INTRODUCTION:

Epilepsy is a common persistent condition characterized by frequent seizures related to neurological homeostasis. These seizures are temporary signs caused by abnormal, excessive neural activity in the brain¹. Around 50 million people worldwide have epilepsy at any given time. Epilepsy has been treated with medication but cannot be completely cured. Therefore, surgery can be considered in difficult circumstances. Despite the best drugs available, more than 30% of the epileptic reported cases had no seizure control. Most symptoms are short-lived and appear at some stage in childhood². Epilepsy is a group of syndromes with very different symptoms, but they are episodically abnormal electrical activity in the brain.

Pregabalin is one of the most effective drugs used to treat epilepsy. It is the most preferred second generation anti-epileptic drug (AED) that eliminates seizures without side effects. Pregabalin is an anticonvulsant used to treat neuropathic pain and as an additional therapy for generalized anxiety disorders and partial seizures. Pregabalin is structurally related to the neurotransmitter gamma-amino-butyric acid (GABA) and therefore mimics the same pharmacological effects³. Pregabalin is well absorbed after oral administration and under fasting conditions; its Tmax is 1.5hours. The bioavailability after oral doses is at least 90% (regardless of the dose). Renal clearance in healthy volunteers is 67.0 to 80.9ml/min. For oral absorption, the pH should be between 5.5 and 7.3 The available dosage forms cannot prevent dose discontinuation leading to complications such as lactic acidosis or gastrointestinal complications.⁴ Thus; there was a need to develop novel drug delivery to overcome these challenges⁴.

Nanotechnology has successfully established itself in this area. Successful advances have been made in the development of a variety of nanocarrier based drug delivery and drug targeting systems to minimize drug loss through degradation and inactivation in the biological environment, minimize side effects, and increase drug bioavailability and targeting of vesicles.⁵ Niosomes are nanovesicles that are obtained by the hydration of nonionic surfactants with or without suitable fluidiser. The fluidiser like cholesterol or other lipids helps in gaining the spherical shape and proper physical form. Niosomes promote the selective distribution of drugs, improve medicinal value, and reduce the side effects.^6,7 The aim of the present study was to develop the niosome vesicles that incorporate drug to enable effective delivery over a longer period of time. Therefore, the main goal is to formulate and develop a pregabalin loaded niosomal dispersion to avoid serious side effects like lactic acidosis or gastrointestinal side effects while reducing the dosage of pregabalin and providing a dosage form that meets the needs of patients.

MATERIALS AND METHODS:

Materials used:

Pregabalin was a gift sample from Wockhardt Labs (Aurangabad). Cholesterol was purchased from J.P. Pharmaceuticals Chemicals (Pune). All non-ionic surfactant and other reagents were of analytical grade.

Preparation of Niosomes:

Numerous techniques have been explored, including the use of supercritical fluid to develop niosomes.^6-8 However, here the pregabalin niosomes were made by thin layer hydration as this is the most suitable technique for scaling^9,10. A rotary flash evaporator was used in which a round bottom flask (RBF) was loaded with an accurately weighed amount of cholesterol, nonionic surfactant and GMS in 10mL of chloroform. The flask was rotated 1.5cm over a water bath at 60±20°C under reduced pressure until all of the organic phase had evaporated and a thin layer had formed on the wall of the round bottom flask. Then an accurately weighed amount of the drug (100mg) was dissolved in 10mL of distilled water. The dried film hydrated with this drug solution, and the mixture was rotated by immersing it in a water bath at 60±2°C for 1 hour until a good dispersion mixture was obtained. The unilamellar vesicles obtained by sonication of the dispersion obtained. Firstly, we screened different ratio of diverse surfactants with cholesterol in a ratio of 1: 1 for their suitability in the development of niosomes^11-12. The study revealed that Span 80 is most suitable surfactant for optimization purpose. Table 1 summarizes the batches suggested by the design expert based on combination and permutation of the independent variables.

Table 1: Box-Behnken experimental design with measured responses

	Factor 1	Factor 2	Factor 3	Response 1	Response 2
Run	A: Cholesterol	B: Span 80	C:GMS	Particle Size	Entrapment Efficiency
				nm	%
1	0	0	0	600	92.2
2	0	-1	-1	720	74.2
3	-1	0	1	440	76.4
4	1	0	-1	690	68
5	1	1	0	530	82.2
6	-1	-1	0	500	62.3
7	0	1	1	460	62.8
8	0	0	0	610	91.8
9	-1	0	-1	660	62.1
10	1	-1	0	520	78.3
11	0	0	0	600	94.4
12	0	0	0	610	94.7
13	0	1	-1	680	73.5
14	1	0	1	480	88.1
15	0	0	0	620	94.8
16	0	-1	1	490	72.4
17	-1	1	0	510	67.2

Optimization of formulation by design of Experiments:

Based on the preliminary assessment of shape and size by phase contrast microscopy, span 80 selected as a surfactant in the final optimization trials. The study examined the effects of three independent variables, viz conc. of soya lecithin, GMS and cholesterol for optimization of the formulation¹³. The design summary is as shown in table 2

Table 2: Design Summary

*File Version*	10.0.8.0
Design Wizard	Optimization > Factorial / RSM > No HTC > All Numeric > 2 factors, 10 runs, alternative design
Study Type	Response Surface	Subtype	Randomized
Design Type	Box-Behnken	Runs:	17
Design Model	Quadratic	Blocks	No Blocks	Build Time (ms)	15.00

Characterization of niosomes:

Particle size distribution and zeta potential determination:

The initial observations during development stage were carried out using a phase contrast microscope however, the optimized batches were analysed using Malvern Nano Zetasizer Ver. 601. The samples were diluted serially to a factor of 50 so that there will be no interference from the agglomerates and aggregates. The surface morphology of the optimized batch was predicted by subjecting sample to SEM.^14-15

Determination of drug entrapment efficiency:

Entrapment efficiency was determined using a sample after subjecting it to a high-speed centrifugation. The content of free medication was determined. The repeated washings with PBS were given to the mass obtained at the bottom of the centrifuge tube and centrifuged again for 1 h. The percent of entrapment efficiency (% EE) was determined according to Eq. (1).¹⁶

% EE = (C-Cf) / C X 100 (1)

Where,

C is the total amount of pregabalin and Cf is the amount of free drug present in supernatant. All the measurements are in triplicate.

In vitro Release Studies:

In vitro release studies of pregabalin and lyophilized pregabalin niosomal dispersion were carried out using the USP dissolution device I. Enteric-coated gelatin capsule (Size 5), filled with the plain API and Lyophilized powder (separate capsule) were placed in the basket 900mL dissolution medium with thermostat at 37.0±0.5°C and stirring at 100rpm. The release of the drug was recorded for 2 hours in simulated gastric fluid (SGF, pH 1.2) and for 24 hours in simulated intestinal fluid (SIF, pH 7.4). At the predetermined intervals, aliquots of the solution (10mL) were removed and analyzed for the active substance content with a UV spectrophotometer at 215nm.^17-18 All experiments were carried out in triplicate.

In vitro permeation studies:

The in vitro permeation study of niosomes was studied using the dialysis bag. An accurately measured volume of the niosomal dispersion produced by re-dispersing the centrifuged slug was added to the semisynthetic dialysis bag. The bag was placed in 20mL of buffer solution and kept on an orbital shaker to maintain sink condition. An aliquot was removed at a fixed interval, replacing fresh buffer of the same amount in the conical flask. The aliquot was sufficiently diluted and estimated for drug content at 215nm.^19-20

Osmotic Shock:

Formulations were treated with hypotonic (0.5% NaCl), hypertonic (1 M NaCl), and normal saline (0.9% NaCl) solutions and examined under a phase contrast microscope.²¹

Viscosity:

The dispersion viscosity was examined using a Brookfield viscometer to check the behavior of the dispersion when stress was applied.^22-23

Stability Studies:

A three-month stability study was performed on all formulated niosome dispersions. The divided portions of formulated niosomes were subjected to three different conditions. The first, second and third portions were kept at refrigeration temperature (4±1°C), room temperature and accelerated temperature/relative humidity respectively.^24-28

Anticonvulsant Activity:

Animals:

Male Swiss mice, wistar strain weighing 20-25g were lodged in groups of six under standard laboratory conditions of temperature, relative humidity (55.5%) and lighting with food (Lipton India Ltd. pellets) and water freely accessible.^29-30 The anticonvulsant activity of pregabalin niosome was controlled by PTZ-induced convulsions in mice³¹. The grouping and dosing parameters are described in Table 5.

Compliance with ethical standards:

The project was partially funded by Sharadchandra Pawar college of Pharmacy, Otur, District Pune Maharashtra India. Good clinical practice guidelines were followed for the care. The protocol was approved by the Institutional Animal Ethical Committee (IAEC) of Sharadchandra Pawar College of Pharmacy, Otur, Dist. Pune (IRB No. SPCOP/IAEC/2016-2017/02).

RESULTS AND DISCUSSION:

Preformulation studies with the help of FTIR and DSC revealed that there was no interaction and the excipients were found suitable for the formulation development. The formulated niosomal dispersion revealed optimum viscosity as per the Quality target product profile, spherical shape with a normal size distribution when observed under a phase contrast microscope after subjecting to osmotic shock, as shown in Fig. 1.

Fig. 1. Particle size and shape of the optimized batch (N₁) by phase contrast microscopy

Optimization using Design Expert:

Response for percentage Entrapment Efficiency:

The suitability of the model was decided based on the F-value of 0.87 that implies the model is significant relative to the noise. The model terms are also significant as the model revealed Values of "Prob > F" less than 0.0500. There are no significant model terms for the response on percentage Entrapment Efficiency. Lack of Fit is not significant relative to the pure error as it revealed a valiue of 1.82 for "Lack of Fit F-value". The difference between the "Pred R-Squared" and “Adj R-Squared" values is less than 0.2 reveals that these are in reasonable agreement which was calculated from the 0.6676 and 0.7296 values for "Pred R-Squared" and “Adj R-Squared". "Adeq Precision" value of 6.316 indicates an adequate signal so that the model could be used for determining the design space^32-35. The equations in terms of coded factor is as follows:

E.E. = +89.38+ 6.07A-0.19B+2.74C- 0.25AB+ 1.45AC-2.23BC-6.98A²- 9.90B²-8.75C²... (1)

From the equation (1), the positive terms shows a positive impact on the values of percentage Entrapment Efficiency, whereas the negative values of B represents that it will have a negative impact. The increase in the value of Span 80 would lead to decrease in the entrapment whereas increase in the concentration of cholesterol and GMS would lead to increase in the entrapment of the drug ³¹. The contour plots for the response on Box- Behnken design are shown in Fig 2.

Fig 2: The design illustrating relationship between span 80 and cholesterol concentration for effect of Particle size and Entrapment efficiency

Response: Particle Size

The Model F-value of 12.50 implies the model is significant. There is only a 0.15% chance that an F-value this large could occur due to noise. The P value less than 0.0500 indicate model terms are significant^32-35. The "Pred R-Squared" and “Adj R-Squared" values of 0.7676 and 0.8661 respectively are in reasonable agreement with each other; i.e. the difference is less than 0.2. "Adeq Precision" value of 10.884 indicates an adequate signal so that the model can be used for design space³¹.Third important term is Lack of Fit, which is 0.27, implies the Lack of Fit is not significant relative to the pure error. A signal to noise ratio greater than 4 is desirable. The equations in terms of coded factor is as follows:

Particle size= +588 +13.75A-6.25B-110C+2.50AC+2.50 BC-46.50A²-26.50B²+26C²…..(2)

From equation (2), the conc of cholestrol alone will have a postive impact on the particle size³¹.

Fig 3: The responses illustrating desirability and the relationship between the factors to achieve optimization

The solutions suggested by the software were considered while developing the optimized batch. The results of the validation batch characterization are summarized in table 3. The responses illustrating desirability and the relationship between the factors to achieve optimization are shown in Fig 3.

Table 3: Results for validation batch

Batch	Particle Size	Èntrapment Efficiency
Suggested Values	491.07nm	90%
Practical values	490nm	92.4%

The SEM analysis of the redispersed lyophilized niosomes revealed spherical shape with single lamellae vesicles of 10.8µm as shown in Fig. 4.

Fig. 4. SEM image of redispersed niosome formulation after lyophilization

The use of SPAN 80 presented the highest entrapment of the pregabalin in the niosome vesicles when used with cholesterol. Furthermore, the percentage drug entrapment increased with the decrease in the sonication time. Therefore, the sonication time was kept contant to a value of 15 min. No further trials were conducted to attempt for reduction in vesicle size by increasing the sonication time.³⁶

The formulations were subjected to drug release in vitro by USP dissolution tester type I and permeation using the dialysis bag method^37-38. Most prevalent problem with sustained-release formulations is dose dumping wherein the amount of the active substance released at early time points under in vitro conditions is the crucial parameter. The European Medicines Agency (EMEA) limit for release to prevent dose dumping is 20 to 30% in the first 2 h³⁹. The problem underlying the present work was to provide release over longer duration that would not release more than 30% of the total amount of active substance in less than 2 h and to offer a better bioavailability leading to minimization of toxicity. The amount of pregabalin released was estimated by the spectral method at 215 nm in the optimized batch N1. The enteric coated capsules containing a constant dose (100 mg) of pregabalin or lyophilized pregabalin niosomes were subjected to drug release study for 2 h in simulated gastric fluid (SGF) and 24 h in SIF, The data obtained during the first 2 h in SGF are reported in Fig. 5.

Fig. 5. Percentage Drug release from Pregabalin and lyophilized niosomal dispersion filled in enteric coated capsule in SGF

The percentage amount of free drug released from the pregabalin filled capsuled was 20.04% in 2.0 h. In fact, the results were as expected, due to hydrophillic nature of the drug. The plain pregabalin showed very high dissolution rate exceeding 99% of the dissolved drug within 30 min as shown in Fig 6. On the other hand, the drug entraped in niosomal vesicles ensured the desired sustained release of the drug upto 24 hrs.

Fig. 6. Percentage drug release from pregabalin and lyophilized niosomal dispersion filled in enteric-coated capsule in SIF

On average, 85% release was obtained with lyophilized pregabalin dispersion in 24 h. These results showed that niosomal pregabalin was able to maintain the release up to 24 h. This is because of the slow diffusion of the drug across the cholesterol bilayer in niosomal pregabalin. The in vitro permeation studies revealed the ability of the drug to permeate very easily across the membrane. The initial high concentration of the drug was observed due to the free drug in the niosomal dispersion that permeated across the membrane. Subsequently, the release was dependent on the drug release from the vesicles.⁴⁰ The aliquots collected at intervals also showed the presence of intact niosomes crossing the membrane owing to their small size, but even the drug concentration reached its peak in 24 h due to the controlled release from the niosomal dispersion. The drug permeation profile, as shown in Fig. 7, is the comparison of plain pregabalin and niosomal dispersion. The drug content of 97.23% at the end of 3 months under refrigerated conditions revealed greater stability whereas storage under room temperature, at accelerated stability condition for three months showed an entrapment efficiency of only 78.17%. Therefore, the final storage conditions finalized was storage under refrigation.

Fig. 7. Drug permeation from pregabalin and optimized niosomal dispersion (N₁)

The drug content was determined on each batch to correlate the stability of batches under different conditions. The results are summarized in Table 4. The high resolution magnification of the sample also revealed the spherical shape even after 3 month for refrigerated and room temperature conditions as shown in Fig 4. There was slight distortion of the niosome shape was observed in the samples kept under accelerated conditions.

Table 4: Drug Content determination before and after stability study

Batch No.	Drug Content	Drug content after Stability Test (3 Month)
Batch No.	Drug Content	*Room Temp*	Refrigeration	Accelerated Condition
N₁	92.3 %	92.3 %	92.3 %	86.2 %

The anticonvulsant activity of pregabalin niosome was examined by PTZ-induced convulsions in mice. PTZ is well recognized to cause its convulsing effect by demonstrating binding of PTZ at GABA receptor complex and other sites in the brain.^41-42

Table 5 shows that in the PTZ model, positive control group treated with diazepam (4 mg/kg, i.p.) demonstrated 100% protection, however the niosomal dispersion showed 26.11, 35.85 and 51.05% of protection from clonic seizures in the animals at a dose of 10, 20 and 40 mg/kg, respectively. However, the niosomal dispersion significantly decreased the seizure latency and reduced the seizure duration in the animals with the increase in the dose. There was no death recorded for diazepam, but a 16.66% mortality was recorded for mice treated with 10 mg/kg of pregabalin loaded niosomal dispersion. In PTZ-induced convulsions, niosome of pregabalin showed moderate (p<0.05) activity in comparison with reference standard diazepam.

CONCLUSION:

Pregabalin-loaded niosomal dispersion was prepared as prospective vehicle for a prolonged drug release by oral route. Niosomal dispersion based on non ionic surfactant (Span 80): Cholesterol (1:1) mixtures was selected as the optimized formulations (N₁) in terms of vesicle dimensions, polydispersity index, zeta potential and % drug entrapment efficiency, and were evaluated for drug release profile. The release profile of the developed formulation was compared to the drug, which was filled into enteric-coated capsule. The developed formulation revealed moderate protection in PTZ induced convulsions as compared to diazepam. From the above studies, it is concluded that pregabalin encapsulated niosomal dispersion showed prolonged release, thereby achieving better therapeutic action.

Current and Future Developments:

The niosomal dispersion are widely gaining a place in drug delivery system specially in the topical delivery but at the same time scientist are exploring the different routes of delivery to take the advantage to the fullest in achieving best bioavailable fraction. The future work involves the attachment of ligands to make them target the organs to increase bioavailability at that site.

Ethics Approval and Consent to Participate Human And Animal Rights:

The study was performed after getting study protocol approved by the Institutional Animal Ethical Committee of Sharadchandra Pawar College of Pharmacy. The study followed all ethical principles while carrying out the research study.

CONFLICT OF INTEREST:

The authors of the paper declare that they have no conflict of interest.

ACKNOWLEDGEMENTS:

The authors are thankful to Sharadchandra Pawar college of Pharmacy, Otur for providing necessary facilities and partial funding of the project.

AUTHOR’S CONTRIBUTIONS:

PNG and SSA designed the project, conducted research, provided research materials, and analysed the data. PNG and AYM performed the experimental work. The draft of the manuscript was prepared by AYM and PNG. SSA did the finalization of the manuscript and communication. All authors have critically reviewed and approved the Manuscript.

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Received on 15.02.2021 Modified on 07.09.2021

Research J. Pharm. and Tech 2022; 15(9):3912-3918.

DOI: 10.52711/0974-360X.2022.00655

S. N.	Group	Dose (mg/kg) and route	Clonic convulsions		Tonic convulsions		Mortality in 24 h (%)
S. N.	Group	Dose (mg/kg) and route	Latency (sec.)	% Protection	Latency (sec.)	% Protection	Mortality in 24 h (%)
1.	Negative Control	10 mL, i.p.	42.50±3.68	----	31.10±3.13	----	--
2.	Diazepam (Positive Control)	4, i.p.	00	100	00	100	00
3.	Niosomal dispersion	10, i.p.	31.40±1.96^**	26.11	26.60±2.23	14.46	16.66
4.	Niosomal dispersion	20, i.p.	27.26±1.92^**	35.85	25.90±2.33	16.72	00
5.	Niosomal dispersion	40, i.p.	20.80±1.89^**	51.05	22.70±1.92^*	27.00	00