Novel analogues of a nonnucleoside SARS-CoV-2 RdRp inhibitor as potential antivirotics.

The RNA-dependent RNA polymerase (RdRp) represents a prominent target in the discovery and development of new antivirotics against RNA viruses, inhibiting the replication process. One of the most targeted RNA viruses of the last years is, without doubt, SARS-CoV-2, the cause of the recent COVID-19 pandemic. HeE1-2Tyr, a known inhibitor of flaviviral RdRp, has been discovered to also have antiviral potency against this coronavirus. In this study, we report three distinct modifications of HeE1-2Tyr: conversion of the core from a benzothiazole to a benzoxazole moiety and two different scaffold simplifications, respectively. We provide a novel synthetic approach and, in addition, evaluate the final molecules in an in vitro polymerase assay for biological activity.

A Helquat-like Compound as a Potent Inhibitor of Flaviviral and Coronaviral Polymerases.

Positive-sense single-stranded RNA (+RNA) viruses have proven to be important pathogens that are able to threaten and deeply damage modern societies, as illustrated by the ongoing COVID-19 pandemic. Therefore, compounds active against most or many +RNA viruses are urgently needed. Here, we present PR673, a helquat-like compound that is able to inhibit the replication of SARS-CoV-2 and tick-borne encephalitis virus in cell culture. Using in vitro polymerase assays, we demonstrate that PR673 inhibits RNA synthesis by viral RNA-dependent RNA polymerases (RdRps). Our results illustrate that the development of broad-spectrum non-nucleoside inhibitors of RdRps is feasible.

Structural and functional insights in flavivirus NS5 proteins gained by the structure of Ntaya virus polymerase and methyltransferase.

Flaviviruses are single-stranded positive-sense RNA (+RNA) viruses that are responsible for several (re)emerging diseases such as yellow, dengue, or West Nile fevers. The Zika epidemic highlighted their dangerousness when a relatively benign virus known since the 1950s turned into a deadly pathogen. The central protein for their replication is NS5 (non-structural protein 5), which is composed of the N-terminal methyltransferase (MTase) domain and the C-terminal RNA-dependent RNA-polymerase (RdRp) domain. It is responsible for both RNA replication and installation of the 5' RNA cap. We structurally and biochemically analyzed the Ntaya virus MTase and RdRp domains and we compared their properties to other flaviviral NS5s. The enzymatic centers are well conserved across Flaviviridae, suggesting that the development of drugs targeting all flaviviruses is feasible. However, the enzymatic activities of the isolated proteins were significantly different for the MTase domains.

Diclofenac sodium entrapment and release from halloysite nanotubules.

UNLABELLED: Halloysite was found to have interesting nanotubular geometry viable for the entrapment of various active agents. In this experiment, the ability of hollow halloysite cylinders to entrap the anionic model drug diclofenac sodium and to retard drug dissolution rate was investigated. Drugs could be incorporated into layered tubules via three different mechanisms: adsorption, intercalation and tubular entrapment. Based on the adsorption studies, some diclofenac sodium was shown to be adsorbed to the polyionic mineral surface despite its permanent negative charge. The X-ray powder diffraction analysis (XRPD) results did not prove any intercalation reaction to occur. The most important drug-loading mechanism involved the tubular entrapment with encapsulation efficiency 48.1%. The drug release from halloysite was prolonged in comparison with the dissolution of pure drug. Halloysite itself as well as halloysite loaded with the drug proved to be appropriate material to form pellets by extrusion /spheronization method. KEYWORDS: halloysite diclofenac sodium drug entrapment pellets prolonged drug release.

Optimization of diclofenac sodium profile from halloysite nanotubules.

Halloysite, aluminosilicate clay with the particle shape of multilayered hollow nanotubes, used in various non-medical applications, e.g. in ceramic industry, was discovered for pharmaceutical purposes in recent years. Several drugs of hydrophilic and lipophilic nature have been successfully encapsulated into halloysite tubules in order to modify their dissolution profile. The main goal of this experiment was to optimize the dissolution profile of diclofenac sodium - a drug with problematic solubility - from halloysite tubules using various polymers. Loading of the drug together with povidone or Eudragit® RS did not lead to drug burst effect reduction and its slower dissolution. In the case of povidone, drug improved wettability and solubilization rather than viscosity increasing expectations were observed. Eudragit® RS formed a solid dispersion with diclofenac sodium and thus the solvent/drug solution penetration through the polymer and not the drug solubility was the dissolution rate limiting factor. Reduction of the burst effect and further prolongation of drug release was achieved by coating the drug-loaded halloysite with chitosan. This formulation exhibited a diffusion-controlled prolonged release following Higuchi kinetic model.

Oxycellulose beads with drug exhibiting pH-dependent solubility.

The aim of this study was to develop novel hydrogel-based beads and characterize their potential to deliver and release a drug exhibiting pH-dependent solubility into distal parts of gastrointestinal (GI) tract. Oxycellulose beads containing diclofenac sodium as a model drug were prepared by the ionotropic external gelation technique using calcium chloride solution as the cross-linking medium. Resulting beads were characterized in terms of particle shape and size, encapsulation efficacy, swelling ability and in vitro drug release. Also, potential drug-polymer interactions were evaluated using Fourier transform infrared spectroscopy. The particle size was found to be 0.92-0.96 mm for inactive (oxycellulose only) and 1.47-1.60 mm for active (oxycellulose-diclofenac sodium) beads, respectively. In all cases, the sphericity factor was between 0.70 and 0.81 with higher values observed for samples containing higher polymer and drug concentrations. The swelling of inactive beads was found to be strongly influenced by the pH and composition (i.e. Na(+) concentration) of the selected media (simulated gastric fluid vs. phosphate buffer pH 6.8). The encapsulation efficiency of the prepared particles ranged from 58% to 65%. Results of dissolution tests showed that the drug loading inside of the particles influenced the rate of its release. In general, prepared particles were able to release the drug within 12-16 h after a lag time of 4 h. Fickian diffusion was found as the predominant drug release mechanism. Thus, this novel particulate system showed a good potential to deliver drugs specifically to the distal parts of the human GI tract.

Non-Nucleotide RNA-Dependent RNA Polymerase Inhibitor That Blocks SARS-CoV-2 Replication.

SARS-CoV-2 has caused an extensive pandemic of COVID-19 all around the world. Key viral enzymes are suitable molecular targets for the development of new antivirals against SARS-CoV-2 which could represent potential treatments of the corresponding disease. With respect to its essential role in the replication of viral RNA, RNA-dependent RNA polymerase (RdRp) is one of the prime targets. HeE1-2Tyr and related derivatives were originally discovered as inhibitors of the RdRp of flaviviruses. Here, we present that these pyridobenzothiazole derivatives also significantly inhibit SARS-CoV-2 RdRp, as demonstrated using both polymerase- and cell-based antiviral assays.

Tumor Marker B7-H6 Bound to the Coiled Coil Peptide-Polymer Conjugate Enables Targeted Therapy by Activating Human Natural Killer Cells.

Targeted cancer immunotherapy is a promising tool for restoring immune surveillance and eradicating cancer cells. Hydrophilic polymers modified with coiled coil peptide tags can be used as universal carriers designed for cell-specific delivery of such biologically active proteins. Here, we describe the preparation of pHPMA-based copolymer conjugated with immunologically active protein B7-H6 via complementary coiled coil VAALEKE (peptide E) and VAALKEK (peptide K) sequences. Receptor B7-H6 was described as a binding partner of NKp30, and its expression has been proven for various tumor cell lines. The binding of B7-H6 to NKp30 activates NK cells and results in Fas ligand or granzyme-mediated apoptosis of target tumor cells. In this work, we optimized the expression of coiled coil tagged B7-H6, its ability to bind activating receptor NKp30 has been confirmed by isothermal titration calorimetry, and the binding stoichiometry of prepared chimeric biopolymer has been characterized by analytical ultracentrifugation. Furthermore, this coiled coil B7-H6-loaded polymer conjugate activates NK cells in vitro and, in combination with coiled coil scFv, enables their targeting towards a model tumor cell line. Prepared chimeric biopolymer represents a promising precursor for targeted cancer immunotherapy by activating the cytotoxic activity of natural killer cells.

Soluble filler as a dissolution profile modulator for slightly soluble drugs in matrix tablets.

The purpose of this experimental work was the development of hydrophilic-lipophilic matrix tablets for controlled release of slightly soluble drug represented here by diclofenac sodium (DS). Drug dissolution profile optimization provided by soluble filler was studied. Matrix tablets were based on cetyl alcohol as the lipophilic carrier, povidone as the gel-forming agent, and common soluble filler, that is lactose or sucrose of different particle size. Physical properties of tablets prepared by melt granulation and drug release in a phosphate buffer of pH 6.8 were evaluated. In vitro studies showed that used filler type, filler to povidone ratio and sucrose particle size influenced the drug release rate. DS dissolution profile could be changed within a wide range from about 50% per 24 hours to almost 100% in 10 hours. The release constant values confirmed that DS was released from matrices by the diffusion and anomalous transport. The influence of sucrose particle size on the drug release rate was observed. As the particle size decreased, the drug release increased significantly and its dissolution profile became more uniform. Soluble fillers participated in the pore-forming process according to their solubility and particle size. Formulations containing 100 mg of the drug, 80 mg of cetyl alcohol, 40 mg of povidone, and 80 mg of either lactose or sucrose (particle size 250-125 microm) were considered optimal for 24-hour lasting dissolution of DS.

Pellet starters in layering technique using concentrated drug solution.

Characteristics of inert starters in drug solution layering are important for successful active pellet formation. Four types of starters composed of sucrose or microcrystalline cellulose (MCC) or lactose and MCC were compared in our study. The active pellets were prepared using Wurster type apparatus. Yield and pellet quality parameters were determined. The highest yield (85.66-89.41%) was obtained for cores composed of MCC due to their insolubility in water (the drug solvent) and good mechanical properties. On the contrary, soluble and brittle sucrose cores dissolved partially during the process forming undesirable agglomerates and giving lower yield (76.2%). All pellet samples showed good flow properties and drug content from 82.4 to 94.5% of the theoretical drug amount.

Differences in characteristics of pellets prepared by different pelletization methods.

Pellets are currently a very popular dosage form for oral application. They can be prepared by several pelletization techniques. Extrusion/spheronization, commonly used in the pharmaceutical industry, and modern agglomeration in a rotoprocessor were the methods chosen for pellet preparation in our study. Theophylline (in 10% to 65% concentration) was the model drug, lactose monohydrate was used as filler, microcrystalline cellulose Avicel PH 101 was thespheronization enhancer, and the wetting agent was purified water. Both techniques led to the formation of pellets of appropriate shape and mechanical properties. Pellets of a higher density, hardness, lower friability, and slightly slower dissolution profiles were obtained by extrusion/spheronization. This method of pelletization also led to production of particles with narrower size distribution and bigger yield of pellets with the requested size.

The influence of drug solubility and particle size on the pellet formulation in a rotoprocessor.

Pellets containing drugs of different properties were prepared in a Rotoprocessor in order to study changes in the formulation process and resulting pellet characteristics. Diltiazem hydrochloride, diclofenac sodium, and theophylline were chosen as model drugs. Pellet size distribution, sphericity, density, hardness, friability, and repose angle were determined using standard methods. The amount of water as a wetting agent necessary for successful pellet formulation was observed for each sample and changed depending on drug solubility, concentration, and particle size. The pelletization of freely soluble diltiazem hydrochloride required 24.8-23.1% of the wetting agent and its amount decreased as the drug concentration increased. The demand for water in the formulation of theophylline pellets was 31.0-34.4% and it increased with increasing drug concentration. The pellet samples containing both drugs were easy to prepare. However, the cohesion of micronized diclofenac sodium particles negatively influenced both the pellet size distribution and the formulation process itself. When the drug concentration exceeded 40%, it was not possible to produce pellets of an appropriate size and the process was not reproducible.