[Component identification and analysis in vivo of Sanhan Huashi formula].

Sanhan Huashi formula(SHF) is the intermediate of a newly approved traditional Chinese medicine(TCM) Sanhan Huashi Granules for the treatment of COVID-19 infection. The chemical composition of SHF is complex since it contains 20 single herbal medicines. In this study, UHPLC-Orbitrap Exploris 240 was used to identify the chemical components in SHF and in rat plasma, lung and feces after oral administration of SHF, and heat map was plotted for characterizing the distribution of the chemical components. Chromatographic separation was conducted on a Waters ACQUITY UPLC BEH C_(18)(2.1 mm×100 mm, 1.7 µm) using 0.1% formic acid(A)-acetonitrile(B) as mobile phases in a gradient elution. Electrospray ionization(ESI) source was used to acquire data in positive and negative mode. By reference to quasi-molecular ions and MS/MS fragment ions and in combination with MS spectra of reference substances and compound information in literature reports, 80 components were identified in SHF, including 14 flavonoids, 13 coumarins, 5 lignans, 12 amino-compounds, 6 terpenes and 30 other compounds; 40 chemical components were identified in rat plasma, 27 in lung and 56 in feces. Component identification and characterization of SHF in vitro and in vivo lay foundations for disclosure of its pharmacodynamic substances and elucidation of the scientific connotation.

Dose-Dependent Production of Anti-PEG IgM after Intramuscular PEGylated-Hydrogenated Soy Phosphatidylcholine Liposomes, but Not Lipid Nanoparticle Formulations of DNA, Correlates with the Plasma Clearance of PEGylated Liposomal Doxorubicin in Rats.

PEGylated lipid nanoparticle-based Covid-19 vaccines, including Pfizer's BNT162b2 and Moderna's mRNA-1273, have been shown to stimulate variable anti-PEG antibody production in humans. Anti-PEG antibodies have the potential to accelerate the plasma clearance of PEGylated therapeutics, such as PEGylated liposomes and proteins, and compromise their therapeutic efficacy. However, it is not yet clear whether antibody titers produced by PEGylated Covid-19 vaccines significantly affect the clearance of PEGylated therapeutics. This study examined how anti-PEG IgM levels affect the pharmacokinetics of PEGylated liposomal doxorubicin (PLD) and compared the immunogenicity of a lipid nanoparticle formulation of linear DNA (DNA-LNP) to standard PEG-HSPC liposomes. DNA-LNP was prepared using the same composition and approach as Pfizer's BNT162b2, except linear double-stranded DNA was used as the genetic material. PEGylated HSPC-based liposomes were formulated using the lipid rehydration and extrusion method. Nanoparticles were dosed IM to rats at 0.005-0.5 mg lipid/kg body weight 1 week before evaluating the plasma pharmacokinetics of clinically relevant doses of PLD (1 mg/kg, IV) or PEGylated interferon α2a (Pegasys, 5 µg/kg, SC). Plasma PEG IgM was compared between pre- and 1-week post-dose blood samples. The results showed that anti-PEG IgM production increased with increasing PEG-HSPC liposome dose and that IgM significantly correlated with the plasma half-life, clearance, volume of distribution, and area under the curve of a subsequent dose of PLD. The plasma exposure of Pegasys was also significantly reduced after initial delivery of 0.005 mg/ml PEG-HSPC liposome. However, a single 0.05 mg/kg IM dose of DNA-LNP did not significantly elevate PEG IgM and did not alter the IV pharmacokinetics of PLD. These data showed that PEGylated Covid-19 vaccines are less immunogenic compared to standard PEGylated HSPC liposomes and that there is an antibody threshold for accelerating the clearance of PEGylated therapeutics.

Cepharanthine Dry Powder Inhaler for the Treatment of Acute Lung Injury.

Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) induces a severe cytokine storm that may cause acute lung injury/acute respiratory distress syndrome (ALI/ARDS) with high clinical morbidity and mortality in infected individuals. Cepharanthine (CEP) is a bisbenzylisoquinoline alkaloid isolated and extracted from Stephania cepharantha Hayata. It exhibits various pharmacological effects, including antioxidant, anti-inflammatory, immunomodulatory, anti-tumor, and antiviral activities. The low oral bioavailability of CEP can be attributed to its poor water solubility. In this study, we utilized the freeze-drying method to prepare dry powder inhalers (DPI) for the treatment of acute lung injury (ALI) in rats via pulmonary administration. According to the powder properties study, the aerodynamic median diameter (Da) of the DPIs was 3.2 µm, and the in vitro lung deposition rate was 30.26; thus, meeting the Chinese Pharmacopoeia standard for pulmonary inhalation administration. We established an ALI rat model by intratracheal injection of hydrochloric acid (1.2 mL/kg, pH = 1.25). At 1 h after the model's establishment, CEP dry powder inhalers (CEP DPIs) (30 mg/kg) were sprayed into the lungs of rats with ALI via the trachea. Compared with the model group, the treatment group exhibited a reduced pulmonary edema and hemorrhage, and significantly reduced content of inflammatory factors (TNF-α, IL-6 and total protein) in their lungs (p < 0.01), indicating that the main mechanism of CEP underlying the treatment of ALI is anti-inflammation. Overall, the dry powder inhaler can deliver the drug directly to the site of the disease, increasing the intrapulmonary utilization of CEP and improving its efficacy, making it a promising inhalable formulation for the treatment of ALI.

Novel SARS-CoV-2 entry inhibitors, 2-anilinoquinazolin-4(3H)-one derivatives, show potency as SARS-CoV-2 antivirals in a human ACE2 transgenic mouse model.

The ongoing COVID-19 has not only caused millions of deaths worldwide, but it has also led to economic recession and the collapse of public health systems. The vaccines and antivirals developed in response to the pandemic have improved the situation markedly; however, the pandemic is still not under control with recurring surges. Thus, it is still necessary to develop therapeutic agents. In our previous studies, we designed and synthesized a series of novel 2-anilinoquinazolin-4(3H)-one derivatives, and demonstrated inhibitory activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and MERS-CoV in vitro. We then conducted in vivo studies using modified compounds that are suitable for oral administration. These compounds demonstrated no toxicity in rats and inhibited viral entry. Here, we investigated the in vivo efficacy of these drug candidates against SARS-CoV-2. Three candidate drugs, 7-chloro-2-((3,5-dichlorophenyl)amino)quinazolin-4(3H)-one (1), N-(7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-N-(3,5-dichlorophenyl)acetamide (2), and N-(7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-N-(3,5-difluorophenyl)acetamide (3) were administered orally to hACE2 transgenic mice at a dose of 100 mg/kg. All three drugs improved survival rate and reduced the viral load in the lungs. These results show that the derivatives possess in vivo antiviral efficacy similar to that of molnupiravir, which is currently being used to treat COVID-19. Overall, our data suggest that 2-anilinoquinazolin-4(3H)-one derivatives are promising as potential oral antiviral drug candidates against SARS-CoV-2 infection.

Evaluation of the Antiviral Efficacy of Subcutaneous Nafamostat Formulated with Glycyrrhizic Acid against SARS-CoV-2 in a Murine Model.

The ongoing COVID-19 pandemic highlights the urgent need for effective antiviral agents and vaccines. Drug repositioning, which involves modifying existing drugs, offers a promising approach for expediting the development of novel therapeutics. In this study, we developed a new drug, MDB-MDB-601a-NM, by modifying the existing drug nafamostat (NM) with the incorporation of glycyrrhizic acid (GA). We assessed the pharmacokinetic profiles of MDB-601a-NM and nafamostat in Sprague-Dawley rats, revealing rapid clearance of nafamostat and sustained drug concentration of MDB-601a-NM after subcutaneous administration. Single-dose toxicity studies showed potential toxicity and persistent swelling at the injection site with high-dose administration of MDB-601a-NM. Furthermore, we evaluated the efficacy of MDB-601a-NM in protecting against SARS-CoV-2 infection using the K18 hACE-2 transgenic mouse model. Mice treated with 60 mg/kg and 100 mg/kg of MDB-601a-NM exhibited improved protectivity in terms of weight loss and survival rates compared to the nafamostat-treated group. Histopathological analysis revealed dose-dependent improvements in histopathological changes and enhanced inhibitory efficacy in MDB-601a-NM-treated groups. Notably, no viral replication was detected in the brain tissue when mice were treated with 60 mg/kg and 100 mg/kg of MDB-601a-NM. Our developed MDB-601a-NM, a modified Nafamostat with glycyrrhizic acid, shows improved protectivity against SARS-CoV-2 infection. Its sustained drug concentration after subcutaneous administration and dose-dependent improvements makes it a promising therapeutic option.

The Prophylactic Effect of Vitamin C and Vitamin B12 against Ultraviolet-C-Induced Hepatotoxicity in Male Rats.

Ultraviolet C (UVC) devices are an effective means of disinfecting surfaces and protecting medical tools against various microbes, including coronavirus. Overexposure to UVC can induce oxidative stress, damage the genetic material, and harm biological systems. This study investigated the prophylactic efficacy of vitamin C and B12 against hepatotoxicity in UVC-intoxicated rats. Rats were irradiated with UVC (725.76, 967.68, and 1048.36 J/cm2) for 2 weeks. The rats were pretreated with the aforementioned antioxidants for two months before UVC irradiation. The prophylactic effect of vitamins against UVC hepatotoxicity was evaluated by monitoring the alteration of liver enzyme activities, antioxidant status, apoptotic and inflammatory markers, DNA fragmentation, and histological and ultrastructural alterations. Rats exposed to UVC showed a significant increase in liver enzymes, oxidant-antioxidant balance disruption, and increased hepatic inflammatory markers (TNF-α, IL-1ß, iNOS, and IDO-1). Additionally, obvious over-expression of activated caspase-3 protein and DNA fragmentation were detected. Histological and ultrastructural examinations verified the biochemical findings. Co-treatment with vitamins ameliorated the deviated parameters to variable degrees. In conclusion, vitamin C could alleviate UVC-induced hepatotoxicity more than vitamin B12 by diminishing oxidative stress, inflammation, and DNA damage. This study could provide a reference for the clinical practice of vitamin C and B12 as radioprotective for workers in UVC disinfectant areas.

The ecology of viruses in urban rodents with a focus on SARS-CoV-2.

Wild animals are naturally infected with a range of viruses, some of which may be zoonotic. During the human COVID pandemic there was also the possibility of rodents acquiring SARS-CoV-2 from people, so-called reverse zoonoses. To investigate this, we sampled rats (Rattus norvegicus) and mice (Apodemus sylvaticus) from urban environments in 2020 during the human COVID-19 pandemic. We metagenomically sequenced lung and gut tissue and faeces for viruses, PCR screened for SARS-CoV-2, and serologically surveyed for anti-SARS-CoV-2 Spike antibodies. We describe the range of viruses that we found in these two rodent species. We found no molecular evidence of SARS-CoV-2 infection, though in rats we found lung antibody responses and evidence of neutralization ability that are consistent with rats being exposed to SARS-CoV-2 and/or exposed to other viruses that result in cross-reactive antibodies.

Pathogen Prevalence Estimates and Diagnostic Methodology Trends in Laboratory Mice and Rats from 2003 to 2020.

Rodents used in biomedical research are maintained as specific pathogen-free (SPF) by employing biosecurity measures that eliminate and exclude adventitious infectious agents known to confound research. The efficacy of these practices is assessed by routine laboratory testing referred to as health monitoring (HM). This study summarizes the results of HM performed at Charles River Research Animal Diagnostic Services (CR-RADS) on samples submitted by external (non-Charles River) clients between 2003 and 2020. Summarizing this vast amount of data has been made practicable by the recent introduction of end-user business intelligence tools to Excel. HM summaries include the number of samples tested and the percent positive by diagnostic methodology, including direct examination for parasites, cultural isolation and identification for bacteria, serology for antibodies to viruses and fastidious microorganisms, and polymerase chain reaction (PCR) assays for pathogen-specific genomic sequences. Consistent with comparable studies, the percentages of pathogen-positive samples by diagnostic methodology and year interval are referred to as period prevalence estimates (%PE). These %PE substantiate the elimination of once common respiratory pathogens, such as Sendai virus, and reductions in the prevalence of other agents considered common, such as the rodent coronaviruses and parvoviruses. Conversely, the %PE of certain pathogens, for example, murine norovirus (MNV), Helicobacter, Rodentibacter, and parasites remain high, perhaps due to the increasing exchange of genetically engineered mutant (GEM) rodents among researchers and the challenges and high cost of eliminating these agents from rodent housing facilities. Study results also document the growing role of PCR in HM because of its applicability to all pathogen types and its high specificity and sensitivity; moreover, PCR can detect pathogens in samples collected antemortem directly from colony animals and from the environment, thereby improving the detection of host-adapted, environmentally unstable pathogens that are not efficiently transmitted to sentinels by soiled bedding.

Protective effects of vitamin D3 (cholecalciferol) on vancomycin-induced oxidative nephrotoxic damage in rats.

CONTEXT: Vancomycin (VCM), an important antibiotic against refractory infections, has been used to treat secondary infections in severe COVID-19 patients. Regrettably, VCM treatment has been associated with nephrotoxicity. Vitamin D3 can prevent nephrotoxicity through its antioxidant effect. OBJECTIVE: This study tests the antioxidant effect of vitamin D3 in the prevention of VCM-induced nephrotoxicity. MATERIALS AND METHODS: Wistar Albino rats (21) were randomly divided into 3 groups: (A) control; (B) VCM 300 mg/kg daily for 1 week; and (C) VCM plus vitamin D3 500 IU/kg daily for 2 weeks. All the rats were sacrificed and serum was separated to determine kidney function parameters. Their kidneys were also dissected for histological examination and for oxidative stress markers. RESULTS: Lipid peroxidation, creatinine, and urea levels decreased significantly (p < 0.0001) in the vitamin D3-treated group (14.46, 84.11, 36.17%, respectively) compared to the VCM group that was given VCM (MIC<2 µg/mL) only. A significant increase was observed in superoxide dismutase levels in the vitamin D3-treated group (p < 0.05) compared to rats without treatment. Furthermore, kidney histopathology of the rats treated with vitamin D3 showed that dilatation, vacuolization and necrosis tubules decreased significantly (p < 0.05) compared with those in the VCM group. Glomerular injury, hyaline dystrophy, and inflammation improved significantly in the vitamin D3 group (p < 0.001, p < 0.05, p < 0.05, respectively) compared with the VCM group. DISCUSSION AND CONCLUSIONS: Vitamin D3 can prevent VCM nephrotoxicity. Therefore, the appropriate dose of this vitamin must be determined, especially for those infected with COVID-19 and receiving VCM, to manage their secondary infections.

Pressure overload induces ISG15 to facilitate adverse ventricular remodeling and promote heart failure.

Inflammation promotes adverse ventricular remodeling, a common antecedent of heart failure. Here, we set out to determine how inflammatory cells affect cardiomyocytes in the remodeling heart. Pathogenic cardiac macrophages induced an IFN response in cardiomyocytes, characterized by upregulation of the ubiquitin-like protein IFN-stimulated gene 15 (ISG15), which posttranslationally modifies its targets through a process termed ISGylation. Cardiac ISG15 is controlled by type I IFN signaling, and ISG15 or ISGylation is upregulated in mice with transverse aortic constriction or infused with angiotensin II; rats with uninephrectomy and DOCA-salt, or pulmonary artery banding; cardiomyocytes exposed to IFNs or CD4+ T cell-conditioned medium; and ventricular tissue of humans with nonischemic cardiomyopathy. By nanoscale liquid chromatography-tandem mass spectrometry, we identified the myofibrillar protein filamin-C as an ISGylation target. ISG15 deficiency preserved cardiac function in mice with transverse aortic constriction and led to improved recovery of mouse hearts ex vivo. Metabolomics revealed that ISG15 regulates cardiac amino acid metabolism, whereas ISG15 deficiency prevented misfolded filamin-C accumulation and induced cardiomyocyte autophagy. In sum, ISG15 upregulation is a feature of pathological ventricular remodeling, and protein ISGylation is an inflammation-induced posttranslational modification that may contribute to heart failure development by altering cardiomyocyte protein turnover.

Effects of maternal social isolation on adult rodent offspring cognition.

Prenatal experiences can influence offspring physiology and behaviour through the lifespan. Various forms of prenatal stress impair adult learning and memory function and can lead to increased occurrence of anxiety and depression. Clinical work suggests that prenatal stress and maternal depression lead to similar outcomes in children and adolescents, however the long-term effects of maternal depression are less established, particularly in well controlled animal models. Social isolation is common in depressed individuals and during the recent COVID-19 pandemic. Accordingly, for this study we were interested in the effects of maternal stress induced via social isolation on adult offspring cognitive functions including spatial, stimulus-response, and emotional learning and memory that are mediated by different networks centered on the hippocampus, dorsal striatum, and amygdala, respectively. Tasks included a discriminative contextual fear conditioning task and cue-place water task. Pregnant dams in the social isolation group were single housed prior to and throughout gestation. Once offspring reached adulthood the male offspring were trained on a contextual fear conditioning task in which rats were trained to associate one of two contexts with an aversive stimulus and the opposing context remained neutral. Afterwards a cue-place water task was performed during which they were required to navigate to both a visible and invisible platform. Fear conditioning results revealed that the adult offspring of socially isolated mothers, but not controls, were impaired in associating a specific context with a fear-inducing stimulus as assessed by conditioned freezing and avoidance. Results from the water task indicate that adult offspring of mothers that were socially isolated showed place learning deficits but not stimulus-response habit learning on the same task. These cognitive impairments, in the offspring of socially isolated dams, occurred in the absence of maternal elevated stress hormone levels, anxiety, or altered mothering. Some evidence suggested that maternal blood-glucose levels were altered particularly during gestation. Our results provide further support for the idea that learning and memory networks, centered on the amygdala and hippocampus are particularly susceptible to the negative impacts of maternal social isolation and these effects can occur without elevated glucocorticoid levels associated with other forms of prenatal stress.

Hand instrumentation provides improved tissue response over ultrasonic scaler and substantiates safe dental practice: An in vivo study in rats.

OBJECTIVE: The aim of this study was to evaluate the effectiveness of hand debridement (HD) versus ultrasonic dental scaler (UDS) for the treatment of experimental periodontitis (EP) in rats. MATERIAL AND METHODS: Thirty 3-month-old male rats were used. EP was induced around the mandibular first molars (right and left). Seven days after induction, the treatments with either HD (n = 30) or UDS (n = 30) were randomly performed in each molar. Euthanasia were performed at 7, 15, and 30 days after treatment. Histometric (percentage of bone in the furcation [PBF]), histopathological, and immunohistochemical (for detection of tartrate-resistant acid phosphatase [TRAP] and osteocalcin [OCN]). Parametric data (PBF and TRAP) was analyzed by One-way ANOVA followed by Tukey's post-test. OCN was analyzed by Kruskal-Wallis followed by Student-Newman-Keuls post-test. The level of significance was 5%. RESULTS: Group HD presented higher PBF and lower TRAP-immunolabeling at 30 days as compared with UDS in the same period (p≤0.05). Group HD presented higher OCN immunolabeling at 30 days as compared with 7 and 15 days (p≤0.05). Persistent and exacerbated inflammatory process was observed in some specimens from group UDS at 30 days, as well as the bone trabeculae presented irregular contour, surrounded by many active osteoclasts. CONCLUSION: Nonsurgical periodontal therapy with HD resulted in higher PBF and lower expression of TRAP as compared with UDS. Also, HD increased the expression of OCN over time.

Biochemical and histopathological evaluation of ChAdOx1-S and BBIBP-CorV vaccination in normal and streptozotocin-induced diabetic Wistar rats.

Infection with Covid-19 has been associated with some medical complications namely diabetes, thrombosis, and hepatic and renal dysfunction among others. This situation has created some concern about the use of relevant vaccines which might cause similar complications. In that regard, we planned to evaluate the impact of two of the relevant vaccines namely ChAdOx1-S and BBIBP-CorV on some of the blood biochemical factors and also on liver and kidneys functions following the immunization of healthy and streptozotocin-induced diabetic rats. Evaluation of the level of neutralizing antibody among the rats indicated that immunization with ChAdOx1-S induced a higher level of neutralizing antibody among both the healthy and diabetic rats compared to the BBIBP-CorV vaccine. Furthermore, the neutralizing antibody levels against both types of vaccines were significantly lower in diabetic rats than in healthy ones. On the other hand, no alterations were observed in the rats' sera biochemical factors, coagulation values and histopathological images of the liver and kidneys. Altogether these data besides of confirming the effectiveness of both vaccines, indicate that both vaccines have no hazardous side effects on rats and probably humans though clinical investigations are required to validate our present data.

Myricetin possesses the potency against SARS-CoV-2 infection through blocking viral-entry facilitators and suppressing inflammation in rats and mice.

BACKGROUND: Myricetin (3,5,7-trihydroxy-2-(3,4,5-tri hydroxyphenyl)-4-benzopyrone) is a common flavonol extracted from many natural plants and Chinese herb medicines and has been demonstrated to have multiple pharmacological activities, such as anti-microbial, anti-thrombotic, neuroprotective, and anti-inflammatory effects. Previously, myricetin was reported to target Mpro and 3CL-Pro-enzymatic activity to SARS-CoV-2. However, the protective value of myricetin on SARS-Cov-2 infection through viral-entry facilitators has not yet been comprehensively understood. PURPOSE: The aim of the current study was to evaluate the pharmacological efficacy and the mechanisms of action of myricetin against SARS-CoV-2 infection both in vitro and in vivo. METHODS: The inhibitory effects of myricetin on SARS-CoV-2 infection and replication were assessed on Vero E6 cells. Molecular docking analysis and bilayer interferometry (BLI) assays, immunocytochemistry (ICC), and pseudoviruses assays were performed to evaluate the roles of myricetin in the intermolecular interaction between the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein and angiotensin-converting enzyme 2 (ACE2). The anti-inflammatory potency and mechanisms of myricetin were examined in THP1 macrophages in vitro, as well as in carrageenan-induced paw edema, delayed-type hypersensitivity (DTH) induced auricle edema, and LPS-induced acute lung injury (ALI) animal models. RESULTS: The results showed that myricetin was able to inhibit binding between the RBD of the SARS-CoV-2 S protein and ACE2 through molecular docking analysis and BLI assay, demonstrating its potential as a viral-entry facilitator blocker. Myricetin could also significantly inhibit SASR-CoV-2 infection and replication in Vero E6 cells (EC50 55.18 µM), which was further validated with pseudoviruses containing the RBD (wild-type, N501Y, N439K, Y453F) and an S1 glycoprotein mutant (S-D614G). Moreover, myricetin exhibited a marked suppressive action on the receptor-interacting serine/threonine protein kinase 1 (RIPK1)-driven inflammation and NF-kappa B signaling in THP1 macrophages. In animal model studies, myricetin notably ameliorated carrageenan-induced paw edema in rats, DTH induced auricle edema in mice, and LPS-induced ALI in mice. CONCLUSION: Our findings showed that myricetin inhibited HCoV-229E and SARS-CoV-2 replication in vitro, blocked SARS-CoV-2 virus entry facilitators and relieved inflammation through the RIPK1/NF-κB pathway, suggesting that this flavonol has the potential to be developed as a therapeutic agent against COVID-19.

The mechanism of Qingwen Gupi decoction on pulmonary fibrosis based on metabolomics and intestinal flora.

AIMS: To evaluate the effects of the Qingwen Gupi decoction (QGT) in a rat model of bleomycin-induced pulmonary fibrosis (PF), and explore the underlying mechanisms by integrating UPLC-Q-TOF/MS metabolomics and 16S rDNA sequencing of gut microbiota. METHODS AND RESULTS: The animals were randomly divided into the control, PF model, pirfenidone-treated, and low-, medium-, and high-dose QGT groups. The lung tissues were examined and the expression of TGF-ß, SMAD-3, and SMAD-7 mRNAs in the lung tissues were analyzed. Metabolomic profiles were analyzed by UPLC-QTOF/MS, and the intestinal flora were examined by prokaryotic 16 rDNA sequencing. Pathological examination and biochemical indices revealed that QGT treatment improved the symptoms of PF by varying degrees. Furthermore, QGT significantly downregulated TGF-ß1 and Smad-3 mRNAs and increased the expression levels of Smad-7. QGT-L in particular increased the levels of 18 key metabolic biomarkers that were associated with nine gut microbial species and may exert antifibrosis effects through arachidonic acid metabolism, glycerophospholipid metabolism, and phenylalanine metabolism. CONCLUSIONS: QGT alleviated PF in a rat model through its anti-inflammatory, antioxidant, and anti-fibrotic effects, and by reversing bleomycin-induced gut dysbiosis.This study lays the foundation for further research on the pathological mechanisms of PF and the development of new drug candidates.

Broad immunity to SARS-CoV-2 variants of concern mediated by a SARS-CoV-2 receptor-binding domain protein vaccine.

BACKGROUND: The SARS-CoV-2 global pandemic has fuelled the generation of vaccines at an unprecedented pace and scale. However, many challenges remain, including: the emergence of vaccine-resistant mutant viruses, vaccine stability during storage and transport, waning vaccine-induced immunity, and concerns about infrequent adverse events associated with existing vaccines. METHODS: We report on a protein subunit vaccine comprising the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 spike protein, dimerised with an immunoglobulin IgG1 Fc domain. These were tested in conjunction with three different adjuvants: a TLR2 agonist R4-Pam2Cys, an NKT cell agonist glycolipid α-Galactosylceramide, or MF59® squalene oil-in-water adjuvant, using mice, rats and hamsters. We also developed an RBD-human IgG1 Fc vaccine with an RBD sequence of the immuno-evasive beta variant (N501Y, E484K, K417N). These vaccines were also tested as a heterologous third dose booster in mice, following priming with whole spike vaccine. FINDINGS: Each formulation of the RBD-Fc vaccines drove strong neutralising antibody (nAb) responses and provided durable and highly protective immunity against lower and upper airway infection in mouse models of COVID-19. The 'beta variant' RBD vaccine, combined with MF59® adjuvant, induced strong protection in mice against the beta strain as well as the ancestral strain. Furthermore, when used as a heterologous third dose booster, the RBD-Fc vaccines combined with MF59® increased titres of nAb against other variants including alpha, delta, delta+, gamma, lambda, mu, and omicron BA.1, BA.2 and BA.5. INTERPRETATION: These results demonstrated that an RBD-Fc protein subunit/MF59® adjuvanted vaccine can induce high levels of broadly reactive nAbs, including when used as a booster following prior immunisation of mice with whole ancestral-strain spike vaccines. This vaccine platform offers a potential approach to augment some of the currently approved vaccines in the face of emerging variants of concern, and it has now entered a phase I clinical trial. FUNDING: This work was supported by grants from the Medical Research Future Fund (MRFF) (2005846), The Jack Ma Foundation, National Health and Medical Research Council of Australia (NHMRC; 1113293) and Singapore National Medical Research Council (MOH-COVID19RF-003). Individual researchers were supported by an NHMRC Senior Principal Research Fellowship (1117766), NHMRC Investigator Awards (2008913 and 1173871), Australian Research Council Discovery Early Career Research Award (ARC DECRA; DE210100705) and philanthropic awards from IFM investors and the A2 Milk Company.

Urinary liver-type fatty acid binding protein is increased in the early stages of the disease with a risk of acute kidney injury induced by histone.

AIM: Urinary liver-type fatty acid binding protein (L-FABP) has potential utility as an early prognostic biomarker ahead of traditional severity scores in coronavirus disease 2019 and sepsis, however, the mechanism of elevated urinary L-FABP in the disease has not been clearly elucidated. We investigated the background mechanisms of urinary L-FABP excretion through non-clinical animal model focusing on histone, which is one of the aggravating factors in these infectious diseases. METHODS: Male Sprague-Dawley rats were placed in central intravenous catheters, and these rats were given a continuous intravenous infusion of 0.25 or 0.5 mg/kg/min calf thymus histones for 240 min from caudal vena cava. RESULTS: After the administration of histone, urinary L-FABP and gene expression of an oxidative stress marker in the kidney increased in a histone dose-dependent manner before increased serum creatinine. Upon further investigation, fibrin deposition in the glomerulus was observed and it tended to be remarkable in the high dose administrated groups. The levels of coagulation factor were significantly changed after the administration of histone, and these were significantly correlated with the levels of urinary L-FABP. CONCLUSIONS: Firstly, it was suggested that histone is one of the causative agents for the urinary L-FABP increase at an early stage of the disease with a risk of acute kidney injury. Secondly, urinary L-FABP could be a marker reflecting the changes of coagulation system and microthrombus caused by histone in the early stage of acute kidney injury before becoming severely ill and maybe a guide to early treatment initiation.

A Phase 1 study for safety and pharmacokinetics of BIO101 (20-hydroxyecdysone) in healthy young and older adults.

BACKGROUND: Sarcopenia is an age-related skeletal muscle disorder characterized by loss of muscle mass and strength leading to mobility disability. 20-Hydroxyecdysone (20E) is a polyhydroxylated plant steroid that demonstrates pharmacological effects in many disease animal models including ageing/sarcopenia. BIO101 is a 20E purified investigational drug (≥97%) that previously demonstrated good toxicology profiles in rat and dog. BIO101 is evaluated in healthy young and older adults in a Phase 1 study. METHODS: This study is a Single Ascending Dose (SAD) followed by a 14-day Multiple Ascending Dose (MAD). In SAD, BIO101 was administered orally to 16 young adults at doses from 100 to 1400 mg and to 8 older adults (age ≥65 years) at 1400 mg. In MAD, doses of 350 mg once daily (qd), 350 mg twice daily (bid) and 450 mg bid were administered to 10 older adults. The primary objective was to evaluate safety and pharmacokinetics (PK), including dosing of circulating metabolites. Pharmacodynamic effects were investigated with regard to myostatin, procollagen-III-amino-terminal propeptide (PIIINP), myoglobin, creatine-kinase Muscle Brain (CKMB), renin and aldosterone plasma/serum levels. RESULTS: BIO101 showed a good safety profile with only mild to moderate adverse events and a satisfactory pharmacokinetic profile. In SAD, at 100 mg to 1400 mg, mean Cmax and areas under the curve increased less than dose-proportionally. Mean half-life was short (2.4-4.9 h), and mean renal clearance was comparable in all doses (4.05-5.05 L/h). Mean plasma exposure was slightly lower in older adults (22% lower for Cmax and 13%-15% lower for AUCs) compared with young subjects. In MAD, 350 and 450 mg bid led to a slight accumulation over 14 days (mean ratio of accumulation [Rac] of 1.31 in both cohorts). Reduction of biomarkers (myoglobin, CK-MB) mean serum levels (vs. baseline) was observed at 450 mg bid. Two major metabolites of 20E (14-deoxy-20-hydroxyecdysone and 14-deoxypoststerone) were identified and quantified. CONCLUSIONS: BIO101 shows a good safety and pharmacokinetic profile that led to the selection of doses for the subsequent interventional clinical trials of Phase 2 in age-related sarcopenia (SARA-INT) and Phase 3 in Covid-19 (COVA).

Phytocompounds from Anvillea radiata as promising anti-Covid-19 drugs: in silico studies and in vivo safety assessment.

As an alternative strategy in combating the COVID-19 pandemic, phytoconstituents from medicinal plants are getting attention worldwide. The current investigation focused on the efficacy of the essential phytocompounds identified in Anvillea radiata to target the main protease (Mpro) of SARS-COV-2 through molecular docking and dynamic analyses; in addition to the safety assessment of this herb in vivo. In silico, the 6LU7 structure of Mpro was prepared as a target by Discovery Studio 2020. The virtual screening of phytocompounds from Anvillea radiata was performed through iGEMDOCK program, followed by an evaluation of the potential inhibitors based on the docking scores calculated using AutoDock Vina and MGL Tools programs, as well as complexes stability assessment through MD simulation. In vivo toxicity studies of Anvillea radiata aqueous extract were also conducted in Wistar rats. Among the phytocompounds evaluated in this study, 3,5-Dicaffeoylquinic acid, Spinacetin, 9α-Epoxyparthenolide, Hispidulin, Quercetin, jaceosidin, Nepetin, and isorhamnetin were predicted to have the highest binding affinity for the Main protease (Mpro) target of SARS-CoV-2. The aqueous extract of Anvillea radiata did not induce any signs of toxicity. 3,5-Dicaffeoylquinic acid, Spinacetin, 9α-Epoxyparthenolide, jaceosidin, and isorhamnetin from Anvillea radiata were selected as potential inhibitors of SARS-Cov-2 to develop new drugs anti-COVID-19.