A small-molecule microtubule-stabilizing agent safely reduces Aß plaque and tau pathology in transgenic mouse models of Alzheimer's disease.

INTRODUCTION: Intraneuronal inclusions composed of tau protein are found in Alzheimer's disease (AD) and other tauopathies. Tau normally binds microtubules (MTs), and its disengagement from MTs and misfolding in AD is thought to result in MT abnormalities. We previously identified triazolopyrimidine-containing MT-stabilizing compounds that provided benefit in AD mouse models and herein describe the characterization and efficacy testing of an optimized candidate, CNDR-51997. METHODS: CNDR-51997 underwent pharmacokinetic, pharmacodynamic, safety pharmacology, and mouse tolerability testing. In addition, the compound was examined for efficacy in 5XFAD amyloid beta (Aß) plaque mice and PS19 tauopathy mice. RESULTS: CNDR-51997 significantly reduced Aß plaques in 5XFAD mice and tau pathology in PS19 mice, with the latter also showing attenuated axonal dystrophy and gliosis. CNDR-51997 was well tolerated at doses that exceeded efficacy doses, with a good safety pharmacology profile. DISCUSSION: CNDR-51997 may be a candidate for advancement as a potential therapeutic agent for AD and/or other tauopathies. Highlights There is evidence of microtubule alterations (MT) in Alzheimer's disease (AD) brain and in mouse models of AD pathology. Intermittent dosing with an optimized, brain-penetrant MT-stabilizing small-molecule, CNDR-51997, reduced both Aß plaque and tau inclusion pathology in established mouse models of AD. CNDR-51997 attenuated axonal dystrophy and gliosis in a tauopathy mouse model, with a strong trend toward reduced hippocampal neuron loss. CNDR-51997 is well tolerated in mice at doses that are meaningfully greater than required for efficacy in AD mouse models, and the compound has a good safety pharmacology profile.

Correction of microtubule defects within Aß plaque-associated dystrophic axons results in lowered Aß release and plaque deposition.

The hallmark pathologies of the Alzheimer's disease (AD) brain are amyloid beta (Aß)-containing senile plaques and neurofibrillary tangles formed from the microtubule (MT)-binding tau protein. Tau becomes hyperphosphorylated and disengages from MTs in AD, with evidence of resulting MT structure/function defects. Brain-penetrant MT-stabilizing compounds can normalize MTs and axonal transport in mouse models with tau pathology, thereby reducing neuron loss and decreasing tau pathology. MT dysfunction is also observed in dystrophic axons adjacent to Aß plaques, resulting in accumulation of amyloid precursor protein (APP) and BACE1 with the potential for enhanced localized Aß generation. We have examined whether the brain-penetrant MT-stabilizing compound CNDR-51657 might decrease plaque-associated axonal dystrophy and Aß release in 5XFAD mice that develop an abundance of Aß plaques. Administration of CNDR-51657 to 1.5-month-old male and female 5XFAD mice for 4 or 7 weeks led to decreased soluble brain Aß that coincided with reduced APP and BACE1 levels, resulting in decreased formation of insoluble Aß deposits. These data suggest a vicious cycle whereby initial Aß plaque formation causes MT disruption in nearby axons, resulting in the local accumulation of APP and BACE1 that facilitates additional Aß generation and plaque deposition. The ability of a MT-stabilizing compound to attenuate this cycle, and also reduce deficits resulting from reduced tau binding to MTs, suggests that molecules of this type hold promise as potential AD therapeutics.

Design, synthesis and evaluation of photoactivatable derivatives of microtubule (MT)-active [1,2,4]triazolo[1,5-a]pyrimidines.

The [1,2,4]triazolo[1,5-a]pyrimidines comprise a promising class of non-naturally occurring microtubule (MT)-active compounds. Prior studies revealed that different triazolopyrimidine substitutions can yield molecules that either promote MT stabilization or disrupt MT integrity. These differences can have important ramifications in the therapeutic applications of triazolopyrimidines and suggest that different analogues may exhibit different binding modes within the same site or possibly interact with tubulin/MTs at alternative binding sites. To help discern these possibilities, a series of photoactivatable triazolopyrimidine congeners was designed, synthesized and evaluated in cellular assays with the goal of identifying candidate probes for photoaffinity labeling experiments. These studies led to the identification of different derivatives that incorporate a diazirine ring in the amine substituent at position 7 of the triazolopyrimidine heterocycle, resulting in molecules that either promote stabilization of MTs or disrupt MT integrity. These photoactivatable candidate probes hold promise to investigate the mode of action of MT-active triazolopyrimidines.

Characterization of Brain-Penetrant Pyrimidine-Containing Molecules with Differential Microtubule-Stabilizing Activities Developed as Potential Therapeutic Agents for Alzheimer's Disease and Related Tauopathies.

The microtubule (MT)-stabilizing protein tau disengages from MTs and forms intracellular inclusions known as neurofibrillary tangles in Alzheimer's disease and related tauopathies. Reduced tau binding to MTs in tauopathies may contribute to neuronal dysfunction through decreased MT stabilization and disrupted axonal transport. Thus, the introduction of brain-penetrant MT-stabilizing compounds might normalize MT dynamics and axonal deficits in these disorders. We previously described a number of phenylpyrimidines and triazolopyrimidines (TPDs) that induce tubulin post-translational modifications indicative of MT stabilization. We now further characterize the biologic properties of these small molecules, and our results reveal that these compounds can be divided into two general classes based on the cellular response they evoke. One group composed of the phenylpyrimidines and several TPD examples showed a bell-shaped concentration-response effect on markers of MT stabilization in cellular assays. Moreover, these compounds induced proteasome-dependent degradation of α- and ß-tubulin and caused altered MT morphology in both dividing cells and neuron cultures. In contrast, a second group comprising a subset of TPD molecules (TPD+) increased markers of stable MTs in a concentration-dependent manner in dividing cells and in neurons without affecting total tubulin levels or disrupting MT architecture. Moreover, an example TPD+ compound was shown to increase MTs in a neuron culture model with induced tau hyperphosphorylation and associated MT deficits. Several TPD+ compounds were shown to be both brain penetrant and orally bioavailable, and a TPD+ example increased MT stabilization in the mouse brain, making these compounds potential candidate therapeutics for neurodegenerative tauopathies such as Alzheimer's disease.

Design, Synthesis, and Evaluation of An Anti-trypanosomal [1,2,4]Triazolo[1,5-a]pyrimidine Probe for Photoaffinity Labeling Studies.

Studies have shown that depending on the substitution pattern, microtubule (MT)-targeting 1,2,4-triazolo[1,5-a]pyrimidines (TPDs) can produce different cellular responses in mammalian cells that may be due to these compounds interacting with distinct binding sites within the MT structure. Selected TPDs are also potently bioactive against the causative agent of human African trypanosomiasis, Trypanosoma brucei, both in vitro and in vivo. So far, however, there has been no direct evidence of tubulin engagement by these TPDs in T. brucei. Therefore, to enable further investigation of anti-trypanosomal TPDs, a TPD derivative amenable to photoaffinity labeling (PAL) was designed, synthesized, and evaluated in PAL experiments using HEK293 cells and T. brucei. The data arising confirmed specific labeling of T. brucei tubulin. In addition, proteomic data revealed differences in the labeling profiles of tubulin between HEK293 and T. brucei, suggesting structural differences between the TPD binding site(s) in mammalian and trypanosomal tubulin.

The microtubule-stabilizing agent, epothilone D, reduces axonal dysfunction, neurotoxicity, cognitive deficits, and Alzheimer-like pathology in an interventional study with aged tau transgenic mice.

Neurodegenerative tauopathies, such as Alzheimer's disease (AD), are characterized by insoluble deposits of hyperphosphorylated tau protein within brain neurons. Increased phosphorylation and decreased solubility has been proposed to diminish normal tau stabilization of microtubules (MTs), thereby leading to neuronal dysfunction. Earlier studies have provided evidence that small molecule MT-stabilizing drugs that are used in the treatment of cancer may have utility in the treatment of tauopathies. However, it has not been established whether treatment with a small molecule MT-stabilizing compound will provide benefit in a transgenic model with pre-existing tau pathology, as would be seen in human patients with clinical symptoms. Accordingly, we describe here an interventional study of the brain-penetrant MT-stabilizing agent, epothilone D (EpoD), in aged PS19 mice with existing tau pathology and related behavioral deficits. EpoD treatment reduced axonal dystrophy and increased axonal MT density in the aged PS19 mice, which led to improved fast axonal transport and cognitive performance. Moreover, the EpoD-treated PS19 mice had less forebrain tau pathology and increased hippocampal neuronal integrity, with no dose-limiting side effects. These data reveal that brain-penetrant MT-stabilizing drugs hold promise for the treatment of AD and related tauopathies, and that EpoD could be a candidate for clinical testing.

Microtubule-Stabilizing 1,2,4-Triazolo[1,5-a]pyrimidines as Candidate Therapeutics for Neurodegenerative Disease: Matched Molecular Pair Analyses and Computational Studies Reveal New Structure-Activity Insights.

Microtubule (MT)-stabilizing 1,2,4-triazolo[1,5-a]pyrimidines (TPDs) hold promise as candidate therapeutics for Alzheimer's disease (AD) and other neurodegenerative conditions. However, depending on the choice of substituents around the TPD core, these compounds can elicit markedly different cellular phenotypes that likely arise from the interaction of TPD congeners with either one or two spatially distinct binding sites within tubulin heterodimers (i.e., the seventh site and the vinca site). In the present study, we report the design, synthesis, and evaluation of a series of new TPD congeners, as well as matched molecular pair analyses and computational studies, that further elucidate the structure-activity relationships of MT-active TPDs. These studies led to the identification of novel MT-normalizing TPD candidates that exhibit favorable ADME-PK, including brain penetration and oral bioavailability, as well as brain pharmacodynamic activity.

Aminothienopyridazine inhibitors of tau aggregation: evaluation of structure-activity relationship leads to selection of candidates with desirable in vivo properties.

Previous studies demonstrated that members of the aminothienopyridazine (ATPZ) class of tau aggregation inhibitors exhibit a promising combination of in vitro activity as well as favorable pharmacokinetic properties (i.e., brain-penetration and oral bioavailability). Here we report the synthesis and evaluation of several new analogues. These studies indicate that the thienopyridazine core is essential for inhibition of tau fibrillization in vitro, while the choice of the appropriate scaffold decoration is critical to impart desirable ADME-PK properties. Among the active, brain-penetrant ATPZ inhibitors evaluated, 5-amino-N-cyclopropyl-3-(4-fluorophenyl)-4-oxo-3,4-dihydrothieno[3,4-d]pyridazine-1-carboxamide (43) was selected to undergo maximum tolerated dose and one-month tolerability testing in mice. The latter studies revealed that this compound is well-tolerated with no notable side-effects at an oral dose of 50mg/kg/day.

Epothilone D improves microtubule density, axonal integrity, and cognition in a transgenic mouse model of tauopathy.

Neurons in the brains of those with Alzheimer's disease (AD) and many frontotemporal dementias (FTDs) contain neurofibrillary tangles comprised of hyperphosphorylated tau protein. Tau normally stabilizes microtubules (MTs), and tau misfolding could lead to a loss of this function with consequent MT destabilization and neuronal dysfunction. Accordingly, a possible therapeutic strategy for AD and related "tauopathies" is treatment with a MT-stabilizing anti-cancer drug such as paclitaxel. However, paclitaxel and related taxanes have poor blood-brain barrier permeability and thus are unsuitable for diseases of the brain. We demonstrate here that the MT-stabilizing agent, epothilone D (EpoD), is brain-penetrant and we subsequently evaluated whether EpoD can compensate for tau loss-of-function in PS19 tau transgenic mice that develop forebrain tau inclusions, axonal degeneration and MT deficits. Treatment of 3-month-old male PS19 mice with low doses of EpoD once weekly for a 3 month period significantly improved CNS MT density and axonal integrity without inducing notable side-effects. Moreover, EpoD treatment reduced cognitive deficits that were observed in the PS19 mice. These results suggest that certain brain-penetrant MT-stabilizing agents might provide a viable therapeutic strategy for the treatment of AD and FTDs.

The characterization of microtubule-stabilizing drugs as possible therapeutic agents for Alzheimer's disease and related tauopathies.

Tau, a protein that is enriched in neurons of the central nervous system (CNS), is thought to play a critical role in the stabilization of microtubules (MTs). Several neurodegenerative disorders referred to as tauopathies, including Alzheimer's disease and certain types of frontotemporal lobar degeneration, are characterized by the intracellular accumulation of hyperphosphorylated tau fibrils. Tau deposition into insoluble aggregates is believed to result in a loss of tau function that leads to MT destabilization, and this could cause neurodegeneration as intact MTs are required for axonal transport and normal neuron function. This tau loss-of-function hypothesis has been validated in a tau transgenic mouse model with spinal cord tau inclusions, where the MT-stabilizing agent, paclitaxel, increased spinal nerve MT density and improved motor function after drug absorption at neuromuscular junctions. Unfortunately, paclitaxel is a P-glycoprotein substrate and has poor blood-brain barrier permeability, making it unsuitable for the treatment of human tauopathies. We therefore examined several MT-stabilizing compounds from the taxane and epothilone natural product families to assess their membrane permeability and to determine whether they act as substrates or inhibitors of P-glycoprotein. Moreover, we compared brain and plasma levels of the compounds after administration to mice. Finally, we assessed whether brain-penetrant compounds could stabilize mouse CNS MTs. We found that several epothilones have significantly greater brain penetration than the taxanes. Furthermore, certain epothilones cause an increase in CNS MT stabilization, with epothilone D demonstrating a favorable pharmacokinetic and pharmacodynamic profile which suggests this agent merits further study as a potential tauopathy drug candidate.

Evaluation of the Structure-Activity Relationship of Microtubule-Targeting 1,2,4-Triazolo[1,5-a]pyrimidines Identifies New Candidates for Neurodegenerative Tauopathies.

Studies in tau and Aß plaque transgenic mouse models demonstrated that brain-penetrant microtubule (MT)-stabilizing compounds, including the 1,2,4-triazolo[1,5-a]pyrimidines, hold promise as candidate treatments for Alzheimer's disease and related neurodegenerative tauopathies. Triazolopyrimidines have already been investigated as anticancer agents; however, the antimitotic activity of these compounds does not always correlate with stabilization of MTs in cells. Indeed, previous studies from our laboratories identified a critical role for the fragment linked at C6 in determining whether triazolopyrimidines promote MT stabilization or, conversely, disrupt MT integrity in cells. To further elucidate the structure-activity relationship (SAR) and to identify potentially improved MT-stabilizing candidates for neurodegenerative disease, a comprehensive set of 68 triazolopyrimidine congeners bearing structural modifications at C6 and/or C7 was designed, synthesized, and evaluated. These studies expand upon prior understanding of triazolopyrimidine SAR and enabled the identification of novel analogues that, relative to the existing lead, exhibit improved physicochemical properties, MT-stabilizing activity, and pharmacokinetics.

A brain-penetrant triazolopyrimidine enhances microtubule-stability, reduces axonal dysfunction and decreases tau pathology in a mouse tauopathy model.

BACKGROUND: Alzheimer's disease (AD) and related tauopathies are neurodegenerative diseases that are characterized by the presence of insoluble inclusions of the protein tau within brain neurons and often glia. Tau is normally found associated with axonal microtubules (MTs) in the brain, and in tauopathies this MT binding is diminished due to tau hyperphosphorylation. As MTs play a critical role in the movement of cellular constituents within neurons via axonal transport, it is likely that the dissociation of tau from MTs alters MT structure and axonal transport, and there is evidence of this in tauopathy mouse models as well as in AD brain. We previously demonstrated that different natural products which stabilize MTs by interacting with ß-tubulin at the taxane binding site provide significant benefit in transgenic mouse models of tauopathy. More recently, we have reported on a series of MT-stabilizing triazolopyrimidines (TPDs), which interact with ß-tubulin at the vinblastine binding site, that exhibit favorable properties including brain penetration and oral bioavailability. Here, we have examined a prototype TPD example, CNDR-51657, in a secondary prevention study utilizing aged tau transgenic mice. METHODS: 9-Month old female PS19 mice with a low amount of existing tau pathology received twice-weekly administration of vehicle, or 3 or 10 mg/kg of CNDR-51657, for 3 months. Mice were examined in the Barnes maze at the end of the dosing period, and brain tissue and optic nerves were examined immunohistochemically or biochemically for changes in MT density, axonal dystrophy, and tau pathology. Mice were also assessed for changes in organ weights and blood cell numbers. RESULTS: CNDR-51657 caused a significant amelioration of the MT deficit and axonal dystrophy observed in vehicle-treated aged PS19 mice. Moreover, PS19 mice receiving CNDR-51657 had significantly lower tau pathology, with a trend toward improved Barnes maze performance. Importantly, no adverse effects were observed in the compound-treated mice, including no change in white blood cell counts as is often observed in cancer patients receiving high doses of MT-stabilizing drugs. CONCLUSIONS: A brain-penetrant MT-stabilizing TPD can safely correct MT and axonal deficits in an established mouse model of tauopathy, resulting in reduced tau pathology.

Isoprostane F2alpha-VI, a new marker of oxidative stress, increases following light damage to the mouse retina.

PURPOSE: A number of studies have suggested that retinal light damage involves oxidative stress. These include demonstration of protection by antioxidants, immunohistochemical detection of oxidative stress markers, and upregulation of antioxidant enzymes. Recently a new specific marker of lipid peroxidation (LPO), isoprostane F(2alpha)-VI, has been developed. This prostaglandin isomer is produced by nonenzymatic oxidation of membrane-linked arachidonic acid. Because it provides an unusually stable and specific measure of LPO, we sought to determine whether its levels would increase following retinal light damage. METHODS: Balb/c mice were exposed to bright fluorescent light for 7 h. Twenty-eight h after light exposure, photoreceptor death was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis. Isoprostane F(2alpha)-VI was quantified in retinal extracts by gas chromatography/mass spectrometry. Retinal isoprostane was localized by immunofluorescence. RESULTS: TUNEL analysis demonstrated photoreceptor cell death after light exposure. Compared with controls, retina extracts from mice exposed to fluorescent light had a significant increase in isoprostane F(2alpha)-VI levels following light damage. Immunohistochemistry confirmed an increase in retinal isoprostane. CONCLUSIONS: Elevated levels of isoprostane F(2alpha)-VI, a stable, highly specific marker of lipid peroxidation, confirm earlier reports of light-mediated retinal lipid peroxidation, potentially an important mechanism of retinal degeneration. Further, since levels of isoprostane F(2alpha)-VI are readily quantified, its measurement provides a new means to specifically monitor retinal oxidative damage caused by prooxidants such as light.

A novel thromboxane receptor antagonist and synthase inhibitor, BM-573, reduces development and progression of atherosclerosis in LDL receptor deficient mice.

Atherosclerosis is a chronic inflammatory disease of the vasculature influenced by a variety of mediators. Among them, prostanoids, which include prostacyclin and thromboxane (Tx) A(2), have recently received a lot of attention. Previous studies demonstrated that antagonism or deletion of the receptor for TxA(2) retards early atherogenesis in apolipoprotein E-deficient mice, but no data are available in low-density lipoprotein (LDL) receptor deficient mice. In our study, we tested the effect of a novel TxA(2) receptor (TP) antagonist and synthase inhibitor, BM-573, on atherosclerosis development and progression in LDL receptor deficient mice. To this end, the effect of 12 weeks treatment with BM-573 on early or established aortic atherosclerotic lesions of these mice was assessed. In both treatments, while BM-573 did not affect body weight, systolic blood pressure, total plasma cholesterol or triglycerides levels, it partially reduced TxA(2) but did not affect prostacyclin biosynthesis. Moreover, BM-573 significantly decreased early atherogenesis and prevented progression of established atherosclerotic lesions. These results show for the first time that this dual Tx inhibitor is effective in reducing atherogenesis in the LDL receptor deficient mice. They also demonstrate the novel concept that this therapeutic approach halts the progression of the disease and influences the cellular composition of the atherosclerotic plaques.

Elevated cerebrospinal fluid F2-isoprostane levels indicating oxidative stress in healthy siblings of multiple sclerosis patients.

Lipid peroxidation has been implicated in the pathogenesis of multiple sclerosis (MS). Isoprostanes, isomers of prostaglandins, are produced by free radical-mediated peroxidation of fatty acids in vivo and can be quantified in biological fluids. This study examines the levels of cerebrospinal fluid (CSF) F2-isoprostanes (F2-iPs) in MS patients (n=46), their healthy siblings (n=46) and unrelated controls (n=50). The median CSF F2-iP concentration (range) was significantly higher in siblings of MS patients, as compared to healthy controls (40.0 [7.1-68.7] and 29.1 [6.4-60.3] pg/mL, respectively, p=0.031). MS patients demonstrated F2-iP levels intermediate between siblings and controls. F2-iP levels in MS patients and siblings correlated significantly (R=0.360, p=0.012). These results suggest that siblings of MS patients have an increased oxidative stress response to environmental and/or genetic factors that may be involved in MS pathogenesis.

Elevated lipid peroxidation biomarkers and low antioxidant status in atherosclerotic patients with increased carotid or iliofemoral intima media thickness.

OBJECTIVES: Lipid peroxidation plays an important role in the development of atherosclerosis, a chronic, age-related disease process of the arterial wall with onset decades prior to its clinical manifestations. The aim of the study was to assess the association between the intima media thickness (IMT) of the major arteries as a clinical marker of atherosclerosis and markers of lipid peroxidation along with the antioxidant status in humans. DESIGN: Case-control study. SETTING: A university-affiliated outpatient clinic. SUBJECTS: Thirty patients (22 males, 8 females; 70.4 +/- 7.3 years) with atherosclerosis of the carotid or iliofemoral arteries and 62 healthy controls (30 males, 32 females; 68.3 +/- 4.3 years). METHODS: Plasma levels of 8,12-isoprostane F2alpha-VI (8,12-IPF2alpha-VI) were measured by gas chromatography/mass spectrometry, whereas levels of malondialdehyde (MDA), vitamins A (retinol) and E (alpha- and gamma-tocopherol), and carotenoids were determined by high-performance liquid chromatography. The IMT was measured by B-mode ultrasonography. RESULTS: Patients showed, independent of fruit and vegetable intake, significantly lower plasma levels of retinol, alpha-tocopherol, and all carotenoids excluding beta-cryptoxanthin compared with controls. On the contrary, plasma 8,12-IPF2alpha-VI levels were almost doubled (p < .001) and MDA levels increased by one-third (p < .01) in atherosclerotic patients compared with controls. CONCLUSIONS: The analyses of isoprostanes and antioxidant nutrients in plasma as markers of oxidative stress and the parallel evaluation of IMT as a structural marker of atherosclerosis are suitable tools for investigating the role of antioxidants and oxidative stress in atherosclerosis.

Evaluation of Oxetan-3-ol, Thietan-3-ol, and Derivatives Thereof as Bioisosteres of the Carboxylic Acid Functional Group.

The oxetane ring serves as an isostere of the carbonyl moiety, suggesting that oxetan-3-ol may be considered as a potential surrogate of the carboxylic acid functional group. To investigate this structural unit, as well as thietan-3-ol and the corresponding sulfoxide and sulfone derivatives, as potential carboxylic acid bioisosteres, a set of model compounds has been designed, synthesized, and evaluated for physicochemical properties. Similar derivatives of the cyclooxygenase inhibitor, ibuprofen, were also synthesized and evaluated for inhibition of eicosanoid biosynthesis in vitro. Collectively, the data suggest that oxetan-3-ol, thietan-3-ol, and related structures hold promise as isosteric replacements of the carboxylic acid moiety.

Multitargeted Imidazoles: Potential Therapeutic Leads for Alzheimer's and Other Neurodegenerative Diseases.

Alzheimer's disease (AD) is a complex, multifactorial disease in which different neuropathological mechanisms are likely involved, including those associated with pathological tau and Aß species as well as neuroinflammation. In this context, the development of single multitargeted therapeutics directed against two or more disease mechanisms could be advantageous. Starting from a series of 1,5-diarylimidazoles with microtubule (MT)-stabilizing activity and structural similarities with known NSAIDs, we conducted structure-activity relationship studies that led to the identification of multitargeted prototypes with activities as MT-stabilizing agents and/or inhibitors of the cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) pathways. Several examples are brain-penetrant and exhibit balanced multitargeted in vitro activity in the low µM range. As brain-penetrant MT-stabilizing agents have proven effective against tau-mediated neurodegeneration in animal models, and because COX- and 5-LOX-derived eicosanoids are thought to contribute to Aß plaque deposition, these 1,5-diarylimidazoles provide tools to explore novel multitargeted strategies for AD and other neurodegenerative diseases.

Involvement of thromboxane receptor in the proatherogenic effect of isoprostane F2alpha-III: evidence from apolipoprotein E- and LDL receptor-deficient mice.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease of the arterial wall, where it associates with oxidative stress and formation of oxidized lipids. The lipid oxidation product isoprostane iPF2alpha-III, also known as 8-isoPGF2alpha and 15-F2t-IsoP, is elevated in patients with cardiovascular disease and present in atherosclerotic lesions. Several proatherogenic biological effects have been attributed to this isoprostane, suggesting that it could be an active factor in the pathogenesis of the disease. METHODS AND RESULTS: In this study we show that iPF2alpha-III directly promotes atherogenesis in 2 different mouse models (ie, apolipoprotein E [apoE]- and LDL receptor-deficient mice) by activating the thromboxane receptor (TP). This effect is mediated by potent proinflammatory vascular reactions but is independent of thromboxane A2 levels, changes in blood pressure, or lipid profile. Pharmacological antagonism of TP suppresses the vascular proatherogenic effects of iPF2alpha-III. Endothelial cells genetically lacking TP show reduced inflammatory responses when stimulated with this product of lipid oxidation but not other oxidized lipids. CONCLUSIONS: Our results demonstrate that in atherosclerosis iPF2alpha-III is not only a biomarker of oxidative stress but also an active mediator of its vascular phenotype. We conclude that in a clinical setting in which both thromboxane A2 and iPF2alpha-III are elevated, suppression of the first alone would not provide the most benefit for patients because the coincidental presence of the isoprostane will still have a proatherogenic effect.

Altered vascular phenotype in autism: correlation with oxidative stress.

BACKGROUND: Autism is a neurologic disorder characterized by impaired communication and social interaction. Results of previous studies showed biochemical evidence for abnormal platelet reactivity and altered blood flow in children with autism. OBJECTIVE: To evaluate the vascular phenotype in children with autism. DESIGN AND MAIN OUTCOME MEASURES: Urinary levels of isoprostane F(2alpha)-VI, a marker of lipid peroxidation; 2,3-dinor-thromboxane B(2), which reflects platelet activation; and 6-keto-prostaglandin F(1alpha), a marker of endothelium activation, were measured by means of gas chromatography-mass spectrometry in subjects with autism and healthy control subjects. SETTING AND SUBJECTS: Children with a clinical diagnosis of autism attending the Pfeiffer Treatment Center. RESULTS: Compared with controls, children with autism had significantly higher urinary levels of isoprostane F(2alpha)-VI, 2,3-dinor-thromboxane B(2), and 6-keto-prostaglandin F(1alpha). Lipid peroxidation levels directly correlated with both vascular biomarker ratios. CONCLUSION: Besides enhanced oxidative stress, platelet and vascular endothelium activation also could contribute to the development and clinical manifestations of autism.