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.

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.

SARS-CoV-2 Exposure in Norway Rats (Rattus norvegicus) from New York City.

Millions of Norway rats (Rattus norvegicus) inhabit New York City (NYC), presenting the potential for transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to rats. We evaluated SARS-CoV-2 exposure among 79 rats captured from NYC during the fall of 2021. Our results showed that 13 of the 79 rats (16.5%) tested IgG- or IgM-positive, and partial SARS-CoV-2 genomes were recovered from all 4 rats that were qRT-PCR (reverse transcription-quantitative PCR)-positive. Genomic analyses suggest these viruses were associated with genetic lineage B, which was predominant in NYC in the spring of 2020 during the early pandemic period. To further investigate rat susceptibility to SARS-CoV-2 variants, we conducted a virus challenge study and showed that Alpha, Delta, and Omicron variants can cause infections in wild-type Sprague Dawley (SD) rats, including high replication levels in the upper and lower respiratory tracts and induction of both innate and adaptive immune responses. Additionally, the Delta variant resulted in the highest infectivity. In summary, our results indicate that rats are susceptible to infection with Alpha, Delta, and Omicron variants, and wild Norway rats in the NYC municipal sewer systems have been exposed to SARS-CoV-2. Our findings highlight the need for further monitoring of SARS-CoV-2 in urban rat populations and for evaluating the potential risk of secondary zoonotic transmission from these rat populations back to humans. IMPORTANCE The host tropism expansion of SARS-CoV-2 raises concern for the potential risk of reverse-zoonotic transmission of emerging variants into rodent species, including wild rat species. In this study, we present both genetic and serological evidence for SARS-CoV-2 exposure to the New York City wild rat population, and these viruses may be linked to the viruses that were circulating during the early stages of the pandemic. We also demonstrated that rats are susceptible to additional variants (i.e., Alpha, Delta, and Omicron) that have been predominant in humans and that susceptibility to infection varies by variant. Our findings highlight the reverse zoonosis of SARS-CoV-2 to urban rats and the need for further monitoring of SARS-CoV-2 in rat populations for potential secondary zoonotic transmission to humans.

Ultrasmall and highly biocompatible carbon dots derived from natural plant with amelioration against acute kidney injury.

BACKGROUND: Acute kidney injury (AKI) refers to a tricky clinical disease, known by its high morbidity and mortality, with no real specific medicine for AKI. The carbonization product from Pollen Typhae (i.e., Pu-huang in China) has been extensively employed in clinic, and it is capable of relieving the renal damage and other diseases in China since acient times. RESULTS: Inspired by the carbonization process of Traditional Chinese Medicine (TCM), a novel species of carbon dots derived from Pollen Typhae (PT-CDs) was separated and then collected using a one-pot pyrolysis method. The as-prepared PT-CDs (4.85 ± 2.06 nm) with negative charge and abundant oxygenated groups exhibited high solubility, and they were stable in water. Moreover, the rhabdomyolysis (RM)-induced AKI rat model was used, and it was first demonstrated that PT-CDs had significant activity in improving the level of BUN and CRE, urine volume and kidney index, and histopathological morphology in RM-induced AKI rats. It is noteworthy that interventions of PT-CDs significantly reduced degree of inflammatory reaction and oxidative stress, which may be correlated with the basial potential mechanism of anti-AKI activities. Furthermore, cytotoxicity assay and biosafety evaluation exhibited high biocompatibility of PT-CDs. CONCLUSION: This study offers a novel relieving strategy for AKI based on PT-CDs and suggests its potential to be a related candidate for clinical applications.

Spike Protein Impairs Mitochondrial Function in Human Cardiomyocytes: Mechanisms Underlying Cardiac Injury in COVID-19.

BACKGROUND: COVID-19 has a major impact on cardiovascular diseases and may lead to myocarditis or cardiac failure. The clove-like spike (S) protein of SARS-CoV-2 facilitates its transmission and pathogenesis. Cardiac mitochondria produce energy for key heart functions. We hypothesized that S1 would directly impair the functions of cardiomyocyte mitochondria, thus causing cardiac dysfunction. METHODS: Through the Seahorse Mito Stress Test and real-time ATP rate assays, we explored the mitochondrial bioenergetics in human cardiomyocytes (AC16). The cells were treated without (control) or with S1 (1 nM) for 24, 48, and 72 h and we observed the mitochondrial morphology using transmission electron microscopy and confocal fluorescence microscopy. Western blotting, XRhod-1, and MitoSOX Red staining were performed to evaluate the expression of proteins related to energetic metabolism and relevant signaling cascades, mitochondrial Ca2+ levels, and ROS production. RESULTS: The 24 h S1 treatment increased ATP production and mitochondrial respiration by increasing the expression of fatty-acid-transporting regulators and inducing more negative mitochondrial membrane potential (Δψm). The 72 h S1 treatment decreased mitochondrial respiration rates and Δψm, but increased levels of reactive oxygen species (ROS), mCa2+, and intracellular Ca2+. Electron microscopy revealed increased mitochondrial fragmentation/fission in AC16 cells treated for 72 h. The effects of S1 on ATP production were completely blocked by neutralizing ACE2 but not CD147 antibodies, and were partly attenuated by Mitotempo (1 µM). CONCLUSION: S1 might impair mitochondrial function in human cardiomyocytes by altering Δψm, mCa2+ overload, ROS accumulation, and mitochondrial dynamics via ACE2.

Preclinical evaluation of immunogenicity, efficacy and safety of a recombinant plant-based SARS-CoV-2 RBD vaccine formulated with 3M-052-Alum adjuvant.

Cost-effective, and accessible vaccines are needed for mass immunization to control the ongoing coronavirus disease 2019 (COVID-19), especially in low- and middle-income countries (LMIC).A plant-based vaccine is an attractive technology platform since the recombinant proteins can be easily produced at large scale and low cost. For the recombinant subunit-based vaccines, effective adjuvants are crucial to enhance the magnitude and breadth of immune responses elicited by the vaccine. In this study, we report a preclinical evaluation of the immunogenicity, efficacy and safety of a recombinant plant-based SARS-CoV-2 RBD vaccine formulated with 3M-052 (TLR7/8 agonist)-Alum adjuvant. This vaccine formulation, named Baiya SARS-CoV-2 Vax 2, induced significant levels of RBD-specific IgG and neutralizing antibody responses in mice. A viral challenge study using humanized K18-hACE2 mice has shown that animals vaccinated with two doses of Baiya SARS-CoV-2 Vax 2 established immune protection against SARS-CoV-2. A study in nonhuman primates (cynomolgus monkeys) indicated that immunization with two doses of Baiya SARS-CoV-2 Vax 2 was safe, well tolerated, and induced neutralizing antibodies against the prototype virus and other viral variants (Alpha, Beta, Gamma, Delta, and Omicron subvariants). The toxicity of Baiya SARS-CoV-2 Vax 2 was further investigated in Jcl:SD rats, which demonstrated that a single dose and repeated doses of Baiya SARS-CoV-2 Vax 2 were well tolerated and no mortality or unanticipated findings were observed. Overall, these preclinical findings support further clinical development of Baiya SARS-CoV-2 Vax 2.

Intradermal delivery of a quadrivalent cell-based seasonal influenza vaccine using an adjuvanted skin patch vaccination platform.

All seasonal influenza vaccines for 2021-2022 in the US were quadrivalent and the market continues to be dominated by intramuscular delivery of non-adjuvanted, virion-derived antigens grown in chicken eggs. Up to four new egg-adapted production influenza vaccine strains must be generated each year. The introduction in 2012 of Flucelvax®, which is grown in mammalian suspension cell culture and uses vaccine production strains without adaptive mutations for efficient growth in eggs, represented a major advance in vaccine production technology. Here we demonstrate that Flucelvax can be reformulated and combined with a liposomal adjuvant containing QS-21 (Verndari Adjuvant System 1.1, VAS1.1) or QS-21 and 3D-PHAD (VAS1.2) for intradermal administration using a painless skin patch, VaxiPatch™. VAS1.2 is similar to AS01B, the adjuvant system used in Shingrix® and Mosquirix™. We show that Flucelvax, when reformulated and concentrated using tangential flow filtration (TFF), maintains hemagglutination and single radial immunodiffusion (SRID) potency. Loading the reformulated Flucelvax material onto VaxiPatch arrays conferred high levels of resistance to heat stress and room temperature stability. TFF enriched vaccine antigens were combined with VAS1.1 or VAS1.2 and dispensed in 10nL drops into the pockets of 36 (total 360 nL) stainless steel microneedles arranged in a microarray 1.2 cm in diameter. Using VaxiPatch delivery of 2 µg of antigen, we demonstrated intramusuclar-comparable IgG and hemagglutination inhibition (HAI) immune responses in Sprague Dawley® rats. With addition of VAS1.2, antigen-specific IgG titers were increased as much as 68-fold (47-fold for VAS1.1) with improvements in seroconversion for three of four strains (all four were improved by VAS1.1). TFF-reformulated antigens combined with VAS1.1 or VAS1.2 and delivered by VaxiPatch showed only minor skin reactogenicity after 1 h and no skin reactogenicity after 24 h. These data indicate that VaxiPatch and the VAS system have the potential to be transformative for vaccine delivery.

Tocotrienol-Rich Fractions Offer Potential to Suppress Pulmonary Fibrosis Progression.

Although pulmonary fibrosis (PF) is considered a rare disease, the incidence thereof has increased steadily in recent years, while a safe and effective cure remains beyond reach. In this study, the potential of tocotrienol-rich fractions (TRF) and carotene to alleviate PF was explored. PF was induced in Sprague-Dawley rats via a single intratracheal bleomycin (BLM) (5 mg/kg) instillation. These rats were subsequently treated with TRF, carotene, pirfenidone (Pir) and nintedanib (Nin) for 28 days via gavage administration, whereafter histopathological performance, biochemical functions and molecular alterations were studied in the lung tissues. Our results showed that TRF, carotene, Nin and Pir all ameliorated PF by reducing inflammation and resisting oxidative stress to varying degrees. The related mechanisms involved the TGF-ß1/Smad, PI3K/Akt and NF-κB signaling pathways. Ultimately, our findings revealed that, when combined with TRF, the therapeutic effects of Nin and Pir on PF were enhanced, indicating that TRF may, indeed, provide promising potential for use in combination therapy in the treatment of PF.

Congenital Rift Valley fever in Sprague Dawley rats is associated with diffuse infection and pathology of the placenta.

Rift Valley fever (RVF) is a disease of animals and humans associated with abortions in ruminants and late-gestation miscarriages in women. Here, we use a rat model of congenital RVF to identify tropisms, pathologies, and immune responses in the placenta during vertical transmission. Infection of late-gestation pregnant rats resulted in vertical transmission to the placenta and widespread infection throughout the decidua, basal zone, and labyrinth zone. Some pups from infected dams appeared normal while others had gross signs of teratogenicity including death. Histopathological lesions were detected in placenta from pups regardless of teratogenicity, while teratogenic pups had widespread hemorrhage throughout multiple placenta layers. Teratogenic events were associated with significant increases in placental pro-inflammatory cytokines, type I interferons, and chemokines. RVFV displays a high degree of tropism for all placental tissue layers and the degree of hemorrhage and inflammatory mediator production is highest in placenta from pups with adverse outcomes. Given the potential for RVFV to emerge in new locations and the recent evidence of emerging viruses, like Zika and SARS-CoV-2, to undergo vertical transmission, this study provides essential understanding regarding the mechanisms by which RVFV crosses the placenta barrier.

A novel RBD-protein/peptide vaccine elicits broadly neutralizing antibodies and protects mice and macaques against SARS-CoV-2.

The development of safe and effective vaccines to respond to COVID-19 pandemic/endemic remains a priority. We developed a novel subunit protein-peptide COVID-19 vaccine candidate (UB-612) composed of: (i) receptor binding domain of SARS-CoV-2 spike protein fused to a modified single-chain human IgG1 Fc; (ii) five synthetic peptides incorporating conserved helper and cytotoxic T lymphocyte (Th/CTL) epitopes derived from SARS-CoV-2 structural proteins (three from S2 subunit, one from membrane and one from nucleocapsid), and one universal Th peptide; (iii) aluminum phosphate as adjuvant. The immunogenicity and protective immunity induced by UB-612 vaccine were evaluated in four animal models: Sprague-Dawley rats, AAV-hACE2 transduced BALB/c mice, rhesus and cynomolgus macaques. UB-612 vaccine induced high levels of neutralizing antibody and T-cell responses, in all animals. The immune sera from vaccinated animals neutralized the SARS-CoV-2 original wild-type strains and multiple variants of concern, including Delta and Omicron. The vaccination significantly reduced viral loads, lung pathology scores, and disease progression after intranasal and intratracheal challenge with SARS-CoV-2 in mice, rhesus and cynomolgus macaques. UB-612 has been tested in primary regimens in Phase 1 and Phase 2 clinical studies and is currently being evaluated in a global pivotal Phase 3 clinical study as a single dose heterologous booster.

Desformylflustrabromine (dFBr), a positive allosteric modulator of α4ß2 nicotinic acetylcholine receptors decreases voluntary ethanol consumption and preference in male and female Sprague-Dawley rats.

Alcohol use disorder is a medical condition that impacts millions of individuals worldwide. Although there are a few pharmacotherapeutic options for alcohol-dependent individuals; there is a need for the development of novel and more effective therapeutic approaches. Alcohol and nicotine are commonly co-abused, and there is evidence that neuronal nicotinic acetylcholine receptors (nAChRs) play a role in both alcohol and nicotine dependence. Desformylflustrabromine (dFBr), a positive allosteric modulator of the α4ß2 nAChRs has been shown to reduce nicotine intake, compulsive-like behavior and neuropathic pain in animal models. dFBr has also been previously shown to cross the blood-brain-barrier. We have recently shown that dFBr can attenuate the response to an acute, hypnotic dose of ethanol, via ß2 nAchR. Here, we have investigated the effect of dFBr in modulating ethanol consumption using the intermittent access two-bottle choice (IA2BC) model of voluntary ethanol consumption in male and female Sprague Dawley rats. We show that dFBr selectively reduced ethanol but not sucrose consumption in the IA2BC model. Furthermore, dFBr decreased preference for ethanol in both male and female rats. No rebound increase in ethanol intake was observed after the washout period after dFBr treatment. The ability of dFBr to decrease ethanol consumption, along with its previously demonstrated ability to decrease nicotine self-administration in rodents, suggest that dFBr is an attractive therapeutic candidate to target both nicotine and alcohol abuse.

Biotransformation and transplacental transfer of the anti-viral remdesivir and predominant metabolite, GS-441524 in pregnant rats.

BACKGROUND: Remdesivir was the first prodrug approved to treat coronavirus disease 2019 (COVID-19) and has the potential to be used during pregnancy. However, it is not known whether remdesivir and its main metabolite, GS-441524 have the potential to cross the blood-placental barrier. We hypothesize that remdesivir and predominant metabolite GS-441524may cross the blood-placental barrier to reach the embryo tissues. METHODS: To test this hypothesis, ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) coupled with multisite microdialysis was used to monitor the levels of remdesivir and the nucleoside analogue GS-441524 in the maternal blood, fetus, placenta, and amniotic fluid of pregnant Sprague-Dawley rats. The transplacental transfer was evaluated using the pharmacokinetic parameters of AUC and mother-to-fetus transfer ratio (AUCfetus/AUCmother). FINDINGS: Our in-vivo results show that remdesivir is rapidly biotransformed into GS-441524 in the maternal blood, which then readily crossed the placenta with a mother-to-fetus transfer ratio of 0.51 ± 0.18. The Cmax and AUClast values of GS-441524 followed the order: maternal blood > amniotic fluid > fetus > placenta in rats. INTERPRETATION: While remdesivir does not directly cross into the fetus, however, its main metabolite, GS-441524 readily crosses the placenta and can reside there for at least 4 hours as shown in the pregnant Sprague-Dawley rat model. These findings suggest that careful consideration should be taken for the use of remdesivir in the treatment of COVID-19 in pregnancy. FUNDING: Ministry of Science and Technology of Taiwan.

Synthesis and Anti-Influenza Virus Effects of Novel Substituted Polycyclic Pyridone Derivatives Modified from Baloxavir.

In this work, a series of novel substituted polycyclic pyridone derivatives were designed and synthesized as potent anti-influenza agents. The cytopathic effect (CPE) assay and cytotoxicity assay indicated that all of the compounds possessed potent anti-influenza virus activity and relatively low cytotoxicity; some of them inhibited the replication of influenza A virus (IAV) at picomolar concentrations. Further studies revealed that, at a concentration of 3 nM, three compounds (10a, 10d, and 10g) could significantly reduce the M2 RNA amounts and M2 protein expression of IAV and inhibit the activity of RNA-dependent RNA polymerase (RdRp). Among them, (R)-12-(5H-dibenzo[a,d][7]annulen-5-yl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (10a) was found to be a promising anti-influenza drug candidate with good human liver microsomal stability, as well as with better selectivity index and oral bioavailability than Baloxavir.

Comparative pharmacokinetics of 24 major bioactive components in normal and ARDS rats after oral administration of Xuanfei Baidu granules.

ETHNOPHARMACOLOGICAL RELEVANCE: Xuanfei Baidu prescription, consisting of 13 Chinese medicines, was formulated by academicians Boli Zhang and Professor Qingquan Liu based on their experience in first-line clinical treatment of COVID-19. Xuanfei Baidu granules (XFBD granules) are a proprietary Chinese medicine preparation developed based on Xuanfei Baidu prescription. It is recommended for the treatment of patients with the common wet toxin and lung stagnation syndrome of COVID-19. However, the pharmacokinetic characteristics of its major bioactive components in rats under different physiological and pathological conditions are unclear. MATERIALS AND METHODS: A rapid and sensitive analytical method, ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS/MS), was developed and applied to 24 major bioactive components in normal and ARDS rats after oral administration of XFBD granules. We studied the metabolic process of XFBD granules in vivo to compare the differences in pharmacokinetic parameters between normal and model metabolic processes. RESULTS: This method was successfully applied to the pharmacokinetic investigation of 24 major components of XFBD granules following oral administration in normal and ARDS rats. Eight components, including ephedrine and amygdalin, were more highly absorbed and had shorter Tmax values than the model group; the absorption of six components, such as rhein, decreased in ARDS rats, and there was no significant difference in the absorption of ten components, such as verbenalin and naringin, between the normal and ARDS rats. The results showed that the peak times of other analytes were very short, and 80% of these target constituents were eliminated in both normal and ARDS rats within 6 h except for liquiritigenin and 18ß-glycyrrhetinic acid. CONCLUSIONS: In this study, a rapid and sensitive UPLC-MS/MS analytical method was developed and applied to 24 major bioactive components in normal and ARDS rats after the oral administration of XFBD granules. This will serve to form the basis for further studies on the pharmacokinetic-pharmacodynamic correlation of XFBD granules.

Impact of Azithromycin on Forsythiaside Pharmacokinetics in Rats: A Population Modeling Method.

OBJECTIVE: Lianhuaqingwen and Shuanghuanglian are drug treatment options for Corona Virus Disease 2019 (COVID-19). In China, use of traditional Chinese medicine with Shuanghuanglian or Lianhuaqingwen (for them, forsythiaside is the active antiviral and antibacterial component) in combination with azithromycin is common for the treatment of pediatric pneumonia. It is important to understand the reason why the combination of these compounds is better than a single drug treatment. This study aimed to explore the pharmacokinetic interaction between forsythiaside and azithromycin. METHODS: Twelve male Sprague-Dawley rats were randomly divided into an experimental group (Forsythia suspensa extract and azithromycin) and a control group (a single dose of Forsythia suspensa extract in 5% glucose solution). Plasma samples were collected at scheduled time points, and the high-performance liquid chromatography combined with ultraviolet method was used to determine the plasma forsythiaside concentration. Non-compartmental analysis and population pharmacokinetic methods were used to investigate the forsythiaside pharmacokinetic difference between the experimental and control group. RESULTS: Compared with a single administration, the area under the curve and half-life of forsythiaside increased, and forsythiaside clearance decreased significantly after co-administration with azithromycin. The in vivo behavior of forsythiaside could be described by the one compartment model. The forsythiaside clearance decreased when combined with azithromycin. Visual evaluation and bootstrap results suggested that the final model was precise and stable. CONCLUSION: Co-administration of azithromycin can significantly decrease the forsythiaside clearance and increase drug exposure. A lower dose of azithromycin can obtain sufficient forsythiaside concentration to provide antiviral and antibacterial activity.

Potential effect of amniotic fluid-derived stem cells on hyperoxia-induced pulmonary alveolar injury.

BACKGROUND: With the widespread of Coronavirus Disease 2019 pandemic, in spite of the newly emerging vaccines, mutated strains remain a great obstacle to supportive and preventive measures. Coronavirus 19 survivors continue to face great danger of contacting the disease again. As long as no specific treatment has yet to be approved, a great percentage of patients experience real complications, including among others, lung fibrosis. High oxygen inhalation especially for prolonged periods is per se destructive to the lungs. Nevertheless, oxygen remains the first line support for such patients. In the present study we aimed at investigating the role of amniotic fluid-mesenchymal stem cells in preventing versus treating the hyperoxia-induced lung fibrosis in rats. METHODS: The study was conducted on adult albino rats; 5 pregnant female rats were used as amniotic fluid donors, and 64 male rats were randomly divided into two groups: Control group; where 10 rats were kept in normal atmospheric air then sacrificed after 2 months, and hyperoxia-induced lung fibrosis group, where 54 rats were exposed to hyperoxia (100% oxygen for 6 h/day) in air-tight glass chambers for 1 month, then randomly divided into the following 5 subgroups: Hyperoxia group, cell-free media-treated group, stem cells-prophylactic group, stem cells-treated group and untreated group. Isolation, culture and proliferation of stem cells were done till passage 3. Pulmonary function tests, histological examination of lung tissue under light and electron microscopes, biochemical assessment of oxidative stress, IL-6 and Rho-A levels, and statistical analysis of data were performed. F-test (ANOVA) was used for normally distributed quantitative variables, to compare between more than two groups, and Post Hoc test (Tukey) for pairwise comparisons. RESULTS: Labelled amniotic fluid-mesenchymal stem cells homed to lung tissue. Stem cells administration in the stem cells-prophylactic group succeeded to maintain pulmonary functions near the normal values with no significant difference between their values and those of the control group. Moreover, histological examination of lung tissues showed that stem cells-prophylactic group were completely protected while stem cells-treated group still showed various degrees of tissue injury, namely; thickened interalveolar septa, atelectasis and interstitial pneumonia. Biochemical studies after stem cells injection also showed decreased levels of RhoA and IL-6 in the prophylactic group and to a lesser extent in the treated group, in addition to increased total antioxidant capacity and decreased malondialdehyde in the stem cells-injected groups. CONCLUSIONS: Amniotic fluid-mesenchymal stem cells showed promising protective and therapeutic results against hyperoxia-induced lung fibrosis as evaluated physiologically, histologically and biochemically.

Prenatal alcohol and cannabis exposure can have opposing and region-specific effects on parvalbumin interneuron numbers in the hippocampus.

BACKGROUND: We recently showed that alcohol and cannabis can interact prenatally, and in a recent review paper, we identified parvalbumin-positive (PV) interneurons in the hippocampus as a potential point of convergence for these teratogens. METHODS: A 2 (Ethanol [EtOH], Air) × 2 (tetrahydrocannabinol [THC], Vehicle) design was used to expose pregnant Sprague-Dawley rats to either EtOH or air, in addition to either THC or the inhalant vehicle solution, during gestational days 5-20. Immunohistochemistry was performed to detect PV interneurons in 1 male and 1 female pup from each litter at postnatal day 70. RESULTS: Significant between-group and subregion-specific effects were found in the dorsal cornu ammonis 1 (CA1) subfield and the ventral dentate gyrus (DG). In the dorsal CA1 subfield, there was an increase in the number of PV interneurons in both the EtOH and EtOH +THC groups, but a decrease with THC alone. There were fewer changes in interneuron numbers overall in the DG, though there was a sex difference, with a decrease in the number of PV interneurons in the THC-exposed group in males. There was also a greater cell layer volume in the DG in the EtOH +THC group than the control group, and in the CA1 region in the EtOH group compared to the control and THC groups. CONCLUSIONS: Prenatal exposure to alcohol and THC differentially affects parvalbumin-positive interneuron numbers in the hippocampus, indicating that both individual and combined exposure can impact the balance of excitation and inhibition in a structure critically involved in learning and memory processes.

Pharmacokinetics of Nafamostat, a Potent Serine Protease Inhibitor, by a Novel LC-MS/MS Analysis.

Nafamostat, a synthetic serine protease inhibitor, has been used for the treatment of inflammatory diseases such as pancreatitis. Recently, an increasing number of studies have shown the promising antiviral effects of nafamostat for the treatment of coronavirus disease-19 (COVID-19). This study aimed to develop a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and to characterize the pharmacokinetics of nafamostat in rats. Nafamostat in the rat plasma was extracted by solid phase extraction, and 13C6-nafamostat was used as an internal standard. The quantification limit of nafamostat in the rat plasma was 0.5 ng/mL. The LC-MS/MS method was fully validated and applied to characterize the pharmacokinetics of nafamostat in rats. Following intravenous injection (2 mg/kg), nafamostat in the plasma showed a multiexponential decline with an average elimination half-life (t1/2) of 1.39 h. Following oral administration of nafamostat solutions (20 mg/kg) in 10% dimethyl sulfoxide (DMSO) and in 10% DMSO with 10% Tween 80, nafamostat was rapidly absorbed, and the average oral bioavailability was 0.95% and 1.59%, respectively. The LC-MS/MS method and the pharmacokinetic information of nafamostat could be helpful for the further preclinical and clinical studies of nafamostat.

Potential "Therapeutic" Effects of Tocotrienol-Rich Fraction (TRF) and Carotene "Against" Bleomycin-Induced Pulmonary Fibrosis in Rats via TGF-ß/Smad, PI3K/Akt/mTOR and NF-κB Signaling Pathways.

BACKGROUND: Pulmonary fibrosis (PF) is a chronic, progressive, and, ultimately, terminal interstitial disease caused by a variety of factors, ranging from genetics, bacterial, and viral infections, to drugs and other influences. Varying degrees of PF and its rapid progress have been widely reported in post-COVID-19 patients and there is consequently an urgent need to develop an appropriate, cost-effective approach for the prevention and management of PF. AIM: The potential "therapeutic" effect of the tocotrienol-rich fraction (TRF) and carotene against bleomycin (BLM)-induced lung fibrosis was investigated in rats via the modulation of TGF-ß/Smad, PI3K/Akt/mTOR, and NF-κB signaling pathways. DESIGN/METHODS: Lung fibrosis was induced in Sprague-Dawley rats by a single intratracheal BLM (5 mg/kg) injection. These rats were subsequently treated with TRF (50, 100, and 200 mg/kg body wt/day), carotene (10 mg/kg body wt/day), or a combination of TRF (200 mg/kg body wt/day) and carotene (10 mg/kg body wt/day) for 28 days by gavage administration. A group of normal rats was provided with saline as a substitute for BLM as the control. Lung function and biochemical, histopathological, and molecular alterations were studied in the lung tissues. RESULTS: Both the TRF and carotene treatments were found to significantly restore the BLM-induced alterations in anti-inflammatory and antioxidant functions. The treatments appeared to show pneumoprotective effects through the upregulation of antioxidant status, downregulation of MMP-7 and inflammatory cytokine expressions, and reduction in collagen accumulation (hydroxyproline). We demonstrated that TRF and carotene ameliorate BLM-induced lung injuries through the inhibition of apoptosis, the induction of TGF-ß1/Smad, PI3K/Akt/mTOR, and NF-κB signaling pathways. Furthermore, the increased expression levels were shown to be significantly and dose-dependently downregulated by TRF (50, 100, and 200 mg/kg body wt/day) treatment in high probability. The histopathological findings further confirmed that the TRF and carotene treatments had significantly attenuated the BLM-induced lung injury in rats. CONCLUSION: The results of this study clearly indicate the ability of TRF and carotene to restore the antioxidant system and to inhibit proinflammatory cytokines. These findings, thus, revealed the potential of TRF and carotene as preventive candidates for the treatment of PF in the future.

Non-clinical immunogenicity, biodistribution and toxicology evaluation of a chimpanzee adenovirus-based COVID-19 vaccine in rat and rhesus macaque.

Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 has rapidly expanded into a serious global pandemic. Due to the high morbidity and mortality of COVID-19, there is an urgent need to develop safe and effective vaccines. AdC68-19S is an investigational chimpanzee adenovirus serotype 68 (AdC68) vector-based vaccine which encodes the full-length spike protein of SARS-CoV-2. Here, we evaluated the immunogenicity, biodistribution and safety profiles of the candidate vaccine AdC68-19S in Sprague Dawley (SD) rat and rhesus macaque under GLP conditions. To characterize the biodistribution profile of AdC68-19S, SD rats were given a single intramuscular injection of AdC68-19S 2 × 1011 VP/dose. Designated organs were collected on day 1, day 2, day 4, day 8 and day 15. Genomic DNA was extracted from all samples and was further quantified by real-time quantitative polymerase chain reaction (qPCR). To characterize the toxicology and immunogenicity profiles of AdC68-19S, the rats and rhesus macaques were injected intramuscularly with AdC68-19S up to 2 × 1011vp/dose or 4 × 1011vp/dose (2 and fourfold the proposed clinical dose of 1 × 1011vp/dose) on two or three occasions with a 14-day interval period, respectively. In addition to the conventional toxicological evaluation indexes, the antigen-specific cellular and humoral responses were evaluated. We proved that multiple intramuscular injections could elicit effective and long-lasting neutralizing antibody responses and Th1 T cell responses. AdC68-19S was mainly distributed in injection sites and no AdC68-19S related toxicological reaction was observed. In conclusion, these results have shown that AdC68-19S could induce an effective immune response with a good safety profile, and is a promising candidate vaccine against COVID-19.