Deuterium-Stabilized (R)-Pioglitazone, PXL065, for Treatment of X-Linked Adrenoleukodystrophy (ALD)

Background and aims: X-linked Adrenoleukodystrophy (ALD) is a rare neurometabolic disorder caused by ABCD1- gene mutations, leading to Very-Long-Chain Fatty Acids (VLCFA; in particular C26:0) accumulation, inflammation, mitochondrial impairment and demyelination. PXL065, a clinical-stage deuterium-stabilized(R)-stereoisomer of pioglitazone, retains pioglitazone non-genomic actions but lacks PPARγ activity. As pioglitazone exhibits beneficial effects in ALD models and PXL065 may avoid PPARγ- related side effects, we investigated PXL065 effects of in preclinical models. Methods: Patient-derived fibroblasts and lymphocytes and Abcd1-KO mouse glial cells were exposed to PXL065 (5-10μM) and pioglitazone (10μM) for 7 days. VLCFA content was measured by mass spectrometry, selected gene expression by RT-qPCR, and mitochondrial function using a Seahorse Analyzer (after 72hr). PXL065 or pioglitazone (15mg/kg QD) were administered to 6-8-week or 13-month old Abcd1-KO mice for 8 and 12 weeks, respectively. VLCFA content (mass spectrometry), sciatic nerve axonal morphology (electronic microscopy), and locomotor function (open field test) were measured. Results: In patient and mouse glial cells, PXL065 and pioglitazone corrected C26:0, improved mitochondrial function, increased compensatory Abcd2-3 transporter gene expression, and decreased inflammatory gene expression. In Abcd1-KO mice, C26:0 levels were normalized in plasma and decreased in spinal cord (-55%, p\u3c0.01) and brain (-49%, p\u3c0.0001). Pioglitazone had no effect in spinal cord. Following PXL065 and pioglitazone treatment, abnormal axonal morphology (stellate-shaped cells) was improved but only PXL065 showed significantly improved locomotor test results. Conclusion: Despite reduced PPARγ activity, PXL065 showed substantial signs of efficacy and superior therapeutic potential vs. pioglitazone (in vivo) supporting clinical development for ALD. A Phase 2a study is planned in 2022

Biomater. Sci.

Several tissue engineering approaches are based on the ability of mesenchymal cells to endogenously synthesize an extracellular matrix (ECM) in vitro, which can be seen as a form of biomaterial. Accordingly, the inter-donor variability of cell-assembled extracellular matrix (CAM) production is a key parameter to understand in order to progress towards clinical applications, especially for autologous strategies. In this study, CAMs were produced, under good manufacturing process conditions, from skin fibroblasts of 21 patients as part of a clinical trial to evaluate a tissue-engineered vascular graft. The inter-donor variability of CAM strength, thickness, hydroxyproline, and glycosaminoglycan was substantial (coefficient of variability of 33%, 19%, 24%, and 19%, respectively), but a significant correlation was observed between all four properties (Pearson r: 0.43 to 0.70; p-value ≤ 0.05). A CAM matrisome analysis, performed by mass spectrometry, revealed the presence of 70 ECM-related proteins. Our study shows that the relative abundance of 16 proteins (15 non-collagenous) correlated with CAM thickness. These proteins also correlated with CAM hydroxyproline content, as well as 21 other proteins that included fibrillar collagens and non-collagenous proteins. However, data demonstrated that only the relative abundance of type I collagen subunit alpha-1 was correlated to CAM strength. This study is the most extensive evaluation of CAM inter-donor variability to date and will help tissue engineers working with this type of biomaterial to design strategies that take into account this variability, especially for autologous tissue manufacturing

Biomaterials

Cell-assembled extracellular matrix (CAM) has been used to produce vascular grafts. While these completely biological vascular grafts performed well in clinical trials, the in vivo remodeling and inflammatory response of this truly "bio" material has not yet been investigated. In this study, human CAM yarns were implanted subcutaneously in nude rats to investigate the innate immune response to this matrix. The impact of processing steps relevant to yarn manufacturing was evaluated (devitalization, decellularization, gamma sterilization, and twisting). We observed that yarns were still present after six months, and were integrated into a non-inflamed loose connective tissue. The CAM was repopulated by fibroblastic cells and blood vessels. While other yarns caused minor peripheral inflammation at an early stage (two weeks of implantation), gamma sterilization triggered a more intense host response dominated by the presence of M1 macrophages. The inflammatory response was resolved at six months. Yarn mechanical strength was decreased two weeks after implantation except for the more compact "twisted" yarn. While the strength of other yarns was stable after initial remodeling, the gamma-sterilized yarn continued to lose mechanical strength over time and was weaker than devitalized (control) yarns at six months. This is the first study to formally demonstrate that devitalized human CAM is very long-lived in vivo and does not trigger a degradative response, but rather is very slowly remodeled. This data supports a strategy to produce human textiles from CAM yarn for regenerative medicine applications where a scaffold with low inflammation and long-term mechanical properties are critical

Direct AMPK activation corrects NASH in rodents through metabolic effects and direct action on inflammation and fibrogenesis

No approved therapies are available for nonalcoholic steatohepatitis (NASH). Adenosine monophosphate–activated protein kinase (AMPK) is a central regulator of cell metabolism; its activation has been suggested as a therapeutic approach to NASH. Here we aimed to fully characterize the potential for direct AMPK activation in preclinical models and to determine mechanisms that could contribute to efficacy for this disease. A novel small-molecule direct AMPK activator, PXL770, was used. Enzyme activity was measured with recombinant complexes. De novo lipogenesis (DNL) was quantitated in vivo and in mouse and human primary hepatocytes. Metabolic efficacy was assessed in ob/ob and high-fat diet–fed mice. Liver histology, biochemical measures, and immune cell profiling were assessed in diet-induced NASH mice. Direct effects on inflammation and fibrogenesis were assessed using primary mouse and human hepatic stellate cells, mouse adipose tissue explants, and human immune cells. PXL770 directly activated AMPK in vitro and reduced DNL in primary hepatocytes. In rodent models with metabolic syndrome, PXL770 improved glycemia, dyslipidemia, and insulin resistance. In mice with NASH, PXL770 reduced hepatic steatosis, ballooning, inflammation, and fibrogenesis. PXL770 exhibited direct inhibitory effects on pro-inflammatory cytokine production and activation of primary hepatic stellate cells. Conclusion: In rodent models, direct activation of AMPK is sufficient to produce improvements in all core components of NASH and to ameliorate related hyperglycemia, dyslipidemia, and systemic inflammation. Novel properties of direct AMPK activation were also unveiled: improved insulin resistance and direct suppression of inflammation and fibrogenesis. Given effects also documented in human cells (reduced DNL, suppression of inflammation and stellate cell activation), these studies support the potential for direct AMPK activation to effectively treat patients with NASH

Reactive oxygen-derived free radicals are key to the endothelial dysfunction of diabetes.

Vascular complications are an important pathological issue in diabetes that lead to the further functional deterioration of several organs. The balance between endothelium-dependent relaxing factors and endothelium-dependent contracting factors (EDCFs) is crucial in controlling local vascular tone and function under normal conditions. Diabetic endothelial dysfunction is characterized by reduced endothelium-dependent relaxations and/or enhanced endothelium-dependent contractions. Elevated levels of oxygen-derived free radicals are the initial source of endothelial dysfunction in diabetes. Oxygen-derived free radicals not only reduce nitric oxide bioavailability, but also facilitate the production and/or action of EDCFs. Thus, the endothelial balance tips towards vasoconstrictor responses over the course of diabetes. © 2009 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Blackwell Publishing Asia Pty Ltd.postprin

Developmental changes in mesenteric artery reactivity in embryonic and newly hatched chicks

At birth, the intestine becomes the sole site for nutrient absorption requiring a dramatic increase in blood flow. The vascular changes accompanying this transition have been partly characterized in mammals. We investigated, using wire myography, the developmental changes in chick mesenteric artery (MA) reactivity. Rings of the MA from 15-day (E15) and 19-day (E19) chicken embryos (total incubation 21 days) as well as non-fed 0–3-h-old (NH3h) and first-fed 1-day-old (NH1d) newly hatched chicks contracted in response to KCl, norepinephrine (NE), U46619, and endothelin (ET)-1 and relaxed in response to acetylcholine (ACh), sodium nitroprusside (SNP), and forskolin indicating the presence of electro- and pharmaco-mechanical coupling as well as cGMP- and cAMP-mediated relaxation. In ovo development and transition to ex ovo life was accompanied by alterations in the response of the MAs, but a different developmental trajectory was observed for each reactivity pathway tested. Thus, the contractile efficacy of KCl underwent a linear increase (E15 < E19 < NH3h < NH1d). The efficacy of NE and U46619 increased in ovo, but not ex ovo (E15 < E19 = NH3h = NH1d) and the efficacy of ET-1 peaked at E19 (E15 < E19 > NH3h = NH1d). The relaxations elicited by ACh (endothelium-dependent), SNP, and forskolin did not undergo significant developmental changes. In conclusion, the ability of chick MAs to constrict in response to pharmacological stimuli increases during the embryonic period, but no dramatic changes are induced by hatching or the first feeding. Maturation of vasodilator mechanisms precedes that of vasoconstrictor mechanisms. Alterations of the delicate balance between vasoconstrictors and vasodilators may play an important role in perinatal intestinal diseases

Hypertension Is Associated with Marked Alterations in Sphingolipid Biology: A Potential Role for Ceramide

Background Hypertension is, amongst others, characterized by endothelial dysfunction and vascular remodeling. As sphingolipids have been implicated in both the regulation of vascular contractility and growth, we investigated whether sphingolipid biology is altered in hypertension and whether this is reflected in altered vascular function. Methods and Findings In isolated carotid arteries from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats, shifting the ceramide/S1P ratio towards ceramide dominance by administration of a sphingosine kinase inhibitor (dimethylsphingosine) or exogenous application of sphingomyelinase, induced marked endothelium-dependent contractions in SHR vessels (DMS: 1.4±0.4 and SMase: 2.1±0.1 mN/mm; n = 10), that were virtually absent in WKY vessels (DMS: 0.0±0.0 and SMase: 0.6±0.1 mN/mm; n = 9, p Conclusions Hypertension is associated with marked alterations in vascular sphingolipid biology such as elevated ceramide levels and signaling, that contribute to increased vascular tone

Endothelial dysfunction and vascular disease

The endothelium can evoke relaxations (dilatations) of the underlying vascular smooth muscle, by releasing vasodilator substances. The best characterized endothelium-derived relaxing factor (EDRF) is nitric oxide (NO). The endothelial cells also evoke hyperpolarization of the cell membrane of vascular smooth muscle (endothelium-dependent hyperpolarizations, EDHF-mediated responses). Endothelium-dependent relaxations involve both pertussis toxin-sensitive G i (e.g. responses to serotonin and thrombin) and pertussis toxin-insensitive G q (e.g. adenosine diphosphate and bradykinin) coupling proteins. The release of NO by the endothelial cell can be up-regulated (e.g. by oestrogens, exercise and dietary factors) and down-regulated (e.g. oxidative stress, smoking and oxidized low-density lipoproteins). It is reduced in the course of vascular disease (e.g. diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively loose the pertussis toxin-sensitive pathway for NO release which favours vasospasm, thrombosis, penetration of macrophages, cellular growth and the inflammatory reaction leading to atherosclerosis. In addition to the release of NO (and causing endothelium-dependent hyperpolarizations), endothelial cells also can evoke contraction (constriction) of the underlying vascular smooth muscle cells by releasing endothelium-derived contracting factor (EDCF). Most endothelium-dependent acute increases in contractile force are due to the formation of vasoconstrictor prostanoids (endoperoxides and prostacyclin) which activate TP receptors of the vascular smooth muscle cells. EDCF-mediated responses are exacerbated when the production of NO is impaired (e.g. by oxidative stress, ageing, spontaneous hypertension and diabetes). They contribute to the blunting of endothelium-dependent vasodilatations in aged subjects and essential hypertensive patients. © 2008 Scandinavian Physiological Society.postprin

Crystal Structure and Functional Analysis of the SARS-Coronavirus RNA Cap 2′-O-Methyltransferase nsp10/nsp16 Complex

Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2′-O)-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 Å resolution, which shows nsp10 bound to nsp16 through a ∼930 Å2 surface area in nsp10. Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F)/nsp16, providing the first structural insight into the regulation of RNA capping enzymes in (+)RNA viruses

Estudo biológico e comportamental de lagartas de Spodoptera frugiperda visando à produção de Baculovírus spodoptera

A utilização de bioinseticida a base de Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) possui potencial para o controle de Spodoptera frugiperda (Lepidoptera: Noctuidae), porém sua obtenção em larga escala depende da maximização da produção in vivo. Assim, alguns fatores biológicos e comportamentais devem ser estudados para aperfeiçoar a produção de SfMNPV com intuito de disponibilizar um bioinseticida eficiente, economicamente viável e que possa ser usado no manejo de S. frugiperda nos mais diversos sistemas agrícolas. Entre os fatores relacionados ao hospedeiro, a temperatura e a idade para inoculação do vírus são de extrema importância, pois interferem diretamente no ciclo de vida e na replicação viral. O comportamento também deve ser avaliado, para evitar condições de criação do hospedeiro que favoreçam o canibalismo e causa prejuízo na multiplicação in vivo do SfMNPV. Assim, objetivou-se determinar a melhor condição térmica para criar as lagartas e a idade ideal, para inocular e multiplicar o vírus no hospedeiro, bem como, verificar a ocorrência do comportamento canibal em lagartas de S. frugiperda. Os experimentos foram conduzidos no Laboratório de Controle Microbiano de Insetos do Núcleo de Desenvolvimento Científico e Tecnológico em Manejo Fitossanitário de Pragas e Doenças (NUDEMAFI), localizado no Centro de Ciências Agrárias da UFES, em Alegre, Espírito Santo, Brasil. A pesquisa foi desenvolvida em duas etapas, a primeira para determinar a condição térmica e a idade ideais para criar e inocular, respectivamente, o hospedeiro com o vírus, para multiplicação in vivo de SfMNPV. A segunda etapa foi para avaliar o comportamento canibal de lagartas da espécie S. frugiperda criadas a 22, 25 e 31°C, inoculadas com SfMNPV quando com idades de 10, 8 e 4 dias, respectivamente, e mantidas em diferentes densidades populacionais (5, 10, 25 e 50 lagartas por recipiente). A mortalidade diminuiu com o aumento da temperatura e da idade do hospedeiro nas temperaturas de 25, 28 e 31 °C. O aumento na taxa de canibalismo foi 12 diretamente proporcional à densidade populacional quando as lagartas foram criadas a 22 °C, inoculadas aos 10 dias de idade e 25 ºC, inoculadas aos 8 dias e atingiram 63,5 e 62,5%, respectivamente na densidade populacional de 50 lagartas. Mas, quando as lagartas foram criadas a 31ºC e inoculadas com idade de 4 dias, a densidade populacional não afetou o comportamento canibal, taxa média de 24%, inferior aos outros tratamentos com 50 lagartas por recipiente. Demonstrando que é viável para a multiplicação viral, criar lagartas a 31 °C e aos 4 dias de idade inocular o vírus, podendo a partir de então colocar até 50 lagartas por recipiente, o que reduz a mão-de-obra necessária para individualizar as lagartas e otimiza o espaço físico em uma biofábrica. Portanto, se para otimizar o processo produção viral e o serviço em uma biofábrica, é preciso maximizar a produção viral, reduzir o tempo de multiplicação do vírus e o canibalismo entre as lagartas, com ausência de contaminação da criação, a temperatura e idade ideais para criação massal de S. frugiperda e inoculação do vírus nas lagartas, respectivamente, visando produção de baculovírus em larga escala são de 31 ºC e 4 dias