Orally Bioavailable Metal Chelators and Radical Scavengers: Multifunctional Antioxidants for the Coadjutant Treatment of Neurodegenerative Diseases.

Neurodegenerative diseases are associated with oxidative stress that is induced by the presence of reactive oxygen species and the abnormal cellular accumulation of transition metals. Here, a new series of orally bioavailable multifunctional antioxidants (MFAO-2s) possessing a 2-diacetylamino-5-hydroxypyrimidine moiety is described. These MFAO-2s demonstrate both free radical and metal attenuating properties that are similar to the original published MFAO-1s that are based on 1-N,N'-dimethylsulfamoyl-1-4-(2-pyrimidyl)piperazine. Oral bioavailability studies in C57BL/6 mice demonstrate that the MFAO-2s accumulate in the brain at significantly higher levels than the MFAO-1s while achieving similar neural retina levels. The MFAO-2s protect human neuroblastoma and retinal pigmented epithelial cells against hydroxyl radicals in a dose-dependent manner by maintaining cell viability and intracellular glutathione levels. The MFAO-2s outperform clioquinol, a metal attenuator that has been investigated for the treatment of Alzheimer's disease.

Effects of multifunctional antioxidants on mitochondrial dysfunction and amyloid-ß metal dyshomeostasis.

BACKGROUND: Redox-active metal dyshomeostasis and oxidative stress are associated with mitochondrial dysfunction and amyloid-ß (Aß) neurotoxicity that are linked to both the development of age-related macular degeneration (AMD) and Alzheimer's disease (AD). As potential therapeutic agents, orally active multifunctional antioxidants (MFAOs) possessing two independent functional groups capable of binding redox-active metals and scavenging free radicals have been synthesized. OBJECTIVE: To determine whether MFAOs affect mitochondrial function and reduce the presence of Aß plaque formation. METHODS: The MFAOs were evaluated in cultured SH-SY5Y cells and ARPE-19 cells. MFAO effects on mitochondrial function were investigated using rhodamine 123 staining after 2 hour exposure to MnCl2. MFAO effects on Aß:Zn complex formation were evaluated with Zinquin staining and the ability of the Aß:Zn complex to be degraded by matrix metalloproteinase-2 (MMP-2). The ability of MFAOs to reduce Aß plaque in the brain was determined by orally feeding MFAO for one year to B6;129-Psen1tm1Mpm Tg(AßPPSwe,tauP301L) 1Lfa/Mmjax transgenic mice. Aß levels were determined by ELISA. RESULTS: MFAOs neither adversely affected mitochondrial signaling nor labile cytoplasmic zinc levels. MFAOs protected cells against Mn2+-induced mitochondrial dysfunction. MFAOs also removed zinc from the Aß:Zn complex so that Aß plaque could be degraded by MMP-2. Zinquin staining indicated that the removed zinc was present in the cytoplasm as labile zinc. Orally administered MFAOs reduced the brain levels of both Aß40 and Aß42 isoforms of Aß. CONCLUSION: These studies demonstrate that these MFAOs have metal attenuating properties with therapeutic potential in the treatment of both AMD and AD.

Topical nutraceutical Optixcare EH ameliorates experimental ocular oxidative stress in rats.

PURPOSE: Based on the hypothesis that oral nutraceuticals do not adequately reach all ocular tissues in the anterior segment, we evaluated the ability of a 3% concentration of the ingredients in a topical nutraceutical antioxidant formulation called Optixcare Eye Health (Optixcare EH) to ameliorate oxidative stress in rat models of age-related ocular diseases. METHODS: Diabetes was induced by tail-vein injection of streptozotocin, and the development of cataracts was monitored by slit lamp. Young rats were exposed to ultraviolet (UV) light, and the reduction in lens glutathione (GSH) levels and increase in 4-hydroxynonenol (4-HNE) were measured. Oxidative stress in the neural retina was generated by exposure of dark-adapted rats to 1,000 lx of light, and oxidative stress markers were measured. Dry eye was induced in rats by twice daily (b.i.d.) subcutaneous scopolamine injections. Topical Optixcare EH was administered b.i.d. and compared in select experiments to the multifunctional antioxidant JHX-4, the topical aldose reductase inhibitor (ARI) Kinostat™, oral Ocu-GLO™, and the topical ocular comfort agents Optixcare Eye Lube, Optixcare Eye Lube + Hyaluron, and Idrop Vet Plus hyaluronic acid. RESULTS: In diabetic rats, topical ARI treatment prevented cataract formation while the nutraceuticals delayed their development with Optixcare EH>Ocu-GLO. In UV-exposed rats, the reduction of GSH and increase in 4-HNE in the lens were normalized in order JHX-4>Optixcare EH>Ocu-GLO. In the retina, oxidative stress markers were reduced better by oral JHX-4 compared with topical Optixcare EH. In the scopolamine-induced dry-eye rats, tear flow was maintained by Optixcare EH treatment, while none of the comfort agents examined altered tear flow. CONCLUSIONS: Topical administration of a 3% concentration of the ingredients in Optixcare EH reduces experimentally induced reactive oxygen species in rats exposed to several sources of ocular oxidative stress. In addition, Optixcare EH maintains tear volume in scopolamine-induced dry eye. This suggests that in the anterior segment, the ingredients in Optixcare EH may have clinical potential against ocular oxidative stress.

Novel transgenic mouse models develop retinal changes associated with early diabetic retinopathy similar to those observed in rats with diabetes mellitus.

Retinal capillary pericyte degeneration has been linked to aldose reductase (AR) activity in diabetic retinopathy (DR). Since the development of DR in mice and rats has been reported to differ and that this may be linked to differences in retinal sorbitol levels, we have established new murine models of early onset diabetes mellitus as tools for investigating the role of AR in DR. Transgenic diabetic mouse models were developed by crossbreeding diabetic C57BL/6-Ins2(Akita)/J (AK) with transgenic C57BL mice expressing green fluorescent protein (GFP), human aldose reductase (hAR) or both in vascular tissues containing smooth muscle actin-α (SMAA). Changes in retinal sorbitol levels were determined by HPLC while changes of growth factors and signaling were investigated by Western Blots. Retinal vascular changes were quantitatively analyzed on elastase-digestion flat mounts. Results show that sorbitol levels were higher in neural retinas of diabetic AK-SMAA-GFP-hAR compared to AK-SMAA-GFP mice. AK-SMAA-GFP-hAR mice showed induction of the retinal growth factors VEGF, IGF-1, bFGF and TGFß, as well as signaling changes in P-Akt, P-SAPK/JNK, and P-44/42 MAPK. Increased loss of nuclei per capillary length and a significant increase in the percentage of acellular capillaries presented in 18 week old AK-SMAA-GFP-hAR mice. These changes are similar to those observed in streptozotocin-induced diabetic rats. Retinal changes in both mice and rats were prevented by inhibition of AR. These studies confirm that the increased expression of AR in mice results in the development of retinal changes associated with the early stages of DR that are similar to those observed in rats.

Novel diabetic mouse models as tools for investigating diabetic retinopathy.

OBJECTIVE: Mouse models possessing green fluorescent protein (GFP) and/or human aldose reductase (hAR) in vascular tissues have been established and crossed with naturally diabetic Akita mice to produce new diabetic mouse models. RESEARCH DESIGN AND METHODS: Colonies of transgenic C57BL mice expressing GFP (SMAA-GFP), hAR (SMAA-hAR) or both (SMAA-GFP-hAR) in vascular tissues expressing smooth muscle actin were established and crossbred with C57BL/6-Ins2(Akita)/J (AK) mice to produce naturally diabetic offspring AK-SMAA-GFP and AK-SMAA-GFP-hAR. Aldose reductase inhibitor AL1576 (ARI) was administered in chow. Retinal and lenticular sorbitol levels were determined by HPLC. Retinal functions were evaluated by electroretinography (ERGs). Growth factor and signaling changes were determined by Western Blots using commercially available antibodies. Retinal vasculatures were isolated from the neural retina by enzymatic digestion. Flat mounts were stained with PAS-hematoxylin and analyzed. RESULTS: Akita transgenics developed DM by 8 weeks of age with blood glucose levels higher in males than females. Sorbitol levels were higher in neural retinas of AK-SMAA-GFP-hAR compared to AK-SMAA-GFP mice. AK-SMAA-GFP-hAR mice also had higher VEGF levels and reduced ERG scotopic b-wave function, both of which were normalized by AL1576. AK-SMAA-GFP-hAR mice showed induction of the retinal growth factors bFGF, IGF-1, and TGFß, as well as signaling changes in P-Akt, P-SAPK/JNK and P-44/42 MAPK that were also reduced by ARI treatment. Quantitative analysis of flat mounts in 18 week AK-SMAA-GFP-hAR mice revealed increased loss of nuclei/capillary length and a significant increase in the percentage of acellular capillaries present which was not seen in AK-SMAA-GFP-hAR treated with ARI. CONCLUSIONS/SIGNIFICANCE: These new mouse models of early onset diabetes may be valuable tools for assessing both the role of hyperglycemia and AR in the development of retinal lesions associated with diabetic retinopathy.

Orally active multi-functional antioxidants are neuroprotective in a rat model of light-induced retinal damage.

BACKGROUND: Progression of age-related macular degeneration has been linked to iron dysregulation and oxidative stress that induce apoptosis of neural retinal cells. Since both antioxidants and chelating agents have been reported to reduce the progression of retinal lesions associated with AMD in experimental animals, the present study evaluates the ability of multi-functional antioxidants containing functional groups that can independently chelate redox metals and quench free radicals to protect the retina against light-induced retinal degeneration, a rat model of dry atrophic AMD. METHODS/RESULTS: Proof of concept studies were conducted to evaluate the ability of 4-(5-hydroxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 4) and 4-(5-hydroxy-4,6-dimethoxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 8) to reduce retinal damage in 2-week dark adapted Wistar rats exposed to 1000 lx of light for 3 hours. Assessment of the oxidative stress markers 4- hydroxynonenal and nitrotyrosine modified proteins and Thioredoxin by ELISA and Western blots indicated that these compounds reduced the oxidative insult caused by light exposure. The beneficial antioxidant effects of these compounds in providing significant functional and structural protection were confirmed by electroretinography and quantitative histology of the retina. CONCLUSIONS/SIGNIFICANCE: The present study suggests that multi-functional compounds may be effective candidates for preventive therapy of AMD.

Effect of 5-S-GAD on UV-B-induced cataracts in rats.

PURPOSE: 5-S-Glutathionyl-N-beta-alanyl-3,4-dihydroxyphenylalanine (5-S-GAD) is a novel antibacterial substance purified from Sarcophaga peregrina (flesh fly) that has both a radical scavenging activity and antioxidative activity. This is a report of an investigation of the effect of 5-S-GAD (eyedrops) on UVB-induced cataracts in rats. METHODS: Brown Norway male rats (n = 32; 7 weeks old) were treated with either 5-S-GAD 0.1%, 5-SGAD 1%, astaxanthin (AST) 0.1% suspension eyedrops or the vehicle alone (the solution without 5-S-GAD) three times a day (three doses at 5-min intervals each time). The treatment was scheduled 2 days before UV-B exposure and 2 days after UV-B exposure. Exposure to 100-200 mJ/cm(2) UV-B was performed once a week between drug treatments for 9 consecutive weeks, with a total dose of 1200 mJ/cm(2) UV-B. Ocular penetration of 5-S-GAD was analyzed using high-pressure liquid chromatography (HPLC). Cataract formation was documented by an anterior eye segment analysis system once a week under mydriasis. The light-scattering intensity (LSI) of the anterior superficial cortex region was measured. RESULTS: In the eighth to ninth week after the start of UV-B exposure, the LSI of anterior subcapsular lenses of 5-S-GAD-treated groups, as detected by HPLC, was significantly lower (P < 0.05) than that of the control, whereas no such difference was found in the AST-treated group. CONCLUSION: 5-S-GAD eyedrop application may delay the progression of UV-B-induced cataract in rats.

A new and efficient synthetic method for 15N3-labeled cytosine nucleosides: Dimroth rearrangement of cytidine N3-oxides.

The treatment of (15)N(4)-labeled cytidine N(3)-oxide and (15)N(4)-labeled 2'-deoxycytidine N(3)-oxide, prepared from the appropriate unprotected uridines in three reaction steps, with benzyl bromide in the presence of excess lithium methoxide allowed the smooth occurrence of their Dimroth rearrangement even under mild conditions leading to the corresponding (15)N(3)-labeled uridine 4-O-benzyloximes which can easily undergo the reductive N-O bond cleavage to give the desirable (15)N(3)-labeled cytosine nucleosides in high total yields.