SARS-CoV-2 inhibition using a mucoadhesive, amphiphilic chitosan that may serve as an anti-viral nasal spray

Abstract

There are currently no cures for coronavirus infections, making the prevention of infections the only course open at the present time. The COVID-19 pandemic has been difficult to prevent, as the infection is spread by respiratory droplets and thus effective, scalable and safe preventive interventions are urgently needed. We hypothesise that preventing viral entry into mammalian nasal epithelial cells may be one way to limit the spread of COVID-19. Here we show that N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ), a positively charged polymer that has been through an extensive Good Laboratory Practice toxicology screen, is able to reduce the infectivity of SARS-COV-2 in A549^ACE2+ and Vero E6 cells with a log removal value of - 3 to - 4 at a concentration of 10-100 μg/ mL (p < 0.05 compared to untreated controls) and to limit infectivity in human airway epithelial cells at a concentration of 500 μg/ mL (p < 0.05 compared to untreated controls). In vivo studies using transgenic mice expressing the ACE-2 receptor, dosed nasally with SARS-COV-2 (426,000 TCID₅₀/mL) showed a trend for nasal GCPQ (20 mg/kg) to inhibit viral load in the respiratory tract and brain, although the study was not powered to detect statistical significance. GCPQ's electrostatic binding to the virus, preventing viral entry into the host cells, is the most likely mechanism of viral inhibition. Radiolabelled GCPQ studies in mice show that at a dose of 10 mg/kg, GCPQ has a long residence time in mouse nares, with 13.1% of the injected dose identified from SPECT/CT in the nares, 24 h after nasal dosing. With a no observed adverse effect level of 18 mg/kg in rats, following a 28-day repeat dose study, clinical testing of this polymer, as a COVID-19 prophylactic is warranted.

Figure 1

GCPQ and HTCC.

Figure 2

Cytotoxicity of GCPQs in vitro. Cell viability was assessed using an XTT assay on Vero E6 cells (A) and A549^ACE2+ cells (B). Relative viability of cells (percentage of the untreated control) is shown on y-axis. All assays were performed in triplicate, and average values with standard errors are presented. The letters a to d refer to the GCPQs shown in Table 2. All assays performed in triplicate and analysed by one-way ANOVA with Tukey's post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.The IC50 values for GCPQa, GCPQb, GCPQc and GCPQd in the Vero E6 cell line are 100 μg/mL, > 200 μg/mL, > 200 μg/mL and > 200 μg/mL respectively. The IC50 values for GCPQa, GCPQb, GCPQc and GCPQd in the A549^ACE2+ cell line are > 25 μg/mL, > 200 μg/mL, > 100 μg/mL and > 200 μg/mL respectively.

Figure 3

Anti-viral activity of GCPQs against SARS-CoV-2. Virus replication was evaluated using RT-qPCR. The data are presented as a number of RNA copies per mL of the original sample (left) or as Log Removal Value (LRV) compared to untreated samples (right). Non-toxic concentrations were tested (10 μg/ml of GCPQa, 25 μg/ml for GCPQc, and 200 μg/ml for GCPQb and GCPQd). The assay was performed in triplicate, and average values with standard errors are presented. For the RNA copies per mL data GCPQa and GCPQc are significantly different from PBS treated cells (p < 0.0001) and for the log removal value, GCPQa is significantly different from PBS treated samples in the A549 and Vero E6 cell lines (p < 0.05).

Figure 4

Replication of SARS-CoV-2 in fully differentiated tissue cultures of the human respiratory epithelium (HAE) in the presence or absence of GPCQ. Virus replication was evaluated using RT-qPCR. The data are presented as a number of viral copies per ml. The assay was performed in triplicate, and median values with range are presented. At the 48 and 72 h time points, GCPQa at 500 μg/ mL is significantly different from PBS treated samples (p < 0.05).

Figure 5

Sagittal SPECT/CT images of radiolabelled GCPQ (10 mg/kg) at 30 min, 2 h 30 min and 24 h after nasal administration to male mice (a–c), the nasal delivery device (Naltos device) that may be used to deliver the prophylactic GCPQ powder, permission from Alchemy Pharmatech Ltd. (d).

Figure 6

Viral titres in transgenic mice expressing the human ACE2 protein under the human cytokeratin 18 promoter (n = 10–14) following the nasal dosing of GCPQ once daily (20 mg/kg) and intramuscular remdesivir at a single daily dose of 25 mg/kg.