Inhibition of the SARS-CoV-2 Main Protease by Isoquercitrin γ-Cyclodextrin Inclusion Complex Formulations: A Biochemical and In Silico Study

The main protease (Mpro) of SARS-CoV-2 is a well-established drug target for rational drug design of COVID-19 inhibitors. To address the serious challenge of COVID-19, we have performed biochemical inhibition screens with recombinantly expressed SARS-CoV-2 main protease (Mpro). A fluorescent assay was used to identify the flavonoid isoquercitrin as an Mpro inhibitor. Both isoquercitrin encapsulated in γ-cyclodextrin (inclusion complex formulations) and alone inhibited SARS-CoV-2 Mpro. For isoquercitrin, a Ki value of 32 μM (IC50 = 63 μM) was obtained. Isoquercitrin γ-cyclodextrin inclusion complex formulations additionally inhibited Zika virus NS2B-NS3pro leading to an IC50 value of 98 μM. Formulations containing the other flavonoid compounds diosmetin-7-O-glucoside, hesperetin-7-O-glucoside, and naringenin-7-O-glucoside did not inhibit SARS-CoV-2 Mpro. Steady-state kinetics indicate that the inhibition mechanism of Mpro by isoquercitrin is potentially competitive. Molecular modeling studies carried out with MM/PBSA confirm the likely modes of isoquercitrin binding to both proteases. These modeling results can be used in the development of structural analogs of isoquercitrin with better inhibitory profiles and potential candidates for anti-coronavirus drugs. Since the targeted proteases are essential for viral activity, the delivery isoquercitrin-cyclodextrin inclusion complex formulations could be of great interest for the development of future antiviral drugs to target intracellular virus proteins or other components.

Evaluation on the inclusion behavior of ß-cyclodextrins with lycorine and its hydrochloride.

Lycorine (Lyc) and its hydrochloride (Lyc∙HCl) as effective drugs can fight against many diseases including novel coronavirus (COVID-19) based on their antiviral and antitumor mechanism. Beta-cyclodextrin (ß-CD) is considered a promising carrier in improving its efficacy while minimizing cytotoxicity due to the good spatial compatibility with Lyc. However, the detailed mechanism of inclusion interaction still remains to be further evaluated. In this paper, six inclusion complexes based on ß-CDs, Lyc and Lyc∙HCl were processed through ultrasound in the mixed solvent of ethanol and water, and their inclusion behavior was characterized after lyophilization. It was found that the inclusion complexes based on sulfobutyl-beta-cyclodextrin (SBE-ß-CD) and Lyc∙HCl had the best encapsulation effect among prepared inclusion complexes, which may be attributed to the electrostatic interaction between sulfonic group of SBE-ß-CD and quaternary amino group of Lyc∙HCl. Moreover, the complexes based on SBE-ß-CD displayed pH-sensitive drug release property, good solubilization, stability and blood compatibility, indicating their potential as suitable drug carriers for Lyc and Lyc∙HCl.

The Role of Cyclodextrins in COVID-19 Therapy-A Literature Review.

Coronavirus disease-19 (COVID-19) emerged in December 2019 and quickly spread, giving rise to a pandemic crisis. Therefore, it triggered tireless efforts to identify the mechanisms of the disease, how to prevent and treat it, and to limit and hamper its global dissemination. Considering the above, the search for prophylactic approaches has led to a revolution in the reglementary pharmaceutical pipeline, with the approval of vaccines against COVID-19 in an unprecedented way. Moreover, a drug repurposing scheme using regulatory-approved antiretroviral agents is also being pursued. However, their physicochemical characteristics or reported adverse events have sometimes limited their use. Hence, nanotechnology has been employed to potentially overcome some of these challenges, particularly cyclodextrins. Cyclodextrins are cyclic oligosaccharides that present hydrophobic cavities suitable for complexing several drugs. This review, besides presenting studies on the inclusion of antiviral drugs in cyclodextrins, aims to summarize some currently available prophylactic and therapeutic schemes against COVID-19, highlighting those that already make use of cyclodextrins for their complexation. In addition, some new therapeutic approaches are underscored, and the potential application of cyclodextrins to increase their promising application against COVID-19 will be addressed. This review describes the instances in which the use of cyclodextrins promotes increased bioavailability, antiviral action, and the solubility of the drugs under analysis. The potential use of cyclodextrins as an active ingredient is also covered. Finally, toxicity and regulatory issues as well as future perspectives regarding the use of cyclodextrins in COVID-19 therapy will be provided.

Biotechnological Approach to Increase Oxyresveratrol Production in Mulberry In Vitro Plants under Elicitation.

Morus alba L. is used for a range of therapeutic purposes in Asian traditional medicine, and its extracts are reported to be effective against lipidemia, diabetes, and obesity, as well as being hepatoprotective and tyrosinase-inhibitory. They are also included in cosmetic products as anti-aging and skin-whitening agents. Stilbenes, the major bioactive compounds found in M. alba, have received renewed attention recently because of their putative activity against COVID-19. In this study M. alba plants were established in vitro, and the effect of elicitation on plant growth and stilbene accumulation, specifically oxyresveratrol and trans-resveratrol, was investigated. Different concentrations of the elicitors including methyl jasmonate and cyclodextrins were applied, and stilbene levels were determined in leaves, roots, and the culture medium. Elicitation of the M. alba plants with 5 mM cyclodextrins, alone or in combination with 10 µM methyl jasmonate, significantly increased the total phenolic content in the culture medium and leaves after 7 days of treatment. The higher total phenolic content in the roots of control plants and those treated only with methyl jasmonate indicated that cyclodextrins promoted metabolite release to the culture medium. Notably, the cyclodextrin-treated plants with the highest levels of oxy- and trans-resveratrol also had the highest total phenolic content and antioxidant capacity. These results indicate that elicited M. alba in vitro plants constitute a promising alternative source of bioactive stilbenes to supply pharmaceutical and cosmeceutical industries.

Quercitrin loaded cyclodextrin based nanosponge as a promising approach for management of lung cancer and COVID-19.

Lung cancer and pandemic acute respiratory disease, COVID-19, are examples of the most worldwide widespread diseases. The aim of the current study is to develop cyclodextrin based nanosponge (CD-NS) for loading the flavonoid drug, quercitrin (QCT). This is to improve its solubility in an attempt to enhance its activity against lung cancer as well as SARS-CoV-2 virus responsible for COVID-19. Preparation of CD-NS was performed by ultrasound-assisted synthesis method. Two CDs were employed, namely, ß cyclodextrin (ßCD) and 2-hydroxy propyl-ß-cyclodextrin (2-HPßCD) that were crosslinked with diphenyl carbonate, one at a time. QCT loaded CD-NS revealed entrapment efficiency and particle size ranged between 94.17 and 99.03% and 97.10-325.90 nm, respectively. QCT loaded 2-HPßCD-NS revealed smaller particle size compared with that of QCT loaded ßCD-NS. Zeta potential absolute values of the prepared formulations were >20 mV, indicating physically stable nanosystems. The selected formulations were investigated by Fourier transform infrared spectroscopy, X-ray powder diffraction and scanning electron microscopy which proved the formation of QCT loaded CD-NS exhibiting porous structure. QCT exhibited partial and complete amorphization in ßCD-NS and 2-HPßCD-NS, respectively. In vitro release revealed an improved release of QCT from CD-NS formulations. The biological activity of free QCT and QCT loaded CD-NS was investigated against lung cancer cell line A549 as well as SARS-CoV-2 virus. The results revealed that IC50 values of free QCT against lung cancer cell line A549 and SARS-CoV-2 were higher than those exhibited by QCT loaded CD-NS by 1.57-5.35 and 5.95-26.95 folds, respectively. QCT loaded 2-HPßCD-NS revealed enhanced in vitro release and superior biological activity compared with QCT loaded ßCD-NS.

Water-soluble inclusion complexes for a novel anti-viral agent with low toxicity;Oseltamivir with the β-cyclodextrins

An innovative sonication method has been developed to produce inclusion complexes (ICs) of Oseltamivir (OTV) which is a potentially water-soluble anti-viral agent with lesser cytotoxicity. Proton signals and chemical shifts of OTV without any ambiguity confirm the formation of ICs with β-Cyclodextrin (B-CD) and Hydroxypropyl-β-cyclodextrin (H-CD). ICs are also supported by their atomic percentages as secondary evidence using XPS analysis. Analysis of drug release at three pH levels revealed the slow release of the OTV from ICs and also suitable for viral inactivation. A very less cytotoxic ability on cancer cell lines and enhanced the viral inactivation of OTV after being made into water-soluble ICs. © 2022 Elsevier B.V.

A novel and water-soluble material for coronavirus inactivation from oseltamivir in the cavity of methyl and sulfated-ß-cyclodextrins through inclusion complexation.

A potentially active water-soluble anti-viral with lesser toxic material from the Oseltamivir (OTV) has been produced by the sonication method. The formed material has been further characterized by UV-visible, FT-IR, powder XRD, SEM, TGA/DTA, ROESY, XPS, AFM and etc., The results of DFT calculation have proven that inclusion complexes (ICs) are theoretically and energetically more advantageous models and structures have also been proposed based on the results. Analysis of drug release has been carried out at three pH levels, and it is revealed the analysis is most helpful at acidic pH levels for the ICs with S-CD over H-CD. Over OTV without CDs, OTV:S-CD-ICs exhibited a very less cytotoxic ability on cancer cell lines than ICs with M-CD. ICs enhanced the coronavirus inactivation nature of OTV. This study provides for the first time a full characterization of ICs of OTV with CDs and highlights the impact of complexation on pharmacological activity.

Cyclodextrins in Antiviral Therapeutics and Vaccines

The present review describes the various roles of cyclodextrins (CDs) in vaccines against viruses and in antiviral therapeutics. The first section describes the most commonly studied application of cyclodextrins—solubilisation and stabilisation of antiviral drugs;some examples also refer to their beneficial taste-masking activity. The second part of the review describes the role of cyclodextrins in antiviral vaccine development and stabilisation, where they are employed as adjuvants and cryopreserving agents. In addition, cyclodextrin-based polymers as delivery systems for mRNA are currently under development. Lastly, the use of cyclodextrins as pharmaceutical active ingredients for the treatment of viral infections is explored. This new field of application is still taking its first steps. Nevertheless, promising results from the use of cyclodextrins as agents to treat other pathologies are encouraging. We present potential applications of the results reported in the literature and highlight the products that are already available on the market.

Remdesivir interactions with sulphobutylether-beta-cyclodextrins: A case study using selected substitution patterns

Modified cyclodextrins (CDs) consist of a distribution of different structures with different number and location of the substituted groups. Among the most important applications of these molecules is their use as an enabling excipient in pharmaceutical formulations to provide the necessary solubility, stability and bioavailability for a drug to be effectively used. The most typical interaction mechanism of small molecular groups with CDs is the formation of host-guest inclusion complexes. The thermodynamic affinity constant between CDs and drugs should not be too strong, since then the biological activity could be negated by the formation of the complex. In the opposite scenario, if the affinity constant is too weak, the complex is barely formed and the amount of CD required in the formulation may become too great. Thus, a balance between the affinity of the CD and the drug is necessary for an optimal formulation. Additionally in the case of modified CDs and specific drug complexes there are further questions concerning the effect that the locations and number of substitutions plays in complexation. In the present work, this question is explored by using sulphobutylether-beta-cyclodextrin and remdesivir, the only antiviral medication currently approved by the United States Food and Drug Administration for treating COVID-19, as a case study. This paper presents results from an orthogonal study using isothermal titration calorimetry measurements and biased molecular dynamics simulations that provide complementary information. Isothermal titration calorimetry delves into the global impact of the species distribution while molecular dynamics simulations deals with specific chemical structures. The goal is to provide useful information to optimize pharmaceutical formulations based on modified CDs, specifically in the case of remdesivir, used to treat SARS-CoV-2 infection, although the main conclusions could be extended to the interaction of other drugs with modified cyclodextrins. (C) 2021 The Authors. Published by Elsevier B.V.

Cyclodextrins in Antiviral Therapeutics and Vaccines.

The present review describes the various roles of cyclodextrins (CDs) in vaccines against viruses and in antiviral therapeutics. The first section describes the most commonly studied application of cyclodextrins-solubilisation and stabilisation of antiviral drugs; some examples also refer to their beneficial taste-masking activity. The second part of the review describes the role of cyclodextrins in antiviral vaccine development and stabilisation, where they are employed as adjuvants and cryopreserving agents. In addition, cyclodextrin-based polymers as delivery systems for mRNA are currently under development. Lastly, the use of cyclodextrins as pharmaceutical active ingredients for the treatment of viral infections is explored. This new field of application is still taking its first steps. Nevertheless, promising results from the use of cyclodextrins as agents to treat other pathologies are encouraging. We present potential applications of the results reported in the literature and highlight the products that are already available on the market.

Targeting Lipid Rafts as a Strategy Against Coronavirus.

Lipid rafts are functional membrane microdomains containing sphingolipids, including gangliosides, and cholesterol. These regions are characterized by highly ordered and tightly packed lipid molecules. Several studies revealed that lipid rafts are involved in life cycle of different viruses, including coronaviruses. Among these recently emerged the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The main receptor for SARS-CoV-2 is represented by the angiotensin-converting enzyme-2 (ACE-2), although it also binds to sialic acids linked to host cell surface gangliosides. A new type of ganglioside-binding domain within the N-terminal portion of the SARS-CoV-2 spike protein was identified. Lipid rafts provide a suitable platform able to concentrate ACE-2 receptor on host cell membranes where they may interact with the spike protein on viral envelope. This review is focused on selective targeting lipid rafts components as a strategy against coronavirus. Indeed, cholesterol-binding agents, including statins or methyl-ß-cyclodextrin (MßCD), can affect cholesterol, causing disruption of lipid rafts, consequently impairing coronavirus adhesion and binding. Moreover, these compounds can block downstream key molecules in virus infectivity, reducing the levels of proinflammatory molecules [tumor necrosis factor alpha (TNF-α), interleukin (IL)-6], and/or affecting the autophagic process involved in both viral replication and clearance. Furthermore, cyclodextrins can assemble into complexes with various drugs to form host-guest inclusions and may be used as pharmaceutical excipients of antiviral compounds, such as lopinavir and remdesivir, by improving bioavailability and solubility. In conclusion, the role of lipid rafts-affecting drugs in the process of coronavirus entry into the host cells prompts to introduce a new potential task in the pharmacological approach against coronavirus.

The Use of Cyclodextrin or its Complexes as a Potential Treatment Against the 2019 Novel Coronavirus: A Mini-Review.

Severe Acute Respiratory Syndrome Coronavirus 2 has spread rapidly since its discovery in December 2019 in the Chinese province of Hubei, reaching this day all the continents. This scourge is, unfortunately, in lineage with various dangerous outbreaks such as Ebola, Cholera, Spanish flu, American seasonal flu. Until today, the best solution for the moment remains prevention (Social distancing, hand disinfection, use of masks, partial or total sanitary containment, etc.); there is also the emergence of drug treatment (research and development, clinical trials, use on patients). Recent reviews emphasized the role of membrane lipids in the infectivity mechanism of SARS-COV-2. Cholesterol-rich parts of cell membranes serve as docking places of host cells for the viruses. Coronavirus 2 is a member of a virus family with lipid envelope that fuses with host cell through endocytosis, internalizing its components in the cell. In vitro cell models have shown that depletion of cholesterol by cyclodextrin, and particularly methyl beta cyclodextrin disturb the host cell membrane lipid composition this way, reducing the attachment of the virus to the protein receptors. This review aims to summarize the state of the art of research concerning the use of cyclodextrin or its complexes as a potential treatment against this new virus and update work already published.

Tuning the Topological Landscape of DNA-Cyclodextrin Nanocomplexes by Molecular Design.

Original molecular vectors that ensure broad flexibility to tune the shape and surface properties of plasmid DNA (pDNA) condensates are reported herein. The prototypic design involves a cyclodextrin (CD) platform bearing a polycationic cluster at the primary face and a doubly linked aromatic module bridging two consecutive monosaccharide units at the secondary face that behaves as a topology-encoding element. Subtle differences at the molecular level then translate into disparate morphologies at the nanoscale, including rods, worms, toroids, globules, ellipsoids, and spheroids. In vitro evaluation of the transfection capabilities revealed marked selectivity differences as a function of nanocomplex morphology. Remarkably high transfection efficiencies were associated with ellipsoidal or spherical shapes with a lamellar internal arrangement of pDNA chains and CD bilayers. Computational studies support that the stability of such supramolecular edifices is directly related to the tendency of the molecular vector to form noncovalent dimers upon DNA templating. Because the stability of the dimers depends on the protonation state of the polycationic clusters, the coaggregates display pH responsiveness, which facilitates endosomal escape and timely DNA release, a key step in successful transfection. The results provide a versatile strategy for the construction of fully synthetic and perfectly monodisperse nonviral gene delivery systems uniquely suited for optimization schemes.

The Lord of the NanoRings: Cyclodextrins and the battle against SARS-CoV-2.

A handful of singular structures and laws can be observed in nature. They are not always evident but, once discovered, it seems obvious how to take advantage of them. In chemistry, the discovery of reproducible patterns stimulates the imagination to develop new functional materials and technological or medical applications. Two clear examples are helical structures at different levels in biological polymers as well as ring and spherical structures of different size and composition. Rings are intuitively observed as holes able to thread elongated structures. A large number of real and fictional stories have rings as inanimate protagonists. The design, development or just discovering of a special ring has often been taken as a symbol of power or success. Several examples are the Piscatory Ring wore by the Pope of the Catholic Church, the NBA Championship ring and the One Ring created by the Dark Lord Sauron in the epic story The Lord of the Rings. In this work, we reveal the power of another extremely powerful kind of rings to fight against the pandemic which is currently affecting the whole world. These rings are as small as ~1 nm of diameter and so versatile that they are able to participate in the attack of viruses, and specifically SARS-CoV-2, in a large range of different ways. This includes the encapsulation and transport of specific drugs, as adjuvants to stabilize proteins, vaccines or other molecules involved in the infection, as cholesterol trappers to destabilize the virus envelope, as carriers for RNA therapies, as direct antiviral drugs and even to rescue blood coagulation upon heparin treatment. "One ring to rule them all. One ring to find them. One ring to bring them all and in the darkness bind them." J. R. R. Tolkien.

COVID-19: A Recommendation to Examine the Effect of Mouthrinses with ß-Cyclodextrin Combined with Citrox in Preventing Infection and Progression.

Considered to be a major portal of entry for infectious agents, the oral cavity is directly associated with the evolutionary process of SARS-CoV-2 in its inhalation of ambient particles in the air and in expectorations. Some new generations of mouth rinses currently on the market have ingredients that could contribute to lower the SARS-CoV-2 viral load, and thus facilitate the fight against oral transmission. If chlorhexidine, a usual component of mouth rinse, is not efficient to kill SARS-CoV-2, the use of a mouth rinses and/or with local nasal applications that contain ß-cyclodextrins combined with flavonoids agents, such as Citrox, could provide valuable adjunctive treatment to reduce the viral load of saliva and nasopharyngeal microbiota, including potential SARS-CoV-2 carriage. We urge national agencies and authorities to start clinical trials to evaluate the preventive effects of ßCD-Citrox therapeutic oral biofilm rinses in reducing the viral load of the infection and possibly disease progression.