INTRODUCTION:

Cancer is a major public health problem in the world. There were 14.1 million new cancer cases and 8.2 million cancer deaths in 2012 worldwide. If these rates do not change, the global cancer burden is expected to nearly double to 21.4 million cases and 13.5 million deaths by 2030. Breast cancer is the most common cancer among women worldwide, with nearly 1.7 million new cases diagnosed in 2012 (the second most common cancer). This represents about 12% of all new cancer cases and 25% of all cancers in women. Cancer is the second leading cause of death worldwide, and was responsible for 8.8 million deaths in 2015. As per WHO, nearly 1 in 6 deaths is due to cancer.

A typical example for Topoisomerase inhibitors is etoposide and it is a first line of chemotherapeutic agents that are used in the treatment of many types of cancer. The mechanism of action of Etoposide by forming a ternary complex with topoisomerase II and DNA, causing DNA breaks and cell death (1). In addition to this, there are many side effects related to the drug (2–4), the administration of etoposide is rate limited by its low solubility in aqueous solutions (5, 6). Therefore, finding an effective approach to facilitate the transport of drugs and to improve the bioavailability of therapeutics is necessary.

The drug candidate etoposide has variable oral bioavailability and ranging from 24-74% and has terminal half-life of 1.5 hours by intravenous route and 0.44 hours by oral route. The conventional oral therapy has drawback of low bio availability and parenteral therapy causes inconvenience and pain to the patients as it has to be given through a continuous IV infusion over 24-34 hour. Hence the objective of present study was made for the pharmacokinetic profile (bioavailability) of etoposidepolymeric nanoparticles in comparison with pure and marketed formulation using albino rats as animal model.

MATERIALS AND METHODS:

Materials:

Etoposide was a gift sample from Biocon Limited, Bangalore, India; Eudragit® EPO and HPMC K-15 were gifts from Cipla Pharmaceuticals, Mumbai, India. Pluronic® F-68 gifted from Alembic pharmaceuticals, Mumbai, India. HPLC grade of Methanol, water and acetonitrile was purchased from Qualigens fine chemicals Mumbai, India. All other reagents and chemicals used in this study were of Analytical Grade.

Preparation of Eudragit EPO based Nanoparticle Suspension:

Nanoparticles suspensions were prepared by nanoprecipitation method. Dissolved the drug and specific amount of Eudragit®-EPO in 15 ml of methanol. The organic solution quickly injected to 40 ml aqueous solution containing Pluronic® F-68 under stirring at 2000 rpm. Stirring was continued for 2 hours at 40°C for complete evaporation of methanol. The volume was adjusted up to 40 ml with aqueous solution of 200 mg of HPMC K-15 to obtain a nanoparticle suspension. The optimized nanoparticles suspension was lyophilized at–42°C for 72 hours and which also redispersed in water to get aqueous nanoparticles suspension⁷.

In previous work, 3² factorial design was used to optimize the process parameters like polymer concentration,stabilizer concentration. two dependent variable’s drug content, entrapment efficiency were measured as responses. Mathematical equations and response surface plots were used to relate the dependent and independent variables. The optimization model of particle size of 131.4±0.057nm, entrapment efficiency of about 94.28±0.198% and drug content of 88.36±0.075 %. The observed responses were in close agreement with the predicted values of the optimized process. The prepared nanoparticle was characterized by Fourier transform infrared spectroscopy, XRD and morphological studies, in-vitro drug release studies, kinetic modeling and stability studies. The prepared nanoparticle was showed good sustained release of drug upto 24 h⁸. Based on in-vitro parameters, the optimized subjected to pharmacokinetic studies compare with marketed formulation.

Pharmacokinetic Studies:

The study protocol was approved to carry out pharmacokinetic studies was obtained from the Institutional Animal Ethical Committee, Adhiparasakthi College of Pharmacy, Melmaruvathur (Approval No. 409/01/a CPCSEA dated on 20.07.2015). These studies were performed on pure drug, optimized Polymeric nanoparticles and marketed formulation (Etosid®). Healthy albino rats of either sex (weighing 200-250 g) were stores under standard laboratory conditions (temperature 25±2°C and relative humidity of 55±5%). The albino rats were allowed for free access to standard laboratory diet. They were fasted for the period of 12 h for observations of any unwanted effects. Albino rats were prevented from coprophagy by fitting muzzles during the night. The dose for the rabbit was calculated according to the surface area ratio method. The albino rats were divided into 3 groups, (n=6) and treated orally as shown below

Group I: Etoposide (9 mg/kg) in 0.5 % CMC.

Group II: Optimized nanoparticles containing equivalent amount of Etoposide

(9 mg/kg) in 0.5 % CMC.

Group III: containing equivalent amount of Etoposide (9 mg/kg) in 0.5 % CMC.

After mild ether anaesthetization, serial blood samples (0.5 ml) was collected by retro orbital sinus puncture technique at predetermined time intervals 0 (pre-dose), 0.5, 1, 2, 4, 6 and 8 h (post- dose). The serum was separated immediately by cold centrifugation at 13,000 rpm for 5 min. The separated serum was stored at -40oC until drug analysis. The drug analysis was carried out using high performance liquid chromatographic (HPLC) method using PK solutions 2.0.68. software.

HPLC Analysis:

The plasma concentration of etoposide was determined by reported HPLC assay procedure. Podophyllotoxin (50 ng/ml dissolved in methanol) and 1.2 ml of tert-butyl methyl ether is mixed with a 0.1 ml aliquot of the plasma sample in a 2.0 ml polypropylene microtube. The resulting mixture was mixed vigorously with a vortex-mixer for 1 min and was centrifuged at 13,000 rpm for 10 min with a high-speed micro centrifuge. A 1.1 ml aliquot of the upper layer was transferred to another clean microtube and evaporated under nitrogen gas at 38°C in an MG 2100 Eyela dry thermo bath. The residue was dissolved in 0.2 ml of 50% methanol in deionized water, and a 50 μl aliquot of the solution is injected into the HPLC system. The HPLC can equip with a Waters 1515 isocratic HPLC Pump, a Waters 717 plus autosampler and a Waters TM 474 scanning fluorescence detector. Data are acquired and processed with breeze™ Software (Version 3.2) (Waters Co.). Chromatographic separations achieved using a Symmetry® C18 column (4.6×150 mm, 5 μm, Waters Co.) and a μBondapak™ C18 HPLC Precolumn (10 μm, Waters Co.). The mobile phase consisted of methanol-deionized water-acetic acid (50:50:0.5, v/v/v) and run at a flow-rate of 1.0 ml/min. Chromatography performed at 30˚C, which is set by a HPLC column temperature controller (Phenomenex Inc., CA, USA). The fluorescence detector is operated at an excitation wavelength of 230 nm with an emission wavelength of 330 nm. Podophyllotoxin and etoposide eluted with retention times of 5.4 and 11.1 min, respectively⁹ as per the reported method.

Statistical analysis:

Student’s t-test was employed to analyse the results (Graph Pad Prism Software). Difference below the probability level of 0.05 was considered statistically significant. The pharmacokinetics parameters were calculated by using PK solutions 2.0^TMNoncompartmental pharmacokinetic data analysis software.

RESULTS AND DISCUSSION:

The pharmacokinetic parameters (Table 1) were calculated from plasma concentration-time curve (Fig.1). Etoposide absorption after oral administration rapid with three groups as indicated by low Tmax value about 1.00 h. However, the Cmaxvalue high with nanoparticles of etoposide indicating maximum absorption of drug. The elimination half- life (t_1/2) of etoposide with nanoparticles less indicating the drug is eliminated from the body rapidly. It further supported by high elimination rate constant value (Ke) of nanoparticles formulation in comparison with pure drug and marketed formulation. The prepared nanoparticles showed high area under the curve (AUC) value indicating the greater bioavailability of drug than pure drug and marketed formulation. This supports the higher values of Cmax observed with etoposide nanoparticles. Hence the pharmacokinetic study indicates rapid and higher absorption in addition to higher bioavailability of drug from nanoparticles in comparison with pure drug and marketed formulation.

Fig.1. Plasma drug concentration-time curve. The points represent mean ± S.D. values, n=3.

Table 1: Pharmacokinetic parameters of orally administered pure etoposide, marketed formulation and optimised nanoparticles

Parameters	Pure drug	Marketed Formulation	Optimised Nanoparticles
Cmax (μg/mL)	1.23±0.01	1.37±0.01	1.63±0.01*
Tmax (h)	1.00±0.00	1.00±0.00	1.00±0.00
t_1/2 (h)	2.60±0.06	2.37±0.37	1.05±0.06*
AUC₀_→₈ (μg-h/mL)	5.77±0.66	6.89±0.37	7.11±0.15*
Ke (h ^-1)	0.267±0.005	0.294±0.04	0.652±0.03*
Fr (%)	-	119.41	123.22

All values are expressed as mean ±S.D., n=6; Cmax: maximum plasma concentration; Tmax: time for maximum plasma concentration; t_1/2: biological half-life; AUC:area under the curve; Ke: elimination rate constant; Fr: relative bioavailability.

*Significant (p < 0.05) compared to pure drug.

CONCLUSION:

This study revealed significantly greater extent of absorption of etoposide polymeric nanoparticles than pure drug and marketed formulation (p < 0.05).The absorption of etoposide from polymeric nanoparticles and marketed formulation resulted in 1.23 and 1.19 fold increases in bioavailability as compared to pure drug. The result of the study concludes that polymeric nanoparticles technique can be successfully used for enhancement of oral bioavailability of etoposide.

The increased bioavailability of etoposide from polymeric nanoparticles may bring about reduction of gastrointestinal side effects. It could be considered a better alternative for new chemical entity. Further dose range study is in the pipeline.

ACKNOWLEDGEMENTS:

The authors are very much thankful to his holiness Arulthiru Bangaru Adigalar, President, with respect to Thirumathi V. Lakshmi Bangaru Adigalar, Vice President for providing entire facilities for the research work. Authors are also thankful to all those who helped us in direct or indirect way to carry out this study successfully.

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Received on 07.02.2018 Modified on 28.02.2018

Research J. Pharm. and Tech 2018; 11(6): 2538-2540.

DOI: 10.5958/0974-360X.2018.00468.7