Novel Prodrugs and Self-Assembling Nanocarriers to Improve Drug Delivery
Abstract
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
Degree
Ph.D.