Vibha Krishnan a, S. Sasikumar b, Febin Prabhu Dass c, R. Vijayaraghavan b* Introduction
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1 Trends Biomater. Artif. Organs, Floating Vol 24(3), Alginate pp Drug (21) Delivery System - An In Vitro Study Effect of Pore Forming Agents on the Physical Characteristics and Release Kinetics of Levofloxacin Hemihydrate from Floating Alginate Drug Delivery System - An In Vitro Study Vibha Krishnan a, S. Sasikumar b, Febin Prabhu Dass c, R. Vijayaraghavan b* a School of Bio Sciences and Technology, b Materials Division, School of Advanced Sciences, c Bioinformatics Division, School of Bio Sciences and Technology, VIT University, Vellore , Tamil Nadu, India Corresponding author: R. Vijayaraghavan (rvijayaraghavan@vit.ac.in) Received 16 February 21; Accepted 9 May 21 Floating Drug Delivery Systems (FDDS) provides a comprehensive solution for the retention of drug in stomach than the conventional dosage forms. In this study, floating alginate beads were prepared using sodium bicarbonate or potassium carbonate as a gas-forming agent and methyl cellulose as a binder. The effect of pore forming agents on the morphology, porosity, density and drug release kinetics of the beads were studied. Levofloxacin Hemihydrate was used as a model drug for in-vitro studies. The average pore size of the beads was measured using optical microscope which shows the pore size of the beads is in the range of 1ìm to 3ìm. The result indicates the better pore forming ability of sodium bicarbonate over potassium bicarbonate hence the potassium carbonate beads shows sustained release than the sodium bicarbonate beads. Introduction In oral drug delivery system bioavailability of the drug depends on gastric residence time (GRT) of the drug. Absorption of the drug in gastrointestinal tract is a complex procedure and it depends on many variables which includes contact time and transit time. Several parameters are taken care in designing a controlled release system for better absorption and enhanced bioavailability. One such difficulty is the inability to deliver the dosage form in the desired area of the gastrointestinal tract within the stipulated time. In order to resolve this problem the gastro retentive dosage forms (GRDF) are used which can remain in the gastric region for several hours and hence significantly prolong the gastric residence time of drugs[1]. The mechanisms like mucoadhesion, sedimentation, expansion and flotation controls the gastric retention of the delivery systems which delays the gastric emptying [2]. The advantages of GRDF are increase in bioavailability[3], sustained release of the drug[4], low dosage formulation and increase in the solubility of the drugs that are less soluble in a high ph environment. In addition to this the toxicity from the drug can be minimized which can otherwise be a crucial limiting parameter owing to the repeated doses of the drug administered. Several approaches were made to increase the gastric residence time of GRDF and majority of the work was carried out by employing lowdensity polymeric substances which can float in the gastric juice[5]. Studies have shown that floating units which remain buoyant on gastric juice cannot be easily expelled from the stomach when compared with the non-floating units which stay in antrum region and easily moved by the peristaltic waves. Hence lot of research is carried out in floating drug delivery systems (FDDS) with different low-density
2 14 V. Krishnan, S. Sasikumar, F.P. Dass, R. Vijayaraghavan organic substances as a matrix to load the drug. Alginate beads are stable in acidic media and easily depredated in alkaline media. Hence, floating alginate beads are effective against the harmful stomach bacteria s such as H. pylori[6]. These properties have enabled widespread use of alginate beads for sustained release of drugs[7]. In the case of alginate beads as FDDS, it has a lower density than the gastric juice and hence remains buoyant and the mode of release of drug from the matrix is speculated to be a combination of both diffusion and dissolution [8]. As a result of buoyancy, the entire delivery system does not sink in the gastric juice hence the drug is released only in the interface between gastric juice and the dosage form by dissolution. The drug that is trapped in the alginate matrix comes out from the centre of the bead where it is trapped in the alginate loops to the periphery through the process of diffusion. Due to the less area of contact the release from alginate beads is found to be sustained [9]. When the floating and non-floating dosage units were compared it is found that regardless of their sizes the floating dosage units remained buoyant on the gastric contents throughout their residence in the gastrointestinal tract, while the nonfloating dosage units sank and remained in the lower part of the stomach. Floating drug delivery systems can be prepared by means of the reaction between carbonate salts present in alginate matrix and acetic acid which produces carbon dioxide gas. The carbondioxide evolved permeates through the alginate leaving gas bubbles or pores. The most commonly used pore forming agents are NaH and Ca [1-12]. The present work is aimed to develop a FDDS which can form a dual role of sustained release and gastric emptying. The objective of the work is to study the effects of pore forming agents on the morphology, density, porosity and release kinetics of the FDDS system. Modifications have been introduced in the current work so as to improve on the methods that have already been attempted by using pore-forming agents. Materials and Methods Preparation of alginate beads A solution of drug carrier was prepared by dissolving 3g of sodium alginate and.1g of methyl cellulose in 1ml of distilled water. Stock solution of drug was prepared by adding.1g of Levofloxacin hemihydrate in 1ml distilled water. 3ml of the Sodium Alginate- Methyl Cellulose mixture and 5ml of the drug solution was mixed together with a consistent stirring using magnetic stirrer. To the sodium alginate and drug mixture varying amounts of pore forming agent (NaH / K 2 ) was added. The various ratios of pore-forming agent to alginate prepared are 1:1, 1:2, 1:3, 1:4. The resultant mixture was degassed using sonicator which results in the formation of highly viscous alginate mixture containing poreforming agent and drug. 1% BaCl 2 solution containing 1% acetic acid solution was prepared using demineralized water. The viscous alginate mixture was added drop wise using 24G syringe into the BaCl 2 - acetic acid solution. The solution containing suspended beads was stirred with a magnetic stirrer for 1 min to improve the mechanical strength of the beads and allowed to complete the reaction by means of producing gas bubbles. The floating beads were kept in their respective BaCl 2 solutions for 3 minutes to improve its strength. The beads were filtered and kept in a hot air oven at 6 o C for drying. The dry beads were taken out and stored at dark for further analysis. Preparation of gastric juice In 2 ml of deionized water analytical grade chemicals of 3.5g of Glucose, 2.5g of NaCl,.6g of KH 2 PO 4,.11g of CaCl 2 and.37g of KCl were dissolved. The solution was sterilized and ph of the solution was brought down to 2. by adding 1M HCl and then volume was made up to one litre. In vitro release studies In a 5-mL Erlenmeyer flask with a ground-glass stopper, containing 5 ml of gastric juice and beads was kept over a magnetic stirrer at the temperature of 37 ± 1ºC with a stirring speed of 1 rpm. At regular time intervals, 5 ml of the buffer medium was collected and the
3 Floating Alginate Drug Delivery System - An In Vitro Study 141 levofloxacin hemihydrate concentration was measured by UV Spectroscopy by following the characteristic l max peak (293.7 nm). The content of flask was replenished with 5 ml of fresh gastric juice after each withdrawal. Characterization Morphology and pore dimensions of the beads were measured using Zeiss Metallurgical microscope-axios Kop 4 MAT. The amount of drug released was estimated using Hitachi, U- 28 Spectrophotometer, Japan. Results and Discussion Alginate was chosen as a matrix to carry the drug as it is a biocompatible and biodegradable polysaccharide which forms a bio-adhesive and stable gel with divalent cations such as Ca 2+, Sr 2+, and Ba 2+. Sodium bicarbonate and potassium carbonate were used as the pore forming agents as the mechanism of the reaction differs for bicarbonate ion and carbonate ion with acetic acid. The mechanism of reaction for pore-forming agent is as follows NaH + CH 3 COOH ---> CH 3 COONa + H 2 O + CO 2 K CH 3 COOH ---> CH 3 COOK + H 2 O + CO 2 Due to the different mechanism and weight ratio the ability to form the pores and the number of pores per unit area formed will differ for any two different pore forming agents. Due to this difference it is expected to form beads with different density, porosity and morphology. When the alginate and pore-forming agent mixture was added to BaCl 2 solution the hardening of beads takes place and the acetic acid reacts with pore-forming agent and forms pores in the beads. It is reported that the size and shape of the beads affects the gastric emptying. Studies showed that dosage forms with lesser dimension shows a better gastric residence time when compared with the one having larger dimensions. In the present study the beads obtained were spherical in shape and uniform in shape and size which is evident from Fig. 1. Average size of the beads before drying and after drying was measured using micrometer screw gauge and listed in Table 1. Average Table 1: Size of beads Pore-forming agent to alginate ratio Size of wet NaH beads cm Size of wet K 2 beads cm 1: : : :4.3.3 Table 2: Weight of beads Pore-forming agent to alginate ratio Weight of wet NaH beads g / 5 beads Weight of wet K 2 beads g / 5 beads 1: : : : weight of the wet beads and dry beads was carried out in electronic balance and listed in Table 2. The beads prepared using different pore-forming agents are comparable in size but not by means of weight. Table 1 shows the size of wet beads prepared using NaH is slightly smaller than the beads prepared using K 2. But Table 2 shows the weight of the beads prepared using NaH is less than 3% weight of beads prepared using K 2 as poreforming agent. This shows the density of the beads prepared using NaH is comparatively lesser than the one prepared using K 2 as pore-forming agent The change in density may be either due to the bigger pore size or more number of pores present in the beads. The number of pores per bead depends on the reaction betweenn the pore forming agent and acetic acid solution which releases CO 2. As the concentration of NaH decreases, the amount of CO 2 produced will be lesser and the beads formed will be less porous with increase in weight. From the measurements it is observed that the average bead size of wet beads is nearly times as that of the dry beads whereas the weight of wet beads is approximately 25 times of dry beads. Change in porosity will effect the drug release kinetics as bigger pores will have fast kinetics as the mechanism involves is dissolution whereas the smaller pores involves dissolution and diffusion together. This interpretation is in
4 142 V. Krishnan, S. Sasikumar, F.P. Dass, R. Vijayaraghavan Fig 1: Buoyancy of alginate beads (a) without pore forming agents (b) sodium bicarbonate as the pore forming agent 1 8 Sodium Bicarbonate Potassium Carbonate 1 75 Sodium Bicarbonate Potassium Carbonate % of Shrinkage Weight Loss :1 1:2 1:3 1:4 Ratio of alginate to pore-forming agent 1:1 1:2 1:3 1:4 Ratio of alginate to pore-forming agent Fig 2: Shrinkage of beads prepared using different pore-forming agent Fig 3: Weight loss of beads prepared using different pore-forming agents agreement with the values in Table 2 as the beads prepared using NaH shows only a small change in weight for higher concentration whereas the K 2 shows more change in weight at higher concentration when compared to the lower concentration. This may be due to increase in porosity of K 2 beads whereas due to highly porous nature of NaH beads further increase is restricted. Figure 2 shows the shrinkage of beads due to drying of beads is found to be higher in K 2 beads than NaHCO- 3 beads. This may be due to the less porosity of K 2 beads so that it undergoes maximum shrinkage whereas NaHCO- 3 beads were highly porous hence the shrinkage is comparatively less. Figure 3 shows the weight loss of beads due to drying is found to be higher in NaHCO- 3 beads than K 2 beads. This may be due to the less porosity of K 2 beads because of which it absorbs small quantity of water and hence, has a lesser weight loss during drying. On the other hand, NaH beads have greater porosity which results in more water absorption and hence, it has a higher weight loss. Optical microscope images (Fig. 4) of alginatesodium bicarbonate beads are in agreement with the physical characteristics of the beads.
5 Floating Alginate Drug Delivery System - An In Vitro Study 143 Fig 4: Optical microscope images of sodium bicarbonate - alginate beads Fig 5: Optical microscopy images of potassium carbonate - alginate beads The bead with higher pore-forming agent concentration shows bigger pores in the range of 4 to 42 um. With the decrease in the concentration of pore forming agent the pore size is found to be decreasing. For the lowest concentration of pore-forming agent the pores formed are found to be one third of the size of higher concentration beads.
6 144 V. Krishnan, S. Sasikumar, F.P. Dass, R. Vijayaraghavan :1 1:2 1:3 1: :1 1:2 1:3 1:4 % of release % of release Time (min.) Fig 6: Release kinetics of Levofloxacin hemihydrate from the beads prepared using sodium bicarbonate as the pore forming agent Time (Min.) Fig 7: Release kinetics of Levofloxacin hemihydrate beads prepared using potassium carbonate as the pore forming agent Optical microscope images (Fig. 5) of alginatepotassium carbonate beads shows comparatively smaller pores when compared with sodium bicarbonate - sodium alginate beads. The bead with higher pore-forming agent concentration shows bigger pores in the range of 2 to 22 ìm which is found to be half the size of pores formed by NaH. With the decrease in the concentration of pore-forming agent the pore size is found to be further decreasing. For the lowest concentration of pore-forming agent the pores formed are found to be one fifth of the size of higher concentration beads. The matrix of the beads are found to be more denser and compact than beads with NaH. From the optical microscopic images it is seen that NaH forms bigger pores where as the pores formed by K 2 are found to be smaller. Even though the pores formed by K 2 are smaller the number of pores formed per bead is more which is evident from the image (Fig. 5) of 1:4 ratio beads. The bigger size of pores formed by NaH may be due to the combination of smaller pores together to form a bigger size pore. The release kinetics (Fig. 6) of sodium bicarbonate beads shows a rapid release of Levofloxacin hemihydrate for higher concentration beads and sustained release for lower concentration beads. Due to the larger size of pores the higher concentration beads results in more dissolution than diffusion hence it shows a rapid release in the first few minutes. The lower concentration beads shows follows both dissolution and diffusion mechanism hence the release is found to be sustained. The release kinetics is found to be almost same for the beads with any concentration with lesser than 5% of NaH. Maximum of 25% of the concentration of the drug is eluted in 1 hour from the beads with higher pore-forming agent concentration and a maximum 12% of drug is eluted from the beads with lower pore-forming agent concentration. When compared to the sodium bicarbonate beads, potassium carbonate beads shows a better sustained release. From the release kinetics graph (Fig. 7) it is evident that the 1:1 beads shows a fast release due to larger pore size in comparison with the other ratios. The release kinetics is found to be slightly lower for every lower concentration which may be due to the lesser pore size. This may be due to the smaller pore size which will have lesser dissolution and more diffusion of drug and hence the release is sustained. Maximum of 15% of the concentration of the drug is eluted in 1 hour from the beads with higher pore-forming
7 Floating Alginate Drug Delivery System - An In Vitro Study 145 agent concentration and a maximum 7% of drug is eluted from the beads with lower poreforming agent concentration. Conclusion For the first time potassium carbonate is employed as a pore-forming agent and from the results it is concluded that both potassium carbonate and sodium bicarbonate are better pore forming agents. The release kinetics of sodium bicarbonate bead is rapid when compared to potassium carbonate bead hence it is recommended to use potassium carbonate beads for sustained release. From the results it is found that there is a direct correlation between the concentration of the pore forming agents and the size, weight, pore size and drug release kinetics of the beads. Pore volume of sodium bicarbonate beads is found to be higher than the potassium carbonate beads. Hence, sodium bicarbonate beads have lesser density and more buoyant. In terms of drug release kinetics, sodium bicarbonate beads shows faster release and potassium carbonate beads shows sustained release. Acknowledgements The authors thank VIT University management and DRDO, Grant in aid scheme, Government of India, for financial assistance and Technology Business Incubator, VIT for UV- Visible measurements. References 1. Murphy, C.S., Pillay, V., Choonara, Y.E., Du Toit, L.C.Gastroretentive drug delivery systems: Current developments in novel system design and evaluation (29) Current Drug Delivery, 6 (5), Arora, S., Ali, J., Ahuja, A., Khar, R.K., Baboota, S.Floating drug delivery systems: A review (25) AAPS PharmSciTech, 6 (3), art. no Menon, A., Ritschel, W.A., Sakr, A. Development and evaluation of a monolithic floating dosage form for furosemide (1994) Journal of Pharmaceutical Sciences, 83 (2), pp Singh, B.N., Kim, K.H. Floating drug delivery systems: An approach to oral controlled drug delivery via gastric retention (2) Journal of Controlled Release, 63 (3), pp Patel, A., Ray, S., Thakur, R.S.Invitro evaluation and optimization of controlled release Floating Drug Delivery System of metformin hydrochloride (26) Daru, 14 (2), pp Ishak, R.A.H., Awad, G.A.S., Mortada, N.D., Nour, S.A.K. Preparation, in vitro and in vivo evaluation of stomach-specific metronidazole-loaded alginate beads as local anti-helicobacter pylori therapy (27) Journal of Controlled Release, 119 (2), pp Gadad, A.P., Patil, M.B., Naduvinamani, S.N., Mastiholimath, V.S., Dandagi, P.M., Kulkarni, A.R. Sodium alginate polymeric floating beads for the delivery of cefpodoxime proxetil (29) Journal of Applied Polymer Science, 114 (3), pp Kikuchi, A., Kawabuchi, M., Watanabe, A., Sugihara, M., Sakurai, Y., Okano, T. Effect of Ca2+-alginate gel dissolution on release of dextran with different molecular weights (1999) Journal of Controlled Release, 58 (1), pp Badwan, A.A., Abumalooh, A., Sallam, E. A sustained release drug delivery system using calcium alginate beads (1985) Drug Development and Industrial Pharmacy, 11 (2-3), pp Gadad, A.P., Patil, M.B., Naduvinamani, S.N., Mastiholimath, V.S., Dandagi, P.M., Kulkarni, A.R. Sodium alginate polymeric floating beads for the delivery of cefpodoxime proxetil(29) Journal of Applied Polymer Science, 114 (3), pp Shishu, Gupta, N., Aggarwal, N. Stomach-specific drug delivery of 5-fluorouracil using floating alginate beads (27) AAPS PharmSciTech, 8 (2), art. no. 48,. 12. Choi, B.Y., Park, H.J., Hwang, S.J., Park, J.B. Preparation of alginate beads for floating drug delivery system: Effects of CO 2 gas-forming agents (22) International Journal of Pharmaceutics, 239 (1-2), pp
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