Month: May 2022

Synthesis of “Nereid,” a new phenol-free detergent to replace Triton X-100 in virus inactivation

In the 1980s, virus inactivation steps were implemented into the manufacturing of biopharmaceuticals in response to earlier unforeseen virus transmissions. The most effective inactivation process for lipid-enveloped viruses is the treatment by a combination of detergents, often including Triton X-100 (TX-100). Based on recent environmental concerns, the use of TX-100 in Europe will be ultimately banned, which forces the pharmaceutical industry, among others, to switch to an environmentally friendly alternative detergent with fully equivalent virus inactivation performance such as TX-100. In this study, a structure–activity relationship study was conducted that ultimately led to the synthesis of several new detergents. One of them, named “Nereid,” displayed inactivation activity fully equivalent to TX-100. The synthesis of this replacement candidate has been optimized to allow for the production of several kg of detergent at lab scale, to enable the required feasibility and comparison virus inactivation studies needed to support a potential future transition. The 3-step, chromatography-free synthesis process described herein uses inexpensive starting materials, has a robust and simple work-up, and allows production in a standard organic laboratory to deliver batches of several hundred grams with >99% purity.

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To minimize the risk of transmitting infectious lipid-enveloped viruses like the hepatitis B or C viruses (HBV, HCV), Human immunodeficiency virus (HIV), and also emerging and unknown viruses, S/D treatment is still recognized as the most effective and robust method.3 Frequently, this mild chemical treatment contains a mixture of the non-ionic detergent TX-100 (I), tri-n-butyl-phosphate (TNBP), and often also Polysorbate 80 (PS80). (J Med Virol. 2021;93:3880–3889.)

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Triphenylphosphonium Moiety Modulates Proteolytic Stability and Potentiates Neuroprotective Activity of Antioxidant Tetrapeptides in Vitro

Although delocalized lipophilic cations have been identified as effective cellular and mitochondrial carriers for a range of natural and synthetic drug molecules, little is known about their effects on pharmacological properties of peptides. The effect of triphenylphosphonium (TPP) cation on bioactivity of antioxidant tetrapeptides based on the model opioid YRFK motif was studied. Two tetrapeptide variants with l-arginine (YRFK) and d-arginine (YrFK) were synthesized and coupled with carboxyethyl-TPP (TPP-3) and carboxypentyl-TPP (TPP-6) units. The TPP moiety noticeably promoted YRFK cleavage by trypsin, but effectively prevented digestion of more resistant YrFK attributed, respectively, to structure-organizing and shielding effects of the TPP cation on conformational variants of the tetrapeptide motif. The TPP moiety enhanced radical scavenging activity of the modified YRFK in a model Fenton-like reaction, whereas decreased reactivity was revealed for both YrFK and its TPP derivative. The starting motifs and modified oligopeptides, especially the TPP-6 derivatives, suppressed acute oxidative stress in neuronal PC-12 cells during a brief exposure similarly with glutathione. The effect of oligopeptides was compared upon culturing of PC-12 cells with CoCl2, l-glutamic acid, or menadione to mimic physiologically relevant oxidative states. The cytoprotective activity of oligopeptides significantly depended on the type of oxidative factor, order of treatment and peptide structure. Pronounced cell-protective effect was established for the TPP-modified oligopeptides, which surpassed that of the unmodified motifs. The protease-resistant TPP-modified YrFK showed the highest activity when administered 24 h prior to the cell damage. Our results suggest that the TPP cation can be used as a modifier for small therapeutic peptides to improve their pharmacokinetic and pharmacological properties.

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The tripeptide glutathione (γ-Glu-Cys-Gly, GSH) and histidine-containing dipeptides, namely carnosine (β-Ala-His) and its homologs, are predominant in mammals and the most studied oligopeptides with intrinsic ROS scavenging and antioxidative functions. Arginine-related bicyclic guanidinium oligomer, which is characterized by enhanced intracellular and intramitochondrial accumulation, was previously developed. Recently, a series of D-tetrapeptides containing two tyrosine residues were synthesized, and N-terminally trifluoroacetylated Tyr-Tyr-His-Pro-HN2 and Tyr-Tyr-Pro-His-NH2 sequence motifs were shown to have highest ability to scavenge ROS and inhibit membrane lipid peroxidation, which was comparable to that of Trolox.

Bioinspired artificial tetrapeptides comprising alternating aromatic (tyrosine, phenylalanine) and cationic (arginine, lysine) amino acids, called Szeto–Schiller (SS) peptides, were earlier developed as dermorphin analogs with pronounced scavenging action against different ROS (Schiller et al., 1989Szeto, 2006a). The aromatic-cationic structure of these peptides, i.e., Tyr-DArg-Phe-Lys-NH2 (starting sequence) and DArg-Dmt-Lys-Phe-NH2 (SS-31 sequence), allows them to pass the plasma membrane and concentrate at the inner membrane of mitochondria (Szeto, 2006a). Cytoprotective and antiapoptotic effects of the SS-peptides were shown to provide therapeutic benefit, for instance, upon exposure to cell-damaging agents (Zhao et al., 2005), ischemia-reperfusion injury (Szeto, 2008), hypertensive cardiomyopathy (Dai et al., 2011), neurodegeneration. (Front Pharmacol. 2018; 9: 115.)

Multiple Beneficial Health Effects of Natural Alkylglycerols from Shark Liver Oil

Alkylglycerols (alkyl-Gro) are ether lipids abundant in the liver of some elasmobranch fish species such as ratfishes and some sharks. Shark liver oil from Centrophorus squamosus (SLO), or alkyl-Gro mix from this source, have several in vivo biological activities including stimulation of hematopoiesis and immunological defences, sperm quality improvement, or anti-tumor and anti-metastasis activities. Several mechanisms are suggested for these multiple activities, resulting from incorporation of alkyl-Gro into membrane phospholipids, and lipid signaling interactions. Natural alkyl-Gro mix from SLO contains several alkyl-Gro, varying by chain length and unsaturation. Six prominent constituents of natural alkyl-Gro mix, namely 12:0, 14:0, 16:0, 18:0, 16:1 n-7, and 18:1 n-9 alkyl-Gro, were synthesized and tested for anti-tumor and anti-metastatic activities on a model of grafted tumor in mice (3LL cells). 16:1 and 18:1 alkyl-Gro showed strong activity in reducing lung metastasis number, while saturated alkyl-Gro had weaker (16:0) or no (12:0, 14:0, 18:0) effect. Multiple compounds and mechanisms are probably involved in the multiple activities of natural alkyl-Gro.

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The usual composition of alkyl chains in alkyl-Gro from Greenland shark (Centrophorus squamosus) liver oil (SLO) is as follow: 12:0, 1–2%. 14:0, 1–3%; 16:0, 9–13%; 16:1n-7, 11–13%; 18:0, 1–5%; 18:1n-9, 54–68%; 18:1n-7, 4–6%, and minor species (<1%).

Beneficial effects of SLO on health have been recognized in traditional medicine of northern countries involved in fishing, such as Japan, Norway or Iceland. In these countries, the ancestral use of SLO was empirical as strengthening or wound healing medication.  Lipids and phospholipids have prominent roles in cell-to-cell communication and in intracellular signals as well. Many lipid-derived bioactive molecules are agonists of specific membrane receptors: besides prostanoids and leukotriens, recent discoveries of new lipid mediators include docosanoids and many other eicosanoids such as isoprostanes, anandamide, 2-arachidonoylglycerol and other natural lipids acting as cannabinoid receptor agonists. Phospholipidic mediators are as well numerous, ubiquitous and of first rank interest; they include Platelet-activating Factor (PAF), lysophosphatidic acid (Lyso-PA), lysophosphatidylcholine (Lyso-PC), and shingosine-1-P (SP-1P). Several lipids act as intracellular second messengers (diacylglycerols, free fatty acids) or as regulators of transcription factors (free fatty acids, Lyso-PA).

Synthetic short chain alkyl-Gro such as 1-O-pentyl sn glycerol are interesting compounds, which transiently open the blood brain barrier (BBB), allowing increased crossing of the BBB by therapeutic molecules. Parenteral 1-O-decyl-sn-glycerol may also increase carbamazepine crossing through BBB. Oxidation of the natural unsaturated alkyl-Gro on the double bound could lead to short-chain alkyl-Gro with putative similar activities. (Mar Drugs. 2010; 8(7): 2175–2184.)