Dioleoylphosphatidylserine Research Articles

Nanoscale lipid domains determine the dynamic molecular portraits of mixed DOPC/DOPS bilayers in a fluid phase: A computational insight

Lateral heterogeneity, or mosaicity, is a fundamental property inherent to cell membranes that is crucial for their functioning. While microscopic inhomogeneities (e.g. rafts) are easily detected experimentally, lipid domains with nanoscale dimensions (nanoclusters of nanodomains, NDs) resist reliable characterization by instrumental methods. In such a case, important insight can be gained via computer modeling. Here, NDs composed of lipid's head groups in the mixed zwitterionic dioleoylphosphatidylcholine (DOPC) and negatively charged dioleoylphosphatidylserine (DOPS) bilayers were studied by molecular dynamics. A new algorithm has been developed to identify NDs. Unlike most similar methods, it implicitly considers the heterogeneous distribution of lipid head atomic density and does not require subjectively chosen parameters. In DOPS-rich membranes, lipids form more compact and stable NDs due to strong interlipid interactions. In DOPC-rich systems, NDs arise due to the “packing” effect of weakly bound lipid heads. The clustering picture is related to the physical properties of the bilayer surface: DOPS-rich systems show more pronounced surface heterogeneity of hydrophilic/hydrophobic regions compared to DOPC-rich ones. The results obtained are important for the effective quantitative characterization of the “dynamic molecular portrait” of a membrane surface – its “fingerprint” characterizing dynamical distribution of its physicochemical properties.

Phospholipids Differentially Regulate Ca2+ Binding to Synaptotagmin-1.

Synaptotagmin-1 (Syt-1) is a calcium sensing protein that is resident in synaptic vesicles. It is well established that Syt-1 is essential for fast and synchronous neurotransmitter release. However, the role of Ca2+ and phospholipid binding in the function of Syt-1, and ultimately in neurotransmitter release, is unclear. Here, we investigate the binding of Ca2+ to Syt-1, first in the absence of lipids, using native mass spectrometry to evaluate individual binding affinities. Syt-1 binds to one Ca2+ with a KD ∼ 45 μM. Each subsequent binding affinity (n ≥ 2) is successively unfavorable. Given that Syt-1 has been reported to bind anionic phospholipids to modulate the Ca2+ binding affinity, we explored the extent that Ca2+ binding was mediated by selected anionic phospholipid binding. We found that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and dioleoylphosphatidylserine (DOPS) positively modulated Ca2+ binding. However, the extent of Syt-1 binding to phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) was reduced with increasing [Ca2+]. Overall, we find that specific lipids differentially modulate Ca2+ binding. Given that these lipids are enriched in different subcellular compartments and therefore may interact with Syt-1 at different stages of the synaptic vesicle cycle, we propose a regulatory mechanism involving Syt-1, Ca2+, and anionic phospholipids that may also control some aspects of vesicular exocytosis.

X-ray reflectivity study of the heat shock protein Hsp70 interaction with an artificial cell membrane model

Membrane-bound heat shock protein 70 (Hsp70) apart from its intracellular localization was shown to be specifically expressed on the plasma membrane surface of tumor but not normal cells. Although the association of Hsp70 with lipid membranes is well documented the exact mechanisms for chaperone membrane anchoring have not been fully elucidated. Herein, we addressed the question of how Hsp70 interacts with negatively charged phospholipids in artificial lipid compositions employing the X-ray reflectivity (XRR) studies. In a first step, the interactions between dioleoylphosphatidylcholine (DOPC) in the presence or absence of dioleoylphosphatidylserine (DOPS) and Hsp70 had been assessed using Quartz crystal microbalance measurements, suggesting that Hsp70 adsorbs to the surface of DOPC/DOPS bilayer. Atomic force microscopy (AFM) imaging demonstrated that the presence of DOPS is required for stabilization of the lipid bilayer. The interaction of Hsp70 with DOPC/DOPS lipid compositions was further quantitatively determined by high energy X-ray reflectivity. A systematic characterization of the chaperone-lipid membrane interactions by various techniques revealed that artificial membranes can be stabilized by the electrostatic interaction of anionic DOPS lipids with Hsp70.

Feasibility of the preparation of cochleate suspensions from naturally derived phosphatidylserines.

Cochleates are cylindrical particles composed of dehydrated phospholipid bilayers. They are typically prepared by addition of calcium ions to vesicles composed of negatively charged phospholipids such as phosphatidylserines (PS). Due to their high physical and chemical stability, they provide an interesting alternative over other lipid-based drug formulations for example to improve oral bioavailability or to obtain a parenteral sustained-release formulation. In the present study, the feasibility to prepare cochleate suspensions from soy lecithin-derived phosphatidylserines (SPS) was investigated and compared to the "gold standard" dioleoyl-phosphatidylserine (DOPS) cochleates. The SPS lipids covered a large range of purities between 53 and >96% and computer-controlled mixing was evaluated for the preparation of the cochleate suspensions. Electron microscopic investigations were combined with small-angle x-ray diffraction (SAXD) and Laurdan generalized polarization (GP) analysis to characterize particle structure and lipid organization. Despite some differences in particle morphology, cochleate suspensions with similar internal lipid structure as DOPS cochleates could be prepared from SPS with high headgroup purity (≥96%). Suspensions prepared from SPS with lower purity still revealed a remarkably high degree of lipid dehydration and well-organized lamellar structure. However, the particle shape was less defined, and the typical cochleate cylinders could only be detected in suspensions prepared with higher amount of calcium ions. Finally, the study proves the feasibility to prepare suspensions of cochleates or cochleate-like particles directly from a calcium salt of soy-PS by dialysis.

CSIG-12. BXQ-350 INDUCES MITOPHAGY IN GBM CELLS LEADING TO CELL DEATH

Abstract BACKGROUND Mitophagy is an autophagic phenomenon where defective mitochondria are selectively degraded. When mitochondria need recycling, the cell upregulates the production of the lipid C-18 ceramide on the mitochondria and lipoprotein LC3B-II on the lysosome, which transitions to an autophagolysosome. This creates a signal causing the degradation of mitochondria. Here we show that BXQ-350, a novel protein-lipid small molecule consisting of the ceramide creating protein Saposin C and the lipid Dioleoyl Phosphatidylserine (DOPS), induces glioblastoma cells into performing mitophagy. Increased production of mitophagy associated proteins, C-18, and LC3B-II leads to the degradation of healthy mitochondria followed by cell death. METHODS The GBM cell lines, Gli36 and U87 cells, were used to determine if increased mitophagy was occurring in response to BXQ-350 treatment. Protein abundance was measured using SILAC proteomics. Ceramide levels were analyzed using LC/MS-MS. Western Blot analysis was used to measure differences in LC3B-II levels. Mitophagy was measured using Mtphagy Dye from Dojindo and the BioTek Cytation 5 microscope. RESULTS Proteomics showed cells treated with BXQ-350 had an increase in proteins associated with overexpression of C-18 ceramide or autophagolysosome maturation. Western blots analysis showed a marked increase in LC3B-II in BXQ-350 treated cells. Furthermore, cells treated with BXQ-350 had increased levels of C-18 ceramide. The Mtphagy dye showed that as BXQ-350 concentrations increased, the percentage of cells positive for Mitophagy increased. To determine if mitophagy occurs in association with BXQ-350 cytotoxicity, medias with varying BXQ-350 cytotoxicity were used. The Mtphagy dye showed cytotoxic BXQ-350 medias increased levels of mitophagy, while noncytotoxic medias display no increase in mitophagy. CONCLUSION BXQ-350 causes the increase of mitophagy signaling proteins, C-18 ceramide and LC3B-II production, resulting in the formation of autophagolysosomes which track towards the mitochondria initiating mitophagy. This leads to aberrant mitophagy where cells degrade healthy mitochondria leading to mitophagic cell death.

Lipid droplet-hitchhiking probe creates Trojan foam cells for fluorescence/photoacoustic imaging of atherosclerotic plaques

Since atherosclerosis, a disease characterized by abnormal arterial lipid deposition, may lead to fatal cardiovascular diseases, imaging of atherosclerotic plaques is of great value for their pathological assessment. In this study, we propose a lipid droplet (LD)-hitchhiking strategy to in situ create Trojan foam cells for fluorescence/photoacoustic imaging of atherosclerotic plaques via homologous targeting effect. In our design, functional liposomes (DCP liposomes) composed of phospholipid dioleoylphosphatidylserine (DOPS), a novel LD inducer we found, and Cypate-PC, a synthesized lipid-like molecular probe, have demonstrated great capability of inducing LDs in monocytes/macrophages while being enveloped into the resulting Trojan foam cells. Taking advantage of homologous targeting effect, the imaging probe hitchhikes on the LDs in Trojan foam cells for targeted transport to the plaque sites. Moreover, the confinement in highly hydrophobic LDs endows the imaging probe with high efficiency in light absorption, enabling greatly intensified fluorescence/photoacoustic signals. The DCP liposomes have shown great potency in inducing the generation of Trojan foam cells, and eventually ex vivo fluorescence imaging and in vivo photoacoustic imaging of atherosclerotic plaques. The proposed strategy provides more insights into the design of targeted imaging methodologies, and also an effective avenue to facilitate the evaluation and subsequent treatment of atherosclerotic plaques.

Pharmacokinetics, biodistribution, and activity of Amphotericin B-loaded nanocochleates on the Leishmania donovani murine visceral leishmaniasis model

Amphotericin B (AmB) is an effective drug to treat visceral leishmaniasis but its use is limited by its poor oral bioavailability. This article describes the in-vivo evaluation of AmB-loaded, lipid-based cochleate systems designed for the oral route. Two different cochleate formulations were studied: one based on the synthetic phospholipid dioleoylphosphatidylserine (DOPS) and another optimized formulation based on a naturally occurring phosphatidylserine (Lipoid PSP70) that would render the formulation more affordable in developing countries. Their antiparasitic activity was evaluated in a mouse model of visceral leishmaniasis. Limited efficacy was observed for the DOPS-based cochleates after three doses of AmB at 1 mg/kg. The Lipoid PSP70-based cochleates were administered either as a buffered suspension or in enteric-coated capsules. AmB-loaded cochleates administered as a suspension at a high dose (3 × 20 mg/kg) exhibited significant antiparasitic activity while AmB-loaded cochleates in enteric-coated capsules at a lower dose (3 × 5 mg/kg) presented a slightly higher significant activity. A pharmacokinetic and biodistribution study in rats was performed with the Lipoid PSP70-based cochleates, with a single oral dose of 7.5 mg AmB/kg. Cochleates in both administration forms led to lower concentrations of Amphotericin B in the plasma than intravenous AmBisome®. However, more accumulation in the organs of interest (liver, spleen) was observed for both presentations of cochleates than for AmBisome® by the oral route. Therefore, cochleate formulations of AmB that could be produced at a cost accessible for developing countries show promise for the treatment of visceral leishmaniasis.

Cryo-EM of the ATP11C flippase reconstituted in Nanodiscs shows a distended phospholipid bilayer inner membrane around transmembrane helix 2

ATP11C is a member of the P4-ATPase flippase family that mediates translocation of phosphatidylserine (PtdSer) across the lipid bilayer. In order to characterize the structure and function of ATP11C in a model natural lipid environment, we revisited and optimized a quick procedure for reconstituting ATP11C into Nanodiscs using methyl-β-cyclodextrin as a reagent for the detergent removal. ATP11C was efficiently reconstituted with the endogenous lipid, or the mixture of endogenous lipid and synthetic dioleoylphosphatidylcholine (DOPC)/dioleoylphosphatidylserine (DOPS), all of which retained the ATPase activity. We obtained 3.4 Å and 3.9 Å structures using single-particle cryo-electron microscopy (cryo-EM) of AlF- and BeF-stabilized ATP11C transport intermediates, respectively, in a bilayer containing DOPS. We show that the latter exhibited a distended inner membrane around ATP11C transmembrane helix 2, possibly reflecting the perturbation needed for phospholipid release to the lipid bilayer. Our structures of ATP11C in the lipid membrane indicate that the membrane boundary varies upon conformational changes of the enzyme and is no longer flat around the protein, a change that likely contributes to phospholipid translocation across the membrane leaflets.

SapC-DOPS as a Novel Therapeutic and Diagnostic Agent for Glioblastoma Therapy and Detection: Alternative to Old Drugs and Agents.

Glioblastoma multiforme (GBM), the most common type of brain cancer, is extremely aggressive and has a dreadful prognosis. GBM comprises 60% of adult brain tumors and the 5 year survival rate of GBM patients is only 4.3%. Standard-of-care treatment includes maximal surgical removal of the tumor in combination with radiation and temozolomide (TMZ) chemotherapy. TMZ is the “gold-standard” chemotherapy for patients suffering from GBM. However, the median survival is only about 12 to 18 months with this protocol. Consequently, there is a critical need to develop new therapeutic options for treatment of GBM. Nanomaterials have unique properties as multifunctional platforms for brain tumor therapy and diagnosis. As one of the nanomaterials, lipid-based nanocarriers are capable of delivering chemotherapeutics and imaging agents to tumor sites by enhancing the permeability of the compound through the blood–brain barrier, which makes them ideal for GBM therapy and imaging. Nanocarriers also can be used for delivery of radiosensitizers to the tumor to enhance the efficacy of the radiation therapy. Previously, high-atomic-number element-containing particles such as gold nanoparticles and liposomes have been used as radiosensitizers. SapC–DOPS, a protein-based liposomal drug comprising the lipid, dioleoylphosphatidylserine (DOPS), and the protein, saposin C (SapC), has been shown to be effective for treatment of a variety of cancers in small animals, including GBM. SapC–DOPS also has the unique ability to be used as a carrier for delivery of radiotheranostic agents for nuclear imaging and radiotherapeutic purposes. These unique properties make tumor-targeting proteo-liposome nanocarriers novel therapeutic and diagnostic alternatives to traditional chemotherapeutics and imaging agents. This article reviews various treatment modalities including nanolipid-based delivery and therapeutic systems used in preclinical and clinical trial settings for GBM treatment and detection.

Three-Phase Coexistence in Binary Charged Lipid Membranes in a Hypotonic Solution

We investigated the phase separation of dioleoylphosphatidylserine (DOPS) and dipalmitoylphosphatidylcholine (DPPC) in giant unilamellar vesicles in hypotonic solution using fluorescence and confocal laser scanning microscopy. Although phase separation in charged lipid membranes is generally suppressed by the electrostatic repulsion between the charged headgroups, osmotic stress can promote the formation of charged lipid domains. Interestingly, we observed three-phase coexistence even in DOPS/DPPC binary lipid mixtures. The three phases were DPPC-rich, dissociated DOPS-rich, and nondissociated DOPS-rich phases. The two forms of DOPS were found to coexist owing to the ionization of the DOPS headgroup, such that the system could be regarded as quasi-ternary. The three formed phases with differently ionized DOPS domains were successfully identified experimentally by monitoring the adsorption of positively charged particles. In addition, coarse-grained molecular dynamics simulations confirmed the stability of the three-phase coexistence. Attraction mediated by hydrogen bonding between protonated DOPS molecules and reduction of the electrostatic interactions at the domain boundaries stabilized the three-phase coexistence.

Targeting of elevated cell surface phosphatidylserine with saposin C-dioleoylphosphatidylserine nanodrug as individual or combination therapy for pancreatic cancer.

Pancreatic cancer is one of the deadliest of cancers with a five-year survival of roughly 8%. Current therapies are: surgery, radiation and chemotherapy. Surgery is curative only if the cancer is caught very early, which is rare, and the latter two modalities are only marginally effective and have significant side effects. We have developed a nanosome comprised of the lysosomal protein, saposin C (SapC) and the acidic phospholipid, dioleoylphosphatidylserine (DOPS). In the acidic tumor microenvironment, this molecule, SapC-DOPS, targets the phosphatidylserine cancer-biomarker which is predominantly elevated on the surface of cancer cells. Importantly, SapC-DOPS can selectively target pancreatic tumors and metastases. Furthermore, SapC-DOPS has exhibited an impressive safety profile with only a few minor side effects in both preclinical experiments and in phase I clinical trials. With the dismal outcomes for pancreatic cancer there is an urgent need for better treatments and SapC-DOPS is a good candidate for addition to the oncologist’s toolbox.

Cardiolipin interacts with beta-2-glycoprotein I and forms an open conformation-Mechanisms analyzed using hydrogen/deuterium exchange.

Beta‐2‐glycoprotein I (β2GPI) is the major antigen for the antiphospholipid antibodies in the antiphospholipid syndrome. The exposed epitope in domain I of β2GPI can be recognized by the anti‐β2GPI antibody. Here, we prepared the anionic di‐oleoyl‐phosphatidylserine (DOPS) and cardiolipin (CL) liposomes to interact with the β2GPI. The conformational changes of β2GPI upon binding with the liposomes were analyzed using hydrogen/deuterium exchange mass spectrometry. The exchange level of sequences 21–27 significantly increased after β2GPI had interacted with DOPS. This change indicated a reduced interaction between domain I and domain V, inferring to a protrusion of the sequences 21–27 from the ring conformation. After β2GPI had interacted with CL for 30 min, the exchange levels in 4 of the 5 domains increased significantly. The deuteration levels of sequences 1–20, 21–27, 196–205, 273–279 and 297–306 increased, suggesting that these regions had become more exposed, and the domain I was no longer in contact with domain V. The increasing deuteration levels in sequences 70–86, 153–162, 191–198, 196–205 and 273–279 indicated β2GPI undergoing conformational changes to expose these inner regions, suggesting a structural transition. Overall, DOPS and CL induced minor conformational changes of β2GPI at sequences 21–27 and forms an intermediate conformation after 10 min of interaction. After a complete protein–lipid interaction, high negatively charged CL membrane induced a major conformation transition of β2GPI.

Effect of packing density of lipid vesicles on the Aβ42 fibril polymorphism

The aggregation of amyloid-β 1–42 (Aβ42) on lipid membranes is closely related to the pathology of Alzheimer’s disease (AD). Herein, we demonstrated the effect of the packing density of lipid vesicles on the Aβ42 fibrillation kinetics and fibril morphology. We used three distinct phosphatidylcholine (PC) lipids, containing different numbers of cis-double bonds in acyl chains, and therefore, a different packing density in the lipid vesicles. Our results showed that the fibrillation of Aβ42 was greatly enhanced and the formed fibrils became shorter as the number of double bonds in lipids increased. Due to the low-density characteristics of dioleoyl phosphatidylcholine (DOPC), Aβ42 monomers were able to interact with the hydrophobic acyl chain of lipids exposed to the aqueous phase, thereby inducing rapid fibrillation and short fibril morphologies. Furthermore, the effects of the anionic lipids dioleoyl phosphatidylserine (DOPS) and dioleoyl phosphatidylglycerol (DOPG), and mixed vesicles of DOPC/DOPS and DOPC/DOPG on Aβ42 fibrillations were investigated. The tight binding of Aβ42 to the lipid head groups via electrostatic interactions was able to suppress the modulation of Aβ42 fibrillations compared to accelerated fibrillations on loosely packed membranes. Our proposed mechanism regarding the influence of lipid packing density on Aβ42 fibrillations provides an advanced understanding of lipid-associated amyloid fibrillations.

Study of Resveratrol's Interaction with Planar Lipid Models: Insights into Its Location in Lipid Bilayers.

Resveratrol, a polyphenolic molecule found in edible fruits and vegetables, shows a wide range of beneficial effects on human health, including anti-microbial, anti-inflammatory, anti-cancer, and anti-aging properties. Due to its poor water solubility and high liposome-water partition coefficient, the biomembrane seems to be the main target of resveratrol, although the mode of interaction with membrane lipids and its location within the cell membrane are still unclear. In this study, using electrophysiological measurements, we study the interaction of resveratrol with planar lipid membranes (PLMs) of different composition. We found that resveratrol incorporates into palmitoyl-oleoyl-phosphatidylcholine (POPC) and POPC:Ch PLMs and forms conductive units unlike those found in dioleoyl-phosphatidylserine (DOPS):dioleoyl-phosphatidylethanolamine (DOPE) PLMs. The variation of the biophysical parameters of PLMs in the presence of resveratrol provides information on its location within a lipid double layer, thus contributing to an understanding of its mechanism of action.

DIPG-11. A PHASE I DOSE ESCALATION STUDY OF BXQ-350 IN CHILDREN AND YOUNG ADULTS WITH RELAPSED SOLID TUMORS

BXQ-350 is a novel agent composed of the multifunctional, lysosomal activator protein Saposin C (SapC) and dioleoyl- phosphatidylserine (DOPS). BXQ-350 demonstrated antitumor effects in vitro and in vivo. Many tumors, including diffuse intrinsic pontine glioma (DIPG), and cells of tumor vasculature have aberrantly-exposed PS-rich domains on the cell surface. BXQ-350 is an anti-tumor agent in development from Bexion Pharmaceuticals, Inc. that selectively targets tumor cell PS, particularly those translocated to the outer leaflet of the plasma membrane in tumor cells. BXQ-350 activates and participates in various cellular processes, including apoptosis and necrosis, and may also exhibit novel mechanisms leading to cell death that require further investigation. An adult Phase I trial with BXQ-350 completed enrollment in 2019 having dosed 86 recurrent solid tumor patients, including glioblastoma, with only one serious infusion-related reaction. The highest planned dose of 2.4 mg/kg was achieved and seven patients remain on study with multiple cases demonstrating an objective response. A Phase I pediatric dose escalation trial in recurrent solid tumors, including central nervous system (CNS) tumors, also completed enrollment in 2019. The highest planned dose of 3.2 mg/kg was achieved and there have been no BXQ-350 related serious adverse events. Eight patients (7 CNS and 1 non-CNS) completed at least one cycle with one DIPG patient completing cycle five. A pediatric Phase I trial in newly diagnosed DIPG and diffuse midline glioma (DMG) is planned for 2nd quarter 2020.

Effects of valinomycin doping on the electrical and structural properties of planar lipid bilayers supported on polyelectrolyte multilayers

Supported Lipid Bilayers (SLBs) on Polyelectrolyte Multilayers (PEMs) have large potential as models for developing sensor devices. SLBs can be designed with receptors and channels, which benefit from the biological environment of the lipid layers, to create a sensing interface for ions and biomarkers. PEMs assembled by the Layer-by-Layer (LBL) technique and used as supports for a lipid bilayer enable an easy integration of the bilayer on almost any surface and device. For electrochemical sensors, LBL assembly enables nanoscale tunable separation of the lipid bilayer from the electrode surface, avoiding undesired effects of the electrode surface on the lipid bilayers. We study the fabrication of valinomycin-doped SLBs on PEMs as a model system for biophysical studies and for selective ion sensing. SLBs are fabricated from dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylserine (DOPS) 50:50 vesicles doped with valinomycin, as a K+-selective carrier. SLBs were deposited on electrodes coated with poly(allyl amine hydrochloride) (PAH) and poly(styrene sodium sulfonate) (PSS) multilayers. Lipid bilayer formation was monitored by using Quartz Crystal Microbalance with Dissipation (QCMD) technique and Atomic Force Microscopy (AFM). Electrochemical impedance spectroscopy (EIS) and potentiometric measurements were performed to assess K+ selectivity over other ions and the potential of valinomycin-doped SLBs for K+-sensing.

CTNI-78. A PEDIATRIC AND YOUNG ADULT PHASE I DOSE ESCALATION SAFETY STUDY OF BXQ-350 FOR REFRACTORY SOLID AND CENTRAL NERVOUS SYSTEM TUMORS

Abstract BACKGROUND BXQ-350 is a novel agent composed of the multifunctional, lysosomal activator protein Saposin C (SapC) and dioleoyl- phosphatidylserine (DOPS) and has demonstrated antitumor effects in both in vitro and in vivo preclinical models. Many tumors, including high-grade glioma and diffuse intrinsic pontine glioma (DIPG), and cells of tumor vasculature have aberrantly-exposed phosphatidylserine (PS)-rich domains on the cell surface. BXQ-350 is an anti-tumor agent in development from Bexion Pharmaceuticals, Inc. that selectively targets tumor cell PS, particularly those translocated to the outer leaflet of the plasma membrane in tumor cells. BXQ-350 activates and participates in various cellular processes, including apoptosis and necrosis, and may also exhibit novel mechanisms leading to cell death that require further investigation. METHODS Nine refractory solid (2) and central nervous system (7) tumor patients (5F:4M, age 4–23 years of age) were enrolled in a 2-site dose escalation Phase I first-in-pediatric trial (NCT03967093) which completed in 2019. All patients received at least one dose of BXQ-350 which was administered as an intravenous infusion. Dosing began at 1.8 mg/kg and escalated to the highest planned dose level of 3.2mg/kg. RESULTS There were no BXQ-350-related serious adverse events, dose limiting toxicities or withdrawals. The highest planned dose of 3.2 mg/kg was achieved safely but a maximum tolerated dose was not established. One osteosarcoma patient had progressive disease prior to completing cycle one of treatment and was removed from trial. Eight patients (DIPG-3, HGG-1, GBM-1, Pineoblasotoma-1, Ependymoma-1, Osteosarcoma-1) completed at least one cycle, with one DIPG patient completing cycle five. CONCLUSION BXQ-350 was well tolerated with no significant dose-limiting toxicities at the highest planed dose level. A pediatric Phase I trial in newly diagnosed patients is planned for 3rd quarter 2020.

Tolerability and preliminary efficacy of BXQ-350 for refractory solid tumors and high-grade gliomas: First-in-human, first-in-class phase I trial.

3505 Background: BXQ-350 is a first-in-class agent comprised of Saposin C (SapC) and dioleoyl phosphatidylserine (DOPS). SapC, a multifunctional lysosomal-activator glycoprotein that preferentially interacts with tumor cell phospholipids, has demonstrated anti-tumor effects in both in vitro and in vivo preclinical models. The tolerability and preliminary efficacy of BXQ-350 in the first-in-human study are summarized here. Methods: Eighty-six refractory solid tumor (ST) or high-grade glioma (HGG) patients age ≥18 (36F:50M, age 24-81) were enrolled in a 3-part first-in-human trial (NCT02859857) from 2016-2019 and received at least one dose of BXQ-350. Doses were administered via intravenous infusion during 28-day cycles until disease progression occurred. The previously reported part 1 dose escalation portion of the study (9 HGG, 9 ST patients) established the highest planned dose of 2.4mg/kg as safe but did not identify a maximum tolerated dose. The part 2 expansion cohort treated 37 patients (18 HGG and 19 ST) and an additional part 3 cohort treated 31 ST gastrointestinal (GI) patients, both at the 2.4 mg/kg dose level. Preliminary antitumor activity was evaluated (RECISTv1.1 or RANO). Results: There were no BXQ-350-related serious adverse events, dose limiting toxicities or withdrawals with the exception of 1 allergic type reaction. Three patients (Glioblastoma, Ependymoma, Appendiceal) demonstrated a partial response per RECIST/RANO. Two HGG patients with progressive radiologic enhancement were seen to have treatment effect at surgery, and hence considered to have stable disease. Seven patients (2 HGG, 3 GI, 2 other ST) remain on study and have received treatment for 9+ to 41+ months, with 5 patients treated for > 1 year. A continuing treatment protocol is planned in order to allow these patients to remain on BXQ-350 treatment. Conclusions: BXQ-350 was well tolerated with no significant dose-limiting toxicities at the highest planed dose level. Preliminary results indicate this novel agent demonstrated possible anti-tumor activity in refractory solid tumors and HGG. Clinical trial information: NCT03967093) .

A pediatric and young adult phase I dose escalation study of BXQ-350 for solid and central nervous system tumors.

2541 Background: BXQ-350 is a novel agent composed of the multifunctional, lysosomal activator protein Saposin C (SapC) and dioleoyl- phosphatidylserine (DOPS) and has demonstrated antitumor effects in both in vitro and in vivo preclinical models. Many tumors, including high-grade glioma and diffuse intrinsic pontine glioma (DIPG), and cells of tumor vasculature have aberrantly exposed phosphatidylserine (PS)-rich domains on the cell surface. BXQ-350 is an anti-tumor agent in development from Bexion Pharmaceuticals, Inc. that selectively targets tumor cell PS, particularly those translocated to the outer leaflet of the plasma membrane in tumor cells. BXQ-350 activates and participates in various cellular processes, including apoptosis and necrosis, and may also exhibit novel mechanisms leading to cell death that require further investigation. Methods: Nine refractory solid (2) and central nervous system (7) tumor patients (5F:4M, age 4-23 years of age) were enrolled in a 2-site dose escalation phase I first-in-pediatric trial ( NCT03967093 ) which completed in 2019. All patients received at least one dose of BXQ-350 which was administered as an intravenous infusion. Dosing began at 1.8 mg/kg and escalated to the highest planned dose level of 3.2mg/kg. Results: There were no BXQ-350-related serious adverse events, dose limiting toxicities, or withdrawals. The highest planned dose of 3.2 mg/kg was achieved safely but a maximum tolerated dose was not established. One osteosarcoma patient had progressive disease prior to completing cycle one of treatment and was removed from trial. Eight patients (DIPG-3, HGG-1, GBM-1, Pineoblasotoma-1, Ependymoma-1, Osteosarcoma-1) completed at least one cycle, with one DIPG patient completing cycle five. Conclusions: BXQ-350 was well tolerated with no significant dose-limiting toxicities at the highest planed dose level. A pediatric phase I trial in newly diagnosed patients is planned for 2nd quarter 2020. Clinical trial information: NCT03967093 .

X-Ray Reflectivity and Diffraction Studies of Doxorubicin Binding to Model Lipid Membranes

The effect of anticancer antibiotic doxorubicin on structural organization of anionic lipid monolayers has been studied. X-ray reflectivity and grazing incidence diffraction techniques were applied to monitor the changes in 2D structure and electron density distribution of Langmuir monolayer composed of negatively charged dipalmitoylphosphatidylglycerol (DPPG) and dioleoylphosphatidylserine (DOPS). For comparison, monolayer of zwitterionic dipalmitoylphosphatidylethanolamine (DPPE) also was investigated. The presented experimental results suggest that doxorubicin interaction with anionic lipid monolayers (DPPG and DOPS) proceeds preferentially via electrostatic attraction—positively charged amino groups of doxorubicin bind to negatively charged head groups of phospholipid molecules. Based on the obtained data, the penetration of doxorubicin into the hydrophobic part of anionic lipid monolayers does not occur. X-ray measurements on DPPE monolayer indicated that doxorubicin did not cause any significant alterations of molecular packing in condensed monolayer of zwitterionic DPPE molecules.