In search of preventive strategies: novel high-CBD Cannabis sativa extracts modulate ACE2 expression in COVID-19 gateway tissues.

With the current COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an urgent need for new therapies and prevention strategies that can help curtail disease spread and reduce mortality. The inhibition of viral entry and thus spread is a plausible therapeutic avenue. SARS-CoV-2 uses receptor-mediated entry into a human host via the angiotensin-converting enzyme 2 (ACE2), which is expressed in lung tissue as well as the oral and nasal mucosa, kidney, testes and gastrointestinal tract. The modulation of ACE2 levels in these gateway tissues may be an effective strategy for decreasing disease susceptibility. Cannabis sativa, especially those high in the anti-inflammatory cannabinoid cannabidiol (CBD), has been found to alter gene expression and inflammation and harbour anti-cancer and anti-inflammatory properties. However, its effects on ACE2 expression remain unknown. Working under a Health Canada research license, we developed over 800 new C. sativa cultivars and hypothesized that high-CBD C. sativa extracts may be used to down-regulate ACE2 expression in target COVID-19 tissues. Using artificial 3D human models of oral, airway and intestinal tissues, we identified 13 high-CBD C. sativa extracts that decrease ACE2 protein levels. Some C. sativa extracts down-regulate serine protease TMPRSS2, another critical protein required for SARS-CoV-2 entry into host cells. While our most effective extracts require further large-scale validation, our study is important for future analyses of the effects of medical cannabis on COVID-19. The extracts of our most successful novel high-CBD C. sativa lines, pending further investigation, may become a useful and safe addition to the prevention/treatment of COVID-19 as an adjunct therapy.

Formulation and Evaluation of a Mucoadhesive Thermoresponsive System Containing Brazilian Green Propolis for the Treatment of Lesions Caused by Herpes Simplex Type I.

The aim of the present work was to develop a topical delivery system that contains Brazilian green propolis extract (PE-8) to increase efficiency and convenience when applied to herpetic lesions. The cytotoxicity and antiherpetic activity was determined in vitro and in vivo. The PE-8 was added to a system that contained poloxamer 407 and carbopol 934P. The in vitro characterization of the system included rheological studies, texture profile analysis, and mucoadhesion analysis. The PE-8 inhibited the virus during the phase of viral infection, induced virion damage, and exhibited an ability to protect cells from viral infection. The system had advantageous mucoadhesive properties, including a suitable gelation temperature of approximately 25°C for topical delivery, a desirable textural profile, and pseudoplastic behavior. The in vitro release study showed a rapid initial release of the PE-8 in the first 3 h, and the rate of drug release remained constant for up to 24 h. The system appeared to be macroscopically and microscopically innocuous to skin tissue. Therefore, the mucoadhesive thermoresponsive system that contained the PE-8 appears to be promising for increasing bioavailability and achieving prolonged release of the PE-8 when applied to skin lesions caused by herpes simplex virus type 1.

Smokeless tobacco increases aneuploidy in oral HPV16 E6/E7-transformed keratinocytes in vitro.

BACKGROUND: The scope of this work was to study synergism between human papillomavirus (HPV) infection and tobacco in vitro, both known to be independent risk factors for oral cancer. METHODS: HPV-positive and HPV-negative oral keratinocytes and oral HPV-negative fibroblasts were exposed to smokeless tobacco extract (STE) prepared from the Scandinavian (STE1) and US-type (STE2) snuff. Cell cycle profiles were determined with flow cytometry, and HPV E6/E7 mRNA expression in HPV-positive cells was assayed using RT-qPCR. RESULTS: The exposure of HPV-positive keratinocytes with STE2 increased the number of aneuploid cells from 27% to 80% of which 44% were in S-phase, while none of the diploid cells were in S-phase. The changes after STE1 exposure were less than seen after STE2: from 27% to 31% of which 34% were in S-phase. STE had no effect on HPV16 E6/E7 expression in HPV-positive keratinocytes. In oral spontaneously transformed, HPV-negative keratinocytes, the number of aneuploid cells at G2-M stage increased after STE1 and STE2 exposure from 3% to 9% and 7%, respectively. In HPV-negative oral fibroblasts, the number of cells at G2-M phase increased from 11% to 21% after STE1 and 29% after STE2 exposure. CONCLUSIONS: The effect of STE varied in the cell lines studied. STE2 increased significantly the proportion of aneuploid cells in HPV-positive oral keratinocytes, but not HPV16 E6/E7 expression. This indicates that tobacco products may enhance the effects of HPV 16 and the risk of DNA aneuploidy increasing risk to malignant transformation.

Antiviral effects of blackberry extract against herpes simplex virus type 1.

OBJECTIVE: The aim of this study was to evaluate antiviral properties of blackberry extract against herpes simplex virus type 1 (HSV-1) in vitro. STUDY DESIGN: HSV-infected oral epithelial (OKF6) cells and cell-free virus suspensions were treated with blackberry extract (2.24-1,400 µg/mL), and virus yield and infectivity were quantified by direct plaque assay. RESULTS: Blackberry extract ≥56 µg/mL inhibited HSV-1 replication in oral epithelial cells by >99% (P < .005). Concentrations ≥280 µg/mL were antiviral when the extract was added after virus adsorption and entry. Exposure of cell-free virus to ≥280 µg/mL blackberry extract for 15 minutes at room temperature was virucidal (P = .0002). The virucidal effects were not due to pH changes at concentrations up to 1,500 µg/mL. CONCLUSIONS: Blackberry extract inhibited the early stages of HSV-1 replication and had potent virucidal activity. These properties suggest that this natural fruit extract could provide advantage as a topical prophylactic/therapeutic agent for HSV infections.

Influence of regular black tea consumption on tobacco associated DNA damage and HPV prevalence in human oral mucosa.

Black tea is more widely consumed than green tea worldwide, particularly in India. Therefore, it is necessary to focus attention on black tea with respect to its health promoting and anti-cancer actions. In order to establish the concept that black tea is a potential candidate for cancer prevention, it is important to provide epidemiological evidence derived from investigations of human populations. In view of this, the objective of the present study was to determine the correlation between nature of black tea consumption and DNA damage in normal subjects with or without tobacco habit and oral cancer patients, taking the latter as positive controls. Much experimental evidence points to associations between tobacco habit and HPV 16 and HPV 18 (Human Papilloma virus) infection. But no studies have taken into account the possible confounding effect of black tea consumption on DNA damage along with HPV infection. A pilot study was therefore undertaken. Comet assay was used to evaluate the DNA damage among normal subjects including tobacco users (n = 86), non-tobacco users (n = 45) and Oral cancer patients (n = 37). Percentage of damaged cells was scored in the buccal squamous cells of all subjects mentioned above. HPV analysis was performed on 79 samples (including 37 oral cancer patients). The evaluation of various confounding factors like age, tenure of tobacco habit and tea habit showed significant associations with DNA damage. The observations strongly indicate that regular intake of black tea at least above four cups can reduce tobacco associated DNA damage among normal tobacco users. HPV prevalence was not seen to be associated with age, tenure of tobacco habit or the tea drinking habit.

A randomized, double-blind, placebo-controlled trial of cidofovir gel for the treatment of acyclovir-unresponsive mucocutaneous herpes simplex virus infection in patients with AIDS.

The safety and efficacy of cidofovir gel for treatment of acyclovir-unresponsive herpes simplex virus infections in AIDS patients was evaluated in a randomized, double-blind, multicenter trial. Cidofovir (0.3% or 1%) or placebo gel was applied once daily for 5 days. Ten of 20 cidofovir-treated and none of 10 placebo-treated patients had complete healing or >50% decreased area (P = .008); 30% of cidofovir-treated patients versus 0 placebo recipients had complete healing (P = .031). Viral shedding ceased in 13 (87%) of 15 cidofovir-treated and 0 of 9 placebo-treated patients (P = .00004). For cidofovir-treated patients, median time to complete or good response was 21 days, and median time to negative viral culture was 2 days (P = .025, P = .0001, respectively). Median lesion area decreases were 58% for cidofovir-treated versus 0 for placebo-treated patients (P = .005), and mean pain score changes were -1.84 versus -0.34 (P = .042). Application site reactions occurred in 25% of cidofovir-treated and 20% of placebo-treated patients; none was dose-limiting. Cidofovir therapy provided significant benefits in lesion healing, virologic effect, and pain reduction.

Attenuation of the natural course of herpes simplex virus infection in human oral epithelial cell cultures by smokeless tobacco extracts suggests the possibility of a synergistic mechanism for carcinogenesis.

High prevalence of both tobacco use and latent herpes simplex virus type 1 suggests the opportunity for synergism between these agents as cocarcinogens. In this study, postprimary human oral epithelial cell cultures were infected with herpes simplex virus type 1 pretreated with 2% extracts of either loose leaf, moist, or dry snuffs. Cultures were subsequently periodically exposed to the tobacco. Parameters measured included percentage of cultures undergoing active virus production, onset and time course of cytopathic effects, and concentration of virus released into the media over time. Results showed inhibition of both herpes simplex virus-mediated cell lysis and viral replication by tobacco extracts. This is the first time that these phenomena have been demonstrated in normal human oral epithelial cells. The work described here provides evidence to support a hypothesis that herpes simplex virus type 1 and smokeless tobacco may act synergistically in oral carcinogenesis.