Reversible hydrophobic ion-paring complex strategy to minimize acylation of octreotide during long-term delivery from PLGA microparticles.

Acylation of peptide has been reported for a number of peptides and proteins during release from polymers comprising of lactide and glycolide. We hypothesize that reversible hydrophobic ion-pairing (HIP) complex may minimize octreotide acylation during release. Sodium dodecyl sulfate (SDS), dextran sulfate A (DSA, Mw 9-20 kDa) and dextran sulfate B (DSB, Mw 36-50 kDa) were selected as ion-pairing agents to prepare reversible HIP complex with octreotide. Complexation efficiency was optimized with respect to the mole ratio of ion-pairing agent to octreotide to achieve 100% complexation of octreotide. Dissociation studies suggested that DSA-octreotide and DSB-octreotide complexes dissociate completely at physiological pH in presence of counter ions unlike SDS-octreotide complex. DSA-octreotide and DSB-octreotide complex encapsulated PLGA microparticles (DSAMPs and DSBMPs) were prepared using the S/O/W emulsion method. Entrapment efficiencies for DSAMPs and DSBMPs were 74.7±8.4% and 81.7±6.3%, respectively. In vitro release of octreotide was performed by suspending MPs in gel. A large fraction of peptide was released in chemically intact form and <7% was acylated from DSAMPs and DSBMPs in gel over 55 days. Therefore, HIP complexation could be a viable strategy to minimize acylation of peptides and proteins during extended release from lactide and glycolide based polymers.

Novel dexamethasone-loaded nanomicelles for the intermediate and posterior segment uveitis.

Development and characterization of dexamethasone (DEX)-encapsulated polymeric nanomicelles have been reported. A low molecular weight di-block copolymer was synthesized and characterized for its structure, molecular weights, critical micelle concentration (CMC), and cytotoxicity in ocular cells. In order to delineate the effects of drug-polymer interactions on drug solubilization in micelle core, a response surface methodology was generated with the help of SAS 9.02 (exploratory model). The method for preparing micelle was modified based on the results obtained from exploratory model. The formulation was optimized by response surface methodology (optimization model) to achieve DEX solubility of above 1 mg/mL. The optimized formulation was characterized for DEX solubility, nanomicelle size, polydispersity index, surface morphology, in vitro transport across conjunctival cell line, and ex vivo transport across excised rabbit sclera. Nanomicelles exhibited average sizes in range of 25-30 nm with unimodel size distribution and low polydispersity of 0.125. Nanomicelles increased DEX permeability by 2 times across conjunctival cell line and by 2.5 times across the excised rabbit sclera as compared to DEX suspension. A design of experiment (DOE) strategy was successfully applied to understand the effects of drug-polymer interaction on drug solubility. DOE was also employed to achieve optimal formulation with high DEX solubility. Nanomicellar formulation significantly enhanced DEX permeability across the excised rabbit sclera. Therefore, nanomicellar formulation may provide therapeutic levels in the back of the eye following topical administration.

Amino Acid Prodrugs: An Approach to Improve the Absorption of HIV-1 Protease Inhibitor, Lopinavir.

Poor systemic concentrations of lopinavir (LPV) following oral administration occur due to high cellular efflux by P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRPs) and extensive metabolism by CYP3A4 enzymes. In this study, amino acid prodrugs of LPV were designed and investigated for their potential to circumvent efflux processes and first pass effects. Three amino acid prodrugs were synthesized by conjugating isoleucine, tryptophan and methionine to LPV. Prodrug formation was confirmed by the LCMS/MS and NMR technique. Interaction of LPV prodrugs with efflux proteins were carried out in P-gp (MDCK-MDR1) and MRP2 (MDCK-MRP2) transfected cells. Aqueous solubility studies demonstrated that prodrugs generate higher solubility relative to LPV. Prodrugs displayed higher stability under acidic conditions and degraded significantly with rise in pH. Uptake and transport data suggested that prodrugs carry significantly lower affinity towards P-gp and MRP2 relative to LPV. Moreover, prodrugs exhibited higher liver microsomal stability relative to LPV. Hence, amino acid prodrug modification might be a viable approach for enhancing LPV absorption across intestinal epithelial and brain endothelial cells which expresses high levels of P-gp and MRP2.

Monocarboxylate transporter mediated uptake of moxifloxacin on human retinal pigmented epithelium cells.

OBJECTIVES: This work was aim to determine in vitro interaction of moxifloxacin with monocarboxylate transporter (MCT) using a human retinal pigment epithelium cells (ARPE-19). METHODS: In vitro moxifloxacin uptakes were performed at 37°C across ARPE-19 cells. Concentration-dependent uptake of moxifloxacin was performed to delineate moxifloxacin kinetics with MCT. Effects of MCT substrates, MCT inhibitors, pH and metabolic inhibitors on moxifloxacin uptake were conducted to delineate mechanism of moxifloxacin influx via MCT. KEY FINDINGS: Moxifloxacin uptake was found to exhibit saturable kinetics (K(m) = 1.56 ± 0.32 µM and V(max) = 0.58 ± 0.16 µM/min/mg protein). Higher uptake of moxifloxacin was observed at acidic pH. MCT substrates such as salicylic acid, ofloxacin and L-lactic acid significantly inhibited the uptake of moxifloxacin. Furthermore, moxifloxacin uptake was significantly reduced in the presence of metabolic and MCT inhibitors. Overall, this study demonstrated an interaction of moxifloxacin with Na⁺ and H⁺-coupled transporter, most likely MCT1. CONCLUSIONS: Apart from the lipophilicity, we anticipate that lowest vitreal half-life of intravitreal moxifloxacin compared with other fluoroquinolones may be due to its interaction with MCT. This information might be crucial in clinical settings and can be further explored to improve vitreous half-life and therapeutic efficacy of moxifloxacin.

In vitro moxifloxacin drug interaction with chemotherapeutics: implications for retinoblastoma management.

Retinoblastoma (RB) is a common malignant intraocular tumor primarily affecting children. Multidrug resistance (MDR) proteins (P-gp and MRPs) mediated chemoresistance have been considered as a major cause of treatment failure in treatment of RB. Ocular cells have shown good tolerability against moxifloxacin (MFX). Hence, the aim of present study was to investigate the effect of moxifloxacin on the functionality of MDR proteins. Furthermore, we have also examined an interaction of MFX with anticancer agents (Topotecan, etoposide and vinblastine) for RB treatment. For interaction of MFX with efflux transporter, model cell lines transfected with the efflux transporters (MDCK-MDR1 and MDCK-MRP2) were used to perform uptake and bi-directional transport experiments. Modulation of anticancer induced cell cytotoxicity, pro-inflammatory cytokines (IL-6 and IL-8) release and caspase-3 enzyme activity in presence of MFX was also evaluated. Result indicates that MFX is a substrate of both MDR1 and MRP2 efflux transporters. Furthermore elevation of anticancer uptake and bi-directional transport, reduction in IC50 cytotoxic value and modulation of antiproliferative and cytokines release in presence of MFX by anticancer agents was observed. Our results demonstrate that MFX may not only modulate the permeability of anticancer agents at efflux sites but it may also potentiate antiproliferative activity of anticancer agents in retinoblastoma cells. This study may be further extended to explore in vivo outcome of this finding.

Microvascular complications and diabetic retinopathy: recent advances and future implications.

Retinal microvascular alterations have been observed during diabetic retinopathy (DR) due to the retinal susceptibility towards subtle pathological alterations. Therefore, retinal microvascular pathology is essential to understand the nature of retinal degenerations during DR. In this review, the role of retinal microvasculature complications during progression of DR, along with recent efforts to normalize such alterations for better therapeutic outcome, will be underlined. In addition, current therapeutics and future directions for advancement of standard treatment for DR patients will be discussed.

Functional characterization and molecular expression of large neutral amino acid transporter (LAT1) in human prostate cancer cells.

The primary objective of this study is to functionally characterize and provide molecular evidence of large neutral amino acid transporter (LAT1) in human derived prostate cancer cells (PC-3). We carried out the uptake of [3H]-tyrosine to assess the functional activity of LAT1. Reverse transcription-polymerase chain reaction (RT-PCR) analysis is carried out to confirm the molecular expression of LAT1. [3H]-tyrosine uptake is found to be time dependent and linear up to 60 min. The uptake process does not exhibit any dependence on sodium ions, pH and energy. However, it is temperature dependent and found maximal at physiological temperature. The uptake of [3H]-tyrosine demonstrates saturable kinetics with K(m) and V(max) values of 34 ± 3 µM and 0.70 ± 0.02 nanomoles/min/mg protein, respectively. It is strongly inhibited by large neutral (phenylalanine, tryptophan, leucine, isoleucine) and small neutral (alanine, serine, cysteine) but not by basic (lysine and arginine) and acidic (aspartic and glutamic acid) amino acids. Isoleucine-quinidine (Ile-quinidine) prodrug generates a significant inhibitory effect on [3H]-tyrosine uptake suggesting that it is recognized by LAT1. RT-PCR analysis provided a product band at 658 and 840 bp, specific to LAT1 and LAT2, respectively. For the first time, this study demonstrates that LAT1, primarily responsible for the uptake of large neutral amino acids, is functionally active in PC-3 cells. Significant increase in the uptake generated by Ile-quinidine relative to quinidine suggests that LAT1 can be utilized for enhancing the cellular permeation of poor cell permeable anticancer drugs. Furthermore, this cell line can be utilized as an excellent in vitro model for studying the interaction of large neutral amino acid conjugated drugs with LAT1 transporter.

Mitochondrial localization of P-glycoprotein and peptide transporters in corneal epithelial cells--novel strategies for intracellular drug targeting.

This study was designed to investigate functional localization of both efflux (P-glycoprotein, P-gp) and influx (peptide) transporters in the mitochondrial membrane of cultured rabbit primary corneal epithelial cells (rPCECs). Isolation and purification of mitochondria was performed by optimized cell fractionation method. Mitochondrial integrity was measured by JC-1 uptake experiment. The efflux activity of P-gp was assessed by performing in vitro uptake studies on isolated mitochondria with Rhodamine 123 (Rho-123) alone and in the presence of P-gp inhibitors (quinidine and cyclosporine A) using fluorimetry and flow cytometry analysis. Functional activity of peptide transporter was assessed by performing in vitro uptake studies of [3H] Gly-sar on isolated mitochondria in the presence or absence of peptide transporter substrate (Val-Val). Molecular characterization of P-gp and peptide transporter was assessed by western blot and confocal analysis. Enhanced JC-1 accumulation in the isolated fraction confirmed mitochondrial membrane integrity. Significantly higher uptake of Rho-123 on isolated mitochondria was observed in the presence of quinidine (75 and 100 µM) and cyclosporine A (10 µM). Significantly lower uptake of [3H] Gly-sar was observed in the presence of val-val due to competitive inhibition of peptide transporter on isolated mitochondria. Western blot and confocal analysis further confirmed the presence of P-gp and peptide transporter on the mitochondrial membrane of rPCECs. The present study demonstrates the functional and molecular characterization of P-gp and peptide transporters in the mitochondrial membranes of rPCECs. This knowledge of mitochondrial existence of P-gp and peptide transporter will aid in the development of subcellular ocular drug delivery strategies.

Design and evaluation of a novel nanoparticulate-based formulation encapsulating a HIP complex of lysozyme.

Formulation development of protein therapeutics using polymeric nanoparticles has found very little success in recent years. Major formulation challenges include rapid denaturation, susceptibility to lose bioactivity in presence of organic solvents and poor encapsulation in polymeric matrix. In the present study, we have prepared hydrophobic ion pairing (HIP) complex of lysozyme, a model protein, using dextran sulfate (DS) as a complexing polymer. We have optimized the process of formation and dissociation of HIP complex between lysozyme and DS. The effect of HIP complexation on enzymatic activity of lysozyme was also studied. Nanoparticles were prepared and characterized using spontaneous emulsion solvent diffusion method. Furthermore, we have also investigated release of lysozyme from nanoparticles along with its enzymatic activity. Results of this study indicate that nanoparticles can sustain the release of lysozyme without compromising its enzymatic activity. HIP complexation using a polymer may also be employed to formulate sustained release dosage forms of other macromolecules with enhanced encapsulation efficiency.

Ocular toxicity from systemically administered xenobiotics.

INTRODUCTION: The eye is considered as the most privileged organ because of the blood-ocular barrier that acts as a barrier to systemically administered xenobiotics. However, there has been a significant increase in the number of reports on systemic drug-induced ocular complications. If such complications are left untreated, then it may cause permanent damage to vision. Hence, knowledge of most recent updates on ever-increasing reports of such toxicities has become imperative to develop better therapy while minimizing toxicities. AREAS COVERED: The article is mainly divided into anterior and posterior segment manifestations caused by systemically administered drugs. The anterior segment is further elaborated on corneal complications where as the posterior segment is focused on optic nerve, retinal and vitreous complications. Furthermore, this article includes recent updates on acute and chronic ocular predicaments, in addition to discussing various associated symptoms caused by drugs. EXPERT OPINION: Direct correlation of ocular toxicities due to systemic drug therapy is evident from current literature. Therefore, it is necessary to have detailed documentation of these complications to improve understanding and predict toxicities. We made an attempt to ensure that the reader is aware of the characteristic ocular complications, the potential for irreversible drug toxicity and indications for cessation.

Transporter-targeted lipid prodrugs of cyclic cidofovir: a potential approach for the treatment of cytomegalovirus retinitis.

Cidofovir (CDF) and its cyclic analogue (cCDF) have shown potential in vitro and in vivo antiviral activity against cytomegalovirus (CMV) retinitis. However, hydrophilic nature of CDF may affect cell permeation across lipophilic epithelium and thus limit its effectiveness in the treatment of CMV retinitis. In the present study, we have tested a novel hypothesis, which involves chemical derivatization of cCDF into lipophilic transporter-targeted prodrug [via conjugation with different carbon chain length of lipid raft and targeting moiety (biotin) for sodium-dependent multivitamin transporter (SMVT)]. We have synthesized and characterized three derivatives of cCDF including biotin B-C2-cCDF, B-C6-cCDF, and B-C12-cCDF. Physicochemical properties such as solubility, partition coefficient (n-octanol/water and ocular tissue), bioreversion kinetics, and interaction with SMVT transporter have been determined. Among these novel conjugates, B-C12-cCDF has shown higher interaction to SMVT transporter with lowest half maximal inhibitory concentration value, higher cellular accumulation, and high tissue partitioning. Improvement in physicochemical properties, lipophilicity, and interaction with transporter was observed in the trend of increasing the lipid chain length, that is, B-C12-cCDF > B-C6-cCDF > B-C2-cCDF. These results indicate that transporter-targeted lipid analogue of cCDF exhibits improved cellular accumulation along with higher transporter affinity and hence could be a viable strategy for the treatment of CMV retinitis.

Recent overview of ocular patents.

Ocular drug therapy has always been considered as a major challenge in the field of drug delivery. The presence of blood ocular barriers and efflux pumps has imposed a great concern as well. Various vision threatening disorders require a long term therapy of drug molecules, especially for the diseases that affect the posterior segment. Pharmaceutical companies and other research institutes have adopted a multidisciplinary approach to meet the current challenges which is evidenced by the trends seen in the published and filed U.S. patents. Various strategies have been employed to achieve long term sustained and targeted delivery for both the anterior and the posterior segments of the ocular diseases. These strategies include formulating drugs into implant, micro or nanoparticulate systems and hydrogel-based systems. Transporter targeted approach has also allowed scientists to deliver drugs to both the segments of the eye. Recent developments such as delivery of drugs utilizing ultrasound, iontophoresis and microneedle based devices have been promising. Genebased therapeutics has opened a new avenue for vision threatening disorders. In all, the current developments in the entire field have been very exciting for finding out new strategies to treat vision threatening disorders.

Prodrug strategies in ocular drug delivery.

Poor bioavailability of topically instilled drug is the major concern in the field of ocular drug delivery. Efflux transporters, static and dynamic ocular barriers often possess rate limiting factors for ocular drug therapy. Different formulation strategies like suspension, ointment, gels, nanoparticles, implants, dendrimers and liposomes have been employed in order to improve drug permeation and retention by evading rate limiting factors at the site of absorption. Chemical modification such as prodrug targeting various nutrient transporters (amino acids, peptide and vitamin) has evolved a great deal of interest to improve ocular drug delivery. In this review, we have discussed various prodrug strategies which have been widely applied for enhancing therapeutic efficacy of ophthalmic drugs. The purpose of this review is to provide an update on the utilization of prodrug concept in ocular drug delivery. In addition, this review will highlight ongoing academic and industrial research and development in terms of ocular prodrug design and delivery.

Mitochondrial dysfunction in retinal diseases.

The mitochondrion is a vital intracellular organelle for retinal cell function and survival. There is growing confirmation to support an association between mitochondrial dysfunction and a number of retinal degenerations. Investigations have also unveiled mitochondrial genomic instability as one of the contributing factors for age-related retinal pathophysiology. This review highlights the role of mitochondrial dysfunction originating from oxidative stress in the etiology of retinal diseases including diabetic retinopathy, glaucoma and age-related macular degeneration (AMD). Moreover, mitochondrial DNA (mtDNA) damage associated with AMD due to susceptibility of mtDNA to oxidative damage and failure of mtDNA repair pathways is also highlighted in this review. The susceptibility of neural retina and retinal pigment epithelium (RPE) mitochondria to oxidative damage with ageing appears to be a major factor in retinal degeneration. It thus appears that the mitochondrion is a weak link in the antioxidant defenses of retinal cells. In addition, failure of mtDNA repair pathways can also specifically contribute towards pathogenesis of AMD. This review will further summarize the prospective role of mitochondria targeting therapeutic agents for the treatment of retinal disease. Mitochondria based drug targeting to diminish oxidative stress or promote repair of mtDNA damage may offer potential alternatives for the treatment of various retinal degenerative diseases.

Effect of emergence of fluoroquinolone resistance on intrinsic expression of P-glycoprotein phenotype in corneal epithelial cells.

PURPOSE: Multidrug resistance (MDR) represents a major obstacle to the success of antimicrobial fluoroquinolone (FQ) therapy. MDR-associated efflux protein pumps antimicrobial agents out of the corneal cells, leading to suboptimal eradication of microbes. This article examines whether long-term FQ (levofloxacin, ofloxacin, and gatifloxacin) therapy can modify the MDR phenotype (P-glycoprotein [P-gp]) on corneal epithelial cells (Statens Seruminstitut Rabbit Cornea [SIRC]). METHODS: To study the effect of FQ, SIRC cells without any exposure to FQ (control) were compared with the cells exposed to ofloxacin, levofloxacin, and gatifloxacin at a concentration of 10 µg/mL for 3 weeks. Efflux activity of P-gp was assessed by in vitro uptake studies (fluorescent and radioactive), flow cytometry, and quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: In the presence of FQ, elevated P-gp expression was noted with uptake, flow cytometry, and qRT-PCR analyses. This study confirms that long-term exposure to antibiotics, particularly FQ, can induce overexpression of P-gp efflux transporter present on the corneal cells. P-gp overexpression is commonly noticed in anticancer drug resistance cell lines; however, for the first time, this report describes overexpression of P-gp due to FQ exposure. CONCLUSIONS: Based on this result, it is suggested that strategies should be developed and implemented not only to overcome resistance to ocular pathogen but also to FQ-induced cellular resistance.

Identification of a small molecule class to enhance cell-cell adhesion and attenuate prostate tumor growth and metastasis.

Expression of calcitonin (CT) and its receptor (CTR) is elevated in advanced prostate cancer, and activated CT-CTR autocrine axis plays a pivotal role in tumorigenicity and metastatic potential of multiple prostate cancer cell lines. Recent studies suggest that CT promotes prostate cancer metastasis by reducing cell-cell adhesion through the disassembly of tight and adherens junctions and activation of beta-catenin signaling. We attempted to identify a class of molecules that enhances cell-cell adhesion of prostate cells and reverses the disruptive actions of CT on tight and adherens junctions. Screening several compounds led to the emergence of phenyl-methylene hydantoin (PMH) as a lead candidate that can augment cell-cell adhesion and abolish disruptive actions of CT on junctional complexes. PMH reduced invasiveness of PC-3M cells and abolished proinvasive actions of CT. Importantly, PMH did not display significant cytotoxicity on PC-3M cells at the tested doses. I.p. administered PMH and its S-ethyl derivative remarkably decreased orthotopic tumor growth and inhibited the formation of tumor micrometastases in distant organs of nude mice. PMH treatment also reduced the growth of spontaneous tumors in LPB-Tag mice to a significant extent without any obvious cytotoxic effects. By virtue of its ability to stabilize cell junctions, PMH could reverse the effect of CT on junctional disruption and metastasis, which strengthens the possibility of using PMH as a potential drug candidate for CT-positive androgen-independent prostate cancers.

Cadherin switching and activation of beta-catenin signaling underlie proinvasive actions of calcitonin-calcitonin receptor axis in prostate cancer.

Calcitonin, a neuroendocrine peptide, and its receptor are localized in the basal epithelium of benign prostate but in the secretory epithelium of malignant prostates. The abundance of calcitonin and calcitonin receptor mRNA displays positive correlation with the Gleason grade of primary prostate cancers. Moreover, calcitonin increases tumorigenicity and invasiveness of multiple prostate cancer cell lines by cyclic AMP-dependent protein kinase-mediated actions. These actions include increased secretion of matrix metalloproteinases and urokinase-type plasminogen activator and an increase in prostate cancer cell invasion. Activation of calcitonin-calcitonin receptor autocrine loop in prostate cancer cell lines led to the loss of cell-cell adhesion, destabilization of tight and adherens junctions, and internalization of key integral membrane proteins. In addition, the activation of calcitonin-calcitonin receptor axis induced epithelial-mesenchymal transition of prostate cancer cells as characterized by cadherin switch and the expression of the mesenchymal marker, vimentin. The activated calcitonin receptor phosphorylated glycogen synthase kinase-3, a key regulator of cytosolic beta-catenin degradation within the WNT signaling pathway. This resulted in the accumulation of intracellular beta-catenin, its translocation in the nucleus, and transactivation of beta-catenin-responsive genes. These results for the first time identify actions of calcitonin-calcitonin receptor axis on prostate cancer cells that lead to the destabilization of cell-cell junctions, epithelial-to-mesenchymal transition, and activation of WNT/beta-catenin signaling. The results also suggest that cyclic AMP-dependent protein kinase plays a key role in calcitonin receptor-induced destabilization of cell-cell junctions and activation of WNT-beta-catenin signaling.