Intranasal Dose Research Articles

131. Antiviral NL-CVX1 Efficiently Blocks Infection of SARS-CoV-2 Viral Variants of Concern (VOC)

BackgroundUsing a computational approach, NL-CVX1 was developed by Neoleukin Therapeutics, Inc. to create a de novo protein that both blocks SARS-CoV-2 infection and is highly resilient to viral escape. In this study we evaluated the efficacy of NL-CVX1 against variants of the original SARS-CoV-2 strain, including important viral variants of concern (VOC) such as B.1.1.7, B.1.351, and P.1.MethodsThe relative binding affinity of NL-CVX1 to the SARS-CoV-2 viral spike protein of VOC was measured using biolayer interferometry (Octet). A competitive ELISA measured the ability of NL-CVX1 to compete with hACE2 for binding to the receptor binding domain (RBD) of the SARS-CoV-2 S protein from the original strain and VOC. The activity of NL-CVX1 in preventing viral infection was assessed by evaluating the cytopathic effects (CPE) of SARS-CoV-2 in a transmembrane protease, serine 2-expressing Vero E6 cell line (Vero E6/TMPRSS2) and determining the viral load using quantitative real-time reverse transcriptase polymerase chain reaction in infected cells. A K18hACE2 mouse model of SARS CoV-2 infection was used to study the dose-response of NL-CVX1 anti-viral activity in vivo.ResultsNL-CVX1 binds the RBD of different VOC of SARS-CoV-2 at low nanomolar concentrations (Fig 1; Kd < 1-~5 nM). When competing with hACE2, NL-CVX1 achieved 100% inhibition against hACE2 binding to the RBD of different VOC with IC50s values ranging from 0.7-53 nM (Fig 2). NL-CVX1 neutralized the B.1.1.7 variant as efficiently as the original strain in Vero E6/TMPRSS2 cells, with EC50 values of 16 nM and 101.2 nM, respectively (Fig 3). In mice, we found that a single intranasal dose of 100 µg NL-CVX1 prevented clinically significant SARS-CoV-2 infection and protected mice from succumbing to infection. Results from additional in vitro and in vivo experiments to be conducted this summer will be presented. Figure 1. NL-CVX1 binds the RBD from multiple strains of SARS-CoV-2 at low nanomolar concentrations. Figure 2. NL-CVX1 achieves 100% inhibition against all strains tested, including SARS-CoV-2 VOC. Figure 3. NL-CVX1 neutralizes the B.1.1.7 variant as efficiently as the original SARS-CoV-2 strain.Conclusion In vitro and in vivo data (Fig 4) demonstrate that NL-CVX1 is a promising drug candidate for the prevention and treatment of COVID-19. As a hACE2 mimetic, it is resilient to antibody escape mutations found in SARS-CoV-2 VOC. NL-CVX1 further demonstrates the power and utility of de novo protein design for developing proteins as human therapeutics. Figure 4. NL-CVX1 is effective in preventing clinically significant SARS-CoV-2 viral infection in a K18hACE2 mouse model.Disclosures Matthew Walker, PhD, Neoleukin Therapeutics, Inc. (Employee, Other Financial or Material Support, Ownership options and stock.) Laurie Tatalick, DVM, PhD, DACVP, Neoleukin Therapeutics, Inc. (Consultant, Other Financial or Material Support, Ownership options and stock.) Marianne Riley, BS, Neoleukin Therapeutics, Inc. (Employee, Other Financial or Material Support, Ownership options and stock.) Kevin Yu, BS, MS, Neoleukin Therapeutics, Inc. (Employee, Other Financial or Material Support, Ownership options and stock.) Luis M. Blancas-Mejia, PhD, Neoleukin Therapeutics, Inc. (Employee, Other Financial or Material Support, Ownership options and stock.) Daniel-Adriano Silva, PhD, Neoleukin Therapeutics, Inc. (Advisor or Review Panel member, Other Financial or Material Support, Ownership of Neoleukin options and stock) David Shoultz, PhD, MBA, Neoleukin Therapeutics, Inc. (Employee, Other Financial or Material Support, Ownership options and stock.) Goncalo Bernardes, PhD, Neoleukin Therapeutics, Inc. (Consultant, Advisor or Review Panel member, Shareholder) Hui-Ling Yen, PhD, Neoleukin Therapeutics, Inc. (Grant/Research Support)Saiba AG (Other Financial or Material Support, Received donation from Saiba AG)

The optimal dose of oral midazolam with or without intranasal S-ketamine for premedication in children: a randomised, double blinded, sequential dose-finding trial.

BackgroundOral administration of midazolam syrup is one of the most favorable methods of premedication, the optimal dose of midazolam and midazolam with S-ketamine for preschool children has not been determined. This prospective, double-blind, randomized, sequential dose-finding study was designed to estimate the 90% effective doses of oral midazolam with and without intranasal S-ketamine in a grade III child medical center.MethodsEighty successive children were recruited and randomly allocated to midazolam group and midazolam with S-ketamine group. The initial oral doses of midazolam were 0.25 mg/kg in both groups, and the dose of midazolam for the next child was based on the response of the preceding child as the biased coin up-and-down designed. The primary outcome was parental separation anxiety score = 1 throughout the period of transferring from premedication center to the operation room 30 min after premedication. Secondary outcomes were the preoperative and post-operative observations. Finally, the 90% effective dose and 95% confidence intervals were estimated by isotonic regression.ResultsThe 90% effective dose of oral midazolam or oral midazolam with intranasal S-ketamine was 0.461 mg/kg (95% confidence interval: 0.425–0.488) and 0.253 mg/kg (95% confidence interval: 0.242–0.278), respectively. Oral midazolam with intranasal S-ketamine was quicker onset (8.9±3.8 vs. 19.7±7.4 min, P<0.001), had less incidence of behavioral changes (7.5% vs. 32.5%, P=0.010) and faster recovery (21.6±14.1 vs. 31.6±13.5 min, P=0.002) than solely oral midazolam.ConclusionsA suggestion of oral midazolam 0.3 mg/kg with intranasal small dose of S-ketamine could be used as premedication for preschool children.Trial RegistrationChinese Clinical Trial Registry.

Intranasal Administration of Chitosan-Nanoparticles Conjugated with Imipramine and its Effect on Stroke-Induced Secondary Neurodegeneration: A Research Protocol

Introduction: Stroke is the second leading cause of death and the third leading cause of permanent disability worldwide. Notably, the recovery period post-stroke is crucial as there is a risk of stroke-induced secondary neurodegeneration. Stroke-induced secondary neurodegeneration is the inevitable loss of viable brain tissue at sites distal from the initial infarct. It shares similarities with neurodegenerative diseases and results in neurological deficits, further complicating stroke recovery. Intranasal administration of chitosan-nanoparticles conjugated with imipramine will be tested to determine if they elicit a synergistic effect in mitigating disease processes associated with stroke-induced secondary neurodegeneration. Methods: Treatment and testing will be conducted in 30 male Wistar rats aged 12 months. Stroke will be induced by occluding the middle cerebral artery. Rats will be divided into three groups of 10 and will receive an intranasal dose of either saline (Control, C), 20mg/kg of imipramine (Treatment 1, T1), or 20mg/kg of chitosan-nanoparticles conjugated with imipramine (Treatment 2, T2). Statistical analysis using analysis of variance will determine if chitosan-nanoparticles conjugated with imipramine can mitigate the effects of stroke-induced secondary neurodegeneration determined by the proposed tests. Results: T2 given 20mg/kg of chitosan-nanoparticles conjugated with imipramine is proposed to spend more time exploring the unfamiliar object in the novel object recognition test. Lesser evidence of Alzheimer’s disease in T2 is expected, as measured by the fludeoxyglucose positron emission tomography imaging. A higher serum brain-derived neurotrophic factor measured by an enzyme-linked immunosorbent assay is also expected to be present in T2. Discussion: It is anticipated that chitosan-nanoparticles conjugated with imipramine will exhibit a synergistic effect in mitigating disease processes accompanying stroke-induced secondary neurodegeneration because of properties associated with neuroplasticity and enhanced drug targeting efficacy. Conclusion: This research protocol aims to elucidate a novel treatment that can be applied to stroke recovery to mitigate stroke-induced secondary neurodegeneration, which tends to complicate this crucial period. Our proposal could have implications in the prognosis and management of stroke and post-stroke recovery, respectively, and inspire a framework for the discovery of novel post-stroke therapeutic interventions.

Osteoclast-mediated bone loss observed in a COVID-19 mouse model.

The consequences of SARS-CoV-2 infection on the musculoskeletal system represent a dangerous knowledge gap. Aging patients are at added risk for SARS-CoV-2 infection; therefore, a greater understanding of the resulting musculoskeletal sequelae of SARS-CoV-2 infection may help guide clinical strategies. This study examined fundamental bone parameters among mice treated with escalating viral loads. Male C57BL/6J (WT, n = 17) and B6.Cg-Tg(K18-ACE2)2Prlmn/J mice (K18-hACE2 transgenic mice, n = 21) expressing human ACE2 (TG) were divided into eight groups (n = 4–6/group) and subjected to intranasal dosing of 0, 1 × 103, 1 × 104, and 1 × 105 PFU (plaque forming units) of human SARS-CoV-2. Animal health was assessed daily by veterinary staff using established and validated scoring criteria (activity, posture, body condition scores and body weight). We report here that mock and WT infected mice were healthy and completed the study, surviving until 12–14 days post infection (dpi). In contrast, the TG mice infected with 1 × 105 PFU all experienced severe health declines that necessitated early euthanasia (6–7 dpi). For TG mice infected with 1 × 104 PFU, 2 mice were also euthanized after 7 dpi, while 3 mice showed signs of moderate disease at day 6 dpi, but recovered fully by day 11 dpi. Four of the 5 TG mice that were infected with 1 × 103 PFU remained healthy throughout the study. This suggests that our study mimics what is seen during human disease, where some patients develop severe disease resulting in death, while others have moderate to severe disease but recover, and others are asymptomatic. At necropsy, femurs were extracted and analyzed by μCT. No difference was found in μCT determined bone parameters among the WT groups. There was, however, a significant 24.4% decrease in trabecular bone volume fraction (p = 0.0009), 19.0% decrease in trabecular number (p = 0.004), 6.2% decrease in trabecular thickness (p = 0.04), and a 9.8% increase in trabecular separation (p = 0.04) among surviving TG mice receiving any viral load compared to non-infected controls. No differences in cortical bone parameters were detected. TRAP staining revealed surviving infected mice had a significant 64% increase in osteoclast number, a 27% increase in osteoclast surface, and a 38% increase in osteoclasts per bone surface. While more studies are needed to investigate the long-term consequences of SARS-CoV-2 infection on skeletal health, this study demonstrates a significant reduction in several bone parameters and corresponding robust increases in osteoclast number observed within 2 weeks post-infection in surviving asymptomatic and moderately affected mice.

Intranasal oxytocin drives coordinated social approach

Coordinated responses to challenge are essential to survival for bonded monogamous animals and may depend on behavioral compatibility. Oxytocin (OT) context-dependently regulates social affiliation and vocal communication, but its role in pair members’ decision to jointly respond to challenge is unclear. To test for OT effects, California mouse females received an intranasal dose of OT (IN-OT) or saline after bonding with males either matched or mismatched in their approach response to an aggressive vocal challenge. Pair mates were re-tested jointly for approach response, time spent together, and vocalizations. Females and males converged in their approach after pairing, but mismatched pairs with females given a single dose of IN-OT displayed a greater convergence that resulted from behavioral changes by both pair members. Unpaired females given IN-OT did not change their approach, indicating a social partner was necessary for effects to emerge. Moreover, IN-OT increased time spent approaching together, suggesting behavioral coordination beyond a further increase in bonding. This OT-induced increase in joint approach was associated with a decrease in the proportion of sustained vocalizations, a type of vocalization that can be associated with intra-pair conflict. Our results expand OT’s effects on behavioral coordination and underscore the importance of emergent social context.

Development of a single-dose intranasal testosterone administration paradigm for use in men and women

For over two decades, researchers in the field of human social neuroendocrinology have been using single-dose pharmacological challenge protocols to determine the causal effects of testosterone on psychological, behavioural, and neural processes. Most of these single-dose administration studies have so far used (1) single-sex samples and (2) varying modes of testosterone administration (intramuscular, transdermal, sublingual, and intranasal) that produced vastly different dose-response curves. Moreover, whereas studies with male participants increased men's testosterone concentrations within the high normal physiological range, studies with women typically increased testosterone concentrations to supraphysiological levels. The purpose of this study was to develop a single-dose administration protocol using intranasal testosterone that would produce a proportionally similar rise in testosterone for both sexes. We found that an 11 mg intranasal testosterone dose in men and a 0.3 mg dose in women raised testosterone concentrations to the high normal physiological range for each sex, producing similar dose-response dynamics in both sexes. This paradigm will allow researchers to design studies with mixed-sex samples that test physiologically plausible sex differences/similarities in the causal effects of testosterone. It will also provide a replicable protocol to examine the possible adaptive functions of acute increases in testosterone in both sexes.

A Phase I, Open-label, Randomized, 2-Way Crossover Study to Evaluate the Relative Bioavailability of Intranasal and Oral Varenicline

PurposeTo estimate the systemic bioavailability of OC-01 (varenicline) nasal spray, an investigational treatment for dry eye disease, relative to oral varenicline approved for smoking cessation. MethodsThe Study to Evaluate the Relative Bioavailability of Varenicline Administered as OC-01 (Varenicline) Nasal Spray as Compared to Varenicline Administered Orally as Chantix (ZEN study) was a Phase I, open-label, randomized, single-center, 2-way crossover study. On day 1, 22 healthy participants were randomized 1:1 to a single intranasal dose of varenicline 0.12 mg in OC-01 nasal spray or a single oral dose of varenicline 1 mg. On day 15, all participants crossed over to receive a single dose of the alternate treatment. Plasma samples were collected for 6 days after each dose, and pharmacokinetic parameters were estimated using noncompartmental analysis. Tolerability was monitored throughout. FindingsAfter a single dose of intranasal varenicline 0.12 mg in OC-01 nasal spray, peak systemic exposure (mean plasma Cmax) was 0.34 ng/mL, which occurred at a median Tmax of 2.0 hours. In comparison, mean plasma Cmax after oral varenicline 1 mg was 4.63 ng/mL at a median Tmax of 3.0 hours. On the basis of geometric mean ratio point estimates, peak exposure (Cmax) and total exposure (AUC0–∞) after intranasal varenicline 0.12 mg were 7.0% and 7.5%, respectively, of the systemic exposure associated with oral varenicline 1 mg. Dose-normalized Cmax and AUC0–∞ for intranasal varenicline remained 39% and 33% lower versus oral varenicline, respectively. No new or unexpected tolerability signals were detected. ImplicationsAt its highest intended single dose in OC-01 nasal spray, intranasal varenicline delivered less drug to the systemic circulation than oral varenicline at its highest approved single dose. ClinicalTrials.gov identifier: NCT04072146.

Single-Dose Intranasal Administration of AdCOVID Elicits Systemic and Mucosal Immunity against SARS-CoV-2 and Fully Protects Mice from Lethal Challenge.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry. The current intramuscular vaccines elicit systemic immunity but not necessarily high-level mucosal immunity. Here, we tested a single intranasal dose of our candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (AdCOVID) in inbred, outbred, and transgenic mice. A single intranasal vaccination with AdCOVID elicited a strong and focused immune response against RBD through the induction of mucosal IgA in the respiratory tract, serum neutralizing antibodies, and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. A single AdCOVID dose resulted in immunity that was sustained for over six months. Moreover, a single intranasal dose completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge, preventing weight loss and mortality. These data show that AdCOVID promotes concomitant systemic and mucosal immunity and represents a promising vaccine candidate.

A recombinant bovine adenoviral mucosal vaccine expressing mycobacterial antigen-85B generates robust protection against tuberculosis in mice

SummaryAlthough the BCG vaccine offers partial protection, tuberculosis remains a leading cause of infectious disease death, killing ∼1.5 million people annually. We developed mucosal vaccines expressing the autophagy-inducing peptide C5 and mycobacterial Ag85B-p25 epitope using replication-defective human adenovirus (HAdv85C5) and bovine adenovirus (BAdv85C5) vectors. BAdv85C5-infected dendritic cells (DCs) expressed a robust transcriptome of genes regulating antigen processing compared to HAdv85C5-infected DCs. BAdv85C5-infected DCs showed enhanced galectin-3/8 and autophagy-dependent in vitro Ag85B-p25 epitope presentation to CD4 T cells. BCG-vaccinated mice were intranasally boosted using HAdv85C5 or BAdv85C5 followed by infection using aerosolized Mycobacterium tuberculosis (Mtb). BAdv85C5 protected mice against tuberculosis both as a booster after BCG vaccine (>1.4-log10 reduction in Mtb lung burden) and as a single intranasal dose (>0.5-log10 reduction). Protection was associated with robust CD4 and CD8 effector (TEM), central memory (TCM), and CD103+/CD69+ lung-resident memory (TRM) T cell expansion, revealing BAdv85C5 as a promising mucosal vaccine for tuberculosis.

Ketamine in depression and electroconvulsive therapy.

The antidepressant effect of subanesthetic doses of ketamine was recognized 20 years ago. This review briefly summarizes the current understanding of the antidepressant mechanisms and the available clinical research on the use of racemic ketamine and enantiomer esketamine for depression. The antidepressant effect of subanesthetic doses of ketamine is currently considered to be predominantly mediated by improved neuroplasticity in cortico-limbic areas in the brain. Single dose of 0.5 mg/kg of ketamine infused intravenously over 40 min, or single intranasal dose of esketamine cause rapid antidepressant and antisuicidal effects within hours of administration, and the antidepressant effect may last up to a week. Repeated administration of nasal spray esketamine is considered to prevent relapse of depression. Longitudinal studies are currently insufficient. When used in various doses for anesthetic induction for electroconvulsive therapy, ketamine improves seizure quality and may possibly diminish posttherapy cognitive impairment. A rapid onset antidepressive effect of ketamine and esketamine has been proven conclusively. The results of extensive basic science research of the mechanism of action of low-dose ketamine doses has led to an alternative hypothesis of the pathophysiology of depression and the development of a novel neurotrophic concept of depression. Further longitudinal studies are warranted to determine the safety and efficacy of repeated administration of ketamine and its analogs to prevent relapse and recurrence of depression.

Single-chain Fragment variable antibody targeting cholecystokinin-B receptor for pain reduction

The cholecystokinin B receptor and its neuropeptide ligand are upregulated in chronic neuropathic pain models. Single-chain Fragment variable antibodies were generated as preferred non-opioid targeting therapy blocking the cholecystokinin B receptor to inhibit chronic neuropathic pain models in vivo and in vitro. Engineered antibodies of this type feature binding activity similar to monoclonal antibodies but with stronger affinity and increased tissue penetrability due to their smaller size. More importantly, single-chain Fragment variable antibodies have promising biotherapeutic applications for both nervous and immune systems, now recognized as interactive in chronic pain. A mouse single-chain Fragment variable antibody library recognizing a fifteen amino acid extracellular peptide fragment of the cholecystokinin B receptor was generated from immunized spleens. Ribosome display, a powerful cell-free technology, was applied for recombinant antibody selection. Antibodies with higher affinity, stability, solubility, and binding specificity for cholecystokinin B not A receptor were selected and optimized for in vivo and in vitro efficacy. A single dose of the lead candidate reduced mechanical and cold hypersensitivity in two rodent models of neuropathic pain for at least seven weeks. Continuing efficacy was evident with either intraperitoneal or intranasal dosing. Likewise, the lead single-chain Fragment variable antibody totally prevented development of anxiety- and depression-like behaviors and cognitive deficits typical in the models. Reduction of neuronal firing frequency was evident in trigeminal ganglia primary neuronal cultures treated in vitro with the cholecystokinin B receptor antibody. Immunofluorescent staining intensity in the trigeminal neuron primary cultures was significantly reduced incrementally after overnight binding with increasingly higher dilutions of the single-chain Fragment variable antibody. While it is reported that single-chain Fragment variable antibodies are removed systemically within 2–6 h, Western blot evidence indicates the His-tag marker remained after 7 weeks in the trigeminal ganglia and in the dorsolateral medulla, providing evidence of brain and ganglia penetrance known to be compromised in overactivated states. This project showcases the in vivo efficacy of our lead single-chain Fragment variable antibody indicating its potential for development as a non-opioid, non-addictive therapeutic intervention for chronic pain. Importantly, studies by others have indicated treatments with cholecystokinin B receptor antagonists suppress maintenance and reactivation of morphine dependence in place preference tests while lowering tolerance and dose requirements. Our future studies remain to address these potential benefits that may accompany the cholecystokinin B receptor biological therapy. Both chronic sciatic and orofacial pain can be unrelenting and excruciating, reducing quality of life as well as diminishing physical and mental function. An effective non-opiate, non-addictive therapy with potential to reduce chronic neuropathic pain long term is greatly needed.

An intranasal vaccine durably protects against SARS-CoV-2 variants in mice.

SummarySARS-CoV-2 variants that attenuate antibody neutralization could jeopardize vaccine efficacy. We recently reported the protective activity of an intranasally administered spike protein-based chimpanzee adenovirus-vectored vaccine (ChAd-SARS-CoV-2-S) in animals, which has advanced to human trials. Here, we assessed its durability, dose response, and cross-protective activity in mice. A single intranasal dose of ChAd-SARS-CoV-2-S induced durably high neutralizing and Fc effector antibody responses in serum and S-specific IgG and IgA secreting long-lived plasma cells in the bone marrow. Protection against a historical SARS-CoV-2 strain was observed across a 100-fold vaccine dose range and over a 200-day period. At 6 weeks or 9 months after vaccination, serum antibodies neutralized SARS-CoV-2 strains with B.1.351, B.1.1.28, and B.1.617.1 spike proteins and conferred almost complete protection in the upper and lower respiratory tracts after challenge with variant viruses. Thus, in mice, intranasal immunization with ChAd-SARS-CoV-2-S provides durable protection against historical and emerging SARS-CoV-2 strains.

Universal influenza vaccine based on conserved antigens provides long-term durability of immune responses and durable broad protection against diverse challenge virus strains in mice

Current influenza vaccines rely on inducing antibody responses to the rapidly evolving hemagglutinin (HA) and neuraminidase (NA) proteins, and thus need to be strain-matched. However, predictions of strains that will circulate are imperfect, and manufacturing of new vaccines based on them takes months. As an alternative, universal influenza vaccines target highly conserved antigens. In proof of concept studies of universal vaccine candidates in animal models challenge is generally conducted only a short time after vaccination, but protective immunity lasting far longer is important for the intended public health impact. We address the challenge of providing long-term protection. We demonstrate here broad, powerful, and long-lasting immune protection for a promising universal vaccine candidate. A single intranasal dose of recombinant adenoviruses (rAd) expressing influenza A nucleoprotein (A/NP) and matrix 2 (M2) was used. Extending our previous studies of this type of vaccine, we show that antibody and T-cell responses persist for over a year without boosting, and that protection against challenge persists a year after vaccination and remains broad, covering both group 1 and 2 influenza A viruses. In addition, we extend the work to influenza B. Immunization with influenza B nucleoprotein (B/NP)-rAd also gives immune responses that last a year without boosting and protect against challenge with influenza B viruses of mismatched HA lineages. Despite host immunity to adenoviral antigens, effective readministration is possible a year after primary vaccination, as shown by successful immunization to a transgene product the animals had not seen before. Protection against challenge with divergent and highly pathogenic A/H7N9 virus was weaker but was enhanced by a second dose of vaccine. Thus, this mucosal vaccination to conserved influenza antigens confers very long-lasting immune protection in animals against a broad range of influenza A and B viruses.

Flurbiprofen sodium microparticles and soft pellets for nose-to-brain delivery: Serum and brain levels in rats after nasal insufflation

Neuroinflammation in Alzheimer’s disease (AD) revamped the role of a preventive therapeutic action of non steroidal anti-inflammatory drugs; flurbiprofen could delay AD onset, provided its access to brain is enhanced and systemic exposure limited. Nasal administration could enable direct drug access to central nervous system (CNS) via nose-to-brain transport. Here, we investigated the insufflation, deposition, dissolution, transmucosal permeation, and in vivo transport to rat brain of flurbiprofen from nasal powders combined in an active device.Flurbiprofen sodium spray-dried microparticles as such, or soft pellets obtained by agglomeration of drug microparticles with excipients, were intranasally administered to rats by the pre-metered insufflator device. Blood and brain were collected to measure flurbiprofen levels.Excipient presence in soft pellets lowered the metered drug dose to insufflate. Nevertheless, efficiency of powder delivery by the device, measured as emitted fraction, was superior with soft pellets than microparticles, due to their coarse size. Both nasal powders resulted into rapid flurbiprofen absorption. Absolute bioavailability was 33% and 58% for microparticles and pellets, respectively. Compared to intravenous flurbiprofen, the microparticles were more efficient than soft pellets at enhancing direct drug transport to CNS. Direct Transport Percentage index evidenced that more than 60% of the intranasal dose reached the brain via direct nose-to-brain transport for both powders. Moreover, remarkable drug concentrations were measured in the olfactory bulb after microparticle delivery. Bulb connection with the entorhinal cortex, from where AD initiates, makes flurbiprofen sodium administration as nasal powder worth of further investigation in an animal model of neuroinflammation.

Ultrapotent miniproteins targeting the SARS-CoV-2 receptor-binding domain protect against infection and disease

Despite the introduction of public health measures and spike protein-based vaccines to mitigate the COVID-19 pandemic, SARS-CoV-2 infections and deaths continue to have a global impact. Previously, we used a structural design approach to develop picomolar range miniproteins targeting the SARS-CoV-2 spike receptor-binding domain. Here, we investigated the capacity of modified versions of one lead miniprotein, LCB1, to protect against SARS-CoV-2-mediated lung disease in mice. Systemic administration of LCB1-Fc reduced viral burden, diminished immune cell infiltration and inflammation, and completely prevented lung disease and pathology. A single intranasal dose of LCB1v1.3 reduced SARS-CoV-2 infection in the lung when given as many as 5 days before or 2 days after virus inoculation. Importantly, LCB1v1.3 protected in vivo against a historical strain (WA1/2020), an emerging B.1.1.7 strain, and a strain encoding key E484K and N501Y spike protein substitutions. These data support development of LCB1v1.3 for prevention or treatment of SARS-CoV-2 infection.

Liquid Film Translocation Significantly Enhances Nasal Spray Delivery to Olfactory Region: A Numerical Simulation Study

Previous in vivo and ex vivo studies have tested nasal sprays with varying head positions to enhance the olfactory delivery; however, such studies often suffered from a lack of quantitative dosimetry in the target region, which relied on the observer’s subjective perception of color changes in the endoscopy images. The objective of this study is to test the feasibility of gravitationally driven droplet translocation numerically to enhance the nasal spray dosages in the olfactory region and quantify the intranasal dose distribution in the regions of interest. A computational nasal spray testing platform was developed that included a nasal spray releasing model, an airflow-droplet transport model, and an Eulerian wall film formation/translocation model. The effects of both device-related and administration-related variables on the initial olfactory deposition were studied, including droplet size, velocity, plume angle, spray release position, and orientation. The liquid film formation and translocation after nasal spray applications were simulated for both a standard and a newly proposed delivery system. Results show that the initial droplet deposition in the olfactory region is highly sensitive to the spray plume angle. For the given nasal cavity with a vertex-to-floor head position, a plume angle of 10° with a device orientation of 45° to the nostril delivered the optimal dose to the olfactory region. Liquid wall film translocation enhanced the olfactory dosage by ninefold, compared to the initial olfactory dose, for both the baseline and optimized delivery systems. The optimized delivery system delivered 6.2% of applied sprays to the olfactory region and significantly reduced drug losses in the vestibule. Rheological properties of spray formulations can be explored to harness further the benefits of liquid film translocation in targeted intranasal deliveries.

Nasal delivery of an IgM offers broad protection from SARS-CoV-2 variants.

Resistance represents a major challenge for antibody-based therapy for COVID-191-4. Here we engineered an immunoglobulinM (IgM) neutralizing antibody (IgM-14) to overcome the resistance encountered by immunoglobulinG (IgG)-based therapeutics. IgM-14 is over 230-fold more potent than its parental IgG-14 in neutralizing SARS-CoV-2. IgM-14 potently neutralizes the resistant virus raised by its corresponding IgG-14, three variants of concern-B.1.1.7 (Alpha, which first emerged in the UK), P.1 (Gamma, which first emerged in Brazil) and B.1.351 (Beta, which first emerged in South Africa)-and 21 other receptor-binding domain mutants, many of which are resistant to the IgG antibodies that have been authorized for emergency use. Although engineering IgG into IgM enhances antibody potency in general, selection of an optimal epitope is critical for identifying the most effective IgM that can overcome resistance. In mice, a single intranasal dose of IgM-14 at 0.044mg per kg body weight confers prophylactic efficacy and a single dose at 0.4mg per kg confers therapeutic efficacy against SARS-CoV-2. IgM-14, but not IgG-14, also confers potent therapeutic protection against the P.1 and B.1.351 variants. IgM-14 exhibits desirable pharmacokinetics and safety profiles when administered intranasally in rodents. Our results show that intranasal administration of an engineered IgM can improve efficacy, reduce resistance and simplify the prophylactic and therapeutic treatment of COVID-19.

Mucosal delivery of a polyanhydride nanovaccine incorporating CpG and the post-fusion F/ G proteins induces protective immunity against BRSV infection in neonatal calves.

Abstract Objective: Previously, immunization with a single intranasal dose of a BRSV post-fusion F/G nanovaccine resulted in partial protection from a BRSV challenge (McGill et al. 2019, McGill et al. 2018). Knowledge gained from these previous studies was used to optimize the vaccine by incorporating CpG-ODN and to investigate the impact of mucosal vs. systemic vaccination routes. In this study a prime-boost immunization regimen was used. Method: 36 neonatal, mixed-sex, Holstein calves were divided into 6 groups. Group 1 received a heterologous regime with saline and group 2 a homologous boost of the ‘empty’ nanovaccine CPG. Groups 3 and 4 received a homologous mucosal regime of the nanovaccine ± CpG, while groups 5 and 6 received a heterologous regime of the nanovaccine ± CpG. Vaccine-induced responses were monitored and 6 weeks after boost, all calves were challenged with BRSV. Nasopharyngeal swabs were collected for viral shedding and calves monitored for clinical signs and euthanized on day 7 after infection. Lungs were scored for gross pathology. Blood, nasal secretions and lung tissue samples were analyzed for BRSV specific immune responses. Results: Unvaccinated controls and CpG-only nanovaccine controls developed lung pathology consistent with a severe BRSV infection. We observed no evidence of vaccine enhanced disease in any vaccinated group. Calves that received the homologous vaccination of the nanovaccine + CPG had significantly less lung pathology and viral burden in the lungs compared to control calves. Conclusions: These results indicate that an intranasal homologous vaccination regimen with the post-fusion F/G + CpG nanovaccine has the capacity to reduce BRSV disease in neonatal calves with preexisting maternal antibodies.

The Endocannabinoid Anandamide Attenuates Acute Respiratory Distress Syndrome by Downregulating miRNA that Target Inflammatory Pathways.

Acute respiratory distress syndrome (ARDS) is defined as a type of respiratory failure that is caused by a variety of insults such as pneumonia, sepsis, trauma and certain viral infections. In this study, we investigated the effect of an endocannabinoid, anandamide (AEA), on ARDS induced in the mouse by Staphylococcus Enterotoxin B (SEB). Administration of a single intranasal dose of SEB in mice and treated with exogenous AEA at a dose of 40 mg/kg body weight led to the amelioration of ARDS in mice. Clinically, plethysmography results indicated that there was an improvement in lung function after AEA treatment accompanied by a decrease of inflammatory cell infiltrate. There was also a significant decrease in pro-inflammatory cytokines IL-2, TNF-α, and IFN-γ, and immune cells including CD4+ T cells, CD8+ T cells, Vβ8+ T cells, and NK+ T cells in the lungs. Concurrently, an increase in anti-inflammatory phenotypes such as CD11b + Gr1+ Myeloid-derived Suppressor Cells (MDSCs), CD4 + FOXP3 + Tregs, and CD4+IL10 + cells was observed in the lungs. Microarray data showed that AEA treatment in ARDS mice significantly altered numerous miRNA including downregulation of miRNA-23a-3p, which caused an upregulation of arginase (ARG1), which encodes for arginase, a marker for MDSCs, as well as TGF-β2, which induces Tregs. AEA also caused down-regulation of miRNA-34a-5p which led to induction of FoxP3, a master regulator of Tregs. Transfection of T cells using miRNA-23a-3p or miRNA-34a-5p mimics and inhibitors confirmed that these miRNAs targeted ARG1, TGFβ2 and FoxP3. In conclusion, the data obtained from this study suggests that endocannabinoids such as AEA can attenuate ARDS induced by SEB by suppressing inflammation through down-regulation of key miRNA that regulate immunosuppressive pathways involving the induction of MDSCs and Tregs.

Polyvalent Bacterial Lysate Protects Against Pneumonia Independently of Neutrophils, IL-17A or Caspase-1 Activation.

Polyvalent bacterial lysates have been in use for decades for prevention and treatment of respiratory infections with reported clinical benefits. However, besides claims of broad immune activation, the mode of action is still a matter of debate. The lysates, formulated with the main bacterial species involved in respiratory infections, are commonly prepared by chemical or mechanical disruption of bacterial cells, what is believed influences the biological activity of the product. Here, we prepared two polyvalent lysates with the same composition but different method of bacterial cell disruption and evaluated their biological activity in a comparative fashion. We found that both bacterial lysates induce NF-kB activation in a MyD88 dependent manner, suggesting they work as TLR agonists. Further, we found that a single intranasal dose of any of the two lysates, is sufficient to protect against pneumococcal pneumonia, suggesting that they exert similar biological activity. We have previously shown that protection against pneumococcal pneumonia can also be induced by prior S. pneumoniae sub lethal infection or therapeutic treatment with a TLR5 agonist. Protection in those cases depends on neutrophil recruitment to the lungs, and can be associated with increased local expression of IL-17A. Here, we show that bacterial lysates exert protection against pneumococcal pneumonia independently of neutrophils, IL-17A or Caspase-1/11 activation, suggesting the existence of redundant mechanisms of protection. Trypsin-treated lysates afford protection to the same extent, suggesting that just small peptides suffice to exert the protective effect or that the molecules responsible for the protective effect are not proteins. Understanding the mechanism of action of bacterial lysates and deciphering the active components shall allow redesigning them with more precisely defined formulations and expanding their range of action.