Chronic cholesterol administration to the brain supports complete and long-lasting cognitive and motor amelioration in Huntington's disease.

Evidence that Huntington's disease (HD) is characterized by impaired cholesterol biosynthesis in the brain has led to strategies to increase its level in the brain of the rapidly progressing R6/2 mouse model, with a positive therapeutic outcome. Here we tested the long-term efficacy of chronic administration of cholesterol to the brain of the slowly progressing zQ175DN knock-in HD mice in preventing ("early treatment") or reversing ("late treatment") HD symptoms. To do this we used the most advanced formulation of cholesterol loaded brain-permeable nanoparticles (NPs), termed hybrid-g7-NPs-chol, which were injected intraperitoneally. We show that one cycle of treatment with hybrid-g7-NPs-chol, administered in the presymptomatic ("early treatment") or symptomatic ("late treatment") stages is sufficient to normalize cognitive defects up to 5 months, as well as to improve other behavioral and neuropathological parameters. A multiple cycle treatment combining both early and late treatments ("2 cycle treatment") lasting 6 months generates therapeutic effects for more than 11 months, without severe adverse reactions. Sustained cholesterol delivery to the brain of zQ175DN mice also reduces mutant Huntingtin aggregates in both the striatum and cortex and completely normalizes synaptic communication in the striatal medium spiny neurons compared to saline-treated HD mice. Furthermore, through a meta-analysis of published and current data, we demonstrated the power of hybrid-g7-NPs-chol and other strategies able to increase brain cholesterol biosynthesis, to reverse cognitive decline and counteract the formation of mutant Huntingtin aggregates. These results demonstrate that cholesterol delivery via brain-permeable NPs is a therapeutic option to sustainably reverse HD-related behavioral decline and neuropathological signs over time, highlighting the therapeutic potential of cholesterol-based strategies in HD patients. DATA AVAILABILITY: This study does not include data deposited in public repositories. Data are available on request to the corresponding authors.

Toxicology of 3-monochloropropane-1,2-diol and its esters: a narrative review.

3-Monochloropropane-1,2-diol (3-MCPD) is a chiral molecule naturally existing as a racemic mixture of (R)- and (S)-enantiomers. It was thoroughly investigated during the 1970s as a male antifertility drug until research was abandoned because of the side effects observed in toxicity studies. More than 20 years later, 3-MCPD, both in the free form and esterified to the fatty acids, was detected in vegetable oil and discovered to be a widespread contaminant in different processed foods. This review summarises the main toxicological studies on 3-MCPD and its esters. Current knowledge shows that the kidney and reproductive system are the primary targets of 3-MCPD toxicity, followed by neurological and immune systems. Despite uncertainties, in vivo studies suggest that renal and reproductive toxicity is mediated by toxic metabolites, leading to inhibition of glycolysis and energy depletion. Few acute, short-term, and subchronic toxicity studies have investigated the 3-MCPD esters. The pattern of toxicity was similar to that of free 3-MCPD. Some evidence suggests that the toxicity of 3-MCPD diesters may be milder than 3-MCPD, likely because of an incomplete enzymatic hydrolysis in the equivalent free form in the gastrointestinal tract. Further research to clarify absorption, metabolism, and long-term toxicity of 3-MCPD esters would be pivotal to improve the risk assessment of these compounds via food.

The mode of dexamethasone decoration influences avidin-nucleic-acid-nano-assembly organ biodistribution and in vivo drug persistence.

Avidin-Nucleic-Acid-NanoASsemblies (ANANAS) possess natural tropism for the liver and, when loaded with dexamethasone, reduce clinical progression in an autoimmune hepatitis murine model. Here, we investigated the linker chemistry (hydrazide-hydrazone, Hz-Hz, or carbamate hydrazide-hydrazone, Cb-Hz bond) and length (long, 5 kDa PEG, or short, 5-6 carbons) in biotin-dexamethasone conjugates used for nanoparticle decoration through in vitro and in vivo studies. All four newly synthesized conjugates released the drug at acidic pH only. In vitro, the Hz-Hz and the PEG derivatives were less stable than the Cb-Hz and the short chain ones, respectively. Once injected in healthy mice, dexamethasone location in the PEGylated ANANAS outer layer favors liver penetration and resident macrophages uptake, while drug Hz-Hz, but not Cb-Hz, short spacing prolongs drug availability. In conclusion, the tight modulation of ANANAS decoration can significantly influence the host interaction, paving the way for the development of steroid nanoformulations suitable for different pharmacokinetic profiles.

Plasma and Brain Concentrations of Doxycycline after Single and Repeated Doses in Wild-Type and APP23 Mice.

Repurposing doxycycline for the treatment of amyloidosis has recently been put forward because of the antiaggregating and anti-inflammatory properties of the drug. Most of the investigations of the therapeutic potential of doxycycline for neurodegenerative amyloidosis, e.g., prion and Alzheimer disease (AD), have been carried out in mouse models, but surprisingly no data are available regarding the concentrations reached in the brain after systemic administration. We filled this gap by analyzing the pharmacokinetic profile of doxycycline in plasma and brain after single and repeated intraperitoneal injections of 10 and 100 mg/kg, in wild-type mice and the APP23 mouse model of AD. The main outcomes of our study are: 1) Peak plasma concentrations ranged from 2 to10 µg/ml, superimposable to those in humans; 2) brain-to-plasma ratio was ∼0.2, comparable to the cerebrospinal fluid/serum ratios in humans; 3) brain Cmax 4-6 hours after a single dose was ∼0.5 (10 mg/kg) and ∼5 µM (100 mg/kg). Notably, these concentrations are lower than those required for the drug's antiaggregating properties as observed in cell-free studies, suggesting that other features underlie the positive cognitive effects in AD mice; 4) elimination half-life was shorter than in humans (3-6 vs. 15-30 hours), therefore no significant accumulation was observed in mouse brain following repeated treatments; and 5) there were no differences between doxycycline concentrations in brain areas of age-matched wild-type and APP23 mice. These data are useful for planning preclinical studies with translational validity, and to identify more reliably the mechanism(s) of action underlying the central in vivo effects of doxycycline.

Identification of Sulfonated and Hydroxy-Sulfonated Polychlorinated Biphenyl (PCB) Metabolites in Soil: New Classes of Intermediate Products of PCB Degradation?

In this paper we describe the identification of two classes of contaminants: sulfonated-PCBs and hydroxy-sulfonated-PCBs. This is the first published report of the detection of these chemicals in soil. They were found, along with hydroxy-PCBs, in soil samples coming from a site historically contaminated by the industrial production of PCBs and in background soils. Sulfonated-PCB levels were approximately 0.4-0.8% of the native PCB levels in soils and about twice the levels of hydroxy-sulfonated-PCBs and hydroxy-PCBs. The identification of sulfonated-PCBs was confirmed by the chemical synthesis of reference standards, obtained through the sulfonation of an industrial mixture of PCBs. We then reviewed the literature to investigate for the potential agents responsible for the sulfonation. Furthermore, we predicted their physicochemical properties and indicate that, given the low pKa of sulfonated- and hydroxy-sulfonated-PCBs, they possess negligible volatility, supporting the case for in situ formation from PCBs. This study shows the need of understanding their origin, their role in the degradation path of PCBs, and their fate, as well as their (still unknown) toxicological and ecotoxicological properties.

Brain Disposition of cis-para-Methyl-4-Methylaminorex (cis-4,4'-DMAR) and Its Potential Metabolites after Acute and Chronic Treatment in Rats: Correlation with Central Behavioral Effects.

para-Methyl-4-methylaminorex (4,4'-DMAR) is a phenethylamine derivative with psychostimulant activity whose abuse has been associated with several deaths and a wide range of adverse effects. We recently validated a high-performance liquid chromatography-tandem mass spectrometry method to measure the compound's concentrations in plasma, and we applied it to describe the pharmacokinetic properties of 4,4'-DMAR after a single dose in rats. In this study, we investigated the brain disposition and metabolism of cis-4,4'-DMAR after intraperitoneal injection as well as its central behavioral effects. Locomotor activity increased after a single injection of 10 mg/kg, peaking at 2 hours and disappearing at 5 hours; in these conditions, brain absorption was very rapid, (tmax = 30-60 minutes) and large (brain-to-plasma ratio = 24); the half-life was approximately 50 minutes. After 14 daily doses, the compound's effect on locomotor activity was greater (approximately 20% compared with the effect after the first dose), but not for pharmacokinetic reasons. Using high-resolution mass spectrometry, we also identified four metabolites of cis-4,4'-DMAR in the plasma and brain of treated rats. Semiquantitative analysis indicated low brain permeability and very low brain concentrations, suggesting that these metabolites do not contribute to central behavioral effects; however, the metabolite originating from oxidation of the para-methyl group (M2) persisted in the plasma longer and at higher concentrations than the parent molecule and could be used to evaluate drug intake in human consumers. Finally, we describe the rewarding effect of cis-4,4'-DMAR in the conditioning place preference test, suggesting a high risk of addiction in humans.

Automated online solid-phase extraction-liquid chromatography mass spectrometric analysis of dithianon in water.

An automated online solid-phase extraction-liquid chromatography mass spectrometry (SPE-LC/MS) method was developed for the quantification of dithianon in surface water samples, using warfarin as internal standard. The method was developed on a liquid chromatography (LC) system with Flexible Cube interfaced to a quadrupole time-of-flight (Q-TOF) mass spectrometer. A small volume of acidified water (1 mL) was spiked with internal standard, pre-concentrated online on polymeric cartridges and analyzed by full-scan MS in high-resolution conditions. The quantitative data were obtained by [M]-• of dithianon and [M - H]- of warfarin, used as internal standard. The chromatographic separation was performed on a C18 column with a gradient mobile phase consisting of acetonitrile and water containing 0.05% acetic acid. The method was validated to measure concentrations of dithianon in the range of 0.010-4 µg L-1 in surface water samples. Twenty real water samples, collected from Torrente Novella, Val di Non (TN, Northern Italy), during fungicide treatments of large apple orchards, were analyzed. All samples were kept in glass bottles and stored in the lab at -20°C until analysis. It was found that in all samples dithianon was undetectable: if it is present, its concentration was lower than the limit of detection (LOD) (0.008 µg L-1.To investigate the stability of dithianon, a series of water samples were spiked at different concentrations and analyzed after different storage conditions. Results suggested that dithianon is not stable in water stored at -20°C at neutral or basic pH, but the addition of acetic acid to pH = 3.5 increases its stability to at least two weeks.

Brain Uptake of Tetrahydrohyperforin and Potential Metabolites after Repeated Dosing in Mice.

Tetrahydrohyperforin (IDN-5706) is a semisynthetic derivative of hyperforin, one of the main active components of Hypericum perforatum extracts. It showed remarkable positive effects on memory and cognitive performances in wild-type mice and in a transgenic mouse model of Alzheimer's disease, but little was known about the concentrations it can reach in the brain. The investigations reported herein show that repeated treatment of mice with tetrahydrohyperforin (20 mg/kg intraperitoneally, twice daily for 4 days and once on the fifth day) results in measurable concentrations in the brain, up to 367 ng/g brain (∼700 nM) 6 h after the last dose; these concentrations have significant effects on synaptic function in hippocampal slices. The other main finding was the identification and semiquantitative analysis of tetrahydrohyperforin metabolites. In plasma, three hydroxylated/dehydrogenated metabolites were the largest (M1-3) and were also formed in vitro on incubation of tetrahydrohyperforin with mouse liver microsomes; the fourth metabolite in abundance was a hydroxylated/deisopropylated derivative (M13), which was not predicted in vitro. These metabolites were all detected in the brain, with peak areas from 10% (M1) to ∼1.5% (M2, M3, and M13) of the parent compound. In summary, repeated treatment of mice with tetrahydrohyperforin gave brain concentrations that might well underlie its central pharmacological effects. We also provide the first metabolic profile of this compound.

Chemical characterization, antioxidant and cytotoxic activities of the methanolic extract of Hymenocrater longiflorus grown in Iraq.

Hymenocrater longiflorus was collected from northern Iraq, and the chemical composition and antioxidant and cytotoxic activities of this plant were investigated. Ten compounds detected by HPLC-ESI/MS were identified as flavonoids and phenolic acids. The free radical scavenging activity of the 70% methanol extract was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The antioxidant activities of the extract may be attributed to its polyphenolic composition. The cytotoxicity of the plant extract against the osteosarcoma (U2OS) cell line was assessed with the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The extract significantly reduced the viability of cells in a concentration- and time-dependent manner. Cells were arrested during the S-phase of the cell cycle, and DNA damage was revealed by antibodies against histone H2AX. The apoptotic features of cell shrinkage and decrease in cell size were also observed. Western blot analysis revealed cleavage of poly (ADP-ribose)-polymerase 1 (PARP-1), in addition to increases in the proteins p53, p21, and γ-H2AX. Collectively, our findings demonstrate that the H. longiflorus extract is highly cytotoxic to U2OS cells, most likely due to its polyphenolic composition.

Validated LC-MS/MS Assay for the Quantitative Determination of Fenretinide in Plasma and Tumor and Its Application in a Pharmacokinetic Study in Mice of a Novel Oral Nanoformulation of Fenretinide.

We describe the development and validation of a HPLC-MS/MS method to assess the pharmacokinetics and tumor distribution of fenretinide, a synthetic retinoid chemically related to all-trans-retinoic acid, after administration of a novel oral nanoformulation of fenretinide, called bionanofenretinide (BNF). BNF was developed to overcome the major limitation of fenretinide: its poor aqueous solubility and bioavailability due to its hydrophobic nature. The method proved to be reproducible, precise and highly accurate for the measurement of the drug and the main metabolites. The lower limit of quantification resulted in 1 ng/mL. The curve range of 1-500 ng/mL and 50-2000 ng/mL, for plasma and tumor homogenate, respectively, was appropriate for the analysis, as demonstrated by the accuracy of between 96.8% and 102.4% for plasma and 96.6 to 102.3% for the tumor. The interdays precision and accuracy determined on quality controls at three different levels were in the ranges of 6.9 to 7.5% and 99.3 to 101.0%, and 0.96 to 1.91% and 102.3 to 105.8% for plasma and tumor, respectively. With the application of the novel assay in explorative pharmacokinetic studies, following acute and chronic oral administration of the nanoformulation, fenretinide was detected in plasma and tumor tissue at a concentration higher than the IC50 value necessary for in vitro inhibitory activity (i.e., 1-5 µM) in different cancer cells lines. We were also able to detect the presence in plasma and tumor of active and inactive metabolites of fenretinide.

Structure-guided optimization of 3-hydroxybenzoisoxazole derivatives as inhibitors of Aldo-keto reductase 1C3 (AKR1C3) to target prostate cancer.

AKR1C3 is an enzyme that is overexpressed in several types of radiotherapy- and chemotherapy-resistant cancers. Despite AKR1C3 is a validated target for drug development, no inhibitor has been approved for clinical use. In this manuscript, we describe our study of a new series of potent AKR1C3-targeting 3-hydroxybenzoisoxazole based inhibitors that display high selectivity over the AKR1C2 isoform and low micromolar activity in inhibiting 22Rv1 prostate cancer cell proliferation. In silico studies suggested proper substituents to increase compound potency and provided with a mechanistic explanation that could clarify their different activity, later confirmed by X-ray crystallography. Both the in-silico studies and the crystallographic data highlight the importance of 90° rotation around the single bond of the biphenyl group, in ensuring that the inhibitor can adopt the optimal binding mode within the active pocket. The p-biphenyls that bear the meta-methoxy, and the ortho- and meta-trifluoromethyl substituents (in compounds 6a, 6e and 6f respectively) proved to be the best contributors to cellular potency as they provided the best IC50 values in series (2.3, 2.0 and 2.4 µM respectively) and showed no toxicity towards human MRC-5 cells. Co-treatment with scalar dilutions of either compound 6 or 6e and the clinically used drug abiraterone led to a significant reduction in cell proliferation, and thus confirmed that treatment with both CYP171A1-and AKR1C3-targeting compounds possess the potential to intervene in key steps in the steroidogenic pathway. Taken together, the novel compounds display desirable biochemical potency and cellular target inhibition as well as good in-vitro ADME properties, which highlight their potential for further preclinical studies.

Environmental fate of sulfonated-PCBs: Soil partitioning properties, bioaccumulation, persistence, and mobility.

Two new classes of PCB metabolites were recently discovered: sulfonated-polychlorinated biphenyls (sulfonated-PCBs) and hydroxy-sulfonated-polychlorinated biphenyls (OH-sulfonated-PCBs). These metabolites, originating from PCB degradation, seem to possess more polar characteristics than their parent compounds. However, no other information, such as their chemical identity (CAS number) or their ecotoxicity or toxicity, is available so far, although more than about one hundred different chemicals were observed in soil samples. In addition, their physico-chemical properties are still uncertain since only estimations are available. Here we show the first evidence on the fate of these new classes of contaminants in the environment, producing results from several experiments, to evaluate sulfonated-PCBs and OH-sulfonated-PCBs soil partition coefficients, degradation in soil after 18 months of rhizoremediation, uptake into plant roots and earthworms, as well as a preliminary analytical method to extract and concentrate these chemicals from water. The results give an overview of the expected environmental fate of these chemicals and open questions for further studies.

Synthesis of a new sulfonated-hexachlorobiphenyl standard for environmental analysis, ecotoxicological, and toxicological studies.

Sulfonated-polychlorinated biphenyls (sulfonated-PCBs) are a newly discovered class of PCB metabolites. They were observed for the first time in polar bear serum and lately, in soil, together with hydroxy-sulfonated-PCBs. Their presence is ubiquitous in soils, and their estimated physical chemical properties show high mobility in water, compared to the parent compounds. However, no single pure standards exist so far and therefore their quantification in the environmental matrices is not accurate. Additionally, pure standards are needed to experimentally determine their physical chemical properties, as well as the ecotoxicological and toxicological characteristics. In the present work, the challenging goal of preparing a polychlorinated biphenyl monosulfonic acid was achieved exploring different synthetic approaches, along which the selection of the starting material resulted in a crucial point. Using PCB-153 (2,2'-4,4'-5,5'-hexachloro-1,1'-biphenyl) the synthesis afforded, as the major species, a side compound. On the contrary, the use of PCB-155 (2,2'-4,4'-6,6'-hexachloro-1,1'-biphenyl), a symmetric hexachlorobiphenyl derivative showing chlorine atoms at all the ortho positions, gave the target sulfonated-PCB compound. In this case, sulfonation was successfully carried out through a two-step procedure, involving chlorosulfonylation and the subsequent hydrolysis of the chlorosulfonyl intermediate.

Bioaccumulation of PCBs and their hydroxy and sulfonated metabolites in earthworms: Comparing lab and field results.

Sulfonated and hydroxy-sulfonated PCBs were recently discovered by our group as new PCB soil contaminants, constituting about 1% of their parent compounds in soil. Here we investigate for the first time the bioaccumulation of these metabolites as well as hydroxy-PCBs and native PCBs in earthworms. A sequence of three experiments, at increasing complexity and ecological realism, were performed with four different earthworm species (Eisenia foetida Savigny, Lumbricus terrestris L, Allolobophora chlorotica Savigny and Aporrectodea caliginosa Savigny) exposed to contaminated soils. The first experiment confirmed that when exposing earthworms to soil contaminated with a single hexa-chlorinated congener (PCB 155), no formation of polar metabolites in earthworms could be detected. This allowed to plan the following two experiments, using a soil from a PCB contaminated site and rich in relatively high levels (10-130 µg kg-1) of hydroxy-, sulfonated-, and hydroxy-sulfonated-PCBs. Bioaccumulation factors (BAFs) and bioconcentration factors (BCFs) were then obtained in the second and third experiments, to compare the accumulation behavior of these chemicals in laboratory and natural conditions. Regressions between BAF/BCF and Log Kow/Log D, produced a variety of results, being generally significant between BCF and PCBs and not significant in the other cases. In general, the metabolites accumulated in earthworms with detectable concentrations in their tissues (8-115 µg kg-1), although sulfonated and hydroxy-sulfonated PCBs showed BAF and BCF values lower (up to two orders of magnitude) than those calculated for the parent PCBs, given their lower lipophilicity.

Development of a Nanoparticle-Based Approach for the Blood-Brain Barrier Passage in a Murine Model of Amyotrophic Lateral Sclerosis.

The development of nanoparticles (NPs) to enable the passage of drugs across blood-brain barrier (BBB) represents one of the main challenges in neuropharmacology. In recent years, NPs that are able to transport drugs and interact with brain endothelial cells have been tested. Here, we investigated whether the functionalization of avidin-nucleic-acid-nanoassembly (ANANAS) with apolipoprotein E (ApoE) would allow BBB passage in the SOD1G93A mouse model of amyotrophic lateral sclerosis. Our results demonstrated that ANANAS was able to transiently cross BBB to reach the central nervous system (CNS), and ApoE did not enhance this property. Next, we investigated if ANANAS could improve CNS drug delivery. To this aim, the steroid dexamethasone was covalently linked to ANANAS through an acid-reversible hydrazone bond. Our data showed that the steroid levels in CNS tissues of SOD1G93A mice treated with nanoformulation were below the detection limit. This result demonstrates that the passage of BBB is not sufficient to guarantee the release of the cargo in CNS and that a different strategy for drug tethering should be devised. The present study furthermore highlights that NPs can be useful in improving the passage through biological barriers but may limit the interaction of the therapeutic compound with the specific target.

Dihydroorotate dehydrogenase inhibition reveals metabolic vulnerability in chronic myeloid leukemia.

The development of different generations of BCR-ABL1 tyrosine kinase inhibitors (TKIs) has led to the high overall survival of chronic myeloid leukemia (CML) patients. However, there are CML patients who show resistance to TKI therapy and are prone to progress to more advanced phases of the disease. So, implementing an alternative approach for targeting TKIs insensitive cells would be of the essence. Dihydroorotate dehydrogenase (DHODH) is an enzyme in the de novo pyrimidine biosynthesis pathway that is located in the inner membrane of mitochondria. Here, we found that CML cells are vulnerable to DHODH inhibition mediated by Meds433, a new and potent DHODH inhibitor recently developed by our group. Meds433 significantly activates the apoptotic pathway and leads to the reduction of amino acids and induction of huge metabolic stress in CML CD34+ cells. Altogether, our study shows that DHODH inhibition is a promising approach for targeting CML stem/progenitor cells and may help more patients discontinue the therapy.

Targeting Acute Myelogenous Leukemia Using Potent Human Dihydroorotate Dehydrogenase Inhibitors Based on the 2-Hydroxypyrazolo[1,5-a]pyridine Scaffold: SAR of the Aryloxyaryl Moiety.

In recent years, human dihydroorotate dehydrogenase inhibitors have been associated with acute myelogenous leukemia as well as studied as potent host targeting antivirals. Starting from MEDS433 (IC50 1.2 nM), we kept improving the structure-activity relationship of this class of compounds characterized by 2-hydroxypyrazolo[1,5-a]pyridine scaffold. Using an in silico/crystallography supported design, we identified compound 4 (IC50 7.2 nM), characterized by the presence of a decorated aryloxyaryl moiety that replaced the biphenyl scaffold, with potent inhibition and pro-differentiating abilities on AML THP1 cells (EC50 74 nM), superior to those of brequinar (EC50 249 nM) and boosted when in combination with dipyridamole. Finally, compound 4 has an extremely low cytotoxicity on non-AML cells as well as MEDS433; it has shown a significant antileukemic activity in vivo in a xenograft mouse model of AML.

An integrated approach, based on mass spectrometry, for the assessment of imidacloprid metabolism and penetration into mouse brain and fetus after oral treatment.

Imidacloprid is an insecticide belonging to neonicotinoids, a class of agonists of the nicotinic acetylcholine receptors that shows higher affinities in insects compared to mammals. However, recent evidence show that neonicotinoids can bind to the mammalian receptors, leading to detrimental responses in cultured neurons. We developed an analytical strategy which uses mass spectrometry with multiple reaction monitoring (targeted approach) and high-resolution acquisitions (untargeted approach), which were applied to quantify imidacloprid and to identify its metabolites in biological tissues after oral treatments of mice. Mouse dams were treated with doses from 0.118 mg/kg bw day up to 41 mg/kg day between gestational days 6-9. Results showed quantifiable levels of imidacloprid in plasma (from 30.48 to 5705 ng/mL) and brain (from 20.48 to 5852 ng/g) of treated mice, proving the passage through the mammalian blood-brain barrier with a high correspondence between doses and measured concentrations. Untargeted analyses allowed the identification of eight metabolites including imidacloprid-olefin, hydroxy-imidacloprid dihydroxy-imidacloprid, imidacloprid-nitrosimine, desnitro-imidacloprid, 6-chloronicotinic acid, 5-(methylsulfanyl)pyridine-2-carboxylic acid and N-imidazolidin-2-ylidenenitramide in plasma and brain. Moreover, analysis of embryonic tissues after oral treatment of mouse dams showed detectable levels of imidacloprid (816.6 ng/g after a dose of 4.1 mg/Kg bw day and 5646 ng/g after a dose of 41 mg/Kg bw day) and its metabolites, proving the permeability of the placenta barrier. Although many studies have been reported on the neurotoxicity of neonicotinoids, our study paves the way for a risk assessment in neurodevelopmental toxicity, demostrating the capability of imidacloprid and its metabolites to pass the biological barriers.

Tumor vascular remodeling by thrombospondin-1 enhances drug delivery and antineoplastic activity.

The disorganized and inefficient tumor vasculature is a major obstacle to the delivery and efficacy of antineoplastic treatments. Antiangiogenic agents can normalize the tumor vessels, improving vessel function and boosting the distribution and activity of chemotherapy. The type III repeats (T3R) domain of thrombospondin-1 contains different potential antiangiogenic sequences. We therefore hypothesized that it might affect the tumor vasculature. Ectopic expression of the T3R domain by the tumor cells or by the host, or administration of recombinant T3R, delayed the in vivo growth of experimental tumors. Tumors presented marked reorganization of the vasculature, with abundant but smaller vessels, associated with substantially less necrosis. Mechanistically, the use of truncated forms of the domain, containing different active sequences, pointed to the FGF2/FGFR/ERK axis as a target for T3R activity. Along with reduced necrosis, the expression of T3R promoted tumor distribution of chemotherapy (paclitaxel), with a higher drug concentration and more homogeneous distribution, as assessed by HPLC and MALDI imaging mass spectrometry. T3R-expressing tumors were more responsive to paclitaxel and cisplatin. This study shows that together with its known role as a canonical inhibitor of angiogenesis, thrombospondin-1 can also remodel tumor blood vessels, affecting the morphological and functional properties of the tumor vasculature. The ability of T3R to reduce tumor growth and improve the response to chemotherapy opens new perspectives for therapeutic strategies based on T3R to be used in combination therapies.

Brain Kynurenine Pathway and Functional Outcome of Rats Resuscitated From Cardiac Arrest.

Background Brain injury and neurological deficit are consequences of cardiac arrest (CA), leading to high morbidity and mortality. Peripheral activation of the kynurenine pathway (KP), the main catabolic route of tryptophan metabolized at first into kynurenine, predicts poor neurological outcome in patients resuscitated after out-of-hospital CA. Here, we investigated KP activation in hippocampus and plasma of rats resuscitated from CA, evaluating the effect of KP modulation in preventing CA-induced neurological deficit. Methods and Results Early KP activation was first demonstrated in 28 rats subjected to electrically induced CA followed by cardiopulmonary resuscitation. Hippocampal levels of the neuroactive metabolites kynurenine, 3-hydroxy-anthranilic acid, and kynurenic acid were higher 2 hours after CA, as in plasma. Further, 36 rats were randomized to receive the inhibitor of the first step of KP, 1-methyl-DL-tryptophan, or vehicle, before CA. No differences were observed in hemodynamics and myocardial function. The CA-induced KP activation, sustained up to 96 hours in hippocampus (and plasma) of vehicle-treated rats, was counteracted by the inhibitor as indicated by lower hippocampal (and plasmatic) kynurenine/tryptophan ratio and kynurenine levels. 1-Methyl-DL-tryptophan reduced the CA-induced neurological deficits, with a significant correlation between the neurological score and the individual kynurenine levels, as well as the kynurenine/tryptophan ratio, in plasma and hippocampus. Conclusions These data demonstrate the CA-induced lasting activation of the first step of the KP in hippocampus, showing that this activation was involved in the evolving neurological deficit. The degree of peripheral activation of KP may predict neurological function after CA.