Safety and target engagement profile of two oxaloacetate doses in Alzheimer's patients.

INTRODUCTION: Brain bioenergetics are defective in Alzheimer's disease (AD). Preclinical studies find oxaloacetate (OAA) enhances bioenergetics, but human safety and target engagement data are lacking. METHODS: We orally administered 500 or 1000 mg OAA, twice daily for 1 month, to AD participants (n = 15 each group) and monitored safety and tolerability. To assess brain metabolism engagement, we performed fluorodeoxyglucose positron emission tomography (FDG PET) and magnetic resonance spectroscopy before and after the intervention. We also assessed pharmacokinetics and cognitive performance. RESULTS: Both doses were safe and tolerated. Compared to the lower dose, the higher dose benefited FDG PET glucose uptake across multiple brain regions (P < .05), and the higher dose increased parietal and frontoparietal glutathione (P < .05). We did not demonstrate consistent blood level changes and cognitive scores did not improve. CONCLUSIONS: 1000 mg OAA, taken twice daily for 1 month, is safe in AD patients and engages brain energy metabolism.

Oxaloacetate Protects Rat Liver From Experimental Warm Ischemia/Reperfusion Injury by Improving Cellular Energy Metabolism.

Liver ischemia/reperfusion injury (IRI) is an important cause of liver damage especially early after liver transplantation, following liver resection, and in other clinical situations. Using rat experimental models, we identified oxaloacetate (OAA) as a key metabolite able to protect hepatocytes from hypoxia and IRI. In vitro screening of metabolic intermediates beneficial for hepatocyte survival under hypoxia was performed by measures of cell death and injury. In vivo, the effect of OAA was evaluated using the left portal vein ligation (LPVL) model of liver ischemia and a model of warm IRI. Liver injury was evaluated in vivo by serum transaminase levels, liver histology, and liver weight (edema). Levels and activity of caspase 3 were also measured. In vitro, the addition of OAA to hepatocytes kept in a hypoxic environment significantly improved cell viability (P < 0.01), decreased cell injury (P < 0.01), and improved energy metabolism (P < 0.01). Administration of OAA significantly reduced the extent of liver injury in the LPVL model with lower levels of alanine aminotransferase (ALT; P < 0.01), aspartate aminotransferase (AST; P < 0.01), and reduced liver necrosis (P < 0.05). When tested in a warm IRI model, OAA significantly decreased ALT (P < 0.001) and AST levels (P < 0.001), prevented liver edema (P < 0.001), significantly decreased caspase 3 expression (P < 0.01), as well as histological signs of cellular vesiculation and vacuolation (P < 0.05). This was associated with higher adenosine triphosphate (P < 0.05) and energy charge levels (P < 0.01). In conclusion, OAA can significantly improve survival of ischemic hepatocytes. The hepatoprotective effect of OAA was associated with increased levels of liver bioenergetics both in vitro and in vivo. These results suggest that it is possible to support mitochondrial activity despite the presence of ischemia and that OAA can effectively reduce ischemia-induced injury in the liver.

Oxaloacetate activates brain mitochondrial biogenesis, enhances the insulin pathway, reduces inflammation and stimulates neurogenesis.

Brain bioenergetic function declines in some neurodegenerative diseases, this may influence other pathologies and administering bioenergetic intermediates could have therapeutic value. To test how one intermediate, oxaloacetate (OAA) affects brain bioenergetics, insulin signaling, inflammation and neurogenesis, we administered intraperitoneal OAA, 1-2 g/kg once per day for 1-2 weeks, to C57Bl/6 mice. OAA altered levels, distributions or post-translational modifications of mRNA and proteins (proliferator-activated receptor-gamma coactivator 1α, PGC1 related co-activator, nuclear respiratory factor 1, transcription factor A of the mitochondria, cytochrome oxidase subunit 4 isoform 1, cAMP-response element binding, p38 MAPK and adenosine monophosphate-activated protein kinase) in ways that should promote mitochondrial biogenesis. OAA increased Akt, mammalian target of rapamycin and P70S6K phosphorylation. OAA lowered nuclear factor κB nucleus-to-cytoplasm ratios and CCL11 mRNA. Hippocampal vascular endothelial growth factor mRNA, doublecortin mRNA, doublecortin protein, doublecortin-positive neuron counts and neurite length increased in OAA-treated mice. (1)H-MRS showed OAA increased brain lactate, GABA and glutathione thereby demonstrating metabolic changes are detectable in vivo. In mice, OAA promotes brain mitochondrial biogenesis, activates the insulin signaling pathway, reduces neuroinflammation and activates hippocampal neurogenesis.

Acetyl-L-carnitine and oxaloacetate in post-treatment against LTP impairment in a rat ischemia model. An in vitro electrophysiological study.

A high proportion of research relating to cerebral ischemia focuses on neuroprotection. The application of compounds normally present in the organism is popular, because they do not greatly influence the synaptic activity by receptor modulation, and can be administered without serious side effects. Oxaloacetate (OxAc) and acetyl-L-carnitine (ALC) are such favorable endogenous molecules. ALC can exert a protective effect by improving the energy state of the neurons under ischemic conditions. A promising neuroprotective strategy is glutamate scavenging, which can be achieved by the intravenous administration of OxAc. This study involved the possible protective effects of ALC and OxAc in different post-treatment protocols against long-term potentiation (LTP) impairment. Ischemia was induced in rats by 2-vessel occlusion, which led to a decreased LTP relative to the control group. High-dose (200 mg/kg) ALC or OxAc post-treatment resulted in a higher potentiation relative to the 2VO group, but it did not reach the control level, whereas low-dose ALC (100 mg/kg) in combination with OxAc completely restored the LTP function. Many previous studies have concluded that ALC can be protective only as pretreatment. The strategy described here reveals that ALC can also be neuroprotective when utilized as post-treatment against ischemia.

Combined Treatment of an Amyotrophic Lateral Sclerosis Rat Model with Recombinant GOT1 and Oxaloacetic Acid: A Novel Neuroprotective Treatment.

BACKGROUND/AIM: The sporadic form of the disease affects the majority of amyotrophic lateral sclerosis (ALS) patients. The role of glutamate (Glu) excitotoxicity in ALS has been extensively documented and remains one of the prominent hypotheses of ALS pathogenesis. In light of this evidence, the availability of a method to remove excess Glu from brain and spinal cord extracellular fluids without the need to deliver drugs across the blood-brain barrier and with minimal or no adverse effects may provide a major therapeutic asset, which is the primary aim of this study. METHODS: The therapeutic efficacy of the combined treatment with recombinant Glu-oxaloacetate-transaminase (rGOT) and its co-factor oxaloacetic acid (OxAc) has been tested in an animal model of sporadic ALS. RESULTS: We found that OxAc/rGOT treatment provides significant neuroprotection to spinal cord motor neurons. It also slows down the development of motor weakness and prolongs survival. CONCLUSION: In this study we bring evidence that the administration of Glu scavengers to rats with sporadic ALS inhibited the massive death of spinal cord motor neurons, slowed the onset of motor weakness and prolonged survival. This treatment may be of high clinical significance for the future treatment of chronic neurodegenerative diseases.

MRS of brain metabolite levels demonstrates the ability of scavenging of excess brain glutamate to protect against nerve agent induced seizures.

This study describes the use of in vivo magnetic resonance spectrocopy (MRS) to monitor brain glutamate and lactate levels in a paraoxon (PO) intoxication model. Our results show that the administration of recombinant glutamate-oxaloacetate transaminase (rGOT) in combination with oxaloacetate (OxAc) significantly reduces the brain-accumulated levels of glutamate. Previously we have shown that the treatment causes a rapid decrease of blood glutamate levels and creates a gradient between the brain and blood glutamate levels which leads to the efflux of excess brain glutamate into the blood stream thereby reducing its potential to cause neurological damage. The fact that this treatment significantly decreased the brain glutamate and lactate levels following PO intoxication suggests that it could become a new effective neuroprotective agent.

Blood glutamate scavengers prolong the survival of rats and mice with brain-implanted gliomas.

L-Glutamate (Glu) plays a crucial role in the growth of malignant gliomas. We have established the feasibility of accelerating a naturally occurring brain to-blood Glu efflux by decreasing blood Glu levels with intravenous oxaloacetate, the respective Glu co-substrate of the blood resident enzyme humane glutamate­oxaloacetate transaminase(hGOT). We wished to demonstrate that blood Glu scavenging provides neuroprotection in the case of glioma.We now describe the neuroprotective effects of blood Glu scavenging in a fatal condition such as brain-implanted C6 glioma in rats and brain-implanted human U87 MG glioma in nude mice. Rat (C-6) or human (U87) glioma cells were grafted stereotactically in the brain of rats or mice. After development of tumors, the animals were drinking oxaloacetate with or without injections of hGOT. In addition, mice were treated with combination treatment, which included drinking oxaloacetate with intracutaneous injections of hGOT and intraperitoneal injection of Temozolomide. Animals drinking oxaloacetate with or without injections of hGOT displayed a smaller tumor volume, reduced invasiveness and prolonged survival than control animals drinking saline. These effects were significantly enhanced by Temozolomide in mice, which increased survival by 237%. This is the first demonstration of blood Glu scavenging in brain cancer, and because of its safety, is likely to be of clinical significance for the future treatment of human gliomas. As we demonstrated, the blood glutamate scavenging treatment in combination with TMZ could be a good candidate or as an alternative treatment to the patients that do not respond to TMZ.

Blood glutamate scavengers increase pro-apoptotic signaling and reduce metastatic melanoma growth in-vivo.

Inhibition of extracellular glutamate (Glu) release decreases proliferation and invasion, induces apoptosis, and inhibits melanoma metastatic abilities. Previous studies have shown that Blood-glutamate scavenging (BGS), a novel treatment approach, has been found to be beneficial in attenuating glioblastoma progression by reducing brain Glu levels. Therefore, in this study we evaluated the ability of BGS treatment to inhibit brain metastatic melanoma progression in-vivo. RET melanoma cells were implanted in C56BL/6J mice to induce brain melanoma tumors followed by treatment with BGS or vehicle administered for fourteen days. Bioluminescent imaging was conducted to evaluate tumor growth, and plasma/CSF Glu levels were monitored throughout. Immunofluorescence staining of Ki67 and 53BP1 was used to analyze tumor cell proliferation and DNA double-strand breaks. In addition, we analyzed CD8, CD68, CD206, p-STAT1 and iNOS expression to evaluate alterations in tumor micro-environment and anti-tumor immune response due to treatment. Our results show that BGS treatment reduces CSF Glu concentration and consequently melanoma growth in-vivo by decreasing tumor cell proliferation and increasing pro-apoptotic signaling in C56BL/6J mice. Furthermore, BGS treatment supported CD8+ cell recruitment and CD68+ macrophage invasion. These findings suggest that BGS can be of potential therapeutic relevance in the treatment of metastatic melanoma.

Manipulation of health span and function by dietary caloric restriction mimetics.

After nearly a century of rigorous investigation and testing, dietary caloric restriction (CR) remains the most robust and reproducible method for slowing aging and maintaining health, function, and vitality. This intervention has been applied to species across the evolutionary spectrum, but for a number of reasons, practical applicability to humans has been questioned. To overcome these issues, we initiated the field of CR mimetics in 1998 and have observed its development into a full-fledged antiaging industry. Basically, strategies that enable individuals to obtain the biological benefits of CR without reducing actual food intake can be considered CR mimetics, whether functional, pharmaceutical, nutraceutical, or other. Some of the best known candidates include resveratrol and related agents, the antidiabetic drug metformin, and rapamycin and other mTOR regulators. While the mechanisms of action vary, these and essentially all CR mimetic candidates work through at least some of the same pathways as actual CR. While the entire field continues to evolve rapidly, the current status will be reviewed here, with particular focus on recent developments, the most practical relevance and applicability for potential consumers, and new strategies for the future.

Effect of alpha-ketoglutarate and oxaloacetate on brain mitochondrial DNA damage and seizures induced by kainic acid in mice.

The effects of alpha-ketoglutarate and oxaloacetate on brain mitochondrial DNA (mtDNA) damage and seizures induced by kainic acid were examined both in vivo and in vitro. An intraperitoneal (ip) injection of kainic acid (45 mg/kg) produced broad-spectrum limbic and severe sustained seizures in all of the treated mice. The seizures were abolished when alpha-ketoglutarate (2 g/kg) or oxaloacetate (1 g/kg) was injected intraperitoneally in the animals 1 min before kainic acid administration. In addition, the administration of kainic acid caused damage to mtDNA in brain frontal and middle cortex of mice. These effects were completely abolished by the ip preinjection of alpha-ketoglutarate (2 g/kg) or oxaloacetate (1 g/kg). In vitro exposure of kainic acid (0.25, 0.5 or 1.0 mM) to brain homogenate inflicted damage to mtDNA in a concentration-dependent manner. The damage of mtDNA induced by 1.0 mM kainic acid was attenuated by the co-treatment with alpha-ketoglutarate (2.5 or 5.0 mM) or oxaloacetate (0.75 or 1.0 mM). Furthermore, in vivo and in vitro exposure of kainic acid elicited an increase in lipid peroxidation. However, the increased lipid peroxidation was completely inhibited by cotreatment of alpha-ketoglutarate or oxaloacetate. These results suggest that alpha-keto acids such as alpha-ketoglutarate and oxaloacetate play a role in the inhibition of seizures and subsequent mtDNA damage induced by the excitotoxic/neurotoxic agent, kainic acid.

Human recombinant glutamate oxaloacetate transaminase 1 (GOT1) supplemented with oxaloacetate induces a protective effect after cerebral ischemia.

Blood glutamate scavenging is a novel and attractive protecting strategy to reduce the excitotoxic effect of extracellular glutamate released during ischemic brain injury. Glutamate oxaloacetate transaminase 1 (GOT1) activation by means of oxaloacetate administration has been used to reduce the glutamate concentration in the blood. However, the protective effect of the administration of the recombinant GOT1 (rGOT1) enzyme has not been yet addressed in cerebral ischemia. The aim of this study was to analyze the protective effect of an effective dose of oxaloacetate and the human rGOT1 alone and in combination with a non-effective dose of oxaloacetate in an animal model of ischemic stroke. Sixty rats were subjected to a transient middle cerebral artery occlusion (MCAO). Infarct volumes were assessed by magnetic resonance imaging (MRI) before treatment administration, and 24 h and 7 days after MCAO. Brain glutamate levels were determined by in vivo MR spectroscopy (MRS) during artery occlusion (80 min) and reperfusion (180 min). GOT activity and serum glutamate concentration were analyzed during the occlusion and reperfusion period. Somatosensory test was performed at baseline and 7 days after MCAO. The three treatments tested induced a reduction in serum and brain glutamate levels, resulting in a reduction in infarct volume and sensorimotor deficit. Protective effect of rGOT1 supplemented with oxaloacetate at 7 days persists even when treatment was delayed until at least 2 h after onset of ischemia. In conclusion, our findings indicate that the combination of human rGOT1 with low doses of oxaloacetate seems to be a successful approach for stroke treatment.

Neuroprotection by glutamate oxaloacetate transaminase in ischemic stroke: an experimental study.

As ischemic stroke is associated with an excessive release of glutamate into the neuronal extracellular space, a decrease in blood glutamate levels could provide a mechanism to remove it from the brain tissue, by increasing the brain-blood gradient. In this regard, the ability of glutamate oxaloacetate transaminase (GOT) to metabolize glutamate in blood could represent a potential neuroprotective tool for ischemic stroke. This study aimed to determine the neuroprotective effects of GOT in an animal model of cerebral ischemia by means of a middle cerebral arterial occlusion (MCAO) following the Stroke Therapy Academic Industry Roundtable (STAIR) group guidelines. In this animal model, oxaloacetate-mediated GOT activation inhibited the increase of blood and cerebral glutamate after MCAO. This effect is reflected in a reduction of infarct size, smaller edema volume, and lower sensorimotor deficits with respect to controls. Magnetic resonance spectroscopy confirmed that the increase of glutamate levels in the brain parenchyma after MCAO is inhibited after oxaloacetate-mediated GOT activation. These findings show the capacity of the GOT to remove glutamate from the brain by means of blood glutamate degradation, and suggest the applicability of this enzyme as an efficient and novel neuroprotective tool against ischemic stroke.

Kynurenate production by cultured human astrocytes.

In the rodent brain, astrocytes are known to be the primary source of kynurenate (KYNA), an endogenous antagonist of both the glycine(B) and the alpha7 nicotinic acetylcholine receptor. In the present study, primary human astrocytes were used to examine the characteristics and regulation of de novo KYNA synthesis in vitro. To this end, cells were exposed to KYNA's bioprecursor L-kynurenine, and newly formed KYNA was recovered from the extracellular milieu. The production of KYNA was stereospecific and rose with increasing L-kynurenine concentrations, reaching a plateau in the high microM range. In an analogous experiment, astrocytes also readily produced and liberated the potent, specific glycine(B) receptor antagonist 7-chlorokynurenate from L-4-chlorokynurenine. KYNA synthesis was dose-dependently reduced by L-leucine or L-phenylalanine, two amino acids that compete with L-kynurenine for cellular uptake, and by aminooxyacetate, a non-specific aminotransferase inhibitor. In contrast, KYNA formation was stimulated by 5 mM pyruvate or oxaloacetate, which act as co-substrates of the transamination reaction. Aglycemic or depolarizing (50 mM KCl or 100 microM veratridine) conditions had no effect on KYNA synthesis. Subsequent studies using tissue homogenate showed that both known cerebral kynurenine aminotransferases (KAT I and KAT II) are present in astrocytes, but that KAT II appears to be singularly responsible for KYNA formation under physiological conditions. Taken together with previous results, these data suggest that very similar mechanisms control KYNA synthesis in the rodent and in the human brain. These regulatory events are likely to influence the neuromodulatory effects of astrocyte-derived KYNA in the normal and diseased human brain.