Absorption-Enhancing Mechanisms of Capryol 90, a Novel Absorption Enhancer, for Improving the Intestinal Absorption of Poorly Absorbed Drugs: Contributions to Trans- or Para-Cellular Pathways.

PURPOSE: We have previously reported that Capryol 90 improves the intestinal absorption of insulin, a peptide drug, without causing serious damage to the intestinal epithelium. However, the effects of Capryol 90 and its related formulations on the intestinal absorption of other drugs, and their absorption-enhancing mechanisms are still unclear. The aim of this study is to evaluate the effects of Capryol 90 and its related formulations on the intestinal absorption of drugs and elucidate their absorption-enhancing mechanisms. METHODS: The intestinal absorption of 5(6)-carboxyfluorescein, fluorescein isothiocyanate-dextrans, and alendronate was evaluated using an in situ closed loop method. Brush border membrane vesicles (BBMVs) were labeled with fluorescent probes, and the fluidity of membrane was evaluated by a fluorescence depolarization method. The expression levels of tight junction (TJ) proteins were measured using a Western blot method and immunofluorescence staining. RESULTS: Among the tested excipients, Capryol 90 significantly improved the small and large intestinal absorption of drugs. In mechanistic studies, Capryol 90 increased the membrane fluidity of lipid bilayers in BBMVs. Additionally, Capryol 90 decreased the expression levels of TJ-associated proteins, namely claudin-4, occludin, and ZO-1. CONCLUSIONS: Capryol 90 is an effective absorption enhancer for improving the intestinal absorption of poorly absorbed drugs via both transcellular and paracellular pathways.

Photoreceptor rescue of pigment epithelium-derived factor-impregnated nanoparticles in Royal College of Surgeons rats.

PURPOSE: To investigate the protective effect of intravitreal injection of pigment epithelium-derived factor-impregnated nanoparticles (PEDF-NPs) against photoreceptor degeneration in Royal College of Surgeons (RCS) rats. METHODS: Three-week-old RCS rats received an intravitreal injection of PBS, blank NPs, PEDF (2.5 µg), or PEDF-NPs (2.5 µg). Eyes were assessed with morphological, immunohistochemical, and physiologic analysis over the following 8 weeks. Cell death was examined using the terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) assay. RESULTS: In RCS rats, the a- and b-wave amplitudes on electroretinograms in eyes treated with PEDF-NPs were greater than those in retinas receiving other treatment. Immunocytochemistry showed consistently greater opsin preservation in eyes treated with PEDF-NPs. A significantly higher number of photoreceptors and significantly fewer TUNEL-positive cells were present after treatment with PEDF-NPs, compared to PEDF-treated eyes. CONCLUSIONS: The results suggest that intravitreally injected PEDF-NPs delayed photoreceptor degeneration by inhibiting apoptosis in the RCS rat retina due to targeting and sustained release of PEDF.

Progression of age-related macular degeneration in eyes with abnormal fundus autofluorescence in a Japanese population: JFAM study report 3.

PURPOSE: To evaluate the progression of early age-related macular degeneration to neovascular age-related macular degeneration (nAMD), and identify the abnormal fundus autofluorescence (FAF) patterns and markers of choroidal neovascularization (CNV) in fellow eyes of patients with unilateral nAMD. METHODS: Sixty-six patients with unilateral nAMD who developed abnormal FAF in the fellow eyes were enrolled in this multicenter, prospective, observational study, and followed-up for 5 years. FAF images on Heidelberg Retina Angiogram Digital Angiography System (HRA) or HRA2 were classified into eight patterns based on the International Fundus Autofluorescence Classification Group system. The patients in which the fellow eyes progressed to advanced nAMD, including those who did not develop nAMD, were assessed based on the following factors: baseline FAF patterns, age, sex, visual acuity, drusen, retinal pigmentation, baseline retinal sensitivity, family history, smoking, supplement intake, hypertension, body mass index, and hematological parameters. RESULTS: Of the 66 patients, 20 dropped out of the study. Of the remaining 46 patients, 14 (30.42%, male: 9, female: 5) progressed to nAMD during the 5-year follow-up. The most common (50% eyes) FAF pattern in the fellow eyes was the patchy pattern. According to the univariate analysis, CNV development was significantly associated with age, supplement intake, and low-density lipoprotein levels (p<0.05). Multivariable analysis revealed that patients who showed non-compliance with the supplement intake were more likely to develop nAMD (p<0.05). No significant association was found between the patchy pattern and CNV development (p = 0.86). CONCLUSION: The fellow eyes (with abnormal FAF) of patients with unilateral nAMD may progress from early to advanced nAMD. However, no FAF pattern was found that predicted progression in nAMD.

Five-year follow-up of fundus autofluorescence and retinal sensitivity in the fellow eye in exudative age-related macular degeneration in Japan.

PURPOSE: To assess the 5-year change in abnormal fundus autofluorescence (FAF) patterns and retinal sensitivity in the fellow eye of Japanese patients with unilateral exudative age-related macular degeneration (AMD). METHODS: Patients with unilateral exudative AMD who developed abnormal FAF in the fellow eyes were enrolled. FAF imaging and microperimetry were performed at baseline and follow-ups. FAF findings were classified into 8 patterns based on the International Fundus Autofluorescence Classification Group to assess retinal sensitivity. Forty-five points covering the central 12 degrees on microperimetry were superimposed onto the FAF images. Each point was classified depending on the distance from the abnormal FAF. "Close" was defined as the portion within 1 degree from the border of any abnormal FAF, and "Distant" was defined as the portion over 1 degree from the border of abnormal FAF. To investigate the association between the retinal sensitivity and distance from the abnormal FAF, hierarchical linear mixed-effect models were used with the distance, time and time squared from baseline (months), and angle (degrees) as fixed effects. Differences among patients, eyes, and test point locations were considered successively nested random effects. RESULTS: We studied 66 fellow eyes with abnormal FAF. Twenty-seven eyes were followed-up during the 5 years. In the 13 of 27 eyes (48%), the abnormal FAF patterns had changed during the 5 years. We found retinal sensitivity was associated significantly with the distance from the abnormal FAF ("Distant": p<0.001, time2 from baseline: p<0.001, angle: p<0.001). The mean retinal sensitivity of the "Close" tended to deteriorate after the third year and eventually showed the similar sensitivity as the portion within the abnormal FAF. CONCLUSION: FAF patterns can change about half during the 5 years and the retinal sensitivity near abnormal FAF tends to deteriorate after the third year.

Fundus autofluorescence and fate of glycoxidized particles injected into subretinal space in rabbit age-related macular degeneration model.

PURPOSE: Abnormal fundus autofluorescence (FAF) is associated with the incidence or progression of dry and wet age-related macular degeneration (AMD). We previously developed a rabbit AMD model with drusen and type-1 choroidal neovascularization (CNV) that mimics the accumulation of lipofuscin using artificial glycoxidized particles. The objective of the current study was to investigate in vitro effects of glycoxidized particles on retinal pigment epithelial (RPE) cells, and the FAF and fate of injected particles in this model. METHODS: Glycoxidized particles were prepared by a 4-day incubation of water-in-oil emulsions of serum albumin and glycolaldehyde to allow glycoxidation and consequent cross-linking. After particles were added in the culture medium of confluent human RPE cells, cell viability, adhesion activity, and proliferation activity were assessed by cell counting. In anesthetized rabbits, 250 microg of glycoxidized particles were injected into the subretinal space to induce experimental AMD. FAF measurement and angiography with sodium fluorescein and indocyanine green were performed periodically using the Heidelberg Retina Angiograph 2 (HRA2). The eyes enucleated, and the lung and the spleen, excised at week 4 or 12, were histologically evaluated by light and fluorescence microscopy. RESULTS: Glycoxidized particles phagocytosed did not impair the cell viability, adhesion, and proliferation of RPE cells, as compared with RPE cells in controls. HRA2 showed different patterns of abnormal FAF in the area with the implanted glycoxidized particles, similar to pathological FAF patterns in aging human eyes with or without AMD. Histologic examination showed accumulated glycoxidized particles and large lipofuscin granules with green autofluorescence in and under the RPE and at the margins of or beneath drusen, possibly associated with abnormal FAF. In addition, some particles were detected in the lung and the spleen. CONCLUSIONS: Glycoxidized particles phagocytosed might stay in RPE cells without any acute biological reaction. Our rabbit model of AMD simulated abnormal FAF patterns observed in aging human eyes with or without AMD. Glycoxidized particles phagocytosed by RPE cells could be deposited on Bruch's membrane in rabbits, possibly excreted in part into choroidal circulation. This model may be useful for understanding various patterns of abnormal FAF histologically, and for elucidating the pathogenesis of AMD.

Systemic expression of Alu RNA in patients with geographic atrophy secondary to age-related macular degeneration.

Geographic atrophy (GA) secondary to age-related macular degeneration (AMD) is characterized by irreversible loss of macular retinal tissue and retinal pigment epithelium (RPE) cells. Several studies have revealed that accumulation of Alu RNA in RPE cell causes RPE cell degeneration in AMD. In the present study, systemic Alu RNA expression levels were determined in 33 subjects with GA and 40 control subjects using a proprietary Alu RNA quantification method. It was observed that the expression level of Alu RNA was not significantly different between GA and Control groups (median = 21.3 in both GA and Control groups, P = 0.251). In addition, the systemic level of Alu RNA was not associated with subject characteristics, such as GA lesion size and SNP profiles of complement factors associated with increased risk of AMD. In conclusion, the usability of systemic Alu RNA expression level as a biomarker of GA secondary to AMD could not be established in this study.

Fundus autofluorescence and retinal sensitivity in fellow eyes of age-related macular degeneration in Japan.

PURPOSE: Abnormal fundus autofluorescence (FAF) potentially precedes onset of late age-related macular degeneration (AMD) in Caucasian patients. Many differences exist between Asian and Caucasian patients regarding AMD types and severity, gender, and genetic backgrounds. We investigated the characteristics of abnormal FAF and retinal sensitivity in the fellow eyes of Japanese patients with unilateral neovascular AMD. METHODS: Sixty-six patients with unilateral neovascular AMD and abnormal FAF in the fellow eye were enrolled in this multicenter, prospective, observational study. The best-corrected visual acuity, fundus photographs, FAF images, and retinal sensitivity on microperimetry were measured periodically for 12 months. The FAF images were classified into eight patterns based on the International Fundus Autofluorescence Classification Group. The points measured by microperimetry were superimposed onto the FAF images and fundus photographs and classified as "within," "close," and "distant," based on the distance from the abnormal FAF and other findings. The relationship between the location of the baseline abnormal FAF and retinal sensitivity was investigated. RESULTS: In Japanese patients, patchy (33.3%) and focally increased (30.3%) patterns predominated in the abnormal FAF. Intermediate-to-large drusen was associated predominantly with hyperfluorescence and hypofluorescence. Neovascular AMD developed within 1 year in six (9.1%) eyes, the mean baseline retinal sensitivity of which was 12.8 ± 4.7 dB, significantly (p<0.002) lower than the other eyes. In 44 of the other 60 eyes, microperimetry was measurable at baseline and month 12 and the mean retinal sensitivity improved significantly from 13.5 ± 4.4 to 13.9 ± 4.8 dB (p<0.001), possibly associated with lifestyle changes (e.g., smoking cessation, antioxidant and zinc supplementation). The mean retinal sensitivities of points within and close to the abnormal FAF were 9.9 and 11.7 dB, respectively, which were significantly lower than the 14.0 dB of the points distant from the abnormal FAF. CONCLUSION: In Japanese patients, patchy and focally increased patterns predominated in the abnormal FAF. The retinal sensitivity was lower close to/within the abnormal FAF. FAF and microperimetry are useful to assess macular function before development of neovascular AMD or geographic atrophy.

Prolonged protective effect of basic fibroblast growth factor-impregnated nanoparticles in royal college of surgeons rats.

PURPOSE: To investigate the protective effect of intravitreal injection of basic fibroblast growth factor-impregnated nanoparticles (bFGF-NPs) against photoreceptor degeneration in Royal College of Surgeons (RCS) rats. METHODS: Three-week-old RCS rats received intravitreal injection of PBS, blank NPs, bFGF (2.5 microg), or bFGF-NPs (2.5 microg). Eyes were assessed by morphologic, immunohistochemical, and physiological analyses for the following 8 weeks. Cell death was examined using the TUNEL assay, and bFGF protein levels in the retina were measured by Western blot analysis. Rhodamine (Rh)-labeled bFGF-NPs were injected intravitreally and visualized by confocal microscopy to determine the localization of the nanoparticles in the retina. RESULTS: Intravitreally injected Rh-labeled bFGF-NPs were found in the outer nuclear layer 6 and 8 weeks after injection. ERG a- and b-wave amplitudes in bFGF-NP-treated retinas were greater than amplitudes in retinas receiving other treatment. Immunocytochemical analysis showed consistently greater opsin preservation in bFGF-NP-treated retinas, and a significantly higher number of photoreceptors and significantly fewer TUNEL-positive cells were present after bFGF-NP treatment than after bFGF treatment. Western blot analysis showed a significant increase in the bFGF level in bFGF-NP-treated retinas. CONCLUSIONS: The results suggest that intravitreally injected bFGF-NPs prevent photoreceptor degeneration by inhibiting apoptosis in the RCS rat retina because of targeting and sustained release of bFGF. This novel drug delivery system for bFGF may serve as a potential short-term treatment for photoreceptor degeneration in humans.

Effect of isolated AMP deaminase deficiency on skeletal muscle function.

Mutation of the AMP deaminase 1 (AMPD1) gene, the predominate AMPD gene expressed in skeletal muscle, is one of the most common inherited defects in the Caucasian population; 2-3% of individuals in this ethnic group are homozygous for defects in the AMPD1 gene. Several studies of human subjects have reported variable results with some studies suggesting this gene defect may cause symptoms of a metabolic myopathy and/or easy fatigability while others indicate individuals with this inherited defect are completely asymptomatic. Because of confounding problems in assessing muscle symptoms and performance in human subjects with different genetic backgrounds and different environmental experiences such as prior exercise conditioning and diet, a strain of inbred mice with selective disruption of the AMPD1 was developed to study the consequences of muscle AMPD deficiency in isolation. Studies reported here demonstrate that these animals are a good metabolic phenocopy of human AMPD1 deficiency but they exhibit no abnormalities in muscle performance in three different exercise protocols.

Validation of Na,K-ATPase pump function of corneal endothelial cells for corneal regenerative medicine.

Tissue-engineering approaches to cultivate corneal endothelial cells (CECs) or induce CECs from stem cells are under investigation for the treatment of endothelial dysfunction. Before clinical application, a validation method to determine the quality of these cells is required. In this study, we quantified the endothelial pump function required for maintaining the corneal thickness using rabbit CECs (RCECs) and a human CEC line (B4G12). The potential difference of RCECs cultured on a permeable polyester membrane (Snapwell), B4G12 cells on Snapwell, or B4G12 cells on a collagen membrane (CM6) was measured by an Ussing chamber system, and the effect of different concentrations of ouabain (Na,K-ATPase specific inhibitor) was obtained. A mathematical equation derived from the concentration curve revealed that 2 mM ouabain decreases pump function of RCECs to 1.0 mV, and 0.6 mM ouabain decreases pump function of B4G12 on CM6 to 1.0 mV. Ouabain injection into the anterior chamber of rabbit eyes at a concentration of <2 mM maintained the corneal thickness, while those over 3 mM significantly increased the corneal thickness. B4G12 cell sheets transplanted into rabbit eyes treated with 0.6 mM ouabain maintained the corneal thickness, while 3.5 mM ouabain significantly increased the corneal thickness. Taken together, pump function >1.0 mV is required to maintain the corneal thickness. These results can be used for standardization of CEC pump function and validation of tissue-engineered CEC sheets for clinical use.

Pharmacokinetics of bevacizumab after topical, subconjunctival, and intravitreal administration in rabbits.

PURPOSE: To investigate the pharmacokinetics of bevacizumab in rabbits for three different routes of administrations: intravitreal injection, subconjunctival injection, and eye drops. METHODS: Pigmented rabbits received bevacizumab in one eye by topical eye drops (1.25 mg/0.05 mL six times daily for the first 7 days), single subconjunctival injection (1.25 mg/0.05 mL), or single intravitreal injection (1.25 mg/0.05 mL). Bevacizumab concentrations in plasma and ocular tissues in the treated and fellow eyes were determined by sandwich enzyme-linked immunosorbent assay at 1, 2, 4, and 12 weeks after administration. RESULTS: After intravitreal injection in the treated eye, the mean maximum concentrations (C(max)) of bevacizumab in the iris/ciliary body and retina/choroid were 109,192.6, and 93,990.0 ng/g, respectively, whereas after subconjunctival injection, the C(max) was 1418.7 and 295.8 ng/g, respectively. In the fellow eyes, when the drug was administered by intravitreal injection, the C(max) was 753.6 ng/g in the iris/ciliary body and 224.2 ng/g in the retina/choroid and by subconjunctival injection was 1192.9 and 187.0 ng/g, respectively. With eye drops, only a small level of bevacizumab was detected in the iris/ciliary body and retina/choroid. Systemic exposure to bevacizumab was at the same level when administered by intravitreal or subconjunctival injection. CONCLUSIONS: Intravitreal injection of bevacizumab was the most effective route of administration for intraocular tissue. Also, bevacizumab injected subconjunctivally was transported into the intraocular tissues of the treated eyes at an effective level. Both intravitreal and subconjunctival injections of bevacizumab resulted in high plasma concentrations. Bevacizumab was distributed into the intraocular tissues in fellow eyes via the systemic circulation. This treatment may be effective for blocking vascular endothelial growth factor activity.

EFEITOS DA SUPLEMENTAÇÃO PROTÉICA NO CONTROLE DA VERMINOSE E NAS CARACTERÍSTICAS DE CARCAÇA DE OVINOS SANTA INÊS

O objetivo deste trabalho foi o de avaliar os efeitos da suplementação protéica na infecção por endoparasitas e nas características de carcaça de ovinos Santa Inês (n=24),submetidos a pasto, com dois níveis de suplementação protéica: alta proteína (AP=19% PB) e baixa proteína (BP=11% PB), os quais foram subdivididos (n=6) em vermifugados (v) e não-vermifugados (n). Mensalmente realizavam-se a colheita de fezes, a pesagem dos animais e a análise do pasto. O abate foi realizado após oito meses e meio, quando se tomaram as medidas da carcaça com análise da 12a costela e contagem dos endoparasitas. O grupo APn apresentou menor número de ovos por grama de fezes em relação ao BPn. Na contagem total de endoparasitas, os tratamentos com AP apresentaram médias inferiores em relação aos dos tratamentos com BP. Os ovinos de APv apresentaram índices superiores para peso vivo, peso ao abate,peso da carcaça quente, gordura de cobertura, comprimento externo da carcaça, área do músculo Longissimus, proporção de gordura e extrato etéreo em relação aos demais tratamentos. Os resultados dos cortes comerciais do grupo APv foram superiores aos observados no grupo BPn (P

Efeitos da suplementação protéica no controle da verminose e nas características de carcaça de ovinos santa inês

O objetivo deste trabalho foi o de avaliar os efeitos da suplementação protéica na infecção por endoparasitas e nas características de carcaça de ovinos Santa Inês (n=24),submetidos a pasto, com dois níveis de suplementação protéica: alta proteína (AP=19% PB) e baixa proteína (BP=11% PB), os quais foram subdivididos (n=6) em vermifugados (v) e não-vermifugados (n). Mensalmente realizavam-se a colheita de fezes, a pesagem dos animais e a análise do pasto. O abate foi realizado após oito meses e meio, quando se tomaram as medidas da carcaça com análise da 12a costela e contagem dos endoparasitas. O grupo APn apresentou menor número de ovos por grama de fezes em relação ao BPn. Na contagem total de endoparasitas, os tratamentos com AP apresentaram médias inferiores em relação aos dos tratamentos com BP. Os ovinos de APv apresentaram índices superiores para peso vivo, peso ao abate,peso da carcaça quente, gordura de cobertura, comprimento externo da carcaça, área do músculo Longissimus, proporção de gordura e extrato etéreo em relação aos demais tratamentos. Os resultados dos cortes comerciais do grupo APv foram superiores aos observados no grupo BPn (P