From Anionic Ring-Opening Polymerization of β-Butyrolactone to Biodegradable Poly(hydroxyalkanoate)s: Our Contributions in This Field

The feasibility of synthesis of functionalized poly(3-hydroxybutanoic acid) analogue and its copolymers via ring-opening polymerization of β-butyrolactone mediated by activated anionic initiators is presented. Using these new synthetic approaches, polyesters with a defined chemical structure of the end groups, as well as block, graft, and random copolymers, have been obtained and characterized by modern instrumental techniques, with special emphasis on ESI-MS. The relationship between the structure and properties of the prepared polymeric materials is also discussed.

Synthesis and Characterization of Multifunctional Nanovesicles Composed of POPC Lipid Molecules for Nuclear Imaging

The integration of nuclear imaging analysis with nanomedicine has tremendously grown and represents a valid and powerful tool for the development and clinical translation of drug delivery systems. Among the various types of nanostructures used as drug carriers, nanovesicles represent intriguing platforms due to their capability to entrap both lipophilic and hydrophilic agents, and their well-known biocompatibility and biodegradability. In this respect, here we present the development of a labelling procedure of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)-based liposomes incorporating an ad hoc designed lipophilic NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) analogue, derivatized with an oleic acid residue, able to bind the positron emitter gallium-68(III). Based on POPC features, the optimal conditions for liposome labelling were studied with the aim of optimizing the Ga(III) incorporation and obtaining a significant radiochemical yield. The data presented in this work demonstrate the feasibility of the labelling procedure on POPC liposomes co-formulated with the ad hoc designed NOTA analogue. We thus provided a critical insight into the practical aspects of the development of vesicles for theranostic approaches, which in principle can be extended to other nanosystems exploiting a variety of bioconjugation protocols.

Access to New Cytotoxic Triterpene and Steroidal Acid-TEMPO Conjugates by Ugi Multicomponent-Reactions

Multicomponent reactions, especially the Ugi-four component reaction (U-4CR), provide powerful protocols to efficiently access compounds having potent biological and pharmacological effects. Thus, a diverse library of betulinic acid (BA), fusidic acid (FA), cholic acid (CA) conjugates with TEMPO (nitroxide) have been prepared using this approach, which also makes them applicable in electron paramagnetic resonance (EPR) spectroscopy. Moreover, convertible amide modified spin-labelled fusidic acid derivatives were selected for post-Ugi modification utilizing a wide range of reaction conditions which kept the paramagnetic center intact. The nitroxide labelled betulinic acid analogue 6 possesses cytotoxic effects towards two investigated cell lines: prostate cancer PC3 (IC50 7.4 ± 0.7 μM) and colon cancer HT29 (IC50 9.0 ± 0.4 μM). Notably, spin-labelled fusidic acid derivative 8 acts strongly against these two cancer cell lines (PC3: IC50 6.0 ± 1.1 μM; HT29: IC50 7.4 ± 0.6 μM). Additionally, another fusidic acid analogue 9 was also found to be active towards HT29 with IC50 7.0 ± 0.3 μM (CV). Studies on the mode of action revealed that compound 8 increased the level of caspase-3 significantly which clearly indicates induction of apoptosis by activation of the caspase pathway. Furthermore, the exclusive mitochondria targeting of compound 18 was successfully achieved, since mitochondria are the major source of ROS generation.

Phase II study of SM-88 in Ewing's and other sarcomas.

e23505 Background: Sarcomas are rare heterogeneous malignancies. Once recurrent, cure is uncommon. SM-88 (racemetyrosine) is an amino acid analogue with no known cross resistance to typical sarcoma regimens. Based on previous anecdotal experience in Ewing’s (EWS) we initiated a Phase 2 trial (HopES) in EWS and other sarcomas (Ss) after >1 prior systemic therapy. We now report preliminary data after having met prespecified continuation criteria. Methods: Open label prospective trial in 2 separate cohorts (EWS and Ss) of oral SM-88 used with MPS conditioning agents (SM-88 920 mg, methoxsalen 10 mg, phenytoin 50 mg, sirolimus 0.5 mg) all daily until progression. Results: As of Feb 5 2021, 10 pts with incurable sarcomas were enrolled; 4 had high risk but stable EWS. Average age 43.9 yrs (13–77); 70% white; 20% female. Median number of prior regimens 4 (1–9); 70% received prior RT; 50% prior surgery. Median time from initial diagnosis 39.5 months with 50% T2 (40% unknown), 30% M1 (30% unknown). Prespecified futility stopping was exceeded (i.e., >1 of first 5 subjects/cohort) upon achieving clinical benefit in each. Stable disease was achieved in 75.0% (6/8 with available data). Time on treatment (TTx) exceeded last known TTx in 80% (95% CI 44.4–97.5). Median SM-88 TTx was 4.9 vs 2.9 mo for prior TTx (logrank HR 0.53; p=0.12). One EWS subject had unresectable disease that became resectable, was completely resected, and remained disease-free for ≥ 6 months. Prior to SM-88, longest TTx was 12 mo (on IT*) and shortest TTx 1 mo (on IEV*) vs SM-88 TTx of 11.9 mo. An angiosarcoma subject had a 21% reduction in the sum of all target lesions and exceeded all prior TTx (including 8 mo on Ap/N* with 12+ mo duration of treatment of SM-88). There were no serious drug-related AEs. ECOG performance remained stable for all. Conclusions: SM-88 has exceeded pre-specified futility in both cohorts (EWS maintenance and Ss salvage). HopES continues to enroll toward the planned total of 12 subjects to more precisely define its benefit in this ultra-orphan, extremely recalcitrant disease. This trial now confirms the previously reported clinical utility of oral SM-88 in EWS and other high-risk sarcomas. Based on durable response (>6mo), SD and prolonged TTx, SM-88 warrants additional investigation in this setting. Clinical trial information: NCT03778996. [Table: see text]

A synthetic RNA-mediated evolution system in yeast

Laboratory evolution is a powerful approach to search for genetic adaptations to new or improved phenotypes, yet either relies on labour-intensive human-guided iterative rounds of mutagenesis and selection, or prolonged adaptation regimes based on naturally evolving cell populations. Here we present CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE) of genomic loci using evolving chimeric donor gRNAs continuously delivered from an error-prone T7 RNA polymerase, and directly introduced as RNA repair donors into genomic targets under either Cas9 or dCas9 guidance. We validate CRAIDE by evolving novel functional variants of an auxotrophic marker gene, and by conferring resistance to a toxic amino acid analogue in baker's yeast Saccharomyces cerevisiae with a mutation rate >3,000-fold higher compared to spontaneous native rate, thus enabling the first demonstrations of in vivo delivery and information transfer from long evolving RNA donor templates into genomic context without the use of in vitro supplied and pre-programmed repair donors.