Topical administration of nepafenac inhibits diabetes-induced retinal microvascular disease and underlying abnormalities of retinal metabolism and physiology.
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Pharmacologic treatment of diabetic retinopathy via eyedrops could have advantages but has not been successful to date. We explored the effect of topical Nepafenac, an anti-inflammatory drug known to reach the retina when administered via eyedrops, on the development of early stages of diabetic retinopathy and on metabolic and physiologic abnormalities that contribute to the retinal disease. Streptozotocin-induced diabetic rats were assigned to three groups (0.3% Nepafenac eyedrops, vehicle eyedrops, and untreated control) for comparison to age-matched nondiabetic control animals. Eyedrops were administered in both eyes four times per day for 2 and 9 months. At 2 months of diabetes, insulin-deficient diabetic control rats exhibited significant increases in retinal prostaglandin E(2), superoxide, vascular endothelial growth factor (VEGF), nitric oxide (NO), cyclooxygenase-2, and leukostasis within retinal microvessels. All of these abnormalities except NO and VEGF were significantly inhibited by Nepafenac. At 9 months of diabetes, a significant increase in the number of transferase-mediated dUTP nick-end labeling-positive capillary cells, acellular capillaries, and pericyte ghosts were measured in control diabetic rats versus nondiabetic controls, and topical Nepafenac significantly inhibited all of these abnormalities (all P < 0.05). Diabetes-induced activation of caspase-3 and -6 in retina was partially inhibited by Nepafenac (all P < 0.05). Oscillatory potential latency was the only abnormality of retinal function reproducibly detected in these diabetic animals, and Nepafenac significantly inhibited this defect (P < 0.05). Nepafenac did not have a significant effect on diabetes-induced loss of cells in the ganglion cell layer or in corneal protease activity. Topical ocular administration of Nepafenac achieved sufficient drug delivery to the retina and diabetes-induced alterations in retinal vascular metabolism, function, and morphology were inhibited. In contrast, little or no effect was observed on diabetes-induced alterations in retinal ganglion cell survival. Local inhibition of inflammatory pathways in the eye offers a novel therapeutic approach toward inhibiting the development of lesions of diabetic retinopathy. (+info)
The use of a cyclooxygenase-2 inhibitor (Nepafenac) in an ocular and metastatic animal model of uveal melanoma.
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The expression of cyclooxygenase-2 (COX-2) has been reported as an indicator of poor prognosis in a wide variety of human tumors, including colon, breast and uveal melanoma (UM). COX-2 inhibitors have shown promise in controlling the malignancy of several types of tumors. Previous studies have demonstrated the efficacy of a COX-2 inhibitor on the proliferation rates of human UM cells. The goal of this experiment was to investigate the efficiency of Nepafenac, a topically administered COX-2 inhibitor, in a rabbit model of UM. The animals were divided into two groups of 14 animals for the duration of the 12-week experiment. One animal per group was killed each week to evaluate disease progression and for histopathological studies. The experimental group received drops containing 0.3% Nepafenac solution. Intraocular tumor growth was evaluated weekly by fundoscopic examination and each animal was weighed prior to examination. Blood samples were taken weekly from all rabbits to detect circulating malignant cells (CMCs) throughout the experiment. After the second week of inoculation, the experimental group weighed significantly more than the control group. The control group developed more intraocular tumors and presented with metastases and higher detectable levels of CMCs before the treated group. These results indicate that the topical administration of a COX-2 inhibitor delayed the progression of this malignancy in our animal model. A clinical trail using an anti-COX-2 inhibitor for patients with UM should be considered. (+info)
Application of (methyl-11C)-methionine in the multicellular spheroid system.
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Human glioma (U-343 MGa) and human colon carcinoma (HT-29) cell lines were cultured as multicellular spheroids, and the accumulations of the L- and D-enantiomers of 11C-methionine were investigated. The accumulation of radioactivity in the spheroids was expressed as relative counts, by dividing the radioactivity measured in the spheroid with the radioactivity of the same volume of the incubation medium. The experiments were verified using 14C-labeled L- and D-methionine. The influence of spheroid volume, specific activity, incubation time, washing time, and the environmental temperatures were investigated. The spheroid model was used to determine the effect of the lipoxygenase inhibitors BW A4C and AA-861, the ether-phospholipid type PAF-antagonist CV-6209 and the protein synthesis inhibitor cycloheximide on methionine uptake. The results showed that 11C-L-methionine can be applied in the study of drug effects on multicellular tumor cell aggregates. (+info)
A novel multivalent ligand that bridges the allosteric and orthosteric binding sites of the M2 muscarinic receptor.
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THRX-160209 is a potent antagonist at the M(2) muscarinic acetylcholine (ACh) receptor subtype that was designed using a multivalent strategy, simultaneously targeting the orthosteric site and a nearby site known to bind allosteric ligands. In this report, we describe three characteristics of THRX-160209 binding that are consistent with a multivalent interaction: 1) an apparent affinity of the multivalent ligand for the M2 receptor subtype (apparent pK(I) = 9.51 +/- 0.22) that was several orders of magnitude greater than its two monovalent components (apparent pK(I) values < 6.0), 2) specificity of THRX-160209 for the M2 receptor subtype compared with the closely related M4 (apparent pK(I) = 8.78 +/- 0.24) and M1, M3, and M5 receptors (apparent pK(I) values 10-fold) of the dissociation rate of tritium-labeled THRX-160209 from M2 receptors by competing monovalent ligands that are known to interact with either the orthosteric site (e.g., atropine) or a well characterized allosteric site (e.g., obidoxime) on the receptor. In complementary kinetic studies assessing allosteric modulation of the receptor, unlabeled THRX-160209 retarded dissociation of [3H]N-methyl scopolamine (NMS). The effects of THRX-160209 on retardation of [3H]NMS dissociation were competitively inhibited by obidoxime, suggesting that obidoxime and THRX-160209 bind to an overlapping region coincident with other typical muscarinic allosteric agents, such as 3-methyl-5-[7-[4-[(4S)-4-methyl-1,3-oxazolidin-2-yl]phenoxy]heptyl]-1,2-oxazole (W84) and gallamine. Taken together, these data are consistent with the hypothesis that THRX-160209 binds in a multivalent manner to the M2 receptor, simultaneously occupying the orthosteric site and a spatially distinct allosteric site. (+info)
Acute amphetamine exposure selectively desensitizes kappa-opioid receptors in the nucleus accumbens.
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In the present study, we investigated the effects of psychostimulant exposure on kappa-opioid peptide (KOP) receptor signaling in the rat mesolimbic system. A single subcutaneous (s.c.) injection of amphetamine (2.5 mg/kg) reduced the KOP receptor-mediated inhibition of glutamate release in the nucleus accumbens shell, as a consequence of KOP receptor desensitization. This effect was blocked by dopamine (DA) receptor antagonists or the nonselective opioid antagonist, naltrexone (1 mg/kg, s.c.), and mimicked by the KOP receptor agonists U69593 (0.32 mg/kg, s.c.) and dynorphin (1 microM), indicating that an amphetamine-induced release of dynorphin is producing a long-lasting desensitization of the KOP receptor. Despite the fact that amphetamine also increases dynorphin release in the ventral tegmental area (VTA), KOP receptor function in this region was not affected by amphetamine; there was no difference in the KOP receptor-mediated change in firing rate or resting membrane potential measured in VTA neurons from saline- or amphetamine-treated animals. This study demonstrates that amphetamine can produce regionally selective adaptations in KOP receptor signaling, which may, in turn, alter the effects of subsequent drug exposure. (+info)
Identification and characterization of UK-201844, a novel inhibitor that interferes with human immunodeficiency virus type 1 gp160 processing.
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More than 10(6) compounds were evaluated in a human immunodeficiency virus type 1 (HIV-1) high-throughput antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (UK-201844). UK-201844 exhibited antiviral activity against HIV-1 NL4-3 in MT-2 and PM1 cells, with 50% effective concentrations of 1.3 and 2.7 microM, respectively, but did not exhibit measurable antiviral activity against the closely related HIV-1 IIIB laboratory strain. UK-201844 specifically inhibited the production of infectious virions packaged with an HIV-1 envelope (Env), but not HIV virions packaged with a heterologous Env (i.e., the vesicular stomatitis virus glycoprotein), suggesting that the compound targets HIV-1 Env late in infection. Subsequent antiviral assays using HIV-1 NL4-3/IIIB chimeric viruses showed that HIV-1 Env sequences were critical determinants of UK-201844 susceptibility. Consistent with this, in vitro resistant-virus studies revealed that amino acid substitutions in HIV-1 Env are sufficient to confer resistance to UK-201844. Western analysis of HIV Env proteins expressed in transfected cells or in isolated virions showed that UK-201844 inhibited HIV-1 gp160 processing, resulting in the production of virions with nonfunctional Env glycoproteins. Our results demonstrate that UK-201844 represents the prototype for a unique HIV-1 inhibitor class that directly or indirectly interferes with HIV-1 gp160 processing. (+info)
Assessment of a new brand of determinants for skin testing in a large group of patients with suspected beta-lactam allergy.
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BACKGROUND: Skin testing with major and minor determinants of benzylpenicillin is recommended standard practice for the evaluation of patients with immediate hypersensitivity reactions to beta-lactams. However, commercial reagents for this purpose were recently dropped from the European market. OBJECTIVE: In the present study, we assessed a new brand of reagents for use in skin testing in patients with suspected penicillin allergy. METHODS: Prick tests and intradermal tests were performed with benzylpenicilloyl polylysine (PPL) and minor determinant mixture (MDM). Penicillin G, amoxicillin, and the culprit beta-lactam were also tested. If skin tests were negative, a single-blind oral challenge test was performed with the culprit active principle or penicillin. If both skin tests and challenge tests were negative, the same procedure was repeated between 2 and 4 weeks later. RESULTS: A total of 636 patients were assessed. The allergy study was positive in 69 patients. Skin tests with PPL were positive in 30 patients (46.8%) and with MDM in 28 (43.7%). Sixteen patients displayed a positive reaction to both PPL and MDM (25%), while 42 patients (65.6%) had a positive reaction to either PPL or MDM alone. Thirty-two patients had positive skin test reactions to penicillin G or another p-lactam antibiotic. Five patients in whom a negative result was obtained in skin tests had a positive reaction to oral challenge. CONCLUSIONS: Our results indicate that a new brand of determinants that is commercially available in Europe is a reliable and useful tool for the diagnosis of beta-lactam allergy. The new reagents are a safe alternative to the previously available brand. (+info)
Induction of apoptosis in human hepatocellular carcinoma cells by synthetic antineoplaston A10.
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Antineoplaston A10 (3-phenylacetylamino-2,6-piperidinedion) is a naturally occurring substance and was the first antineoplaston in the human body to be chemically identified. The effect of antineoplaston A10 on human hepatocellular carcinoma cell lines HepG2 and HLE has been examined. Antineoplaston A10 displayed anti-proliferative action inhibiting cell growth in a dose- and time-dependent manner in vitro as measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays. Incubation with antineoplaston A10 for 48 h induced apoptotic events such as a typical apoptotic morphology, formation of a characteristic ladder pattern of DNA migration and accumulation of sub-G1 phase cells. Next, hepatoma xenografts in nude mice were employed to study the antitumor effects of antineoplaston A10 in vivo. Oral administration of antineoplaston A10 delayed the growth of HepG2 and HLE cells in the mice without a reduction in body weight. A higher proportion of apoptotic cells in xenografts was observed by means of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. In addition, the level of expression of apoptotic marker p53 increased while that of anti-apoptotic protein bcl-2 decreased, as evaluated with immunohistochemical staining in the xenografts. These results suggested that antineoplaston A10 may inhibit the growth of human hepatoma cells through the induction of apoptosis. (+info)