Labile Linker Research Articles

Detectability of Biotin Tags by LC-MS/MS.

The high affinity of biotin to streptavidin has made it one of the most widely used affinity tags in proteomics. Early methods used biotin for enrichment alone and mostly ignored the biotin-labeled peptide. Recent advances in labeling have led to an increase in biotinylation efficiency and shifted the interest to the detection of the site of biotinylation. This has increased the confidence in identification and provided additional structural information, yet it requires the efficient release of the biotinylated protein/peptide and the sensitive separation and detection of biotinylated peptides by LC-MS/MS. Despite its long use in affinity proteomics, the effect of biotinylation on the chromatographic, ionization, and fragmentation behavior and the ultimate detection of peptides is not well understood. To address this, we compare two commercially available biotin labels, EZ-Link Sulfo-NHS-Biotin and Sulfo-NHS-SS-Biotin, the latter containing a labile linker to efficiently release biotin to determine the effects of peptide modification on peptide detection. We describe an increase in the hydrophobicity and charge reduction with an increasing number of biotin labels attached. On the basis of our data, we recommend gradient optimization to account for more hydrophobic biotinylated peptides and include singly charged precursors to account for charge reduction by biotin.

Activatable polymer nanoagonist for second near-infrared photothermal immunotherapy of cancer

Nanomedicine in combination with immunotherapy offers opportunities to treat cancer in a safe and effective manner; however, remote control of immune response with spatiotemporal precision remains challenging. We herein report a photothermally activatable polymeric pro-nanoagonist (APNA) that is specifically regulated by deep-tissue-penetrating second near-infrared (NIR-II) light for combinational photothermal immunotherapy. APNA is constructed from covalent conjugation of an immunostimulant onto a NIR-II semiconducting transducer through a labile thermo-responsive linker. Upon NIR-II photoirradiation, APNA mediates photothermal effect, which not only triggers tumor ablation and immunogenic cell death but also initiates the cleavage of thermolabile linker to liberate caged agonist for in-situ immune activation in deep solid tumor (8 mm). Such controlled immune regulation potentiates systemic antitumor immunity, leading to promoted cytotoxic T lymphocytes and helper T cell infiltration in distal tumor, lung and liver to inhibit cancer metastasis. Thereby, the present work illustrates a generic strategy to prepare pro-immunostimulants for spatiotemporal regulation of cancer nano-immunotherapy.

Adsorption Properties of Soft Hydrophobically Functionalized PSS/MA Polyelectrolytes

We investigated the adsorption properties of the newly synthesized, hydrophobically functionalized polyelectrolyte (HF-PE), poly(4-styrenesulfonic-co-maleic acid) copolymer (PSS/MA). The hydrophobic alkyl side chains (C12 or C16) were incorporated into the polyelectrolyte backbone via the labile amid linker to obtain the soft HF-PE product with the assumed amount of 15% and 40% degree of grafting for every length of the alkyl chain, i.e., PSS/MA-g-C12NH2 (15% or 40%) as well as PSS/MA-g-C16NH2 (15% or 40%). In the present contribution, we determined both the effect of grafting density and the length of alkyl chain on adsorption at water/air and water/decane interfaces, as well as on top of the polyelectrolyte multilayer (PEM) deposited on a solid surface. The dependence of the interfacial tension on copolymer concentration was investigated by the pendant drop method, while the adsorption at solid surface coated by poly(diallyldimethylammonium chloride)/poly(styrene sulphonate) PEM by the quartz crystal microbalance with dissipation (QCM-D), attenuated total reflection Fourier transform infrared spectroscopy (FTIR-ATR) and contact angle analysis. We found that surface activity of the hydrophobized copolymer was practically independent of the grafting ratio for C16 side chains, whereas, for C12, the copolymer with a lower grafting ratio seemed to be more surface active. The results of QCM-D and FTIR-ATR experiments confirmed the adsorption of hydrophobized copolymer at PEM along with the modification of water structure at the interface. Finally, it can be concluded that the hydrophobically modified PSS/MA can be successfully applied either as the efficacious emulsifier for the formation of (nano)emulsions for further active substances encapsulation using the sequential adsorption method or as one of the convenient building blocks for the surface modification materials.

Encapsulation and Controlled Release of a Camptothecin Prodrug from Nanocarriers and Microgels: Tuning Release Rate with Nanocarrier Excipient Composition.

Nanocarriers (NCs) are an attractive class of vehicles for drug delivery with the potential to improve drug efficacy and safety, particularly for intravenous parenteral delivery. Many therapeutics remain challenging to formulate in NCs due to their intrinsic solubilities that frustrate NC loading or result in too rapid release in vivo. Therapeutic conjugate approaches that alter the solubility of a conjugate "prodrug" have been used to enable NC formation and controlled release from NCs using labile linker chemistry. A limitation of this approach has been that a different linker chemistry must be used to produce an adjustable release rate for a single therapeutic. We report on a new approach where the therapeutic conjugate hydrolysis rates are varied by adjusting the excipient formulation of the NC core, not the conjugate linker chemistry. A hydrophobic therapeutic conjugate of camptothecin (PROCPT) is synthesized by conjugating camptothecin (CPT) with an acid derivative of α-tocopherol (vitamin E). The PROCPT compound can be loaded to 50% wt in poly(lactic acid)-block-poly(ethylene glycol) (PLA-b-PEG)-stabilized NCs produced by Flash NanoPrecipitation with particle diameters between 60 and 80 nm. Co-loading a zwitterionic lipid, 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, from 0 to 67% core loading tunes the PROCPT hydrolysis from no observable therapeutic release over 200 h to therapeutic conjugate half-life times of 31 h. For a single therapeutic conjugate molecule, the hydrolysis rate can be tuned by modifying the NC formulation with different excipient concentrations. NCs containing a 50% core loading of PROCPT were lyophilized and encapsulated in a PEG hydrogel matrix to make microparticles for depot delivery with an average diameter of 65 ± 10 μm that provide a sustained, first-order release of CPT with a therapeutic conjugate half-life of 240 h. These results demonstrate a new approach to the formulation of therapeutic NCs with variable release profiles using a single molecular entity therapeutic conjugate.

Multifunctional cationic surfactants with a labile amide linker as efficient antifungal agents-mechanisms of action.

Our research aimed to expand the knowledge of relationships between the structure of multifunctional cationic dicephalic surfactants with a labile linker-N,N-bis[3,3-(dimethylamine)propyl]alkylamide dihydrochlorides and N,N-bis[3,3-(trimethylammonio)propyl]alkylamide dibromides (alkyl: n-C9H19, n-C11H23, n-C13H27, n-C15H31)-and their possible mechanism of action on fungal cells using the model organism Saccharomyces cerevisiae. General studies performed on surfactants suggest that in most cases, their main mechanism of action is based on perforation of the cell membranes and cell disruption. Experiments carried out in this work with cationic dicephalic surfactants seem to modify our understanding of this issue. It was found that the investigated compounds did not cause perforation of the cell membrane and could only interact with it, increasing its permeability. The surfactants tested can probably penetrate inside the cells, causing numerous morphological changes, and contribute to disorders in the lipid metabolism of the cell resulting in the formation of lipid droplet aggregates. This research also showed that the compounds cause severe oxidative stress within the cells studied, including increased production of superoxide anion radicals and mitochondrial oxidative stress. Dicephalic cationic surfactants due to their biodegradability do not accumulate in the environment and in the future may be used as effective antifungal compounds in industry as well as medicine, which will be environmentally friendly. KEY POINTS: • Dicephalic cationic surfactants do not induce disruption of the cell membrane. • Surfactants could infiltrate into the cells and cause accumulation of lipids. • Surfactants could cause acute oxidative stress in yeast cells. • Compounds present multimodal mechanism of action. Graphical abstract.

Novel N-Methylated Cyclodepsipeptide Prodrugs for Targeted Cancer Therapy.

Coibamide A (1) is a highly N-methylated cyclodepsipeptide with low nanomolar antiproliferative activities against various cancer cell lines. In previous work, we discovered a simplified analogue, [MeAla3-MeAla6]-coibamide (1a), which exhibited the same inhibitory abilities as coibamide A. Herein, to reduce the whole-body toxicity and improve the solubility of 1a, two novel peptide-drug conjugates RGD-SS-CA (2) and RGD-VC-CA (3) were designed, synthesized, and evaluated. Composed of cyclodepsipeptide 1a, a tumor-homing RGD motif, and a conditionally labile linker, the conjugates are expected to release 1a tracelessly in specific tumor microenvironments. Compared with RGD-VC-CA (3), RGD-SS-CA (2) proved to be superior in in vitro drug release and cytotoxicity tests. Notably, intravenous injection of RGD-SS-CA (2) into mice-bearing human tumor xenografts induced significant tumor growth suppression with negligible toxicity. Therefore, as a novel prodrug of the coibamide A analogue, conjugate 2 has great potential for further exploration in cancer drug discovery.

Antibacterial performance of fully biobased chitosan-grafted-polybenzoxazine films: Elaboration and properties of released material

A fully biobased benzoxazine monomer, V-fa (using vanillin and furfurylamine) was grafted onto chitosan (CS) at different weight ratios (CXVY) using "grafting to" benign Schiff base chemistry. Incorporation of V-fa onto CS increased the tensile strength and improved chemical resistance of the CS-graft-V-fa films. Reversible labile linkages, expansion of CS galleries and leaching out of phenolic species from biobased polymer films led to an improved antibacterial activity against Staphylococcus aureus, which is ∼125 times higher than the bare CS film, V-fa and oligomeric V-fa. The leached out species from films were analyzed extensively by NMR, FTIR, GPC, ABTS and HRMS analysis. Oxidative-stress seems to be responsible for antibacterial activity. Current work illustrates an attractive synthetic approach and the improved antibacterial performance of biobased CS-graft-poly(V-fa) films which may hold as a potential alternative for wound-healing and implant applications in future.

Dynamic Bonds Mediate π-π Interaction via Phase Locking Effect for Enhanced Heat Resistant Thermoplastic Polyurethane

Stimulus-responsive polymers containing dynamic bonds enable fascinating properties of self-healing, recycling and reprocessing due to enhanced relaxation of polymer chain/network with labile linkages. Here, we study the structure and properties of a new type of thermoplastic polyurethanes (TPUs) with trapped dynamic covalent bonds in the hard-phase domain and report the frustrated relaxation of TPUs containing weak dynamic bond and π-π interaction in hard segments. As detected by rheometry, the aromatic TPUs with alkyl disulfide in the hard segments possess the maximum network relaxation time in contrast to those without dynamic bonds and alicyclic TPUs. In situ FTIR and small-angle scattering results reveal that the alkyl disulfide facilitates stronger intermolecular interaction and more stable micro-phase morphology in π-π interaction based aromatic TPUs. Molecular dynamics simulation for pure hard segments of model molecules verify that the presence of disulfide bonds leads to stronger π-π stacking of aromatic rings due to both enhanced assembling thermodynamics and kinetics. The enhanced π-π packing and micro-phase structure in TPUs further kinetically immobilize the dynamic bond. This kinetically interlocking between the weak dynamic bonds and strong molecular interaction in hard segments leads to much slower network relaxation of TPU. This work provides a new insight in tuning the network relaxation and heat resistance as well as molecular self-assembly in stimulus-responsive dynamic polymers by both molecular design and micro-phase control toward the functional applications of advanced materials.

Dual-Location Dual-Acid/Glutathione-Degradable Cationic Micelleplexes through Hydrophobic Modification for Enhanced Gene Silencing.

Gene therapy holds great promise for the treatment of acquired genetic disorders such as cancer with reduced side effects compared to chemotherapy. For gene therapy to be successful, it is crucial to develop efficient and nontoxic gene carriers to overcome the poor in vivo stability and low cellular uptake of nucleic acid-based therapeutic agents. Here, we report a new and versatile approach exploring a combination of hydrophobic modifications and dual-stimuli-responsive degradation (SRD) for controlled gene delivery with amphiphilic block copolymer-based nanocarriers. The block copolymer, synthesized by atom transfer radical polymerization, is designed with an acid-labile acetal linkage at the block junction and a pendant disulfide group in the hydrophobic block. The incorporation of labile linkages enables both disulfide-core-cross-linking and dual-location dual-acid/reduction-responsive degradation (DL-DSRD). Furthermore, the disulfide linkages integrated as hydrophobic moieties facilitate the nucleic acids to condense into nanometer-sized micelleplexes through electrostatic interactions of pendant dimethylamino groups with the anionic phosphate groups of the nucleic acids. Our preliminary results demonstrate that the DL-DSRD approach through hydrophobic modification is a robust platform in the development of gene delivery systems with enhanced colloidal stability, reduced cytotoxicity, and improved gene transfection efficiency.

Abstract 1715: Anti-cancer activity of a SN-38 nanoparticle, DEP® irinotecan, in human colon cancer xenograft models

Abstract Irinotecan (Camptosar®) is a water-soluble prodrug of the potent topoisomerase I inhibitor SN-38 used clinically to treat multiple cancers. Irinotecan must be metabolized to its active metabolite SN-38 by carboxylesterases in hepatic and tumor cells. This metabolism in humans is inefficient with significant interpatient variability and leads to serious toxicities, notably diarrhea and myelosuppression. The narrow therapeutic window of irinotecan makes it an ideal candidate for improvement using dendrimer nanomedicine delivery. Starpharma's novel dendrimer nanoparticle DEP® platform has broad applicability in drug delivery, enhancing the therapeutic utility of drugs through improved solubility, efficacy and pharmacokinetics, reductions in certain toxicities (e.g. bone marrow toxicity) and providing novel intellectual property. The DEP platform has shown reproducible advantages across a wide range of drug classes including small molecule, peptides and proteins. Starpharma's DEP irinotecan is a PEGylated polylysine dendrimer with SN-38 conjugated to the surface via a hydrolytically labile linker. The incorporation of the active metabolite, SN-38 avoids the need for hepatic conversion and thereby minimizes variability in SN-38 levels. In preclinical studies, DEP irinotecan has shown to be well tolerated in animals. Efficacy assessments have been conducted, using a range of xenograft cancer models [SW620 (colon), HT-29 (colon), MDA-MB-231 (Breast) and CAPAN-1 (pancreatic)]. These studies demonstrated significant efficacy and survival benefits of DEP irinotecan compared to standard irinotecan. DEP irinotecan also showed significant benefit when dosed in combination with current standard of care therapies such as the anti-EGFR antibody, cetuximab, and the PARP inhibitor, olaparib. DEP irinotecan in combination with olaparib, significantly improved anti-tumor efficacy leading to tumor regression and extended median survival by 28 days compared to DEP irinotecan alone (P<0.0001, Mantel Cox Log rank test) whereas olaparib alone had minimal activity. This result highlights the synergistic combination benefits that can be achieved using DEP technology. DEP irinotecan is Starpharma's third internal DEP candidate to enter the clinic. The fourth DEP candidate, AZD0466 is a promising Bcl-2/Bcl-xL inhibitor and is partnered with AstraZeneca. AZD0466, is currently transitioning into clinical trials in the US. In summary, DEP irinotecan is well tolerated and allows for the delivery of SN-38 by successfully utilizing dendrimer drug delivery technology. The dendrimer nanoparticle shows significant efficacy and survival benefits compared to current standard of care therapies when used either as a monotherapy or in combination. DEP irinotecan is currently in a phase 1 / 2 adaptive trial at leading UK hospitals; The Christie, The Royal Marsden and The Newcastle Upon Tyne. Citation Format: Brian D. Kelly, Victoria McLeod, Rachael Walker, Jeannette Schreuders, Susan Jackson, Michael Giannis, Christine Dietinger, Shirley Xia, Anne Cargill, Aynaz Seta, Richard Hufton, Graham Heery, Carleen Cullinane, David J. Owen. Anti-cancer activity of a SN-38 nanoparticle, DEP® irinotecan, in human colon cancer xenograft models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1715.

Abstract 56: AZD0466, a nanomedicine of a potent dual Bcl-2/Bcl-xL inhibitor, exhibits anti-tumor activity in a range of hematological and solid tumor models

Abstract The induction of apoptosis in tumor cells represents a promising approach to the treatment of cancer. In tumor cells, the B cell lymphoma 2 (Bcl-2) protein family promotes cell survival through upregulation of anti-apoptotic Bcl-2 proteins, such as Bcl-2, Bcl-xL, Mcl-1 and Bcl-w. Clinical activity of the Bcl-2 inhibitor venetoclax has validated the approach of targeting this class of molecules, but additional value remains in jointly targeting Bcl-2 with other family members. AZD0466 is a novel drug-dendrimer conjugate, where the active moiety, AZD4320, is chemically conjugated to Starpharma's DEP® dendrimer platform, a 5-generation PEGylated poly-lysine dendrimer via a hydrolytically labile linker. AZD4320 is a potent dual Bcl-2/Bcl-xL inhibitor, with nanomolar affinity for both proteins1. AZD0466 has been optimized to maintain efficacy whilst mitigating anticipated on-target toxicities of AZD4320. The active moiety, AZD4320, was profiled in an unbiased 72 h cell proliferation screen of 764 cancer cell lines. The greatest degree of sensitivity to AZD4320 (IC50 value ≤0.1 µM) was observed in hematological and small cell lung cancer (SCLC) cell lines. AZD0466 demonstrated greater monotherapy activity than platinum/etoposide chemotherapy regimen or venetoclax monotherapy in 6 out of 11 SCLC PDX models. AZD0466 was also evaluated at different doses in the RS4;11 B-ALL xenograft model. Weekly intravenous dose of AZD0466 resulted in complete tumor regression at 34 and 103 mg/kg doses. Administration of a single dose of AZD0466 produced dose dependent induction of cleaved caspase 3 in tumors as measured by MSD ELISA, which was consistent with the concentrations of released AZD4320 measured in the tumor. All treatments were well tolerated. Anti-tumor activity of AZD0466 was also evaluated in the disseminated luciferase-tagged Ri-1-DLBCL tumor model. AZD0466 dosed weekly IV at 34 mg/kg showed approximately 82% inhibition of bioluminescence compared to vehicle treated animals, whereas 103 mg/kg and 340 mg/kg showed complete inhibition of bioluminescence. In the SUDHL-4 GCB DLBCL model, 103 mg/kg AZD0466 with 10 mg/kg Rituximab resulted in complete and durable regressions in 5/6 animals. Finally, combination of 103 mg/kg AZD0466 with 12.5 mg/kg BID Acalabrutinib, a Bruton's Tyrosine kinase inhibitor, was investigated in OCI-LY10 DLBCL model. While neither agent showed any demonstrable monotherapy activity the combination resulted in complete regressions in 8/8 mice in this model. These data show that AZD0466 has monotherapy activity and a differentiated response from Venetoclax in SCLC models. AZD0466 also has therapeutic potential as monotherapy and a combinatorial agent to increase the depth and duration of response to standard of care and BTK inhibitors in hematological tumors. 1Cidado, J; et al. AACR (2018) Citation Format: Srividya B. Balachander, Areya Tabatabai, Shenghua Wen, Francis D. Gibbons, Giulia Fabbri, Guangnong Sunny Zhang, Justin Cidado, Lorraine Graham, Marianne Ashford, Barry Davies. AZD0466, a nanomedicine of a potent dual Bcl-2/Bcl-xL inhibitor, exhibits anti-tumor activity in a range of hematological and solid tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 56.

Abstract 1718: Design and optimization of a dendrimer-conjugated dual Bcl-2/Bcl-xL inhibitor, AZD0466, with improved therapeutic index

Abstract Dual Bcl-2/Bcl-xL inhibitors are expected to deliver therapeutic benefit in many hematological and solid tumors, but their clinical application has been limited by tolerability issues, including thrombocytopenia. AZD4320, a potent dual Bcl-2/Bcl-xL inhibitor, showed good efficacy but encountered dose limiting cardiovascular toxicity in preclinical species, and had challenging physicochemical properties which prevented its clinical development. Nanocarriers can provide prolonged circulation time, controlled release, tumor accumulation and retention. Consequently, they have been explored to improve the therapeutic index of small molecules in oncology. This work describes the design and development of AZD0466, a novel drug-dendrimer conjugate, where AZD4320 is chemically conjugated to Starpharma's DEP® dendrimer platform, a 5-generation PEGylated poly-lysine dendrimer via a hydrolytically labile linker. Release of AZD4320 is through hydrolytic cleavage of the linker, which is characterized by a “release half-life”, defined as the time to release 50% of the active moiety. This release half-life can be modified through linker design. Initially, three drug-dendrimer conjugates with a range of AZD4320 release half-lives (from 1.7 h to 217 h) were synthesized and efficacy was investigated in C.B-17 SCID mice bearing RS4;11 tumors while cardiovascular parameters and tolerance were assessed in a telemetered rat model. A mathematical model was developed and used to optimize the desired release rate of the active moiety, AZD4320, from the dendrimer conjugate for maximal therapeutic index in terms of preclinical anti-tumor efficacy and cardiovascular profile. Based on this modeling, AZD0466, with a release half-life of 25.5 h, was synthesized and selected for further in vivo efficacy and tolerability assessment. Efficacy studies in the RS4;11 xenograft model showed similar efficacy to AZD4320, while cardiovascular studies in rat and dog demonstrated that AZD0466 was tolerated at doses of active-moiety that are more than ten-fold higher. In addition, it can be easily formulated for intravenous administration due to significantly enhanced solubility. The AZD4320-dendrimer conjugate, AZD0466, identified in this study has resulted in an improved therapeutic index and enabled progression of this promising Bcl-2/Bcl-xL inhibitor into clinical development. Citation Format: Marianne B. Ashford, Srividya B. Balachander, Lorraine Graham, Iain Grant, Francis D. Gibbons, Kathryn J. Hill, Alexander R. Harmer, Sonya Gales, Sean Redmond, Brian Kelly, William McCoull, Shenghua Wen, Martin Wild, Eric Gangl, David J. Owen, Barry R. Davies. Design and optimization of a dendrimer-conjugated dual Bcl-2/Bcl-xL inhibitor, AZD0466, with improved therapeutic index [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1718.

Abstract 1716: Anticancer activity of the taxane nanoparticles, DEP® docetaxel and DEP® cabazitaxel

Abstract Docetaxel (Taxotere®) and cabazitaxel (Jevtana®) are mitotic inhibitors that function as effective cytotoxic agents and are widely used in many chemotherapy regimens. However, treatment with taxanes is limited by serious adverse toxicities, notably bone marrow toxicity (neutropenia, leukopenia and anemia) and hepatotoxicity. Taxanes are poorly water soluble and must be formulated with surfactants such as polysorbate, which can cause systemic adverse events (e.g. anaphylaxis and fluid retention) requiring predosing with corticosteroids. These combined drug and excipient toxicities limit clinical use and make them ideal candidates for improvement using dendrimer technology. Starpharma's novel dendrimer nanoparticle DEP platform has broad applicability in drug delivery through improved drug solubility, efficacy and pharmacokinetics, reductions in certain toxicities (e.g. bone marrow toxicity) and generation of intellectual property. The DEP platform has shown reproducible benefits across a wide range of drug classes including small molecules, peptides and proteins. Currently there are three DEP candidates in the clinic; DEP docetaxel (DEP DTX), DEP cabazitaxel (DEP CTX), and DEP irinotecan. The fourth DEP candidate, AZD0466, is a promising Bcl-2/Bcl-xL inhibitor, partnered with AstraZeneca, and is currently transitioning into clinical trials in the US. Starpharma's DEP DTX and DEP CTX are both PEGylated polylysine dendrimers with the drug conjugated to the surface via a hydrolytically labile linker. Both products have demonstrated superior efficacy and survival compared to the standard drug formulations in a range of xenograft cancer models in immunocompromised mice: breast (MDA-MB-2231), prostate (DU145), and pancreatic (CAPAN-1). In the CAPAN-1 pancreatic xenograft model, the DEP taxanes were superior to Nab paclitaxel (Abraxane®) when dosed either as monotherapy or in combination with gemcitabine. Abraxane inhibited tumor growth by 85% (monotherapy) and 81% (combination with gemcitabine) compared to complete inhibition for DEP CTX and DEP DTX treated groups (at day 37). DEP CTX induced complete regression of tumors over the duration of the study. Treatment with DEP taxanes significantly extended survival compared to Abraxane as monotherapy (P= 0.004, DEP DTX; P <0.0001, DEP CTX), and in combination with gemcitabine (P<0.0001, DEP DTX; P<0.0001, DEP CTX). In summary, both DEP taxanes are well tolerated and show significant efficacy and survival benefits compared to current standard of care therapies when used either as a monotherapy or in combination. DEP CTX and DEP DTX are two of four clinical stage products from Starpharma's DEP platform. DEP DTX is currently in a P2 clinical trial at Guy's and St Thomas', NUTH, The Leeds Teaching Hospital, UCLH, The Christies and The Beatson while DEP CTX is in a phase 1/2 adaptive trial at Guy's and St Thomas', UCLH, Velindre Cancer Centre and ICLH. Citation Format: Brian D. Kelly, Victoria McLeod, Rachael Walker, Jeannette Schreuders, Susan Jackson, Michael Giannis, Christine Dietinger, Pauline Reitano, Rashmi Pathak, Shirley Xia, Anne Cargill, Aynaz Seta, Richard Hufton, Graham Heery, Carleen Cullinane, David J. Owen. Anticancer activity of the taxane nanoparticles, DEP® docetaxel and DEP® cabazitaxel [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1716.

Synthesis and Identification of a Novel Lead Targeting Survivin Dimerization for Proteasome-Dependent Degradation

Survivin, a homodimeric member of the Inhibitor of Apoptosis Protein (IAP) family, is required for cancer cell survival and overexpressed in almost all solid tumors. However, targeting survivin has been challenging due to its "undruggable" nature. Recently, we used a novel approach to target the dimerization interface and identified inhibitors of two scaffolds that can directly bind to and inhibit survivin dimerization. One of the scaffolds, represented by the compound LQZ-7, contains an undesirable labile hydrazone linker and a potentially nonfunctional furazanopyrazine ring that we attempted to eliminate in this study. We found one compound, 7I, that is more active than the parent compound, LQZ-7, and when given orally effectively inhibits xenograft tumor growth and induces survivin loss in tumors. These findings indicate that 7I with a stable linker and a quinoxaline ring can be used as a lead for further optimization of this novel class of survivin inhibitors.

Self-assembly of oxidation-responsive polyethylene glycol-paclitaxel prodrug for cancer chemotherapy

Amphiphilic drug conjugates can self-assemble into nanovehicles for cancer drug delivery, but the key is to design stable yet intracellular labile drug linkers for drug retention during blood circulation but fast intracellular drug release. The conjugation of paclitaxel (PTX) is generally via the ester of its 2′-hydroxyl group, but the ester is either too stable to release PTX in the cytosol or so labile that hydrolyzes during circulation. Herein, we report a p-(boronic ester)benzyl-based tumor-specifically cleavable linker for preparing PTX-conjugate with polyethylene glycol (PEG, Mw = 5000 Da) (PEG-B-PTX). The amphiphilic PEG-B-PTX self-assembled into micelle with an average size of ~50 nm and a PTX loading content of 13.3 wt%. The PEG-B-PTX micelles were very stable at the normal physiological environment and thus circulated long in the blood compartment, but fast dissociated and released PTX in response to the elevated reactive‑oxygen species (ROS) level in tumors. The conjugate micelles showed significantly improved antitumor efficiency in vitro and in vivo against human glioma and breast cancer cells, and reduced toxicity compared to the clinically used Taxol. Thus, the PTX-conjugate micelles were characteristic of well-characterized chemical structure and nanostructure, precise and reproducible drug loading efficiency (i.e., 100%) and fixed loading content, high PTX loading content due to PTX itself as part of the carrier, no burst drug release, and easy and reproducible fabrication of the micelles, which are all essential for clinical translation.

Natural Nanofiber Shuttles for Transporting Hydrophobic Cargo into Aqueous Solutions.

Hydrophobic biomolecules realize their functions in vivo in aqueous environments, often through a delicate balance of amphiphilicity and chaperones. Introducing exogenous hydrophobic biomolecules into in vivo aqueous systems is a challenge in drug delivery and regenerative medicine, where labile linkers, carriers, and fusions or chimeric molecules are often designed to facilitate such aqueous interfaces. Here, we utilize naturally derived silk nanofiber shuttles with the capacity to transport hydrophobic cargos directly into aqueous solutions. These nanofibers disperse in organic solvents and in aqueous solutions because of their inherent amphiphilicity, with enriched hydrophobicity and strategically interspersed negatively charged groups. Hydrophobic molecules loaded on these shuttles in organic solvent-water systems separated from the solvent after centrifugation. These concentrated hydrophobic molecule-loaded nanofibers could then be dispersed into aqueous solution directly without modification. These shuttle systems were effective for different hydrophobic molecules such as drugs, vitamins, and dyes. Improved biological stability and functions of hydrophobic cargos after loading on these nanofibers suggest potential applications in drug delivery, cosmetology, medical diagnosis, and related health fields, with a relatively facile process.

Doxorubicin-Loaded Delta Inulin Conjugates for Controlled and Targeted Drug Delivery: Development, Characterization, and In Vitro Evaluation.

Delta inulin, also known as microparticulate inulin (MPI), was modified by covalently attaching doxorubicin to its nanostructured surface for use as a targeted drug delivery vehicle. MPI is readily endocytosed by monocytes, macrophages, and dendritic cells and in this study, we sought to utilize this property to develop a system to target anti-cancer drugs to lymphoid organs. We investigated, therefore, whether MPI could be used as a vehicle to deliver doxorubicin selectively, thereby reducing the toxicity of this antibiotic anthracycline drug. Doxorubicin was covalently attached to the surface of MPI using an acid–labile linkage to enable pH-controlled release. The MPI-doxorubicin conjugate was characterized using FTIR and SEM, confirming covalent attachment and indicating doxorubicin coupling had no obvious impact on the physical nanostructure, integrity, and cellular uptake of the MPI particles. To simulate the stability of the MPI-doxorubicin in vivo, it was stored in artificial lysosomal fluid (ALF, pH 4.5). Although the MPI-doxorubicin particles were still visible after 165 days in ALF, 53% of glycosidic bonds in the inulin particles were hydrolyzed within 12 days in ALF, reflected by the release of free glucose into solution. By contrast, the fructosidic bonds were much more stable. Drug release studies of the MPI-doxorubicin in vitro, demonstrated a successful pH-dependent controlled release effect. Confocal laser scanning microscopy studies and flow cytometric analysis confirmed that when incubated with live cells, MPI-doxorubicin was efficiently internalized by immune cells. An assay of cell metabolic activity demonstrated that the MPI carrier alone had no toxic effects on RAW 264.7 murine monocyte/macrophage-like cells, but exhibited anti-cancer effects against HCT116 human colon cancer cells. MPI-doxorubicin had a greater anti-cancer cell effect than free doxorubicin, particularly when at lower concentrations, suggesting a drug-sparing effect. This study establishes that MPI can be successfully modified with doxorubicin for chemotherapeutic drug delivery.

437TiP - CNS penetration and preliminary efficacy of sacutizumab govitecan in breast brain metastasis and glioblastoma: A surgical study

BackgroundSacituzumab govitecan (SG) is an antibody drug conjugate (ADC) that targets Trop-2 for the selective delivery of SN-38 to tumors. SG carries SN-38, a topoisomerase inhibitor active in the nanomolar range for most cells (including TNBC and GBM) and freely cross the blood brain barrier. SN-38 is conjugated to SG by a linker designated CL2A which is sensitive to acidic and conditions. SG was evaluated in a single-arm, multicenter trial with relapsed/refractory metastatic triple negative breast cancer (TNBC) with impressive results including a confirmed objective response rate of 30%, with responses occurring early (median onset of 1.9 months) and being durable (median duration 8.9m). SG has since been granted priority review designation by the FDA, with approval anticipated. Brain metastases is a significant concern in this patient population, but whether this agent is able to target the CNS through the blood brain barrier is unknown. Based upon the characteristics of this specific ADC, including the use of a pH labile linker and a payload with good CNS penetration, it is our specific hypothesis that the SG can achieve intratumoral concentrations of SN-38 sufficient to achieve therapeutic benefit in patients with neoplastic involvement of the brain. We further hypothesize that while total concentration of SN-38 will correlate with expression of trop2, free SN-38 will correlate more strongly with intratumoral hypoxia. Trial designSingle center, non-randomized, prospective study of SG in subjects with CNS involvement and planned surgical resection. SG is given as single dose at 10mg/kg administered pre-operatively on Day-1. Surgery will be followed by post-operative treatment with sacituzumab govitecan given intravenously with standard dose of 10mg/kg on day1 and day 8 of 21-day cycle, until disease progression. Approximately 20 patients, 2 cohorts of 10 patients each with GBM and breast brain tumors from TNBC.Tumors will be analyszed for total antibody (hRS7 + hRS7-SN-38), free SN-38, and total SN-38 (free SN-38 + hRS7-SN-38) concentrations in tumor tissue. Correlations will be made to Trop2 expression and hypoxia. Legal entity responsible for the studyUniversity of Texas Health San Antonio. FundingMays Cancer Center, Kolitz Endowment. DisclosureA.J. Brenner: Research grant / Funding (institution): Immunomedics. V. Kaklamani: Research grant / Funding (institution): Immunomedics. V. Madhusudanan-Kunnuparampil: Research grant / Funding (institution): Immunomedics. All other authors have declared no conflicts of interest.

Influence of side-chain length on long-term release kinetics from poly(2-oxazoline)-drug conjugate networks

Four drug-conjugated poly(2-alkyl-2-oxazoline) (PAOx) networks with different hydrophobicity were synthesized via copolymerization of either 2-methyl-, 2-ethyl-, 2-propyl- or 2-butyl-2-oxazoline with the functional monomer, 2-dec-9-enyl-2-oxazoline. The incorporation of a labile ester linkage between the polymer and the drug benazepril allowed for sustained drug release over periods of months with the release rates strongly depending on the hydrophobicity of the polymer pendant groups. Drug loading of 13 ± 2 wt% was used with 10 mol% crosslinking sites simply by tuning the thiol-ene stoichiometry. The networks exhibited negligible cell toxicity but cell repulsion was observed for hydrogels based on poly(2-methyl-2-oxazoline) and poly(2-ethyl-2-oxazoline) while those based on poly(2-n-propyl-2-oxazoline) and poly(2-n-butyl-2-oxaoline) showed cell adhesion. These results suggest that PAOx networks have great potential as drug delivery devices for long-lasting drug release applications.

TRLS-08. CNS PENETRATION AND PRELIMINARY EFFICACY OF SACUTIZUMAB GOVITECAN IN BREAST BRAIN METASTASIS AND GLIOBLASTOMA: A SURGICAL STUDY

Sacituzumab govitecan (SG) is an antibody drug conjugate (ADC) that targets Trop-2 for the selective delivery of SN-38 to tumors. SG carries SN-38, a topoisomerase inhibitor active in the nanomolar range for most cells (including TNBC and GBM) and freely cross the blood brain barrier. SN-38 is conjugated to SG by a linker designated CL2A which is sensitive to acidic conditions. SG has since been granted priority review designation by the FDA, with approval anticipated for triple negative breast cancer. Brain metastases is a significant concern in this patient population, but whether this agent is able to target the CNS through the blood brain barrier is unknown. Based upon the characteristics of this specific ADC, including the use of a pH labile linker and a payload with good CNS penetration, it is our specific hypothesis that the SG can achieve intratumoral concentrations of SN-38 sufficient to achieve therapeutic benefit in patients with neoplastic involvement of the brain. We further hypothesize that while total concentration of SN-38 will correlate with expression of trop2, free SN-38 will correlate more strongly with intratumoral hypoxia. To address this, we are performing a non-randomized, prospective study of SG in subjects with CNS involvement and planned surgical resection. SG is given as single dose at 10mg/kg pre-operatively on Day-1. Surgery will be followed by post-operative treatment with sacituzumab govitecan given intravenously with standard dose of 10 mg/kg on day1 and day 8 of 21-day cycle, until disease progression. Approximately 20 patients, 2 cohorts of 10 patients each with GBM and breast brain tumors, will be enrolled. Tumors will be analyzed for total antibody, free SN-38, and total SN-38 (free SN-38 + Antibody-SN38) concentrations in tumor tissue. Correlations will be made to Trop2 expression and hypoxia. Interim results will be presented.