Synergism of dual AAV gene therapy and rapamycin rescues GSDIII phenotype in muscle and liver.

Glycogen storage disease type III (GSDIII) is a rare metabolic disorder due to glycogen debranching enzyme (GDE) deficiency. Reduced GDE activity leads to pathological glycogen accumulation responsible for impaired hepatic metabolism and muscle weakness. To date, there is no curative treatment for GSDIII. We previously reported that 2 distinct dual AAV vectors encoding for GDE were needed to correct liver and muscle in a GSDIII mouse model. Here, we evaluated the efficacy of rapamycin in combination with AAV gene therapy. Simultaneous treatment with rapamycin and a potentially novel dual AAV vector expressing GDE in the liver and muscle resulted in a synergic effect demonstrated at biochemical and functional levels. Transcriptomic analysis confirmed synergy and suggested a putative mechanism based on the correction of lysosomal impairment. In GSDIII mice livers, dual AAV gene therapy combined with rapamycin reduced the effect of the immune response to AAV observed in this disease model. These data provide proof of concept of an approach exploiting the combination of gene therapy and rapamycin to improve efficacy and safety and to support clinical translation.

Mesenchymal stromal cell-derived extracellular vesicles therapy openings new translational challenges in immunomodulating acute liver inflammation.

Inflammation plays a critical role in conditions such as acute liver failure, acute-on-chronic liver failure, and ischemia-reperfusion-induced liver injury. Various pathogenic pathways contribute to liver inflammation, involving inflammatory polarization of macrophages and Küpffer cells, neutrophil infiltration, dysregulation of T cell subsets, oxidative stress, and activation of hepatic stellate cells. While mesenchymal stromal cells (MSCs) have demonstrated beneficial properties, their clinical translation is limited by their cellular nature. However, MSC-derived extracellular vesicles (MSC-EVs) have emerged as a promising cell-free therapeutic approach for immunomodulation. MSC-EVs naturally mirror their parental cell properties, overcoming the limitations associated with the use of MSCs. In vitro and in vivo preclinical studies have demonstrated that MSC-EVs replicate the beneficial effects of MSCs in liver injury. This includes the reduction of cell death and oxidative stress, improvement of hepatocyte function, induction of immunomodulatory effects, and mitigation of cytokine storm. Nevertheless, MSC-EVs face challenges regarding the necessity of defining consistent isolation methods, optimizing MSCs culture conditions, and establishing quality control measures for EV characterization and functional assessment. By establishing standardized protocols, guidelines, and affordable cost mass production, clinicians and researchers will have a solid foundation to conduct further studies, validate the therapeutic efficacy of MSC-EVs, and ultimately pave the way for their clinical implementation in acute liver injury.

Deleterious effect of sustained neuroinflammation in pediatric traumatic brain injury.

INTRODUCTION: Despite improved management of traumatic brain injury (TBI), it still leads to lifelong sequelae and disability, particularly in children. Chronic neuroinflammation (the so-called tertiary phase), in particular, microglia/macrophage and astrocyte reactivity, is among the main mechanisms suspected of playing a role in the generation of lesions associated with TBI. The role of acute neuroinflammation is now well understood, but its persistent effect and impact on the brain, particularly during development, are not. Here, we investigated the long-term effects of pediatric TBI on the brain in a mouse model. METHODS: Pediatric TBI was induced in mice on postnatal day (P) 7 by weight-drop trauma. The time course of neuroinflammation and myelination was examined in the TBI mice. They were also assessed by magnetic resonance, functional ultrasound, and behavioral tests at P45. RESULTS: TBI induced robust neuroinflammation, characterized by acute microglia/macrophage and astrocyte reactivity. The long-term consequences of pediatric TBI studied on P45 involved localized scarring astrogliosis, persistent microgliosis associated with a specific transcriptomic signature, and a long-lasting myelination defect consisting of the loss of myelinated axons, a decreased level of myelin binding protein, and severe thinning of the corpus callosum. These results were confirmed by reduced fractional anisotropy, measured by diffusion tensor imaging, and altered inter- and intra-hemispheric connectivity, measured by functional ultrasound imaging. In addition, adolescent mice with pediatric TBI showed persistent social interaction deficits and signs of anxiety and depressive behaviors. CONCLUSIONS: We show that pediatric TBI induces tertiary neuroinflammatory processes associated with white matter lesions and altered behavior. These results support our model as a model for preclinical studies for tertiary lesions following TBI.

MRI volumetry and diffusion tensor imaging for diagnosis and follow-up of late post-traumatic injuries.

BACKGROUND: Traumatic Brain Injury (TBI) is a major cause of acquired disability and can cause devastating and progressive post-traumatic encephalopathy. TBI is a dynamic condition that continues to evolve over time. A better understanding of the pathophysiology of these late lesions is important for the development of new therapeutic strategies. OBJECTIVES: The primary objective was to compare the ability of fluid-attenuated reversion recovery (FLAIR) and diffusion tensor imaging (DTI) magnetic resonance imaging (MRI) markers to identify participants with a Glasgow outcome scale extended (GOS-E) score of 7-8, up to 10 years after their original TBI. The secondary objective was to study the brain regionalization of DTI markers. Finally, we analyzed the evolution of late-developing brain lesions using repeated MRI images, also taken up to 10 years after the TBI. METHODS: In this retrospective study, participants were included from a cohort of people hospitalized following a severe TBI. Following their discharge, they were followed-up and clinically assessed, including a DTI-MRI scan, between 2012 and 2016. We performed a cross-sectional analysis on 97 participants at a median (IQR) of 5 years (3-6) post-TBI, and a further post-TBI longitudinal analysis over 10 years on a subpopulation (n = 17) of the cohort. RESULTS: Although the area under the curve (AUC) of FLAIR, fractional anisotropy (FA), and mean diffusivity (MD) were not significantly different, only the AUC of FA was statistically greater than 0.5. In addition, only the FA was correlated with clinical outcomes as assessed by GOS-E score (P<10-4). On the cross-sectional analysis, DTI markers allowed study post-TBI white matter lesions by region. In the longitudinal subpopulation analysis, the observed number of brain lesions increased for the first 5 years post-TBI, before stabilizing over the next 5 years. CONCLUSIONS: This study has shown for the first time that post-TBI lesions can present in a two-phase evolution. These results must be confirmed in larger studies. French Data Protection Agency (Commission nationale de l'informatique et des libertés; CNIL) study registration no: 1934708v0.

Identification of unknown impurities J, RRT 2.2, 2.4, 2.6 and 3.4 in tetralysal® capsules.

Tetralysal® is a Galderma oral drug product (DP) marketed for the treatment of acne. Tetralysal® is sold in capsules containing either 150 mg or 300 mg of the drug substance. In the British Pharmacopoeia monograph for Lymecycline Capsules, the impurities already specified in the drug substance (A-G), visible in the European Pharmacopoeia 〈1654〉, are also specified together with an unidentified impurity at RRT 1.6 (Impurity J). Based on both monographs Galderma has focused on characterizing most of specified and unspecified impurities to better understand the stability and degradation processes of the formulation. In this manuscript, through both formal synthesis, preparative LCMS and formal degradation studies, we are the first group to confirm the structural identities of 5 unidentified impurities (Impurity J (RRT 1.6), RRT 2.2, 2.4, 2.6 and 3.4), conditions which exacerbate the formation of all 5 impurities and response factors for RRT 2.2, 2.6 and 3.4.

Therapeutic potentials of mesenchymal stromal cells-derived extracellular vesicles in liver failure and marginal liver graft rehabilitation: a scoping review.

Liver failure includes distinct subgroups of diseases: Acute liver failure (ALF) without preexisting cirrhosis, acute-on-chronic liver failure (ACLF) (severe form of cirrhosis associated with organ failures and excess mortality), and liver fibrosis (LF). Inflammation plays a key role in ALF, LF, and more specifically in ACLF for which we have currently no treatment other than liver transplantation (LT). The increasing incidence of marginal liver grafts and the shortage of liver grafts require us to consider strategies to increase the quantity and quality of available liver grafts. Mesenchymal stromal cells (MSCs) have shown beneficial pleiotropic properties with limited translational potential due to the pitfalls associated with their cellular nature. MSC-derived extracellular vesicles (MSC-EVs) are innovative cell-free therapeutics for immunomodulation and regenerative purposes. MSC-EVs encompass further advantages: pleiotropic effects, low immunogenicity, storage stability, good safety profile, and possibility of bioengineering. Currently, no human studies explored the impact of MSC-EVs on liver disease, but several preclinical studies highlighted their beneficial effects. In ALF and ACLF, data showed that MSC-EVs attenuate hepatic stellate cells activation, exert antioxidant, anti-inflammatory, anti-apoptosis, anti-ferroptosis properties, and promote regeneration of the liver, autophagy, and improve metabolism through mitochondrial function recovery. In LF, MSC-EVs demonstrated anti-fibrotic properties associated with liver tissue regeneration. Normothermic-machine perfusion (NMP) combined with MSC-EVs represents an attractive therapy to improve liver regeneration before LT. Our review suggests a growing interest in MSC-EVs in liver failure and gives an appealing insight into their development to rehabilitate marginal liver grafts through NMP.

Efficacy, pharmacokinetics, and safety in the mouse and primate retina of dual AAV vectors for Usher syndrome type 1B.

Gene therapy of Usher syndrome type 1B (USH1B) due to mutations in the large Myosin VIIA (MYO7A) gene is limited by the packaging capacity of adeno-associated viral (AAV) vectors. To overcome this, we have previously developed dual AAV8 vectors which encode human MYO7A (dual AAV8.MYO7A). Here we show that subretinal administration of 1.37E+9 to 1.37E+10 genome copies of a good-manufacturing-practice-like lot of dual AAV8.MYO7A improves the retinal defects of a mouse model of USH1B. The same lot was used in non-human primates at doses 1.6× and 4.3× the highest dose proposed for the clinical trial which was based on mouse efficacy data. Long-lasting alterations in retinal function and morphology were observed following subretinal administration of dual AAV8.MYO7A at the high dose. These findings were modest and improved over time in the low-dose group, as also observed in other studies involving the use of AAV8 in non-human primates and humans. Biodistribution and shedding studies confirmed the presence of vector DNA mainly in the visual pathway. Accordingly, we detected human MYO7A mRNA expression predominantly in the retina. Overall, these studies pave the way for the clinical translation of subretinal administration of dual AAV vectors in USH1B subjects.

Completion of the impurity profile of lymecycline: Formal identification of impurities E and F.

Lymecycline is the drug substance (DS) used in the Galderma drug product Tetralysal® capsules with 7 impurities currently described in the pharmacopeia labelled as A-G. In the current monograph, the structural identity of all impurities except E and F have been formally identified. In this manuscript, through both formal synthesis and preparative chromatography, we are the first group to confirm the structural identity, response factor of Impurity F and conditions which exacerbate the formation of both impurities.

Liver-Directed Adeno-Associated Virus-Mediated Gene Therapy for Mucopolysaccharidosis Type VI.

BACKGROUND: Mucopolysaccharidosis type VI (MPS VI) is an inherited multisystem lysosomal disorder due to arylsulfatase B (ARSB) deficiency that leads to widespread accumulation of glycosaminoglycans (GAG), which are excreted in increased amounts in urine. MPS VI is characterized by progressive dysostosis multiplex, connective tissue and cardiac involvement, and hepatosplenomegaly. Enzyme replacement therapy (ERT) is available but requires life-long and costly intravenous infusions; moreover, it has limited efficacy on diseased skeleton and cardiac valves, compromised pulmonary function, and corneal opacities. METHODS: We enrolled nine patients with MPS VI 4 years of age or older in a phase 1/2 open-label gene therapy study. After ERT was interrupted, patients each received a single intravenous infusion of an adeno-associated viral vector serotype 8 expressing ARSB. Participants were sequentially enrolled in one of three dose cohorts: low (three patients), intermediate (two patients), or high (four patients). The primary outcome was safety; biochemical and clinical end points were secondary outcomes. RESULTS: The infusions occurred without severe adverse events attributable to the vector, meeting the prespecified end point. Participants in the low and intermediate dose cohorts displayed stable serum ARSB of approximately 20% of the mean healthy value but returned to ERT by 14 months after gene therapy because of increased urinary GAG. Participants in the high-dose cohort had sustained serum ARSB of 30% to 100% of the mean healthy value and a modest urinary GAG increase that did not reach a concentration at which ERT reintroduction was needed. In the high-dose group, there was no clinical deterioration for up to 2 years after gene therapy. CONCLUSIONS: Liver-directed gene therapy for participants with MPS VI did not have a dose-limiting side-effect and adverse event profile; high-dose treatment resulted in ARSB expression over at least 24 months with preliminary evidence of disease stabilization. (Funded by the Telethon Foundation ETS, the European Commission Seventh Framework Programme, and the Isaac Foundation; ClinicalTrials.gov number, NCT03173521; EudraCT number, 2016-002328-10.)

Novel library synthesis of 3,4-disubstituted pyridin-2(1H)-ones via cleavage of pyridine-2-oxy-7-azabenzotriazole ethers under ionic hydrogenation conditions at room temperature.

In our hands, efficient access to the 4-amino-3-carboxamide disubstituted pyridine-2(1H)-one kinase hinge-binder motif proved to be more challenging than anticipated requiring a significant investment in route scouting and optimization. This full paper focuses on the synthesis issues that we encountered during our route exploration and the original solutions we found that helped us to identify two optimized library-style processes to prepare our large kinase inhibitor library.

Rational Drug Design of Topically Administered Caspase 1 Inhibitors for the Treatment of Inflammatory Acne.

The use of an interleukin ß antibody is currently being investigated in the clinic for the treatment of acne, a dermatological disorder affecting 650M persons globally. Inhibiting the protease responsible for the cleavage of inactive pro-IL1ß into active IL-1ß, caspase-1, could be an alternative small molecule approach. This report describes the discovery of uracil 20, a potent (38 nM in THP1 cells assay) caspase-1 inhibitor for the topical treatment of inflammatory acne. The uracil series was designed according to a published caspase-1 pharmacophore model involving a reactive warhead in P1 for covalent reversible inhibition and an aryl moiety in P4 for selectivity against the apoptotic caspases. Reversibility was assessed in an enzymatic dilution assay or by using different substrate concentrations. In addition to classical structure-activity-relationship exploration, topical administration challenges such as phototoxicity, organic and aqueous solubility, chemical stability in solution, and skin metabolic stability are discussed and successfully resolved.

Sulfoximines as potent RORγ inverse agonists.

Progress in the identification of suitable RORγ inverse agonists as clinical candidates has been hampered by the high lipophilicity that seems required for high potency on this nuclear receptor. In this context, we decided to focus on the replacement of the hydroxymethyl group found on known modulators to determine if more polarity could be tolerated in this position. SAR of the replacement of this moiety is presented in this article leading to the identification of sulfoximine derivatives as potent modulators with pharmacological activity in the in vivo mouse Imiquimod psoriasis model.

Discovery and Characterization of CD12681, a Potent RORγ Inverse Agonist, Preclinical Candidate for the Topical Treatment of Psoriasis.

With possible implications in multiple autoimmune diseases, the retinoic acid receptor-related orphan receptor RORγ has become a sought-after target in the pharmaceutical industry. Herein are described the efforts to identify a potent RORγ inverse agonist compatible with topical application for the treatment of skin diseases. These efforts culminated in the discovery of N-(2,4-dimethylphenyl)-N-isobutyl-2-oxo-1-[(tetrahydro-2H-pyran-4-yl)methyl]-2,3-dihydro-1H-benzo[d]imidazole-5-sulfonamide (CD12681), a potent inverse agonist with in vivo activity in an IL-23-induced mouse skin inflammation model.

Discovery of phenoxyindazoles and phenylthioindazoles as RORγ inverse agonists.

Targeting the IL17 pathway and more specifically the nuclear receptor RORγ is thought to be beneficial in multiple skin disorders. The Letter describes the discovery of phenoxyindazoles and thiophenoxy indazoles as potent RORγ inverse agonists. Optimization of the potency and efforts to mitigate the phototoxic liability of the series are presented. Finally, crystallization of the lead compound revealed that the series bound to an allosteric site of the nuclear receptor. Such compounds could be useful as tool compounds for understanding the impact of topical treatment on skin disease models.