Chronomodulated Drug Delivery System of Salmeterol Fluticasone Nlcs Loaded Tablets: Preparation, Characterization, Stability and Drug Release Studies for Management of Asthma

Mohammed Waseem A¹*, Ajin P Kurian¹, Dhanapal Y²

¹Department of Pharmaceutics, Sri Ramakrishna Institute of Paramedical Science, Coimbatore – 04.

²Department of Pharmaceutical Analysis PSG College of Pharmacy, Coimbatore – 04.

*Corresponding Author E-mail: mw304279@gmail.com, ajin.p.kurian@gmail.com, dhanapalpharma1994@gmail.com

ABSTRACT:

Objective: The purpose of this study was to develop salmeterol, fluticasone nano-lipid carriers to estimate as potentials of oral delivery system for poorly water soluble drugs. Nano-lipid carriers applied to chronomodulated pulsatile drug delivery system maintain the concentration level by releasing the drug at predetermined time interval throughout the management of asthma. Method: The particle size analysis revealed that all the formulations were within the nanometer range of 150.0±2.4nm. Percentage of entrapment efficiency and drug loading were found to be 69.5±4.4 - 85.3±1.3 and 9.358±2.2-10.45±8.1, respectively. The SLM-FCN nano-lipid carrier’s optimized formulation showed spherical morphology with smooth surface under the transmission electron microscope (TEM), the crystalline characterization of drug in NLC was investigated by X-ray diffraction and differential scanning calorimetric (DSC). The ex-vivo permeation study showed many folds increment in the SLM-FCN NLCs compared to powder SLM-FCN 96.0±2.55 and pulsing plugs in-vivo drug released effectively in pre-determine time intervals. Conclusion: The progression concludes that chronomodulated programming pulsatile release was achieved with modified pulsing bilayerd plugged of salmeterol, fluticasone propionate NLCs, formulation remarkably improved oral bioavailability. we promise that finding in this investigations suggest practicability of the dosage form system can be taken after at bedtime then it will be delivered in the early morning which maintains the drug concentration throughout to control asthma.

KEYWORDS: Chronomodulated, Liposome’s, Salmeterol, Fluticasone Propionate, Pulsing Erodible layer.

INTRODUCTION:

The general considered for the oral route drug delivery is favorite for drug administration. Most conventional drug delivery system for time-controlled release is based on single (or) multiple matrix systems^1,3. This system designed in the program to deliver constant drug levels at an extended period.

Chronomodulated system is a time-controlled drug delivery system that has designed to mimic the cardiac rhythm of the biological system and deliver the drug at a specific time (in after a design Log-phase), as the physiological conditions of disease required^4,5.

NEED OF STUDY:

Asthma is characterized by inflammation in airways of the lungs and it makes breathing difficult, it is the most common chronic condition. Bronchitis asthma symptoms arising early morning and late afternoon like coughing, wheezing and whistling sound made when breathing. Salmeterol is a long-acting beta-2 androgen-receptor agonist its continually binding and releasing beta-2 receptor stimulation in the lung resulting relaxation of bronchial smooth muscle and increased bronchial airflow, fluticasone propionate is activating glucocorticoid receptor and inhibit the lung eosinophilia inflammation. Currently available marketed formulations of these combinations are inhalers and nebulizers, oral dosage forms are not available because of low lipophilicity and high protein binding. The inhalers, nebulizers have lots of limitations such as require a certain inspiratory flow to accurate medicine, difficult for some people to coordinate particularly young children, mentally challenge elders and induce serious side effects includes throat irritations^6,7. So emerged the need for effective drug delivery in chronic asthma treatment and better patient compliance.

In this present study, we develop liposome-encapsulated salmeterol, fluticasone loaded chronomodulated pulsatile drug delivery system bilayer tablets.

MATERIALS AND METHODS:

Salmeterol, fluticasone was purchased from Mdpharma.co (Mumbai, India),  Ethyl cellulose  (SD Fine-Chem. Limited ), HPMC (sigma-Aldrich), Sodium starch glycolate (Himedia Laboratories), Sodium bicarbonate (Thermo Fisher Scientific India Pvt. LTD), Citric acid (Nice chemicals private limited), Microcrystalline cellulose (Otto chemicals ), Magnesium stearate (Himedia Laboratories) Talc, stearic acid, elaichi acid, (Himedia Laboratories) Sodium hydroxide (Merck) Potassium dihydrogen phosphate (Himedia Laboratories), acetonitrile, methanol (Sigma- Aldrich.co). All the chemicals and reagents were used in analytical grades.

METHOD:

Preformulation studies:

The pre-formulation studies were performed to determine the solubility of SLM-FCN in the various components used in the formulations. Solid lipids, liquid lipids, and surfactants, co-surfactant are subjected to this study to optimize and select the most compatible out of each category⁽⁷⁾, results shown in (Table: 1).

Table: 1 Composition of different SLM-FCN loaded NLCs formulation

Formulation- code	Stearic acid: elaichi oil	Polysorbate 80 (%, v/v)	PEG4000 (%)	Water (%)
FSF1	40:60	5	10	50
FSF2	50:50	5	10	50
FSF3	70:30	5	10	50
FSF4	40:60	5	10	50
FSF5	60:40	5	10	50
FSF6	35:65	5	10	50

Note:  all the compositions taken (Mg)

Preparation of Salmeterol, Fluticasone loaded NLCs:

Salmeterol, fluticasone loaded NLC was prepared by using the microencapsulation method followed by the Ultrasonication technique. The aqueous phase was prepared by mixing water with polysorbate-80 (surfactant), PEG-4000 (co-surfactant) separately and Different concentrations of lipid phase (steric: elaichi oil) were heated at 80˚C in a separate beaker. Added with aqueous phase drop wise slowly in lipid phase at constant string room temperature. The mixture was allowed for 20min to form a stable emulsion, further this emulsion was subjected to probe sonication for five cycles and cooled. The NLC is solidified after NLC dispersion was subjected to freeze-drying using the freeze-dryer (labconco) at chamber pressure (90) and cold temperature (-30˚C) in these total process to obtain a solid form of the NLC. Prior to the drying process NLC was frozen in a freezer for 8hrs. Additionally, mannitol (2%) was added as a cryoprotectant to avoid the lysis of the nanoparticles⁸.

The entrapment efficiency of drug loading:

SLM-FCN NLCs desired amount was dehydrated with PBS (pH 7.4) and these centrifuge for 15000rpm in 15min at (4˚C) to remove the unbounded drug from the samples. The supernatant aqueous phase pipette out transfer into a centrifuge tube and methanol was added to destroy  structure of NLCs. Then the suspension solution was centrifuged for 15000rpm in 15min at 4˚C, after centrifuge remove the aqueous phase and  the precipitate was then entrapped with SLM-FCN (Emanuela Fabiola et, al 2018). The contents of SLM-FCN were determined by high-performance liquid chromatography (Waters 515 HPLC, made in the US), column sun fire (C18 (4.6x150mm) 3.5µm), the mobile phase methanol: water: formic acid (80:10:2v/v/v) flow rate 1ml/min and wavelength at (262nm)^9,10. 

The drug loading DL (%) and entrapment efficiency (EE %) was calculated by the following formulas.

DL%= weight of the SLM-FCN encapsulated NLCs/weight of SLM-FCN NLCs × 100

EE%= the calculate DL/ theoretical DL× 100

Determination Particle size:

The particle size and size distribution of SLM-FCN NLCs was determined by the dynamic light scattering method using a Malvern Zetasizer (Malvern Zetasizer Nano ZS, Instrument UK). The samples were diluted with distilled water before measurement⁶.

Morphologic feature (TEM):

A selected best formulation was chosen for the morphological examination of the NLCs. A drop of the NLCs suspension was applied on a carbon-coated grid. The suspension was left for 2min, allowed to absorb in the carbon film, and the excess liquid was drawn off

Table: 2 The composition of Bilayer tablet plugs

Ingredients	CT1	CT2	CT3	CT4	CT5	CT6	CT7	CT8	CT9
SLN- NLCs (equivalent weight)	40	40	40	40	40	40	40	40	40
FCN-NLCs (equivalent weight)	60	60	60	60	60	60	60	60	60
Sodium starch glycolate	10	15	10	---	---	---	5	7.5	10
NaHCO3: citric acid (1:1)	---	---	---	10	10	20	5	7.5	---
Microcrystalline cellulose (Avicel PH 102)	40	35	30	40	30	30	40	25	30
Magnesium stearate	1	1	1	1	1	1	1	1	1
Talc	1	1	1	1	1	1	1	1	1
HPMC	150	---	160	150	---	---	150	160	---
Natural polymer (jack fruit)	---	160	---	---	160	150	---	---	160

Note: all the compositions taken (Mg)

with filter paper and uranyl acetate was used as a negative stain. Samples were examined by TEM operating at an accelerating voltage of 80kV⁷.

Compatibility Studies:

A mixture consisting of different ratios of lipid vehicles and either alone were analyzed using FTIR (Shimadzu 8400 S).

Preparation of core tablet and assembly the erodible pluggings:

Briefly, core tablets were prepared by using a direct compression method. SLM-FCN NLCs mixture of microcrystalline cellulose (MCC, Avicel PH-102), sodium starch glycolate, effervescent agent (NaHCO3: citric acid) was blended for 20 minutes and followed by addition of magnesium sterate and talc. The powder mixture was further blended for 10minutes and compressed with 6mm concave punches by using a tablet compression machine (Rimik mini press). The core tablets were assembled by coating with 150mg and 150mg of coating granules (HPMC, JACK FRUIT PULB). Half the quantity of the coating material was weighed and transferred into a 10mm die. Then the core tablet was placed at the center. The remaining half of the coating material was filled into the die and compressed by Rimik Mini Press made in the US  the composition of all formulations is provided⁹. (table:2)

Evaluation of core and pulsed tablets:

Weight Variation Test:

To study weight variation, 20 tablets of formulation were weighed using an electronic balance and the test was performed according to the USP official limits 

Tablet Thickness:

Thickness of tablets was important for uniformity of tablet size. Ten tablets were selected randomly and thickness was measured by using vernier-caliper scale, which permits accurate measurement.

Hardness of Tablet:

Hardness of the tablet is the indication of its strength against resistance of tablet to capping, abrasion or breakage under conditions of storage, temperature and handling before usage. Hardness is the measure of the force required to break the tablet using a specific device. The hardness of 10 tablets (randomly) was determined by Monsanto hardness tester. Hardness measured in kg/cm2.⁹

Friability Test:

The friability of the tablet was determined by Roche friabilator. Accurately weigh the tablets and place them in the friabilator. These tablets were subjected to friability test at 25rpm for 4 minutes (100 rotations).

In vitro release kinetics:

The study was used to measure the amount of drug released in specific time interval were carried out in dissolution test apparatus (LABINDIA DS 8000, XXIII-type 2 Paddle), using  900ml Phosphate buffer (pH 6.8) as dissolution  media  and  maintained at temperature 37°C±0.5ºC. The tablets are directly placed in the medium and immediately operate the apparatus at 50 rpm. At different time intervals (0.5, 1, 2, 3, 4, 5, 6hr) samples were drawn off and replacing it with a fresh medium. The collected samples were filtered and diluted, further analyzed by developed RP–HPLC method. The mean cumulative amount of drug release at each time point was calculated. The analyzed data were fitted into different kinetic models such as zero order, first order, Higuchi, Hixson–Crowell, and Korsmeyer–Pappas model and best fit model was determined based on the regression coefficient (R2) value (7).

Ex vivo transport study:

The transport study of SLM-FCN NLCs was performed by diffusion cell method, diffusion cell having an area of 1.5cm, using specified Phosphate buffer (pH 7.4) as the receptor media. A small quantity of NLCs (equivalent to 50mg of SLM-FCN) was placed on the egg yolk membrane surface, at specific time interval (0.5, 1, 2, 3, 4, 5, 6, 12hr), sampling was performed by removing the media from receptor compartment and replacing it with fresh medium. The collected sample was filtered and diluted, further analyzed using HPLC. The mean cumulative amount of drug release at each time point was calculated.^10,11.

Stability studies:

Stability study was established that long term stability testing should be done at 25°C/60% RH; stress testing should be done at 40°C/75% RH for 6 months. According to ICH (Q₁) guidelines, if significant change occurs at these stress conditions, then the formulation should be tested at an intermediate condition i.e. 30°C / 75% RH.

Statistical analysis and mathematical fittings:

Statistic optimized was performed using a design expert prism software (graphical pade 5.0.0. inc.).

RESULT AND DISCUSSION:

The maximum solubility of SLM-FCN in solid and liquid lipids of stearic acid (123.0mg/ml) elaichi oil (139.2mg/ml) were selected as lipids respectively, (fig-1) were surfactant selected as polysorbate 80(5%) and co-surfactant (10%) respectively based on the stability of prepared formulation with different surfactants.

Encapsulation efficiency and drug loading:

NLCs as drug carriers and their capacity for drug encapsulation was an important parameter. As summarized results show in (table:3), SLM-FCN shows  highest entrapment of 86.65±5.32% in the formulation (F3) and lowest in the 70.23±3.62% formulation (F6) as compared to the other formulations. The encapsulation increase was observed by increasing oil content, the percentage of encapsulated drug increase as the drug shows more solubility in lipid blend. High EE% values observed in this study indicate that the lipid and surfactant compositions employed were satisfactory for SLM-FCN NLCs. The drug loading was done for all developed formulation; each formulation was evaluated for the free and total drug. The drug loading for SLM-FCN NLCs dispersion was found to be in the range of 7.52±1.31 – 11.46±0.36%. These values of parameters can be explained based on high solubility of SLM-FCN both in the oil (oleic acid) and in the lipid (stearic acid).

Particle size analysis:

The NLCs lipid particles were found in the mean size range 150.13 to 245.63nm (table:3) the particle size variations show it particle size statistics, particle size depends on the amount of lipid encapsulated in the formulation. The PDI measure which indicates the unimodal size distribution was within acceptable limits for all the formulations. Especially, a small value of PDI indicates a homogenous population, while a large PDI value means heterogeneity in particle size results shown in (fig-2).

Figure 1: Solubility of SLM-FCN in various solid and liquid lipids.

Table 3: Physicochemical evaluation parameters of SLM FCN- NLCs formulation

Formulation	Particle Size (nm)	EE (%)	PDI (%)	DL (%)	DR (%)	DP(µg/ml)
FSF1	170.4±1.4	82.4±3.6	0.483	11.45±6.4	53.1±9.01	894.3±9.6
FSF2	185.5±3.6	72.9±9.1	0.495	9.358±2.2	64.2±581	801.4±1.2
FSF3	190.3±1.4	84.6±6.4	0.364	12.89±9.2	69.6±3.29	908.6±3.4
FSF4	150.0±2.4	85.3±1.3	0.447	11.98±4.5	72.2±1.36	866.1±8.1
FSF5	176.0±9.4	69.5±4.4	0.468	10.45±8.1	70.5±6.75	820.8±6.5
FSF6	166.3±2.2	78.7±1.1	0.401	11.86±3.9	68.4±2.58	850.6±8.5

Note: Data presented as mean ±standard deviation. EE- Encapsulation Efficiency, PDI- Polydispersity index, DL- Drug Loading, DR- Drug release, DP- Drug permeate

Figure: 2 Particle size peak of SLM-FCN NLCs

Preparation Evaluation of core and pulsed tablets:

Chronomodulated SLM-FCN NLCs loaded tablets were prepared by direct compression method. The compressed dosage form was evaluated for weight variation test, friability, hardness and thickness for resting all formulations (F1 to F6). No significant difference was observed in the weight of individual tablets from the average weight. The hardness of tablets of all formulations was within acceptable limits (range 5.2-6.2kg/cm2). All the formulations showed % friability less than1%, which indicates the ability of tablets to withstand shock. No significant difference was observed in the thickness of individual tablets from the average. The % drug content of all the tablets was found to be in the range of 98.64% - 100.38%. All the results show in a (table:4).

Morphological feature:

To investigate the morphology of the SLM-FCN-NLC, a transmission electron microscope (TEM) was used to depict the image of SLM-FCN-NLC Figure (3). It is almost spherical and non adherent to each other on a scale. The images showed uniform size distribution of NLC having a coarsely spherical shape, displaying a sealed structure. As indicated in the figure, particle diameters were consistent with the results obtained by DLS characterization and depicted a mono-dispersed spheroid-like appearance with a distinct boundary between each particle.

Differential scanning calorimetry (DSC): The DSC thermograms of pure SLM-FCN, stearic acid, and lyophilized SLM-FCN-NLC were shown in (Figure.4) The thermal curve of SLM-FCN and stearic acid showed an endothermic peak at 66.28°C and 119.28°C, respectively. The melting endothermic peak of the lyophilized SLM-FCN-NLC (Figure.4C) shifted to a lower temperature (61.94°C). The decrease in melting temperature of NLC compared with stearic acid alone has been attributed to their small size (nanometer range), the dispersed state of the lipid, and the presence of surfactants. This indicated that CAR was not in the crystalline state, but rather present in amorphous state and drug was completely entrapped within the nanoparticles (Agnihotri and Vavia., 2009; Raj et al., 2015).

Table: 4 The evaluation parameters data of compressed pulsatile tablets

Formulation code	Average weight of 20 tablets (mg)±SD	Friability (%)	Hardness Mean±SD Kg/cm2	Thickness Mean±SD (mm)
PF1	300.1±1.18	0.8%	4.53±0.15	0.43±0.05
PF2	301.6±1.18	0.7%	4.36±0.2	0.5±0.1
PF3	299.8±1.47	0.6%	4.33±0.05	0.43±0.05
PF4	299.6±1.56	0.7%	4.9±0.1	0.46±0.05
PF5	298.9±2.19	0.5%	5.4±0.2	0.43±0.05
PF6	299.5±1.23	0.7%	5.13±0.15	0.4±0.1

Note:  Average SD± of (n=5) determinations

Figure: 3 Surface morphology of SLM-FCN NLCs transmission electron microscopy (TEM)

Figure: 4 SLM-FCN NLCs with polymers DSC image (A) Sabutamol, fluticasone (B) SLM-FCN NLCs (C) stearic, elaichic acid

Figure: 5 X-ray powder diffraction studies of SLM-FCN NLCs and its additives, (A)Salmulatol, fluticasone (B) placebo NLCs, (C) lypholized SLM-FCN  NLCs.

X-ray diffraction study:

To find out the physical state of SLM-FCN, stearic acid, blank NLC was compared with that obtained XRD diffractogram of lyophilized SLM-FCN-NLC. The diffraction pattern of SLM-FCN showed that it is highly crystalline as indicated by its numerous distinctive peaks with the major characteristic diffraction pattern appearing at a diffraction angle. The XRD interpretation of SLM-FCN-NLC formulation shows more of an amorphous nature as compared to the pure SLM-FCN has seen in (Figure 5). Some sharp peaks were also observed which may be due to the presence of mannitol in the NLCs which is crystalline. The principle peak of SLM-FCN was absent in NLC formulation, which may be due to the incorporation of SLM-FCN between parts of the crystal lattice of the lipid leading to change in the crystallinity of SLM-FCN NLCs. Further, it was observed that the lipid matrix is less crystalline, it may be expected that the amorphous portion would accommodate drug as there would be enough space where the drug would be incorporated, compared with their physical mixture. As a result, drug entrapment efficiency in SLM-FCN-NLC would be experienced in the above-mentioned lipid matrix.

In vitro release kinetics:

The kinetics drug release from NLC suspension and the erodible pulsatile layer was plotted against time as concentration, results shown in (table:5) a Drug release showing was biphasic burst release (23.42% in 4h) followed by sustained release, 87.56% drug release in the next 6h. The initial burst release occurred due to the presence of the free SLM-FCN in the external phase and on the surface of the NLC. The burst release rate was affected by the change of concentration of lipid and surfactant in the external phase. The release rate decreased this may be due to the higher concentration of drug presence in the inner core ^(12)(15). The lipophilic nature of the SLM-FCN could be the reason for the sustained release of the drug from an internal lipid phase after the initial burst release. Drug suspension was just taken to show that enhancement of solubility of the drug by any means does not have any effect on the achievement of a sustained-release profile of the drug. To propose the possible release mechanism, the release data were evaluated to check the goodness of fit for various kinetic models (Higuchi, 1963; Korsmeyer, 1983; Peppas, 1985). The goodness of fit was evaluated by R2 (correlation coefficient) values and the model showing the highest value of R2 was considered as the best model for release kinetics. The highest value of the correlation coefficient (R2¼0.996 was observed for Korsmeyer–Peppas model (1983), followed by the first-order (R2¼0.378), Higuchi’s (R2¼0.639) and zero-order (R2¼0.475) models. The correlation coefficients obtained after fitting the in vitro release data to the respective model equations indicates that the best fit was obtained with the Korsmeyer–Peppas model, results shown in (table:5, 5a)^16,17.

Table: 5 Percentage cumulative drug release data of SLM-FCN loaded pulsatile tablet formulations

Time (hr)	Cumulative percentage amount of drug release (%)
	PF1	PF2	PF3	PF4	PF5	PF6
1	00.00±0.00	00.00±0.00	00.00±0.00	00.00±0.00	00.00±0.00	00.00±0.00
2	00.00±0.00	00.00±0.00	00.00±0.00	00.00±0.00	00.00±0.00	00.00±0.00
3	00.00±0.00	00.00±0.00	00.00±0.00	00.00±0.00	00.00±0.00	00.00±0.00
4	9.2±2.26	28.2±1.78	21.6±3.32	00.00±0.00	14.6±3.34	00.00±0.00
5	72.7±1.46	43.9±9.33	55.0±4.76	27.9±4.33	57.6±9.78	58.5±0.36
6	90.7±0.83	86.0±1.43	93.6±1.32	96.0±2.55	93.6±3.48	90.8±4.51

Note: Average SD± of (n=5) determinations

Table: 5a Kinetic order drug release of pulsatile formulations

S. No	Model Equation Plot	Plot X axis Y axis		R 2 value
Zero order	M0–M = kt	Fraction of drug released	Time	0.475
First order	m = kt Log %	Log % drug remaining	Time	0.378
Higuchi model	M0–M = kt1/2	Fraction of drug released	√ time	0.639
Koremeyer Peppas	log (M0–M) = log k + n log	Log fraction of drug released	Log time	0.999

Where, mo, and m, is initial drug content at time. t,drug content at time respectively.

Table: 6 Stability results of formulated pulsatile tablets

Time durations	Drugs	% Assay of drugs recovered			% RSD
		1 month	3 month	6 month
THERMAL/HUMIDITY/30 ^˚C/75%RH	SLM	99.0±1.4	98.8±5.4	98.2±9.0	0.93
	FCN	98.3±9.0	98.0±0.3	97.9±7.9	1.69
THERMAL/HUMIDITY/40 ^˚C/75%RH	SLM	99.1±4.5	98.9±2.9	98.2±1.4	0.38
	FCN	96.8±5.8	96.2±1.2	96.0±3.9	2.37
Room temperature (28^˚C)	SLM	98.9±4.4	98.4±3.4	98.2±6.7	1.35
	FCN	99.4±7.1	99.1±6.5	98.9±5.6	0.93

Stability studies:

The stability studies results of SLM-FCN NLCs pulsing drug delivery system as showed no significant changes concerning the physical appearance, drug content and in vitro drug release at the end of six months results show in the table(6). Aging did not alter the drug release profile and drug content of any formulations significantly at the end of the storage period¹⁸.

CONCLUSION:

In the present study, chronomodulated time-controlled pulsatile drug delivery system of SLM-FCN NLCs compressed tablet for sustained release after the drug lag time was successfully developed. SLM-FCN NLCs formulation developed by microencapsulation ultrasonication technique using stearic acid and elaichi acid as the lipid phases. The formulated nanolipid carriers with lowest particle size, and highest encapsulation efficiency, loading efficiency chosen on Ex-vivo transport studies. The overall progression proves an alternate drug delivery system for increasing the SLM-FCN bioavailability through the nanostructure lipid carriers (NLCs) methodology and we are promising that pulsing dosage form of SLM-FCN could be effectively control the asthma throughout the day in respect to release of pulses. Hence this delivery system would be suitable for asthma patient to manage breathing difficult.

ACKNOWLEDGEMENT:

I thank Sri Ramakrishna institute of paramedical science for providing all the working facilities for performing this study.

CONFLICTS OF INTREST:

No conflict of interest

AUTHORS CONTRIBUTION

M.W conceived the study and designs the experiments. M.W. performed Formulation experiments D.Y HPLC analysis and. A.P.K help to wrote the manuscript.

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Received on 25.06.2020            Modified on 19.10.2020

Accepted on 12.01.2021      ©Asian Pharma Press All Right Reserved

Asian J. Res. Pharm. Sci. 2021; 11(2):95-102.

DOI: 10.52711/2231-5659.2021-11-2-1