Rabbit Aqueous Humor Research Articles

Positively Charged Polymeric Nanoparticle Reservoirs of Terbinafine Hydrochloride: Preclinical Implications for Controlled Drug Delivery in the Aqueous Humor of Rabbits

Frequent instillation of terbinafine hydrochloride (T HCl) eye drops (0.25%, w/v) is necessary to maintain effective aqueous humor concentrations for treatment of fungal keratitis. The current approach aimed at developing potential positively charged controlled-release polymeric nanoparticles (NPs) of T HCl. The estimation of the drug pharmacokinetics in the aqueous humor following ocular instillation of the best-achieved NPs in rabbits was another goal. Eighteen drug-loaded (0.50%, w/v) formulae were fabricated by the nanopreciptation method using Eudragit® RS100 and chitosan (0.25%, 0.5%, and 1%, w/v). Soybean lecithin (1%, w/v) and Pluronic® F68 (0.5%, 1%, and 1.5%, w/v) were incorporated in the alcoholic and aqueous phases, respectively. The NPs were evaluated for particle size, zeta potential, entrapment efficiency percentage (EE%), morphological examination, drug release in simulated tear fluid (pH 7.4), Fourier-transform IR (FT-IR), X-ray diffraction (XRD), physical stability (2 months, 4°C and 25°C), and drug pharmacokinetics in the rabbit aqueous humor relative to an oily drug solution. Spherical, discrete NPs were successfully developed with mean particle size and zeta potential ranging from 73.29 to 320.15 nm and +20.51 to +40.32 mV, respectively. Higher EE% were achieved with Eudragit® RS100-based NPs. The duration of drug release was extended to more than 8 h. FT-IR and XRD revealed compatibility between inactive formulation ingredients and T HCl and permanence of the latter's crystallinity, respectively. The NPs were physically stable, for at least 2 months, when refrigerated. F5-NP suspension significantly (P<0.05) increased drug mean residence time and improved its ocular bioavailability; 1.657-fold.

Promising ion-sensitive in situ ocular nanoemulsion gels of terbinafine hydrochloride: Design, in vitro characterization and in vivo estimation of the ocular irritation and drug pharmacokinetics in the aqueous humor of rabbits

Terbinafine hydrochloride (T-HCl) is recommended for the management of fungal keratitis. To maintain effective aqueous humor concentrations, frequent instillation of T-HCl drops is necessary. This work aimed to develop alternative controlled-release in situ ocular drug-loaded nanoemulsion (NE) gels. Twelve pseudoternary-phase diagrams were constructed using oils (isopropyl myristate/Miglyol 812), surfactants (Tween 80/Cremophor EL), a co-surfactant (polyethylene glycol 400) and water. Eight drug-loaded (0.5%, w/v) NEs were evaluated for thermodynamic stability, morphology, droplet size and drug release in simulated tear fluid (pH 7.4). Following dispersion in gellan gum solution (0.2%, w/w), the in situ NE gels were characterized for transparency, rheological behavior, mucoadhesive force, drug release and histopathological assessment of ocular irritation. Drug pharmacokinetics of sterilized F31 [Miglyol 812, Cremophor EL: polyethylene glycol 400 (1:2) and water (5, 55 and 40%, w/w, respectively)] in situ NE gel and oily drug solution were evaluated in rabbit aqueous humor. The NEs were thermodynamically stable and have spherical droplets (<30 nm). The gels were transparent, pseudoplastic, mucoadhesive and showed more retarded zero-order drug release rates. F31 in situ NE gel showed the least ocular irritation potential and significantly (P<0.01) higher C(max), delayed T(max), prolonged mean residence time and increased bioavailability.

Homemade lyophilized cross linking amniotic sustained-release drug membrane with anti-scarring role after filtering surgery in rabbit eyes.

To investigate the antifibrotic effect of the freeze-dried bilayered fibrin-binding amniotic membrane as a drug delivery system on glaucoma surgery in rabbit model. The aim of this study was to prepare a novel local delivery system for the sustained and controllable release of 5-Fu. Twenty-four Japanese white rabbits were randomized into three groups: the experimental group (ocular trabeculectomy in combination with 5-Fu loaded freeze-dried bilayered fibrin-binding amniotic membrane transplantation), the control group (ocular trabeculectomy in combination with 5-Fu) and the blank group (single trabeculectomy). HE staining, massion staining and immunohistochemistry for α-SMA were performed on days 7, 14, 21 and 30 following surgery. The concentration of 5-Fu in rabbit aqueous humor was examined by high performance liquid chromatography (HPLC) 3 days after the surgery. Statistical differences were noted in intraocular pressure among groups on day 7, 14, 21 and 30 following surgery. Histology further demonstrated that trabeculectomy in combination with freeze-dried bilayered fibrin-binding amniotic membrane yielded well wound healing and no scar formation and was beneficial for long term effect. HPLC showed a good slow-release effect with freeze-dried bilayered fibrin-binding amniotic membrane.

UPLC/Q-TOF-MS/MS METHOD FOR EVALUATION OF MOXIFLOXACIN LOADED NANOPLEXES AS VEHICLES FOR OCULAR DRUG DELIVERY

A simple, sensitive, and selective ultra-performance liquid chromatographic (UPLC) method with quadrupole-time of flight-mass spectrometric (Q-TOF-MS/MS) detection was developed for the determination of moxifloxacin (moxi) in rabbit aqueous humor. After a simple protein-precipitation by acetonitrile, the post-treatment samples were separated on a UPLC Bridged Ethyl Hybrid (BEH) C-18 column with 0.1% formic acid in water as a mobile phase and analyzed in positive ion mode. The method developed was validated in rabbit aqueous humor with a daily working range of 0.1–200 ng/mL with correlation coefficient, r2 > 0.999 and a sensitivity of 0.12 ng/mL as limit of quantification. The method proved to be accurate (recovery, 96.0–99.3%), precise (%RSD, ≤0.71%), and specific and was applied to evaluate the moxifloxacin loaded nanoplexes (moxi-NPX) as vehicles for ocular drug delivery. Moxi-NPX (4037 ± 45.9 ng/mL hr) provided approximately five-fold increase in the relative ocular bioavailability compared with moxi solution (806.5 ± 32.5 ng/mL hr). In conclusion, moxi-NPX increases ocular bioavailability of moxi, prolongs its residence time in the eye, and are efficient ocular drug delivery vehicles. Supplemental Materials are available for this article. Go to the publisher's online edition of Journal of Liquid Chromatography & Related Technologies to view the free supplemental file.

Modeling Approach To Characterize Intraocular Doripenem Pharmacokinetics after Intravenous Administration to Rabbits, with Tentative Extrapolation to Humans

The aim of this study was to determine the penetration of doripenem administered intravenously into the rabbit aqueous and vitreous humors. Nineteen New Zealand White rabbits received a 20-mg dose of doripenem intravenously over 60 min. Specimens of aqueous humor, vitreous humor, and blood were obtained 30 min (n = 5), 1 h (n = 5), 2 h (n = 5), and 3 h (n = 4) after the beginning of the infusion and analyzed by high-performance liquid chromatography (HPLC). A pharmacokinetic (PK) model was developed to fit the experimental data. Doripenem concentrations in aqueous humor were lower than those in plasma ultrafiltrates at all sampling times, with an average aqueous humor-to-plasma ultrafiltrate area under the concentration-time curve ratio estimated as 8.3%. A pharmacokinetic model with peripheral elimination described the data adequately and was tentatively used to predict concentration-versus-time profiles and pharmacokinetic-pharmacodynamic (PK-PD) target attainment in patients under various dosing regimens. In conclusion, systematically administered doripenem does not seem to be a promising approach for the treatment of intraocular infections, especially since it could not be detected in the vitreous humor. However, this study has provided an opportunity to develop a new PK modeling approach to characterize the intraocular distribution of doripenem administered intravenously to rabbits, with tentative extrapolation to humans.

Lysophospholipids and lysophospholipase D in rabbit aqueous humor following corneal injury

We previously found that lysophosphatidic acid (LPA)-like activity eliciting Cl− currents in Xenopus oocytes is increased in rabbit aqueous humor (AH) following corneal freeze wounds. The purpose of this study was to examine whether actual levels of LPA in AH from wounded eyes are higher than those from control eyes, and to determine the sources and enzymatic pathways of AH LPA in control and wounded conditions. Lysophospholipase D (lysoPLD) activity was measured by the enzymatic determination of choline following incubation of AH samples with exogenous lysophosphatidylcholines (LPCs). The molecular species compositions of LPA and LPC in fresh and incubated AH were determined by liquid chromatography–tandem mass spectrometry. A high, but similar activity of lysoPLD in the samples from both control and freeze-wounded eyes was detected. Its enzymatic properties resemble those of plasma lysoPLD, identified as autotaxin. Levels of LPCs, predominant substrates of lysoPLD in AH, were several times higher in the AH samples from injured eyes than those from the control eyes. Our results suggest that lysoPLD is constitutively released from corneal tissues and/or ciliary body into the AH, with no injury-induced increase in release following freeze-wounding. They also suggest that wound-induced increases in LPA-like biological activity are due to linoleoyl species-rich molecular composition in AH from wounded eyes. A possible mechanism of the altered molecular composition is an increase in the AH concentrations of LPCs, linoleoyl species of which are preferentially converted to corresponding unsaturated LPA by the constitutively active lysoPLD.

Development and validation of a liquid chromatography/electrospray ionization tandem mass spectrometry method for the quantification of latanoprost free acid in rabbit aqueous humor and ciliary body

A rapid, selective and sensitive method for quantification of latanoprost free acid in rabbit aqueous humor (AH) and ciliary body (CB) using reverse phase-high performance liquid chromatography coupled with electrospray ionization (ESI)-mass spectrometry/mass spectrometry has been developed and validated. Quantification in AH and CB was achieved by stable isotope dilution employing tetra-deuterated analog of latanoprost free acid, used as internal standard. Sample preparation was based on protein precipitation with methanol in AH, and on liquid extraction with a mixture of ethyl acetate and isopropanol 60:40 (v/v) in CB. Elution was achieved on an octylsilica (C8) column, using an isocratic elution method. Detection was performed on a triple quadrupole mass spectrometer, using ESI in positive ion selected reaction monitoring mode. Calibration curves were linear in the validated concentration ranges of 10-160 ng/mL in AH and 80-1280 ng/g in CB. The accuracy and precision values, obtained from three different sets of quality control samples, each analyzed in triplicate on three different days, were within the generally accepted criteria for analytical methods (< 15%). The limit of detection was 30.66 pg/mL in AH and 237.75 pg/g in CB. The assay proved to be accurate and precise when applied to the in vivo study of latanoprost free acid in rabbit AH and CB after single administration of an eye drops containing latanoprost.

Pharmacokinetics comparison of amphotericin B using three administrating approach in a rabbit model

Background Intracameral or intracorneal administration of amphotericin B (AMB) can achieve significant therapeutic efficacy in the treatment of recalcitrant fungal keratitis in cases that do not respond to conventional antifungal therapy. However, the ocular pharmacokinetics of the two routes of administration is unclear.Objective The goal of this study was to investigate the level of amphotericin B in cornea and aqueous humor of rabbits after administration of AMB via three different routes. Methods Forty-five healthy domestic rabbits were randomly divided into three groups. 1% amphotericin B of 10 μg was intrastromally or intracamerally injected into 15 rabbits, respectively,in group A and group B. Topical 0. 25% amphotericin B was topically administered to the eyes with corneal epithelial debridement (group C). Experimental animals were sacrificed and the corneas and aqueous humor samples were obtained for the detection of levels of amphotericin B at 30 minutes,6 hours, 1 day,3 and 7 days by high-performance liquid chromatography (HPLC). Results Calibration curves were linear over the range of 0. 10-100. 00 mg/L. The concentration of 0. 10 mg/L was the lowest quantifiable limit. The recovery of amphotericin B ranged from 89. 1% -95.7% from aqueous humor samples and 81.4% -83.6% from the cornea samples. After a single injection,effective drug levels were achieved and maintained for 7 days in cornea in group A, exceeding the minimum inhibitory concentration at which 90% of isolates are inhibited (MIC90) for a wide spectrum of fungi and molds with significant differences in comparison with group B and group C ( P＜0. 05 ). Effective drug levels were achieved in the aqueous humor in group B at 30 minutes after a single injection, but drug levels decreased dramatically within 6 hours. The evident differences were found between group B and group A or group C (P＜ 0.05). A considerable amount of amphotericin B was detected in the cornea and aqueous humor in group C within 1 day.Conclusion Effective high drug levels can be reached in rabbit cornea and aqueous humor after intrastromal and intracameral injection, respectively. Penetration of topical amphotericin B was greatly elevated after epithelial debridement. Key words: Amphotericin B; Chromatography; High pressure liquid; Pharmacokinetics

Aqueous Humor Pharmacokinetics of Dorzolamide Loaded PLGAChitosan Nanoparticles by Ultra Performance Liquid Chromatography

Ocular pharmacokinetic analysis of drugs is useful for evaluation of efficacy of novel ophthalmic formulations. A simple and sensitive ultra performance liquid chromatographic (UPLC) method with ultraviolet (UV) detection was developed and validated for the quantification of dorzolamide (DZA) in rabbit aqueous humor. The aqueous humor samples were deproteinized with acetonitrile prior to UPLC analysis. A chromatographic separation was achieved on a Waters Acquity BEH C18 (100 mm mm, 1.7 μm) column with a mobile phase consisting of 0.1% triethylamine (TEA) in water and acetonitrile (50:50, v/v) at a flow rate of 0.25 ml min-1. Detection of DZA was carried out at 254 nm. The method developed was linear (r2 = 0.9996) over the concentration range of 5 - 800 ng mL-1. Accuracy ranged from 98.23 to 99.53% and precision was between 0.12% and 0.36%. Limit of detection and quantitation of DZA in aqueous humor were 5 and 10 ng mL-1, respectively. The method was applied to determine pharmacokinetics of DZA in aqueous humor, following topical instillation of DZA-solution (DZA-SOL) and DZA nanoparticles (DZA-NPs) to rabbit eyes. The pharmacokinetic results showed that DZA-NPs enhances the ocular bioavailability of DZA by 6-fold compared to DZA-SOL. Keywords: Aqueous humor, Dorzolamide, Nanoparticles, Ocular pharmacokinetics, Ultra performance liquid chromatography, Ultraviolet (UV) detection, Ocular bioavailability, Limit of detection, Limit of quantification, Assay validation

Determination of puerarin in rabbit aqueous humor by liquid chromatography tandem mass spectrometry using microdialysis sampling after topical administration of puerarin PAMAM dendrimer complex

To study pharmacokinetic properties of puerarin poly(amido amine) (PAMAM) dendrimer complex, a sensitive liquid chromatography tandem mass spectrometry method (LC-MS/MS) was developed and validated to determine puerarin in rabbit aqueous humor using microdialysis sampling. Astilbin was used as the internal standard. The linear range for puerarin was from 2 to 1000 ng/mL ( r = 0.9986) based on 20 μL of aqueous humor. The coefficients of variations for intra-day and inter-day precisions were less than 10.0%, and the relative error of accuracy was within ±6.3%. The mean extraction recovery of puerarin varied from 80.4% to 85.5%. Microdialysis provides a complete concentration versus time profile. A significant difference was observed in main pharmacokinetic parameters of C max, AUC and t 1/2 between puerarin solution and puerarin PAMAM dendrimer complex. Complex formation resulted in an obvious increase in bioavailability of puerarin after topical administration to rabbit according to the above LC-MS/MS assay method.

Metabolomics of Rabbit Aqueous Humor after Administration of Glucocorticosteroid

Purpose: The purpose of this study was to evaluate metabolic changes in the aqueous humor of rabbits after the administration of glucocorticosteroids.Methods: Twenty-four New Zealand white rabbits were divided into the following three groups: control (intravitreal injection of sterile saline solution), TA (intravitreal injection of triamcinolone acetonide), and DEX (subconjunctival injection of dexamethasone). Intraocular pressure (IOP), flash electroretinogram (FERG), and flash visual evoked potential (FVEP) were tested. The aqueous humor was acquired pre-injection and at 1 and 30 days post-injection. The samples were detected by 1H NMR, and then the spectrum was analyzed by principal component analysis (PCA).Results: A significant elevation of IOP was observed in the TA group (1, 7, 14, 21, and 30 days post-injection) and the DEX group (14, 21, and 30 days post-injection) compared to the controls (P < 0.01). In FERG, the amplitudes were decreased, and the implicit time was prolonged in the TA group at 7, 14, and 30 days post-injection compared to the controls (P < 0.05). In FVEP, the amplitudes were decreased, and the implicit time was prolonged in the TA group at 30 days post-injection (P < 0.05). The concentrations of 12 metabolites (including lipid, protein, sugar, antioxidant, and acute phase reaction protein) were significantly changed after intravitreal TA, while 11 metabolites were changed after subconjunctival DEX, compared to the controls. PCA showed that alanine and acetate were the highest value contributions, while sugar metabolism was the major metabolic difference between TA and DEX.Conclusions: The main metabolic change in the aqueous humor of rabbits after the administration of glucocorticosteroids is sugar metabolism, and the function of the retina and the optic nerve was impaired after intravitreal TA.

Ocular Pharmacokinetic Study of l-Carnosine and N-Acetyl-l-carnosine in Rabbit by Microdialysis Coupled with UPLC-MS-MS

In order to provide useful information for rational drug design, the ocular pharmacokinetics of l-carnosine (CAR) and its acetylized prodrug N-acetyl-l-carnosine (NAC) were investigated. The in vivo microdialysis sampling coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) was developed for continuously simultaneous monitoring of CAR and NAC in rabbit aqueous humor. The measured in vitro recoveries of the probe were 61.3% for CAR and 65.8% for NAC, while in vivo recoveries decreased to 43.1% for CAR and 43.0% for NAC, respectively. The method was sensitive with LLOQ 20.5 ng mL−1 for CAR and 20.4 ng mL−1 for NAC. The initial data indicated that the value of C max and AUC(0–∞) of NAC were higher than these of CAR (C max 2305 vs. 1,802 ng mL−1), (AUC(0–∞) 1,337 vs. 1,891 ng h mL−1), which indicated that the NAC exhibited better ocular bioavailability and duration. The method was rapid, specific and sensitive for continuously monitoring of aqueous humor and it was successfully applied to pharmacokinetic studies of CAR and NAC.

Aqueous humor penetration of topical bimatoprost 0.01% and bimatoprost 0.03% in rabbits: response to authors

We read with great interest the recent article by Ogundele and Jasek,1 in which the authors concluded that bimatoprost ophthalmic solution 0.01% (Lumigan®; Allergan, Inc, Irvine, CA) produced lower bimatoprost acid concentration than bimatoprost ophthalmic solution 0.03% (Lumigan; Allergan, Inc) in the aqueous humor of rabbits. This conclusion was made based on two treatment time points (30 and 90 minutes) with a small sample size (n = 4) at each time point and with large variability. In comparing pharmacokinetic profiles of two formulations, it is a general practice that the study design would support assessment of the speed of onset (ie, time to maximum exposure [Tmax]), and the extent of absorption (ie, maximum concentration and area under the concentration-time curve). Therefore, a more complete temporal profile would be necessary. An erroneous conclusion could be drawn based on two seemingly arbitrary time points. For example, it is unclear whether these two time points reside in the ascending or descending portion of the temporal profile or if they reside one in each portion and the Tmax is missing. Based on two data points, Ogundele and Jasek1 hypothesized that bimatoprost 0.01% might have compromised the intraocular pressure (IOP)-lowering effect. Not only is there no reported correlation of animal pharmacokinetic and/or metabolism results to clinical efficacy, this hypothesis directly contradicts existing evidence. A multicenter, 12-month, randomized, controlled trial demonstrated equivalent efficacy of bimatoprost 0.01% and bimatoprost 0.03% based on predetermined IOP criteria (limits of the 95% confidence interval of the between-group difference in mean IOP within ±1.5 mmHg at all time points and within ±1 mmHg at most time points).2 In addition, Ogundele and Jasek1 speculated that bimatoprost 0.01% might have increased the risk of ocular toxicity, based on prior publications of in vitro and animal studies that may not be relevant to patients, case series, or open-label clinical studies. This evidence is considered lower level compared with randomized, controlled trials. Katz et al2 conducted a randomized, controlled trial and reported bimatoprost 0.01% had (a) significantly lower overall incidence of treatment-related adverse events (P < 0.03); (b) significantly reduced conjunctival hyperemia (P < 0.044), skin pigmentation (P < 0.02), and eye pruritus (P < 0.035); and (c) significantly lower discontinuation rates than bimatoprost 0.03% (P = 0.043). Based on clinical trial evidence, the claims of ocular toxicity are unfounded for at least 12 months of administration as monotherapy.2 In addition, a recent clinical study comparing ocular surface tolerability of topical prostaglandin analogs demonstrated no statistical differences following 3 months of treatment in either corneal staining or conjunctival hyperemia between latanoprost, which has 200 ppm benzalkonium chloride (BAK), similar to bimatoprost 0.01%, and travoprost ophthalmic solution 0.004% (Travatan Z®; Alcon Laboratories, Inc, Fort Worth, TX) (BAK free, preserved with sofZia® [Alcon Laboratories, Inc]).3 Bimatoprost 0.01%, which offers improved ocular tolerability while maintaining the established efficacy of bimatoprost 0.03%, recently gained approval by the European Medicines Evaluation Agency (2009) and the US Food and Drug Administration (2010) as first-line therapy in patients with open-angle glaucoma or ocular hypertension. Any ophthalmologist or patient concerns raised by the incomplete and potentially misleading conclusion published by Ogundele and Jasek1 can be addressed with a carefully constructed pharmacokinetic study with a complete time-concentration profile. We are currently preparing a manuscript that reports the findings of such a study design.

Analysis of protein composition of rabbit aqueous humor following two different cataract surgery incision procedures using 2-DE and LC-MS/MS.

BackgroundThe aqueous humor (AH), a liquid of the anterior and posterior chamber of the eye, comprises many proteins with various roles and important biological functions. Many of these proteins have not been identified yet and their functions in AH are still unknown. Recently, our laboratory published the protein database of AH obtained from healthy rabbits which expanded known protein identifications by 65%. Our present study extends our previous work and analyses AH following two types of cataract surgery incision procedures (clear corneal and limbal incisions) by using two dimensional gel electrophoresis (2-DE) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Although both incision protocols are commonly used during cataract surgeries, the difference in protein composition and their release into AH following each surgery has never been systematically compared and remains unclear. The first step, which is the focus of this work, is to assess the scale of the protein change, at which time does maximum release occurs and when possible, to identify protein changes.ResultsSamples of AH obtained prior to surgery and at different time points (0.5, 2, 12, 24 and 48 hours) following surgery (n = 3/protocol) underwent protein concentration determination, 2-DE and LC-MS/MS. There was a large (9.7 to 31.2 mg/mL) and rapid (~0.5 hour) influx of proteins into AH following either incision with a return to baseline quantities after 12 hours and 24 hours for clear corneal and limbal incision, respectively. We identified 80 non-redundant proteins, and compared to our previous study on healthy AH, 67.5% of proteins were found to be surgery-specific. In addition, 51% of those proteins have been found either in clear corneal (20%) or limbal incision (31%) samples.ConclusionsOur results imply that a mechanism of protein release into AH after surgery is a global response to the surgery rather than increase in amount of protective proteins found in healthy AH and a mechanism of protein release for each type of incision procedure could be different. Although the total protein concentration was increased (at 0.5 and 2 hour time points and between types of surgery) many of 2-DE protein spots were similar based on 2-DE and MS analyses, and only a small number of protein spots changed with either the time points or surgical conditions (0.4 -1.9%). This suggests that the high protein content is due to an increase in the concentration of the same proteins with only a few unique proteins being altered per time point and with the different surgery type. This is the first report on the comparison of AH protein composition following two different cataract surgery procedures and it establishes the basis for better understanding of protein release into AH during events such as cataract surgery or other possible intervention to the eyes.

Preparation and evaluation of solid lipid nanoparticles of baicalin for ocular drug delivery system in vitro and in vivo

Purpose: To prepare and evaluate the solid lipid nanoparticles of baicalin (BA-SLN) for ocular drug delivery system. Methods: The BA-SLN was prepared by emulsification/ultrasonication method. The appearance of BA-SLN was examined by the negative stain method. The mean diameter and zeta potential of BA-SLN were determined using a Zetasizer. The entrapment efficiency of BA-SLN was determined by Sephadex-G50 column. And the solid-state characterization of BA-SLN was analyzed by DSC and X-ray. The release of drug from BA-SLN was evaluated using dialysis bag diffusion technique. The effects of SLN on corneal permeability of baicalin were investigated in vitro, using isolated rabbit corneas. The in vivo ocular irritation of BA-SLN was tested by pathological section observation using rabbits. The pharmacokinetics was evaluated by microdialysis in the rabbit aqueous humors. Results: The results showed that the BA-SLN had an average diameter of 91.42 ± 1.02 nm with a zeta potential of –33.5 ± –1.28 mV and the entrapment efficiency of 62.45 ± 1.67%. In vitro release studies indicated that the BA-SLN retained the drug entity better than the baicalin ophthalmic solutions (BA-SOL). In the pharmacokinetics studies, the AUC value of BA-SLN was 4.0-fold versus the BA-SOL (P < 0.01), and the Cmax value of BA-SLN versus the BA-SOL was 5.3-fold (P < 0.01). Conclusion: SLN can be used as a carrier to enhance ocular bioavailability of baicalin.

Surface Tension of Aqueous Humor

To measure and compare the surface tension of aqueous humor in patients with and without glaucoma. The surface tension of aqueous humor was measured using a commercially available instrument and software that were validated by using a known fluid (deionized water and methanol). Analysis of aqueous and vitreous samples obtained from 20 rabbit eyes showed that the system could be used successfully for small amounts of ocular fluid. The effect of glaucoma drugs on the surface tension of aqueous humor was then studied in a rabbit model. Comparison of aqueous humor from 66 patients with glaucoma and 53 patients with cataracts but no glaucoma was carried out. The surface tension of rabbit aqueous humor was 65.9 ± 1.2; vitreous, 60.6 ± 2.6; and balanced salt solution, 70.7 ± 0.9. Timolol and latanoprost did not alter the surface tension of the aqueous humor in the rabbit model. The average surface tension of human aqueous humor was 63.33 ± 4.0 (glaucomatous eyes) and 66.19 ± 2.64 (nonglaucomatous eyes with cataracts) (P=0.0001). A technique of measuring the surface tension from small quantities of aqueous humor is validated. Surface tension of the aqueous humor in glaucoma patients was less than that of cataract patients.

Cyclodextrin solubilization of carbonic anhydrase inhibitor drugs: Formulation of dorzolamide eye drop microparticle suspension

Topically applied carbonic anhydrase inhibitors (CAIs) are commonly used to treat glaucoma. However, their short duration of action requiring multiple daily dosing can hamper patient compliance. The aim of this study was to develop novel aqueous CAI eye drop formulation containing self-assembled drug/cyclodextrin (D/CD) microparticles that enhance and prolong drug delivery to the eye. Phase-solubility of each drug tested (i.e. methazolamide, brinzolamide and dorzolamide HCl) was determined in either pure water or an aqueous eye drop medium. The pH was adjusted to maximize the fraction of unionized drug. Dorzolamide had the highest affinity for γ-cyclodextrin (γCD) and, thus, was selected for further investigation. Hydroxypropyl methylcellulose (HPMC) was the most effective polymer tested for stabilization of the dorzolamide/γCD complexes and gave the highest mucoadhesion at 0.5% w/v concentration. Thus, the dorzolamide eye drop vehicle containing γCD (18% w/v) and HPMC (0.5% w/v) was developed. The physicochemical properties of this formulation complied with the specifications of the eye drop suspension monograph of the European Pharmacopoeia. The in vivo testing of the formulation showed that the drug was delivered to the aqueous humor in rabbits for at least 24 h with the maximum drug concentration at 4 h. Furthermore, this formulation delivered the drug to the posterior segment of the eye after topical administration. These results indicate that this CAI eye drop formulation has the potential of being developed into a once-a-day product.

Sustained Anti-Staphylococcal Effect of Lysostaphin in the Rabbit Aqueous Humor

Purpose: Staphylococcus aureus is an important cause of ocular infections including endophthalmitis. The purpose of this study was to determine the ability of a relatively large molecule, such as lysostaphin, to remain in the rabbit aqueous humor for extended periods while retaining its bactericidal activity.Methods: Lysostaphin, gatifloxacin, or Tris-buffered saline (TBS) was injected into the rabbit anterior chamber. Aqueous humor was sampled at 0.5, 1, 2, 3, and 4 days after injection, and then assayed for bactericidal activity against S. aureus. The anterior chamber of treated eyes was also challenged 2 days after treatment by an infection of S. aureus. The surviving bacteria were quantified to determine bactericidal effectiveness in the anterior chamber. The aqueous humor of lysostaphin injected eyes was assayed by western blot for the presence of the molecule 2 days post-injection.Results: The bactericidal activity of lysostaphin was confirmed by lysis of S. aureus and sensitivity to zinc. Eyes injected with lysostaphin showed no adverse reactions. Aqueous humor of gatifloxacin injected eyes demonstrated no greater effectiveness than that of TBS injected eyes in vitro at any time point assayed, whereas lysostaphin injected eyes retained potent bactericidal activity for at least 3 days. In an in vivo challenge, the anterior chamber of lysostaphin injected eyes retained significant bactericidal activity for at least 2 days after treatment, whereas gatifloxacin injected eyes demonstrated no significant difference from those injected with TBS. Western blot analysis demonstrated the presence of lysostaphin in the aqueous humor 2 days post-injection.Conclusions: Lysostaphin demonstrated the ability to remain in the aqueous humor for days while maintaining its bactericidal activity, an indication that a high molecular weight antimicrobial can provide prolonged prophylactic protection of the anterior chamber.

Simultaneous quantification of carteolol and dorzolamide in rabbit aqueous humor and ciliary body by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

A rapid, sensitive and selective method for the simultaneous quantification of carteolol and dorzolamide in rabbit aqueous humor (AH) and ciliary body (CB) has been developed and validated using reversed phase-high performance liquid chromatography (RP-HPLC) with isocratic elution coupled with atmospheric pressure chemical ionization mass spectrometry/mass spectrometry (APCI-MS/MS). The analytes and nadolol (used as internal standard, IS) were purified from AH by protein precipitation. The sample preparation from CB was based on a two steps extraction procedure at different pH, utilizing a liquid–liquid extraction with a mixture of ethyl acetate, toluene and isopropanol 50:40:10 (v/v) at pH 8, followed by a second extraction with ethyl acetate at pH 11. The combined organic extracts were then back extracted into 0.1% aqueous trifluoroacetic acid (TFA). The accuracy and precision values, calculated from three different sets of quality control samples analyzed in sestuplicate on three different days, were within the generally accepted criteria for analytical methods (<15%). The assay proved to be accurate and precise when applied to the in vivo study of carteolol and dorzolamide in rabbit AH and CB after single administration of an eye drops containing both drugs.

Aqueous humor penetration of topical bimatoprost 0.01% and bimatoprost 0.03% in rabbits

PurposeThe purpose of this study was to compare the aqueous humor concentrations of bimatoprost acid after topical instillation in rabbits of bimatoprost ophthalmic solution 0.01% and bimatoprost ophthalmic solution 0.03%, two commercially available intraocular pressure-lowering medications.MethodsMale Dutch Belted rabbits were divided into two treatment groups (four rabbits/eight eyes per group): bimatoprost 0.01% and bimatoprost 0.03%. Thirty microliters (μL) of study medication was to pically instilled into both eyes of each animal. Thirty minutes and 90 minutes after instillation, aqueous humor samples were collected. These samples were analyzed by reverse-phase high-performance liquid chromatography for bimatoprost acid concentration.ResultsFollowing a single topical ocular instillation, the bimatoprost 0.01% formulation had a lower mean aqueous humor concentration of bimatoprost acid than the bimatoprost 0.03% formulation at both 30 minutes (11.5 ± 2.1 ng/mL versus 37.8 ± 28.8 ng/mL; P = 0.17) and 90 minutes (20.8 ± 5.7 ng/mL versus 45.8 ± 14.3 ng/mL; P = 0.03) after topical instillation.ConclusionsTopical ocular instillation of bimatoprost 0.01% produced significantly lower bimatoprost acid concentration in the aqueous humor of rabbits than bimatoprost 0.03%, despite the 4-fold increase of benzalkonium chloride contained in bimatoprost 0.01%.