gellan gum based mucoadhesive microspheres of almotriptan for nasal administration formulation optimization using factorial design characterization and in vitro evaluation

O riginal ArticleGellan gum-based mucoadhesive microspheres of almotriptan for nasal administration: Formulation optimization using factorial design, characterization, and in vitro eva

O riginal Article

Gellan gum-based mucoadhesive microspheres of almotriptan for nasal administration: Formulation optimization using

factorial design, characterization, and in vitro

evaluation Zaheer Abbas, Sachin Marihal

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 23 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

KEY WORDS: Almotriptan malate, emulsification cross‑linking technique, factorial design, gellan gum, intranasal drug delivery, mucoadhesive microspheres

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Website:

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DOI:

10.4103/0975-7406.142959

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.

Research Scientist,

Formulation Development

Department, Apotex

Research Private Limited,

Bangalore - 560 099,

India

Address for correspondence:

Mr Zaheer Abbas,

E-mail: zaheergcp@gmail.com

Received : 02-02-14

Review completed : 07-05-14

Accepted : 16-05-14

M igraine is a recurrent incapacitating neurovascular

disorder characterized by attacks of debilitating pain

associated with photophobia, phonophobia, nausea, and

vomiting.[1] Almotriptan malate (ALM), a triptan derivative

is a novel selective 5-hydroxytryptamine1B/1D receptor agonist indicated for the acute treatment of migraine with or without aura in adults.[2] 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.[3]

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%.[4] 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.[5,6]

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.[7-9] 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.[10] 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.[11]

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.[12] 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.[13]

Gellan gum (GG) is an extracellular polysaccharide produced

by aerobic fermentation of the bacterium Sphingomonas elodea/

Pseudomonas elodea.[14] 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.[15] 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.[16-19]

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.[20-22] 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 23 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 (CaCl2) 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 CaCl2 as cross-linking agent.[23,24] Gellan solution was prepared by dissolving the GG in double-distilled water

Abbas and Marihal: Intranasal delivery of almotriptan microspheres

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 CaCl2 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

Design of experiments employing factorial design

Various batches of ALM-loaded GG microspheres were prepared

by employing 23 factorial design The independent variables

chosen were drug to polymer ratio (X1), CaCl2 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.[25-27] 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:

Percentage drug incorporation efficiency=

Practical drug conteent

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.[28] 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).[29] 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

Dmean= ∑

∑

nd n 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.[30]

Table 1: Formulation of the microspheres employing a 2 3

factorial design

Formulation code X1 X2 X3 Y1 * Y2 *

*Values are expressed as mean±SD Y1 and Y2 are particle size and

in vitro mucoadhesion, respectively SD: Standard deviation

Table 2: Factorial design parameters and experimental

conditions

Factors Levels used, actual (coded)

Low (−1) High (+1)

X1=Drug to polymer ratio 0.5:1 1:1

X2=Concentration of CaCl2 (%) 2 4

CaCl2: Calcium chloride

In vitro mucoadhesion studies

The in vitro mucoadhesion study of microspheres was assessed

using falling liquid film technique.[31-33] 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

Percentagemucoadhesion =

Weight of sample-weight

of detached partiicles

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.[34] The degree of swelling was arrived at using

the following formula:

W

s o

o

Where, α is the degree of swelling; Wo is the weight of

microspheres before swelling and Ws is the weight of

microspheres after swelling

Thermal analysis

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.[35]

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.[36]

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.[37]

In vitro nasal diffusion study was carried out using nasal diffusion cell, having three openings each for sampling, thermometer and donor tube chamber.[38] 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 cm2 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.[39-41]

Stability studies

Stability studies of the select formulations were carried out as per ICH guidelines.[42] 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

Abbas and Marihal: Intranasal delivery of almotriptan microspheres

a multiple linear regression model to a 23 factorial design gave a

predictor equation which was a first-order polynomial, having

the form:

Y b b X b X b X b X X b X s b X X

b X X X

+

0 1 1 2 2 3 3 12 1 2 13 1 3 23 2 3

123 1 2 3

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.[43]

Linearmodel : Y b X= 1 1+b X b X2 2+ 3 3

12 1 2 13 1 3 23

b X X b X X b

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 CaCl2 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

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.[44] The SEM photographs of the optimized formulation (AGM1) taken by SEM are depicted

in Figure 1a and b

Table 3: Characteristics of the prepared ALM‑loaded alginate microspheres

Formulation code yield % % drug incorporation efficiency* Zeta potential (mV) Degree of swelling*

AGM1 93.16 91.65±1.13 −31.6 1.206±0.199 AGM2 90.24 76.41±1.65 −32.7 1.113±0.208 AGM3 83.54 86.72±1.97 −31.7 0.996±0.123 AGM4 86.99 73.54±1.08 −33.9 0.940±0.168 AGM5 89.95 87.69±2.10 −34.2 0.906±0.746 AGM6 92.16 74.37±1.67 −32.1 0.844±0.176 AGM7 91.62 85.48±0.95 −35.1 0.791±0.196 AGM8 90.73 71.65±1.09 −35.5 0.754±0.421

*Values are expressed as mean±SD SD: Standard deviation, ALM: Almotriptan malate

Figure 1: Scanning electron microscope microphotograph of

formulation AGM1 at low (a) and high (b) Magnification

b a

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 CaCl2 concentration as well as

cross-linking time The addition of higher amount of Ca2+ 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.[45] 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

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 CaCl2 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.[46] 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 CaCl2 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

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

Figure 2: Zeta potential distribution curve of microsphere formulation

AGM1

Abbas and Marihal: Intranasal delivery of almotriptan microspheres

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

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 5a As the concentration

of CaCl2 and cross-linking time increased, percentage drug

dissolved decreased The in vitro drug diffusion study from

formulations AGM5 to AGM8 are presented in Figure 5b

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

Ca2+ 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

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 6a 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

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

Figure 3: Differential scanning calorimetry thermograms of (a) Pure

almotriptan malate (b) Blank microspheres and (C) Drug - loaded

microspheres

c

b

a

Figure 4: Powder X-ray diffractograms of (a) Pure almotriptan malate

(b) Blank microspheres and (c) Drug - loaded microspheres

a b c

variables (X1, X2, and X3) demonstrated that the model was

significant for both the response variables (Y1 and Y2)

It was observed that all the three independent variables namely

X1 (drug: Polymer ratio), X2 (concentration of CaCl2) 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 Figures 7 and 8

for the studied responses that is, particle size and in vitro

mucoadhesion, respectively Figures 7a depicts response

surface plot for the effect of drug: Polymer ratio (X1) and CaCl2

Table 4: Release kinetics parameters of ALM loaded‑GG microspheres

Formulation code Correlation coefficient (R) Korsmeyer‑Peppas

Zero order order First Higuchi matrix Crowell Hixon‑ R N K

AGM1 0.8862 0.8915 0.991 0.8265 0.9514 0.1693 0.4512 AGM2 0.9011 0.9365 0.9875 0.9365 0.9422 0.1651 0.6012 AGM3 0.9114 0.9461 0.9802 0.9465 0.9406 0.1772 0.6613 AGM4 0.9392 0.9462 0.9728 0.9645 0.9707 0.1305 0.4823 AGM5 0.9126 0.9422 0.9811 0.9461 0.9684 0.1654 0.6971 AGM6 0.9288 0.9501 0.9893 0.9089 0.9469 0.1984 0.6429 AGM7 0.9562 0.9399 0.9865 0.9513 0.9654 0.157 0.4562 AGM8 0.9579 0.9466 0.9729 0.9366 0.9644 0.1592 0.6026 GG: Gellan gum, ALM: Almotriptan malate

Table 5: Summary of results of regression analysis for responses Y1 and Y2

Models R2 Adjusted

R2 Predicted

R2 SD % CV P

Response Y1: Linear model 0.9558 0.9227 0.8234 2.65 6.77 0.0036

Response Y2: Interactive model 1.0000 1.0000 0.9997 0.028 0.033 0.0038

SD: Standard deviation, CV: Coefficient of variation, P: Probability

value Regression equations of the fitted linear and interactive model:

Y1=2.19+32.09X1+1.01X2+1.31X3, Y2=+117.74−27.82−1.82X2−

1.56X3+1.86X1X2+1.08X1X3−0.146X2X3

Figure 5: In vitro drug diffusion profile of microsphere formulation AGM1 to AGM4 (a) and AGM5 to AGM8 (b)























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b a























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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)

b a

Abbas and Marihal: Intranasal delivery of almotriptan microspheres

concentration (X2) on particle size, which indicate a linear effect

on particle size of the microspheres The combined effects

of CaCl2 concentration (X2) and cross-linking time (X3) and

drug: Polymer ratio (X1) and cross-linking time (X3) on particle

size, as shown in Figure 7b and c also revealed linearity This

explains that the higher the amount of CaCl2 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

The combined effect of X1 and X2 on in vitro mucoadhesion of

the microspheres was observed to be nonlinear, as in Figure 8a

At low value of drug: Polymer ratio and CaCl2 concentration,

a higher value for in vitro mucoadhesion was observed Similar

effects were observed for factors X2, X3 and X1, X3, as shown in

Figures 8b and c, respectively As the CaCl2 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), CaCl2 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

CONCLUSION

Mucoadhesive and biodegradable ALM loaded GG microspheres were successfully fabricated by w/o emulsification cross-linking technique employing a 23 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

Table 6: Results of analysis of variance for measured responses

F

Particle size

Significant

In vitro mucoadhesion

Significant Residual 1 8.000E‑004 8.000E‑004 ‑

df: Degrees of freedom, SS: Sum of square, MS: Mean sum of square,

F: Fischer’s ratio

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

c

b a

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

c

b a

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 7: The predicted and observed response variables of the

microspheres

Responses Formulation Predicted

value Actual value error* (%) Prediction

*Prediction error (%)=(Actual value−predicted value)/predicted

value×100 Y1 and Y2 are particle size and in vitro mucoadhesion

respectively

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... Abbas Z Preparation and in vitro characterization

of mucoadhesive polyvinyl alcohol microspheres containing amlodipine besylate for nasal administration Ind J Pharm Educ...

33 Sankar C, Mishra B Development and in vitro evaluations of

gelatin A microspheres of ketorolac tromethamine for intranasal administration Acta Pharm 2003;53:101‑10.