorly water-soluble weak bases, as evidenced by PluSLH particles. In this study, three hybrid lipid-based delivery vehicles, engineered by combining colloidal particles and lipid droplets, were designed for encapsulation and improved oral delivery of CIN. The formulation compositions (structure and excipient types) enabled us to determine their ability to inhibit the recrystallization of lipophilic weak bases in the intestinal environment and enhance oral bioavailability. Rationale for the synthesis and analysis of the three CIN formulations used within this study is provided below: (i)

polymer-lipid hybrid (PLH) particles: the solubilisation capacity of liquid mediumchain triglycerides18 (MCT) was combined with the precipitation inhibition effect of polymeric nanoparticles21 (N.B. PLH have not previously been reported for oral delivery);

(ii)

silica-solid lipid hybrid (SSLH) particles: the sustained release mechanism of saturated long-chain length glyceride droplets22 was combined with the stabilising and solubilisation capacity of hydrophilic porous silica particles;23 and,

(iii)

silica-stabilised lipid cubosomes (SSLC): the nanostructured, liquid crystalline properties of mono-unsaturated glycerides,24 which induce the controlled release mechanism of encapsulated drug molecules,25 were combined with the stabilising effect of non-porous silica particles.26

That is, changes in nanostructure and surface chemistry were introduced to the hybrid carrier materials by altering (i) the lipid and solid phase component in each system, and (ii) the colloidal self-assembly fabrication technique used. In each system, the selection and design of conjugated agglomerates was based on previous studies that have indicated the solubilisation potential of each individual component. The aim of this study, was therefore, to systematically study the influence of varying solid-state lipid-based formulation parameters 5 ACS Paragon Plus Environment


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on the oral absorption of CIN. Subsequently, comparisons between the efficacy of the three nanostructured materials revealed novel insights into the solubilisation and absorption mechanism of poorly water-soluble weak bases, which can be used to develop next generation lipid-based formulations with improved oral drug delivery performance. Experimental Section Materials Cinnarizine (CIN), poly(lactic-co-glycolic) acid (PLGA; lactide:glycolide 50:50 MW = 30,000-60,000 Da), poly(vinyl alcohol) (PVA; MW = 30,000-70,000 Da), non-porous hydrophilic silica particles (Ludox SM) with an average particle size of ~7 nm, mannitol, sodium taurodeoxycholate (NaTDC) 99%, egg lecithin, porcine pancreatin extract, 4bromophenylboronic acid (4-BBA), and phosphate buffered saline (PBS) tablets were purchased from Sigma-Aldrich (Australia). Fumed hydrophilic silica (Aerosil 380) existing of primary particles with an average diameter of 7 ± 1 nm that randomly cluster to form aggregates with an estimated pore size of 2-7 nm27 and a specific surface area of 380 ± 30 m2/g were supplied by Evonik Degussa (Germany). Medium-chain triglyceride (MCT; Miglyol 812) was obtained from Hamilton Laboratories (Adelaide, Australia) and soybean lecithin (containing >94% phosphatidycholine and ˂2% triglycerides) was obtained from BDH Merck (Sydney, Australia). Glyceryl monostearate (GMS; Geleol Mono and diglycerides NF) and glyceryl monooleate (GMO) were kindly donated by Gattefosse (Sydney, Australia). All other chemicals were of analytical grade and used as received. High purity (Milli-Q) water was used throughout the study. Synthesis of Hybrid Lipid-Based Delivery Systems Preparation of PLGA-Lipid Hybrid (PLH) Microparticles PLH microparticles were prepared following a two-step process as described previously (i.e. homogenization followed by spray drying of a PLGA nanoparticle-stabilised 6 ACS Paragon Plus Environment

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emulsion).28 Firstly, PLGA nanoparticles, with a particle size of 169 ± 7 nm and a zeta potential of -20.1 ± 2.9 mV, were synthesized via a modified emulsion-diffusion-evaporation method using ethyl acetate as an organic solvent.29 PLGA (500 mg) and CIN (25 mg) were dissolved in ethyl acetate (25 mL) to form the organic phase, which was added dropwise to the aqueous phase containing 0.5% w/v PVA (250 mg in 50 mL). The resulting primary emulsion was stirred at 1000 rpm for 3 h and subsequently homogenised at 1000 bar for 5 min using a high-pressure homogenizer (Avestin EmulsiFlex-C5 Homogenizer, Canada). Water was added with constant stirring to this nanoemulsion to facilitate diffusion and evaporation of ethyl acetate, leading to the nanoprecipitation of PLGA nanoparticles. CIN was also added to the lipid phase of PLH microparticles by dissolving CIN in MCT (Miglyol 812) at its solubility. An oil-in-water emulsion was then prepared by dissolving 0.6% w/w lecithin in 10% w/w drug-MCT and Milli-Q water was added as the continuous phase. The coarse o/w droplets were homogenized under a pressure of 1000 bar for five cycles. PLGA nanoparticles were added to this submicron emulsion at a concentration of 60% w/w (relative to lipid content). A 1% w/v mannitol solution (50 mL) was also added to the PLGA nanoparticle and submicron emulsion mixture as a cryoprotectant. The PLGAnanoparticle stabilized emulsion was then spray dried (Mini Spray Dryer B-290, BÜCHI Labortechnik AG, Switzerland) to form PLH microparticles under the following conditions: emulsion flow rate, 0.5 mL/min; air flow rate, 0.6 m3/min; inlet temperature, 60°C; outlet temperature, 35°C; aspirator setting, 10. Preparation of Silica-Solid Lipid Hybrid (SSLH) Microparticles SSLH microparticles were prepared using a hot homogenization followed by ultrasonication method, described previously.30 Firstly, solid lipid (GMS; 500 mg) was melted at 70°C and CIN (50 mg) was added at its solubility. The drug-solid lipid mixture was stirred at 70°C until a clear solution formed. A 5% w/v porous silica dispersion was added to the hot 7 ACS Paragon Plus Environment


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drug-solid lipid mixture to form a 50:50 silica:solid lipid mixture, which was homogenized at 10,000 rpm for 30 min using a high-speed homogenizer (MICCRA D-1, Germany). The homogenized dispersion was then subjected to an ultrasonic probe (QSonica, USA) fitted with a 3 mm probe using a pulsing mode (3 s on and 2 s off) and 60% power (max 125 watts) for 20 min. The final dispersion was then cooled using an ice water bath and centrifuged at 20,000 rpm for 1 h; the solid mass was collected and placed in a desiccator for 48 h. Preparation of Silica-Stabilised Lipid Cubosomes (SSLC) SSLC were prepared following a method previously described by Bhatt et al.26 CIN was dissolved in molten GMO at its 100% equilibrium solubility by heating the mixture to 80°C, which was then added to 40% w/w Milli-Q water to form a primary cubic phase (based on the binary diagram of GMO-water).31 The mixture was vortexed vigorously for 5 min, followed by heating at 80°C and further vortex for 5 min. The mixture was then allowed to equilibrate for 48 h. The bulk cubic phase was then combined with a 5% w/v non-porous silica dispersion to facilitate a 80:20 lipid:silica ratio. The resultant mixture was dispersed using an ultrasonic probe at pulsing mode (3s on and 3s off) and 40% power for 20 min. SSLC were analysed in their dispersed state. Physicochemical Characterisation of Hybrid Systems Composition The lipid loading content of the three lipid hybrid formulations was determined by thermogravimetric analysis (TGA). The particles were heated at a scanning rate of 10°C/min from 20-600°C under nitrogen purging. The lipid completely decomposed by 500°C. The silica component of SSLH microparticles and SSLC remained thermally stable up to 600°C. For PLH microparticles, the weight loss corresponding to the PLGA component was measured by initially heating spray dried PLGA nanoparticles (in the absence of lipid) within this temperature range, as described previously.28 The weight loss, after correction for drug 8 ACS Paragon Plus Environment

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and water content, was computed using the associated TA Universal Analysis software, which corresponded to the lipid content of the microparticles. The amount of CIN loaded into the microparticles was determined by a solvent extraction method. The encapsulated CIN was extracted by dissolving 10 mg of the formulation in 10 mL acetonitrile, followed by sonication for 15 min and centrifugation at 20,000 rpm for 10 min (Hermle high speed table top centrifuge Z36HK, Germany). The supernatant was diluted with HPLC mobile phase prior to HPLC analysis for CIN content. Solid-state characterisation The particle size and surface morphology of PLH and SSLH particles was analysed using high resolution analytical scanning electron microscopy (SEM, Zeiss, Merlin). Each sample was mounted on double-faced adhesive tape prior to imaging at an accelerating voltage between 1-3 kV. The cubic phase structure of the liquid crystalline lipid dispersion, along with the particle morphology, association and orientation of silica nanoparticles in SSLC were studied using cryogenic transmission electron microscopy (cryo-TEM). SSLC, frozen in liquid nitrogen, were imaged using a Tecnai F30 microscope (FEI, USA) fitted with a 4K Direct Electron LC1100 camera. Briefly, a drop of sample dispersion was placed on the copper EM-grid coated with perforated carbon film and the excess of sample was blotted using the filter paper to form a thin film of ~ 50 nm on the grid. The sample was then vitrified by plunging the grid into liquid ethane held just above its freezing point and stored in liquid nitrogen. The entire sample preparation procedure was performed in the chamber with controlled temperature of 22 ˚C and humidity. Finally, the vitrified samples were examined by the TEM in the cryogenic mode operating at 300 keV/100 K and a working temperature of -180 ˚C.

The degree of crystallinity of encapsulated CIN was monitored by differential scanning calorimetry (DSC Q100, TA Instruments). Approximately 5 mg of each sample was heated at a rate of 10°C/min from 25-150°C, under nitrogen purging (80 mL/min). 9 ACS Paragon Plus Environment


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In Vitro Drug Dissolution Studies CIN solubilisation under non-digesting conditions In vitro dissolution studies were performed in non-digesting conditions using a USP 23 type II apparatus (paddle method, 50 rpm). CIN formulations (equivalent to ~ 10 mg CIN) were dispersed in simulated intestinal fluid (0.1 M PBS, 37°C, pH 7.50). Aliquots (5 mL) were taken at fixed time intervals over the 60 min dissolution period and replaced accordingly with fresh medium. The samples were centrifuged 22,000 rpm for 5 min (37°C) and the supernatants were diluted with the mobile phase and analysed by HPLC for CIN content. Dissolution efficiencies (%DE) were determined from solubilisation profiles by expressing the area under the dissolution curve as a percentage of the area under the rectangle described by 100% dissolution. %DE was calculated using Equation 1,32 below: % =

× 100

(1)

where, y is the percentage of drug dissolved at time t. CIN solubilisation under digesting conditions Solubilisation of CIN under digestive conditions was examined in simulated fasted-state intestinal media in the presence of the digestive enzyme, pancreatic lipase. A known amount of CIN formulation (equivalent to ~ 200 mg triglycerides) was dispersed in 18 mL of buffered fasted state micellar solution (37°C, pH 7.50), as described previously.33 Lipid hydrolysis was initiated by the addition of 2 mL pancreatin extract into the digestion medium. Lipid digestion was monitored over a 60 min period using a pH-stat titration unit (TIM854 Titration Manager, Radiometer, Copenhagen, Denmark) which immediately titrated free fatty acids with 0.6 M NaOH via an autoburette. Aliquots (1 mL) were collected periodically throughout the digestion period and digestive enzymes were inhibited by the addition of 10 µL 4-BBA (0.5 M in methanol solution). Samples were centrifuged at 22,000 rpm for 1 h 10 ACS Paragon Plus Environment

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(37°C). The aqueous phase was collected and diluted suitably with mobile phase solution and analysed by HPLC for CIN content. CIN content within the pellet phase was first extracted with ACN and then diluted with mobile phase. In Vivo Oral Administration of CIN Formulations All animal experiments were approved by the Monash Institute of Pharmaceutical Sciences Animal Ethics Committee (Australia). Twelve male Sprague-Dawley (SD) rats (270-310 g) were divided into three groups and used for each absorption study. Prior to the study, rats were cannulated with 0.96 x 0.58 mm polyethylene cannula (Microtube Extrusion, Australia) under light anaesthesia (2% v/v isoflurane) to facilitate blood collection. Rats were allowed to recover overnight prior to dosing and fasted up to 12 h prior to and 8 h post administration with water provided ad libitum. The rats were administered one of three formulations (equivalent to 10 mg/kg CIN) via oral gavage (after being lightly anaesthetised): (i) PLH, (ii) SSLH, and (iii) SSLC. Each formulation was redispersed in Milli-Q water (equivalent to 10 mg of CIN/mL) immediately prior to dose administration. After each dose administration, blood samples (0.2 mL) were collected up to 24 h with the cannula flushed with 10 IU/mL heparin in normal saline between samples. Blood samples were centrifuged for 5 min at 10,000 rpm to facilitate the collection of plasma. Plasma samples were stored at -20°C until analysis. Results and Discussion Development and characterisation of hybrid CIN formulations Preparation and physicochemical characterisation of PLH, SSLH and SSLC particles Three novel lipid-hybrid delivery systems were developed with varying three-dimensional nanostructures and surface chemistries, which resulted from significantly different colloidal assembly fabrication techniques and starting materials. Firstly, PLH particles were synthesised by spray drying a liquid lipid emulsion partially stabilised by PLGA 11 ACS Paragon Plus Environment


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nanoparticles and mannitol, to form a free-flowing powder consisting of spherical particles 26 µm in size (Table 1). The morphology of PLH particles was smooth, with a highly porous structure where submicron MCT droplets were encapsulated within a PLGA nanoparticle matrix formed through the aggregation of particles and droplets during the water removal process (Figure 1).28

Figure 1: Schematic representation of the fabrication processes for PLH and SSLH particles, and SSLC. SEM images for PLH and SSLH particles, and cryo-TEM images for SSLC illustrate the aggregation behaviour and surface morphologies of each lipid-hybrid system. Table 1: Physicochemical properties of CIN loaded formulations. Formulation

Lipid excipient

Solid particle excipient

Final physical state

Average particle size


CIN loading (% w/w)

Relative lipid concentrationa (% w/w)

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PLH

SSLH SSLC a

Miglyol MCT (liquid lipid) GMS (solid lipid) GMO (solid lipid)

PLGA NPs

Solid

2 – 6 µm

1.25 ± 0.2

25.8 ± 3.2

Porous silica particles Solid silica particles

Solid

30 – 70 µm 50 – 200 nm

2.80 ± 0.3

35.6 ± 1.1

2.16 ± 0.2

80.0 ± 2.5

Liquid

Lipid concentration relative to solid particle excipient.

SSLH particles were engineered through the self-assembly of porous silica nanoparticles and solid lipid droplets.30 Specifically, the ultrasonication of a solid lipid emulsion, partially stabilised by porous silica particles, induced the forced agglomeration of the colloidal particles and droplets, forming relatively large, non-spherical hybrid particles with an average particle size of 30-70 µm (Figure 1). The initial solid lipid droplets were 200-400 nm in size. Subsequently, extensive agglomeration of droplets and particles occurred due to an interfacial interaction between hydrophilic silica particles and submicron lipid droplets,8, 34, 35 which led to submicron lipid droplets being confined within a three-dimensional porous silica network. In contrast, SSLC were administered as a liquid crystalline lipid dispersion, where solid lipid cubic particles approximately 50 nm in size were partially stabilised by non-porous silica nanoparticles (Figure 1). The silica nanoparticles (average diameter = 7 nm) randomly adsorbed and aggregated at the lipid-in-water interface. Previous studies characterised the bulk cubic (Pn3m) phase structure using synchrotron small angle X-ray scattering (sSAXS).36 Cryo-TEM micrographs highlighted a distinctive cross pattern within the cubosomes, which is characteristic of bicontinuous cubic phases.37 Hence, the presence of silica particles did not influence the internal structure of the cubosomes. CIN and lipid loading varied considerably between the three formulations, which can be attributed to the differences in drug solubility capacity within the various excipients, the relative lipid concentration and the hybrid microstructure. Figure 2 illustrates the DSC profiles for the three CIN formulations and the pure drug. Pure CIN exhibited an endothermic peak at 122°C, which correlates to its melting point and confirms its crystalline nature.19 In 13 ACS Paragon Plus Environment


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contrast, endothermic peaks were not observed for CIN when encapsulated within PLH and SSLH, indicating that the CIN was solubilised within each system in an amorphous form. For the SSLC formulation, a minor endotherm is present indicating that the majority of CIN is solubilised and in its amorphous form, with an apparent small fraction of crystalline drug.

Figure 2: DSC profiles of the following cinnarizine (CIN) formulations: pure CIN (purple curve), polymer-lipid hybrid (black curve), silica-solid lipid hybrid (blue curve) and silicastabilised lipid cubosomes (red curve). The arrow, inset, indicates the minor endotherm peak for SSLC. Each test was conducted using approximately 5 mg of sample. In vitro CIN dissolution studies in simulated intestinal media CIN is prone to intestinal precipitation due to its pH-dependent solubility and thus, leads to low dissolution in the GIT after oral administration.38 Subsequently, two in vitro models were used to analyse the precipitation behaviour of CIN in simulated intestinal environment when formulated with the lipid hybrid formulations: (i) dissolution during non-digesting conditions, and (ii) dissolution during fasted-state digesting conditions. By assessing the formulations in such conditions, it is possible to predict the impact of pH and lipolysis on drug precipitation. In vitro CIN dissolution studies under non-digesting conditions The rate and extent of dissolution of CIN in simulated non-digesting intestinal conditions (i.e. pH 7.5 in PBS solution) were enhanced for all lipid hybrid formulations compared to the pure drug (Figure 3). After 60 min dissolution, the degree of CIN solubilisation increased in 14 ACS Paragon Plus Environment

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the following order of formulations: pure CIN (0.12 ± 0.02 µg/mL) ˂ SSLH (0.89 ± 0.45 µg/mL) ˂ SSLC (1.11 ± 0.22 µg/mL) ˂ PLH (4.58 ± 0.58 µg/mL).

Figure 3: In vitro dissolution of cinnarizine (CIN) under non-digesting simulated intestinal conditions, i.e. pH 7.5 with phosphate buffered saline at 37°C (mean ± S.E.M., n = 3), for the following formulations: pure CIN (purple ×), PLH (black ■), SSLH (blue ●) and SSLC particles (red ). Both silica-lipid hybrid formulations exhibited statistically equivalent solubilisation profiles for the first 30 min of dissolution, highlighted by a 7-fold enhancement in CIN solubilisation compared to a saturated aqueous solution of the drug alone. In a recent dissolution study performed by Rao et al.,19 it was highlighted that silica-liquid lipid hybrid particles were unable to inhibit CIN precipitation in simulated intestinal conditions. However, when Pluronic F127 was incorporated to the delivery vehicle, CIN solubilisation was enhanced 8-fold. This was hypothesised due to the solubilisation capacity of Pluronic F127 on CIN, preventing drug precipitation at neutral pH. The solubilisation profiles for both solid lipid systems in this study were equivalent in non-digesting conditions, therefore we propose 15 ACS Paragon Plus Environment


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that the solid lipid matrix acts as a precipitation inhibitor for CIN by limiting the rapid, burst release of CIN and restricting the generation of supersaturation in the dissolution media;22, 30, 39

that is, the metastable state that causes drug to precipitate in its thermodynamically

favourable crystalline state.40, 41 CIN solubilisation was enhanced approximately 4.5-fold when administered with PLH particles, compared to the two silica-lipid delivery systems. PLH particles differed from SSLH and SSLC particles due to the incorporation of CIN within both the polymer and liquid lipid phases, which thereby enabled a multicomponent drug release approach. Both solid lipid nanoparticles and PLGA nanoparticles enable sustained release of poorly water-soluble drugs due to the matrix-dependent nature of release.42-44 Drug release is controlled by the rate of matrix degradation or the diffusion of encapsulated molecules from the dense polymeric matrix. In contrast, release from liquid lipid nanoemulsions is a diffusion-dependent process in non-digesting conditions, and is therefore commonly a fast process in the intestinal environment.45 While CIN dissolution was enhanced within PLH particles, the dual drug release mechanism was not observed from the dissolution curve (Figure 3), which was expected to consist of rapid initial release kinetics (from the lipid phase), followed by a period of sustained CIN release (from the polymer phase). The exact cause of this is unclear. The results indicate that a high portion of CIN ‘encapsulated’ within the PLGA nanoparticles may have been adsorbed to the external surface or loaded within the upper surface layers of the nanoparticles. Desorption of surface bound drug was therefore likely to be rapid and diffusion-dependent, as shown previously for drug release from PLGA nanoparticles.46 Additionally, several other polymeric systems, including cyclodextrins, Pluronics and cellulose derivatives, have exhibited the ability to prevent aqueous precipitation of poorly water-soluble drugs by stabilising supersaturation, and therefore act as suitable PPIs.20,

47

PPIs increase the hydrophobicity of the local microenvironment by which the drug is released 16 ACS Paragon Plus Environment

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and subsequently enhance the solubility of poorly soluble weak bases.19 Since PLGA nanoparticles are mostly hydrophobic, especially compared to hydrophilic silica particles, it is possible that the polymer phase prolonged CIN precipitation in the intestinal conditions. Consequently, it is hypothesised that the enhanced solubilisation of CIN when administered with PLH particles is due to one of, or a combination of the following mechanisms: (i) the multicomponent drug release from the liquid lipid phase and the dense polymeric matrix, and/or (ii) the precipitation inhibitory nature of PLGA nanoparticles and their degradation products (i.e. PLGA polymers and oligomers). For both SSLH and PLH particles, a fast initial rate of CIN dissolution was followed by a small decline in drug solubilisation. This is indicative of drug supersaturation within the simulated intestinal media, which resulted in a fraction of the drug recrystallizing into CINs more thermodynamically favourable form. In contrast, the degree of CIN solubilisation for SSLC increased over time for the entire dissolution period. Thus, the rate of CIN release from SSLC enabled equilibrium solubility to be sustained. In vitro CIN dissolution studies under digesting conditions Digestion-mediated precipitation was investigated by analysing the partition of CIN between the aqueous and pellet phases during lipase-mediated hydrolysis of the lipid-based formulations. A dynamic in vitro lipolysis model in biorelevant media consisting of enzymes, co-enzymes, bile salts and phospholipids was selected to simulate intestinal digestion during the fasted state. A 1.5- and 6-fold improvement in aqueous drug solubilisation was observed when CIN was administered with PLH particles, compared to SSLH particles and SSLC, respectively (Figure 4).



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Figure 4: Partition of cinnarizine (CIN) into the aqueous phase (blue column, left axis) and pellet phase (black column, right axis) during the in vitro lipase-mediated digestion of (A) PLH, (B) SSLH and (C) SSLC particles, under simulated fasted state intestinal conditions. The line graphs (right axis) represent lipid hydrolysis as a function of time for each formulation. Each data point is expressed as the mean ± S.E.M., n = 3. 18 ACS Paragon Plus Environment

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Drug solubilisation from lipid-based delivery vehicles is highly dependent on lipolysis kinetics, as poorly water-soluble drugs partition between the undigested triglycerides and the hydrolysis products depending on the affinity.48, 49 Lipid digestion kinetics, monitored by the production of free fatty acids, were greatest for PLH particles compared to the silica-lipid systems, evidenced by 78.7 ± 3.3% lipolysis over the 60 min period. This is due to the ability for digestive enzymes to more readily hydrolyse shorter chain triglycerides (i.e lipids encapsulated within PLH) compared to long chain triglycerides (i.e. lipids encapsulated within SSLH and SSLC).12, 50 Furthermore, short chain fatty acids form mixed micelles and partition towards the aqueous phase at a greater rate than the more hydrophobic long chain fatty acids, which adsorb to the oil-in-water interface.51 Therefore, this suggests that the improved solubilisation capacity of PLH particles is a result of the hydrophobic drug partitioning towards hydrophobic core of mixed micelles, formed from fatty acids, monoglycerides and bile salts (Figure 5). The higher concentration of digestion products in the aqueous digestion media of PLH particles increased the level of CIN solubilisation. Despite sharing similar dissolution profiles in non-digesting conditions, the degree of solubilisation of CIN in the aqueous phase varied significantly between the two silica-lipid systems in digesting conditions. Quantitatively, 30.1 ± 0.58% of CIN partitioned towards the aqueous phase during the digestion of SSLH particles, compared to 7.50 ± 0.58% for that of SSLC. While drug partitioning varied considerably, lipolysis kinetics were equivalent, given by degrees of lipid digestion of 58.7 ± 3.3% and 54.4 ± 3.3% for SSLH and SSLC, respectively. Thus it is hypothesised that one, or a combination of the following mechanisms caused a difference in aqueous drug solubilisation (Figure 4): (i)

The solubility of CIN was limited in SSLC digestion products: the lipid used for SSLH particles and SSLC was GMS and GMO, respectively. Both lipid excipients have equivalent carbon chain lengths (i.e. C21 if the pre-digestion glycerol is 19 ACS Paragon Plus Environment


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included) but differ by levels of saturation, where GMS is a saturated glyceride and GMO is monounsaturated glyceride due to one double bond within the carbon chain. Based on the aqueous solubilisation profile of CIN during the digestion of SSLH, whereby CIN solubilisation is enhanced and increases incrementally over the lipolysis period, it suggests that CIN was solubilised more avidly by mixed micelles rich in stearic acid. In contrast, approximately 20% of CIN partitions towards the aqueous phase during the digestion of SSLC, but this decreases rapidly over the lipolysis period until only 7.50 ± 0.58% of CIN was retained within the aqueous phase. This may indicate that supersaturation of CIN in the aqueous phase is not supported by the monounsaturated fatty acid, oleic acid, which may be attributed to the role of fatty acid saturation on the self-assembly structure of digestion products.52 (ii)

A synergistic role exists between porous silica and solid lipid: Yasmin et al.30 previously highlighted the benefits of formulating CIN with porous silica particles and solid lipid nanoparticles. For porous silica, drug adsorbs within the pores in its amorphous state,53 leading to improved drug solubilisation. For solid lipid nanoparticles, drug molecularly disperses within the solid lipid matrix and then resolubilises within lipolysis products upon lipase-mediated degradation of the matrix. When both systems were combined to form SSLH particles, a 2.2- and 1.5fold improvement in drug dissolution was observed compared to drug loaded silica particles and solid lipid particles, respectively. Since non-porous silica particles were used to formulate SSLC in the current study, this synergistic effect was absent, restricting the inhibition of precipitation. Furthermore, it is possible that the solid silica surface promoted precipitation through adsorption of CIN onto the negatively charged silanol groups.19 20 ACS Paragon Plus Environment

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Figure 5: Schematic representation of drug solubilisation and the potential gastrointestinal processing pathways for each CIN formulation. In vivo pharmacokinetic study The mean plasma concentration-time profiles of CIN following a single oral dose of PLH, SSLH and SSLC formulations to fasted male SD rats are presented in Figure 6. The corresponding pharmacokinetic data is summarised in Supporting Information Table S1.



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Figure 6: Plasma cinnarizine (CIN) concentration (mean ± S.E.M., n = 4) as a function of time following oral administration of PLH (black ■), SSLH (blue ●) and SSLC particles (red ) at a CIN concentration of 10 mg/kg. Plasma concentrations were dose normalised to 10 mg/kg to account for variation in animal weight. The oral absorption of CIN from PLH microparticles was significantly enhanced compared to the SSLH and SSLC formulations, as highlighted by 2-5 fold improvements in AUC0-last (Figure 7) and 2-3 fold improvements in Cmax (p ˂ 0.05), which related well with the in vitro assessments. Furthermore, Tmax was increased for PLH particles (4.0 ± 2.0 h), indicating that absorption of CIN was more sustained when encapsulated within the polymer and lipid phase. The sustained CIN absorption correlates well with several other studies that have demonstrated the ability for PLGA nanoparticles to control and sustain the release of poorly water-soluble drugs when administered orally.29,

54, 55

Furthermore, previous studies have

shown that co-administering CIN with MCT alone results in a minimal improvement in oral bioavailability,18 highlighting the importance of PLGA nanoparticles within the formulation. Yasmin et al.30 indicated that a strong synergy existed between porous silica particles and 22 ACS Paragon Plus Environment

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solid lipid droplets within SSLH particles. Since the pharmacokinetics of CIN delivered by PLH particles were significantly enhanced compared to the other two hybrid lipid formulations, it is also hypothesised that a synergistic effect exists between PLGA nanoparticles, mannitol and lipid droplets in solubilising and promoting absorption of CIN. However, to unequivocally and quantitatively determine the presence and extent of synergy, future studies are required to analyse the pharmacokinetic performance of PLGA nanoparticles, mannitol and lipid droplets independently.

Figure 7: The area under the curve (AUC0-last) for plasma concentration-time profiles following the single oral dose of PLH (black bar), SSLH (blue bar) and SSLC particles (red bar) at a CIN concentration of 10 mg/kg. A 2-fold improvement in the AUC0-last was observed between SSLH particles and SSLC (p ˂ 0.05). Previous studies have demonstrated the importance of the nanostructured, threedimensional porous silica matrix of SLH microparticles on improving the in vivo performance of co-administered drugs.10,

15, 56

The solid-state structure improves drug

absorption through the following mechanisms: (i) optimised lipolytic digestion of encapsulated lipid colloids,12, 13, 57, 58 (ii) controlled disposition, speciation and self-assembly 23 ACS Paragon Plus Environment


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structure of digestion products,33 and (iii) sustained drug solubilisation within the porous silica matrix.59,

60

Since SSLC were administered as a liquid crystalline lipid dispersion,

stabilised with non-porous silica particles, the formulation benefits of nanostructuring lipid colloids in a silica matrix were not present, resulting in lower drug absorption when compared with SSLH particles. In vitro-in vivo correlations (IVIVC) Strong single-point correlations (R2 > 0.9) existed between in vitro drug dissolution data, under digesting and non-digesting conditions, and in vivo oral absorption of CIN when administered with the hybrid particles (Figure 8). The oral absorption of poorly water-soluble drugs with high permeability (i.e. CIN and other BCS Class II compounds) is rate-limited by the dissolution in the GIT.61 Thus, it is considered that the most useful IVIVC models are correlations between drug dissolution or release profiles and the absorption data.10 In this study, dissolution efficiency (%DE), under digesting and non-digesting intestinal conditions, was correlated to the area under the plasma drug concentration-time curve (AUC0-last) as both variables are estimated by the integration of the area under the curve and are therefore considered useful parameters for determining IVIVC.32


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Figure 8: Single-point correlations of CIN formulated with PLH particles (black ■), SSLH particles (blue ●) and SSLC (red ): (A) %DE60 non-digesting correlated to AUC0-last; (B) %DE60 digesting correlated

to AUC0-last.

Since dissolution efficiency was well correlated to the area under the plasma drug concentration-time curve, it indicates that both in vitro evaluation methods were adequate for predicting the trends in in vivo performance of the formulations. Interestingly, %DE60 digesting

non-

and AUC0-last (R2 = 0.95) was found to be more closely correlated than %DE60 digesting

correlated to AUC0-last (R2 = 0.91) (Figure 8), despite the use of non-biorelevant media in non-digesting conditions. Previous studies have indicated that employing a physiologically 25 ACS Paragon Plus Environment


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relevant media, specifically a fasted-state media containing bile salts and lipolysis products, facilitates higher IVIVC with fewer prediction errors.62 However, it was evident that lipolysis of SSLC in simulated intestinal media induced precipitation of CIN, as seen by a reduction of CIN solubilisation over time, which resulted in weaker IVIVC for the three formulations during digestion conditions. Thus, the variation in the CIN absorption profiles is expected to correlate with the differences in solid particles and lipid excipients used in each formulation. This further highlights the need for careful design of formulation structure and composition when administering complex poorly water-soluble drugs with lipid-based delivery systems.

Conclusions Three novel hybrid lipid-based formulations were fabricated using colloidal self-assembly techniques to determine the optimal parameters for improving the oral absorption of the poorly water-soluble weak base, CIN. PLH particles, composed of PLGA nanoparticles and submicron liquid lipid droplets, demonstrated superior ability to inhibit precipitation of CIN in simulated intestinal conditions in both digesting and non-digesting conditions, compared to SSLH particles and SSLC. This correlated well with enhanced pharmacokinetics following the oral administration to SD rats, evidenced by a 2- to 5-fold improvement in the AUC compared to the silica-lipid formulations. Thus, the ability for PLGA nanoparticles to be incorporated into a solid-state lipid-based formulation and act as a precipitator inhibitor for increased oral absorption of a lipophilic bioactive has been highlighted for the first time. This indicates that PLH particles serve as a promising approach to improve the in vitro and in vivo performance of poorly water-soluble compounds by combining the solubilisation behaviour of lipids and the precipitation inhibition effect of PLGA nanoparticles.


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Acknowledgments: The Australian Research Council’s Centre of Excellence in Convergent Bio-Nano Science and Technology (ARC CE140100036) and UniSA Ventures are greatly acknowledged for research funding and support. Supporting Information: More extensive pharmacokinetic data is provided. This material is available and free of charge via the Internet at http://pubs.acs.org.

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For table of contents only 88x45mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Comparison across Three Hybrid Lipid-Based ... - ACS Publications

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