Rivastigmine Tartrate Solid Lipid Nanoparticles Loaded Transdermal Film: An In vivo study

 

Ravi G*, Vishal Gupta N, Balamuralidhara V

Department of Pharmaceutics, JSS College of Pharmacy, Sri Shivarathreeshwara Nagar, Mysuru,

JSS Academy of Higher Education and Research, JSS Medical Institutions Campus,

Sri Shivarathreeshwara Nagar, Mysuru-570015, Karnataka, India.

 

ABSTRACT:

In the present investigation, Rivastigmine Tartrate incorporated solid lipid nanoparticles (SLN) films were made to enhance its uptake to brain via systemic circulation. SLN were prepared by modified emulsification diffusion method and SLN’s loaded transdermal films were prepared by solvent casting method. The optimized Rivastigmine Tartrate SLN loaded formulation was evaluated for pharmacokinetic study and dermal toxicity study. In vivo studies were performed on New Zealand white rabbits and various pharmacokinetic and dermal toxicity parameters were determined. The pharmacokinetic parameters after administration of Rivastigmine Tartrate loaded SLN film were found to be, T_max 3 h, C_max 116.17 ± 1.5 ng/mL, AUC_{0 - ∞} 1848.29±4.87 ng.h/mL, and K_e 0.18 ± 0.028 h^-1. The dermal toxicity study was carried out for 3 min, 1 h and 4 h respectively with optimized film and no skin irritation or redness was found. The results highlights that the prepared formulation of SLN loaded films were able to deliver a sustained supply of the Rivastigmine Tartrate.

 

 

 

 

INTRODUCTION:

Rivastigmine is a slowly reversible, centrally selective dual inhibitor of butyrylcholinesterase and acetylcholinesterase, which improves neurotransmission and enhances the acetylcholine availability levels. It has established efficacy in the symptomatic treatment of Parkinson disease^1,2,3 and Alzheimer disease⁴ and it was shown to improve the daily activities like living, cognition, behavior, and global function^5,6,7,8. Dose response relationships studies for cholinesterase inhibitors support better enzyme inhibition, in turn leading to higher efficacy and long-term benefits with higher drug doses⁹.

 

Transdermal film is a medicated device that delivers drugs through the skin for systemic effects at a programmed and controlled rate¹⁰. The advantages of transdermal drug delivery is, providing controlled release of the drug to the patient and enabling a steady blood level profile, avoidance of first-pass hepatic metabolism and helping in the rapid termination of therapy¹¹. Furthermore, the dosage form of transdermal film is user friendly, convenient and offers multi-day dosing.

 

In general terms, pharmacokinetic (PK) models of dermal absorption of chemicals have been created and reported in a series of publications^12,13,14. Skin is frequently represented either as a single compartment or by two compartments separately distinguishing the lipophilic and hydrophilic layers of the skin¹⁵. Such modeling attempts were generally used in the toxicology rather than clinical pharmacology fields until recently when Polak et al.¹⁶ reported a mechanistic dermal absorption model using simcyp simulator. Still, there is limited information on dermal absorption models that were put into practical use for simulations of drug concentrations in the clinical setting.

 

In present study in vivo studies for Dermal toxicity study and Pharmacokinetics were performed on New zealand white rabbits for prepared Rivastigmine Tartrate solid lipid nanoparticles loaded transdermal films for Alzheimer’s disease.

 

MATERIALS AND METHODS:

Rivastigmine Tartrate was obtained as a gift sample from Jubilant life sciences Ltd, India. Acetonitrile was procured from Merck Specialities Pvt Ltd, Mumbai, India. All other solvents, reagents and chemicals used were of analytical grade.

 

Study was conducted at Institutional Animals Ethics Committee, JSS College of Pharmacy, Mysuru. (Proposal No. 215/2017).

 

Dermal Toxicity Study^17,18:

The in vivo test was performed initially using one rabbit and applying the following approach. Up to three test films were applied one after another to the rabbit. After 3 minutes the first film was removed and if no serious skin reaction was observed, then a second film was applied at a different site and removed after 1 h and the observations at this stage indicated that exposure can humanely be allowed to extend to four hours, a third film was applied and removed after 4 h, and Dermal toxicity was scored and recorded according to the Draize scoring system (Table 1)

 

Table 1: Draize scoring system

 

If a corrosive effect was observed after any of the three sequential exposures, the test was immediately terminated. If a corrosive effect was not observed after the last film was removed, the animal was observed for 14 days, unless corrosion develops at an earlier time point. In those cases in which the test chemical was not expected to produce corrosion but may be irritating, a single film was applied to one animal for four hours.

 

Pharmacokinetic Study¹⁹:

All experiment rabbits were acclimatised to the laboratory conditions for a period of one week prior to the initiation of experiment. The experimental animals were exposed to test drug or reference drug once only. On the day of experiment the transdermal film  was applied on the shaven back of the rabbit and blood samples were collected at different time intervals of 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h and 24 h from ear vein of the rabbit. Plasma was separated and stored at -20 ^oC until analysis. Plasma concentration of drug was determined using high performance liquid chromatography (HPLC) method.

 

Blood collection method:

Rabbits were placed in rabbit holder; blood was collected in glass tube containing 11% tri sodium citrate (100µl) using heparinised capillary tube. Blood sample was processed by centrifugation (REMI, India) to separate serum and was used for HPLC analysis.

 

HPLC method

Analytical method

A previously reported and validated RP-HPLC method was used to estimate pure Rivastigmine tartrate [20]. The mobile phase consisted mixture of 10 mM concentration of ammonium acetate buffer:Acetonitrile [30: 70% v/v, pH adjusted to 4.0 with orthophosphoric buffer] for 100 mcg/ml with C₁₈ column (250mm ×4.6mm × 5µ). The drug retention time was found to be 4.08 min. Representative chromatogram of Rivastigmine Tartrate obtained at 263 nm. A linear relationship was observed in the concentration range of 50-100 mcg/ml (r²=0.999; n= 3). Typical chromatogram of Rivastigmine Tartrate by RP-HPLC graph is represented in fig. 1. A calibration graph is represented in fig. 2.

 

 

Fig. 1: Typical chromatogram of Rivastigmine Tartrate by RP-HPLC

 

Fig. 2: Standard calibration curve of Rivastigmine Tartrate by RP-HPLC

 

Bio analytical method

The HPLC bio analytical method used for Rivastigmine Tartrate analysis was rapid and simple. Protein precipitation method was used (Acetonitrile) for extraction, the clear supernatant liquid was separated, filtered (0.45μ filter) and injected into the HPLC system. Drug retention time was 4.15 min. A linear relationship was observed in the concentration range of 50-100 mcg/ml (r² = 0.999; n = 3).  The results show the linearity between the peak area and the concentration of the analyte. Typical chromatogram of Rivastigmine Tartrate in rabbit serum by RP-HPLC graph is represented in fig. 3. A calibration graph is represented in fig. 4.

 

 

Fig. 3: Typical chromatogram of Rivastigmine Tartrate in rabbit serum by RP-HPLC

 

Fig. 4: Calibration curve for the estimation of Rivastigmine Tartrate in serum by RP-HPLC method

 

RESULTS AND DISCUSSION:

Dermal toxicity study

Dermal toxicity study of Rivastigmine SLN loaded transdermal films were performed on 3 male New zealand rabbits using 3 different films for 4 h and the results are shown in table 2.

 

Table 2: Dermal toxicity study results for 24 h

 

Pharmacokinetic Study

Pharmacokinetic study of formulation shows significant improvement of C_max and bioavailability of the drug compared to the pure drug.

 

The pharmacokinetic parameters of Rivastigmine Tartrate SLN loaded film absorption are summarized in Table 3. Figure 5 depicts the mean serum concentration profile as a function of time obtained by the pharmacokinetic studies carried out in rabbits for Rivastigmine Tartrate SLN loaded film formulation and pure drug. The serum level profiles were significantly increased for Rivastigmine Tartrate SLN loaded film formulation compared to pure drug.

 

 

 

Table 3: Pharmacokinetic parameters of pure Rivastigmine SLN film and optimized formulation

* mean ± SD, n=3

The pharmacokinetic results of pure drug showed the C_max value 142.26 ng/mL and T_max 2 h. Similarly the C_max value of formulation Rivastigmine Tartrate SLN loaded film showed 116.16 ng/mL and T_max 3h. The Pharmacokinetic study shows that SLN loaded film shows significant improvement of bioavailability of the drug while compared to its pure form.

 

 

 

Figure 5: Plasma drug level profiles of pure Rivastigmine Tartrate and optimized formulation

 

 

CONCLUSION:

Optimized formulation of Rivastigmine Tartrate SLN loaded was subject to dermal toxicity study and Pharmacokinetic studies, successfully done on New zealand white rabbits. No serious skin reaction was observed in dermal toxicity syudy. Rivastigmine Tartrate SLN film shows the retention time of 4.15 min. The analytical method conducted using RP-HPLC was found to be highly sensitive, selective and reproducible for measurement of Rivastigmine Tartrate. The pharmacokinetic results of pure drug Rivastigmine Tartrate showed the C_max value 142.26 ng/mL and T_max 2 h. Similarly the C_max value of optimized SLN loaded film showed 116.16 ng/mL and T_max 3 h. The Pharmacokinetic study shows that SLN loaded film shows significant improvement of bioavailability of the drug while compared to its pure form.

 

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Received on 12.10.2017             Modified on 27.10.2017

Accepted on 20.11.2017           © RJPT All right reserved