Lipid drug conjugate nanoparticle as a potential nanocarrier for the oral delivery of pemetrexed diacid: Formulation design, characterization, ex vivo, and in vivo assessment

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Abstract

The present work was to develop lipid drug conjugated (LDC) nanoparticles for the potential oral delivery of pemetrexed diacid (PTX) and evaluation of its in vitro, ex vivo and in vivo potentials. The LDC was prepared by salt formation of PTX with stearic acid and followed by cold homogenization technique to produce the LDC nanoparticles. FTIR analysis of LDC proved the presence of amide bond in LDC powder indicating the conjugation between drug and lipid. LDC nanoparticles was found to have particle size 121.9 ± 1.85 nm and zeta potential −51.6 m V ± 1.23 and entrapment efficiency 81.0 ± 0.89%. TEM images revealed spherical morphology and were in corroboration with particle size measurements. Ex vivo gut permeation studies revealed a very good enhancement in permeation of drug present in the LDC as compared to plain drug solution and were confirmed by CLSM. MTT assay conformed significant% toxicity at the end of 24 h and 48 h. Furthermore, the AUC0-24 of PTX from the optimized LDC nanoparticels was found to be 4.22 folds higher than that from PTX suspension on oral administration. Thus, LDC has high potential for the oral delivery of PTX in cancer therapy and future prospects for the industrial purpose.

Introduction

Pemetrexed diacid (N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo-[2,3-d]-pyrimidin-5-yl)-ethyl]-benzoyl]-l-glutamic acid belongs to a class of chemotherapeutic drugs known as folate antimetabolites [1], [2]. Pemetrexed diacid (PTX) developed by Eli Lilly and Company, was approved by the United States Food and Drug administration in 2004 for the treatment of malignant pleural mesothelioma in combination with cisplatin, a platinum-containing chemotherapeutic drug and a second line agent for the treatment of advanced or metastatic non-small cell lung cancer. Currently, the drug is used as a single agent or in combination with other chemotherapeutic agents for the treatment of other types of cancer such as breast cancer, bladder cancer, colorectal carcinoma and cervical cancer [3], [4]. It is available in the form of intravenous injection, marketed by Eli Lilly as Pemetrexed disodium Injection (Alimta®)100 mg and 500 mg lyophilized Powder. Dosing Regimen followed is 500 mg/m2 i.v. on day 1 of each 21-day cycle [5]. Oral bioavailability has not been evaluated since pemetrexed is intended for use by short-term i.v. infusion. However, oral absorption was evaluated in mice using 20 mg/kg i.v. and oral doses of 14C radiolabeled pemetrexed. Results from mice indicate that the oral absorption is low with only 13% of an oral dose absorbed in mice [6]. Although it is sparingly soluble (0.0905 mg/ml) in water, the permeation of the drug is low, which may be the cause of low oral bioavailability. Till date no oral formulation of the drug has been available [7]. So, we aimed to formulate an oral dosage form which can solve the problem of solubility as well as permeability in intestine and increase the overall oral bioavailability of the drug which can lead to increase patient compliance for the therapy.

Lipid-based nanoparticles represent an alternative drug carrier system to traditional colloidal carriers, such as liposome, nanoemulsions, polymeric nanoparticles and polymeric microparticles, and also they possess advantages of controlled drug release, drug targeting, increase in intestinal permeability, and increase in bioavailability. Moreover, they avoid the need for use of organic solvents [8], [9], [10]. Hence, the current focus of the research based on the search of bio-compatible lipids as a carrier for low bioavailable drugs to minimize problem of the mammalian tissue toxicity due to use of organic solvents, limited physical stability and leakage of drug during storage [11], [12]. Stearic acid has been already used for the delivery of many drugs as lipid-based nanoformulation as well as Lipid drug conjugate (LDC) nanoparticle [13]. The drug molecule shows the presence of carboxylic and amino groups, which is an ideal condition for linking with lipids such as stearic acid. It has been widely reported that drugs incorporated into lipid and stabilized by the use of various surfactants show high permeability of the intestine because lipids and these surfactants act as a good permeation enhancer of drugs from the gastrointestinal tract by solubilization of the drug in the intestinal milieu and reduce the first-pass metabolism of the drug by transport of the drug through a lymphatic route to the systemic circulation [14], [15]. The surfactants (Labrasol, capryol 90 and Tween 80) which are used for the stabilization of the LDC nanoparticle have their own role in the increase in the permeability of the drug through the intestine by inhibiting the P-glycoprotein efflux pump that is present in the intestinal brush border region [16], [17]. These lipid nanoparticles are becoming one of the most acceptable drug delivery systems for both lipophilic as well as hydrophilic drugs because of ease of production, easy scalability and, most advantageously, the use of lipid excipients with generally regarded as safe (GRAS) status, avoiding the use of organic solvents [18].

The literature search revealed no reports on the LDC nanoparticle and other oral dosage forms for PTX. Therefore, the specific objective of this study was to develop a LDC nanoparticle for the oral delivery of PTX by using stearic acid as a lipid using cold homogenization technique. The role of key independent variables influencing on the dependent variables (particle size, polydispersity index and entrapment efficiency) was determined by constructing a Box–Behnken design. Also the characterization of the optimized LDC nanoparticle was made for various parameters and an ex vivo gut permeation study was performed to determine the apparent permeability coefficient of the formulation in comparison with a plain suspension of the PTX. Finally the efficacy of the developed formulation against cancer cells was evaluated.

Section snippets

Material

Pemetrexed diacid (PTX) was obtained as a gift sample from Dabur research foundation, India. Labrasol and capryol 90 were obtained from gattefosse (Mumbai, india) as gift samples. Stearic acid was purchased from qualikem fine chemicals, India. Tween 80 was purchased from S.D fine chemicals, India. Methanol HPLC grade, ortho-phosphoric acid, triethylamine, HPLC grade water and ethanol (SRL, Mumbai, India) were used. All the other reagents used in the study were of analytical grade.

Excipients screening

Stearic acid

Screening of excipients

Selection of stearic acid was made for the production of LDC nanoparticles due to the presence of carboxyl group which helps in conjugating with amino and hydroxyl group of the drug PTX. The surfactant combination selected was tween 80, capryol 90 and labrasol in the ratio 2:1:1. This was selected on the basis of stability of the coarse dispersions.

Optimization of LDC nanoparticles

Box–Behnken statistical screening design was used to statistically optimize the formulation parameters and evaluate main effects, interaction

Conclusion

LDC nanoparticles of PTX were successfully prepared by the cold homogenization technique by using stearic acid as the lipid which successfully conjugated with the PTX. The formulation was optimized by RSM using Design-Expert software, and the optimized batch was characterized for various parameters. The optimized formulation was found to be efficient as suggested by in vitro, ex vivo and in vivo assessments. A much deeper penetration to the intestine was observed with LDC nanoparticles by using

Conflict of interest

The authors report no conflicts of interest.

Acknowledgments

The authors are thankful to the Central Instrumental Facility (CIF) Laboratory, Jamia Hamdard, New Delhi, India to carry out the present research work. The authors would like to convey their heartiest gratitude to Yub Raj Neupane of the Faculty of Pharmacy, Jamia Hamdard, for providing timely support and suggestions related to formulation development.

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