The development of folate-PAMAM dendrimer conjugates for targeted delivery of anti-arthritic drugs and their pharmacokinetics and biodistribution in arthritic rats

Abstract

The aim of this study was to synthesize folate-dendrimer conjugates as suitable vehicle for site specific delivery of anti-arthritic drug (indomethacin) to inflammatory regions and to determine its targeting efficiency, biodistribution in adjuvant induced arthritic rats. Folic acid was coupled to the surface amino groups of G4-PAMAM dendrimer (G4D) via a carbodiimide reaction and loaded with indomethacin. The conjugates were characterized by ¹H-NMR and IR spectroscopy. The drug content and percent encapsulation efficiency increased with increasing folate content for the dendrimer conjugates. The in vitro release rate was decreased for the folate conjugates when compared with unconjugated dendrimer (DNI). The plasma concentration profile showed a biphasic curve indicating rapid distribution followed by slow elimination. The AUC_0−∞, half-life and residence time of indomethacin in inflamed paw was higher for folate-dendrimer conjugates. The time-averaged relative drug exposure (r_e) of the drug in paw and overall drug targeting efficiency (T_e) were higher for folate conjugate with 21 folate moieties (4.1 and 2.78, respectively) when compared with DNI (1.91 and 1.88, respectively). This study demonstrated the superiority of active targeting over dendrimer mediated passive targeting and also for the first time, folate-mediated targeting of an anti-arthritic drug to the inflammatory tissues.

Introduction

Rheumatoid arthritis (RA) is a chronic auto-immune disease that is characterized by destructive inflammation of both internal organs and joints. It is considered the most common cause of disability in the workforce [1]. Most of the currently available anti-arthritic drugs suffer from limited efficacy and/or undesirable side effects [2]. Targeted drug delivery systems (TDDS) are promising excellent drug carriers that deliver the drug at the target site thereby reducing the accompanying drug toxicity and increasing the therapeutic efficacy at equivalent plasma concentrations.

Folate receptor (FR), a 38 kDa glycosylphosphotidylinositol-anchored protein, exists in three major forms namely FR-α, FR-β and FR-γ. The FR-α form is over expressed in many types of tumor cells including ovarian, endometrial, breast, renal cell carcinomas [3]. The facts that high affinity FR binding is retained when folate is covalently linked via its γ-carboxyl group to a foreign molecule and the prevalence of FR over expression among tumors makes it a good choice for targeted drug delivery to tumors [4]. It has recently been discovered that a functionally active FR-β form is present in the activated (but not resting) synovial macrophages taken from RA patients [5]. These FR isoforms on the activated cell surfaces were also found to bind the folate-linked fluorophores with high affinity. Since tumor tissue also presents an inflammatory micro-environment, the activated macrophages can be selectively targeted with folate conjugates in both cancer tissue and arthritis joints. Low et al. recently demonstrated the folate receptor mediated targeting of imaging agent (EC20) and folate-hapten conjugate to activated macrophages in RA [6]. However, this recent discovery is limited only to imaging agents. Till date no report is available on the folate-mediated delivery of low molecular therapeutic agents/drugs to RA.

An optimal TDDS requires a platform that is able to carry multiple components, such as a drug, a targeting ligand and a fluorescent sensing agent. Poly(amidoamine) (PAMAM) dendrimers offer such a carrier system, having a defined branched chain structure capable of carrying multiple molecular entities that are either linked covalently to its surface or are encapsulated in its interior space [7]. A comparison of the features of dendrimers with those of linear polymers shows that the dendritic architecture can provide several advantages for drug delivery applications. The controlled multivalency of dendrimers can be used to attach several drug molecules, targeting groups and other agents (solublizing/sensing) to the periphery of the dendrimers in a well-defined manner. Dendrimers conjugates have been used for delivering drugs [8], [9], DNA [10], [11], radionucleides [12], MRI contrast agents [13] and boron for neutron capture therapy [14].

Indomethacin is a non-steroidal anti-inflammatory drug used for the management of RA, osteoarthritis and acute gout but has a limited efficacy coupled with gastric intestinal hemorrhage and other side effects such as renal dysfunction with conventional therapies [15]. Site-specific delivery of indomethacin to the target site remains the best choice to overcome the side effects and to increase its efficacy. The main aim of this study was to synthesize folate-dendrimer conjugates as suitable vehicle for carrying indomethacin and to determine its site specific targeting efficiency to inflammatory region in adjuvant induced arthritic rats.