INTRODUCTION:^11,12

Alzheimer's disease is named after Dr. Alois Alzheimer. In 1906, Dr. Alzheimer noticed changes in the brain tissue of a woman who had died of an unusual mental illness. Alzheimer's disease is a progressive, neurodegenerative disease of brain characterized by abnormal clumps (amyloid plaques) and tangled bundles of fibers (neurofibrillary tangles) composed of misplaced proteins. Age is the most important risk factor in Alzheimer's disease. The number of people with the disease doubles every 5 years beyond age 65. Three genes have been discovered that cause early onset Alzheimer's disease. Other genetic mutations that cause excessive accumulation of amyloid protein are associated with age-related (sporadic) Alzheimer's disease.

The incidence of Alzheimer's disease increases with age. In the United States, Alzheimer’s prevalence was estimated to be 1.6% in 2000 both overall and in the 65–74 years age group, with the rate increasing to 19% in the 75–84 years age group and to 42% in the greater than 84years age group. World Health Organization estimated that in 2005, 0.379% of people worldwide had dementia, and that the prevalence would increase to 0.441% in 2015 and to 0.556% in 2030.

RT exhibits action in inhibiting the breakdown of acetylcholine, an important neurotransmitter associated with memory, by blocking the enzyme acetyl cholinesterase. RT is also indicated in the treatment of Parkinson’s disease. RT, having the chemical structure shown in Fig. 1.1 shows rapid onset of action and is metabolized by cholinesterase mediated hydrolysis. However, the therapeutic potential of RT is markedly delayed due to its low oral bioavailability. The low bioavailability of RT results from its first pass metabolism resulting in poor absorption on oral administration. This is due to a large fraction of the drug that remained undissolved to reach absorption site. Rivastigmine tartarate, a potent rapidly-acting cholinesterase inhibitor is practically soluble in water. It belongs to BCS class III and possesses oral bioavailability of 40 %.^12,4

Figure 1: structure of Rivastigmine tartarate^12,13

Formulation approach to avid the first pass metabolism of drug, and also to reduce the particle size of drug, for purposed of crossing BBB as well as enhancing solubility of drug. In view of this, solid lipid nanoparticle (SLN) appear to be an attractive approach for the delivery of hydrophilic drugs such as RT as SLNs have advantages over all other colloidal systems SLNs, liposomes, nanoemulsions, and micro emulsions. The SLN are prepared by various methods like, high pressure homogenization, Ultra sonication/ high seed homogenization, solvent evaporation method, supercritical fluid method, sprays drying method, double emulsion method, precipitation method, etc.

MATERIALS AND METHODS:

Materials

Rivastigmine tartarate was a gift sample from Unichem Ltd, Goa. Precirol ATO 5 was gift sample from Gattefosse, Mumbai Tween 80, and Eudragit RS 100, Eudragit RL 100 were a gratis samples from Loba Chemicals, Mumbai.

Methods

Construction of calibration curve of Rivastigmine tartarate in distilled water

Stock solution was prepared by dissolving 100 mg of rivastigmine tartarate in 100 mL distilled water to obtain working standard solution. Then 1ml standard solution was diluted to 10 mL with distilled water. The solution was analysed spectroscopically to determine ƛ_max using distilled water as blank solution.

Construction of calibration curve of Rivastigmine tartarate in pH 7.4 phosphate buffer

Stock solution was prepared by dissolving 100 mg of rivastigmine tartarate in 100 mL pH 7.4 phosphate buffer to obtain working standard solution. Then 1 mL of standard solution diluted to 10 mL with distilled water. The solution was analysed spectroscopically to determine ƛ_max using pH 7.4 phosphate buffer as blank solution.

Selection of lipids¹⁹

The compatibility of lipids with RT was ascertained by differential scanning calorimetry (Mettler DSC 1star system, Mettler-Toledo, Switzerland), and FT-IR spectroscopy study(IRAffinity-1, Shimadzu, Japan ). The binary lipid mixtures (1:1) were obtained from each lipid (Glyceryl monostearate, Stearic acid, and Compritol 888,Precirol ATO 5). The lipids compatible with RT were selected for further studies.

Drug-lipid solubility determination¹⁹

The lipids (glyceryl monostearate, Compritol 888,Precirol ATO 5, and Stearic acid) were melted 10 ^oC above the melting point of lipid.. To the molten, RT was added in smaller amounts till the precipitate was not obtained. The RT solubility at 10–100 mg in individual lipids (Glyceryl monostearate, Compritol 888,Precirol ATO 5, and Stearic acid). The lipids were able to solubilise up to at 100 mg of RT. The lipids shown maximum solubility of drug were selected for RT-SLN preparation. Solubility of RT in various lipid is shown in table 1.

Table 1 Solubility of rivastigmive tartarate

Solid lipid	Drug solubility (mg/g) in solid lipid
Stearic acid	45±0.51 mg
Glyceryl monostearate	56±0.60 mg
Compritol 888 ATO	70±0.23 mg
Precirol ATO 5	81±0.84 mg

Preparation of RT SLN by high pressure homogenization²⁴.

The RT-SLN were prepared by high pressure homogenization (HPH). Lipid 1 g was accurately weighed and melted, 60 mg RT was dispersed in molten lipid. Separately, 100 mL distilled water was taken in beaker and to it added required quantity of surfactant with continuous stirring on magnetic stirrer. The lipid phase was mixed in aqueous phase at 60 ⁰C for 30minutes and pass through HPH (PANDA2K, GEA Niro Soavi,Italy) at 900 bar pressure for 7 cycles. The pre-emulsion obtained was lyophilized with of 5% mannitol.

Selection of components for the SLN was based on drug Solubility, and compatibility studies of the excipients. Precirol ATO 5, Surfactant phase-tween 80 were selected for further studies. The formulation table 2 shows the composition for preparation of SLN.

Table 2 Formulation table of solid lipid nanoparticles

Sr. No.	Ingredient	F1	F2	F3
1	Rivastigmine tartarate	60 mg	60mg	60 mg
2	Precirol ATO 5	1g	1 g	1 g
3	Tween 80	1%w/v	2%w/v	3%w/v
4	Distilled water	100mL	100 mL	100mL

Freeze-drying of solid lipid nanoparticles²⁴

The SLN dispersion mixed with 2% mannitol as a cryoprotectant was deep freeze under -60 ⁰C overnight. The frozen sample was kept for drying process in freeze-dryer. The drying period was 72 hours by applying vacuum at 100 mtorr. It was used for further analysis and incorporation into transdermal patch. Dried SLN were collected and used for the stability studies, solid state characterisation of SLN and formulation of transdermal patch.

Determination of particle size, zeta potential and polydispersity index¹⁹.

Particle size, zeta potential and polydispersity index of solid lipid nanoparticles loaded with RT was determined using Zetasizer (Beckman Coulter, USA). Prior to the measurements all samples were diluted with double distilled water to produce a suitable scattering intensity. All measurements were performed in triplicate. The photomicroscopy of RT loaded solid lipid nanoparticles were also captured from motic microscope.

Determination of drug encapsulation efficiency of RT¹⁶.

SLN was diluted with double distilled water and then centrifuged for 30 minutes at 50,000 rpm. The entrapment efficiency was then determined by taking absorbance at 263nm by UV spectrophotometer. The entrapment efficiency was calculated by the following equation:

%EE = Amount of drug added - amount of drug in supernatant

Amount of drug added100

Particle morphology¹⁸

The particle size and surface morphology of the formulation was determined by SEM (Zesis, EV-18, Research, Japan). Colloidal suspensions were deposited on a metallic probe, placed in liquid nitrogen for 10 minutes and evaporated under vacuum. SLNs were metalized with gold/palladium with a cathodic pulverizer.

Formulation of RT-SLN loaded transdermal patch^,14

Formulation of transdermal patch using of 3² full factorial design

As shown in table 3, various formulation batches were developed based on two variables as Eudragit RS100 and Eudragit RL100 at low (-1)and high (+1) level and tworesponses folding endurance, and % drug released by using 3² full factorial design.

Table. 3 Independent variablesin transdermal patch preparation¹⁴

Independent	Variable Unit	Optimization level
		Low (-1) High (+1)
Eudragit RS100	(X1) mg	180 900
Eudragit RL100	(X2) mg	900 1620

Table 4 Formulation factor, concentration, and levels¹⁴

Coaded Value	Actual value	Response
	X 1 X 2	Y 1 Y 2
-1	180 900	% Folding Cumulative drug Endurance release
0	540 1260
+1	900 1620

The formulation of RT-SLN transdermal patch is shown in table 5.

Evaluation of transdermal patch

Folding endurance²⁰:

A strip of specific area was cut evenly and repeatedly folded at the same place till it broke. The number of times the film could be folded at the same place without breaking was noted as the folding endurance value.

Thickness of the patch²⁰:

The thickness of the SLN loaded patch was measured in different points by using a Vernier caliper and determined the average thickness.

Weight uniformity²⁰:

A specified area of patch was cut in different parts of patch and weighed in digital balance. The average weight and standard deviation values were calculated from the individual weights.

Drug content uniformity²¹:

An accurately measured portion of film (1 cm²) was dissolved in 100 mL phosphate buffer pH 7.4.The solution was shaken continuously for 15minutes and sonicated. It was subjected to filtration, and appropriate dilution was made for spectrophotometric estimation.

Percentage Moisture content²⁰:

The prepared films were weighed individually and were kept in a desiccators containing fused calcium chloride at room temperature for 24 hours. After 24 hours, the films were reweighed and determined the percentage moisture content from the below mentioned formula.

Percentage moisture content =

[Initial weight- Final weight/ Final weight] ×100.

Percentage Moisture uptake²⁰:

The weighed films were kept in a desiccator at room temperature for 24 hours containing saturated solution of potassium chloride to get 84% relative humidity. After 24 hours,the films were reweighed and determined the percentage moisture uptake using following formula.

Percentage moisture uptake =

[Final weight- Initial weight/ initial weight] ×100.

Percentage Elongation break test²²:

The percentage elongation break was determined by noting the length just before the break point, the percentage elongation was determined using following formula.

Elongation percentage = L1-L2/ L2 ×100

Where, L1is the final length of each strip and L2 is the initial length of each strip.

In vitro skin permeation studies²³:

In in vitro skin permeation study was carried using Franz diffusion cell. Full thickness abdominal skin of male Wistar rats weighing 200 to 250g was used. Hair from the abdominal region was removed carefully using electric clipper. The dermal side of skin was thoroughly cleaned with distilled water to remove any adhering tissues or blood vessels. The skin was equilibrated for an hour in phosphate buffer pH 7.4 before starting skin permeation studies. The isolated rat skin piece was mounted between compartments of the diffusion cell, with the epidermis facing upward into the donor compartment containing pH 7.4 phosphate buffer. The temperature of the cell was maintained at 32±0.5°C using a thermostatically controlled heater. Sample of definite volume was withdrawn from receptor compartment at regular intervals, and an equal volume of fresh medium was replaced. Samples were filtered through filtering medium and were analyzed spectrophotometrically. Flux was determined directly as the slope of the curve between the steady-state values of the amount of drug permeated vs. time in hours and permeability coefficients were deduced by dividing the flux by the initial drug load.

Tensile Strength²⁰

To determine tensile strength, polymeric films were sandwiched separately by corked linear iron plates. One end of the films was kept fixed with the help of an iron screen and other end was connected to a freely movable thread over a pulley. The weights were added gradually to the pan attached with the hanging end of the thread. A pointer on the thread was used to measure elongation of film. The weight just sufficient to break the film was noted. The tensile strength was calculated using following equation;

Tensile strength= F/a.b (1+L/l)

F is the force required to break; a is width of film; bis thickness of film; L is length of film; l isElongation of film at break point.

In vitro release kinetics of RT-SLN²¹

The in vitro drug release studies were performed on optimized RT-SLN formulations using modified Franz diffusion cell. Dialysis membrane (Himedia, Mumbai) having pore size 2.4 nm, molecular weight cut off between 12,000–14,000, was used. Membrane was soaked in double-distilled water for 12 h before mounting in a Franz diffusion cell. SLN formulation containing 4.5 mg of RT was placed in the donor compartment and the receptor compartment was filled with phosphate buffer pH 7.4 (25ml). At fixed time intervals, 2 ml of the sample was withdrawn from receiver compartment through side tube. Fresh dialysis medium was placed to maintain constant volume. Sample was diluted suitably and analyzed by UV spectrophotometry at 263 nm.

Stability studies²⁰

The accelerated stability of optimized formulation study of SLN patch at 40 ± 2 °C / 75 ± 5 % RH for 1 month. The patch was tested for drug content

RESULT AND DISCUSSION:

Calibration curve of rivastigmine tartarate in distilled water

The calibration curve for RT in distilled water is shown in figure 1.5.

Figure 1.5 calibration of RT in distilled water

Calibration curve of Rivastigmine tartarate in pH 7.4 phosphate buffer

The calibration curve for RT in pH 7.4 phosphate buffer is shown in figure 1.6.

Figure 1.6 calibration of RT in phosphate buffer 7.4

Drug excipients compatibility studies

Differential Scanning Calorimetry

Figure 1.7 DSC graph of RT

Figure1.8 DSC graph of physical mixture (RT+Precirol ATO 5)

Figure 1.9 DSC graph of physical mixture (RT+polymer +lipid+mannitol)

In DSC studies rivastigmine tartarate as shown melting peek at 126.26⁰C. The physical mixture of RT with precirol as shown the melting peak at 65.48⁰ C which is similar to the melting peak of precirol at 65.83⁰C.

Whereas the SLN of RT as shown the melting peak at 59.39⁰ C from the above results it can be concluded that the lipid precirol has internal solubilised the drug and hence the reason the peak was disappeared in the DSC thermogram. It indicates the physical compatibility of RT with lipidic material

Evaluation of SLN

Particle size and zeta potential ¹⁴

The three formulation batches PF1, PF2, and PF3 were evaluated for average particle size and polydispersity index (PDI). The formulation batch showing least particle and PDI (batch PF3) were selected. The results obtained in particle size and PDI of batches PF1, PF2, and PF3 are shown in table 13 and figure 1.10.

Table 13 Average particle size and polydispersity index of formulation batches.

Batch code	Average particle size	Polydispersity index
PF1	426.2nm	0.390
PF2	342.3nm	0.542
PF3	214.2nm	0.614

Figure 1.10 Average particle size of formulation batch PF1(426.2 nm)

Figure 1.11 Average particle size of formulation batch PF2(342.3 nm)

Figure 1.12 Average particle size of formulation batch PF3(214.2 nm)

Zeta potential of SLN dispersion was determined to assess the stability of SLN dispersion. The dispersion was found to be stable between -30 to and30mV. Three formulation batches PF1, PF2, and PF3 were evaluated for zeta potential. The formulation batch (PF3) showing zeta potential (-) was selected because it showed good stability according to standard value given for zeta potential. Result is given in table 14 and depicted in figure 1.13, 1.14. and 1.15.

Table 14 zeta potential of all formulation

Sr. No.	Formulation Batch	Zeta potential (mV)
1	PF1	-5.53
2	PF2	-6.78
3	PF3	-10.5

Figure 1.13Zeta potential distribution of batch F1 (-5.53 mV)

Figure 1.14 zeta potential distribution of batch F2 (-6.78mV)

Figure 1.15zeta potential distribution of batch F3 (-10.5mV)

Percent encapsulation efficiency^14,
16

The SLN formulation batches PF1, PF2, and PF3 were evaluated for percent encapsulation efficiency. The formulation batch (PF3) showing a high %EE was selected. The result are shown in table15

Table 15 Percent encapsulation efficiency of all formulation

Formulation code	% Encapsulation efficiency
PF 1	55.02
PF 2	56.67
PF 3	59.23

Figure 1.17 % Encapsulation efficiency of SLN

Scanning electron microscopy

Scanning electron microscopy (SEM) photograph of drug and SLN are shown in figures 1.18 and 1.19.

Figure 1.18 SEM images of rivastigmine tartarate

Figure 1.19 SEM images of lyophilized SLN

SEM images of lyophilized RT -SLN was converted in amorphous form .The RT-SLN nanoparticles has not shown spherical structure.

% Cumulative drug release

The % cumulative drug release of RT SLN formulation batches is shown in table 16. The formulation batch PF5 has shown cumulative drug release 95.70% and folding endurance was found to be 160.

Table 16 Experimental design and parameters for 3²factorial designs

Sr. No.	Formulation batch	Eudragit RS100	Eudragit RL100	%Cumulative release	Folding endurance
1	PF1	900	1260	88.68	146
2	PF2	180	900	84.83	152
3	PF3	540	1260	91.86	132
4	PF4	180	1260	81.80	153
5	PF5	900	900	95.70	160
6	PF6	180	1620	93.46	148
7	PF7	540	900	92.45	155
8	PF8	540	120	91.16	152
9	PF9	900	1620	95.32	158

Ex-vivo Permeation study^11,17

Ex-vivo permeation study of RT in transdermal patch and RT-SLN optimized batch PF5 in transdermal patch is shown in table 17 and figure 1.23. The permeation study has shown 72.13±1.3 and 96.90±0.695% drug permeation. An enhancement of 24.13% in drug permeation was observed in RT-SLN formulation as compared to RT from transdermal patch.

Table 17 Ex-vivo Permeation study

Time (Hours)	Pure drug (% drug permeated)	Formulation batch PF5 (% drug permeated)
1	6.4 ±0.46	2.26±0.56
2	10.83±0.63	6.88±0.36
3	18.47±0.69	10.63±0.42
4	22.45±0.82	13.76±0.96
5	25.42±0.32	17.15±0.41
6	30.48 ± 0.96	26.90±1.2
7	33.18 ±0.78	33.12±1.03
8	35.37±0.17	40.81±0.56
9	38.93 ±0.13	49.60±0.92
10	41.25±0.65	60.88±0.85
11	43.85±0.96	76.53±0.38
12	46.26±1.2	87.20±0.23
24	72.13±1.36	96.90±0.69

Fig 1.23: Ex-vivo Permeation Study

CONCLUSION:

Transdermal drug delivery system containing the solid lipid nano particle entrapped rivastigmine tratarate was designed with an aim to achieve the drug delivery in the brain for treatment of Alzeimer’s disease. DSC, FT-IR study showed there was no incompatibility between drug polymer and lipid carried out for formulation study. The zeta potential value of (-)10.5 mV and average particle size of 214.2 nm was found to be suitable for the brain targeting of drug. Also the drug permeation from transdermal patch of SLN entrapped drug was increased as compared to the pure drug. The in-vitro dissolution study results of optimized formulation showed sustained drug release for a period of 24 hours.

ACKNOWLEDGEMENT

Authors express their thanks Prof. Dr. N. J. Gaikwad Head of Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, for making availability of all the required facilities to carry out my research work successfully and Unichem Laboratory, Goa for providing rivastigmine tartarate. Also like to acknowledge Evonic India, Colorcon, Gattefosse for providing the excipients.

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Received on 23.03.2016 Modified on 14.04.2016

Res. J. Pharm. Dosage Form. and Tech. 2016; 8(2):73-80.

DOI: 10.5958/0975-4377.2016.00011.2

Sr. No.	Ingredient	Formulation Composition
Sr. No.	Ingredient	PF1 PF2 PF3 PF4 PF5 PF6 PF7 PF8 PF9
1	Eudragit RS100	900 180 540 180 900 180 540 540 900
2	Eudragit RL100	1260 900 1260 1260 900 1620 900 1620 1620
3	RT-SLN	127.11 127.11 127.11 127.11 127.11 127.11 127.11 127.11 127.11
4	Mannitol	2 2 2 2 2 2 2 2 2
5	PEG400	0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
6	De-ionized Water	100 100 100 100 100 100 100 100 100