Brief report: Tempol, a novel antioxidant, inhibits both activated T cell and antigen presenting cell derived cytokines in-vitro from COVID-19 patients.

COVID-19 is characterized by a dysregulation of inflammatory cytokines ultimately resulting a cytokine storm that can result in significant morbidity and mortality. We developed an in-vitro assay using activated peripheral blood mononuclear cells (PBMCs) stimulated with lipopolysaccharide (LPS) or CD3 + CD28 to examine secretion of cytokines from antigen presenting cells (APCs) and T cells, respectively, in donor patients with a history of COVID-19 (convalescent) and uninfected negative controls. We hypothesized that a novel antioxidant called Tempol may decrease cytokines from activated peripheral blood cells from both COVID-19 patients and normal donors. Preincubation of immune cells with Tempol resulted in a significant (P < 0.05) decrease in multiple T cell and APC-derived cytokines from both cells of COVID-19 (n = 7) and uninfected donors (n = 7). These preliminary results suggest that Tempol has strong in-vitro anti-cytokine activity and supports additional studies examining the use of Tempol for the treatment of COVID-19.

The Effects of Intramuscular Naloxone Dose on Mu Receptor Displacement of Carfentanil in Rhesus Monkeys.

Naloxone (NLX) is a mu receptor antagonist used to treat acute opioid overdoses. Currently approved doses of naloxone to treat opioid overdoses are 4 mg intranasal (IN) and 2 mg intramuscular (IM). However, higher mu receptor occupancy (RO) may be required to treat overdoses due to more potent synthetic opioids such as fentanyl and carfentanil that have entered the illicit drug market recently. To address this need, a higher dose of NLX has been investigated in a 5 mg IM formulation called ZIMHI but, while the effects of intravenous (IV) and IN administration of NLX on the opioid mu receptor occupancy (RO) have been studied, comparatively little is known about RO for IM administration of NLX. The goal of this study was to examine the effect of IM dosing of NLX on mu RO in rhesus macaques using [11C]carfentanil positron emission tomography (PET) imaging. The lowest dose of NLX (0.06 mg/kg) approximated 51% RO. Higher doses of NLX (0.14 mg/kg, 0.28 mg/kg) resulted in higher mu RO of 70% and 75%, respectively. Plasma levels were 4.6 ng/mL, 16.8 ng/mL, and 43.4 ng/mL for the three IM doses, and a significant correlation between percent RO and plasma NLX level was observed (r = 0.80). These results suggest that higher doses of IM NLX result in higher mu RO and could be useful in combating overdoses resulting from potent synthetic opioids.

Human factors study of a newly approved prefilled syringe of epinephrine for the treatment of anaphylaxis.

BACKGROUND: Epinephrine remains the treatment of choice for acute anaphylaxis. However, currently available autoinjectors are costly, and studies have demonstrated human factor issues that result in incorrect use as well as device failures. OBJECTIVE: A recent U.S. Food and Drug Administration approved prefilled syringe of epinephrine for the treatment of anaphylaxis was examined in a prospective human factors validation study to determine the likelihood that the product would be used effectively by intended users. METHODS: A total of 82 participants were enrolled in this prospective study, including adults with and without epinephrine injector experience, adolescents with and without epinephrine injector experience, and lay caregivers with and without epinephrine injector experience. Half of the participants in each user group were trained to use the newly approved prefilled epinephrine syringe before its first use in the study. Critical tasks that could cause harm and compromise the successful use of epinephrine were assessed and included five categories: (1) open the case, (2) retrieve prefilled syringe, (3) remove needle cap, (4) insert needle in the thigh by using a needle pad, and (5) press plunger until it stops. The participants were scored by an independent observer on the correct use of the device. RESULTS: Of the participants, 100% (82/82) completed category 1, 100% of the participants (82/82) completed category 2, 100% (82/82) completed category 3, 93% (71/76) completed category 4 (six participants were observed to have a device with a bent needle and were taken out of the analysis), and 99% (81/82) completed category 5. CONCLUSION: In this prospective study of human factors that effect correct epinephrine injection, a high rate of participants successfully completed the tasks when using the prefilled syringe, a newly approved epinephrine syringe for the treatment of anaphylaxis. These results indicated that the newly approved prefilled syringe of epinephrine should provide a user-friendly treatment for acute anaphylaxis.

DAS181 for Treatment of Parainfluenza Virus Infections in Hematopoietic Stem Cell Transplant Recipients at a Single Center.

Parainfluenza virus (PIV) causes severe respiratory infections in hematopoietic stem cell transplant (HSCT) recipients. Currently, no effective therapies are available. DAS181 is a novel antiviral agent that inhibits attachment of PIV to respiratory cells, but clinical data on the use of DAS181 for PIV infection are limited to case reports. We report the clinical manifestations and outcomes of 16 HSCT recipients who received DAS181 daily for the treatment of PIV infection through a compassionate-use protocol or a single-arm clinical trial. Of the 16 patients (clinical trial: 9; compassionate use: 7), 13 were allogeneic HSCT recipients and 8 had graft-versus-host disease. PIV types were 3 (n = 7), 4 (n = 5), 1 (n = 3), and type 3 and 4 coinfection (n = 1). Fourteen patients had pneumonia. All patients presented with cough, 14 had dyspnea, 11 had hypoxia, and 8 had a fever. Patients received 5 to 10 days of treatment. Nine patients (56%) had a complete clinical response after DAS181 therapy and 4 (25%) had a partial response. The 3 patients without a clinical response had coinfections with other pathogens. Of the 7 patients with virologic and spirometric data, 5 had >1-log reduction in nasopharyngeal swab PIV viral load and 4 had improved forced expiratory volumes by the end of treatment. Three patients (19%) died within 30 days and 2 of these deaths were related to PIV infection. Our data suggest that DAS181 may be an effective therapy for PIV pneumonia in HSCT recipients. Randomized placebo-controlled trials are needed to better evaluate its efficacy.

A phase II study of DAS181, a novel host directed antiviral for the treatment of influenza infection.

BACKGROUND: DAS181, a novel host-directed antiviral in development for influenza treatment, was assessed in this phase II clinical trial. METHODS: This study was a double-blind, placebo-controlled phase II clinical trial assessing influenza viral load and patient safety in otherwise healthy influenza-infected participants. Participants were randomized to a single-dose, multiple-dose, or placebo group and were followed for safety and virologic outcomes. RESULTS: A total of 177 laboratory-confirmed influenza-infected participants were enrolled in the trial, which encompassed 3 influenza seasons from 2009-2011 in both the Northern and Southern Hemispheres. Thirty-seven percent of participants had confirmed infection with influenza B, 33% with seasonal H3N2, 29% with pandemic 2009 H1N1, and 1 participant was positive for both influenza B and pandemic 2009 H1N1. Significant effects were observed in regard to decreased change from baseline viral load and viral shedding in the multiple-dose group compared with placebo as measured by quantitative polymerase chain reaction (P < .05). No instances of H274Y were observed among viral isolates from this trial. Overall, the drug was generally well tolerated. CONCLUSIONS: DAS181 significantly reduced viral load in participants infected with influenza, thus warranting future clinical development of this novel host-directed therapy. CLINICAL TRIALS.GOV IDENTIFIER: NCT01037205.

Treatment of parainfluenza 3 infection with DAS181 in a patient after allogeneic stem cell transplantation.

Parainfluenza virus (PIV) can cause significant morbidity after allogeneic stem cell transplantation (SCT). We report the first use of inhaled DAS181 for PIV in an allogeneic SCT recipient. Symptoms, oxygenation, and pulmonary function tests improved. Nasopharyngeal samples showed a reduction in viral load. DAS181 should be systematically evaluated for severe PIV infection.

Targeting pandemic influenza: a primer on influenza antivirals and drug resistance.

The emergence of the 2009 H1N1 pandemic influenza A virus, as well as constant antigenic drift of seasonal influenza, underscores the remarkable versatility of this virus in adapting to the human population. While vaccines are the principal public health defence against influenza, rapid vaccine development can be a daunting task. Antiviral drugs offer the promise of inhibiting influenza regardless of its genetic variations. However, the rapid rise of resistance to several antivirals has highlighted the need for developing novel therapeutics with reduced drug resistance potential. In this review, we will summarize the effects of the currently licensed anti-influenza drugs as well as the candidates in development against the seasonal and the 2009 H1N1 pandemic influenza A virus with an emphasis on drug resistance.

An open-label, randomized, single-dose, two-period, two-treatment crossover bioavailability study comparing 5 mg/0.5 mL of intramuscular naloxone hydrochloride to 2 mg/0.4 mL intramuscular naloxone hydrochloride autoinjector in healthy subjects.

Naloxone is an opioid antagonist used for the acute treatment of opioid overdoses. There has been a dramatic increase of deaths due to synthetic opioids such as fentanyl, some requiring multiple doses of naloxone for reversal of opioid tox-icity. Fentanyl appears to differ from other opiates as having a very rapid onset and transport in and out of the central nervous system (CNS). Fentanyl is therefore widely distributed in the CNS. Furthermore, a high range of systemic levels of fentanyl have been observed in overdose victims. Taken together, we believe it is very likely that higher doses of naloxone are needed to combat this new era of overdoses. We examined the bioavailability of an investigational 5 mg intramuscular naloxone in a prefilled syringe (PFS) compared to 2 mg intramuscular naloxone in an autoinjector (AI) at the current approved dose in a crossover design which included 14 healthy subjects. Overall, both doses were well tol-erated with no adverse events noted during the trial. The pharmacokinetic results showed that a higher dose of intra-muscular naloxone hydrochloride increases Cmax, AUC, and t1/2; however, Tmax was similar for both treatments. Statistical analysis indicated that there were statistical differences between the test and reference treatments for Cmax, AUCs, and t1/2 with ratios of test to reference for Cmax of 337.1 percent (CI: 263.3 percent, 431.5 percent), AUC0-t of 277.5 percent (CI: 260.4 percent, 295.7 percent), AUC0-inf of 273.4 percent (CI: 255.6 percent, 292.4 percent), and t1/2 of 110.5 percent (CI: 95.5, 127.9). These results are consistent with the study rationale that indicated higher doses of intramuscular naloxone hy-drochloride would result in higher Cmax and AUCs. These PK characteristics may be desirable for reversing opioid toxicity caused by the higher, more potent synthetic opioids.

Higher naloxone dosing in a quantitative systems pharmacology model that predicts naloxone-fentanyl competition at the opioid mu receptor level.

Rapid resuscitation of an opioid overdose with naloxone, an opioid antagonist, is critical. We developed an opioid receptor quantitative systems pharmacology (QSP) model for evaluation of naloxone dosing. In this model we examined three opioid exposure levels that have been reported in the literature (25 ng/ml, 50 ng/ml, and 75 ng/ml of fentanyl). The model predicted naloxone-fentanyl interaction at the mu opioid receptor over a range of three naloxone doses. For a 2 mg intramuscular (IM) dose of naloxone at lower fentanyl exposure levels (25 ng/ml and 50 ng/ml), the time to decreasing mu receptor occupancy by fentanyl to 50% was 3 and 10 minutes, respectively. However, at a higher fentanyl exposure level (75 ng/ml), a dose of 2 mg IM of the naloxone failed to reduce mu receptor occupancy by fentanyl to 50%. In contrast, naloxone doses of 5 mg and 10 mg IM reduced mu receptor occupancy by fentanyl to 50% in 5.5 and 4 minutes respectively. These results suggest that the current doses of naloxone (2 mg IM or 4 mg intranasal (IN)) may be inadequate for rapid reversal of toxicity due to fentanyl exposure and that increasing the dose of naloxone is likely to improve outcomes.

Prospects for control of emerging infectious diseases with plasmid DNA vaccines.

Experiments almost 20 years ago demonstrated that injections of a sequence of DNA encoding part of a pathogen could stimulate immunity. It was soon realized that "DNA vaccination" had numerous potential advantages over conventional vaccine approaches including inherent safety and a more rapid production time. These and other attributes make DNA vaccines ideal for development against emerging pathogens. Recent advances in optimizing various aspects of DNA vaccination have accelerated this approach from concept to reality in contemporary human trials. Although not yet licensed for human use, several DNA vaccines have now been approved for animal health indications. The rapid manufacturing capabilities of DNA vaccines may be particularly important for emerging infectious diseases including the current novel H1N1 Influenza A pandemic, where pre-existing immunity is limited. Because of recent advances in DNA vaccination, this approach has the potential to be a powerful new weapon in protecting against emerging and potentially pandemic human pathogens.

Higher doses of naloxone are needed in the synthetic opiod era.

There has been a dramatic increase of deaths due to illicit fentanyl. We examined the pharmacology of fentanyl and reviewed data on the number of repeat doses of naloxone used to treat fentanyl overdoses. Multiple sequential doses of naloxone have been required in a certain percentage of opioid overdoses due to fentanyl. In addition, fentanyl appears to differ from other opioids as having a very rapid onset with high systemic levels found in overdose victims. A rapid competition is required by naloxone to out-compete large numbers of opioid receptors occupied by fentanyl in the CNS. Taken together, we propose that higher doses of naloxone are needed to combat the new era of overdoses due to the more potent synthetic opioids such as fentanyl.

Enhanced HIV-1 specific immune response by CpG ODN and HIV-1 immunogen-pulsed dendritic cells confers protection in the Trimera murine model of HIV-1 infection.

We have recently developed a novel small animal model for HIV-1 infection (Ayash-Rashkovsky et al., http://www.fasebj.org/cgi/doi/10.1096/fj.04-3184fje; doi:10.1096/fj.04-3184fje). The mice were successfully infected with HIV-1 for 4-6 wk with different clades of either T- or M-tropic isolates. HIV-1 infection was accompanied by rapid loss of human CD4+ T cells, decrease in CD4/CD8 ratio, and increased T cell activation. HIV specific human humoral and cellular immune responses were observed in all HIV-1 infected animals. In the present study, HIV specific human immune responses, both humoral and cellular, were generated in noninfected Trimera mice, after their immunization with gp120-depleted HIV-1 antigen, presented by autologous human dendritic cells. Addition of CpG ODN to the antigen-pulsed DCs significantly enhanced (by 2- to 30-fold) the humoral and cellular HIV-1 specific immune responses. Only mice immunized with the HIV-1 immunogen and CpG were completely protected from infection with HIV-1 after challenge with high infection titers of the virus. This novel small animal model for HIV-1 infection may thus serve as an attractive platform for rapid testing of candidate HIV-1 vaccines and of adjuvants and may shorten the time needed for the development and final assessment of protective HIV-1 vaccines in human trials.

Enterovirus 68 Infection--Association with Asthma.

A previously sporadic virus called enterovirus 68 (EV-D68) appears to have been associated with asthma-like illness with a predisposition for asthmatics after an outbreak that occurred in North America in 2014. Clinicians should be aware of the clinical associations with EV-D68 particularly its predilection with pre-existing asthma or asthma-like illness as well as the potential association with acute flaccid myelitis. Further elucidation and development of diagnostic and treatments modalities are warranted to better understand and limit the potential public health impact of future outbreaks of EV-D68 infection.

Treatment of resistant influenza virus infection in a hospitalized patient with cystic fibrosis with DAS181, a host-directed antiviral.

We report a cystic fibrosis patient infected with influenza 2009H1N1 who had persistent viral shedding and clinical deterioration despite prolonged treatment with oseltamivir and zanamivir. The patient was diagnosed with H275Y neuraminidase inhibitor resistant influenza during treatment, thus was treated for 10 days with DAS181, an investigational host-directed inhaled sialidase fusion protein. Viral clearance occurred after 5 days of therapy and the patient became eligible for lung transplantation. Although the patient succumbed prior to receiving a transplant, this case exemplifies the potential utility of a host-directed approach against influenza which has potential to become resistant to neuraminidase inhibitors.

Emergence of Hemagglutinin Mutations During the Course of Influenza Infection.

Influenza remains a significant cause of disease mortality. The ongoing threat of influenza infection is partly attributable to the emergence of new mutations in the influenza genome. Among the influenza viral gene products, the hemagglutinin (HA) glycoprotein plays a critical role in influenza pathogenesis, is the target for vaccines and accumulates new mutations that may alter the efficacy of immunization. To study the emergence of HA mutations during the course of infection, we employed a deep-targeted sequencing method. We used samples from 17 patients with active H1N1 or H3N2 influenza infections. These patients were not treated with antivirals. In addition, we had samples from five patients who were analyzed longitudinally. Thus, we determined the quantitative changes in the fractional representation of HA mutations during the course of infection. Across individuals in the study, a series of novel HA mutations directly altered the HA coding sequence were identified. Serial viral sampling revealed HA mutations that either were stable, expanded or were reduced in representation during the course of the infection. Overall, we demonstrated the emergence of unique mutations specific to an infected individual and temporal genetic variation during infection.

The use of sialidase therapy for respiratory viral infections.

DAS181 is an inhaled bacterial sialidase which functions by removing sialic acid (Sia) from the surface of epithelial cells, preventing attachment and subsequent infection by respiratory viruses that utilize Sia as a receptor. DAS181 is typical of bacterial sialidases in cleaving Sia α2-3 and Sia α2-6 linkages, and it also has a demonstrated effect against acetylated and hydroxylated forms of Sia. The potency of the compound has been enhanced by coupling the active sialidase with an amphiregulin tag, allowing a longer duration of action and minimizing spread to the systemic circulation. DAS181 is now in Phase II development for the treatment of influenza, and it has also demonstrated activity in individual cases of parainfluenza in immunosuppressed patients. Continued evaluation of the roles and activities of bacterial sialidases is required to expand the range of successful antiviral therapies targeting Sia or its derivatives.

Perspective: emerging challenges in the treatment of influenza and parainfluenza in transplant patients.

Influenza, respiratory synctial virus, and parainfluenza are common respiratory infections in immunocompromised transplant recipients, causing significant morbidity and mortality in this patient population. This paper focuses on influenza and parainfluenza virus infections in transplant patients with emphasis on the pandemic 2009 H1N1 influenza infection. Current antiviral treatment recommendations for influenza and parainfluenza in immunocompromised patients as well as novel investigational therapeutic approaches currently being tested in the clinic are discussed. In addition to the morbidity and mortality caused by these viruses, the development of multidrug resistance leading to transmission of resistant viruses is of great public health concern. The development of effective new therapies for influenza and parainfluenza in these high-risk patients is needed with randomized placebo-controlled studies to assess their clinical utility.

Phase 1 clinical trials of the safety and immunogenicity of adjuvanted plasmid DNA vaccines encoding influenza A virus H5 hemagglutinin.

BACKGROUND: Development of vaccines against highly pathogenic avian influenza virus H5N1 subtypes posing a pandemic threat remains a priority. Limitations in manufacturing capacity and production time of conventional inactivated vaccines highlight the need for additional approaches. METHODS: We conducted two double-blind, placebo-controlled phase 1 studies involving a total of 103 healthy adults who received two intramuscular injections of Vaxfectin-adjuvanted plasmid DNA vaccine or placebo 21 days apart. Vaccine cohorts received either a monovalent vaccine containing an A/Vietnam/1203/04 H5 hemagglutinin-encoding plasmid or a trivalent vaccine with plasmids encoding H5, NP, and M2 proteins in doses from 0.1 to 1mg of DNA/injection. RESULTS: All doses were well tolerated without vaccine-related serious adverse events or discontinuations. In the monovalent cohorts, hemagglutination inhibition (HI) titers of > or =40 and 4-fold rises from baseline were achieved in 47-67% of subjects and H5-specific T-cell responses in 75-100%. Trivalent cohorts had lower HI response rates (< or = 20%), but 72% of subjects achieved T-cell and/or antibody responses to one or more antigens. CONCLUSIONS: Vaxfectin-adjuvanted monovalent H5 DNA vaccines were well tolerated and induced HI response rates and titers in the reported range of inactivated protein-based H5 vaccines, suggesting that adjuvanted DNA vaccines with rapid vaccine production could be useful for pandemic control.