Prompt treatment with uridine triacetate improves survival and reduces toxicity due to fluorouracil and capecitabine overdose or dihydropyrimidine dehydrogenase deficiency.

Uridine triacetate has been shown to be an effective antidote against mortality and toxicity caused by either overdoses or exaggerated susceptibility to the widely used anticancer agents 5-fluorouracil (5-FU) and capecitabine. However, a direct assessment of efficacy based on when emergency treatment was initiated was not clinically feasible. In this study we used mouse models of 5-FU overdose and of dihydropyrimidine dehydrogenase (DPD) deficiency to compare the efficacy of uridine triacetate in reducing toxicity and mortality when treatment was initiated at time points from 4 to 144 h after administration of 5-FU. We found that uridine triacetate was effective both in the 5-FU overdose and DPD deficiency models. Starting treatment within 24 h was most effective at reducing toxicity and mortality in both models, while treatment starting more than 96 to 120 h after 5-FU was far less effective. Uridine triacetate also reduced mortality in the DPD deficiency model when mice were treated with the 5-FU prodrug capecitabine. The results of this study are supportive of clinical observations and practice, indicating that efficacy declined progressively with later and later treatment initiation. Prompt treatment with uridine triacetate, within 24 h, conferred the greatest protection against 5-FU overexposure.

Uridine prodrug improves memory in Tg2576 and TAPP mice and reduces pathological factors associated with Alzheimer's disease in related models.

Uridine prodrug PN401 has been shown to have neuroprotective effects in models of Parkinson's disease and Huntington's disease. These age-related neurodegenerative diseases including Alzheimer's disease (AD) are associated with mitochondrial dysfunction, oxidative stress, and inflammation. Attenuation of these pathological factors in AD, in addition to amyloid fibrils and neurofibrillary tangles, is critical to prevent cognitive impairment. The effects of PN401 treatment were tested in the Tg2576 and Tg2576 X P301L (TAPP) mouse models of AD. Treatment with PN401 reduced impairments in the Tg2576 mice in contextual fear conditioning and novel object recognition. In the TAPP mice, PN401 reduced the impairments in novel object recognition and social transmission of food preference. PN401 also improved motor behavior and reduced anxiety-like behavior in the TAPP mice. TAPP mouse hippocampal tau phosphorylation and lipid peroxidation were reduced by PN401 treatment. Increased tau phosphorylation was significantly correlated with worsening novel object recognition memory. PN401 did not affect amyloid plaque area in the AD mice. In other AD-related animal studies, PN401 treatment reduced blood-brain barrier damage due to intracortical LPS, elevation of serum TNFα due to systemic LPS, and hippocampal CA1 neuronal loss in the gerbil stroke model. Uridine dose-dependently protected cells from chemical hypoxia and ceramide, and decreased formation of reactive oxygen species and mitochondrial DNA damage due to hydrogen peroxide. These protective effects were achieved by raising uridine levels to at least 25-50 µM and serum uridine levels in this range in humans were obtained with oral PN401.

Oral uridine pro-drug PN401 is neuroprotective in the R6/2 and N171-82Q mouse models of Huntington's disease.

Previously, uridine pro-drug 2',3',5'-tri-O-acetyluridine (PN401) was shown to be protective in the mitochondrial complex II inhibitor 3-nitropropionic acid model of Huntington's disease (HD). In this study, PN401 increased survival and improved motor function on the rotarod in both R6/2 and N171-82Q polyglutamine repeat mouse models of HD. PN401 significantly decreased neurodegeneration in both the piriform cortex and striatum although PN401 decreased huntingtin protein aggregates only in the striatum. Cortical and striatal brain-derived neurotrophic factor (BDNF) protein levels were reduced in the +/- compared to the -/- N171-82Q mice and PN401 treatment significantly increased cortical BDNF in both +/- and -/- mice, but PN401 did not affect striatal BDNF. These results suggest that PN401 may have beneficial effects in the treatment of neurodegenerative diseases such as HD.

Severe cytochrome c oxidase inhibition in vivo does not induce a pyrimidine deficiency; neuroprotective action of oral uridine prodrug PN401 requires supraphysiological levels of uridine.

It has been hypothesized that mitochondrial respiratory chain dysfunction leads to a pyrimidine deficiency since the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase is coupled to the electron transport chain. The uridine prodrug triacetyluridine (PN401) is neuroprotective in several models of neurodegenerative disease involving respiratory chain toxins. Therefore, the therapeutic effects of PN401 might involve the correction of a pyrimidine deficiency secondary to respiratory chain impairment. We infused mice with the cytochrome c oxidase inhibitor azide, which inhibited brain complex IV activity. Chronic infusion of azide for 2 or 14 days induced significant toxicity and mortality but did not cause a pyrimidine deficit in the brain. In contrast, the pyrimidine synthesis inhibitor N-phosphonoacetyl-l-aspartate (PALA) produced a pyrimidine deficit with minimal mortality. Treatment with 6% PN401 decreased mortality and cerebrocortical apoptosis caused by azide. Previously, we found that optimal neuroprotection against mitochondrial complex II inhibition required 4-6% PN401. PN401 at 1, 3, 6 and 10% in chow induced nonlinear increases in plasma uridine with 6% PN401 elevating plasma uridine up to 80 muM, and these higher micromolar uridine levels were also required for neuroprotection in chemical hypoxia models in vitro. Our results indicate that severe complex IV inhibition in vivo does not lead to a pyrimidine deficiency, and therefore the protective effect of PN401 in the azide toxin model is not mediated through the correction of a pyrimidine deficiency. Furthermore, supraphysiological levels of uridine are required to produce optimal protective effects in disorders involving impairment of mitochondrial respiratory complex II or IV.

Oral uridine pro-drug PN401 decreases neurodegeneration, behavioral impairment, weight loss and mortality in the 3-nitropropionic acid mitochondrial toxin model of Huntington's disease.

Huntington's disease (HD) is associated with decreased activity of mitochondrial succinate dehydrogenase (complex II). De novo biosynthesis of uridine nucleotides is directly coupled to the respiratory chain. Cells with impaired mitochondrial function become uridine auxotrophs and can be maintained with high micromolar concentration of uridine and pyruvate. The therapeutic role of pyrimidines and possible changes in uridine content has not been assessed in neurological diseases involving mitochondrial dysfunction in vivo. Oral administration of PN401 delivers much higher levels of uridine to the circulation than oral administration of uridine itself. Administration of complex II inhibitor 3-nitropropionic acid (3NP) induced neuronal damage in the striatum, substantia nigra and/or thalamus in 80% of the mice and led to 38% mortality. Treatment with PN401 almost completely prevented the neuronal damage due to 3NP and completely prevented mortality. In two subsequent experiments, 3NP-induced weight loss, mortality and behavioral impairment in rotarod performance and spontaneous motor activity were attenuated by treatment with oral PN401. 3NP did not reduce forebrain total uridine nucleotides (TUN), though higher doses of PN401 associated with optimal neuroprotection did elevate TUN to supranormal levels. Thus, oral PN401 treatment has neuroprotective effects in a HD model of mitochondrial dysfunction and the mechanism is more complex than correction of a pyrimidine deficit.

No detectable analgesic effects in the formalin test even with one million bovine adrenal chromaffin cells.

The present experiments were conducted to identify analgesic agents for transfection into immortalized adrenal chromaffin cell lines to maximize their analgesic potential. Analgesic agents known to be produced by adrenal chromaffin cells were infused intrathecally at a low dose (0.2 microg) which might conceivably be attained by adrenal chromaffin cell transplants. Numerous agents, administered individually and in two-factor combinations, produced significant analgesic effects in the formalin test. Before assessing the potential additive or synergistic effects of these analgesic agents with adrenal chromaffin cells, studies were conducted to demonstrate analgesic effects with adrenal chromaffin cells alone. Analgesic effects were previously reported in the literature with 80-100k intrathecal bovine adrenal chromaffin (BAC) cells; but in the present study 500k purified BAC cells failed to produce detectable analgesic effects. One million purified BAC cells also failed to produce analgesic effects in the formalin test. In a final study, even nicotine-stimulated release from one million purified BAC cells failed to produce analgesic effects in the formalin test. The fact that even one million nicotine-stimulated BAC cells failed to demonstrate therapeutic potential in these blinded experiments under conditions which were clearly sensitive to the analgesic agents produced by BAC cells, raises serious questions about the clinical utility of this experimental treatment.

Numerous adrenal chromaffin cell preparations fail to produce analgesic effects in the formalin test or in tests of acute pain even with nicotine stimulation.

Previous studies have reported that intrathecal implants of a variety of adrenal chromaffin cell preparations all produce analgesic effects in rodents. The major objective of the present study was to determine if any adrenal chromaffin cell preparations produce more robust analgesic effects than other cell preparations. The present study included adult rat adrenal chromaffin tissue allografts, purified adult bovine chromaffin cells, and polymer-encapsulated calf adrenal chromaffin cells, all prepared according to previously published procedures, as well as purified calf adrenal chromaffin cells. Previous studies have also suggested that immunosuppression may play a role in graft survival, and potentially increase the magnitude of analgesic effects, so the present study included both immunosuppressed and non-immunosuppressed groups (cyclosporin A, 10 mg/kg per day). Behavioral tests included the formalin test; and a dorsal tail-flick, hot-plate, and paw-pinch test, conducted sequentially 2 min after systemic nicotine (0.1 mg/kg) to evoke release from the chromaffin cells, as previously reported. Analgesic effects related to morphine and nicotine were detected, and consistent differences in performance could be detected between individual animals. Surprisingly, no analgesic effects were detectable with any of the four chromaffin cell preparations, with or without immunosuppression, in the formalin test or with nicotine stimulation in tests of acute pain.