Clemastine/tamoxifen hybrids as easily accessible antileishmanial drug leads.

A library of hybrid molecules is developed based on the common chemical features shared by clemastine and tamoxifen both of which are well known for their antileishmanial activities. In the initial screening against Leishmania major and L. amazonensis promastigotes, as well as cytotoxicity assays using HepG2 cells, several hybrids showed submicromolar activity against the parasite and no toxicity against human cells. The compounds with an EC50 < 2 µM against promastigotes of both species and a selectivity index >10 were further characterized against intracellular amastigotes as well as promastigotes of species that cause both visceral and cutaneous leishmaniasis, such as L. infantum and L. braziliensis, respectively. These sequential screenings revealed the high pan-activity of this class of molecules against these species, with several compounds displaying an EC50 ≤ 2 µM against both promastigotes and intracellular amastigotes. Two of them were identified as the potential templates for lead optimization of this series having shown the highest activities against all species in both stages of parasite. The present findings can serve as a good starting point in the search for novel antileishmanial compounds that are easy to access and highly active.

Clemastine and metformin extend the window of NMDA receptor surface expression in ageing oligodendrocyte precursor cells.

In the central nervous system, oligodendrocyte precursor cells (OPCs) proliferate and differentiate into myelinating oligodendrocytes throughout life, allowing for ongoing myelination and myelin repair. With age, differentiation efficacy decreases and myelin repair fails; therefore, recent therapeutic efforts have focused on enhancing differentiation. Many cues are thought to regulate OPC differentiation, including neuronal activity, which OPCs can sense and respond to via their voltage-gated ion channels and glutamate receptors. However, OPCs' density of voltage-gated ion channels and glutamate receptors differs with age and brain region, and correlates with their proliferation and differentiation potential, suggesting that OPCs exist in different functional cell states, and that age-associated states might underlie remyelination failure. Here, we use whole-cell patch-clamp to investigate whether clemastine and metformin, two pro-remyelination compounds, alter OPC membrane properties and promote a specific OPC state. We find that clemastine and metformin extend the window of NMDAR surface expression, promoting an NMDAR-rich OPC state. Our findings highlight a possible mechanism for the pro-remyelinating action of clemastine and metformin, and suggest that OPC states can be modulated as a strategy to promote myelin repair.

The preferential effect of Clemastine on F3/Contactin-1/Notch-1 compared to Jagged-1/Notch-1 justifies its remyelinating effect in an experimental model of multiple sclerosis in rats.

Clemastine (CLM) is repurposed to enhance remyelination in multiple sclerosis (MS) patients. CLM blocks histamine and muscarinic receptors as negative regulators to oligodendrocyte differentiation. These receptors are linked to the canonical and non-canonical Notch-1 signaling via specific ligands; Jagged-1 and F3/Contactin-1, respectively. Yet, there are no previous studies showing the influence of CLM on Notch entities. Herein, the study aimed to investigate to which extent CLM aligns to one of the two Notch-1 arms in experimental autoimmune encephalomyelitis (EAE) rat model. Three groups were utilized where first group received vehicles. The second group was injected by spinal cord homogenate mixed with complete Freund's adjuvant on days 0 and 7. In the third group, CLM (5 mg/kg/day; p.o) was administered for 15 days starting from the day of the first immunization. CLM ameliorated EAE-associated motor and gripping impairment in rotarod, open-field, and grip strength arena beside sensory anomalies in hot plate, cold allodynia, and mechanical Randall-Selitto tests. Additionally, CLM alleviated depressive mood observed in tail suspension test. These findings harmonized with histopathological examinations of Luxol-fast blue stain together with enhanced immunostaining of myelin basic protein and oligodendrocyte lineage gene 2 in corpus callosum and spinal cord. Additionally, CLM enhanced oligodendrocyte myelination and maturation by increasing 2',3'-cyclic nucleotide 3'-phosphodiesterase, proteolipid protein, aspartoacylase as well. CLM restored the level of F3/Contactin-1 in the diseased rats over Jagged-1 level; the ligand of the canonical pathway. This was accompanied by elevated gene expression of Deltex-1 and reduced hairy and enhancer-of-split homologs 1 and 5. Additionally, CLM suppressed microglial and astrocyte activation via reducing the expression of ionized calcium-binding adaptor molecule-1 as well as glial fibrillary acidic protein, respectively. These results outlined the remyelinating beneficence of CLM which could be due to augmenting the non-canonical Notch-1 signaling over the canonical one.

Measuring and predicting the effect of remyelinating therapy in multiple sclerosis: a randomised controlled trial protocol (RESTORE).

INTRODUCTION: Remyelination failure hampers symptomatic recovery in multiple sclerosis (MS), underlining the importance of developing remyelinating therapies. Optic neuritis is currently the most established method of measuring remyelination in MS trials. Complementary more generalisable methods of measuring remyelination are required to confirm treatment efficacy. Measuring internuclear ophthalmoplegia (INO) with infrared oculography provides such a method. Moreover, this method can be expanded with a test for selecting likely treatment responders by using fampridine. The aim of this trial is to investigate the (long-term) remyelinating effects of clemastine fumarate in patients with MS and INO and to evaluate if treatment response can be predicted using fampridine. METHODS AND ANALYSIS: RESTORE is a single-centre double-blind randomised placebo-controlled trial of clemastine fumarate versus placebo. Prior to clemastine treatment improvement in oculographic features of INO after a single 10 mg dose of fampridine is measured in all participants and used to predict the treatment response to clemastine. Eighty individuals with MS and INO will be 1:1 randomised to 4 mg of clemastine fumarate two times a day for 6 months or equivalent placebo. Our primary outcome is improvement in the Versional Dysconjugacy Index-area under the curve, measured by infrared oculography after 6 months of treatment. Participants are assessed for persistent treatment effects 6, 18 and 30 months after end of treatment. Secondary outcome measures include other oculography parameters including double-step saccades, retinal imaging, visual acuities, physical disability, cognition and patient-reported outcomes. ETHICS AND DISSEMINATION: Clemastine is a registered and very well-established drug with well-known safety and side effects. The protocol was approved by the medical ethical committee of the Amsterdam UMC, location VUMC and the Dutch Central Committee on Research Involving Human Subject. Written informed consent is obtained from all participants. The results will be published in peer-reviewed medical scientific journals. TRIAL REGISTRATION NUMBER: EudraCT: 2021-003677-66, ClinicalTrials.gov: NCT05338450.

Clemastine-induced enhancement of hippocampal myelination alleviates memory impairment in mice with chronic pain.

Patients with chronic pain often experience memory impairment, but the underlying mechanisms remain elusive. The myelin sheath is crucial for rapid and accurate action potential conduction, playing a pivotal role in the development of cognitive abilities in the central nervous system. The study reveals that myelin degradation occurs in the hippocampus of chronic constriction injury (CCI) mice, which display both chronic pain and memory impairment. Using fiber photometry, we observed diminished task-related neuronal activity in the hippocampus of CCI mice. Interestingly, the repeated administration with clemastine, which promotes myelination, counteracts the CCI-induced myelin loss and reduced neuronal activity. Notably, clemastine specifically ameliorates the impaired memory without affecting chronic pain in CCI mice. Overall, our findings highlight the significant role of myelin abnormalities in CCI-induced memory impairment, suggesting a potential therapeutic approach for treating memory impairments associated with neuropathic pain.

Clemastine fumarate attenuates tauopathy and meliorates cognition in hTau mice via autophagy enhancement.

Clemastine fumarate, which has been identified as a promising agent for remyelination and autophagy enhancement, has been shown to mitigate Aß deposition and improve cognitive function in the APP/PS1 mouse model of Alzheimer's disease. Based on these findings, we investigated the effect of clemastine fumarate in hTau mice, a different Alzheimer's disease model characterized by overexpression of human Tau protein. Surprisingly, clemastine fumarate was effective in reducing pathological deposition of Tau protein, protecting neurons and synapses from damage, inhibiting neuroinflammation, and improving cognitive impairment in hTau mice. Interestingly, chloroquine, an autophagy inhibitor, had a significant impact on total and Sarkosyl fractions of autophagy, demonstrating that it can interrupt autophagy. Notably, after administration of chloroquine, levels of Tau protein were significantly increased. When clemastine fumarate was co-administered with chloroquine, the protective effects were reversed, indicating that clemastine fumarate indeed triggered autophagy and promoted the degradation of Tau protein, while also inhibiting further Tauopathy-related neuroinflammation and synapse loss to improve cognitive function in hTau mice.

Pro-myelinating clemastine administration improves recording performance of chronically implanted microelectrodes and nearby neuronal health.

Intracortical microelectrodes have become a useful tool in neuroprosthetic applications in the clinic and to understand neurological disorders in basic neurosciences. Many of these brain-machine interface technology applications require successful long-term implantation with high stability and sensitivity. However, the intrinsic tissue reaction caused by implantation remains a major failure mechanism causing loss of recorded signal quality over time. Oligodendrocytes remain an underappreciated intervention target to improve chronic recording performance. These cells can accelerate action potential propagation and provides direct metabolic support for neuronal health and functionality. However, implantation injury causes oligodendrocyte degeneration and leads to progressive demyelination in surrounding brain tissue. Previous work highlighted that healthy oligodendrocytes are necessary for greater electrophysiological recording performance and the prevention of neuronal silencing around implanted microelectrodes over the chronic implantation period. Thus, we hypothesize that enhancing oligodendrocyte activity with a pharmaceutical drug, Clemastine, will prevent the chronic decline of microelectrode recording performance. Electrophysiological evaluation showed that the promyelination Clemastine treatment significantly elevated the signal detectability and quality, rescued the loss of multi-unit activity, and increased functional interlaminar connectivity over 16-weeks of implantation. Additionally, post-mortem immunohistochemistry showed that increased oligodendrocyte density and myelination coincided with increased survival of both excitatory and inhibitory neurons near the implant. Overall, we showed a positive relationship between enhanced oligodendrocyte activity and neuronal health and functionality near the chronically implanted microelectrode. This study shows that therapeutic strategy that enhance oligodendrocyte activity is effective for integrating the functional device interface with brain tissue over chronic implantation period.

Comparing the efficacy in reducing brain injury of different neuroprotective agents following neonatal hypoxia-ischemia in newborn rats: a multi-drug randomized controlled screening trial.

Intrapartum hypoxia-ischemia leading to neonatal encephalopathy (NE) results in significant neonatal mortality and morbidity worldwide, with > 85% of cases occurring in low- and middle-income countries (LMIC). Therapeutic hypothermia (HT) is currently the only available safe and effective treatment of HIE in high-income countries (HIC); however, it has shown limited safety or efficacy in LMIC. Therefore, other therapies are urgently required. We aimed to compare the treatment effects of putative neuroprotective drug candidates following neonatal hypoxic-ischemic (HI) brain injury in an established P7 rat Vannucci model. We conducted the first multi-drug randomized controlled preclinical screening trial, investigating 25 potential therapeutic agents using a standardized experimental setting in which P7 rat pups were exposed to unilateral HI brain injury. The brains were analysed for unilateral hemispheric brain area loss after 7 days survival. Twenty animal experiments were performed. Eight of the 25 therapeutic agents significantly reduced brain area loss with the strongest treatment effect for Caffeine, Sonic Hedgehog Agonist (SAG) and Allopurinol, followed by Melatonin, Clemastine, ß-Hydroxybutyrate, Omegaven, and Iodide. The probability of efficacy was superior to that of HT for Caffeine, SAG, Allopurinol, Melatonin, Clemastine, ß-hydroxybutyrate, and Omegaven. We provide the results of the first systematic preclinical screening of potential neuroprotective treatments and present alternative single therapies that may be promising treatment options for HT in LMIC.

Promyelinating drugs promote functional recovery in an autism spectrum disorder mouse model of Pitt-Hopkins syndrome.

Pitt-Hopkins syndrome is an autism spectrum disorder caused by autosomal dominant mutations in the human transcription factor 4 gene (TCF4). One pathobiological process caused by murine Tcf4 mutation is a cell autonomous reduction in oligodendrocytes and myelination. In this study, we show that the promyelinating compounds, clemastine, sobetirome and Sob-AM2 are effective at restoring myelination defects in a Pitt-Hopkins syndrome mouse model. In vitro, clemastine treatment reduced excess oligodendrocyte precursor cells and normalized oligodendrocyte density. In vivo, 2-week intraperitoneal administration of clemastine also normalized oligodendrocyte precursor cell and oligodendrocyte density in the cortex of Tcf4 mutant mice and appeared to increase the number of axons undergoing myelination, as EM imaging of the corpus callosum showed a significant increase in the proportion of uncompacted myelin and an overall reduction in the g-ratio. Importantly, this treatment paradigm resulted in functional rescue by improving electrophysiology and behaviour. To confirm behavioural rescue was achieved via enhancing myelination, we show that treatment with the thyroid hormone receptor agonist sobetirome or its brain penetrating prodrug Sob-AM2, was also effective at normalizing oligodendrocyte precursor cell and oligodendrocyte densities and behaviour in the Pitt-Hopkins syndrome mouse model. Together, these results provide preclinical evidence that promyelinating therapies may be beneficial in Pitt-Hopkins syndrome and potentially other neurodevelopmental disorders characterized by dysmyelination.

Pharmacological treatment promoting remyelination enhances motor function after internal capsule demyelination in mice.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system characterized by remyelination failure, axonal degeneration, and progressive worsening of motor functions. Animal models of demyelination are frequently used to develop and evaluate therapies for MS. We recently reported that focal internal capsule (IC) demyelination in mice with lysophosphatidylcholine injection induced acute motor deficits followed by recovery through remyelination. However, it remains unknown whether the IC demyelination mouse model can be used to evaluate changes in motor functions caused by pharmacological treatments that promote remyelination using behavioral testing and histological analysis. In this study, we examined the effect of clemastine, an anti-muscarinic drug that promotes remyelination, in the mouse IC demyelination model. Clemastine administration improved motor function and changed forepaw preference in the IC demyelinated mice. Moreover, clemastine-treated mice showed increased mature oligodendrocyte density, reduced axonal injury, an increased number of myelinated axons and thicker myelin in the IC lesions compared with control (PBS-treated) mice. These results suggest that the lysophosphatidylcholine-induced IC demyelination model is useful for evaluating changes in motor functions following pharmacological treatments that promote remyelination.

Insufficient Oligodendrocyte Turnover in Optic Nerve Contributes to Age-Related Axon Loss and Visual Deficits.

Age-related decline in visual functions is a prevalent health problem among elderly people, and no effective therapies are available up-to-date. Axon degeneration and myelin loss in optic nerves (ONs) are age-dependent and become evident in middle-aged (13-18 months) and old (20-22 months) mice of either sex compared with adult mice (3-8 months), accompanied by functional deficits. Oligodendrocyte (OL) turnover is actively going on in adult ONs. However, the longitudinal change and functional significance of OL turnover in aging ONs remain largely unknown. Here, using cell-lineage labeling and tracing, we reported that oligodendrogenesis displayed an age-dependent decrease in aging ONs. To understand whether active OL turnover is required for maintaining axons and visual function, we conditionally deleted the transcription factor Olig2 in the oligodendrocyte precursor cells of young mice. Genetically dampening OL turnover by Olig2 ablation resulted in accelerated axon loss and retinal degeneration, and subsequently impaired ON signal transmission, suggesting that OL turnover is an important mechanism to sustain axon survival and visual function. To test whether enhancing oligodendrogenesis can prevent age-related visual deficits, 12-month-old mice were treated with clemastine, a pro-myelination drug, or induced deletion of the muscarinic receptor 1 in oligodendrocyte precursor cells. The clemastine treatment or muscarinic receptor 1 deletion significantly increased new OL generation in the aged ONs and consequently preserved visual function and retinal integrity. Together, our data indicate that dynamic OL turnover in ONs is required for axon survival and visual function, and enhancing new OL generation represents a potential approach to reversing age-related declines of visual function.SIGNIFICANCE STATEMENT Oligodendrocyte (OL) turnover has been reported in adult optic nerves (ONs), but the longitudinal change and functional significance of OL turnover during aging remain largely unknown. Using cell-lineage tracing and oligodendroglia-specific manipulation, this study reported that OL generation was active in adult ONs and the efficiency decreased in an age-dependent manner. Genetically dampening OL generation by Olig2 ablation resulted in significant axon loss and retinal degeneration, along with delayed visual signal transmission. Conversely, pro-myelination approaches significantly increased new myelin generation in aging ONs, and consequently preserved retinal integrity and visual function. Our findings indicate that promoting OL generation might be a promising strategy to preserve visual function from age-related decline.

Modulation of p38 MAPK and Nrf2/HO-1/NLRP3 inflammasome signaling and pyroptosis outline the anti-neuroinflammatory and remyelinating characters of Clemastine in EAE rat model.

There is vast evidence for the effect of NOD-like receptor protein-3 (NLRP3) inflammasome on multiple sclerosis (MS) pathogenesis. Clemastine (CLM) targets NLRP3 in hypoxic brain injury and promotes oligodendrocyte differentiation. However, no previous study pointed to the link of CLM with inflammasome components in MS. Herein, the study aimed to verify the action of CLM on NLRP3 signaling in experimental autoimmune encephalomyelitis (EAE) as an MS rat model. Homogenate of spinal cord with complete Freund's adjuvant was administered on days 0 and 7 to induce EAE. Rats received either CLM (5 mg/kg/day; p.o.) or MCC950 (2.5 mg/kg/day; i.p) for 15 days starting from the first immunization day. In EAEs' brains, NLRP3 pathway components; total and phosphorylated p38 mitogen-activated protein kinase (MAPK), apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, interleukins 1ß and -18 along with pyroptotic marker; gasdermin D (GSDMD) were upregulated. These were accompanied with diminished nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and total antioxidant capacity levels. CLM improved these perturbations as well as signs of MS; weight loss, clinical scores, and motor disorders observed in the open field, hanging wire and rotarod tests. Histopathological examinations revealed improvement in H&E abnormalities and axonal demyelination as shown by luxol fast blue stain in lumbar sections of spinal cord. These CLM's actions were studied in comparison to MCC950 as a well-established selective blocker of the NLRP3 inflammasome. Conclusively, CLM has a protective role against neuroinflammation and demyelination in EAE via its anti-inflammatory and anti-pyroptotic actions.

Promotion of the genipin crosslinked chitosan-fiber hydrogel loaded with sustained release of clemastine fumarate in diabetic wound repair.

Diabetic skin disorders are lingering and refractory clinical diseases. In this study, a genipin-crosslinked porous chitosan fiber (CSF) hydrogel was fabricated to achieve rapid wound healing. By embedding clemastine fumarate (CF) in the CSF hydrogel pores, we synthesised a CSF/CF hydrogel for the treatment of diabetic wounds. The microstructure, chemical elements, spectral variation, mechanical properties, swelling ratios, degradability, and toxicity of the CSF/CF hydrogels were studied. Compared with the typical CS power hydrogel, the porous CSF hydrogel crosslinked with genipin possesses a stable structure and improved physicochemical properties. Moreover, CF was slowly released from the CSF hydrogel. Molecular simulation also showed that CF was evenly embedded inside the cavity formed by the novel CSF hydrogel. The results suggested that CF can resist damage from high glucose levels and promote proliferation, tube formation, and migration of endothelial cells (ECs) and fibroblasts. The CSF/CF hydrogel promoted wound healing in a rat model. Mechanistically, the beneficial effect of CF on wound healing may be related to activation of the MEK/ERK and PI3K/Akt signalling pathways. In conclusion, genipin-crosslinked CSF/CF hydrogel can accelerate wound healing and may be an effective therapeutic method for treating diabetic skin lesions.

Drug repurposing: Clemastine fumarate and neurodegeneration.

Neurodegenerative diseases have been a weighty problem in elder people who might be stricken with motor or/and cognition defects with lower life quality urging for effective treatment. Drugs are costly from development to market, so that drug repurposing, exploration of existing drugs for novel therapeutic purposes, becomes a wise and popular strategy to raise new treatment options. Clemastine fumarate, different from anti-allergic effect as H1 histamine antagonist, was screened and identified as promising drug for remyelination and autophagy enhancement. Surprisingly, fumarate salt also has similar effect. Hence, whether clemastine fumarate would make a protective impact on neurodegenerative diseases and what contribution fumarate probably makes are intriguing to us. In this review, we summarize the potential mechanism surrounding clemastine fumarate in current literature, and try to distinguish independent or synergistic effect between clemastine and fumarate, aiming to find worthwhile research direction for neurodegeneration diseases.

Validating visual evoked potentials as a preclinical, quantitative biomarker for remyelination efficacy.

Many biomarkers in clinical neuroscience lack pathological certification. This issue is potentially a significant contributor to the limited success of neuroprotective and neurorestorative therapies for human neurological disease-and is evident even in areas with therapeutic promise such as myelin repair. Despite the identification of promising remyelinating candidates, biologically validated methods to demonstrate therapeutic efficacy or provide robust preclinical evidence of remyelination in the CNS are lacking. Therapies with potential to remyelinate the CNS constitute one of the most promising and highly anticipated therapeutic developments in the pipeline to treat multiple sclerosis and other demyelinating diseases. The optic nerve has been proposed as an informative pathway to monitor remyelination in animals and human subjects. Recent clinical trials using visual evoked potential have had promising results, but without unequivocal evidence about the cellular and molecular basis for signal changes on visual evoked potential, the interpretation of these trials is constrained. The visual evoked potential was originally developed and used in the clinic as a diagnostic tool but its use as a quantitative method for assessing therapeutic response requires certification of its biological specificity. Here, using the tools of experimental pathology we demonstrate that quantitative measurements of myelination using both histopathological measures of nodal structure and ultrastructural assessments correspond to visual evoked potential latency in both inflammatory and chemical models of demyelination. Visual evoked potential latency improves after treatment with a tool remyelinating compound (clemastine), mirroring both quantitative and qualitative myelin assessment. Furthermore, clemastine does not improve visual evoked potential latency following demyelinating injury when administered to a transgenic animal incapable of forming new myelin. Therefore, using the capacity for therapeutic enhancement and biological loss of function we demonstrate conclusively that visual evoked potential measures myelin status and is thereby a validated tool for preclinical verification of remyelination.

Effect of Clemastine Fumarate on Perioperative Hemodynamic Instability Mediated by Anaphylaxis During Cardiopulmonary Bypass Surgery.

BACKGROUND Perioperative hemodynamic instability mediated by anaphylaxis is a life-threatening complication in patients undergoing cardiac surgery with cardiopulmonary bypass (CPB). This study aimed to evaluate the effect of clemastine fumarate in this specific patient population. MATERIAL AND METHODS We enrolled 100 participants who met the inclusion criteria and randomly allocated them to the treatment group and the placebo group. Participants in the treatment group and the placebo group were treated separately with an injection of clemastine fumarate and saline, respectively. Plasma histamine concentration and blood pressure were quantified at 5 timepoints during the perioperative period, and differences between the 2 groups were assessed by repeated-measures ANOVA. The postoperative complications and in-hospital mortality also were evaluated. All participants were followed up for 7 days after cardiac surgery. RESULTS Plasma histamine concentrations increased in both groups but were statistically significantly lower in the treatment group during the perioperative period (P=0.007). Diastolic blood pressure (P=0.014) and mean arterial pressure (P=0.024) in the treatment group were significantly higher than in the placebo group during the perioperative period. The coefficients of variation for systolic (13.9±4.2% vs 17.2±4.4%, P<0.01) and diastolic (12.9±4.9% vs 15.3±5.2%, P=0.02) blood pressure were significantly lower in the treatment group compared with the placebo group. CONCLUSIONS Pretreatment with clemastine fumarate restrains the increase in histamine concentration and provides safer hemodynamics in patients undergoing cardiac surgery with CPB.

Clemastine protects against sepsis-induced myocardial injury in vivo and in vitro.

Sepsis-induced myocardial dysfunction (SIMD) is associated with high morbidity and mortality rates; however, it lacks targeted therapies. Modulating cardiomyocyte autophagy maintains intracellular homeostasis during SIMD. Clemastine, a histamine receptor inhibitor, promotes autophagy and other effective biological functions. Nevertheless, the effect of clemastine on SIMD remains unclear. This study aimed to explore the underlying mechanism of clemastine in cardiomyocyte injury in cecum ligation and perforation (CLP)-induced rats and lipopolysaccharide (LPS)-stimulated H9c2 cells. Clemastine (10 mg/kg, 30 mg/kg, and 50 mg/kg) was intraperitoneally injected after 30 min of CLP surgery. Serum cTnI levels and the 7-day survival rate were evaluated. Echocardiograms and H&E staining were used to evaluate cardiac function and structure. TEM was used to detect the mitochondrial ultrastructure and autophagosomes. Clemastine significantly improved the survival rate and reduced cTnI production in serum. Clemastine ameliorated cellular apoptosis, improved mitochondrial ultrastructure both in vivo and in vitro, increased ATP content, decreased dynamin-related protein 1 (DRP1) expression, and decreased mitochondrial ROS levels. Additionally, clemastine treatment increased autophagosome concentration, LC3II/LC3I rate, and Beclin 1 expression. However, 3-methyladenine (3-MA), an autophagy inhibitor, could abolish the effect of clemastine on alleviating myocardial apoptosis. In conclusion, clemastine protected against cardiac structure destruction and function dysfunction, mitochondrial damage, apoptosis, and autophagy in vivo and in vitro. Moreover, clemastine attenuated myocardial apoptosis by promoting autophagy. This study provides a novel favorable perspective for SIMD therapy.

Clemastine Inhibits the Biofilm and Hemolytic of Staphylococcus aureus through the GdpP Protein.

Staphylococcus aureus poses a significant threat to human health due to its virulence and multidrug resistance. In addition, recalcitrant biofilm formation of S. aureus often results in chronic infection and the treatment tolerance toward the traditional antibiotics. Thus, the development of novel antimicrobial agents capable to inhibit or eradicate S. aureus biofilm formation does matter. Here, we demonstrated that clemastine showed slight bacteriostatic activity and enhanced the antibacterial activity of oxacillin against S. aureus. Moreover, the dramatic inhibition of biofilm formation was found in clinical S. aureus strains by clemastine. Clemastine inhibited the release of eDNA during the biofilm formation and decreased the S. aureus hemolytic activity. Moreover, the S. aureus SA113 treated with clemastine displayed the decreased transcriptional level of the biofilm formation relevant genes (fnbB, icaA, and icaB), virulence genes (hlg, hld, lukde, lukpvl, beta-PSM, delta-PSM, and cap5A), and the regulatory genes agrA. The proteomics analysis of SA113 treated with clemastine demonstrated the significant changes in levels of biofilm-related proteins (stress response regulators ClpB and GroS, ATP-binding proteins, and urease metabolism), virulence-related proteins (SspA, superantigen, and VWbp), and methicillin resistance-related proteins (glutamine metabolism). The genetic mutations on gdpP (cyclic di-AMP phosphodiesterase) were found in the clemastine-induced tolerant derivative isolate by whole-genome sequencing. Furthermore, the interaction between clemastine and GdpP protein was demonstrated by the molecular docking, gdpP overexpression experiment, and thermal stability assay. Conclusively, clemastine might exert its inhibitory effects against the biofilm formation and hemolysis in S. aureus through targeting GdpP protein. IMPORTANCE The biofilm formation, which protects bacteria from stresses, including antibiotics and host immune responses, can be commonly found in clinical S. aureus isolates worldwide. Treatment failure of traditional antibiotics in biofilm-associated S. aureus infections remains a serious challenge. The novel anti-biofilm drug is urgently needed to address the looming crisis. In this study, clemastine, which is a histamine receptor H1 (HRH1) antagonist, was found to have a novel role of the significant inhibition against the biofilm formation and hemolytic activity of S. aureus and enhanced antibacterial activity against S. aureus when used in combination with oxacillin by targeting the GdpP protein. The discovery of this study identified novel use and mechanism of action of clemastine as a potential anti-biofilm drug for clinical application for S. aureus infectious.

Clemastine Rescues Chemotherapy-Induced Cognitive Impairment by Improving White Matter Integrity.

With the improvement of cancer treatment techniques, increasing attention has been given to chemotherapy-induced cognitive impairment through white matter injury. Clemastine fumarate has been shown to enhance white matter integrity in cuprizone- or hypoxia-induced demyelination mouse models. However, whether clemastine can be beneficial for reversing chemotherapy-induced cognitive impairment remains unexplored. In this study, the mice received oral administration of clemastine after chemotherapy. The open-field test and Morris water maze test were used to evaluate their anxiety, locomotor activity and cognitive function. Luxol Fast Blue staining and transmission electron microscopy were used to detect the morphological damage to the myelin. Demyelination and damage to the mature oligodendrocytes and axons were observed by immunofluorescence and western blotting. Clemastine significantly improved their cognitive function and ameliorated white matter injury in the chemotherapy-treated mice. Clemastine enhanced myelination, promoted oligodendrocyte precursor cell differentiation and increased the neurofilament 200 protein levels in the corpus callosum and hippocampus. We concluded that clemastine rescues cognitive function damage caused by chemotherapy through improving white matter integrity. Remyelination, oligodendrocyte differentiation and the increase of neurofilament protein promoted by clemastine are potential strategies for reversing the cognitive dysfunction caused by chemotherapy.

Clemastine Enhances Myelination, Delays Axonal Loss and Promotes Functional Recovery in Spinal Cord Injury.

Recent evidence has shown that demyelination occurs along with axonal degeneration in spinal cord injury (SCI) during the secondary injury phase. Oligodendrocyte precursor cells (OPC) are present in the lesions but fail to differentiate into mature oligodendrocytes and form new myelin. Given the limited recovery of neuronal functions after SCI in adults without effective treatment available so far, it remains unknown whether enhancing OPC differentiation and myelination could benefit the recovery of SCI. To show the significance of myelin regeneration after SCI, the injury was treated with clemastine in the rat model. Clemastine is an FDA-approved drug that is potent in promoting oligodendrocyte differentiation and myelination in vivo, for four weeks following SCI. Motor function was assessed using sloping boards and grid walking tests and scored according to the Basso, Beattie, and Bresnahan protocol. The myelin integrity and protein expression were evaluated using transmission electron microscopy and immunofluorescence, respectively. The results indicated that clemastine treatment preserves myelin integrity, decreases loss of axons and improves functional recovery in the rat SCI model. The presented data suggest that myelination-enhancing strategies may serve as a potential therapeutic approach for the functional recovery in SCI.