Formulation of Granules for Site-Specific Delivery of an Antimicrobial Essential Oil to the Animal Intestinal Tract

Journal of Pharmaceutical Sciences xxx (2015) 1e10

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Research Article

Formulation of Granules for Site-Specific Delivery of an Antimicrobial Essential Oil to the Animal Intestinal Tract Yin-Hing Ma 1, 2, Qi Wang 1, Joshua Gong 1, Xiao Yu Wu 2, * 1 2

Guelph Food Research Centre, Agriculture and Agri-Food Canada, Guelph, Ontario, Canada N1G 5C9 Advanced Pharmaceutics and Drug Delivery Laboratory, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S 3M2

a r t i c l e i n f o

a b s t r a c t

Article history: Available online xxx

Owing to proliferation of antibiotic-resistant bacteria, the use of antibiotics for livestock growth promotion is banned in many countries and alternatives to in-feed antibiotics are needed. Cinnamon essential oil exhibits strong in vitro antibacterial activity; however, direct addition of essential oils to animal feed has limited practicality due to their high volatility, odor, fast decomposition, and poor availability in the lower intestines. To solve these problems, we formulated trans-cinnamaldehyde (CIN) with an adsorbent powder and fatty acid via a melt-solidification technique. Core granules of an optimized composition contained up to 48% wt/wt CIN. The granules were then coated with an enteric polymer to impart site-specific release of CIN. CIN was mostly retained in simulated gastric fluid and released rapidly (>80% under 2 h) in simulated intestinal fluids. Rapid CIN autoxidation into cinnamic acid was inhibited by adding 1% vol/vol eugenol, which maintained CIN stability for at least 1 y. The granule formulation increased the antimicrobial activity of CIN against Escherichia coli K88 slightly with a minimum bactericidal concentration of 450 mg/mL for CIN in lauric acidebased granules compared with 550-600 mg/mL for palmitic acidebased granules and free CIN, respectively. These results encourage the potential use of encapsulated CIN for control of animal enteric pathogens by oral in-feed administration. © 2015 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Keywords: formulation site-specific delivery anti-infectives antioxidants coating oral drug delivery stability

Introduction The practice of supplementing antibiotics to animal feed at subtherapeutic levels to promote the growth of food-producing animals and prophylaxis has been banned across EU members1 in an effort to curb spreading of antibiotic resistance among pathogens caused by selective pressure under intensive farming environments.2 Alternative compounds to in-feed antibiotics are thus pressingly needed to help maintain economical livestock production. Many plant-derived essential oils (EOs) exhibiting strong antimicrobial activity in vitro3-5 can be used as alternatives to antimicrobial drugs, shifting the use and resistance development away from more medically important antibiotic drugs. Of various EOs, trans-cinnamaldehyde (CIN), the most abundant component of cinnamon oil, shows antimicrobial effects (ranging

This article contains supplementary material available from the authors by request or via the Internet at http://dx.doi.org/10.1016/j.xphs.2015.10.001. * Correspondence to: Xiao Y. Wu (Telephone þ1-416-978-5272; Fax þ1-416-9788511) E-mail address: [email protected] (X.Y. Wu).

from inhibitory to bactericidal) against various bacterial pathogens at concentrations between 56 and 385 mg/L (mg/mL) in liquid media.4,6,7 In addition, CIN has shown some potential use in dairy cows to affect rumen microbial fermentation.8 Hence, CIN is a good candidate for an alternative antimicrobial agent. Nevertheless, the effectiveness of direct addition of free EOs to the feed is questionable due to several limiting physical-chemical factors such as the high hydrophobicity, volatility, odor, oxygen sensitivity, and low availability of the EOs in the lower gastrointestinal tract (GIT) of animals.9,10 To improve the availability of EOs to exert their antimicrobial activity in vivo, it is desirable to develop a formulation that is amenable to oral delivery to the lower GIT of food-producing animals. For this purpose, solid granules containing liquid EOs would be a good choice as they can be mixed within animal feed more readily than liquid EOs, and can be further processed to reduce volatility and degradation of EOs, and to allow site-specific release of EOs in the GIT by applying a suitable coating to the granules. The granulation and coating can potentially control the effects of EOs on feed palatability for animals by reducing oil volatility and masking odor.11

http://dx.doi.org/10.1016/j.xphs.2015.10.001 0022-3549/© 2015 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

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To obtain EO granules with high loading levels while minimizing the evaporation and odors of the oils, we have designed a new granule formulation in this work by soaking the oil in an adsorbent powder and then incorporating the powder in a melted lipid to form solid granules. Various pharmaceutically acceptable powdery, inert adsorbents were investigated including magnesium aluminum silicate, microcrystalline cellulose, and wheat bran powder. The powder possessing the highest oil retention capability was then selected for further development of granules with a lipid binder. The success of developing solid granules containing lipid and EO-adsorbed powder depends on many factors that influence the granule properties, for example, granule size and strength, EO loading levels, and release kinetics. These factors include the compatibility of the powder with the lipid, the melting temperature of the lipid and its mixture with the EO-adsorbed powder, and the droplet-forming properties that can affect the size and shape of solidified granules. Although meltable lipids have been used to prepare solid lipid particles previously to encapsulate various drugs, such dosage forms were primarily designed for parenteral drug delivery with small (nano) sizes and low drug loading levels, and in the absence of powdery materials.12,13-17 Therefore, we conducted in-depth studies on the molten and resolidified properties of mixtures in relation to the composition of the 3 component system and rationally selected the type of lipid. Simple saturated fatty acids (FAs) with well-defined melting points from processed oils of palm or coconut as well as fatty alcohols were tested, as they have a melting range from 44 C to 70 C and have been successfully applied in melt-pelletization and meltgranulation processes.12,18-20 Despite its good antimicrobial activity, the practical application of CIN as an alternative antibiotic product is limited by its poor stability. CIN is known to undergo rapid oxidation on exposure to atmosphere, converting to nonbioactive cinnamic acid (CA). Therefore, a series of antioxidants were screened to stabilize CIN. Eugenol, an effective antioxidant present in natural EOs, was selected for further study of CIN in granules and its effect on CIN stability was monitored at room temperature and 4 C for 1 y. To protect actives from acidic pH and achieve site-specific drug release in the lower GIT, an enteric polymer was introduced in the core and surface of granules. The surface of core CIN granules was coated using a pH-responsive polymer, Eudragit® (Evonik Industries) L 100. Structurally, this polymer contains acidic monomers (methacrylic acid) copolymerized with nonionizable monomers (methyl or ethyl acrylate) and is identified in the United States Pharmacopeia under the general term “methacrylic acid copolymers.” It dissolves in an aqueous medium at pH > 6. Here, we describe a systematic investigation for the first time on the formulation development, granule production, stability testing, and enteric coating of CIN core granules intended for releasing CIN in the lower GIT regions to target intestinal pathogens. Finally, the antimicrobial activity of CIN granules was examined against a multidrug-resistant bacterial strain Escherichia coli K8821 as compared with free CIN.

Neusilin® US2 and UFL2 (magnesium aluminum silicate) were bulk samples obtained from Fuji Chemical Industries (Fuji Health Science, Burlington, NJ), microcrystalline cellulose Avicel® grades PH102, RC-591, CL-611 were obtained from FMC Biopolymer, and white wheat bran powder (50 mesh) was provided by Hayhoe Mills, Ltd. (Woodbridge, Ontario, Canada). Carvacrol, butylated hydroxy toluene (BHT) and t-butyl methyl phenol were obtained from Sigma-Aldrich chemicals. Determination of Oil-Adsorbing Capacity of Powders Two types of pharmaceutical excipient powders, that is, microcrystalline cellulose powder (Avicel® PH102, Avicel® RC-591, Avicel® CL-611) and magnesium aluminum silicate powder (Neusilin® UFL2, Neusilin® US2), and a food powder (wheat bran) were tested for their oil-adsorbing capacity. Approximately 0.5-1 g of a powder was accurately weighed into a 15-mL conical polypropylene screw-cap tube (BD Falcon), and then, CIN was added until excess was visible. After allowing the oil to completely soak the powder for >10 min, the excess oil was then separated by centrifugation for 10 min at 3000 g. The excess oil was poured off, and a pipette was used to draw any remaining superficial oil after inverting the tube and allowing drainage. After all the excess oil was removed, the tube containing powder and adsorbed oil was weighed again. The weight of adsorbed oil was obtained by weight difference. The oil loading capacity was calculated by the following equation:

%wt=wt oil ¼

weight of oil  100% weight of oil þ weight of powder

Formulation Selection by Phase Diagram Based on the test of oil-adsorbing capacity of powders described previously, Neusilin® US2 powder yielded the highest CIN oil adsorbing capacity among the 6 studied powders and was selected for further formulation development. Oil-loaded core granules were formulated with 3 components: CIN oil, adsorbent powder, and FA. A phase diagram was constructed to better define composition ranges that yielded: (1) at molten temperatures, a mixture in the liquid state for ease of droplet formation in suspension and (2) when cooled to room temperature (23 C), a solid with absence of excess oil on the surface. Compositions (see Supplementary Figure S1) were tested by making 2 g of mixtures containing different weight % of each component in a 10-mL glass vial. Melting and solidification of the mixtures were carried out using a hot water bath and ice water bath, respectively. The CIN oil was mixed with the powder first and then blended into molten FA with a spatula and subsequently solidified by cooling in a water bath. Qualitative observations were made (see Supplementary Table S1) of the mixtures at different temperatures and classified as powder, solid (homogeneous), paste, or liquid. Preparation of Core Granules

Materials and Methods EO compounds trans-CIN (99%) and eugenol (99%), FAsdlauric acid (LA; C12), myristic acid (MA; C14), palmitic acid (PA; C16), stearic acid (SA; C18), and the fatty alcohol palmitic alcohol (C16), were obtained from Sigma-Aldrich (Oakville, Ontario, Canada). Enteric polymers Eudragit® (Evonik Industries) L 100 and S 100 are methacrylic acidemethyl methacrylate copolymers in the ratio 1:1 and 1:2, respectively, obtained in powder form from Almat Pharmachem Inc., (Concord, Ontario, Canada). Oil adsorbent powders

The CIN oil was encapsulated into spherical granules by meltsolidification in an aqueous suspension. To prepare 100 g of oilcontaining granules, CIN oil (40-50 g) was first adsorbed with Neusilin® US2 powder (US2; 0-13 g) by slow addition and mixed gently in a beaker to ensure even distribution of oil. In a separate beaker, the FA was melted in a water bath set at 45 C-67 C (i.e., up to 5 C higher than the melting point of the FA chosen). The oil-powder mixture was then added to the molten FA and mixed with a spatula until it became homogeneous. To stabilize the molten droplets, an

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individual batches were taken for determining loading and stability of CIN contained in individual batches. Due to the oxidation of CIN (lmax ¼ 291 nm) to CA (lmax ¼ 272 nm), a mixture of CIN and CA was present in some cases. Thus, calibration equations of the 2 standards were obtained with both pure compounds and mixtures and then used in determining unknown concentrations of both compounds simultaneously using a simple additive rule.22 Readings were obtained on a UV-vis spectrophotometer (Lambda; Perkin-Elmer). The EO loading efficiency in prepared granules was calculated in the following manner:

Theoretical loading ¼

weight of CIN added total weight of all granule materials  100%

Actual loading ¼

weight of total CIN extracted  100% total weight of granules

Loading efficiency ¼

actual laoding of CIN in granules theoretical loading of CIN granules  100 %

Figure 1. Schematic diagram for granule production process.

aqueous solution of methacrylic acidemethyl methacrylate copolymer (Eudragit® S 100 [Evonik Industries]) neutralized with NaOH to pH 7.5 was used at a final concentration of 4%-5% wt/wt. The aqueous dispersion medium (200-300 mL) was heated to the same temperature as the molten FA (51 C-52 C for LA; 66 C-67 C for PA) in a 60-mL beaker with a water bath to ensure the CIN-US2-FA mixture remained molten when dispersed. Tween 80 (at 0.5% vol/vol of the aqueous phase) was found to slightly improve the round shape of granules. An overhead stirrer (Caframo, Ontario, Canada) was used to disperse the oily phase into the aqueous phase at 150-375 rpm using a stainless steel 4-bladed round-edged propeller to form a 2-phase, liquid-liquid suspension. After droplet size and shape were visually acceptable (~30 s-1 min), stirring was stopped, and the beaker was transferred to an ice-water bath to solidify the droplets into granules. Afterward, granules were suction filtered and washed with small volumes (10 mL) of distilled water, and then allowed to air dry on aluminum pans for 45 min or until free flowing. For PA granules, higher initial temperature was used (67 C-70 C) to maintain molten state before blending with oil-soaked powder. Figure 1 summarizes the steps in the granule production process. Control granules were made with 10% US2:90% FA by physically breaking down the resolidified mixture into small-sized particles. Enteric Coating of Core Granules Core granules were first subcoated with an ethanolic dispersion of Kollicoat IR (polyvinyl alcoholepolyethylene glycol graft copolymer) to resist further evaporation of oil by acting as a sealant layer. The outer enteric polymer coating was applied using a fluid bed machine (Glatt laboratory fluid bed, Binzen, Germany) in 100- to 500-g batches of core granules. The polymer for enteric coating was Eudragit® L 100 (Evonik Industries), which dissolves above pH 6 giving targeted release in the jejunum-ileum regions of the intestinal tract. Assay of CIN and CA Content in Granules Granules were weighed (50-100 mg), and methanol (10-20 mL) was added into glass vials. A sonication bath and shaking were used to disintegrate granules to fully release CIN. Triplicate samples from

In Vitro Release of CIN From Granules Simulated gastric fluid (SGF, adjusted with 0.1-M HCl, 0.2% wt/ vol NaCl) and simulated intestinal fluid (SIF, 50-mM KH2PO4, adjusted with 0.1-M NaOH) were prepared at pH of 1.2 and 6.8, respectively. Release of CIN was monitored by a UV detector at 2 wavelengths (291 and 272 nm) at 10-min intervals via flowthrough cuvettes with up to 6 replicates per run. Granules were tested for 2 h in SGF (~250 mL) and 6 h in SIF (~900 mL), and the CIN concentrations were converted into amounts to calculate % released. Solutions were maintained at 37 C, and stirring rate was 100 rpm using a dissolution testing apparatus (ERWEKA, Germany). Antimicrobial Activity Assay With Pure Culture in Liquid Growth Media Antimicrobial activity of pure compound CIN, its oxidation product CA, and CIN-encapsulated core granules were assessed. A study of the growth inhibition by EO compounds was performed against a pure culture of E. coli K88 strain JG280 undergoing exponential growth at 37 C in tryptic soy broth (TSB) media as described previously.4 The 3 treatments were control (no EO compounds), free EO, and EO granules at different EO concentrations in 5 mL of TSB inoculated with E. coli K88 at a concentration of 104 colony forming units (CFUs)/mL. The bacteria were then allowed to grow with agitation at 200 rpm on an orbital shaker incubator at 37 C. The optical density (OD) of the suspensions was monitored at a wavelength of 600 nm in comparison with the control OD.23 The experiment was stopped when the control OD reached 1.2-1.3 or typically between 5 and 6 h of incubation. The minimum inhibitory concentration (MIC)90 of CIN against this E. coli K88 strain was defined as the lowest concentration of the EO compound that produced a 90% inhibition of the bacteria undergoing log growth.4,24

 Inhibition % ¼ 100 

ODCont  ODEO



ODCont

ODCont represents the OD600 of a bacterial culture grown with the same initial 104 CFU/mL in the absence of any EO (negative

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Table 1 The Oil-Adsorbing Capacity of Various Powders Powder Type

Grade

Oil-Adsorbing Capacity

Avicel® Avicel® Avicel® Neusilin® Neusilin® Wheat bran

PH102 RC-591 CL-611 UFL2 US2 e

1.532 0.609 0.676 4.023 5.074 0.970

± ± ± ± ± ±

% wt/wt CIN

0.083 0.022 0.010 0.018 0.107 0.039

61.66 39.00 41.52 80.86 84.19 50.4

± ± ± ± ± ±

0.99 0.84 0.34 0.07 0.28 1.00

Specific Surfacea (m2/g) 1-1.35 e e 300-339 300-339 e

Values represented in average ± SD (n  3). a Values obtained from Westermarck et al and Hentzschel et al25,26 and product bulletin.

control). The minimum bactericidal concentration (MBC) of CIN against this E. coli K88 strain was taken as the lowest concentration that produced no observable growth after transferring to fresh growth media for up to 6 h, followed by plating dilutions (20-100 mL) onto trypticase soy agar plates incubated overnight (16-24 h) to identify the number of surviving CFUs per milliliter. These experiments were repeated at least twice. Results Formulation and Properties of CIN Core Granules Selection of Adsorbent Powder With Highest Capacity Oil retention capacity of 3 types of pharmaceutical or food grade powders was evaluated to find a suitable powder with the highest oil adsorption. Table 1 compares the oil retention capacity of these powders. Among the tested powders, Neusilin® US2 shows the highest adsorbing capacity of 84%, attributable to its high specific surface area (Table 1), and was thus selected for further study. Although the powders could hold a large amount of oil, once above ~78% wt/wt the powder surface appeared wet with oil due to powder saturation. Furthermore, powders containing oils were fairly soft and weak and appeared almost like a wet paste (Fig. 2), so a third component was added to allow formation into granules that are a meltable FA binder. Selection of FA Binder to Obtain Sufficiently Strong Granules Saturated FAs were chosen as the binder for their well-defined melting point (to allow quick encapsulation by rapid cooling) and their lipophilic property. LA was initially chosen for its low melting point at ~44 C and for being easily resolidified at room temperature. When molten, LA blended easily with the oil-soaked powder and when cooled and dried overnight, spherical granules were obtained. However, LA-based granules were mechanically too weak to withstand further processing in a fluid-bed coating machine due to problems such as attrition and fusing of the granules. To address these issues, 2 approaches were undertaken: blending with higher melting temperature FAs or fully substituting with another FA type. It was found that blending of different FAs yielded mixtures that were more difficult to solidify than pure FA. After experimenting further with other excipients, it was concluded that the granule strength was greatest when a single FA was used instead of mixtures. Optimizing Formulation of Core Granules Via Phase Diagram A phase diagram was used as an aid to optimizing oil loading in the final mixture and to find compositions that would allow uniform, completely resolidified granules that were suitable for further processing, as shown in Figure 3. The phase regions and boundaries were drawn as a result of the processability limits of the mixtures (see Supplementary Table and Figure S1 for actual composition points tested). There were 4 types of phase states that were observed, based on the physical processability of the mixture at

molten temperatures or at room temperature (after cooling and/or resolidification). Regions of phase diagram were classified as follows:  Powder: The mixture was loose and powdery due to undersaturation of the US2 and insufficient interpowder binding. At molten temperature, the mixture was not dispersible in the aqueous continuous phase unless further liquid adsorption occurred. After cooling down, there was insufficient interpowder binding by the FA component to hold the mixture together.  Paste: The mixture appeared wet, weakly held together, and not free flowing, containing oversaturation of the US2 powder by liquid (CIN and FA) at molten temperatures. After the mixture cooled, an excess of liquid CIN remained and weakened binding due to insufficient amount of FA.  Liquid: The mixture was liquidlike, that is, free flowing under its own weight. It was easy to disperse into uniform droplets at above molten temperature.  Solid: A sufficient amount of resolidified FA to hold the mixture together with little to no excess oil visible. On cooling, granules became uniformly solid; when dry, they were free flowing and round in shape due to good dispersability as droplets in the molten state. For ease of dispersibility into small droplets and forming granules, a molten mixture with liquid behavior was preferred corresponding to >80% liquid and <16% powder. When the liquid saturation of US2 was approached or exceeded, the mixture became more easily dispersible into droplets. But in order for resolidification of granules to occur on cooling, sufficient FA needed to be present in the mixture to bind the powder together on resolidification. The region where a paste occurred was between 15% and 20% US2 and 80%-84% liquid, and such mixtures were not processable into droplets and resulted in large globs being formed, even under high-speed stirring (>1000 rpm). The powder region (blue) was where there was 80% liquid and 20% powder, and due to undersaturation of the powder, the mixture was not dispersible within a liquid continuous phase, which was unfavorable for forming CIN-containing droplets. The region of the phase diagram with maximal yield of granules with high EO loading is outlined in red. The type of FA used to make granules did not affect the oil loading properties (i.e., a similar phase diagram could be used for the other FAs). It was observed that use of a higher melting temperature FA resulted in harder granules that could be felt by grinding the particles between fingers because neither the oil nor the powder possessed appreciable binding ability, and hence, the granule solidification was essentially resulting from resolidification of the FA component. Process Conditions and Properties of Core Granules Table 2 presents the process and granule characteristics of batches prepared under a range of conditions. From these studies,

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Figure 4. It was found that when the US2 remained constant (10%), increase in PA concentration from 40% to 45% produced a higher proportion of larger granules; similarly, when the CIN concentration kept constant (45%), increasing the US2 concentration from 10% to 13% produced higher fractions of larger granules, especially those >1400 mm. The encapsulation efficiency was similar among the formulations tested (~93%). The formulation 48% CIN:10% US2:42% PA yielded hard granules, with a high loading efficiency, and had the highest weight fraction of granules in the range of 6001400 mm, which are suitable for processing in fluid-bed coating and are in a product form convenient for mixing with animal feed. Thus, this formulation was selected for further study. Formulations with >10% US2 produced much larger fractions of granules >1400 mm due to the apparent viscosity-enhancing effect of US2 on the molten mixture (and other suspensions in general). Selection of Antioxidant to Stabilize CIN in the Core Granules Against Atmospheric Oxidation (Autoxidation) After incorporating pure CIN in initial batches of granules, the EO was unstable as it underwent autoxidation into CA fairly rapidly on exposure to the atmosphere which led to loss of antimicrobial activity as explained in the following section. As shown in Figure 5a, the rapid conversion of CIN into CA started as soon as granules were exposed to atmospheric oxygen overnight. Without any antioxidant protection, >70% CIN (of the initial loading) degraded into CA within 5 d at room temperature and within 30 d at 4 C; the rate of decomposition was slower (Fig. 5a). To counteract this undesirable degradation, a series of potential antioxidative compounds were screened. Among the tested compounds (BHT, carvacrol, eugenol, and t-butyl methyl phenol), it was found that proton-donating compounds (phenols) were the most effective in slowing down or inhibiting autoxidation. Eugenol was selected for further study on account of its natural presence in cinnamon oils, primarily in the plant leaves.27,28 It was found that a concentration of 1% vol/vol eugenol added to pure CIN was sufficient for protection against CIN autoxidation in granules for at least 1 y at both temperatures. After addition of the antioxidant to CIN, granules made with PA and LA showed the same degree of stability. Coated granules containing CIN with 1% eugenol were also stable for at least 1 y when stored either at room temperature or refrigerator temperatures as seen in Figure 5b. Properties of Coated Granules

Figure 2. Pictures of Neusilin US2 at various CIN oil loading: top 0%, middle 62%, and bottom 78%.

optimized conditions were found and then used to produce 0.8- to 1-kg batch granules for subsequent coating. The shape and size of granules were found to be highly affected by the fluid properties of the molten mixture. Cooling of the mixture after stirring stopped yielding of more spherical granules, whereas addition of cooling water into the suspension disrupted droplet shape during cooling. The sieved size distribution of finished batches prepared at fixed conditions (300 rpm, 150-g batch, 300-mL continuous phase in 600-mL vessel), but different formulation composition is shown in

Two types of core granules produced from LA and PA were coated by a fluid-bed process. The first type of granules made using LA are shown in Figure 6a. The CIN in these granules did not contain an antioxidant, so the CIN content was low in the final product (~6% wt/wt), and granules were solid enough to be coated due to conversion to the solid CA. However, after inclusion of an antioxidant (1% eugenol) in subsequent batches of LA granules, the granules were not strong enough to withstand the fluid-bed coating process; so, higher melting temperature FAs were investigated and used. The stronger granules based on PA were thus produced and coated. The core granules contained 42% CIN and the coated granules contained 20% CIN (with 1% eugenol) due to a weight gain of ~30%35% from coating. Both types of granules were fairly round, and the coating uniform as shown in Figure 6. In Vitro Release of CIN From Core Granules and Coated Granules Figure 7a compares release profiles of CIN from uncoated core granules prepared with different fats in SIF at pH 6.8 at 37 C. LAbased granules showed the highest rate and extent of release,

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Figure 3. Phase diagram for a 3-component mixture at molten (fatty acid) and cooled temperatures. Dot indicates optimized formulation. Outline indicates range of formulation components that resulted in acceptable granules.

followed by MA, PA, SA, and cetyl alcohol (CAl). Without coating, CIN was released rapidly from granules with >80% being released from LA-, MA-, and PA-based granules within 1 h. SA- and CAlbased granules showed lower extent of release probably attributable to their higher hydrophobicity. Core granules were coated with an enteric polymer (Eudragit® L 100 [Evonik Industries]) to suppress the release of CIN in gastric conditions and enable the release after dissolution of the coating polymer at pH > 6. The in vitro release profiles were determined in a 2-stage test: 2 h in SGF at pH 1.2 and then continued in SIF at pH 6.8 for 3 h. For the uncoated granules, the release test was undertaken in SIF. Figure 7b shows pH-dependent release of CIN from the coated granules which is absent for the uncoated core granules. The enteric coating prevented CIN from release in the SGF significantly with >70% of the loaded CIN being released in the SIF. Ultimately, after dissolution of the coating material, >90% of the CIN was released. Antimicrobial Activity of CIN Against E. Coli K88: MIC and MBC Determination The effectiveness of antimicrobials against bacteria growth and viability can be generally expressed in 2 forms: MIC90 and MBC. Inhibition of growth was determined for different concentrations of CIN and its oxidation product CA. The MIC90 of CIN against E. coli K88 was found to be 150 mg/mL, whereas CA showed much lower inhibitory activity (>1000 mg/mL). Hence, no MBC determination for CA was carried out due to the low inhibitory activity against the target strain as shown in Figure 8a. Figure 8a shows the inhibition

of E. coli K88 growth (initial 104 CFU/mL) by CIN at various concentrations in a liquid medium (TSB). The low activity of CA suggested preventative measures against oxidation of CIN to CA using antioxidant. From the curves in Figure 8b, the MBC of free CIN oil and CIN encapsulated into PA and LA granules against E. coli K88 were found to be 600, 550, and 450 mg/mL, respectively. Discussion Importance of Formulation on the Properties of Granules The current results indicate that the melting point of the specific FA used predominately affected granule strength because granules were primarily held together by intermolecular van der Waals forces between FA molecules. As FA chain length increases, its melting temperature increases, and granules become harder at room temperature as compared with those made from a shorterchain FA. As such, PA yielded harder granules than LA due to its higher melting temperature resulting from the longer FA chain length (C16 vs. C12). Thus, the formulation with LA was substituted with PA to obtain harder granules at room temperature. FA blends were not used for making stronger granules because blends did not have a well-defined melting transition temperature compared with pure FAs, possibly due to less

Table 2 Granule Properties and Process Characteristics Properties and variables

Value or range

Oil loading, % wt/wt (actual/theoretical) Oil loading/encapsulation efficiency RPM range Granule batch size range Batch size Process yield Molten:aqueous phase ratio Formulation components ranges

16.9-45/25-48 ~93% 150-375 0.5-5 mm 20-200 g >90% 0.13-0.66 CIN 20%-50% US2 0%-10% PA 40%-60% 48:10:42

Optimum ratio of CIN:US2:PA RPM, rotations per minute.

Figure 4. Effect of formulation composition changes on the size distribution of granules while keeping batch size and stirring rate constant.

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Figure 5. (a) Stability of CIN in uncoated granules with and without antioxidant when stored at 23 C and 4 C. (b) Stability of coated granules with antioxidant (1% EUG) stored at 23 C and 4 C for up to 1 y. EUG, eugenol; RT, room temperature; AX, antioxidant.

effective packing between the dissimilar chain length FA molecules (e.g., LA with SA). With regard to the amount of CIN that could be accommodated in the granules, the independence of FA type could be due to similar volume being occupied by these different FAs in both molten and solid states. Structurally, the different FAsdLA, MA, PA, SA (12, 14, 16, and 18 C)dand fatty alcohol CAl (C16) differ only by a few (-CH2-) groups. When granules were made with 2 components, that is, oil and FA, the loading maximum was about 28% wt/wt for LA, MA, PA, and SA. The CAl, on the other hand, allowed loading up to 40%, without excess oil being visible after cooling. Including Neusilin® US2 in the formulation allowed increased oil loading levels up to 48% wt/ wt due to its high oil-adsorbing capacity. Constructing the 3-component phase diagram allowed us to define optimum ratios of the mixture components for production of core granules with high oil loading, good dispersibility (affected the size and shape of granules) at molten temperatures, and good mechanical strength (hardness) after solidification, which was necessary to endure subsequent processing steps. This analysis led to an optimal weight ratio of each component of CIN:US2:PA at 48:10:42, and this formulation was then used for scale-up production. Granules were coated with Eudragit® L 100 (Evonik Industries) to render pH-dependent release behavior with fast release at pH > 6 at the jejunum, ileum, and cecum sections of the intestines. For some applications, though, enteric coating may not be required where a slow release into the environment is more desirable to reduce pathogens on the feedlot floor surface.29

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The behavior of the mixtures could be explained by the saturation point of the Neusilin® powder component. As shown in Figure 2, Neusilin® powder became saturated around 78%-80% wt/wt liquid. Below this saturation point, the mixture still exists as a powder irrespective of the temperature; hence, it cannot be dispersed well in an aqueous continuous phase without taking up water to become dispersible (not processable). Thus, when at molten temperatures, the 3-component mixture simplifies to a 2phase, liquid (FA and EO) and solid (neusilin US2) mixture and the level of powder saturation becomes dominant in affecting the properties of the mixture. Figure 3 shows that at molten temperatures, the FA and CIN are both miscible, and they penetrated the powder as 1 liquid; depending on the extent of US2 saturation, this mixture behaved as a liquid when near or above saturation of the powder (~84% liquid:~16% powder). It was observed that the granule formulation affected the size distribution primarily by the degree of liquid saturation of the Neusilin® US2 powder (Fig. 4). Granules became larger as the amount of US2 was increased from 10% to 13% while the degree of powder saturation decreased. As the level of available liquid (CIN þ molten FA) saturated the available powder (e.g., >48% CIN, US2 10%), smaller droplets were formed which led to smaller granules as the molten mixture behaved as a low-viscosity liquid. In contrast, as the saturation level of US2 is decreased or if the US2 >10%, the powder exerts a viscosity-enhancing effect on the molten liquid mixture, leading to larger droplets and granules. This effect is one of the properties of magnesium aluminum silicates, and other silicates widely used in cosmetics.30 However, if the powder was oversaturated with oil, this effect appeared less influential in the present method. After cooling to room temperature, the FA resolidified while CIN remained liquid because no chemical reactions among the components were expected or observed. Due to displacement of CIN by molten FA during mixing with US2, the maximum loading in the cooled state was about 48%-50% or less than that without FA. Incorporating more than 50% CIN initially led to excess superficial CIN appearing, and a paste or liquid was obtained at room temperature instead of granules. The loading limit was thus determined to be 50% CIN in the final formulation of granules. PA was chosen in formulating larger batches of granules for offering the best combination of higher granules strength and fast, more complete release of the encapsulated CIN, in addition to the lower temperature required for preparation vs SA. Thus, using a higher melting FA like PA yielded harder granules than LA- and MAbased granules yet released the encapsulated oil better than SA or CAl. However, fatty alcoholebased granules resulted in decreased extent of release possibly due to the less polar nature of the fatty alcohol compared with FAs. Coated granules exhibited only partial resistance to release in SGF with around 27% of the CIN being released from coated granules. The early release of the activity in SGF could have been caused by the migration of the volatile liquid oil from the cores to the coating layers during the fluid-bed process. The long coating times typically used (>6 h) as well as the complex processes occurring during film formation31 could have contributed to this, as well as loss of the activity as the constant fluidization by air could have drawn away some of the volatile CIN from the cores. Further optimization of the fluid-bed coating process or selecting an alternative coating process32 (e.g., dry coating) could be evaluated in future experiments. Antibacterial Activity and Storage Stability of CIN Granules E. coli K88 is an enterotoxigenic strain of E. coli that commonly causes piglet diarrhea during the postweaning period when animals are most susceptible to infection.33 This particular strain of

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Figure 6. (a) Lauric acid granules uncoated (left) and after coating (right) and (b) palmitic acid granules with 42% wt/wt CIN, coated with Eudragit® L100 and subcoated with Kollicoat® IR.

E. coli produces K88 pili that adhere at the jejunum and ileum epithelial regions of the pig intestine, where specific receptor sites are expressed at the mucous surface,34 and then produce toxins that cause tissue damage and diarrhea. Furthermore, compared with other strains of E. coli, K88 is resistant to a spectrum of antibiotics but was found to be effectively inhibited by EOs like CIN.4 Even though CA has little inhibitory activity against E. coli K88, it has good inhibitory activity against a variety of fungi35 and spoilage bacteria36 with MICs in the 1-5 mM (150-750 mg/mL) range. LA-based granules showed an overall better killing effect with a lower MBC occurring at 450 mg/mL compared with the MBC of 600 mg/mL for free CIN oil (Fig. 8b), whereas control granules containing only excipient (US2 and either LA or PA at the equivalent CIN concentrations ~450-1000 mg/mL) did not show significant inhibitory activity against E. coli K88, suggesting that a synergistic activity of the LA-CIN mixture might be happening. This result could be attributed to the antibacterial effect of FAs that were used in the granule formulation37 in addition to the melting point depression effect after mixing with the CIN oil, which allowed the granules to melt and disintegrate at the incubation temperature of 37 C (see Figure S1), allowing dispersal of LA and CIN within the test medium. Further study into this effect could be performed, but due to difficulty in coating LA granules, PA-based granules were chosen for subsequent development.

PA-based granules had the same antibacterial activity as the pure oil and did not exhibit an enhanced effect as LA due to its higher melting temperature and lower solubility. The MBC of CIN obtained in the present study was about 2-6 times higher than previous reports.4,7 This difference could be attributed to variations in some minor EO components normally found in EO products from different sources, and furthermore, EO mixtures can exhibit stronger antibacterial activity than the respective single EO component.38 By using the current strategy of encapsulating CIN (or other EOs) into granules and coating with an enteric polymer, a sufficient concentration (MIC to MBC range) of EO can potentially be delivered to the lower intestinal tract of pigs (site of disease) while minimizing or avoiding the early absorption of EO compounds in the upper GI tract.9 Such granule formulations could be an effective strategy, without using antibiotics, to combat piglet postweaning diarrhea caused by E. coli K88. Because the antibacterial activity of CA was determined to be much less than CIN against the target pathogen, it is necessary to prevent autoxidation of CIN in granules. This work has demonstrated that with addition of antioxidant 1% eugenol to CIN, granules made with PA and LA showed the same degree of stability, with or without coating, which can last for at least 1 y at room temperature or refrigerator temperatures (Fig. 5b). These positive results prompted the exploration of in vivo performance of the

Y.-H. Ma et al. / Journal of Pharmaceutical Sciences xxx (2015) 1e10

Figure 7. (a) Release of CIN in SIF (pH, 6.8) from core granules formulated with different fatty acid types and (b) release of CIN from coated granules under 2-stage dissolution. Values represent average ± SD.

coated CIN granules in a subsequent study which will appear in another publication (Y.H. Ma et al, unpublished data, 2015). Conclusions Granular formulations of CIN, a model EO compound with good antimicrobial activity, were successfully developed with loadings up to 50% wt/wt. The core granules were prepared by a melt dispersionesolidification method using rationally selected oiladsorbing powder Neusilin® US2 and lipids at an optimized ratio of CIN:US2:lipid. The enteric polymer-coated granules based on PA exhibited fast release of CIN at intestinal pH, suitable for sitespecific delivery of CIN in the lower intestines where E. coli reside most. The addition of antioxidant eugenol to the granule formulation significantly increased CIN stability to at least 1 y. The LAencapsulated CIN granules were found more effective in vitro against E. coli K88 than free CIN with about 33% lower MBC but were unsuccessfully coated by fluid bed, while PA-based granules which were coated successfully exhibited an MBC similar to the free oil. The in vitro results obtained here indicate the potential of CINcontaining granules in combating E. coli K88 and other susceptible pathogens. Further in vivo studies are warranted to verify the effectiveness of CIN granules. The formulations and method developed in this work for encapsulation of oils in solid granules are relatively simple and economical and can potentially be used to encapsulate a variety of pure EOs and their mixtures or other lipophilic liquid actives.

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Figure 8. (a) Inhibitory activity of CIN vs CA toward E. coli K88 grown in TSB culture based on change in OD600 over 6 h at 37 C with 200 rpm shaking. (b) Antibacterial activity of CIN oil and granules against E. coli K88 in TSB medium. Data points represent average ± SD.

Acknowledgments The authors thank the Natural Science and Engineering Research Council (grant no. RGPIN 170460-13), Agriculture and Agri-Food Canada (grant no. J-000235.001.04), and the Canadian Poultry Research Council for funding the research. Escherichia coli K88 strain JG280 was a gift from Dr. C. Gyles, University of Guelph.

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