Release and swelling studies of an innovative antidiabetic-bile acid microencapsulated formulation, as a novel targeted therapy for diabetes treatment.

http://informahealthcare.com/mnc ISSN: 0265-2048 (print), 1464-5246 (electronic) J Microencapsul, Early Online: 1–6 ! 2014 Informa UK Ltd. DOI: 10.3109/02652048.2014.958204

RESEARCH ARTICLE

Release and swelling studies of an innovative antidiabetic-bile acid microencapsulated formulation, as a novel targeted therapy for diabetes treatment

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Armin Mooranian1, Rebecca Negrulj1, Hesham S. Al-Sallami2, Zhongxiang Fang3, Momir Mikov4,5, Svetlana Golocorbin-Kon4,5, Marc Fakhoury6, Frank Aruso7 and Hani Al-Salami1 1

Biotechnology and Drug Development Research Laboratory, School of Pharmacy, Curtin Health Innovation Research Institute, Biosciences Research Precinct, Curtin University, Perth, Western Australia, Australia, 2School of Pharmacy, Otago University, Otago, New Zealand, 3 School of Public Health, Curtin University, Perth, Western Australia, Australia, 4Department of Pharmacology, Toxicology and Clinical Pharmacology, Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia, 5Department of Pharmacy, Faculty of Medicine, University of Montenegro, Podgorica, Montenegro, 6Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada, and 7School of Biomedical Sciences, Curtin Health Innovation Research Institute, Biosciences Research Precinct, Curtin University, Perth, Western Australia, Australia Abstract

Keywords

In previous studies carried out in our laboratory, a bile acid formulation exerted a hypoglycaemic effect in a rat model of type 1 diabetes (T1D). When the antidiabetic drug gliclazide was added to the bile acid, it augmented the hypoglycaemic effect. In a recent study, we designed a new formulation of gliclazide–deoxycholic acid (G-DCA), with good structural properties, excipient compatibility and which exhibited pseudoplastic–thixotropic characteristics. The aim of this study is to test the slow release and pH controlled properties of this new formulation. The aim is also to examine the effect of DCA on G release kinetics at various pH values and different temperatures. Microencapsulation was carried out using our Buchi-based microencapsulating system developed in our laboratory. Using sodium alginate (SA) polymer, both formulations were prepared including: G-SA (control) and G-DCA-SA (test) at a constant ratio (1:3:30), respectively. Microcapsules were examined for efficiency, size, release kinetics, stability and swelling studies at pH 1.5, 3, 7.4 and 7.8 and temperatures of 25 C and 37 C. The new formulation is further optimised by the addition of DCA. DCA reduced bead-swelling of the microcapsules at pH 7.8 and 3 at 25 C and 37 C, and even though bead size remains similar after DCA addition, the percentage of G release was enhanced at high pH values (pH 7.4 and 7.8, p50.01). The new formulation exhibits colon-targeted delivery and the addition of DCA prolonged G release suggesting its suitability for the sustained and targeted delivery of G and DCA to the lower intestine.

Artificial-cell microencapsulation, bile acid, diabetes, gliclazide

Introduction Diabetes mellitus (DM) is a serious health issue that is affecting millions of people globally. The prevalence of DM is increasing globally at an alarming rate due to lifestyle changes, aging, urbanisation, increasing obesity and physical inactivity (Wild et al., 2004; Chan et al., 2009). DM is classified as type 1 diabetes (T1D) or type 2 diabetes (T2D). T1D is an autoimmune disease marked by the destruction of b-cells of the pancreas, resulting in a partial or complete lack of insulin production and the inability of the body to control glucose homeostasis (Barbeau et al., 2007). T2D develops due to genetic and environmental factors that lead to tissue desensitisation to insulin (Moore et al., 2003). Antidiabetic drugs are widely used and are effective in

Address for correspondence: Dr. Hani Al-Salami, Senior Lecturer of Pharmaceutics, School of Pharmacy, Curtin University, Perth, WA, Australia. Tel: +61 8 9266 9816. Fax: +61 8 9266 2769. E-mail: [email protected]

History Received 4 April 2014 Revised 23 July 2014 Accepted 4 August 2014 Published online 29 September 2014

minimizing variations between peaks and troughs of blood glucose levels in diabetic patients. However, the risks of hypoglycaemia and toxin build up in the gut remain major issues which may result in compromised absorption and efficacy of many antidiabetic drugs (Cani et al., 2007). Commonly used antidiabetic drugs include insulin for T1D, and gliclazide (G) and metformin as well as many others for T2D. G is a second generation sulphonylurea drug commonly used to treat T2D. G primary mechanism of action is to stimulate insulin secretion by pancreatic beta cells which results in improved glucose regulations (Rendell, 2004; Mikov et al., 2008). However, in addition to its pancreatic effects, G also has many desirable extrapancreatic effects making it a good candidate for T1D (Al-Salami, et al., 2008a, 2008b, 2009, 2012). Such desirable effects include antioxidants, antiradicals, antithrombotic and insulin-mimicking effects, in particular, when coadministered with bile acids and probiotics (Al-Salami et al., 2008c; Mooranian et al., in press). Bile acids are naturally produced in humans and have many beneficial characteristics. Our preliminary data and work either


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published (Mikov et al., 2007; Al-Salami et al., 2008b, 2008d, 2009, 2012; Negrulj et al., 2013; Fakhoury et al., 2014), in press (Mooranian et al., in press a, in press b; Mikov et al., 2007; Al-Salami et al., 2008d; Negrulj et al., 2013; Fakhoury et al., 2014), or currently under review, have demonstrated strong potentials of G–bile acids mixtures in diabetes treatment through better regulation of glucose levels (a hypoglycaemic effect) or reduction of inflammation. In a recent publication, the bile acid deoxycholic acid (DCA) has enhanced G-permeation in our rat model of T1D (Lalic-Popovic et al., 2013). However, this permeation-enhancing effect did not bring about a hypoglycaemic effect. In a more recent study, we successfully designed a novel and complex multicompartmental microcapsules of G-DCA with good compatibility, morphology and excellent rheological characteristics (manuscript is currently under review). Thus, in this study, we aimed at characterising the newly developed G-DCA microcapsules in terms of drug contents, production yield, stability, size and their swelling profiles. We also aimed at investigating the effect of DCA on G release profiles at various pH and temperature values.

Materials and methods Materials G (99.92%), LVSA (99%), and DCA (99%) were purchased from Sigma-Aldrich Co (St Louis, MO). Calcium chloride dihydrate ([CaCl2. 2H2O] 98%) was obtained from Scharlab SL (Barcelona, Spain). All solvents and reagents were supplied by Merck (Darmstadt, Germany), and were of high-performance liquid chromatography (HPLC) grade and used without further purification. Drugs preparations Stock suspensions of G (20 mg/ml) and DCA (1 mg/ml) were prepared by adding the powder to 10% ultra water-soluble gel. The CaCl2 stock solution (2%) was prepared by adding CaCl2 powder to HPLC water. All preparations were mixed thoroughly at room temperature, for 4 h, stored in the refrigerator, and used within 48 h of preparation. Preparation of microcapsules Microcapsules of G-loaded LVSA were prepared using our Buchi-based-microencapsulating system. Parameters that were used include: a frequency range of 1000–1500 Hz, flow rate of 4 ml/min at a consistent air pressure of 1.5 bar. Polymer solutions containing SA and G with or without DCA were made up to a final concentration (of G–DCA–SA) in a ratio of 1:3:30, respectively (Al-Salami et al., 2008d, 2009). Microcapsules were collected from our microencapsulating system and for each formulation three independent batches were prepared and tested separately (n ¼ 3). All microcapsules (G-loaded and G–DCA–SA loaded) were prepared and treated in the exact same way. Microcapsules were dried by using the Stability Chambers (Angelantoni Environmental and Climatic Test Chamber, Italy). The weight of the recovered dry particles was recorded and the G contents, production yield, microencapsulation efficiency, zeta potentials and mean particle size of each preparation were all measured and compared, as described below. Characterisation of loaded microcapsules Drug content, production yield, microencapsulation efficiency and stability studies Drug content, production yield and microencapsulation efficiency. One gram of microcapsules were carefully weighed,

J Microencapsul, Early Online: 1–6

grounded and dissolved in 200 ml of phosphate buffer (pH 7.8) and the suspension was stirred with a magnetic stirrer for 6 h. Two millilitres of the solution were transferred to 100 ml flask and diluted with phosphate buffer (vehicle) to 100 ml. Aliquots of the dissolution medium (2 ml) were withdrawn at predetermined time points (every 200 s) and filtered through Millipore, 0.22 mm filter. Amount of dissolved drug was determined spectrophotometrically at 229 nm against the buffer as blank (Prajapati et al., 2008; Takka and Cali, 2012). The measurements were performed under sink conditions, and average values were calculated. Absorbances were measured using a UV spectrophotometer (Shimatzu UV-vis spectrophotometer 1240, Japan). G concentrations were calculated from the calibration curve. All analyses were carried out in triplicate (n ¼ 3). The drug contents, production yield and microencapsulation efficiency were calculated from the following equations (Barakat et al., 2013). Calculated amount of G in the microcapsules 100 ð1Þ % Drug content ¼ Total weight of microcapsules Total weight of the microcapsules % Production yield ¼ Total weight of the polymer þdrug solution 100 % encapsulation efficiency ¼

Drug content 100 Theoretical content

ð2Þ

ð3Þ

Physical and chemical stability. The stability test was carried out by placing predetermined amounts of freshly prepared microcapsules onto sterile petri dishes (30 microcapsules in each) and storing them in thermostatically controlled ovens at 20 C, 5 C, 25 C and 40 C with relative humidity set at 35% for 3 days. The experiment was conducted using stability chamber (Angelantoni Environmental and Climatic Test Chamber, Italy). A temperature and humidity regulator was used to ensure constant experimental conditions. At the end of the experiment, the microcapsules were analysed for any changes in appearance and for the determination of the amount of drug remaining in each formulation, using a validated UV-Vis stability-indicating method. Briefly, the dosage forms were crushed and dissolved in 200 ml phosphate buffer at pH 7.8. The solution was filtered and the first 20 ml of the solution was removed, and 10 ml of the filtrate was diluted to 100 ml in a volumetric flask with PBS. One millilitre aliquot of the prepared solution was transferred to 10 ml volumetric flask, and the volume was completed with the buffer. A calibration curve was constructed for G in phosphate buffer across the concentration range of 0.1 mg to 40 mg/ml with R2 ¼ 0.99 (data not shown). Zeta-potential and size analysis. To determine the electrokinetic stability and size uniformity of the microcapsules in the dispersion system, zeta potential and size distribution for the microencapsulated formulation of G-SA and for G-DCA-SA were measured by photon correlation spectroscopy using a Zetasizer 3000HS (Malvern Instruments, Malvern, UK) and by Mie and Fraunhofer scattering technique using Mastersizer 2000 (Malvern Instruments). The measurements were performed at 25 C with a detection angle of 90 , and the raw data were subsequently correlated to Z average mean size using a cumulative analysis via OmniSEC-Zetasizer software package. All analyses were performed on samples appropriately diluted with filtered deionised water. All determinations were performed in triplicate and results were reported as mean ± SD.

Antidiabetic-bile acid microencapsulated formulation

DOI: 10.3109/02652048.2014.958204

Drug release studies (in-vitro dissolution test) A weighed samples (2 g) of G and DCA loaded microcapsules were suspended in 200 ml of phosphate buffer solution at pH values of 1.5, 3, 7.4 and 7.8 for 2 h, as appropriate. The dissolution medium was stirred at 200 rpm. Sink conditions were maintained throughout the assay period. All the experiments were carried out at 25 C. All absorbances of the solution were measured every 200 s using our Hewlett Packard-based Time Controlled UV-Spec mounted with close-loop flow system. All analyses were carried out in triplicate (n ¼ 3).


Swelling studies To determine the swelling properties of the beads, 50 mg dry beads were weighed and placed in 20 ml of buffer of two pH values (3 and 7.8) and two temperatures (25 C and 37 C) for 6 h. The swollen beads were then removed at periodically predetermined intervals (hourly). The wet weight of the swollen beads was determined by blotting them with a filter paper to remove moisture adhering to the surface, immediately followed by weighing on an electronic balance. All experiments were done in triplicate. The percentage of swelling of the beads was calculated from the following formula (Barakat et al., 2013): Weight ratio ¼

final weight 100 Initial weight

ð4Þ

Statistical analysis Values are expressed as means ± SD. Drug content, production yield and microencapsulation efficiency were assessed using Student’s t test. Statistical comparisons between G concentrations in different microencapsulated formulations were carried out by repeated measures analysis of variance (ANOVA) using each formulation excipients as fixed terms. Swelling studies were assessed using two-way ANOVA to assess the main effects of microcapsule formulation and pH and their two-way interaction. Tukey HSD post-hoc comparison of means was done only when the associated main effect or interaction was statistically significant. The best fit model was derived using GraphPad Prism software (V6; GraphPad Software, Inc., San Diego, CA). Statistical significance was set at p50.05.

Results and discussion Drug content, production yield, microencapsulation efficiency and stability studies Drug content, production yield and microencapsulation efficiency As shown in Table 1, the results for % drug content for both formulations revealed consistent drug-microcapsule content with very low variations. As expected, there is less G in the

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G-DCA-SA microcapsules compared with G-SA microcapsules (p ¼ 0.04). The total production yield of G-SA and G-DCA-SA microcapsules prepared ranged from 89% to 98% with no significant difference between both formulations. Good levels of G-loading (microencapsulation) efficiency were achieved for all microcapsules, with values averaging 90 ± 10%. Stability, Zeta-potential and size analysis Accelerated stability studies were carried out over 3 days, testing both formulations at 20 C, 5 C, 25 C and 40 C and a relative humidity of 35%. Upon visual examination, both formulations (G-SA and G-DCA-SA) retained their original morphological characteristics (sphericity and homogenous particle size distribution) across the experimental conditions. However, there were some changes in the colour, overall size and quality of the microcapsule surfaces at higher temperature. Specifically, at 20 C, microcapsules retained their original size and some had form agglomerates which were easily re-dispersed. At this temperature, microcapsules were white and spherical and had retained their original quality (soft and flexible). At the higher temperatures, the microcapsules changed colour due to oxidation of the alginate from cream to orange at 5–25 C and brown at 40 C. Whilst retaining their spherical changes and even homogenous particle size distribution, they had shrunk in size by 50%, with the smallest microcapsules being at the highest temperature of 40 C. This may be explained by the loss of moisture content, reducing the overall surface area and volume of each microcapsule. In addition, the microcapsules had become brittle at all temperatures (except at 20 C) owing to the loss of moisture within the microcapsules. Upon UV analysis, the amount of G remaining in freshly prepared microcapsules revealed an average % drug content of 5 for G-SA microcapsules and 3 for G-DCA-SA (Table 1). This complemented the visual characterisation of the microcapsules following accelerated stability testing and confirmed uniformity of drug contents and is in line with the used drug: polymer ratios (G-SA 1:30 & G–DCA–SA 1:3:30). Neither any peaks for a biodegradable polymer nor any alteration of the chromatographic pattern of G was observed, indicating that the experimental conditions for the microencapsulating process did not compromise drug analysis. Furthermore, the results of the drug content and encapsulation efficiency showed minimum variation among repeated samples which confirms the reproducibility of our developed microencapsulation method. The dispersion system formed by the microcapsules is stable as shown by Zeta-potential values ranging from 52 to 66 mV, whilst the mean particle size remained within a narrow range of 1000–1050 mm (Table 1) suggesting significant uniformity in the size distribution of the microcapsules and no significant difference after the incorporation of DCA to G-microcapsules.

Table 1. Drug contents, production yield, encapsulation efficiency, mean particle size and zeta potential of G–SA and G–DCA–SA microcapsule formulations.

Formulation code G–SA G–DCA–SA

*p50.05.

Formula composition Gliclazide–low viscosity sodium alginate microcapsules Gliclazide–deoxycholic acid–low viscosity sodium alginate microcapsules

Encapsulation efficiency (%) ± SD

Production yield (%) ± SD

Mean particle size (mm) ± SD

Zeta-potential (mV) ± SD

5 ± 0.2

98 ± 2

86 ± 8.2

1000 ± 32 (span 0.68)

66.1 ± 1.6

3 ± 0.15*

90 ± 5

83 ± 6.6

1050 ± 63 (span 0.68)

52.4 ± 1.7

Drug content (%) ± SD

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Figure 1. Gliclazide release over time from G–SA and G–DCA–SA microcapsules at pH 3 and 1.5. G–SA: gliclazide–sodium alginate. G–DCA–SA: gliclazide–deoxycholic acid–sodium alginate.

Figure 2. Gliclazide release over time from G–SA and G–DCA–SA microcapsules at pH 7.8 and 7.4. G–SA: gliclazide–sodium alginate. G–DCA–SA: gliclazide–deoxycholic acid–sodium alginate.

Drug release studies and in-vitro dissolution G release from the microcapsules was studied in triplicates across 4 pH values (1.5, 3, 7.4 and 7.8) at 25 C using both formulations G–SA and G–DCA–SA. pH values were chosen based on our previous studies examining the best sites of drug absorption in the GIT (Al-Salami et al., 2008e; Jyothi et al., 2010). The release of G was slower and largely dependent on both the pH and the composition of the coating. As shown in Figures 1 and 2, G release was smaller at low pH values (1.5 and 3) and the bile acid coating in the G–DCA–SA formulation allowed for a slower rate of drug release. As expected, the release of G was increased at higher pH values (especially at pH 7.8) for both formulations. Notably, at pH 7.8, DCA reinforcement allowed for a more controlled and sustained release of G. This has important ramifications for diabetes therapy, as previous work in our laboratory has confirmed the distal ileum as the intended site of G absorption where pH values are in the range of 7–7.8 (Al-Salami et al., 2008e). Our results demonstrate that the DCA-reinforced microcapsules stipulated controlled drug release at the targeted pH of 7.8. The mechanisms responsible for such differences in the release profiles are affected by the pH of the medium. At low (stomach) pH, the hydrated SA is modified to a porous structure known as alginic acid skin (Al-Salami et al., 2008e). This insoluble structure results in the shrinkage of alginate and thus encapsulated drugs are not completely released. However at higher pH (intestine) such as the distal end of the small intestine, the alginic


acid forms a soluble viscous layer due to the rapid dissolution and solubilisation of the alginate matrix resulting in the burst of encapsulated drugs. As evident from the G–SA dissolution graphs (Figure 1 and Figure 2), this release profile is not in a sustained, controlled pattern and may result in reduced bioavailability of G. Whilst there is substantial evidence regarding bile saltreinforced microcapsules used to ensure controlled drug release from microcapsules, little is known about the incorporation of bile acids rather than bile salts in the microencapsulation formulation (Bakatselou et al., 1991; Luner, 2000; Martoni et al., 2008). In this study, the bile acid DCA was used. In our previous work, we used a similar formulation (without microencapsulation) and have shown an antidiabetic effect in an animal model of T1D (Al-Salami et al., 2008e). We have also shown through ex-vivo studies that the bile acid-derivative does enhance G permeation through the ileal mucosa of diabetic rats (Al-Salami et al., 2008e). Our dissolution and release studies using microcapsules from both formulations may provide an explanation as to why the BA-derivative has improved the G absorption through the ileum (Al-Salami et al., 2008e). We therefore hypothesise that the bile acid, DCA, reinforces the soluble polymer alginate matrix by cross-linking with the alginate such that rapid burst of drug is evaded at higher pH values. This brings about a prolonged release targeting the lower intestine, which facilitates better absorption and less gut-metabolism. However, due to the fact that in our previous work, we did not microencapsulate our G–DCA–SA formulation, the improvement of G permeation by DCA was rather limited. Additionally, unlike bile salts which contain both hydrophilic and hydrophobic ends due to their zwitterionic structure, bile acids lack good solubility and thus, resists greater dissolution and solubilisation at higher pH values whilst retaining cross-linking (inotropic bridging) with the alginate formulation of the microcapsules (Al-Salami et al., 2008e; Raskovic et al., 2008; Yang et al., 2009; Pavlovic et al., 2012). Hence, they reinforce the microcapsule wall integrity allowing for a more gradual release of entrapped drugs as they are not as easily solubilised as bile salts (Luner, 2000; Yang et al., 2009). Thus, we anticipate our G-DCA–SA microcapsules to exert a more slow-release properties, better controlled effect and optimised pharmacokinetic and pharmacodynamic characteristics of G when administered to the diabetic animals (in vivo). One of the potential advantages of using bile acids over bile salts is that they are far less soluble whilst still forming cross-links with the matrix and they help stabilise the membrane from rapid disintegration, as shown in Figure 1 and Figure 2. Accordingly, this formulation is promising as a platform for antidiabetic drug delivery. Swelling studies Figure 3 and Figure 4 show that the formulation type, the pH of the medium and the temperature do have an effect on the swelling characteristics of the beads. In line with G release studies, the percentage of bead swelling was reduced by the addition of DCA. DCA reduced bead swelling at low and high pH (at 37 C, Figure 4) and to a less extent, at lower temperature (at 25 C, Figure 3). DCA exerted a stronger reduction in bead swelling, at higher temperature suggesting better control of G release across the stomach and lower proximal site of the intestine, whilst still maintaining targeted delivery at pH 3 and pH 7.8 (Figure 3 and Figure 4). This may be due to the fact that even though alginate has been shown to undergo substantial swelling at higher pH and temperature due to higher water uptake (Bajpai et al., 2006; Pasparakis and Bouropoulos, 2006; Takka and Cali, 2012), DCA reduces such effect and thus brought about a stronger control of G release. Moreover, at high pH values, the porosity and

DOI: 10.3109/02652048.2014.958204

Antidiabetic-bile acid microencapsulated formulation

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Conclusion Our microencapsulation method of G–SA (1:30) and G–DCA–SA at the set ratio of 1:3:30, respectively, produces suitable characteristics for the oral delivery of G and the bile acid DCA, which are useful for the treatment of diabetes. The microcapsules display appropriate stability, release profile, drug-content and production yield and pH-targeted delivery at various pH values and different temperatures. It was also shown that the addition of DCA can help further optimise the new microencapsulation formulation and reduce the bead swelling of microcapsules. An interesting future investigation will examine the suitability of bile acid-incorporated microcapsules, as a platform, for the oral delivery of potential antidiabetic drugs such as Probucol.


Acknowledgements Figure 3. Swelling characteristics of G–SA and G–DCA–SA beads (pH 3 and 7.8) at 25 C. G–SA: gliclazide–sodium alginate. G–DCA–SA: gliclazide–deoxycholic acid–sodium alginate.

The authors acknowledge the CHIRI, Biosciences Research Precinct at Curtin University.

Declaration of interest The authors declare no conflict of interest.

References

Figure 4. Swelling characteristics of G–SA and G–DCA–SA beads (pH 3 and 7.8) at 37 C. G–SA: gliclazide–sodium alginate. G–DCA–SA: gliclazide–deoxycholic acid–sodium alginate.

solubilisation of the polymer matrix are expected to be higher (Wee and Gombotz, 1998; Takka and Acarturk, 1999; Takka and Cali, 2012). Temperature also plays a key part in swelling and water uptake as higher temperatures causes erosion and disintegration of the matrix wall allowing for greater water penetration (Anal et al., 2003; Anal and Stevens, 2005; Bajpai et al., 2006; Pasparakis and Bouropoulos, 2006; Al-Kassas et al., 2007; Nochos et al., 2008). At pH 7.8 and higher temperature (37 C), the G–SA beads (without DCA) experienced greater swelling. Again, at pH 3.0 and 37 C, G–SA–CA beads underwent greater swelling due to enhanced water uptake. This clearly supports our hypothesis that the bile acid used in the formulation has enhanced membrane stabilisation most likely via cross-linking and ionic interactions with the alginate matrix. Another possible explanation is that the free carboxyl groups of secondary bile acids DCA act as water binding sites allowing for greater water uptake and thus weight gain and more bead-swelling capacity (Al-Salami et al., 2008e; Raskovic et al., 2008; Yang et al., 2009; Pavlovic et al., 2012). This swelling characteristic property of bile acidreinforced beads is desirable in forming a controlled release system, especially when it does not adversely influence production yield or system stability (Table 1).

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