Novel Prodrugs and Self-Assembling Nanocarriers to Improve Drug Delivery
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PART I: DIPEPTIDE PRODRUG APPROACH TO IMPROVE INTESTINAL ABSORPTION OF LOPINAVIR Lopinavir (LPV), a highly potent second-generation HIV-1 protease inhibitor, is currently indicated in HIV-1 infection. However, poor systemic exposure following oral LPV dosing is a major concern. One of the major factors limiting intestinal permeability is the high substrate affinity of LPV towards major drug efflux pumps such as P-gp and MRP2. To address these issues, a histidine-leucine-LPV (His-Leu-LPV) dipeptide prodrug was synthesized and evaluated. His-Leu-LPV was identified by 1H-NMR and LCMS/MS techniques. Aqueous solubility generated by this prodrug was markedly higher relative to unmodified LPV. Importantly, His-Leu-LPV displayed significantly lower affinity towards P-gp and MRP2 as evident from higher uptake and transport rates. [3H]-GlySar and [3H] L-His uptake receded to approximately 30% in the presence of His-Leu-LPV supporting the PepT1/PHT1 mediated uptake process. A steady regeneration of LPV and Leu-LPV in Caco-2 cell homogenates indicated His-Leu-LPV undergoes both esterase and peptidase mediated hydrolysis. Based on these results, it appeared that histidine based dipeptide prodrug approach might be an alternative to improve LPV absorption across poorly permeable barriers such as intestinal and blood-brain barriers (BBB). PART II: NANOFORMULATIONS TO IMPROVE OCULAR DELIVERY OF CIDOFOVIR AND OCTREOTIDE Cidofovir (CDF) has demonstrated significant antiviral activity against cytomegalovirus (CMV) and is indicated for the treatment of CMV retinitis. Nonetheless, high water solubility of CDF limits its absorption through passive transcellular transport. While repeated intravitreal (IVT) injections leads to serious adverse events, back of the eye delivery after topical application remains a major challenge. Therefore, a prodrug containing C12 (twelve carbon chain length) lipid linker and biotin (ligand) for targeting sodium dependent multivitamin transporter (SMVT) was formulated with polymeric nanomicelles for topical application. Apart from serving as an inert nanocarrier for hydrophobic therapeutic agents, polymeric nanomicelles being extremely small in size promotes circumvention of mononuclear phagocytic system (MPS) and efflux transporters thereby improving drug bioavailability. Therefore, we prepared polymeric nanomicelles using polyoxyethylene hydrogenated castor oil 40 (HCO-40) and octoxynol 40 (OC-40). In vitro release studies revealed that B-C12-cCDF-loaded nanomicelles released B-C12 cCDF at a faster rate in stimulated tear fluid in comparison to PBS. MTT and LDH assays demonstrated negligible cytotoxicity of B-C12-cCDF-loaded nanomicelles relative to CDF and B-C12-cCDF in D407 (retinal pigment epithelial), SV-40 (immortalized human corneal epithelial) and CCL 20.2 (conjunctival epithelial) cells. Confocal laser scanning microscopy and flow cytometry analyses indicated that B-C12-cCDF-loaded nanomicelles were efficiently internalized into D407 and SV-40 cells in contrast to CDF and B-C12 cCDF. Moreover, little B-C12-cCDF was also observed in the nuclei after 24 h of incubation. Polymeric nanomicelles carrying the transporter targeted prodrug did not produce any cytotoxic effects and were internalized into the cells effectively. Permeability experiments across SV-40 cells further confirmed significant transport of prodrug loaded nanomicelles and their subsequent uptake into D407 cells. These findings indicate that HCO-40/OC-40 based polymeric nanomicelles could become a promising topical delivery system for ocular administration of anti-viral agents. Additionally, octreotide, a somatostatin peptide analogue is a promising therapeutic agent for treating proliferative diabetic retinopathy (PDR) by the activation of pituitary somatostatin receptor (SSTR) and inhibition of the GH-insulin-like growth factor (IGF)-1 axis. However, delivery related issues such as short half-life, low stability due to hydrophilicity, high molecular weight and minimal permeability across blood-retinal barrier are some of the major concerns. To overcome these challenges, we developed self assembling multi-layered nanomicelles composed of two polymers, HCO-40 and OC-40 designed to combine hydrophilic interaction and solvent induced encapsulation of peptides and proteins. HCO-40 and OC-40 polymers are employed to encapsulate peptides and proteins in the core of the organo-nanomicelles with chloroform as a dispersant. The individual organo-nanomicelles are further encapsulated with another layer of the same polymers leading to the formation of an aqueous stable amphiphilic nanomicellar solution. The size of the multi-layered nanomicelles ranged from ~16-20 nm with zeta potential close to neutral (~-2.44-0.39 mV). In vitro release studies revealed that octreotide loaded multi layered nanomicelles released octreotide at much slower rate in STF (~27 days) compared to PBST (~11 days) in its native form. MTT assay demonstrated negligible toxicity of the multi-layered nanomicelles at lower concentrations in HRPE (Human retinal pigment epithelial, D407), CCL 20.2 (Human conjunctival epithelial) and RF/6A (rhesus choroid retinal endothelial) cells. This work demonstrates an efficient peptide delivery platform with significant advantages over existing approaches, as it does not require modification of the peptide, is biodegradable, has small size and a high loading capacity.
Table of Contents
Dipeptide prodrug approach to improve intestinal absorption of lopinavir -- Nanoformulations to improve ocular delivery of cidofovir and octreotide -- Summary and recommendations -- Appendix