Phase 1 clinical trials of DAS181, an inhaled sialidase, in healthy adults.

DAS181, (study drug, Fludase®) was developed for treatment of influenza and parainfluenza infections. Delivered by inhalation, DAS181 cleaves sialic acid receptors from respiratory epithelial cells. Treatment of influenza for three days with DAS181 reduced viral shedding. To increase deposition in the upper airways and decrease systemic absorption, the particle size was increased to 10µm. We conducted two Phase I trials with three cohorts, randomized 2:1, active drug to placebo. The initial cohort got a single 20mg dose of DAS181, or placebo; the second, 20mg DAS181 or placebo for 10days, and the third got 20mg of DAS181 or placebo for 3days. Formulations differed slightly in their excipients. Subjects in the 1- and 3-day cohorts completed dosing without serious adverse events. Two subjects in the 10-day cohort stopped at Day 9 after developing respiratory and systemic symptoms, and a third experienced a decrease in FEV1 (Forced Expiratory Volume in 1s) after the 9th dose and a further decline after the 10th dose. Plasma DAS181, in the 10-day cohort, peaked and began falling before the last dose. Antibodies, predominately IgG with neutralizing activity, were detected in 15/18 subjects by Day 30. The highest IgG concentrations were in the 10-day cohort. The respiratory adverse events occurring after seven days and rapid drug clearance during continued dosing are consistent with the induction of DAS181 antibodies. This could preclude use of this medication for longer than seven days or for repeated courses. (These studies have been registered at ClinicalTrials.gov under registration Nos. NCT 00527865 and NCT 01651494.).

Subventricular zone-mediated ependyma repair in the adult mammalian brain.

The subventricular zone (SVZ) of the adult mouse brain is a narrow stem cell niche that lies along the length of the lateral wall of the lateral ventricles. The SVZ supports neurogenesis throughout adulthood; however, with increasing age, the ventral SVZ deteriorates and only the dorsolateral SVZ remains neurogenic. Associated with the elderly dorsolateral SVZ, we reported previously an increased number of astrocytes interposed within the adjacent ependymal lining. Here, we show that astrocytes integrated within the ependyma are dividing, BrdU-labeled astrocytes that share cellular adherens with neighboring ependymal cells. By tracking BrdU-labeled astrocytes over time, we observed that, as they incorporated within the ependyma, they took on antigenic and morphologic characteristics of ependymal cells, suggesting a novel form of SVZ-supported "regenerative" repair in the aging brain. A similar form of SVZ-mediated ependyma repair was also observed in young mice after mild ependymal cell denudation with low dosages of neuraminidase. Together, this work identifies a novel non-neuronal mechanism of regenerative repair by the adult SVZ.

Concerns of using sialidase fusion protein as an experimental drug to combat seasonal and pandemic influenza.

Sialidase fusion protein is reported to have great potential to combat seasonal and pandemic influenza, because it may prevent influenza virus infection by removing all sialic acid receptors from host cells. Meanwhile, recent studies have demonstrated that absence of alpha2-6 sialic acid does not protect a cell from influenza infection, and influenza virus can infect desialylated cells, suggesting that accessible surface sialic acid is dispensable for influenza virus infection. In addition, studies using animal models have shown that neuraminidase promotes adherence and invasion of Streptococcus pneumoniae, because cleavage of sialic acid from host cells exposes cryptic receptors for S. pneumoniae. The purpose of this article is to comment on the benefits and potential risks of using sialidase fusion protein as an experimental drug to combat seasonal and pandemic influenza.

Influenza virus neuraminidase contributes to secondary bacterial pneumonia.

Secondary bacterial pneumonia is a common cause of death during influenza epidemics. We hypothesized that virus-specific factors could contribute to differences in annual excess mortality. Recombinant influenza viruses with neuraminidases from representative strains from the past 50 years were created and characterized. The specific level of their neuraminidase activity correlated with their ability to support secondary bacterial pneumonia. Recombinant viruses with neuraminidases from 1957 and 1997 influenza strains had the highest level of activity, whereas a virus with the neuraminidase from a 1968 strain had the lowest level of activity. The high level of activity of the neuraminidase from the 1957 strain, compared with that of other neuraminidases, more strongly supported the adherence of Streptococcus pneumoniae and the development of secondary bacterial pneumonia in a mouse model. These data lend support to our hypothesis that the influenza virus neuraminidase contributes to secondary bacterial pneumonia and subsequent excess mortality.

Zanamivir: an influenza virus neuraminidase inhibitor.

Zanamivir is the first of two registered neuraminidase inhibitors for the treatment and prophylaxis of influenza. Relenza, an orally inhaled powder form of zanamivir, is currently approved in 19 countries for treatment, and in two for prophylaxis. Relenza reduces the time to alleviation of symptoms by 1 to 2 days in the influenza-positive population, if taken within 48 h of symptom onset, and in prophylaxis in family settings, it confers an 80% reduction in the odds of contracting influenza. The resistance profile of zanamivir is encouraging in the sense that there are still no reports of patients on acute therapy shedding drug-resistant virus. However, patient uptake of the inhaled drug has been insufficient to conclude that drug resistance will not be an issue in the future. All zanamivir-resistant variants selected in the laboratory so far have diminished viability.

Ependymal denudation, aqueductal obliteration and hydrocephalus after a single injection of neuraminidase into the lateral ventricle of adult rats.

To investigate the role of sialic acid in the ependyma of the rat brain, we injected neuraminidase from Clostridium perfingens into the lateral ventricle of 86 adult rats that were sacrificed at various time intervals. After administration of 10 micrograms neuraminidase, ciliated cuboidal ependymal cells of the lateral ventricles, third ventricle, cerebral aqueduct, and the rostral half of the fourth ventricle died and detached. The ependymal regions sealed by tight junctions such as the choroid plexus and the subcommissural organ were not affected. Debris was removed by infiltrating neutrophils and macrophagic cells. At the same time, after ependymal disappearance, the aqueduct was obliterated. In this region, mitoses were evident and cystic ependymal cells were frequent. Hydrocephalus of the lateral and third ventricles was evident 4 days after neuraminidase injection. Gliosis was restricted to the dorsal telencephalic wall of the injected lateral ventricle. It is thought that cleavage of sialic acid from ependymal surface glycoproteins or glycolipids, likely involved in cell adhesion, led to the detaching and death of the ependymal cells. Thereafter, ependymal loss, together with edema, led to fusion of the lateral walls of the cerebral aqueduct and this in turn provoked hydrocephalus of the third and lateral ventricles. This model of experimental hydrocephalus is compared with other models, in particular those of hydrocephalus after viral invasion of the cerebral ventricles.

Adjuvant immunotherapy of malignant melanoma.

A vaccine made of irradiated Vibrio cholerae neuroaminidase (VCN) treated autochthonous tumor cells plus BCG was utilized in combination with surgery or with chemotherapy for Stage II and Stage III malignant melanoma, respectively. A few patients with Stage I melanoma were treated with surgery and BCG. Most of the studies were carried out on a prospective, randomized protocol. When the results with conventional therapy were compared with the results of conventional therapy plus immunotherapy, no beneficial effects of the immunotherapy were seen. Stratification insured comparability in both immunotherapy and nonimmunotherapy groups. We conclude that VCN treated tumor cells plus BCG, when administered according to the protocol utilized here, offer patients with malignant melanoma no substantial benefit when compared with conventional therapy.

[Neuraminidase induced hemolytic anemia. Experimental and clinical observations (author's transl)]. / Neuraminidase-induzierte Hämolyse. Experimentelle und klinische Untersuchungen

Recently, increasing attention has been focussed on the in vivo action of neuraminidase as possible pathogenetical factor of hemolytic anemia and even hemolytic-uremic syndrome. Neuraminidase action in red cell membranes results in the release of neuraminic acid, and thereby the uncovering of previously hidden receptors, socalled cryptantigens. With special reference to the phythemagglutinin Anti-TAh from the peanut (Arachis hypogae) and the agglutinin Anti-AHP from the albumin gland of the small Helix pomatia we describe some new methods for the detection of these cryptantigens. In addition to the screebubg genagglutination test with Anti-TAh we developed an "Anti-T-consumption test" for quantitative detection of neuraminidase action on red cells. With the purified reagents we developed an indirect fluorescnet antibody method on blood smears for the detection of cryptantigens on single cells. By animal experiments we could show that not only the membranes of red cells but the intima of renal capillaries as well are damaged by neuraminidase. With these new methods we observed 14 patients suffering from hemolytic anemia due to bacterial or viral neuraminidase. Some of these patients developed a hemolytic-uremic syndrome. We believe that the positive reaction with Anti-T Ah should lead to prophylactic heparinization to prevent dissiminated intravascular coagulation. Neuraminidase is the first identified toxin which directly acts on the membranes of red cells and the intima of renal capillaries as well, and thereby in some patients may induce hemolytic-uremic syndrome. Possibly, these results may stimulate the development of further testsystems for the detection of still unknown toxins which are not tested with our reagents, but may equally be involved in the damage of cell membranes.