Corneal delivery of moxifloxacin and dexamethasone combination using drug-eluting mucoadhesive contact lens to treat ocular infections.

The important causes of loss of vision are ocular infections, including keratitis and conjunctivitis. Attaining an adequate concentration of topically applied antibiotics to prevent or treat infections within the cornea is challenging. The study aimed to design and develop a drug-eluting polymeric contact lens for the effective delivery of moxifloxacin (MF) and dexamethasone (DM). The polymeric contact lens was prepared using chitosan, glycerol, and polyethylene glycol. MF and DM were loaded into the contact lens, both separately and in combination. The MF and DM loaded contact lenses were characterized for thickness, swelling index, surface topography, and mucoadhesion strength. Furthermore, studies were performed to understand the in vitro drug release behavior, ex vivo corneal permeation, and in vitro and in vivo antimicrobial activity. The drug-loaded contact lens was compared with the standard drug solutions. The physical characteristics of the polymeric contact lens were similar to the commercially available contact lens. Compared to the topically applied standard drug solutions, the drug-loaded contact lens showed significantly (p < 0.05) greater corneal drug distribution after 24 h incubation. In vitro and in vivo antimicrobial activity of the MF loaded contact lens was superior to the standard drug solution. In vivo drug distribution studies showed greater tissue concentration of MF in cornea, sclera, and aqueous humor with contact lens application compared with drug solutions. Overall, the polymeric contact lens was efficient in delivering MF and DM at required therapeutic concentrations. The findings from the present study show that drug-eluting contact lenses could be used in post-operative conditions to prevent ocular infections.

Effect of Mucoadhesive Polymeric Formulation on Corneal Permeation of Fluoroquinolones.

PURPOSE: The aim of the study was to develop a novel formulation of levofloxacin and besifloxacin to achieve improved mucoadhesion and permeability of besifloxacin and levofloxacin through cornea for the effective treatment of ocular infections. METHODS: A multicomponent hydrogel formulation containing chitosan-polyvinyl alcohol (PVA)-poly(N-vinylpyrrolidone) (PVP) was designed. Lysophosphatidylcholine was used to enhance corneal penetration of the drugs. The hydrogel preparations were characterized for various parameters, including clarity, pH, viscosity, in vitro release kinetics, mucoadhesion, ex vivo human corneal permeation, and antimicrobial efficacy. The formulations were compared with standard drug solution and marketed eye drops (Besix® and Levotop®). RESULTS: Compared to commercial ophthalmic preparations and free drug solutions, hydrogel formulation of both besifloxacin and levofloxacin was found to have 3.5- and 8-fold higher (P < 0.001) mucoadhesion and superior cumulative corneal permeation. The formulations showed superior in vitro anti-infective properties. Incubation of besifloxacin and levofloxacin formulations with Staphylococcus aureus-infected cornea model for 0.5 h showed greater potency of the hydrogel formulations compared to the marketed eye drops and standard solutions. CONCLUSIONS: The results of the study show the multicomponent hydrogel formulations of besifloxacin and levofloxacin to have superior corneal permeation with the potential for being used as topical ophthalmic preparations.

Corneal delivery of besifloxacin using rapidly dissolving polymeric microneedles.

Penetration of antibiotics into and through the cornea is a major limiting factor in the treatment of ocular infections. Several strategies are in vogue to overcome this limitation such as use of fortified drops, gels, and subconjunctival injections. Here, we present the fabrication of rapidly dissolving polymeric microneedle array to effectively deliver besifloxacin through the cornea. Microneedles were prepared using polyvinyl alcohol and polyvinyl pyrrolidone by the micromolding technique. The model fluoroquinolone antibiotic, besifloxacin, was loaded in 36 microneedles arranged in a 6 × 6 array format within a 1 cm2 area. The average height and base width of microneedles was 961 ± 27 and 366 ± 16 µm, respectively. Each microneedle array contained 103.4 ± 8.5 µg of besifloxacin. Cryosectioning and confocal microscopy of excised human cornea revealed that microneedles penetrated to a depth of up to 200 µm. Microneedles were found to completely dissolve in the cornea within 5 min. Application of microneedles for 5 min significantly (p < 0.05) improved the besifloxacin deposition and permeation through the cornea compared with free besifloxacin solution. Similarly, besifloxacin-loaded microneedles showed greater antibacterial activity in Staphylococcus aureus-infected cornea in comparison to free besifloxacin solution. Taken together, rapidly dissolving microneedles can be developed to effectively deliver besifloxacin to treat bacterial infections in the cornea and eye.

Effective Skin Cancer Treatment by Topical Co-delivery of Curcumin and STAT3 siRNA Using Cationic Liposomes.

The aim of the present study was to evaluate the effectiveness of iontophoretic co-delivery of curcumin and anti-STAT3 siRNA using cationic liposomes against skin cancer. Curcumin was encapsulated in DOTAP-based cationic liposomes and then complexed with STAT3 siRNA. This nanocomplex was characterized for the average particle size, zeta-potential, and encapsulation efficiency. The cell viability studies in B16F10 mouse melanoma cells have shown that the co-delivery of curcumin and STAT3 siRNA significantly (p < 0.05) inhibited the cancer cell growth compared with either liposomal curcumin or STAT3 siRNA alone. The curcumin-loaded liposomes were able to penetrate up to a depth of 160 µm inside the skin after iontophoretic (0.47 mA/cm2) application. The in vivo efficacy studies were performed in the mouse model of melanoma skin cancer. Co-administration of the curcumin and STAT3 siRNA using liposomes significantly (p < 0.05) inhibited the tumor progression as measured by tumor volume and tumor weight compared with either liposomal curcumin or STAT3 siRNA alone. Furthermore, the iontophoretic administration of curcumin-loaded liposome-siRNA complex showed similar effectiveness in inhibiting tumor progression and STAT3 protein suppression compared with intratumoral administration. Taken together, cationic liposomes can be utilized for topical iontophoretic co-delivery of small molecule and siRNA for effective treatment of skin diseases.

The curious case of benzbromarone: insight into super-inhibition of cytochrome P450.

Cytochrome P450 (CYP) family of redox enzymes metabolize drugs and xenobiotics in liver microsomes. Isozyme CYP2C9 is reported to be inhibited by benzbromarone (BzBr) and this phenomenon was hitherto explained by classical active-site binding. Theoretically, it was impossible to envisage the experimentally derived sub-nM Ki for an inhibitor, when supra-nM enzyme and 10X KM substrate concentrations were employed. We set out to find a more plausible explanation for this highly intriguing "super-inhibition" phenomenon. In silico docking of various BzBr analogs with known crystal structure of CYP2C9 did not provide any evidence in support of active-site based inhibition hypothesis. Experiments tested the effects of BzBr and nine analogs on CYPs in reconstituted systems of lab-purified proteins, complex baculosomes & crude microsomal preparations. In certain setups, BzBr and its analogs could even enhance reactions, which cannot be explained by an active site hypothesis. Generally, it was seen that Ki became smaller by orders of magnitude, upon increasing the dilution order of BzBr analogs. Also, it was seen that BzBr could also inhibit other CYP isozymes like CYP3A4, CYP2D6 and CYP2E1. Further, amphipathic derivatives of vitamins C & E (scavengers of diffusible reactive oxygen species or DROS) effectively inhibited CYP2C9 reactions in different reaction setups. Therefore, the inhibition of CYP activity by BzBr analogs (which are also surface-active redox agents) is attributed to catalytic scavenging of DROS at phospholipid interface. The current work expands the scope of interpretations of inhibitions in redox enzymes and ushers in a new cellular biochemistry paradigm that small amounts of DROS may be obligatorily required in routine redox metabolism for constructive catalytic roles.

Redox active molecules cytochrome c and vitamin C enhance heme-enzyme peroxidations by serving as non-specific agents for redox relay.

We report that incorporation of very low concentrations of redox protein cytochrome c and redox active small molecule vitamin C impacted the outcome of one-electron oxidations mediated by structurally distinct plant/fungal heme peroxidases. Evidence suggests that cytochrome c and vitamin C function as a redox relay for diffusible reduced oxygen species in the reaction system, without invoking specific or affinity-based molecular interactions for electron transfers. The findings provide novel perspectives to understanding - (1) the promiscuous role of cytochrome b(5) in the metabolism mediated by liver microsomal xenobiotic metabolizing systems and (2) the roles of antioxidant molecules in affording relief from oxidative stress.

Cytochrome P450 reductase: a harbinger of diffusible reduced oxygen species.

The bi-enzymatic system of cytochrome P450 (CYP, a hemoprotein) and cytochrome P450 reductase (CPR, a diflavoenzyme) mediate the redox metabolism of diverse indigenous and xenobiotic molecules in various cellular and organ systems, using oxygen and NADPH. Curiously, when a 1:1 ratio is seen to be optimal for metabolism, the ubiquitous CYP:CPR distribution ratio is 10 to 100:1 or higher. Further, the NADPH equivalents consumed in these in vitro or in situ assemblies usually far exceeded the amount of substrate metabolized. We aimed to find the rationale to explain for these two oddities. We report here that CPR is capable of activating molecular oxygen on its own merit, generating diffusible reduced oxygen species (DROS). Also, in the first instance for a flavoprotein, CPR is shown to deplete peroxide via diffusible radical mediated process, thereby leading to the formation of water (but without significant evolution of oxygen). We also quantitatively demonstrate that the rate of oxygen activation and peroxide depletion by CPR accounts for the major reactivity in the CYP+CPR mixture. We show unambiguously that CPR is able to regulate the concentration of diffusible reduced oxygen species in the reaction milieu. These findings point out that CPR mediated processes are bound to be energetically 'wasteful' and potentially 'hazardous' owing to the unavoidable nature of the CPR to generate and deplete DROS. Hence, we can understand that CPR is distributed at low densities in cells. Some of the activities that were primarily attributed to the heme-center of CYP are now established to be a facet of the flavins of CPR. The current approach of modeling drugs to minimize "uncoupling" on the basis of erstwhile hypothesis stands questionable, considering the ideas brought forth in this work.