|Year : 2014 | Volume
| Issue : 4 | Page : 267-277
Gellan gum-based mucoadhesive microspheres of almotriptan for nasal administration: Formulation optimization using factorial design, characterization, and in vitro evaluation
Zaheer Abbas, Sachin Marihal
Research Scientist, Formulation Development Department, Apotex Research Private Limited, Bangalore - 560 099, India
|Date of Submission||02-Feb-2014|
|Date of Decision||07-May-2014|
|Date of Acceptance||16-May-2014|
|Date of Web Publication||16-Oct-2014|
Research Scientist, Formulation Development Department, Apotex Research Private Limited, Bangalore - 560 099
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Almotriptan malate (ALM), indicated for the treatment of migraine in adults is not a drug candidate feasible to be administered through the oral route during the attack due to its associated symptoms such as nausea and vomiting. This obviates an alternative dosage form and nasal drug delivery is a good substitute to oral and parenteral administration. Materials and Methods: Gellan gum (GG) microspheres of ALM, for intranasal administration were prepared by water-in-oil emulsification cross-linking technique employing a 2 3 factorial design. Drug to polymer ratio, calcium chloride concentration and cross-linking time were selected as independent variables, while particle size and in vitro mucoadhesion of the microspheres were investigated as dependent variables. Regression analysis was performed to identify the best formulation conditions. The microspheres were evaluated for characteristics such as practical percentage yield, particle size, percentage incorporation efficiency, swellability, zeta potential, in vitro mucoadhesion, thermal analysis, X-ray diffraction study, and in vitro drug diffusion studies. Results: The shape and surface characteristics of the microspheres were determined by scanning electron microscopy, which revealed spherical nature and nearly smooth surface with drug incorporation efficiency in the range of 71.65 ± 1.09% - 91.65 ± 1.13%. In vitro mucoadhesion was observed the range of 79.45 ± 1.69% - 95.48 ± 1.27%. Differential scanning calorimetry and X-ray diffraction results indicated a molecular level dispersion of drug in the microspheres. In vitro drug diffusion was Higuchi matrix controlled and the release mechanism was found to be non-Fickian. Stability studies indicated that there were no significant deviations in the drug content, in vitro mucoadhesion and in vitro drug diffusion characteristics. Conclusion: The investigation revealed promising potential of GG microspheres for delivering ALM intranasally for the treatment of migraine.
Keywords: Almotriptan malate, emulsification cross-linking technique, factorial design, gellan gum, intranasal drug delivery, mucoadhesive microspheres
|How to cite this article:|
Abbas Z, Marihal S. Gellan gum-based mucoadhesive microspheres of almotriptan for nasal administration: Formulation optimization using factorial design, characterization, and in vitro evaluation. J Pharm Bioall Sci 2014;6:267-77
|How to cite this URL:|
Abbas Z, Marihal S. Gellan gum-based mucoadhesive microspheres of almotriptan for nasal administration: Formulation optimization using factorial design, characterization, and in vitro evaluation. J Pharm Bioall Sci [serial online] 2014 [cited 2020 Apr 5];6:267-77. Available from: http://www.jpbsonline.org/text.asp?2014/6/4/267/142959
Migraine is a recurrent incapacitating neurovascular disorder characterized by attacks of debilitating pain associated with photophobia, phonophobia, nausea, and vomiting.  Almotriptan malate (ALM), a triptan derivative is a novel selective 5-hydroxytryptamine 1B/1D receptor agonist indicated for the acute treatment of migraine with or without aura in adults.  During an attack, the blood vessels in the brain dilate and then draw together with stimulation of nerve endings near the affected blood vessels. These changes in the blood vasculature may be responsible for the pain. However, the exact cause of migraine still remains unclear whether it is a vascular or a neurological dysfunction. Therapeutic approaches for management of migraine has a strong rationale; however, it is still a poorly understood phenomenon. 
Almotriptan malate is generally given by the oral route and commercially available as a conventional immediate release tablet. ALM is well-absorbed after oral administration, with absolute bioavailability of about 70%.  The optimal dose for ALM is a 12.5 mg at the start of a migraine headache, which may be repeated once in 2 h to a maximum of 25 mg/24 h. Low oral bioavailability, frequent administration due to lower plasma half-life of 3-4 h and associated symptoms such as nausea and vomiting makes oral drug delivery undesirable and justifies a need of an alternate route for drug delivery. ,
In the recent years, nasal route has received special attention as a convenient and reliable method for the systemic delivery of drugs, especially those that are ineffective by the oral route due to their metabolism in the gastrointestinal tract subject to first-pass effect and must be administered by injection. Conventionally, the nasal cavity is used for the treatment of local diseases, such as rhinitis and nasal congestion. However, in the past few decades, nasal drug delivery has been paid much more attention as a promising drug administration route for the systemic therapy as it possesses numerous advantages such as relatively large surface area, porous endothelial basement membrane, highly vascularized epithelial layer, enhanced blood flow, avoiding the first-pass metabolism, and ready accessibility. ,, However, the major limitation of the nasal drug delivery is the nasal mucociliary clearance (NMCC) that determines a limited time available for adsorption within the nasal cavity.
Nasal mucociliary clearance system transports the mucus layer that covers the nasal epithelium towards the nasopharynx by ciliary beating. Its function is to protect the respiratory system from damage by inhaled substances. NMCC transit time in humans has been reported to be 12-15 min. The average rate of nasal clearance is about 8 mm/min, ranging from less than 1 to more than 20 mm/min. NMCC is one of the most important limiting factor for nasal drug delivery as it severely limits the time allowed for drug absorption to occur and effectively rules out the option of sustained nasal drug administration.  Several approaches are discussed in the literature to increase the residence time of drug formulations in the nasal cavity, resulting in improved nasal drug absorption. 
Among the various approaches available to enhance the transnasal delivery of drugs, the mucoadhesive microsphere drug delivery system is an attractive concept that has the ability to control the rate of drug clearance from the nasal cavity as well as to protect the drug from enzymatic degradation.  The microspheres swell in contact with nasal mucosa and form a gel-like layer, which controls the rate of clearance from the nasal cavity. In the presence of microspheres, the nasal mucosa is dehydrated due to moisture uptake by the microspheres. This results in reversible shrinkage of the cells, providing a temporary physical separation of the tight (intercellular) junction, which increase the absorption of the drug. Hence, a formulation that would increase residence time in the nasal cavity and at the same time increased absorption of the drug would be highly beneficial in all respects. 
Gellan gum (GG) is an extracellular polysaccharide produced by aerobic fermentation of the bacterium Sphingomonas elodea/Pseudomonas elodea.  The natural form of GG is a linear anionic heteropolysaccharide, which is based on a tetrasaccharide repeated unit of β-D-glucose, β-D-glucuronic acid and α-L-rhamnose residues in the molar ratio of 2:1:1.  Commercially available GG is a deacetylated product obtained by treatment with an alkali. Due to the characteristic property of cation-induced gelation, the pharmaceutical applications are mainly in the in situ gelling ophthalmic drug delivery and oral controlled release preparations. Due to its ability to form strong clear gels at physiological ion concentration, it can provide a longer contact time for drug transport across the nasal mucosa. The mechanism of gelation involves the formation of double helical junction zones followed by aggregation of double helical segments to form a three-dimensional network by complexation with cations and hydrogen bonding with water. These features along with biodegradability, biocompatibility, and absence of toxicity of the polymer, attracted widespread interest in GG as drug carrier. ,,,
Statistical optimization techniques employing factorial design is a powerful, efficient and systematic tool that shortens the time required for the drug product development and improves research and development work. Factorial designs, where all the factors are studied in all possible combinations are considered to be the most efficient in estimating the influence of individual variables and their interactions using minimum experiments. The application of factorial design in pharmaceutical product development has played a key role in understanding the relationship between the independent variables and the responses to them. The independent variables are controllable, whereas responses are dependent. The response surface plot gives a visual representation of the values of the response. ,, This helps the process of optimization by providing an empirical model equation for the response as a function of the different variables.
The objective of the current investigation was to improve the therapeutic efficacy of ALM by preparing ALM-loaded GG microspheres for intranasal administration. The microspheres were prepared by emulsification cross-linking technique utilizing a 2 3 factorial design. The effect of formula variables, such as drug: polymer ratio, concentration of cross-linking agent and cross-linking time on the particle size and in vitro mucoadhesion was investigated.
| Materials and Methods|| |
Almotriptan malate was obtained as gift sample from Apotex Research Private Limited, Bangalore. GG was generously gifted by Strides Arcolabs Limited, Bangalore. Span-80, n-octanol and calcium chloride (CaCl 2 ) were procured from S.D. Fine Chemicals, Mumbai. All other reagents used were of analytical grade commercially available from Merck Pvt. Ltd., Mumbai, India.
Preparation of mucoadhesive microspheres
Almotriptan malate-loaded GG microspheres were prepared by water-in-oil (w/o) emulsification cross-linking technique employing CaCl 2 as cross-linking agent. , Gellan solution was prepared by dissolving the GG in double-distilled water by heating at 90°C. ALM was uniformly dispersed in Gellan solution with constant agitation (500 rpm) at 40°C until a homogeneous solution was formed. The resultant homogeneous bubble free solution was extruded through a syringe (no. 18) into 100 mL of n-octanol: Water system (20:1 ratio) containing 2% w/v Span-80 with constant agitation at 1800 rpm using a mechanical stirrer (Remi stirrer, Mumbai, India). The resultant w/o emulsion was stirred for 30 min. CaCl 2 solution was then added drop-wise and the dispersion was agitated for another 5 min to provide sufficient mechanical strength. The microspheres were then collected by vacuum filtration, washed twice with isopropyl alcohol followed by double distilled water, dried in a hot air oven at 50°C and stored in a desiccator at room temperature. A total of eight formulations were prepared, and the assigned formulation codes are provided in [Table 1].
|Table 1: Formulation of the microspheres employing a 23 factorial design|
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Design of experiments employing factorial design
Various batches of ALM-loaded GG microspheres were prepared by employing 2 3 factorial design. The independent variables chosen were drug to polymer ratio (X1 ), CaCl 2 concentration (X2 ) and cross-linking time (X3 ). The independent variables and their levels are shown in [Table 2]. Particle size of the microspheres (Y1 ) and in vitro mucoadhesion (Y2 ) were taken as the response parameters and are categorized as dependent variables. [Table 1] represents the independent and dependent variables.
Characterization of almotriptan malate-loaded gellan microspheres
Percentage yield and drug incorporation efficiency
The practical percentage yield was calculated from the weight of dried microspheres recovered from each batch in relation to the sum of the initial weight of starting materials. To determine the percentage drug incorporated, microspheres equivalent to 10 mg of ALM were crushed in a glass mortar and pestle, and the powdered microspheres were suspended in 25 mL of phosphate buffer pH 6.4. After 24 h, the solution was filtered, 1 mL of the filtrate was pipetted out, diluted to 10 mL and analyzed for the drug content using Elico SL-159 ultraviolet (UV) visible spectrophotometer (Elico Limited, Hyderabad, India) at 228 nm. ,, It was confirmed from preliminary UV studies that the presence of dissolved polymers did not interfere with the absorbance of the drug at 228 nm. The drug incorporation efficiency was calculated using the following formula:
Shape and surface morphology
The shape and surface characteristics of the microspheres were evaluated by means of scanning electron microscope (SEM) (JEOL - JSM - 840A, Japan). The samples were prepared by gently sprinkling the microspheres on a double-adhesive tape, which is stuck to an aluminum stub.  The stubs were then coated with gold using a sputter coater (JEOL Fine coat JFC 1100E, ion sputtering device, JEOL Technics Co., Tokyo, Japan) under high vacuum and high voltage to achieve a film thickness of 30 nm. The samples were then imaged using a 20 kV electron beam.
Particle size measurement
Particle size of the microspheres was determined by optical microscopy using an optical microscope Olympus BH2-UMA (Olympus, NWF 10x, India).  The eye piece micrometer was calibrated with the help of a stage micrometer. The particle diameters of more than 300 microspheres were measured randomly. The average particle size was determined by using Edmondson's equation.
Where, n = number of microspheres checked; d = mean size range.
Zeta potential study
Laser Doppler electrophoresis technique was applied to measure particle electrostatic charge. Microspheres AGM1 to AGM8 were subjected to zeta potential measurements using zeta sizer (Nano ZS, Malvern Instruments, UK). The microspheres were dispersed in distilled water and placed into the electrophoretic cells of the instrument and potential of 100 mV was applied. Zeta potential was determined for 25 distinct particles. 
In vitro mucoadhesion studies
The in vitro mucoadhesion study of microspheres was assessed using falling liquid film technique. ,, A strip of sheep nasal mucosa was mounted on a glass slide and 50 mg of accurately weighed microspheres were sprinkled on the nasal mucosa. This glass slide was incubated for 15 min in a desiccator at 90% relative humidity (RH) to allow the polymer to interact with the membrane and finally placed on the stand at an angle of 45°. Phosphate buffered saline of pH 6.4; previously warmed to 37 ± 0.5°C was allowed to flow over the microspheres and membrane at the rate of 1 mL/min for 5 min with the help of a peristaltic pump. At the end of this process, the detached particles were collected and weighed. The percentage mucoadhesion was determined by using the following equation.
In vitro swelling studies
The swellability of microspheres in physiological media was determined by allowing the microspheres to swell in the phosphate buffer saline pH 6.4. 100 mg of accurately weighed microspheres were immersed in little excess of phosphate buffer saline of pH 6.4 for 24 h and washed thoroughly with deionized water.  The degree of swelling was arrived at using the following formula:
Where, α is the degree of swelling; Wo is the weight of microspheres before swelling and Ws is the weight of microspheres after swelling.
Differential scanning calorimetry (DSC) was performed on pure ALM, placebo microspheres and ALM-loaded GG microspheres. DSC measurements were performed on a differential scanning calorimeter (DSC 823, Mettler Toledo, Switzerland). The thermograms were obtained at a scanning rate of 10°C/min over a temperature range of 25-250°C under an inert atmosphere flushed with nitrogen at a rate of 20 mL/min. 
Powder X-ray diffraction studies
The qualitative powder X-ray diffraction studies were performed using an X-ray diffractometer (PANalytical, X Pert Pro, PANalytical B.V., Almelo, The Netherlands). ALM, placebo microspheres and ALM-loaded microspheres were scanned from 0° to 40° diffraction angle (2θ) range under the following measurement conditions: Source, nickel filtered Cu-Kα radiation; voltage 40 kV; current 30 mA; scan speed 0.05/min. Microspheres were triturated to get fine powder before taking the scan. X-ray diffractometry was carried out to investigate the effect of microencapsulation process on crystallinity of the drug. 
In vitro drug diffusion studies
Preparation of the nasal mucosa
Fresh sheep nasal mucosa was collected from a nearby slaughter house. The nasal mucosa of sheep was separated from sub layer bony tissues and stored in distilled water containing few drops of gentamycin injection. After complete removal of blood from mucosal surface, it was attached to the donor chamber tube. 
In vitro nasal diffusion study was carried out using nasal diffusion cell, having three openings each for sampling, thermometer and donor tube chamber.  The receptor compartment has a capacity of 60 mL in which Phosphate buffer, pH 6.4 was taken. Within 80 min of removal, the nasal mucosa measuring an area of 3 cm 2 was carefully cut with a scalpel and tied to the donor tube chamber, and it was placed establishing contact with the diffusion medium in the recipient chamber. Microspheres equivalent to 10 mg of ALM were spread on the sheep nasal mucosa. At hourly intervals, 1 mL of the diffusion sample was withdrawn with the help of a hypodermic syringe, diluted to 10 mL and absorbance was read at 228 nm. Each time, the sample withdrawn was replaced with 1 mL of prewarmed buffer solution (pH 6.4) to maintain a constant volume of the receptor compartment vehicle.
In vitro drug diffusion kinetics
For understanding the mechanism of drug release and release rate kinetics of the drug from the microspheres, the obtained in vitro drug diffusion data was fitted into software (PCP - Disso-V2.08 developed by Poona College of Pharmacy, Pune, India) with zero order, first-order, Higuchi matrix, Hixson-Crowell, Korsmeyer-Peppas model. By analyzing the R (correlation coefficient) values, the best fit model was arrived at. ,,
Stability studies of the select formulations were carried out as per ICH guidelines.  The optimum formulation were packed in amber colored glass containers, closed with air tight closures and stored at 25 ± 2°C/60 ± 5% RH, 30 ± 2°C/65 ± 5% RH and 40 ± 2°C/75 ± 5% RH for 3 months using programmable environmental test chambers (Remi Instruments Ltd., Mumbai, India). Samples were analyzed at the end of 30, 60 and 90 days and they were evaluated for percentage drug incorporation efficiency, in vitro mucoadhesion test and in vitro drug diffusion studies.
Optimization data analysis and model-validation
ANOVA was used to establish the statistical validation of the polynomial equations generated by Design Expert ® software (version 9.0, Stat-Ease Inc., Minneapolis, MN). Fitting a multiple linear regression model to a 2 3 factorial design gave a predictor equation which was a first-order polynomial, having the form:
Where Y is the measured response associated with each factor level combination; b0 is an intercept representing the arithmetic average of all quantitative outcomes of eight runs; b1 to b123 are regression coefficients computed from the observed experimental values of Y. X1 , X2 and X3 are the coded levels of independent variables. The terms X1 X2 , X2 X3 and X1 X3 represent the interaction terms. The main effects (X1 , X2, and X3 ) represent the average result of changing one factor at a time from its low to high value. The interaction terms show how the response changes when two factors are changed simultaneously. The polynomial equation was used to draw conclusions after considering the magnitude of coefficients and the mathematical sign it carries that is, positive or negative. A positive sign signifies a synergistic effect, whereas a negative sign stands for an antagonistic effect.
In the model analysis, the responses: The particle size of the microspheres (Y1 ) and in vitro mucoadhesion (Y2 ) of all model formulations were treated by Design Expert ® software. The best fitting mathematical model was selected based on the comparisons of several statistical parameters including the coefficient of variation (CV), the multiple correlation coefficient (R2 ), adjusted multiple correlation coefficient (adjusted R2 ) and the predicted residual sum of square (PRESS), provided by Design Expert ® software. Among them, PRESS indicates how well the model fits the data and for the selected model it should be small relative to the other models under consideration. Level of significance was considered at P < 0.05. Three-dimensional response surface plots resulting from equations were obtained by the Design Expert ® software. Subsequently, the desirability approach was used to generate the optimum settings for the formulations. 
| Results and Discussion|| |
Almotriptan malate-loaded mucoadhesive GG microspheres were successfully fabricated by emulsification cross-linking method which involved interaction between negatively charged GG with positively charged calcium ions. During the process of microsphere preparation, the drug may partition out into the aqueous phase due to its hydrophilic nature, hence in the present investigation n-octanol was used as the harvesting medium. In this condition, ALM would find it nonfavorable to diffuse out of the microspheres before they harden thus resulting in sufficiently high drug incorporation efficiency.
The practical percentage yield of the microspheres was observed to be in the range of 83.54-93.16%. The low percentage yield in some of the formulations may be due to loss of microspheres during the washing process. As the drug to polymer ratio was varied from 0.5:1 to 1:1, it was observed that the particle size increased, whereas, the drug incorporation efficiency decreased. The drug entrapment efficiency was found to be in the range between 71.65 ± 1.09% and 91.65 ± 1.13% and revealed its dependency on drug loading, amount of cross-linking agent and time of cross-linking. The formulations loaded with higher amount of drug (AGM2, AGM4, AGM6, and AGM8) exhibited decreased incorporation efficiency, which could be attributed to the increase in extent of drug diffusion to the external phase due to greater flux at higher drug content during the emulsification and microsphere formation process. The decrease in drug incorporation efficiency with an increase in the concentration of CaCl 2 and cross-linking time could be related either to an increase in cross-link density, which will reduce the free volume spaces within the polymer matrix or could be due to incomplete emulsification as a result of higher viscosity of the internal phase. The results of percentage practical yield and drug incorporation efficiency of the prepared microspheres are tabulated in [Table 3].
|Table 3: Characteristics of the prepared ALM-loaded alginate microspheres|
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The prepared microspheres were found to be discrete and spherical in shape and had nearly smooth surface morphology. These microspheres had no pores or rupture on the surface, such morphology would result in slow clearance and good deposition pattern in the nasal cavity.  The SEM photographs of the optimized formulation (AGM1) taken by SEM are depicted in [Figure 1]a and b.
|Figure 1: Scanning electron microscope microphotograph of formulation AGM1 at low (a) and high (b) Magnification|
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Particle size of the microspheres is one of the most important characteristics of a nasal drug delivery system. The mean particle size of microspheres ranged from 24.86 ± 1.34 μm to 52.42 ± 1.03 μm, ideal for intranasal absorption. Preliminary studies showed that as the concentration of polymer was increased, the particle size also proportionally increased. Lower GG concentrations (0.5% w/v, 1% w/v and 1.5% w/v) resulted in the clumping of microspheres, whereas high GG concentration (4% w/v) resulted in formation of discrete microspheres with a mean particle size greater than 80 μm which could be attributed to an increase in the relative viscosity at higher concentration of polymer and formation of larger particles during emulsification. Hence an optimum GG concentration of 2% w/v was selected for preparing the different batches of the microspheres. The mean particle size of the microspheres increased with an increase in drug loading. This can be attributed to the corresponding increase in viscosity of drug-polymer dispersion comprising the internal phase of the emulsion. The increase in viscosity within the internal phase results in the generation of a coarser emulsion with larger droplets leading eventually to the formation of larger microspheres. A similar increase in the size of microspheres was also observed with an increase in CaCl 2 concentration as well as cross-linking time. The addition of higher amount of Ca 2 + will result in relatively more cross-linking of the guluronic acid units, thereby leading to the formation of larger microspheres. Similarly, increasing the cross-linking time will increase the extent of cross-linking and thereby increase the particle size. The mean particle size (Y1 ) of the prepared microspheres is presented in [Table 1].
Zeta potential analysis was performed to get the information about the surface properties of the microspheres. Zeta potential values higher than −30 mV show good physical stability, being optimized when they reach approximately −60 mV, exhibiting a very good physical stability during the shelf-life. In this study, zeta potential of AGM1 to AGM8 was in the range of −31.60 to −35.50 mV and are compiled in [Table 3]. The zeta potential distribution curve of optimum formulation (AGM1) is displayed in [Figure 2]. All microspheres prepared were negatively charged, indicating the presence of GG at the surface of all microspheres formed.  Studies have cited that polymers with charged density can serve as good mucoadhesive agents. It has also been reported that anion polymers are more effective bioadhesive than polycations or nonionic polymers.
|Figure 2: Zeta potential distribution curve of microsphere formulation AGM1|
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The results of in vitro mucoadhesion test (Y2 ) are tabulated in [Table 1]. The prepared microspheres had satisfactory mucoadhesive properties ranging from 79.45 ± 1.69% to 95.48 ± 1.27% and could adequately adhere onto the nasal mucosa. The data revealed that, with an increase in polymer ratio, percentage mucoadhesion increased, which could be correlated to the availability of a higher amount of polymer for interaction with mucus. Increase in CaCl 2 concentration and cross-linking time decreased the mucoadhesive property of the microspheres. Most of the studies showed that the prerequisite for a good mucoadhesion is the high flexibility of the polymer backbone structure and its polar functional groups. Such a flexibility of the polymer chains, however, is reduced if the polymer molecules are cross-linked either with each other or with coagulation agents like calcium ions. Although the cross-linked microspheres will absorb water, they are insoluble and will not form a liquid gel on the nasal epithelium but rather a more solid gel-like structure. This decrease in flexibility imposed upon polymer chains by the cross-linking makes it more difficult for cross-linked polymers to penetrate the mucin network.  Thus, cross-linking effectively limits the length of polymer chains that can penetrate the mucus layer and could possibly decrease the mucoadhesion strength of the microspheres. The formulation, AGM1, with highest mucoadhesion (95.48 ± 1.27%) was considered to be an optimum formulation.
The percentage in vitro swelling is an indicative parameter for rapid availability of drug solution for diffusion with greater flux. The rapid fluid uptake from the mucus layer enabling the polymer chain to penetrate mucin network and establish adhesive bond has a key role in mucoadhesion. Linear relationship has been observed between polymer concentration, swelling index and mucoadhesion. It can be concluded from the data presented in [Table 3] that, with an increase in CaCl 2 concentration and cross-linking time, the degree of swelling decreased in the range from 1.206 ± 0.199 to 0.754 ± 0.421. This tendency could be attributed to greater cross-linking degree of the polymer resulting in rigid microspheres, which lowers the solvent transfer rate, reduced swelling and thus reduced mucoadhesiveness. GG microspheres after uptake of fluid transform into gel matrix and as swelling behavior play an important role in the in situ gel formation on the nasal mucosa and hence retard the drug diffusion rate.
In an effort to assess the physical state of the drug in the GG microspheres, we attempted to analyze pure ALM, placebo microspheres and drug-loaded microspheres (AGM1) using DSC. The thermograms are presented in [Figure 3]. For pure ALM a sharp endothermic peak at 169.9°C was observed due to the melting of ALM but, in the case of ALM-loaded microspheres, no characteristic peak was observed at 169.9°C, suggesting that ALM is molecularly dispersed in the matrix.
|Figure 3: Differential scanning calorimetry thermograms of (a) Pure almotriptan malate (b) Blank microspheres and (C) Drug - loaded microspheres|
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XRD studies are useful to investigate the crystallinity of drug in the polymeric microspheres. The X-ray diffractograms recorded for pure ALM, placebo microspheres and drug-loaded microspheres (AGM1) are presented in [Figure 4]. ALM revealed characteristic intense peaks at 2θ of 16°, 17° and 22° which are due to crystalline nature of ALM. However, in case of blank microspheres and drug-loaded microsphere no intense peaks were observed between 2θ of 16°, 17° and 22° indicating amorphous nature of the drug substance after entrapment into GG microspheres. It can be concluded that, drug particles are dispersed at the molecular level in the polymer matrices since no indication about the crystalline nature of the drug was observed in the drug-loaded microspheres.
|Figure 4: Powder X-ray diffractograms of (a) Pure almotriptan malate (b) Blank microspheres and (c) Drug - loaded microspheres|
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The in vitro diffusion of ALM from the prepared microspheres exhibited a biphasic mechanism. The release of ALM from the microspheres was characterized by an initial phase of burst effect due to the presence of drug particles on the surface of the microspheres followed by a second phase of moderate release. The initial burst effect is a desired effect to achieve initial therapeutic plasma concentration of the drug. The in vitro drug diffusion study from formulations AGM1 to AGM4 is presented in [Figure 5]a. As the concentration of CaCl 2 and cross-linking time increased, percentage drug dissolved decreased. The in vitro drug diffusion study from formulations AGM5 to AGM8 are presented in [Figure 5]b. The decrease in percentage drug dissolved could be attributed to increase in the extent of cross-linking in the microsphere with an increase in the amount of cross-linking agent. The Ca 2 + cross-linked microspheres form three-dimensional bonding structures with the GG inside the microspheres. This three-dimensional bonding results in extended cross-linking through the whole microsphere producing hard microcapsules with lower water uptake and thus leading to slow removal of drug in the dissolution media. The release of the drug has been controlled by swelling control release mechanism. In addition, the larger particle size at higher polymer concentration also restricts the total surface area thus resulting in slower drug release over a span of 8 h.
|Figure 5: In vitro drug diffusion profile of microsphere formulation AGM1 to AGM4 (a) and AGM5 to AGM8 (b)|
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In order to investigate the drug diffusion mechanism, the in vitro drug diffusion data were fitted to models representation zero-order, first-order, Higuchi matrix, Hixson-Crowell and Korsmeyer-Peppas model utilizing software (PCP - Disso-V2.08). The drug release kinetic data is compiled in [Table 4]. In all cases, the R values of Higuchi matrix model were close to 1; hence, the drug release follows matrix diffusion controlled kinetics and the plot shown in [Figure 6]a and b revealed linearity; therefore it was concluded that diffusion was the main mechanism of drug release from the microspheres. The n values were in the range of 0.4512-0.6971 indicating that all the prepared formulations followed the Fickian diffusion controlled mechanism of drug release. On the basis of results of characterization of microspheres and in vitro drug diffusion study AGM1 was considered as optimized formulation and found to give satisfactory results, which make it suitable for nasal administration of almotriptan.
|Figure 6: Plot of amount of drug released versus square root of time (Higuchi plot) for formulation AGM1 to AGM4 (a) and AGM5 to AGM8 (b)|
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The stability data for optimum formulation (AGM1) showed that there was no change in the appearance of the microspheres indicating that the formulations were stable at different conditions of storage. The stability study was performed for the prepared formulation as per the ICH guidelines, and it showed that the formulation was stable, with no physical change and also there was no significant reduction in drug content and in vitro drug diffusion profile. Thus, we may conclude that, the drug does not undergo degradation on storage.
Optimization data analysis and model-validation
Fitting of data to the model
The three factors with lower and upper design points in coded and uncoded values are shown in [Table 2]. The ranges of responses Y1 and Y2 were 24.86 ± 1.34-52.42 ± 1.03 μm and 79.45 ± 1.69%-95.48 ± 1.27%, respectively. All the observed responses for eight formulations (AGM1 to AGM8) prepared were fitted to various models using Design-Expert ® software. It was observed that the best-fitted models were linear and interactive. The values of R2 , adjusted R2 , predicted R2 , standard deviation and %CV are given in [Table 5], along with the regression equation generated for each response. The results of ANOVA in are compiled in [Table 6]. The dependent variables (X1 , X2 , and X3 ) demonstrated that the model was significant for both the response variables (Y1 and Y2 ).
|Table 5: Summary of results of regression analysis for responses Y1 and Y2|
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It was observed that all the three independent variables namely X1 (drug: Polymer ratio), X2 (concentration of CaCl 2 ) and X3 (cross-linking time) had a positive effect on particle size (Y1 ), but, a negative effect on in vitro mucoadhesion (Y2 ). The coefficients with more than one factor term in the regression equation represent interaction terms. It also shows that the relationship between the factors and responses evaluated is not always linear. When more than one factor is changed simultaneously and used at different levels in a formulation, a factor can produce different degrees of response. The interaction effects of X1 and X2 ; X1 and X3 were favorable (positive), whereas the interaction effect of X2 and X3 was unfavorable (negative), for response Y2 . From the equations presented in [Table 5], it is evident that the drug to polymer ratio plays an important role in the in vitro mucoadhesion of the microspheres.
Response surface plot analysis
Three-dimensional response surface plots generated by the Design Expert ® software are presented in [Figure 7] and [Figure 8] for the studied responses that is, particle size and in vitro mucoadhesion, respectively. [Figure 7]a depicts response surface plot for the effect of drug: Polymer ratio (X1 ) and CaCl 2 concentration (X2 ) on particle size, which indicate a linear effect on particle size of the microspheres. The combined effects of CaCl 2 concentration (X2 ) and cross-linking time (X3 ) and drug: Polymer ratio (X1 ) and cross-linking time (X3 ) on particle size, as shown in [Figure 7]b and c also revealed linearity. This explains that the higher the amount of CaCl 2 or higher the time of cross-linking, the more will be the cross-linking of the guluronic acid units of GG leading to the formation of larger microspheres.
|Figure 7: Response surface plots for the (a) Effect of drug: Polymer ratio (X1) and calcium chloride (CaCl2) concentration (X2), (b) Effects of CaCl2 concentration (X2) and cross-linking time (X3) and (c) Effect of drug: Polymer ratio (X1) and cross-linking time (X3) On particle size|
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|Figure 8: Response surface plots for the (a) effect of drug: Polymer ratio (X1) and calcium chloride (CaCl2) concentration (X2), (b) Effects of CaCl2 concentration (X2) and cross-linking time (X3) and (c) Effect of drug: Polymer ratio (X1) and cross-linking time (X3) On in vitro mucoadhesion|
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The combined effect of X1 and X2 on in vitro mucoadhesion of the microspheres was observed to be nonlinear, as in [Figure 8]a. At low value of drug: Polymer ratio and CaCl 2 concentration, a higher value for in vitro mucoadhesion was observed. Similar effects were observed for factors X2 , X3 and X1 , X3 , as shown in [Figure 8]b and c, respectively. As the CaCl 2 concentration and cross-linking time increased from low to high, value for in vitro mucoadhesion of the microspheres was decreased.
Optimization and validation
A numerical optimization technique by the desirability approach was used to generate the optimum settings for the formulation. The process was optimized for the dependent (response) variables Y1 and Y2 . The optimum formulation was selected based on the criteria of attaining the minimum value of particle size and maximum value of in vitro mucoadhesion. Formulation AGM1 having drug to polymer ratio (0.5:1), CaCl 2 concentration (2%) and cross-linking time (5 min) fulfilled all the criteria set from desirability approach. To gainsay the reliability of the response surface model, a new optimized formulation (as per formula AGM1) was prepared according to the predicted model and evaluated for the responses. The results presented in [Table 7] illustrate the comparison between the observed and predicted values for both the responses Y1 and Y2 for all the formulations prepared. It can be seen that in all cases there was a reasonable agreement between the predicted and the actual values as prediction error was found to vary between −7.446% and +7.064 for response Y1 and −0.013 to +0.012% for response Y2 . For this reason, it can be concluded that the equations adequately describe the influence of the selected independent variables on the responses under study. This indicates that the optimization technique was an appropriate tool for optimizing the GG microsphere formulation. Thus, the low magnitudes of error as well as the significant values of R2 in the present investigation prove the high prognostic ability of the optimization technique by factorial design.
|Table 7: The predicted and observed response variables of the microspheres|
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| Conclusion|| |
Mucoadhesive and biodegradable ALM loaded GG microspheres were successfully fabricated by w/o emulsification cross-linking technique employing a 2 3 full factorial design. The results of our present study clearly indicated promising potential of GG microspheres for delivering drug intranasally. The microspheres upon contact with the nasal mucosa form viscous gel by withdrawing water, and interaction with cations present in nasal secretions, which eventually leads to decrease in the ciliary clearance rate and as a consequence prolongs the formulation residence time. Furthermore, mucoadhesive microspheres could be exploited for burst release at desired times to affect any required modulation in the drug plasma level. The controlled release profile of ALM from the microspheres may help in decreasing the frequency of dosing and possibly maximize the therapeutic benefit, thereby providing safe, patient friendly, efficacious and economic drug delivery. However, thorough animal studies using different species followed by extensive clinical trials and toxicological evaluation need to be conducted to establish the appropriateness of these formulations in clinical practice.
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[Table 5], [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 6], [Table 7]