Polylysine Dendrimers Research Articles

Poly-basic peptides and polymers as new drug candidates against Plasmodium falciparum

BackgroundPlasmodium falciparum, the malaria-causing parasite, is a leading cause of infection-induced deaths worldwide. The preferred treatment approach is artemisinin-based combination therapy, which couples fast-acting artemisinin derivatives with longer-acting drugs, such as lumefantrine, mefloquine, and amodiaquine. However, the urgency for new treatments has risen due to the parasite's growing resistance to existing therapies. In this study, a common characteristic of the P. falciparum proteome—stretches of poly-lysine residues, such as those found in proteins related to adhesion and pathogenicity—is investigated for its potential to treat infected erythrocytes.MethodsThis study utilizes in vitro culturing of intra-erythrocytic P. falciparum to assess the ability of poly-lysine peptides to inhibit the parasite’s growth, measured via flow cytometry of acridine orange-stained infected erythrocytes. The inhibitory effect of many poly-lysine lengths and modifications were tested this way. Affinity pull-downs and mass spectrometry were performed to identify the proteins interacting with these poly-lysines.ResultsA single dose of these poly-basic peptides can successfully diminish parasitemia in human erythrocytes in vitro with minimal toxicity. The effectiveness of the treatment correlates with the length of the poly-lysine peptide, with 30 lysine peptides supporting the eradication of erythrocytic parasites within 72 h. PEG-ylation of the poly-lysine peptides or utilizing poly-lysine dendrimers and polymers retains or increases parasite clearance efficiency and bolsters the stability of these potential new therapeutics. Lastly, affinity pull-downs and mass-spectrometry identify P. falciparum’s outer membrane proteins as likely targets for polybasic peptide medications.ConclusionSince poly-lysine dendrimers are already FDA-approved for drug delivery and this study displays their potency against intraerythrocytic P. falciparum, their adaptation as anti-malarial drugs presents a promising new therapeutic strategy for malaria.

Abstract C167: An SN38 dendrimer nanoparticle, DEP irinotecan (SN38-SPL9111), demonstrates efficacy in mouse models of gastrointestinal cancer and augments anti-tumor effects of immune checkpoint blockade and PARP inhibition

Abstract Background: Irinotecan is a topoisomerase 1 inhibitor pro-drug used to treat gastrointestinal (GI) cancers including as first line for colorectal cancer (CRC) as part of the FOLFIRI plus monoclonal antibody (mAb) regimen, and for pancreatic cancer in the FOLFIRINOX regimen. However, irinotecan causes severe and dose-limiting side effects, including diarrhea and neutropenia. Starpharma has developed a highly optimized polylysine dendrimer enhanced (DEP) nanoparticle conjugate of the irinotecan active metabolite, SN38 (DEP SN38 or DEP irinotecan). DEP SN38 avoids the need for liver conversion of irinotecan to SN38 and achieves preferential tumor targeting. DEP SN38 is in phase 2 clinical investigation as monotherapy or combination therapy in patients with solid tumors (EudraCT no: 2019-001318-40). SN38 is known to enhance the efficacy of immune checkpoint blockade (ICB) via effects on immune cells in the tumor microenvironment. Here, we evaluated the anti-tumor effects of DEP SN38 alone or in combination with either ICB (anti- programmed cell death-1 [PD1] mAb) or poly adenosine diphosphate (ADP) ribose polymerase (PARP) inhibition (olaparib) in mouse models of GI cancer. Methods: HT29 (human CRC) and CAPAN-1 (human pancreatic cancer) cell line xenografts were established subcutaneously in BALB/c nude or NOD-scid-Gamma immunodeficient mice, respectively. Three doses of DEP SN38 (at 5-10mg/kg SN38) or irinotecan (35-80 mg/kg) were administered intravenously (IV) on days 1, 8, 15. Olaparib (50 mg/kg) was delivered by daily IV injection for 5 days/week over 3 weeks. DEP SN38 (IV on days 1, 8, 15 at 18-35 mg/kg SN38) or irinotecan (75 mg/kg) plus rat anti-PD1 mAb therapy or control mAb (intraperitoneally, 100 mg/kg then 50 mg/kg on days 5, 8, 12) was evaluated in both MC38 (C57BL/6) and CT26 (BALB/c) allograft models of CRC. Toleration of drug was bodyweight loss ≤15%. Results: DEP SN38 treatment of HT29 or CAPAN-1 xenografts led to tumor regressions, prolonged control of tumor growth and improved survival. In contrast, conventional irinotecan had minimal effect on tumor growth versus vehicle control. DEP SN38 was well tolerated by mice with minimal bodyweight loss. Administration of anti-PD1 mAb alone confirmed the known modest sensitivity of MC38, and resistance of CT26 syngeneic tumors, to ICB. DEP SN38 plus anti-PD1 mAb, enhanced anti-tumor responses in the MC38 model beyond those seen for each agent alone and also resulted in tumor eradication and prolonged survival. In the CT26 model, anti-PD1 mAb was also augmented by addition of DEP SN38 but not irinotecan alone, despite resistance of CT26 tumors to anti-PD1 mAb alone. The HT29 CRC model was resistant to Olaparib alone. In contrast, anti-tumor effects and prolonged survival were observed with olaparib plus DEP SN38. Conclusions: DEP SN38 demonstrated superior efficacy compared to irinotecan when used in models of GI cancer. The anti-tumor efficacy of both ICB and PARP inhibition was augmented by DEP SN38 in models of CRC, and these combinations should be investigated clinically. Citation Format: Benjamin J Blyth, Brian D Kelly, Michael Giannis, Anne Cargill, Aynaz Seta, Graham P Heery, Anthony Eglezos, Cameron N Johnstone, Jeremy R A Paull. An SN38 dendrimer nanoparticle, DEP irinotecan (SN38-SPL9111), demonstrates efficacy in mouse models of gastrointestinal cancer and augments anti-tumor effects of immune checkpoint blockade and PARP inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C167.

Abstract A147: A HER2 targeted polylysine dendrimer nanoparticle radiotheranostic demonstrates excellent tumor accumulation, rapid clearance from circulation, and promising performance in PET-CT imaging

Abstract Background: Development of targeted radiotheranostics for human epidermal growth factor receptor type 2 positive (HER2+) tumors has lagged advances made in other receptor systems such as prostate-specific membrane antigen (PSMA) in prostate cancer. Myelosuppression is a dose limiting toxicity in clinical studies of trastuzumab radiotheranostics due to slow clearance of this large radiotherapeutic from circulation. Conjugation of chemotherapeutics such as taxanes and irinotecan/SN38 to highly optimized dendrimer nanoparticles improves pharmacokinetics (PK), tumor uptake and antitumor efficacy in vivo. Dendrimers can be used as targeted radiotheranostics with potential to achieve superior PK, enhanced tumor killing, and reduced toxicity (improved therapeutic index) compared to traditional antibody-based radiotheranostics. A radio-imaging and biodistribution study in mice implanted with HER2+ tumors directly compared the imaging performance, in positron emission tomography-computed tomography (PET-CT), and kinetics of tumor and normal tissue uptake of a HER2-targeted dendrimer radiotheranostic to radiolabeled trastuzumab. Methods: A HER2 targeted VHH (single domain antibody [sdAb], or antigen binding fragment of heavy chain only camelid antibody) was covalently linked to a generation 4 dendrimer with 16 deferoxamine (DFO) chelation groups on its surface (DEP-HER2-DFO). Trastuzumab was covalently linked to DFO (trastuzumab-DFO). DEP-HER2-DFO and trastuzumab-DFO were successfully radiolabeled with 89Zr to high specific activity. Female BALB/c nude mice were inoculated subcutaneously with BT474 HER2+ breast cancer cells. When tumors reached 150mm3, a single dose of DEP-HER2-DFO-89Zr and trastuzumab-DFO-89Zr (3MBq) was administered intravenously. Uptake was evaluated in tumor and normal tissues by ex vivo biodistribution (n=3/group). In vivo biodistribution (n=3/group) was determined using quantified PET-CT data (4h-5d). PET-CT images were obtained on day 2 and 4. Results: Both DEP-HER2-DFO-89Zr and trastuzumab-DFO-89Zr showed sustained tumor accumulation of >20% injected dose per gram (ID/g) over several days with low levels of normal tissue uptake (<5% ID/g), as determined by ex vivo biodistribution. However, DEP-HER2-DFO-89Zr was cleared from the vascular circulation more quickly than trastuzumab-DFO-89Zr resulting in superior tumor/blood, tumor/heart, and tumor/lung ratios. These findings were corroborated by quantified PET-CT data. Excellent signal to noise (tumor to normal tissue) was found for DEP-HER2-DFO-89Zr on whole body PET-CT images. DEP-HER2-DFO-89Zr was stable in serum and not susceptible to radiolysis over 7 days. Conclusions: DEP-HER2-DFO-89Zr achieved high levels of accumulation in tumor, low uptake in normal tissues and relatively fast clearance from circulation, yielding favorable tumor/blood ratios compared with trastuzumab-DFO-89Zr. The dendrimer-based DEP-HER2 radiotheranostic offers advantages over antibody-based radiotheranostics and is under development ahead of a clinical biodistribution study. Citation Format: Cameron N Johnstone, Graham P Heery, Brian D Kelly, James Humphries, Dewan T Akhter, Kristofer J Thurecht, Daniel Yuen, Michael Giannis, Ken Yong, Gareth Chadwick, Helena Barker, Anne Cargill, Anthony Eglezos, Alex Castellarnau, Jeremy R A Paull, Richard Hufton. A HER2 targeted polylysine dendrimer nanoparticle radiotheranostic demonstrates excellent tumor accumulation, rapid clearance from circulation, and promising performance in PET-CT imaging [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A147.

Abstract 6150: AZD0466, a dual BCL-2/XL targeting nanomedicine, is active in small cell lung cancer models

Abstract Small cell lung cancer (SCLC) is an aggressive malignancy with critical need for new therapies. While currently treated as a single disease, SCLC is heterogenous, comprised of several transcriptional subtypes. Each of these subtypes has distinct drivers and may warrant unique therapeutic targets. Two potential therapeutic targets for SCLC are the pro-survival proteins BCL-2 and BCL-XL. BCL-2 is overexpressed in ASCL1 (A) and POUF3 (P) subtypes of SCLC. We therefore sought to evaluate efficacy of the dual BCL-2/XL inhibitor AZD0466 in SCLC models and to determine whether transcriptional subtype would predict response. AZD0466 is a novel drug-dendrimer conjugate. The active moiety, AZD4320, is a potent dual inhibitor of BCL-2 and BCL-XL. AZD4320 is covalently conjugated to a 5th-generation PEGylated poly-lysine dendrimer through a hydrolytically labile linker to make AZD0466. AZD0466 has been optimized to deliver efficacy while mitigating potential Cmax-driven on-target toxicities of AZD4320. AZD4320 was active (IC50 ≤0.1 µM) in 9/27 SCLC cell lines. AZD4320 in vitro sensitivity was enriched in cell lines that represented A and P subtypes of SCLC compared to NEUROD1 and YAP1 subtypes. We next profiled AZD0466 in a panel of SCLC patient-derived models: 14 patient-derived xenografts and 10 circulating tumor cell-derived xenografts. AZD0466 monotherapy dosed weekly IV was active in 12/24 SCLC xenografts, driving regressions in 8 models. AZD0466 drove efficacy and cleaved caspase-3 induction in a dose-dependent manner. Similar to in vitro, AZD0466 in vivo efficacy was enriched in subtype-A, driving responses in 10/14 ASCL1 models (7 regression, 3 stable disease). AZD0466 response also correlated strongly with BCL-2 mRNA expression (P<0.0001). AZD0466 outperformed the selective BCL-2 inhibitor venetoclax in 6/10 models. Notably, AZD0466 was active in models resistant to platinum/etoposide chemotherapy, the standard-of-care for SCLC. Together, these data suggest BCL-2/XL inhibition has therapeutic potential in SCLC. AZD0466 is in clinical development. The first-in-human study treated 9 patients with advanced solid tumors (NCT04214093) at doses from 50-200mg, all of which were well-tolerated. The BOR was SD observed in 3 patients (100mg) with 1 patient receiving treatment for 5.5 months. AZD0466 is now under evaluation in patients with hematologic malignancies (NCT04865419 and NCT05205161). AZD0466 has been dosed in 33 patients up to 2400mg. No DLTs have been reported to date. Initial clinical activity has been observed through reduction of bone marrow blasts following AZD0466 treatment. AZD0466 exhibits linear PK, consistent across solid tumor and leukemia patients. The doses tested are in line with preclinical studies in SCLC. Citation Format: Courtney L. Andersen, Giulia Fabbri, David Jenkins, Zumla Cader, Shringi Sharma, Areya Tabatabai, Srividya Balachander, Jordan Roebuck, Melanie Galvin, Kathryn Simpson, Caroline Dive, Jordi Rodon Ahnert, Jamal Saeh. AZD0466, a dual BCL-2/XL targeting nanomedicine, is active in small cell lung cancer models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6150.

Dye-cored polylysine dendrimer as luminescent nanoplatform for imaging-guided anticancer drug delivery

Dendrimers have numerous applications in imaging and drug delivery. Designing a dendrimer diagnostic platform with a well-defined structure and controlled drug delivery is a formidable challenge. Here, we design dendritic polymer-platinum conjugates (G5-PEG-Pt) as pH-responsive nanovesicles for imaging-guided platinum drug delivery. The G5-PEG-Pt have a well-defined structure, intrinsically bright fluorescence, and acid-responsive drug release. The pH-responsive G5-PEG-Pt could rapidly release the platinum drug at acidic pH (5.0) than neutral pH (7.4). The G5-PEG-Pt could enter SKOV-3 human ovarian cancer cells by the endocytosis pathway and exhibited comparative cytotoxicity to free cisplatin. By virtue of the prolonged blood circulation time and the enhanced permeability and retention (EPR) effect, a 4.4-fold higher tumor platinum uptake than that of free cisplatin was achieved, potentially enhancing the therapeutic indexes of the platinum drug. Therefore, these pH-responsive platinum and fluorescent dendrimer conjugates are expected to be potent in vivo cancer optical imaging and therapy platforms.

Surface chemistry mediates the tumor entrance of nanoparticles probed using single-molecule dual-imaging nanodots.

The active transport of nanoparticles into solid tumors through transcytosis has been recognized as a promising way to enhance tumor accumulation and penetration, but the effect of the physicochemical properties of nanoparticles remains unclear. Herein, we develop a type of single-molecule dual imaging nanodot by divergent growth of perylenediimide (PDI)-dye-cored polylysine dendrimers and internal orthogonal conjugation of Gd(III)-based macrocyclic probes for fluorescence imaging and magnetic resonance imaging (MRI) of surface chemistry-dependent tumor entrance. The MRI and fluorescence imaging show that sixth-generation nanodots with acetylated (G6-Ac) and oligo ethylene glycol (G6-OEG) surfaces exhibit similar high tumor accumulation but different intratumor distribution. Cellular uptake and transport experiments suggest that G6-Ac nanodots have lower lysosomal entrapment (61% vs. 83%) and a higher exocytotic rate (47% vs. 29%) than G6-OEG. Therefore, G6-Ac is more likely to undergo intercellular transport through cell transcytosis, and is able to reach a tumor area distant from blood vessels, while G6-OEG mainly enters the tumor through enhanced permeability and retention (EPR) effect-based passive transport, and is not able to deliver to distant tumor areas. This study suggests that it is possible to boost the tumor entrance of nanoparticles by engineering surface chemistry for active transport.

The Global Characterisation of a Drug-Dendrimer Conjugate - PEGylated poly-lysine Dendrimer

The recent emergence of drug-dendrimer conjugates within pharmaceutical industry research and development introduces a range of challenges for analytical and measurement science. These molecules are very high molecular weight (100-200kDa) with a significant degree of structural complexity. The characteristics and quality attributes that require understanding and definition, and impact efficacy and safety, are diverse. They relate to the intact conjugate, the various building blocks of these complex systems and the level of the free and bound active pharmaceutical ingredient (API). From an analytical and measurement science perspective, this necessitates the measurement of the molecular weight, impurity characterisation, the quantitation of the number of conjugated versus free API molecules, the determination of the impurity profiles of the building blocks, primary structure and both particle size and morphology.Here we report the first example of a global characterisation of a drug-dendrimer conjugate - PEGylated poly-lysine dendrimer currently under development (AZD0466). The impact of the wide variety of analytical and measurement techniques on the overall understanding of this complex molecular entity is discussed, with the relative capabilities of the various approaches compared. The results of this study are an essential platform for the research and development of the future generations of related dendrimer-based medicines.

Molecularly Precise, Bright, Photostable, and Biocompatible Cyanine Nanodots as Alternatives to Quantum Dots for Biomedical Applications

Fluorescent imaging with fluorophores has become a powerful way to explore complex biological systems and visualize nanoparticles for drug delivery. However, it is challenging to develop fluorophores with ideal physical and optical properties. We report a method to synthesize cyanine nanodots with a single-molecule structure, well-defined particle size, customizable fluorescent spectrum, and bright and stable fluorescence. These cyanine nanodots are acquired by the divergent synthesis of cyanine-dye-cored polylysine (PLL) dendrimers. We demonstrated the feasibility of the method by synthesizing cyanine 3 (Cy3), cyanine 5 (Cy5), or cyanine 7 (Cy7) cored single-molecule nanodots up to eight generations with a size of around 11 nm. We show that these cyanine nanodots are capable of multiple biomedical applications, including multicolor cellular tracing and cancer imaging. These cyanine nanodots possess many merits of organic dots and quantum dots that are promising for future application.

Molecularly Precise, Bright, Photostable, and Biocompatible Cyanine Nanodots as Alternatives to Quantum Dots for Biomedical Applications.

Fluorescent imaging with fluorophores has become a powerful way to explore complex biological systems and visualize nanoparticles for drug delivery. However, it is challenging to develop fluorophores with ideal physical and optical properties. We report a method to synthesize cyanine nanodots with a single-molecule structure, well-defined particle size, customizable fluorescent spectrum, and bright and stable fluorescence. These cyanine nanodots are acquired by the divergent synthesis of cyanine-dye-cored polylysine (PLL) dendrimers. We demonstrated the feasibility of the method by synthesizing cyanine 3 (Cy3), cyanine 5 (Cy5), or cyanine 7 (Cy7) cored single-molecule nanodots up to eight generations with a size of around 11 nm. We show that these cyanine nanodots are capable of multiple biomedical applications, including multicolor cellular tracing and cancer imaging. These cyanine nanodots possess many merits of organic dots and quantum dots that are promising for future application.

Synthesis and Self‐Assembly of Stimuli‐Responsive Amphiphilic Dendrimers

AbstractThe synthesis of stimuli‐responsive amphiphilic dendrimers consisting of two or three hydrophobic octadecyl groups linked to either a second or third generation hydrophilic polylysine dendron through disulfide linkage is reported. The polylysine dendrimers functionalized with four or eight tert‐butyloxycarbonyl (Boc)‐protected amino groups are characterized by 1H NMR spectroscopy and matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. With the positively charged versions generated by removing the Boc groups, their ability to form spherical micelles that can be readily disassembled in the presence of glutathione (GSH) is demonstrated. The self‐assembly behavior is monitored by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The amphiphilic dendritic systems containing cleavable disulfide linkage serve as key building blocks for generating stimuli‐responsive supramolecular architectures.

A novel BH3-mimetic, AZD0466, targeting BCL-XL and BCL-2 is effective in pre-clinical models of malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive cancer with treatment limited to Cisplatin and Pemetrexed chemotherapy. Recently, we showed that drugs targeting the BCL-2-regulated apoptosis pathway could kill MPM cell lines in vitro, and control tumor growth in vivo. These studies showed BCL-XL was the dominant pro-survival BCL-2 family member correlating with its high-level expression in cells and patient tumor samples. In this study we show another inhibitor, AZD4320 that targets BCL-XL (and BCL-2), can also potently kill MPM tumor cells in vitro (EC50 values in the 200 nM range) and this effect is enhanced by co-inhibition of MCL-1 using AZD5991. Moreover, we show that a novel nanoparticle, AZD0466, where AZD4320 is chemically conjugated to a PEGylated poly-lysine dendrimer, was as effective as standard-of-care chemotherapy, Cisplatin, at inhibiting tumor growth in mouse xenograft studies, and this effect was enhanced when both drugs were combined. Critically, the degree of thrombocytopenia, an on-target toxicity associated with BCL-XL inhibition, was significantly reduced throughout the treatment period compared to other BCL-XL-targeting BH3-mimetics. These pre-clinical findings provide a rationale for the future clinical evaluation for novel BH3-mimetic formulations in MPM, and indeed, other solid tumor types dependent on BCL-XL.

Design and optimisation of dendrimer-conjugated Bcl-2/xL inhibitor, AZD0466, with improved therapeutic index for cancer therapy

Dual Bcl-2/Bcl-xL inhibitors are expected to deliver therapeutic benefit in many haematological and solid malignancies, however, their use is limited by tolerability issues. AZD4320, a potent dual Bcl-2/Bcl-xL inhibitor, has shown good efficacy however had dose limiting cardiovascular toxicity in preclinical species, coupled with challenging physicochemical properties, which prevented its clinical development. Here, we describe the design and development of AZD0466, a drug-dendrimer conjugate, where AZD4320 is chemically conjugated to a PEGylated poly-lysine dendrimer. Mathematical modelling was employed to determine the optimal release rate of the drug from the dendrimer for maximal therapeutic index in terms of preclinical anti-tumour efficacy and cardiovascular tolerability. The optimised candidate is shown to be efficacious and better tolerated in preclinical models compared with AZD4320 alone. The AZD4320-dendrimer conjugate (AZD0466) identified, through mathematical modelling, has resulted in an improved therapeutic index and thus enabled progression of this promising dual Bcl-2/Bcl-xL inhibitor into clinical development.

Molecular level precision and high molecular weight peptide dendrimers for drug-specific delivery.

Peptide dendrimers have a broad application in biomedical science due to their biocompatibility, diversity, and multifunctionality, but the precision synthesis of high-molecule weight peptide dendrimers remains challenging. We here report the facile and liquid-phase synthesis of molecular level precision and amino-acid built-in polylysine (PLL) dendrimers with molecular weights as high as ∼60 kDa. Three types of polyhedral oligosilsesquioxane (POSS)-cored PLL dendrimers with phenylalanine, tyrosine, or histidine as building blocks were synthesized. The precise structures of the dendrimers were confirmed by MALDI-TOF MS, GPC, and 1H NMR spectroscopy. The interior functionalized peptide dendrimers improved the encapsulation capability of SN38 and sustained the release profiles. Enhanced molecular interactions between the peptide dendrimers and drugs were explored by both NMR experiments and computer simulations. The peptide dendrimer/SN38 formulations showed potent antitumor activity against multiple cancer cell lines. We believe that this strategy can be applied to the synthesis of tailor-made functional peptide dendrimers for drug-specific delivery and other diverse biomedical applications.

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.

Elucidation of anti-HIV mechanism of sulfated cellobiose–polylysine dendrimers

Three new spherical sulfated cellobiose–polylysine dendrimers of increasing generations bearing negatively charged sulfate groups were prepared by sulfating the corresponding cellobiose–polylysine dendrimers. The first, second, and third-generation derivatives exhibited potent anti-HIV activity with EC50 values of 3.7, 0.6, and 1.5 μg/mL, respectively, in constant to sulfated oligosaccharides with low anti-HIV activity, while the second-generation sulfated dendrimer was the most active. Surface plasmon resonance measurements with poly-l-lysine bearing positively charged amino acids as a model of the HIV surface glycoprotein gp120, indicated that the second-generation dendrimer had the lowest dissociation constant (KD = 1.86 × 10−12 M). Both the particle size and ζ potential increased in the presence of poly-l-lysine. It was proven that the moderate distance between the terminal sulfated cellobiose units in the second-generation dendrimer favored the high anti-HIV activity, owing to the electrostatic interactions developed due to the cluster effect.

Tuning the Brightness and Photostability of Organic Dots for Multivalent Targeted Cancer Imaging and Surgery.

Specific labeling of biomarkers with bright and high photostable fluorophores is vital in fluorescent imaging applications. Here, we report a general strategy to develop single-molecule dendritic nanodots with finely tunable optical properties for in vivo fluorescent imaging. The well-defined nanodots are based on the divergent growth of biodegradable polylysine dendrimers with a fluorophore as the core. By tuning the size and surface chemistry, we obtained fluorescent nanodots with excellent brightness and photostability, favorable pharmacokinetics, and multivalent tumor-targeting capability. The nanodots provided robust, stable, long-lasting, and specific fluorescence enhancement in tumor tissue with an in situ tumor-to-normal ratio (TNR) of ∼3 and lasting over 5 days and an ex vivo TNR up to ∼17, holding considerable promise for cancer imaging and image-guided surgery. This strategy significantly improves the in vivo performance of fluorophores and can be applied to other modality imaging probes.

Sugar Functionalized Synergistic Dendrimers for Biocompatible Delivery of Nucleic Acid Therapeutics.

Sugars containing cationic polymers are potential carriers for in vitro and in vivo nucleic acid delivery. Monosaccharides such as glucose and galactose have been chemically conjugated to various materials of synergistic poly-lysine dendrimer systems for efficient and biocompatible delivery of short interfering RNA (siRNA). The synergistic dendrimers, which contain lipid conjugated glucose terminalized lysine dendrimers, have significantly lower adverse impact on cells while maintaining efficient cellular entry. Moreover, the synergistic dendrimers complexed to siRNA induced RNA interference (RNAi) in the cells and profoundly knocked down green fluorescence protein (GFP) as well as the endogenously expressing disease related gene Plk1. The new synergic dendrimers may be promising system for biocompatible and efficient siRNA delivery.