Cetylpyridinium Chloride (CPC) Exhibits Potent, Rapid Activity Against Influenza Viruses in vitro and in vivo

Abstract

Background: There is a continued need for strategies to prevent influenza. While cetylpyridinium chloride (CPC), a broad-spectrum antimicrobial agent, has an extensive antimicrobial spectrum, its ability to affect respiratory viruses has not been studied in detail.

Objectives: Here, we evaluate the ability of CPC to disrupt influenza viruses in vitro and in vivo.

Methods: The virucidal activity of CPC was evaluated against susceptible and oseltamivir-resistant strains of influenza viruses. The effective virucidal concentration (EC) of CPC was determined using a hemagglutination assay and tissue culture infective dose assay. The effect of CPC on viral envelope morphology and ultrastructure was evaluated using transmission electron microscopy (TEM). The ability of influenza virus to develop resistance was evaluated after multiple passaging in sub-inhibitory concentrations of CPC. Finally, the efficacy of CPC in formulation to prevent and treat influenza infection was evaluated using the PR8 murine influenza model.

Results: The virucidal effect of CPC occurred within 10 minutes, with mean EC50 and EC2log ranging between 5 to 20 μg/mL, for most strains of influenza tested regardless of type and resistance to oseltamivir. Examinations using TEM showed that CPC disrupted the integrity of the viral envelope and its morphology. Influenza viruses demonstrated no resistance to CPC despite prolonged exposure. Treated mice exhibited significantly increased survival and maintained body weight compared to untreated mice.

Conclusions: The antimicrobial agent CPC possesses virucidal activity against susceptible and resistant strains of influenza virus by targeting and disrupting the viral envelope. Substantial virucidal activity is seen even at very low concentrations of CPC without development of resistance. Moreover, CPC in formulation reduces influenza-associated mortality and morbidity in vivo.

Keywords: CPC; Cetylpyridinium Chloride; Influenza; Respiratory tract illness; quaternary ammonium compound; respiratory virus.

Conflict of interest statement

TRANSPARENCY DECLARATIONS MAG acts as Chief Scientific Officer and Consultant for ARMS Pharmaceutical LLC. PKM acts as a Consultant for ARMS Pharmaceutical LLC. There is no conflict of interest for the remaining authors regarding the publication of this paper.

Figures

Figure 1.

MDCK cell viability following treatment with increasing concentrations of CPC for 10 minutes. The cell viability was determined using the neutral red assay and the absorbance was measured at 450 nm, n = 4 experimental replicates; data represent mean ± SD.

Figure 2.

Semi-logarithmic time-kill curves of influenza exposed to EC50 concentrations of CPC at increasing exposure times are shown. The resulting percentage of hemagglutination compared to control is shown over time with mean and standard deviation reported at the indicated time post CPC at EC50. The trend line displays the rate of inactivation for each virus. A) Fifty percent of infectious virus was inactivated in the first 5 minutes of exposure for all isolates except for FluB. Isolate 40 is a clinical strain of influenza A (H1N1pdm09) virus propagated from a patient sample. B) Time-kill curve between oseltamivir-resistant and susceptible strains. No difference in CPC susceptibility was seen between strains, n = 3 experimental replicates.

Figure 3.

Transmission electron microscopy (TEM) demonstrating CPC disrupts the integrity of the viral envelope and morphology of influenza virus. (A) Untreated influenza virus, (B, C) influenza virus treated with 50 μg/mL CPC for 5 minutes. Viral particles exposed to CPC demonstrate disrupted envelope or cavitation (arrows) of viral units. The presence of negative stain inside the virions indicates membrane permeabilization. The scale bar is in the lower left corner at 100 nm (A) or 50 nm (B, C). We quantified the number of intact and disrupted viruses after treatment, and found that in CPC-treated samples, 86% (172/200) of the viruses were disrupted while in untreated samples only 4.5% (9/200) were disrupted.

Figure 4.

Influenza nucleoprotein release from the virus in response to increasing concentrations of CPC was measured by ELISA. Exposure to CPC concentrations above 7.5 μg/mL produced significant elevations in nucleoprotein release compared to control, n = 8 experimental replicates, shown as mean ± SD. Mean influenza nucleoprotein levels in the presence of CPC were compared to control (0 μg/mL) using independent samples t test (IBM SPSS ver 24, IBM Corp).

Figure 5.

Exposure of influenza to 3 sub-inhibitory concentrations of CPC for 10 passages shows an absence of resistance to CPC. Influenza A (H1N1, H3N2) and Influenza B strains were continuously grown in 3 sub-inhibitory concentrations of CPC: 0.2, 0.02, and 0.002 μg/mL. Viral titer was determined at the end of each passage by hemagglutination assay to ensure an adequate inoculation for each subsequent passage. Ten passages, with 3–4 days exposure per passage, of each influenza strain were performed for all concentrations of CPC. The EC50 was then determined for each virus at the 10th passage and compared to the original strain (baseline control). No development of resistance to CPC (defined as over 2-fold change in EC50) was seen in any strain of influenza at any tested concentration of CPC, n = 3 experimental replicates, shown as mean ± SD.

Figure 6.

CPC formulation (ARMS-I, 0.1% CPC w/v) reduced influenza-associated pathogenicity in vivo. ARMS-I prophylactic protection from morbidity and mortality was demonstrated by (A) body weight (percentage of initial weight on Day 0) and (B) Kaplan-Meier survival curve, respectively. The ARMS-I and oseltamivir groups were treated 15 minutes before the challenge and then twice a day for 5 consecutive days.

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