The Statistical Fragility of Tranexamic Acid Use in the Orthopaedic Surgery Literature: A Systematic Review of Randomized Controlled Trials.

INTRODUCTION: Randomized controlled trials (RCTs) represent the highest level of evidence in orthopaedic surgery literature, although the robustness of statistical findings in these trials may be unreliable. We used the fragility index (FI), reverse fragility index (rFI), and fragility quotient (FQ) to evaluate the statistical stability of outcomes reported in RCTs that assess the use of tranexamic acid (TXA) across orthopaedic subspecialties. METHODS: PubMed, EMBASE, and MEDLINE were queried for RCTs (2010-present) reporting dichotomous outcomes with study groups stratified by TXA administration. The FI and rFI were defined as the number of outcome event reversals needed to alter the significance level of significant and nonsignificant outcomes, respectively. FQ was determined by dividing the FI or rFI by sample size. Subgroup analyses were conducted based on orthopaedic subspecialty. RESULTS: Six hundred five RCTs were screened with 108 studies included for analysis comprising 192 total outcomes. The median FI of the 192 outcomes was 4 (IQR 2 to 5) with an associated FQ of 0.03 (IQR 0.019 to 0.050). 45 outcomes were reported as statistically significant with a median FI of 1 (IQR 1 to 5) and associated FQ of 0.02 (IQR 0.011 to 0.034). 147 outcomes were reported as nonsignificant with a median rFI of 4 (IQR 3 to 5) and associated FQ of 0.04 (IQR 0.023 to 0.051). The adult reconstruction, trauma, and spine subspecialties had a median FI of 4. Sports had a median FI of 3. Shoulder and elbow and foot and ankle had median FIs of 6. DISCUSSION: Statistical outcomes reported in RCTs on the use of TXA in orthopaedic surgery are fragile. Reversal of a few outcomes is sufficient to alter statistical significance. We recommend reporting FI, rFI, and FQ metrics to aid in interpreting the outcomes reported in comparative trials.

Environmental surface monitoring as a noninvasive method for SARS-CoV-2 surveillance in community settings: Lessons from a university campus study.

Environmental testing of high-touch objects is a potential noninvasive approach for monitoring population-level trends of SARS-CoV-2 and other respiratory viruses within a defined setting. We aimed to determine the association between SARS-CoV-2 contamination on high-touch environmental surfaces, community level case incidence, and university student health data. Environmental swabs were collected from January 2022 to November 2022 from high-touch objects and surfaces from five locations on a large university campus in Florida, USA. RT-qPCR was used to detect and quantify viral RNA, and a subset of positive samples was analyzed by viral genome sequencing to identify circulating lineages. During the study period, we detected SARS-CoV-2 viral RNA on 90.7 % of 162 tested samples. Levels of environmental viral RNA correlated with trends in community-level activity and case reports from the student health center. A significant positive correlation was observed between the estimated viral gene copy number in environmental samples and the weekly confirmed cases at the university. Viral sequencing data from environmental samples identified lineages concurrently circulating in the local community and state based on genomic surveillance data. Further, we detected emerging variants in environmental samples prior to their identification by clinical genomic surveillance. Our results demonstrate the utility of viral monitoring on high-touch environmental surfaces for SARS-CoV-2 surveillance at a community level. In communities with delayed or limited testing facilities, immediate environmental surface testing may considerably inform epidemic dynamics.

Evidence of a Sjögren's disease-like phenotype following COVID-19 in mice and humans.

Sjögren's Disease (SjD) is a systemic autoimmune disease characterized by lymphocytic inflammation of the lacrimal and salivary glands (SG), dry eyes and mouth, and systemic symptoms. SARS-CoV-2 may trigger the development or progression of autoimmune diseases. To test this, we used a mouse model of SARS-CoV-2 infection and convalescent patients' blood and SG in order to understand the development of SjD-like autoimmunity after infection. First, SARS-CoV-2-infected human angiotensin-converting enzyme 2 (ACE2) transgenic mice exhibited decreased salivation, elevated antinuclear antibodies (ANA), and lymphocytic infiltration in the lacrimal and SG. The sera from patients with COVID-19 sera showed increased ANA (i.e., anti-SSA [Sjögren's-syndrome-related antigen A]/anti-Ro52 and anti-SSB [SS-antigen B]/anti-La). Male patients showed elevated anti-SSA compared with female patients, and female patients exhibited diverse ANA patterns. SG biopsies from convalescent COVID-19 patients were microscopically similar to SjD SG with focal lymphocytic infiltrates in 4 of 6 patients and 2 of 6 patients exhibiting focus scores of at least 2. Lastly, monoclonal antibodies produced in recovered patients blocked ACE2/spike interaction and cross-reacted with nuclear antigens. Our study shows a direct association between SARS-CoV-2 and SjD. Hallmark features of SjD-affected SGs were histologically indistinguishable from convalescent COVID-19 patients. The results implicate that SARS-CoV-2 could be an environmental trigger for SjD.

Favipiravir Inhibits Zika Virus (ZIKV) Replication in HeLa Cells by Altering Viral Infectivity.

This study aims to evaluate the antiviral potential of the nucleoside analogue favipiravir (FAV) against ZIKV, an arbovirus for which there are no approved antiviral therapies, in three human-derived cell lines. HeLa (cervical), SK-N-MC (neuronal), and HUH-7 (liver) cells were infected with ZIKV and exposed to different concentrations of FAV. Viral supernatant was sampled daily, and infectious viral burden was quantified by plaque assay. Changes in ZIKV infectivity were quantified by calculating specific infectivity. FAV-related toxicities were also assessed for each cell line in both infected and uninfected cells. Our results demonstrate that FAV activity was most pronounced in HeLa cells, as substantial declines in infectious titers and viral infectivity were observed in this cell type. The decline in infectious virus occurred in an exposure-dependent manner and was more pronounced as FAV exposure times increased. Additionally, toxicity studies showed that FAV was not toxic to any of the three cell lines and, surprisingly, caused substantial improvements in the viability of infected HeLa cells. Although SK-N-MC and HUH-7 cells were susceptible to FAV's anti-ZIKV activity, similar effects on viral infectivity and improvements in cell viability with therapy were not observed. These results indicate that FAV's ability to substantially alter viral infectivity is host cell specific and suggest that the robust antiviral effect observed in HeLa cells is mediated through drug-induced losses of viral infectivity.

Favipiravir Suppresses Zika Virus (ZIKV) through Activity as a Mutagen.

In a companion paper, we demonstrated that the nucleoside analogue favipiravir (FAV) suppressed Zika virus (ZIKV) replication in three human-derived cell lines-HeLa, SK-N-MC, and HUH-7. Our results revealed that FAV's effect was most pronounced in HeLa cells. In this work, we aimed to explain variation in FAV activity, investigating its mechanism of action and characterizing host cell factors relevant to tissue-specific differences in drug effect. Using viral genome sequencing, we show that FAV therapy was associated with an increase in the number of mutations and promoted the production of defective viral particles in all three cell lines. Our findings demonstrate that defective viral particles made up a larger portion of the viral population released from HeLa cells both at increasing FAV concentrations and at increasing exposure times. Taken together, our companion papers show that FAV acts via lethal mutagenesis against ZIKV and highlight the host cell's influence on the activation and antiviral activity of nucleoside analogues. Furthermore, the information gleaned from these companion papers can be applied to gain a more comprehensive understanding of the activity of nucleoside analogues and the impact of host cell factors against other viral infections for which we currently have no approved antiviral therapies.

Combination Therapy with UV-4B and Molnupiravir Enhances SARS-CoV-2 Suppression.

The host targeting antiviral, UV-4B, and the RNA polymerase inhibitor, molnupiravir, are two orally available, broad-spectrum antivirals that have demonstrated potent activity against SARS-CoV-2 as monotherapy. In this work, we evaluated the effectiveness of UV-4B and EIDD-1931 (molnupiravir's main circulating metabolite) combination regimens against the SARS-CoV-2 beta, delta, and omicron BA.2 variants in a human lung cell line. Infected ACE2 transfected A549 (ACE2-A549) cells were treated with UV-4B and EIDD-1931 both as monotherapy and in combination. Viral supernatant was sampled on day three when viral titers peaked in the no-treatment control arm, and levels of infectious virus were measured by plaque assay. The drug-drug effect interaction between UV-4B and EIDD-1931 was also defined using the Greco Universal Response Surface Approach (URSA) model. Antiviral evaluations demonstrated that treatment with UV-4B plus EIDD-1931 enhanced antiviral activity against all three variants relative to monotherapy. These results were in accordance with those obtained from the Greco model, as these identified the interaction between UV-4B and EIDD-1931 as additive against the beta and omicron variants and synergistic against the delta variant. Our findings highlight the anti-SARS-CoV-2 potential of UV-4B and EIDD-1931 combination regimens, and present combination therapy as a promising therapeutic strategy against SARS-CoV-2.

A five-day treatment course of zanamivir for the flu with a single, self-administered, painless microneedle array patch: Revolutionizing delivery of poorly membrane-permeable therapeutics.

Seasonal influenza virus infections cause a substantial number of deaths each year. While zanamivir (ZAN) is efficacious against oseltamivir-resistant influenza strains, the efficacy of the drug is limited by its route of administration, oral inhalation. Herein, we present the development of a hydrogel-forming microneedle array (MA) in combination with ZAN reservoirs for treating seasonal influenza. The MA was fabricated from Gantrez® S-97 crosslinked with PEG 10,000. Various reservoir formulations included ZAN hydrate, ZAN hydrochloric acid (HCl), CarraDres™, gelatin, trehalose, and/or alginate. In vitro permeation studies with a lyophilized reservoir consisting of ZAN HCl, gelatin, and trehalose resulted in rapid and high delivery of up to 33 mg of ZAN across the skin with delivery efficiency of up to ≈75% by 24 h. Pharmacokinetics studies in rats and pigs demonstrated that a single administration of a MA in combination with a CarraDres™ ZAN HCl reservoir offered a simple and minimally invasive delivery of ZAN into the systemic circulation. In pigs, efficacious plasma and lung steady-state levels of ∼120 ng/mL were reached within 2 h and sustained between 50 and 250 ng/mL over 5 days. MA-enabled delivery of ZAN could enable a larger number of patients to be reached during an influenza outbreak.

Structure-Based Design of Potent Iminosugar Inhibitors of Endoplasmic Reticulum α-Glucosidase I with Anti-SARS-CoV-2 Activity.

Enveloped viruses depend on the host endoplasmic reticulum (ER) quality control (QC) machinery for proper glycoprotein folding. The endoplasmic reticulum quality control (ERQC) enzyme α-glucosidase I (α-GluI) is an attractive target for developing broad-spectrum antivirals. We synthesized 28 inhibitors designed to interact with all four subsites of the α-GluI active site. These inhibitors are derivatives of the iminosugars 1-deoxynojirimycin (1-DNJ) and valiolamine. Crystal structures of ER α-GluI bound to 25 1-DNJ and three valiolamine derivatives revealed the basis for inhibitory potency. We established the structure-activity relationship (SAR) and used the Site Identification by Ligand Competitive Saturation (SILCS) method to develop a model for predicting α-GluI inhibition. We screened the compounds against SARS-CoV-2 in vitro to identify those with greater antiviral activity than the benchmark α-glucosidase inhibitor UV-4. These host-targeting compounds are candidates for investigation in animal models of SARS-CoV-2 and for testing against other viruses that rely on ERQC for correct glycoprotein folding.

The Fragility of Tourniquet Use in Total Knee Arthroplasty: A Systematic Review of Randomized Controlled Trials.

BACKGROUND: Physicians utilize P-values to interpret clinical trial data and guide patient-care decisions. Fragility analysis assesses the stability of statistical findings in relation to outcome event reversals. This study assessed the statistical fragility of recent randomized controlled trials (RCTs) investigating tourniquet use in total knee arthroplasty (TKA). METHODS: We queried PubMed, EMBASE, and MEDLINE for RCTs comparing outcomes in TKA based on tourniquet use. Fragility index (FI) and reverse fragility index (reverse FI) were calculated - for significant and nonsignificant outcomes, respectively - as the number of outcome reversals required to change statistical significance. The fragility quotient (FQ) was calculated by dividing the FI or reverse FI by the sample size. Median overall FI and FQ were calculated for all included outcomes, and sub-analyses were performed by reported significance. The literature search yielded 23 studies reporting 91 total dichotomous outcomes. RESULTS: Overall median FI was 4 with an interquartile range (IQR) of 3 to 6. Overall median FQ was 0.0476 (IQR 0.0291 to 0.0867). A total of 11 outcomes were statistically significant with a median FI and FQ of 2 (IQR 1.5 to 5) and 0.0200 (IQR 0.0148 to 0.0484), respectively. There were 80 outcomes that were nonsignificant with a median reverse FI of 4 (IQR 3 to 6). Loss to follow-up was greater than the median FI in 17.6% of outcomes. CONCLUSION: Altering a small number of outcomes is often sufficient to reverse findings in RCTs evaluating tourniquet use in TKA. We recommend including fragility analyses to increase reliability in the interpretation of study conclusions.

Why Molnupiravir Fails in Hospitalized Patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has radically altered daily life. Effective antiviral therapies to combat COVID-19, especially severe disease, remain scarce. Molnupiravir is an antiviral that has shown clinical efficacy against mild-to-moderate COVID-19 but failed to provide benefit to hospitalized patients with severe disease. Here, we explained the mechanism behind the failure of molnupiravir in hospitalized patients and identified alternative dosing strategies that would improve therapeutic outcomes in all patients with COVID-19. We showed that delaying therapy initiation markedly decreased the antiviral effect of molnupiravir, and these results were directly related to intracellular drug triphosphate pools and intracellular viral burden at the start of therapy. The adverse influence of therapeutic delay could be overcome by increasing drug exposure, which increased intracellular molnupiravir triphosphate concentrations that inhibited viral replication. These findings illustrated that molnupiravir must be administered as early as possible following COVID-19 symptom onset to maximize therapeutic efficacy. Higher doses may be effective in patients hospitalized with severe disease, but the safety of high-dose molnupiravir regimens is unknown. Our findings could be extended to design effective regimens with nucleoside analogs for other RNA viruses, especially those with pandemic potential. IMPORTANCE In this study, we showed that early intervention with molnupiravir resulted in a greater antiviral effect, and we explained the mechanism behind this phenomenon. Our results predicted and explained the failure of molnupiravir in hospitalized patients and highlighted the utility of preclinical pharmacodynamic studies to design optimal antiviral regimens for the treatment of viral diseases. This contrasts with the procedure that was implemented early in the pandemic in which clinical studies were conducted in the absence of preclinical experimentation. These findings are significant and demonstrated the importance of experimental approaches in antiviral development for treatments against COVID-19 as well as other viral diseases.

UV-4B potently inhibits replication of multiple SARS-CoV-2 strains in clinically relevant human cell lines.

BACKGROUND: SARS-CoV-2 is the coronavirus responsible for the COVID-19 pandemic. Although it poses a substantial public health threat, antiviral regimens against SARS-CoV-2 remain scarce. Here, we evaluated the antiviral potential of UV-4B, a host targeting antiviral, against SARS-CoV-2 in clinically relevant human cell lines. METHODS: Cells derived from human lung (A549 cells transfected with human angiotensin converting enzyme 2 receptor (ACE2; ACE2-A549)) and colon (Caco-2) were infected with either a wild type or beta variant strain of SARS-CoV-2 and exposed to various concentrations of UV-4B. Supernatant was sampled daily and viral burden was quantified by plaque assay on Vero E6 cells. RESULTS: Therapeutically feasible concentrations of UV-4B inhibited the replication of the wild type strain in ACE2-A549 and Caco-2 cells yielding EC50 values of 2.694 and 2.489 µM, respectively. UV-4B's antiviral effect was also robust against the beta variant in both cell lines (ACE2-A549 EC50: 4.369 µM; Caco-2 EC50: 6.816 µM). CONCLUSIONS: These results highlight UV-4B's antiviral potential against several strains of SARS-CoV-2.

Antiviral Activity of the PropylamylatinTM Formula against the Novel Coronavirus SARS-CoV-2 In Vitro Using Direct Injection and Gas Assays in Virus Suspensions.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of novel coronavirus disease 2019 (COVID-19), has become a severe threat to global public health. There are currently no antiviral therapies approved for the treatment or prevention of mild to moderate COVID-19 as remdesivir is only approved for severe COVID-19 cases. Here, we evaluated the antiviral potential of a Propylamylatin formula, which is a mixture of propionic acid and isoamyl hexanoates. The Propylamylatin formula was investigated in gaseous and liquid phases against 1 mL viral suspensions containing 105 PFU of SARS-CoV-2. Viral suspensions were sampled at various times post-exposure and infectious virus was quantified by plaque assay on Vero E6 cells. Propylamylatin formula vapors were effective at inactivating infectious SARS-CoV-2 to undetectable levels at room temperature and body temperature, but the decline in virus was substantially faster at the higher temperature (15 min versus 24 h). The direct injection of liquid Propylamylatin formula into viral suspensions also completely inactivated SARS-CoV-2 and the rapidity of inactivation occurred in an exposure dependent manner. The overall volume that resulted in 90% viral inactivation over the course of the direct injection experiment (EC90) was 4.28 µls. Further investigation revealed that the majority of the antiviral effect was attributed to the propionic acid which yielded an overall EC90 value of 11.50 µls whereas the isoamyl hexanoates provided at most a 10-fold reduction in infectious virus. The combination of propionic acid and isoamyl hexanoates was much more potent than the individual components alone, suggesting synergy between these components. These findings illustrate the therapeutic promise of the Propylamylatin formula as a potential treatment strategy for COVID-19 and future studies are warranted.

Antiviral Evaluation of UV-4B and Interferon-Alpha Combination Regimens against Dengue Virus.

Dengue virus (DENV) is a flavivirus associated with clinical manifestations ranging in severity from self-limiting dengue fever, to the potentially life threatening condition, severe dengue. There are currently no approved antiviral therapies for the treatment of DENV. Here, we evaluated the antiviral potential of four broad-spectrum antivirals, UV-4B, interferon-alpha (IFN), sofosbuvir (SOF), and favipiravir (FAV) against DENV serotype 2 as mono- and combination therapy in cell lines that are physiologically relevant to human infection. Cell lines derived from human liver (HUH-7), neurons (SK-N-MC), and skin (HFF-1) were infected with DENV and treated with UV-4B, IFN, SOF, or FAV. Viral supernatant was sampled daily and infectious viral burden was quantified by plaque assay on Vero cells. Drug effect on cell proliferation in uninfected and infected cells was also assessed. UV-4B inhibited DENV in HUH-7, SK-N-MC, and HFF-1 cells yielding EC50 values of 23.75, 49.44, and 37.38 µM, respectively. Clinically achievable IFN concentrations substantially reduced viral burden in HUH-7 (EC50 = 102.7 IU/mL), SK-N-MC (EC50 = 86.59 IU/mL), and HFF-1 (EC50 = 163.1 IU/mL) cells. SOF potently inhibited DENV in HUH-7 cells but failed to produce the same effect in SK-N-MC and HFF-1 cells. Finally, FAV provided minimal suppression in HUH-7 and SK-N-MC cells, but was ineffective in HFF-1 cells. The two most potent anti-DENV agents, UV-4B and IFN, were also assessed in combination. UV-4B + IFN treatment enhanced antiviral activity in HUH-7, SK-N-MC, and HFF-1 cells relative to monotherapy. Our results demonstrate the antiviral potential of UV-4B and IFN against DENV in multiple physiologically relevant cell types.

Combination Regimens of Favipiravir Plus Interferon Alpha Inhibit Chikungunya Virus Replication in Clinically Relevant Human Cell Lines.

Chikungunya virus (CHIKV) is an alphavirus associated with a broad tissue tropism for which no antivirals or vaccines are approved. This study evaluated the antiviral potential of favipiravir (FAV), interferon-alpha (IFN), and ribavirin (RBV) against CHIKV as mono- and combination-therapy in cell lines that are clinically relevant to human infection. Cells derived from human connective tissue (HT-1080), neurons (SK-N-MC), and skin (HFF-1) were infected with CHIKV and treated with different concentrations of FAV, IFN, or RBV. Viral supernatant was sampled daily and the burden was quantified by plaque assay on Vero cells. FAV and IFN were the most effective against CHIKV on various cell lines, suppressing the viral burden at clinically achievable concentrations; although the degree of antiviral activity was heavily influenced by cell type. RBV was not effective and demonstrated substantial toxicity, indicating that it is not a feasible candidate for CHIKV. The combination of FAV and IFN was then assessed on all cell lines. Combination therapy enhanced antiviral activity in HT-1080 and SK-N-MC cells, but not in HFF-1 cells. We developed a pharmacokinetic/pharmacodynamic model that described the viral burden and inhibitory antiviral effect. Simulations from this model predicted clinically relevant concentrations of FAV plus IFN completely suppressed CHIKV replication in HT-1080 cells, and considerably slowed down the rate of viral replication in SK-N-MC cells. The model predicted substantial inhibition of viral replication by clinical IFN regimens in HFF-1 cells. Our results highlight the antiviral potential of FAV and IFN combination regimens against CHIKV in clinically relevant cell types.

Modeling the viral dynamics of SARS-CoV-2 infection.

Coronavirus disease 2019 (COVID-19), an infectious disease caused by the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is spreading and causing the global coronavirus pandemic. The viral dynamics of SARS-CoV-2 infection have not been quantitatively investigated. In this paper, we use mathematical models to study the pathogenic features of SARS-CoV-2 infection by examining the interaction between the virus, cells and immune responses. Models are fit to the data of SARS-CoV-2 infection in patients and non-human primates. Data fitting and numerical simulation show that viral dynamics of SARS-CoV-2 infection have a few distinct stages. In the initial stage, viral load increases rapidly and reaches the peak, followed by a plateau phase possibly generated by lymphocytes as a secondary target of infection. In the last stage, viral load declines due to the emergence of adaptive immune responses. When the initiation of seroconversion is late or slow, the model predicts viral rebound and prolonged viral persistence, consistent with the observation in non-human primates. Using the model we also evaluate the effect of several potential therapeutic interventions for SARS-CoV-2 infection. Model simulation shows that anti-inflammatory treatments or antiviral drugs combined with interferon are effective in reducing the duration of the viral plateau phase and diminishing the time to recovery. These results provide insights for understanding the infection dynamics and might help develop treatment strategies against COVID-19.

The effectiveness of antiviral agents with broad-spectrum activity against chikungunya virus varies between host cell lines.

Chikungunya virus (CHIKV) is a mosquito-borne virus that has recently emerged in the Western Hemisphere. Approved antiviral therapies or vaccines for the treatment or prevention of CHIKV infections are not available. This study aims to evaluate the antiviral activity of commercially available broad-spectrum antivirals against CHIKV. Due to host cell-specific variability in uptake and intracellular processing of drug, we evaluated the antiviral effects of each agent in three cell lines. Antiviral activities of ribavirin (RBV), interferon-alfa (IFN-α) and favipiravir (FAV) were assessed in CHIKV-infected Vero, HUH-7, and A549 cells. CHIKV-infected cells were treated with increasing concentrations of each agent for three days and viral burden was quantified by plaque assay on Vero cells. Cytotoxic effects of RBV, FAV and IFN-α were also evaluated. Antiviral activity differed depending on the cell line used for evaluation. RBV had the greatest antiviral effect in HUH-7 cells (EC50 = 2.575 µg/mL); IFN-α was most effective in A549 cells (EC50 = 4.235 IU/mL); and FAV in HUH-7 cells (EC50 = 20.00 µg/mL). The results of our study show FAV and IFN-α are the most promising candidates, as their use led to substantial reductions in viral burden at clinically achievable concentrations in two human-derived cell lines. FAV is an especially attractive candidate for further investigation due to its oral bioavailability. These findings also highlight the importance of cell line selection for preclinical drug trials.

Clinical Regimens of Favipiravir Inhibit Zika Virus Replication in the Hollow-Fiber Infection Model.

Zika virus (ZIKV) infection is associated with serious, long-term neurological manifestations. There are currently no approved therapies for the treatment or prevention of ZIKV infection. Favipiravir (FAV) is a viral polymerase inhibitor with broad-spectrum activity. Our prior studies used static FAV concentrations and demonstrated promising activity. However, the anti-ZIKV activity of dynamic FAV concentrations has never been evaluated in a human cell line. Here we employed the hollow-fiber infection model (HFIM) to simulate the human pharmacokinetic (PK) profiles associated with the clinically utilized FAV dosage regimens against influenza and Ebola viruses and assessed the viral burden profiles. Clinically achievable FAV concentrations inhibited ZIKV replication in HUH-7 cells in a dose-dependent fashion (50% effective concentration = 236.5 µM). The viral burden profiles under dynamic FAV concentrations were predicted by use of a mechanism-based mathematical model (MBM) and subsequently successfully validated in the HFIM. This validated, translational MBM can now be used to predict the anti-ZIKV activity of other FAV dosage regimens in the presence of between-patient variability in pharmacokinetics. This approach can be extended to rationally optimize FAV combination dosage regimens which hold promise to treat ZIKV infections in nonpregnant patients.

Antiviral Effects of Clinically-Relevant Interferon-α and Ribavirin Regimens against Dengue Virus in the Hollow Fiber Infection Model (HFIM).

Dengue virus (DENV) is the most prevalent mosquito-borne viral illness in humans. Currently, there are no therapeutic agents available to prevent or treat DENV infections. Our objective was to fill this unmet medical need by evaluating the antiviral activity of interferon-α (IFN) and ribavirin (RBV) as a combination therapy against DENV. DENV-infected Vero and Huh-7 cells were exposed to RBV and/or IFN, and the viral burden was quantified over time by plaque assay. Drug-drug interactions for antiviral effect were determined by fitting a mathematical model to the data. We then assessed clinically-relevant exposures of IFN plus RBV using the hollow fiber infection model (HFIM) system. RBV monotherapy was only effective against DENV at toxic concentrations in Vero and Huh-7 cells. IFN, as a single agent, did inhibit DENV replication at physiological concentrations and viral suppression was substantial in Huh-7 cells (Half maximal effective concentration (EC50) = 58.34 IU/mL). As a combination therapy, RBV plus IFN was additive for viral suppression in both cell lines; however, enhancement of antiviral activity at clinically-achievable concentrations was observed only in Huh-7 cells. Finally, clinical exposures of RBV plus IFN suppressed DENV replication by 99% even when treatment was initiated 24 h post-infection in the HFIM. Further evaluation revealed that the antiviral effectiveness of the combination regimen against DENV is mostly attributed to activity associated with IFN. These findings suggest that IFN is a potential therapeutic strategy for the treatment of DENV.

A sensitive electrochemical immunosensor for label-free detection of Zika-virus protein.

This work, as a proof of principle, presents a sensitive and selective electrochemical immunosensor for Zika-virus (ZIKV)-protein detection using a functionalized interdigitated micro-electrode of gold (IDE-Au) array. A miniaturized IDE-Au immunosensing chip was prepared via immobilization of ZIKV specific envelop protein antibody (Zev-Abs) onto dithiobis(succinimidyl propionate) i.e., (DTSP) functionalized IDE-Au (electrode gap/width of 10 µm). Electrochemical impedance spectroscopy (EIS) was performed to measure the electrical response of developed sensing chip as a function of ZIKV-protein concentrations. The results of EIS studies confirmed that sensing chip detected ZIKV-protein selectively and exhibited a detection range from 10 pM to 1 nM and a detection limit of 10 pM along with a high sensitivity of 12 kΩM-1. Such developed ZIKV immune-sensing chip can be integrated with a miniaturized potentiostat (MP)-interfaced with a smartphone for rapid ZIKV-infection detection required for early stage diagnostics at point-of-care application.

Utility of a Novel Three-Dimensional and Dynamic (3DD) Cell Culture System for PK/PD Studies: Evaluation of a Triple Combination Therapy at Overcoming Anti-HER2 Treatment Resistance in Breast Cancer.

Background: Emergence of Human epidermal growth factor receptor 2 (HER2) therapy resistance in HER2-positive (HER2+) breast cancer (BC) poses a major clinical challenge. Mechanisms of resistance include the over-activation of the PI3K/mTOR and Src pathways. This work aims to investigate a novel combination therapy that employs paclitaxel (PAC), a mitotic inhibitor, with everolimus (EVE), an mTOR inhibitor, and dasatinib (DAS), an Src kinase inhibitor, as a modality to overcome resistance. Methods: Static (two dimensional, 2D) and three-dimensional dynamic (3DD) cell culture studies were conducted using JIMT-1 cells, a HER2+ BC cell line refractory to HER2 therapies. Cell viability and caspase-3 expression were examined after JIMT-1 cell exposure to agents as monotherapy or in combination using a 2D setting. A pharmacokinetic/pharmacodynamic (PK/PD) combination study with PAC+DAS+EVE was conducted over 3 weeks in a 3DD setting. PAC was administered into the system via a 3 h infusion followed by the addition of a continuous infusion of EVE+DAS 24 h post-PAC dosing. Cell counts and caspase-3 expression were quantified every 2 days. A semi-mechanistic PK/PD model was developed using the 2D data and scaled up to capture the 3DD data. The final model integrated active caspase-3 as a biomarker to bridge between drug exposures and cancer cell dynamics. Model fittings were performed using Monolix software. Results: The triple combination significantly induced caspase-3 activity in the 2D cell culture setting. In the 3DD cell culture setting, sequential dosing of PAC then EVE+DAS showed a 5-fold increase in caspase-3 activity and 8.5-fold decrease in the total cell number compared to the control. The semi-mechanistic PK/PD models fit the data well, capturing the time-course profiles of drug concentrations, caspase-3 expression, and cell counts in the 2D and 3DD settings. Conclusion: A novel, sequential triple combination therapeutic regimen was successfully evaluated in both 2D and 3DD in vitro cell culture systems. The efficacy of this combination at inhibiting the cellular proliferation and re-growth of HER2/mTOR resistant cell line, JIMT-1, is demonstrated. A biomarker-linked PK/PD model successfully captured all time-course data. The latter can be used as a modeling platform for a direct translation from 3DD in vitro settings to the clinic.