Tenebrio molitor as a Simple and Cheap Preclinical Pharmacokinetic and Toxicity Model

The progression of drugs into clinical phases requires proper toxicity assessment in animals and the correct identification of possible metabolites. Accordingly, different animal models are used to preliminarily evaluate toxicity and biotransformations. Rodents are the most common models used to preliminarily evaluate the safety of drugs; however, their use is subject to ethical consideration and elevated costs, and strictly regulated by national legislations. Herein, we developed a novel, cheap and convenient toxicity model using Tenebrio molitor coleoptera (TMC). A panel of 15 drugs-including antivirals and antibacterials-with different therapeutic applications was administered to TMC and the LD50 was determined. The values are comparable with those already determined in mice and rats. In addition, a TMC model was used to determine the presence of the main metabolites and in vivo pharmacokinetics (PK), and results were compared with those available from in vitro assays and the literature. Taken together, our results demonstrate that TMC can be used as a novel and convenient preliminary toxicity model to preliminarily evaluate the safety of experimental compounds and the formation of main metabolites, and to reduce the costs and number of rodents, according to 3R principles

Upcycling Milk Industry Byproducts into Tenebrio molitor Larvae: Investigation on Fat, Protein, and Sugar Composition

Edible insects represent a growing sector of the food industry and have a low carbon footprint. Noteworthy, insects can upcycle different leftovers and byproducts into high-quality nutrients. Herein, the larvae of the edible insect Tenebrio molitor (TML) were fed using local milk industry byproducts. Mozzarella whey and whey permeate obtained in cheese production were used to formulate three alternative diets. Both byproducts are rich in sugars, in particular the disaccharide lactose and the monosaccharides glucose and galactose. Two of the three diets did not interfere with biometric data and vitality, while the use of whey permeate alone significantly reduced development. At the end of the trial, the proximate composition of TML was strongly affected, with an increased protein content of up to +7% and a favorable fat composition. The analysis of secondary metabolites revealed the accumulation of different compounds, in particular monounsaturated fatty acids (MUFAs), amino acids, and the disaccharide trehalose, essential for the correct larval development and pupation. In conclusion, the present study demonstrates that milk industry byproducts can be upcycled as feed for TML, maintaining an optimal nutrient composition and favorably increasing the protein conten

Privileged Scaffold Decoration for the Identification of the First Trisubstituted Triazine with Anti-SARS-CoV-2 Activity

: Current therapy against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) are based on the use of Remdesivir 1, Molnupiravir 2, and the recently identified Nirmatrelvir 3. Unfortunately, these three drugs showed some limitations regarding potency and possible drug-drug interactions. A series of derivatives coming from a decoration approach of the privileged scaffold s-triazines were synthesized and evaluated against SAR-CoV-2. One derivative emerged as the hit of the series for its micromolar antiviral activity and low cytotoxicity. Mode of action and pharmacokinetic in vitro preliminary studies further confirm the role as candidates for a future optimization campaign of the most active derivative identified with this work. © 2022 by the authors

Exploiting the bile acid binding protein as transporter of a Cholic Acid/Mirin bioconjugate for potential applications in liver cancer therapy

Bioconjugation is one of the most promising strategies to improve drug delivery, especially in cancer therapy. Biomolecules such as bile acids (BAs) have been intensively explored as carriers, due to their peculiar physicochemical properties and biocompatibility. BAs trafficking is regulated by intracellular lipid-binding proteins and their transport in the liver can be studied using chicken liver Bile Acid-Binding Proteins (cL-BABPs) as a reference model. Therefore, we conceived the idea of developing a BA-conjugate with Mirin, an exonuclease inhibitor of Mre11 endowed with different anticancer activities, to direct its transport to the liver. Following computational analysis of various BAs in complex with cL-BABP, we identified cholic acid (CA) as the most promising candidate as carrier, leading to the synthesis of a novel bioconjugate named CA-M11. As predicted by computational data and confirmed by X-ray crystallographic studies, CA-M11 was able to accommodate into the binding pocket of BABP. Hence, it can enter BAs trafficking in the hepatic compartment and here release Mirin. The effect of CA-M11, evaluated in combination with varying concentrations of Doxorubicin on HepG2 cell line, demonstrated a significant increase in cell mortality compared to the use of the cytotoxic drug or Mirin alone, thus highlighting chemo-sensitizing properties. The promising results regarding plasma stability for CA-M11 validate its potential as a valuable agent or adjuvant for hepatic cancer therapy

Applying molecular hybridization to design a new class of pyrazolo[3,4-d]pyrimidines as Src inhibitors active in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common type of liver solid tumor and the second leading cause of cancer-related deaths worldwide. Although new treatment options have been recently approved, the development of tumor resistance and the poor prognosis for advanced HCC make the current standard of care unsatisfying. In this scenario, the non-receptor tyrosine kinase (TK) c-Src emerged as a promising target for developing new anti-HCC agents. Our group reported a large library of pyrazolo[3,4-d]pyrimidines active as potent c-Src inhibitors. Starting from these data, we applied a molecular hybridization approach to combine the in-house pyrazolo[3,4-d]pyrimidine SI192 with the approved TK inhibitor (TKI) dasatinib, with the aim of identifying a new generation of Src inhibitors. Enzymatic results prompted us to design second-generation compounds with a better binding profile based on a hit optimization protocol comprised of molecular modeling and on-paper rational design. This investigation led to the identification of a few nanomolar Src inhibitors active toward two HCC cell lines (HepG2 and HUH-7) selected according to their high and low c-Src expression, respectively. In particular, 7e showed an IC50 value of 0.7 nM toward Src and a relevant antiproliferative efficacy on HepG2 cells after 72h (IC50 = 2.47 μM). Furthermore, 7e exhibited a cytotoxic profile better than dasatinib. The ADME profile suggested that 7e deserves further investigation as a promising TKI in cancer therapies. Finally, 7e′s ability to inhibit HepG2 cell proliferation, elicit an irreversible cytotoxic effect, arrest cellular migration, and induce apoptotic-mediated cell death was assessed

Investigation of in vitro and in vivo pharmacokinetics and biological evaluation of pharmacologically active compounds

The first part of this thesis deals with the evaluation of in vitro and in vivo pharmacokinetics of novel broad-spectrum antiviral compounds active against enveloped viruses. The design and synthesis of new series of antiviral compounds with 1H-pyrrol-methylene thioxodihydropyrimidine structure, has been realized by Professor Maurizio Botta's research group, at the University of Siena. Compounds’ antiviral activity has been evaluated on several enveloped viruses, such as ZIKAV, DENV-2 and five influenza strains including the pandemic strain H7N9. The selectivity against enveloped viruses, time of addition and binding experiments confirmed their ability to intercalate in the viral envelope membrane, oxidize phospholipids and alter the fluidity of the lipid bilayer, compromising the efficacy of the virus-cell fusion step and preventing viral entry. With the aim of investigating the in vitro ADME properties, the most active compounds were selected to assay their chemical-physical properties and early select the most promising lead candidate. Thus, membrane permeability, binding to human serum albumin, and stability in human plasma and microsomes have been assayed. Finally, the lead candidate was selected to evaluate preliminary in vivo pharmacokinetic parameters; after formulation studies, the compound was administrated intravenously (iv) at the dose of 25 mg/kg and 12.5 mg/kg. The second chapter of this Ph.D. thesis concerns the investigation of the in vitro biological profile of nitric oxide-donor largazole prodrugs. Two hybrid analogues of largazole, as dual HDAC inhibitor and nitric oxide (NO) donors potentially useful as anticancer agents, have been designed and synthesized thanks to the collaboration between Professor Maurizio Botta’s research group and IRBM. Largazole is a natural product identified as the most potent and selective Class-I deacetylase (HDAC) inhibitor, that showed a broad-spectrum growth-inhibitory activity against epithelial and fibroblastic tumor cell lines and a low cytotoxicity profile. Over the last decades, dual nitric oxide (NO) donors/HDAC inhibitors have been developed as novel anticancer chemical entities, potentially more efficacious than selective HDAC inhibitors, owing to the capability of NO to specifically modulate the function of some HDAC isoforms and to overcome tumor cell resistance to conventional treatments. Thus, after the synthesis, the characterization of derivatives compounds and the in vitro NO release assay performed by Professor Maria Frosini using the Griess method, biological evaluation of their antiproliferative activities against U-2OS, Caco-2, and IMR-32 cell lines have been conducted. To further explain the additive antiproliferative effect of NO-donor compounds vs largazole, their stabilities both in human plasma and in cell culture medium were assessed. The third and last chapter of this Ph.D. thesis deals with the project I participated in during my exchange period at the research group of Professor Per Artursson, who hosted me for 5 months, at Uppsala University. In collaboration with AstraZeneca, a series of antisense oligonucleotide (ASO) conjugates, targeting MALAT1 chosen as a model target, were used to assess and validate their silencing efficiency and enhance/overcome endosomal escape. The MALAT1 silencing efficiencies of lipophilic ASO-conjugates and a peptide-ASO have been determined in the presence and absence of a cyclic cell permeation peptide (CPP) in two human embryonic kidney (HEK293) cell lines of which one overexpressing the target G-protein coupled receptor selected for the study. For this purpose, the expression levels of the MALAT1 gene mRNA were measured using qPCR in a time (0-24-48 hrs) and concentration (0.5-2.5-5 µM of ASOs and 5-10-15 µM of CPP)-dependent manner

A Greener Technique for Microwave-Assisted O-Silylation and Silyl Ether Deprotection of Uridine and Other Substrates

A single clean, good-yielding, environment-friendly microwave-assisted procedure for O-silylation of uridine with tert-butyldimethylsilyl chloride (TBDMSCl), 1,8-Diazabicyclo(5.4.0)undec-7-ene (DBU) and potassium nitrate as catalyst under solvent-free conditions is reported. Subsequent silyl ether deprotection is accomplished with a reusable acidic resin via microwave irradiation. Both the silylation and desilylation protocols have been applied to a panel of alcohols of pharmaceutical interest

Infesting Seaweeds as a Novel Functional Food: Analysis of Nutrients, Antioxidants and ACE Inhibitory Effects

Globalization and climate change are both contributing to an increase in the number of potentially invasive algae in coastal areas. In terms of biodiversity and financial losses, the invasiveness of algae has become a significant issue in Orbetello Lagoon. Indeed, studies from the Tuscany Regional Agency for Environmental Protection show that the reduction in dissolved oxygen caused by algal diffusion is detrimental to fisheries and biodiversity. Considering that wakame and numerous other potentially invasive seaweeds are consumed as food in Asia, we assess the nutritional and nutraceutical qualities of two potentially invasive seaweeds: Valonia aegagrophila and Chaetomorpha linum. We found that both algae are a valuable source of proteins and essential amino acids. Even if the fat content accounts for less than 2% of the dried weight, its quality is high, due to the presence of unsaturated fatty acids. Both algae are rich in antioxidants pigments and polyphenols, which can be exploited as nutraceuticals. Most importantly, human gastrointestinal digestion increased the quantity of polyphenols and originated secondary metabolites with ACE inhibitory activity. Taken together, our data strongly promote the use of Valonia aegagrophila and Chaetomorpha linum as functional foods, with possible application in the treatment of hypertension and cardiovascular diseases

Insects as Source of Nutraceuticals with Antioxidant, Antihypertensive, and Antidiabetic Properties: Focus on the Species Approved in Europe up to 2024

Insects represent a traditional food in different parts of the world, where eating insects is not only related to nutrition, but also results from a variety of sociocultural customs. Insects’ nutritional profiles typically vary by species. Nevertheless, in terms of nutrition, edible insects can be a rich source of protein, dietary fiber, healthy fatty acids, and micronutrients, including minerals and vitamins. Insects have a low carbon footprint and require fewer resources in terms of land, water, and food with respect to animal livestock. Interestingly, insects are a source of bioactive compounds with different pharmacological activities, including antioxidant, antimicrobial, antidiabetic, antiobesity, antihypertensive, and antilipidemic. Among the bioactive compounds, polyphenols, chitosan, and protein hydrolysates are the most important ones, with direct activity on ROS quenching and enzymatic inhibition. Glucosidase, DPP-IV, ACE, and lipases are directly inhibited by insects’ bioactive peptides. Lipids and tocopherols reduce inflammation and lipid peroxidation by acting on LOX and COX-2 enzymes and on ROS quenching. The insects’ nutrient composition, coupled with their easy and economical breeding, is the cause of the growing interest in edible insects. During the last 20 years, the study and development of novel insect-based products increased, with relevant effects on the market. This review focuses on the edible insects currently approved in Europe, namely, Acheta domesticus, Alphitobus diaperinus, Locusta migratoria, and Tenebrio molitor. The nutrient profile and the functional compounds are examined, with an eye on market trends and on the patent applications filed in the last decades

A Greener Technique for Microwave-Assisted O-Silylation and Silyl Ether Deprotection of Uridine and Other Substrates

A single clean, good-yielding, environment-friendly microwave-assisted procedure for O-silylation of uridine with tert-butyldimethylsilyl chloride (TBDMSCl), 1,8-Diazabicyclo(5.4.0)undec-7-ene (DBU) and potassium nitrate as catalyst under solvent-free conditions is reported. Subsequent silyl ether deprotection is accomplished with a reusable acidic resin via microwave irradiation. Both the silylation and desilylation protocols have been applied to a panel of alcohols of pharmaceutical interest