PLGA nanoparticles for oral delivery of cyclosporine: Nephrotoxicity and pharmacokinetic studies in comparison to Sandimmune Neoral®☆
Introduction
Cyclosporine is a potent immunosuppressive agent, which has been widely used for the prevention of graft rejection following organ transplantation such as kidney, liver, heart, lung and pancreas as well as in the treatment of various autoimmune disorders [1]. However, the oral bioavailability of cyclosporine is low and highly variable owing to its troublesome biopharmaceutical properties including poor aqueous solubility and low intestinal permeability due to high molecular weight and rigid cyclic structure. Additionally, P-glycoprotein (P-gp) efflux from the enterocytes and extensive pre-systemic metabolism in the gut wall and liver further reduces its oral bioavailability [2], [3].
Apart from the low and variable oral bioavailability, cyclosporine is a critical dose drug with a narrow therapeutic window [4] and the commercial formulation exhibits dose dependent nephrotoxicity. Cyclosporine can induce different forms of kidney dysfunction, from acute renal failure to chronic renal diseases depending upon the dose and the duration of treatment [1], [5]. The severity of symptoms those were observed outside the therapeutic window of cyclosporine necessitate the development of less toxic cyclosporine formulation that can control the blood levels of cyclosporine in the therapeutic range.
In order to overcome the above-mentioned difficulties, various formulation approaches have been investigated including the incorporation of drug into the particulate carriers. The nanoparticulate formulations have been known to be an efficient approach to achieve better pharmacokinetic profiles and even to increase the oral bioavailability of poorly bioavailabile drugs due to their specialized uptake mechanisms [6], [7]. This system circumvents the P-gp efflux and protects incorporated drug molecules from the gastro-intestinal tract (GIT) degradation as well as gut wall metabolism. The NPs also bypass the liver and prevent the first pass metabolism of the incorporated drug. Since the immunosuppressive activity of cyclosporine is related to its selective action against T lymphocytes, which mainly circulate in the lymphatic system, targeting the lymphatic system through NPs may improve the therapeutic potential of cyclosporine [8]. In addition, the NPs remain in the blood circulation for a longer time and release drug in the plasma in a sustained and continuous manner leading to lower fluctuations in the blood levels that could minimize adverse effects caused by the drug.
Biodegradable NPs, particularly of PLGA have been increasingly used in drug delivery because of its biocompatibility and biodegradability; however, their use in oral administration has been limited. The degradation products formed from the PLGA, which includes lactic acid and glycolic acid, are the normal components of Kreb's cycle and are subsequently eliminated as carbon dioxide and water without affecting the normal cellular functions. Apart from the polymer, the preparation of nanoparticles requires use of stabilizers, which are the surface-active agents by virtue of their nature. It is now well known that surfactants can alter the integrity of the biological membranes [9]. Thus, if they are used in higher concentration, they may induce toxicity. Additionally, the stabilizer like PVA forms interconnected network with the polymer at the nanoparticle interface, which cannot be removed even with repeated washings. The nanoparticles with the higher amount of residual PVA on the nanoparticle surfaces resulted in its lower cellular uptake [10]. Therefore, it is desirable to have a lower stabilizer concentration in the formulation. Recently, our group used DMAB as stabilizer for making positively charged estradiol nanoparticles of about 150 nm size. These particles showed improved oral bioavailability of estradiol in comparison to estradiol suspension. A series of studies in our laboratory suggest DMAB as suitable stabilizer for PLGA NPs intended for oral administration [7], [11], considering the smaller sized particles and the positive zeta potential. In light of the above, the present study was intended to develop cyclosporine-loaded PLGA NPs for improving its oral bioavailability and reducing nephrotoxicity.
Section snippets
Materials
PLGA (Resomer RG 50:50 H; inherent viscosity 0.41 dl/g) and cyclosporine were gift samples from Boehringer Ingelheim (Ingelheim, Germany) and Panacea Biotech (Mumbai, India), respectively. Sandimmune Neoral® (Novartis) was purchased from a local pharmacy store. Naproxen and Cremophor EL® were purchased from Sigma (St. Louis, MO, USA). Didodecyldimethylammonium bromide (DMAB) was purchased from Aldrich (St. Louis, MO, USA). Ethyl acetate and acetonitrile (HPLC grade) were purchased from Rankem
Results and discussion
PLGA NPs containing cyclosporine were prepared using DMAB as stabilizer. Use of DMAB led to smaller sized particles with narrow size distribution. The dispersion of the organic phase due to stirring resulted in irregular-sized globules in equilibrium with continuous phase. Sonication led to reduction in size of globules and addition of water destabilizes the equilibrium to force the organic solvent to diffuse to the continuous phase. This process led to nanoprecipitation resulting in the
Conclusions
The current study revealed the fact that cyclosporine-loaded PLGA NPs can be prepared with high entrapment efficiency using low stabilizer concentration. These particles were efficient in controlling the drug release in vitro and in vivo. The in vitro hemocompatibility studies revealed no significant hemolysis exerted by the NPs upon the incubation with the blood, indicating very good hemocompatibility. The nanoparticulate formulation showed significantly improved intestinal uptake and oral
Acknowledgements
Authors are thankful to Viswanand for his help with the bioavailability study. The work was supported in partial by research grants to MNVRK from Department of Biotechnology (DT/PR5097/BRB/10391/2004) and Department of Science and Technology (No. SR/FTP/CS-32/2004), Government of India. Start-up funds to MNVRK, MS fellowship to JLI, DKB and PhD fellowship to VB from NIPER are gratefully acknowledged. Thanks are due to Rahul Mahajan and Dinesh Singh for providing technical assistance to the
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Indian patent pending (1690/Del/2006).