Oral Favipiravir Exposure and Pharmacodynamic Effects in Outpatient Adults with Acute Influenza.

BACKGROUND: The pharmacokinetics of oral favipiravir and the relationships of plasma concentrations to antiviral effects are incompletely studied in influenza. METHODS: Serial plasma samples were collected from adults with uncomplicated influenza who were randomized to favipiravir (1800 mg BID on day 1, 800 mg BID on days 2 to 5)(N = 827) or placebo (N = 419) in two phase 3 trials. Post hoc analyses assessed the frequency of reaching an average Cmin ≥20ug/ml, its association with antiviral efficacy, and factors associated with reduced favipiravir exposure. RESULTS: Wide inter-individual variability existed in favipiravir concentrations, and this regimen failed to reach an average Cmin >20ug/ml in 41-43% of participants. Those attaining this threshold showed greater reductions in nasopharyngeal infectious virus titers on treatment days 2 and 3 (approximately 0.3-0.4 log10TCID50/ml) and lower viral titer AUCs compared to those who did not. Those with average Cmin <20ug/ml had over 2-fold higher mean ratios of the metabolite T-705-M1 to favipiravir, consistent with greater metabolism, and were more likely to weigh >80 kg (61.5-64%). CONCLUSIONS: Higher favipiravir levels with average Cmin >20ug/ml were associated with larger antiviral effects and more rapid illness alleviation compared to placebo and to favipiravir recipients with lower average Cmin values in uncomplicated influenza.

Favipiravir Treatment of Uncomplicated Influenza in Adults: Results of Two Phase 3, Randomized, Double-Blind, Placebo-Controlled Trials.

BACKGROUND: We conducted double-blind, placebo-controlled trials assessing the efficacy and tolerability of favipiravir in acute influenza. METHODS: Otherwise healthy adults with influenza-like symptoms and fever of ≤48 hours were randomized to favipiravir (1800 mg twice daily [BID] on day 1, 800 mg BID on days 2-5) or placebo tablets (1:1 in US316; 3:1 in US317). The primary efficacy endpoint was the time to illness alleviation when 6 influenza symptoms were self-rated as absent or mild and fever was absent in the intention-to-treat, influenza-infected participants. RESULTS: In US316 (301 favipiravir, 322 placebo), favipiravir was associated with a 14.4-hour reduction (median, 84.2 vs 98.6 hours; P = .004) in time to illness alleviation vs placebo. In US317 (526 favipiravir, 169 placebo), favipiravir did not significantly reduce time to alleviation (median, 77.8 vs 83.9 hours). In both trials favipiravir was associated with reduced viral titers, RNA load area under the curve over days 1-5, and median times to cessation of virus detection (P < .001). Aside from asymptomatic hyperuricemia, no important differences in adverse events were found. CONCLUSIONS: This favipiravir dosing regimen demonstrated significant antiviral efficacy but inconsistent illness alleviation in uncomplicated influenza. Studies of higher doses and antiviral combinations for treating serious influenza and other RNA viral infections are warranted. Clinical Trials Registration. NCT02026349; NCT02008344.

Liposomal formulation of amphiphilic fullerene antioxidants.

Novel amphiphilic fullerene[70] derivatives that are rationally designed to intercalate in lipid bilayers are reported, as well as its vesicular formulation with surprisingly high loading capacity up to 65% by weight. The amphiphilic C(70) bisadduct forms uniform and dimensionally stable liposomes with auxiliary natural phospholipids as demonstrated by buoyant density test, particle size distribution, and (31)P NMR. The antioxidant property of fullerenes is retained in the bipolarly functionalized C(70) derivative, amphiphilic liposomal malonylfullerene[70] (ALM), as well as in its liposomal formulations, as shown by both electron paramagnetic resonance (EPR) studies and in vitro reactive oxygen species (ROS) inhibition experiments. The liposomally formulated ALM efficiently quenched hydroxyl radicals and superoxide radicals. In addition, the fullerene liposome inhibited radical-induced lipid peroxidation and maintained the integrity of the lipid bilayer structure. This new class of liposomally formulated, amphipathic fullerene compounds represents a novel drug delivery system for fullerenes and provides a promising pathway to treat oxidative stress-related diseases.

Uptake and distribution of fullerenes in human mast cells.

Fullerenes are carbon cages of variable size that can be derivatized with various side chain moieties resulting in compounds that are being developed into nanomedicines. Although fullerene use in several preclinical in vitro and in vivo models of disease has demonstrated their potential as diagnostic and therapeutic agents, little is known about how they enter cells, what organelles they target, and the time course for their cellular deposition. Fullerenes (C(70)) that have already been shown to be potent inhibitors of mast cell (MC)-mediated allergic inflammation were conjugated with Texas red (TR) and used in conjunction with confocal microscopy to determine mechanisms of uptake, the organelle localization, and the duration they can be detected in situ. We show that C(70)-TR are nonspecifically endocytosed into MCs, where they are shuttled throughout the cytoplasm, lysosomes, mitochondria, and into endoplasmic reticulum at different times. No nuclear or secretory granule localization was observed. The C(70)-TR remained detectable within cells at 1 week. These studies show that MCs endocytose fullerenes, where they are shuttled to organelles involved with calcium and reactive oxygen species production, which may explain their efficacy as cellular inhibitors. From the clinical editor: Fullerenes are carbon cages of variable size that have already been shown to be potent inhibitors of mast cell (MC)-mediated allergic inflammation. These were conjugated with Texas red (TR) and used in conjunction with confocal microscopy to determine mechanisms of uptake, the organelle localization, and duration, demonstrating that MCs endocytose fullerenes, which are shuttled to organelles involved with calcium and reactive oxygen species production. This intracellular trafficking may explain the efficacy of fullerenes as cellular inhibitors.

Fullerene nanomaterials inhibit phorbol myristate acetate-induced inflammation.

Inflammation is a natural biological response that occurs when vascular tissues are subjected to harmful stimuli. This process may be beneficial to the host during wound healing and infections but can be detrimental if left unchecked. Oxidative stress, the generation of reactive oxygen species, is thought to be one component of this response. Fullerenes can counteract reactive oxygen species due to their potent antioxidant capabilities. Thus, we hypothesized that these molecules may inhibit inflammation. To test this hypothesis we used an in vivo model of phorbol 12-myristate 13-acetate (PMA)-induced inflammation and examined the effects fullerenes have on mitigating this response. We show that PMA-induced inflammation and oedema is dramatically inhibited when fullerenes are given prior to challenge. Thus, fullerene derivatives may be a novel way to blunt certain inflammatory conditions and facilitate faster recovery of damaged tissue.

Fullerene nanomaterials potentiate hair growth.

Hair loss is a common symptom resulting from a wide range of disease processes and can lead to stress in affected individuals. The purpose of this study was to examine the effect of fullerene nanomaterials on hair growth. We used shaved mice as well as SKH-1 "bald" mice to determine if fullerene-based compounds could affect hair growth and hair follicle numbers. In shaved mice, fullerenes increase the rate of hair growth as compared with mice receiving vehicle only. In SKH-1 hairless mice fullerene derivatives given topically or subdermally markedly increased hair growth. This was paralleled by a significant increase in the number of hair follicles in fullerene-treated mice as compared with those mice treated with vehicle only. The fullerenes also increased hair growth in human skin sections maintained in culture. These studies have wide-ranging implications for those conditions leading to hair loss, including alopecia, chemotherapy, and reactions to various chemicals.

Intracellular conversion and in vivo dose response of favipiravir (T-705) in rodents infected with Ebola virus.

During the 2013-2016 Ebola virus (EBOV) outbreak in West Africa, our team at USAMRIID evaluated the antiviral activity of a number of compounds, including favipiravir (T-705), in vitro and in mouse and nonhuman primate (NHP) models of Ebola virus disease. In this short communication, we present our findings for favipiravir in cell culture and in mice, while an accompanying paper presents the results of NHP studies. We confirmed previous reports that favipiravir has anti-EBOV activity in mice. Additionally, we found that the active form of favipiravir is generated in mice in tissues relevant for the pathogenesis of EBOV infection. Finally, we observed that protection can be achieved in mice down to 8 mg/kg/day, which is lower than the dosing regimens previously reported. An accompanying paper reports the results of treating nonhuman primates infected with EBOV or with Marburg virus with oral or intravenous favipiravir.

Efficacy of favipiravir (T-705) in nonhuman primates infected with Ebola virus or Marburg virus.

Favipiravir is a broad-spectrum antiviral agent that has demonstrated efficacy against Ebola virus (EBOV) in rodents. However, there are no published reports of favipiravir efficacy for filovirus infection of nonhuman primates (NHPs). Here we evaluated the pharmacokinetic profile of favipiravir in NHPs, as well as in vivo efficacy against two filoviruses, EBOV and Marburg virus (MARV). While no survival benefit was observed in two studies employing once- or twice-daily oral dosing of favipiravir during EBOV infection of NHPs, an antiviral effect was observed in terms of extended time-to-death and reduced levels of viral RNA. However, oral dosing in biosafety level-4 (BSL-4) presents logistical and technical challenges, and repeated anesthesia events may potentially worsen survival outcome in animals. For the third study of treatment of MARV infection, we therefore made use of catheters, jackets, and tethers for intravenous (IV) dosing and blood collection, which minimized the requirement for repeated anesthesia events. When MARV infection was treated with IV favipiravir, five of six animals (83%) survived infection, while all untreated NHPs succumbed. An accompanying report presents the results of favipiravir treatment of EBOV infection in mice.

Post-exposure efficacy of oral T-705 (Favipiravir) against inhalational Ebola virus infection in a mouse model.

Filoviruses cause disease with high case fatality rates and are considered biological threat agents. Licensed post-exposure therapies that can be administered by the oral route are desired for safe and rapid distribution and uptake in the event of exposure or outbreaks. Favipiravir or T-705 has broad antiviral activity and has already undergone phase II and is undergoing phase III clinical trials for influenza. Here we report the first use of T-705 against Ebola virus. T-705 gave 100% protection against aerosol Ebola virus E718 infection; protection was shown in immune-deficient mice after 14 days of twice-daily dosing. T-705 was also shown to inhibit Ebola virus infection in cell culture. T-705 is likely to be licensed for use against influenza in the near future and could also be used with a new indication for filovirus infection.

A new class of human mast cell and peripheral blood basophil stabilizers that differentially control allergic mediator release.

Treatments for allergic disease block the effects of mediators released from activated mast cells and blood basophils. A panel of fullerene derivatives was synthesized and tested for their ability to preempt the release of allergic mediators in vitro and in vivo. The fullerene C(70)-tetraglycolic acid significantly inhibited degranulation and cytokine production from mast cells and basophils, while C(70)-tetrainositol blocked only cytokine production in mast cells and degranulation and cytokine production in basophils. The early phase of FcepsilonRI inhibition was dependent on the blunted release of intracellular calcium stores, elevations in reactive oxygen species, and several signaling molecules. Gene microarray studies further showed the two fullerene derivatives inhibited late phase responses in very different ways. C(70)-tetraglycolic acid was able to block mast cell-driven anaphylaxis in vivo, while C(70)-tetrainositol did not. No toxicity was observed with either compound. These findings demonstrate the biological effects of fullerenes critically depends on the moieties added to the carbon cage and suggest they act on different FcepsilonRI-specific molecules in mast cells and basophils. These next generation fullerene derivatives represent a new class of compounds that interfere with FcepsilonRI signaling pathways to stabilize mast cells and basophils. Thus, fullerene-based therapies may be a new approach for treating allergic diseases.

A novel class of compounds with cutaneous wound healing properties.

Impaired wound healing is a major complication underlying several disease processes (such as diabetes). Efficient wound healing is hampered by a wide variety of processes including hypoxia (oxygen deprivation), inflammation, infection, and oxidative stress through the generation of harmful reactive oxygen species (ROS). The inherent complexity of the healing wound has resulted in limited efficacy of most therapies that target single parameters involved in the slow healing processes. Fullerenes are carbon nanospheres previously shown to exhibit a wide range of biological activities. Given that these molecules have been shown to be potent anti-inflammatories and antioxidants we hypothesized that fullerenes could aid in wound healing based on these properties. We designed and synthesized a panel of fullerene derivatives and investigated their ability to accelerate wound healing using a modified scratch assay, an ex vivo human skin model, and a mouse model of skin irritation. Several derivatives supported cell migration, induced wound closure in human skin explants, and greatly accelerated the rate at which wound healing occurred in vivo. Therefore, fullerene derivatives represent a potential new class of wound healing therapies that may aid in wound healing treatment.

Hydrochalarones: a novel endohedral metallofullerene platform for enhancing magnetic resonance imaging contrast.

Targeted imaging requires contrast agents that remain in the vasculature for extended periods of time. A new contrast agent is described in which gadolinium is encapsulated within an extremely stable carbon sphere, thus allowing for safe extended residence. Water solubility and small particle size is achieved with novel fullerene chemistry, attaching multiple oligoethylene glycol groups through nitrogen chemistry. These new compounds can be used to visualize tissue architecture in vivo with standard MRI techniques.

Fullerene nanomaterials inhibit the allergic response.

Fullerenes are a class of novel carbon allotropes that may have practical applications in biotechnology and medicine. Human mast cells (MC) and peripheral blood basophils are critical cells involved in the initiation and propagation of several inflammatory conditions, mainly type I hypersensitivity. We report an unanticipated role of fullerenes as a negative regulator of allergic mediator release that suppresses Ag-driven type I hypersensitivity. Human MC and peripheral blood basophils exhibited a significant inhibition of IgE dependent mediator release when preincubated with C(60) fullerenes. Protein microarray demonstrated that inhibition of mediator release involves profound reductions in the activation of signaling molecules involved in mediator release and oxidative stress. Follow-up studies demonstrated that the tyrosine phosphorylation of Syk was dramatically inhibited in Ag-challenged cells first incubated with fullerenes. In addition, fullerene preincubation significantly inhibited IgE-induced elevation in cytoplasmic reactive oxygen species levels. Furthermore, fullerenes prevented the in vivo release of histamine and drop in core body temperature in vivo using a MC-dependent model of anaphylaxis. These findings identify a new biological function for fullerenes and may represent a novel way to control MC-dependent diseases including asthma, inflammatory arthritis, heart disease, and multiple sclerosis.