key: cord-0714654-3nknawgp authors: Galindo-Cardiel, Iván José; Núñez, Adriana Toledo; Fernández, María Celaya; Ramírez Labrada, Ariel; Uranga-Murillo, Iratxe; Cabrero, Maykel Arias; Pardo, Julian; Panzeri, Ezio title: First-described recently discovered non-toxic vegetal-derived furocoumarin preclinical efficacy against SARS-CoV-2: a promising antiviral herbal drug date: 2020-12-07 journal: bioRxiv DOI: 10.1101/2020.12.04.410340 sha: e2b0f9e658062f6e1d5473bad97e8c318030c090 doc_id: 714654 cord_uid: 3nknawgp Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiology of coronavirus disease 2019 (COVID19) pandemic. ICEP4 purified compound (ICEP4) is a recently discovered furocoumarin-related purified compound derived from the roots and seeds of Angelica archangelica (herbal drug). ICEP4-related herbal preparations have been extensively used as active herbal ingredients in traditional medicine treatments in several European countries. Extraction method of patent pending ICEP4 (patent application no. GB2017123.7) has previously shown strong manufacturing robustness, long-lasting stability, and repeated chemical consistency. Here we show that ICEP4 presents a significant in vitro cytoprotective effect in highly virulent-SARS-CoV-2 challenged Vero E6 cellular cultures, using doses of 34.5 and 69 μM. No dose-related ICEP4 toxicity was observed in Vero E6 cells, M0 macrophages, B, CD4+ T and CD8+ T lymphocytes, Natural Killer (NK) or Natural Killer T (NKT) cells. No dose-related ICEP4 inflammatory response was observed in M0 macrophages quantified by IL6 and TNFα release in cell supernatant. No decrease in survival rate was observed after either 24 hr acute or 21-day chronic exposure in in vivo toxicity studies performed in C. elegans. Therefore, ICEP4 toxicological profile has demonstrated marked differences compared to others vegetal furocoumarins. Successful ICEP4 doses against SARS-CoV-2-challenged cells are within the maximum threshold of toxicity concern (TTC) of furocoumarins as herbal preparation, stated by European Medicines Agency (EMA). The characteristic chemical compounding of ICEP4, along with its safe TTC, allow us to assume that the first-observation of a natural antiviral compound has occurred. The potential druggability of a new synthetic ICEP4-related compound remains to be elucidated. However, well-established historical use of ICEP4-related compounds as herbal preparations may point towards an already-safe, widely extended remedy, which may be ready-to-go for large-scale clinical trials under the EMA emergency regulatory pathway. To the best of the authors’ knowledge, ICEP4-related herbal drug can be postulated as a promising therapeutic treatment for COVID19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped non-segmented positive-sense single-stranded RNA virus (genus Betacoronavirus, subfamily Orthocoronavirinae). SARS-CoV-2 is the aetiology of the coronavirus disease 2019 (COVID19) pandemic (1). Social distancing, case identification, contact tracing, quarantine and isolation are postulated as the main strategies to reduce viral spreading. Despite worldwide research efforts and some very promising advances, no effective antiviral drugs, or mitigant sanitary products against SARS-CoV-2 infection currently exist, therefore, current pharmacological therapy is mostly restricted towards mitigating the associated symptoms (2, 3) . There is continuous interest in searching for alternative antiviral drugs among phytochemical extracts, medicinal plants, and aromatic herbs. Discovery and production of novel antiviral drugs frequently occurs from spices, herbal medicines, essential oils (EOs), and distilled natural products (4) . Coumarins comprise a large class of compounds found within medicine herbal preparations (5) (6) (7) . Coumarins are found at high levels in some EOs, particularly cinnamon bark oil, cassia leaf oil, and lavender oil. Coumarin is also found in fruits (e.g. bilberry, cloudberry), green tea, and other foods, such as chicory (8) . Most coumarins occur in higher plants, with the richest sources being the Rutaceae and Umbelliferae. Although distributed throughout all parts of the plant, the coumarins occur at the highest levels in the fruits, followed by the roots, stems, and then leaves. Environmental conditions and seasonal changes may influence the occurrence of coumarins in various parts of the plant (9) . Psoralens are natural products that are linear furanocoumarins (most furanocoumarins can be regarded as derivatives of psoralen or angelicin), which are extremely toxic to a wide variety of prokaryotic and eukaryotic organisms. Some important psoralen derivatives are xanthotoxin, imperatorin, bergapten and nodekenetin (8, 9) . The demonstrated activities of coumarins include anticoagulant, anticancer, antioxidant, antiviral, anti-diabetic, anti-inflammatory, antibacterial, antifungal, and anti-neurodegerative properties as drugs, as well as the ability to act as fluorescent sensors for biological systems (10) . The genus Angelica litoralis is comprised of over 90 species spread throughout most areas of the globe (11) . More than half of these species are used in traditional therapies, while some of them are included in several national and European pharmacopoeias (12) (13) (14) (15) . Bioactive constituents in different Angelica species include coumarins, EOs, polysaccharides, organic acids and acetylenic compounds (16) . In vitro testing confirmed cytotoxic (17, 18) , anti-inflammatory (19) , antibacterial (20) , antifungal (21) , neuroprotective (22) and serotonergic (23) activities for extracts obtained from a range of Angelica species. pág. 4 Reducing viral replication at the beginning of SARS-CoV-2 infection and, subsequently, the associated degree of immunopathological damage, is a critical step to mitigate and cure COVID19 (2). ICEP4 (patent pending, application nº GB2017123.7) is an Angelica archangelica-based purified compound with previous evidence of antiviral and oncolytic in vitro effects (ICE-P Life, data not shown). ICEP4-related herbal preparations have been extensively used as active herbal ingredients in traditional medicine treatments in several countries, including the EU and US (12) (13) (14) (15) For in vivo studies, a 1 mM dose of ICEP4 was added to a 25º C-cultivated infertile strain of C. elegans for acute and chronic studies (10.000 times higher than reference dose). A high-pathogenic strain of SARS-CoV-2 was isolated and cultured from a 72-year old patient at University Clinical Hospital Lozano Blesa (Zaragoza, Spain). Second-passage vials with the SARS-CoV-2 strain were provided by Dr. Julian Pardos (IISA, UNATI, Zaragoza, Spain). Virus was maintained and cultured following UNATI protocols in BSL3 facilities at Zaragoza (WGUSA, Spain). The tissue culture infectious dose 50% (TCID50) was determined to be 1.47 x 10 6 /mL. The same strain and TCID50 was used at UNATI facilities for repeatability and interlaboratory consistency studies. Vero E6 cells were provided by Eugenia Puentes (Biofabri, Porriño, Spain) and cultured following provider´s descriptions. Cellular cultures were maintained at a density of 10 A minimal-disease certified mouse (B6, Charles River, US) was killed by cervical dislocation. The spleen was then carefully extracted and mashed through a cell strainer. Splenocytes were washed with RPMI and centrifuged at 1200 rpm for 5 min. Splenocytes were counted and adjusted to 10 6 cells/mL. Cell viability was analysed by PrestoBlue TM HS (high sensitivity) assay following the manufacturer´s instructions. PrestoBlue TM HS contain resazurin and a propriety buffering system (#P50200, ThermoFisher, US). Absorbance was measured using an iMark™ Microplate Absorbance Reader (BioRad, Germany). Activation of the inflammatory response in macrophages was analysed quantifying the cytokines IL6 and TNFα in cell supernatants by ELISA (Ready-Set-Go kit, eBiosciences) following the manufacturer´s instructions. Worms without treatment served as negative controls. Three different independent survival assays were carried out. Worms were cultured as previously described and treated with different ICEP4 doses. Worms without treatment served as negative controls. Survival assays were carried out for 21 days at 25 pág. 9 °C. Every 7 days, ICEP4 and E. coli OP50 were added to the C. elegans. Chronic toxicity worms were seeded and counted twice a week calculating the percentage of worms that survived with respect to the number of worms at time zero. Three independent experiments were performed. Mean ± SD (Standard Deviation). One-way ANOVA was used to confirm statistical differences among multiple groups between treated and non-treated groups. ICEP4 -TCID50 groups were analysed by two-sample t-test, assuming equal variances, to confirm significant differences. Significant differences are indicated by: *P < 0.05; **P < 0.01; and ***P < 0.001. P < 0.05 was considered as significant. Results from UNATI were analysed together with WGUSA obtained data, in order to check robustness and repeatability. Descriptive statistics including UNATI results are shown in Table 1 . Maximum SD was observed in the TCID50 group, mostly due to outlier results in the first replication of the experiment ( Figure 2 ). After this first trial, more coherent and consistent TCID50 results (5±1,73) were obtained across the rest of the replications. Marked significant differences were found between groups for at least one group as stated by ANOVA of a factor (ICEP4 treatment) ( Table 2 ). No differences were found by analysing the effect of solvents or raw material vs Vero culture in a two-sample t-test assuming equal variances (Table 3) . Marked increases in cell viability were observed when comparing to TCDI50 control were found in SARS-CoV-2-infected ICEP4-treated groups, corresponding to 34.5 µM (WP2) and 69 µM (WP3) doses (Table 4 ). Significant differences were also found between these groups when analysing two-sample t-test results, assuming equal variances, thus confirming preliminary descriptive results. Interpreting these results, ANOVA significance can be directly correlated to cytoprotective effects of ICEP4 treatment. pág. 10 No toxicity on CD4 and CD8 T cells, B cells, or NKT was seen at the tested does of ICEP4 (Tables 5 and 6 ; Figure 3 ). However, increased Annexin-V staining, indicating slight decreases in cellular viability, were observed in NK cells at the 100 µM dose. A similar effect was also found in macrophages, which showed a 30% reduction in cell viability at 100 µM of ICEP4 ( Figure 3) . Remarkably, ICEP4 plant-derived extract did not induce an inflammatory response in M0 macrophages at any of the tested doses, which we were able to verify via the release of IL6 and TNFα after challenge (Table 7) . IL6 and TNFα values were very low and, in some samples, very close to or even below the limit of detection (detection limit IL6: 4 pg/mL, TNFα: 8 pg/mL). As expected, LPS induced a high inflammatory response thereby confirming macrophage functionality. Twenty-four hr acute and 21-day chronic in vivo toxicity studies were performed on ICEP4 in C. elegans ( Figure 4 ). No toxicity was observed from doses of 1 nM to 100 µM in either assay. In both assays, only the highest dose showed toxicity (1 mM), which is presumably due to the higher ethanol concentration (20%) and not to the active ingredient, ICEP4, as confirmed by the SHAM control ( Figure 4 ). Antiviral herbal drugs have been widely used on the clinical frontline against respiratory diseases. (16) . Active principles isolated from these plants mainly include various types of coumarins, acetylenic compounds, chalcones, sesquiterpenes, and polysaccharides (9, 10, 16). Frequently, most of the existing conventional antiviral treatments lead to the development of viral resistance in addition to the problems of side effects, viral re-emergence, and viral dormancy (2) . Therefore, the WHO also supports and welcomes innovations around the world regarding scientifically proven traditional medicine, in order to increase the clinical alternatives of safe antiviral therapies (30). IL1β, and IL6 (Fructus forsythiae) (38) . A psychoneuroimmune mechanism has been highlighted as a possible immune-mediated pathway in COVID19 treatment support (39, 40) . Prophylactic therapeutics with potential immunomodulatory activity have been postulated as add-on treatments for COVID19 (29) . Many medicinal compounds and natural products have exhibited several antiviral mechanisms that prevent early stages of infection, including viral attachment and penetration (36, 41) . Psoralens, the main moiety of furocoumarins, may react directly with pyrimidine nucleotides to form mono-and di-adducts in DNA of even interstrand cross links (6) . Another route of psoralen toxicity derives from the ability of UV-A photoactivated furanocoumarins to react with grand state oxygen, generating toxic oxyradicals capable of inactivating proteins within cells (6, 7) . Due to this reactivity, a broad range of therapeutic applications requiring inhibitors of cell division (main drug targets are the cytochrome P450 superfamily) have been suggested, such as vitiligo, psoriasis, and several type of cancers, including T cell lymphoma (7, (32) (33) (34) . Taking together the data from the cytotoxicity and in vivo assays, we postulate that furocoumarin-derived ICEP4 has shown very little, or negligible evidence of toxicity towards immune and epithelial-derived cells. NK and macrophages showed slight decreases in viability when exposed to the highest dose, probably due to the same effect of solvent stated for the in vivo assays. It is worth mentioning that Angelica-based furocoumarin, the gold standard, is phototoxic and affects cellular viability within the studied doses (32, 34) . Several structural changes, well-established compound concentration and composition differences are expected by meaning of geographic, stational or plant-related issues, many of them can be used as drug template design (27). Therefore, it is postulated that ICEP4, despite being extracted and purified in the same manner as other Angelica-related furocoumarins, possesses differences in chemical or racemic compounding composition relating to the extraction method, which leads to marked differences in terms of toxicity and antiviral efficacy (SARS-CoV-2 infection model). These facts may let us assume that an unknown different furocoumarin-related compound has been discovered or, at least, first observed. The extraction method of patent-pending ICEP4 has showed previously strong manufacturing robustness, long-lasting stability, and repeated chemical consistency. Nonetheless, additional chemical-related data regarding ICEP4 are urgently needed, in order to highlight several concerns regarding its use as a herbal drug. TCM, AM, and EHD-prescribed herbal drugs decrease the severity and mortality rate of COVID19 (28, 35, 38, 41 Table 3 . Two-sample t-test assuming equal variances results of in vitro challenge test for solvents and raw material. Non-treated Vero E6 2% medium culture (CT) group was considered treatmentnegative control (maximum cell viability) for comparison (data no shown). Ethanol 0.15%, 0.75% and 1,5% respectively were considered as solvent control. 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Quality assurance (Iván José Galindo-Cardiel Iván José Galindo-Cardiel and Ezio Panzeri are co-authors of ICEP4-related patent application Maykel Arias is supported by a Juan de la Cierva postdoctoral contract Agencia Estatal de Investigación -SAF2017-83120-C2-1-R-)