Intact NMR Approach Quickly Reveals Synchronized Microstructural Changes in Oil-in-Water Nanoemulsion Formulations.

A soft-core oil-in-water (o/w) nanoemulsion (NE) is composed of nanometer (nm) sized oil droplets, stabilized by a surfactant layer and dispersed in a continuous bulky water phase. Characterization of the o/w NE molecule arrangements non-invasively, particularly the drug phase distribution (DPD) and its correlation to oil globule size (OGS), remains a challenge. Here we demonstrated the analytical methods of intact 19F Nuclear Magnetic Resonance (NMR) and 1H diffusion ordered spectroscopy (DOSY) NMR for their specificity in measuring DPD and OGS, respectively, on three NE formulations containing the active ingredient difluprednate (DFPN) at the same concentration. The results illustrated synchronized molecular rearrangement reflected in the DPD and OGS upon alterations in formulation. Addition of surfactant resulted in a higher DPD in the surfactant layer, and concomitantly smaller OGS. Mechanic perturbation converted most of the NE globules to the smaller thermodynamically stable microemulsion (ME) globules, changing both DPD and OGS to ME phase. These microstructure changes were not observed using 1D 1H NMR; and dynamic light scattering (DLS) was only sensitive to OGS of ME globule in mechanically perturbed formulation. Collectively, the study illustrated the specificity and essential role of intact NMR methods in measuring the critical microstructure attributes of soft-core NE systems quickly, accurately, and non-invasively. Therefore, the selected NMR approach can be a unique diagnostic tool of molecular microstructure or Q3 property in o/w NE formulation development, and quality assurance after manufacture process or excipient component changes.

Difluprednate-Hydroxypropyl-ß-Cyclodextrin-Based Ophthalmic Solution for Improved Delivery in a Porcine Eye Model.

Purpose: Difluprednate (DFP) is an approved corticosteroid, available as an ophthalmic emulsion (Durezol®), used to treat pain and inflammation of the eye following ocular surgeries. This study utilized hydroxypropyl-ß-cyclodextrin (HPBCD)-based DFP ophthalmic solution for improved ocular delivery. Methods: The DFP-HPBCD complex formation was studied in the liquid and solid states. Phase solubility, molecular docking studies, differential scanning calorimetry, and Fourier transform infrared spectroscopy suggested inclusion complexation of DFP and HPBCD. Results: DFP-HPBCD-based eye drops (solution) provided 16 and 26 times higher transcorneal permeation when compared to the suspension (no HPBCD, control) and Durezol, respectively (P < 0.001). In addition, ocular drug distribution studies conducted in continuously perfused whole porcine eyes showed DFP permeated into all of the ocular tissues in significantly higher amounts than Durezol. Conclusions: The solution-based eye drops in this study is iso-osmotic, safe, and more permeable in porcine eyes compared to Durezol.

Structural Phylogeny of Different Allergens May Reveal Common Epitopic Footprint.

BACKGROUND: The incidence of allergy has been increasing at an alarming rate over the last few decades. OBJECTIVE: Our present study aims to find out the structurally homologous motifs present in different proteinaceous allergens. METHODS: Significant number of protein sequences and their corresponding structures of various pollen, fungal, bacterial, and food allergens were retrieved and the sequence and structural identity were analyzed. RESULTS: Intra- and inter-sequence along with their structural analysis of the proteinaceous allergens revealed that no significant relationships exist among them. A few, but not the negligible number of high structural similarities, were observed within different groups of allergens from fungus, angiosperms, and animals (Aves and Mammalia). CONCLUSION: Our in silico study on thirty-six different allergens showed a significant level of structural similarities among themselves, regardless of their sequences.

Understanding drug distribution and release in ophthalmic emulsions through quantitative evaluation of formulation-associated variables.

Establishing bioequivalence (BE) of ophthalmic emulsions in the absence of in vivo data is challenging. In these emulsions, drug release is a complex process due to drug distribution among various phases which are difficult to characterize. The objective of this study is to investigate the process of drug distribution and mechanism of drug release in the context of formulation-associated variables. A previously reported kinetic method for determining drug partitioning was used to quantitatively evaluate the drug distribution within a simplified biphasic (emulsion) system employing cyclosporine and difluprednate as model drugs. The impacts of formulation variables, such as the amount of polysorbate 80, glycerin, and carbomer copolymer as well as the area of oil-water interface were investigated. Polysorbate 80 was found to have the greatest influence on the drug distribution. It enhanced both the rate and extent of the drug distribution from oil to aqueous phase. Glycerin was found to slightly reduce the rate and extent of drug distribution of cyclosporine into the aqueous phase, probably by suppressing the solubilization capability of the micelles. Carbomer slowed down the diffusion of drug into the oil phase and shifted the equilibrium drug distribution towards the aqueous phase. Furthermore, increase in the interfacial area significantly increased the rate of drug diffusion across the oil-aqueous interface but had negligible effect on the extent of drug distribution. It is noteworthy that the experimental setup utilized a planar interface rather than an interface with curvature, which may have slightly underestimated the influence of globule size on equilibrium drug distribution. The findings of this study give insight into the drug distribution and diffusion in complex ophthalmic emulsions and assist with formulation design as well as development of in vitro methods to support BE assessment of ophthalmic emulsions.

A Kinetic Approach to Determining Drug Distribution in Complex Biphasic Systems.

Pharmaceutical emulsions contain multiple components, such as micellar, aqueous, and oil phases, leading to complex drug transfer and equilibrium phenomena. These complex components present challenges for the bioequivalence assessment of the drug products. The objective of the study was to develop a method that can probe the underlying mechanism and process of drug distribution. The concept of drug partitioning into biphasic systems was used to simplify the complex transfer phenomenon. A kinetic method was developed taking into account the biphasic diffusion. Using this approach, both the rate (kinetics) and the extent (equilibrium) of distribution can be determined. For method development purpose, 3 model compounds (triamcinolone acetonide, difluprednate, and cyclosporine), with expected partition coefficient values ranging from 2 to 6, were tested using the kinetic method and the traditional shake-flask method. The values obtained by the 2 methods for all compounds correlated well (r2 = 0.825). Various organic and aqueous solvents which are commonly encountered in formulations were also tested to determine the impact of phase composition on drug distribution. The kinetic method was found to offer more flexibility in terms of solvent composition and can lead to better understanding for drug distribution and potential drug release in complex biphasic systems.

A Simple and Noninvasive DOSY NMR Method for Droplet Size Measurement of Intact Oil-In-Water Emulsion Drug Products.

In a typical oil-in-water emulsion drug product, oil droplets with varied sizes are dispersed in a water phase and stabilized by surfactant molecules. The size and polydispersity of oil droplets are critical quality attributes of the emulsion drug product that can potentially affect drug bioavailability. More critically, to ensure accuracy in characterization of the finished drug product, analytical methods should introduce minimal physical perturbation (e.g., temperature variation or dilution) before the analysis. The classical methods of dynamic light scattering or electron microscopy can be used but they generally require sample dilution or harsh preparation conditions, respectively. By contrast, the size distribution of emulsion formulations can be assessed with a simple and noninvasive solution nuclear magnetic resonance method, namely, two-dimensional Diffusion Ordered SpectroscopY. The two-dimensional Diffusion Ordered SpectroscopY method probed signal decay of methyl resonances from oil and sorbate molecules and was applied to 3 types of U.S.-marketed emulsion drug products, that is, difluprednate, cyclosporine, and propofol, yielding measured droplet sizes of 40-280 nm in diameter. The high precision of ±6 nm of the new nuclear magnetic resonance method allows analytical differentiation of lot-to-lot and brand-to-brand droplet size differences in emulsion drug products, critical for drug-quality development, control, and surveillance.

Application of Ultra-Centrifugation and Bench-Top 19F NMR for Measuring Drug Phase Partitioning for the Ophthalmic Oil-in-Water Emulsion Products.

Generic drug products are expected to have the same active pharmaceutical ingredient (API) (Q1) with the same content (Q2) and microstructure arrangement (Q3) as the innovator product. In complex oil-in-water emulsion drugs, the hydrophobic API is mainly formulated in oil droplets stabilized by surfactant and micelles composed of extra surfactant molecules. The API phase partition in oil and water (mainly micelle) is a critical quality attribute (CQA) of emulsion product in demonstrating physicochemical equivalence using difluprednate (DFPN) emulsion product Durezol® as a model, we developed a novel low-field benchtop NMR method to demonstrate its applicability in measuring DFPN phase partition for ophthalmic oil-in-water emulsion products. Low-field 19F spectra were collected for DFPN in formulation, in water phase and oil phase after separation from ultra-centrifugation. The NMR data showed the mass balance of DFPN before and after phase separation. The average water phase content of different Durezol® lots was 32 ± 3% with 1% variation from method reproducibility test. The partition results were 52 ± 2% for the in-house control products prepared in Q1/Q2 equivalence to Durezol® but by a different process. The significant difference in DFPN-phase partition between Durezol® and the in-house formulation demonstrated manufacture difference readily changed the API partition. The newly developed ultra-centrifugation and 19F NMR by benchtop instrument is a simple, robust, and sensitive analytical method for ophthalmic emulsion drug product development and control.

Nonclinical Development of ENV905 (Difluprednate) Ophthalmic Implant for the Treatment of Inflammation and Pain Associated with Ocular Surgery.

PURPOSE: Topical corticosteroids are widely used in the treatment of inflammation and pain after ocular surgery, but they possess several shortcomings, including frequent dosing and low patient adherence. We evaluated the efficacy and pharmacokinetics of ENV905 (difluprednate or DFBA) Ophthalmic Implant, a single-dose drug delivery system, compared with 0.05% Durezol. METHODS: PRINT® technology was used to fabricate ENV905 implants for either intracameral (IC) or subconjunctival (SCJ) delivery of extended-release DFBA. A postoperative inflammation model and ocular pharmacokinetics studies of ENV905 or Durezol were conducted in albino rabbits for a maximum of 12 weeks. RESULTS: Suppression of ocular inflammation was marked for both IC and SJC ENV905 compared with placebo, and it was superior or equivalent to that observed with QID Durezol. Concentrations of desacetyl difluprednate (DFB, active metabolite) peaked on day 1 and tapered over time for ENV905, with IC ENV905 delivering DFB to the target tissue at the time of greatest inflammation, whereas SJC produced a longer duration of exposure. Durezol eyes demonstrated consistent exposure over time with maximal exposure in the cornea. Although the pharmacokinetic profile differed for the two routes, efficacy was similar. CONCLUSION: ENV905 was well tolerated and demonstrated a robust reduction in ocular inflammation with targeted drug delivery. The results from these studies show that ENV905 provides a sustained therapeutic effect after a single dose. By resolving low patient compliance and eliminating the peaks and troughs in drug concentration, sustained drug delivery via ENV905 may further improve the overall control of postoperative inflammation and pain.

Difluprednate: more than meets the eye.

Difluprednate, a FDA approved topical corticosteroid indicated for the treatment of inflammation and pain associated with ocular surgery, affords three polymorphic crystal forms (one hexagonal, sg. P65, and two distinct orthorhombic, both sg.'s P212121, phases), whose preparation, thermal stability ranges, crystal structures and stereochemical preferences are here reported. Using DSC, single-crystal structural analysis and less conventional ab-initio X-ray powder diffraction methods, the rich structural and thermal behavior of three difluprednate polymorphs have been clarified, and the validity of previous complex and sometimes contradicting literature reports has been challenged. Complementary solution state NMR provided (1)H, (13)C and (19)F chemical shifts full assignment of the corresponding signals. These results allow us to precisely describe the selective isolation pathways toward three distinct crystal phases, and to define their structural and analytical data necessary for identification and easy and accurate quantification, by modern Rietveld analysis, of complex difluprednate polymorphic mixtures, often obtained as a result of poorly controlled (co)-precipitation methods.

Formulation of an ophthalmic lipid emulsion containing an anti-inflammatory steroidal drug, difluprednate.

Preparation of oil-in-water (o/w) type lipid emulsion is one of the approaches to formulate drugs that are poorly water-soluble but can be dissolved in the oil phase of the emulsions. A synthetic glucocorticoid medicine, difluprednate (DFBA), is a water-insoluble compound. We formulated DFBA (0.05%, w/v) ophthalmic lipid emulsion containing 5.0% (w/v) caster oil and 4.0% (w/v) polysorbate 80. The appearance of the emulsion was blue and translucent lipid emulsion, and the median particle size of the lipid emulsion was 104.4 nm. Neither separation nor change in particle size was observed after 6 months at 40 degrees C. Furthermore, when compared with DFBA (0.05%, w/v) ophthalmic suspension, the lipid emulsion showed 5.7-fold higher concentration of DFB that was an active metabolite of DFBA in aqueous humor at 1h after instillation. Ophthalmic lipid emulsion enhances the intraocular penetration of drugs, and it is useful as a delivery system for the ophthalmic preparations of lipophilic drugs.

Effect of chirality at C-20 of methyl 11beta,17alpha,20-trihydroxy-3-oxo-1,4-pregnadien-21-oate derivatives on antiinflammatory activity.

In an effort to determine the C-20 chirality effect on the antiinflammatory activity of 17beta-glycolate esters, methyl 11beta,17alpha,20-trihydroxy-3-oxo-1,4-pregnadien-21-oate and its 9alpha-fluoro analog, their acetonide and their carbonate derivatives were synthesized and evaluated. The agents were tested for their binding potency to the macrophage glucocorticoid receptor, and their effect on LPS-induced nitric oxide generation in RAW 264.7 cells. The acetonide derivatives showed the highest binding affinity while the triols and carbonates bound rather poorly to the receptors. With the exception of the triols, the alpha-isomer in each pair of the agents exhibited higher binding affinity to the receptor than its corresponding beta-isomer, clearly indicating that C-20 chirality has a significant effect on antiinflammatory activity. In addition, the alpha-isomers of the acetonides showed substantially higher binding affinity than the parent compound, prednisolone. In contrast to the high binding activity exhibited by some of the acetonides, all of the agents showed weak inhibitory effect on NO generation. Metabolic inactivation during assessment of NO inhibition may play a role in the divergence noted between receptor affinity and the measured biologic activity resulting from the binding.