Uptake Efficacy Research Articles

Co-delivery of trifluralin and miltefosin with enhanced skin penetration and localization in Leishmania affected macrophages

In this study, Trifluralin (TRF) co-loaded with Miltefosine (MTF) in nano transfersomes (NTs) were prepared aiming their enhanced penetration topically with improved macrophage targeting and antileishmanial effects. Formulations were optimized in a series of experiments based on their particle size, polydispersity index (PDI), drug incorporation and deformability index. TEM analysis was performed to check morphology of NTs. TRF-MTF co-loaded NTs were further incorporated into chitosan gel to obtain better retention. In vitro release and Ex vivo permeation studies of the optimized TRF-MTF co-loaded NTs were conducted to check dissolution behavior and skin permeation of the developed formulation as compared to the drug solutions and TRF-MTF co-loaded NTs gel. Macrophage uptake and antileishmanial efficacy of the developed formulation was also investigated. The optimized formulation had a particle size of 188.5 nm with rounded morphology, PDI of 0.101, and deformability index of 0.987. The drug incorporation of TRF and MTF were respectively found as 91% and 88%. Controlled and significantly enhanced drug release and augmented skin penetration was observed from the TRF-MTF co-loaded NTs as compared to pure drugs. Significant uptake of the TRF-MTF co-loaded NTs by the macrophage proved greater intracellular infiltration via the skin barriers. In vitro anti-leishmanial study exhibited 6-folds and 5-folds decrease in the respective IC50 values of TRF-MTF co-loaded NTs when used against Leishmania (L.) tropica amastigotes and promastigotes. This study exhibited the efficacy of NTs when loaded with dual drugs for enhanced skin penetration and macrophage localization.

Tumor acidic environment directs nanoparticle impacts on cancer cells

Despite impressive progress in nanotechnology-based cancer therapy being made by in vitro research, few nanoparticles (NPs) have been translated into clinical trials. The wide gap between in vitro results and nanomedicine’s clinical translation might be partly due to acidic microenvironment of cancer cells being ignored in in vitro studies. To check this hypothesis, we studied the biological impacts of two different structures of NPs on cancer cells (MDA-MB 231) at acidic (pH: 6.5) low (pH: 7) and physiological pH (pH: 7.4). We uncovered that a slight change in the pH of the cancer cell microenvironment affects the cellular uptake efficacy and toxicity mechanism of nanographene sheets and SPION@silica nanospheres. Both nanostructures exerted more substantial toxic impacts (e. g. apoptosis, necrosis, membrane disruption, and oxidative stress induction) against cancer cells at physiological pH compared to acidic niche. They also differently slowed or arrested phases of the cell cycle at different pH (S and G2/M at normal pH while G0/G1 at acidic/low pH). More specifically, cancer cells expressed higher levels of interleukins involved in cancer cell resistance at acidic pH than those incubated at physiological pH. This study revealed that a slight change in extracellular pH of cancer cells could strongly affect the therapeutic/toxic impact of nanomaterials and therefore, it should be considered in the future cancer nanomedicine research.

Identification of 5-Carboxyfluorescein as a Probe Substrate of SLC46A3 and Its Application in a Fluorescence-Based In Vitro Assay Evaluating the Interaction with SLC46A3.

The therapeutic modalities that involve the endocytosis pathway, including antibody-drug conjugates (ADCs), have recently been developed. Since the drug escape from endosomes/lysosomes is a determinant of their efficacy, it is important to optimize the escape, and the cellular evaluation system is needed. SLC46A3, a lysosomal membrane protein, has been implicated in the pharmacological efficacy of trastuzumab emtansine (T-DM1), a noncleavable ADC used for the treatment of breast cancer, and the cellular uptake efficacy of lipid-based nanoparticles. Recently, we identified the SLC46A3 function as a proton-coupled steroid conjugate and bile acid transporter, which can directly transport active catabolites of T-DM1. Thus, the rapid and convenient assay systems for evaluating the SLC46A3 function may help to facilitate ADC development and to clarify the physiological roles in endocytosis. Here, we show that SLC46A3 dC, which localizes to the plasma membrane owing to lacking a lysosomal-sorting motif, has a great ability to transport 5-carboxyfluorescein (5-CF), a fluorescent probe, in a pH-dependent manner. 5-CF uptake mediated by SLC46A3 was significantly inhibited by compounds reported to be SLC46A3 substrates/inhibitors and competitively inhibited by estrone 3-sulfate, a typical SLC46A3 substrate. The inhibition assays followed by uptake studies revealed that SG3199, a pyrrolobenzodiazepine dimer, which has been used as an ADC payload, is a substrate of SLC46A3. Accordingly, the fluorescence-based assay system for the SLC46A3 function using 5-CF can provide a valuable tool to evaluate the interaction of drugs/drug candidates with SLC46A3.

Novel nanoparticle improves intracellular uptake and cardioprotective efficacy of DJ-1 mimic peptide

Novel nanoparticle improves intracellular uptake and cardioprotective efficacy of DJ-1 mimic peptide

Phenethyl Isothiocyanate-Conjugated Chitosan Oligosaccharide Nanophotosensitizers for Photodynamic Treatment of Human Cancer Cells.

The aim of this study is to synthesize phenethyl-conjugated chitosan oligosaccharide (COS) (abbreviated as ChitoPEITC) conjugates and then fabricate chlorin E6 (Ce6)-incorporated nanophotosensitizers for photodynamic therapy (PDT) of HCT-116 colon carcinoma cells. PEITC was conjugated with the amine group of COS. Ce6-incorporated nanophotosensitizers using ChitoPEITC (ChitoPEITC nanophotosensitizers) were fabricated by dialysis method. 1H nuclear magnetic resonance (NMR) spectra showed that specific peaks of COS and PEITC were observed at ChitoPEITC conjugates. Transmission electron microscope (TEM) confirmed that ChitoPEITC nanophotosensitizers have spherical shapes with small hydrodynamic diameters less than 200 nm. The higher PEITC contents in the ChitoPEITC copolymer resulted in a slower release rate of Ce6 from nanophotosensitizers. Furthermore, the higher Ce6 contents resulted in a slower release rate of Ce6. In cell culture study, ChitoPEITC nanophotosensitizers showed low toxicity against normal CCD986Sk human skin fibroblast cells and HCT-116 human colon carcinoma cells in the absence of light irradiation. ChitoPEITC nanophotosensitizers showed a significantly higher Ce6 uptake ratio than that of free Ce6. Under light irradiation, cellular reactive oxygen species (ROS) production of nanophotosensitizers was significantly higher than that of free Ce6. Especially, PEITC and/or ChitoPEITC themselves contributed to the production of cellular ROS regardless of light irradiation. ChitoPEITC nanophotosensitizers showed significantly higher PDT efficacy against HCT-116 cells than that of free Ce6. These results indicate that ChitoPEITC nanophotosensitizers have superior potential in Ce6 uptake, ROS production and PDT efficacy. In the HCT-116 cell-bearing mice tumor-xenograft model, ChitoPEITC nanophotosensitizers efficiently inhibited growth of tumor volume rather than free Ce6. In the animal imaging study, ChitoPEITC nanophotosensitizers were concentrated in the tumor tissue, i.e., fluorescence intensity in the tumor tissue was stronger than that of other tissues. We suggest that ChitoPEITC nanophotosensitizers are a promising candidate for the treatment of human colon cancer cells.

Nanoaggregates of Disulfide-Decorated TrxR Inhibitor Promote Cellular Uptake, Selective Targeting, and Antitumor Efficacy.

Three self-assembled nanoaggregates (CPUL1-LA NAs, CPUL1-DA NAs, and CPUL1-AA NAs) were constructed through lipoic acid (LA), dithiodipropionic acid (DA), and adipic acid (AA) decorated TrxR inhibitor (CPUL1), respectively. Measurements of DLS, TEM, UV-vis, fluorescence, 1H NMR, ITC, and MTT assays verified disulfide-containing CPUL1-LA NAs and CPUL1-DA NAs spontaneously assembled carrier-free nanoparticles in aqueous solution, which possessed high drug contents, excellent stability, improved cytotoxicity against HUH7 hepatoma cells, and potential biosafety because of low cytotoxicity against L02 normal cells. In contrast, disulfide-free CPUL1-AA NAs happened to aggregate and precipitate after 48 h, which showed distinct instability in aqueous solution. Thus, disulfide units seemed to be crucial for constructing controllable and stable nanoaggregates. While measuring the reduction of nanoaggregates by TrxR/NADPH and GSH/GR/NADPH, cyclic disulfide of LA and linear disulfide of DA were verified to endow the nanoaggregates with targeting ability to respond specifically to TrxR over GSH. Furthermore, by tests of flow cytometry, fluorescence images, and CLSM, both CPUL1-LA NAs and CPUL1-DA NAs displayed a faster cellular uptake characteristic to be internalized by cancer cells and could generate more abundant ROS to induce cell apoptosis than that of free CPUL1, resulting in significantly improved antitumor efficacy against HUH7 cells in vitro.

Increased Tropism of Extracellular Vesicles Derived from Palmitic Acid-Treated Hepatocytes to Activated Hepatic Stellate Cells.

Myofibroblast-like activated hepatic stellate cells (aHSCs), which produce collagen, a major cause of liver fibrosis, are specific target cells for antifibrotic treatment. Recently, several reports have indicated that extracellular vesicles (EVs) play important roles in cell-to-cell communication through their tropism for specific cells or organs. Therefore, the present study aimed to identify aHSC-directed EVs by focusing on cell-to-cell interactions in the liver under pathological conditions. EVs were derived from the hepatocyte cell line AML12 treated with or without palmitic acid (PA) and evaluated for their physical properties and uptake by the aHSC cell line LX-2. AML12-derived EVs had a mean particle diameter of 110–130 nm, negative charge, and expressed the exosomal makers CD9 and CD63. PA-treated AML12 cells released larger EVs with higher protein levels than those without PA treatment. The intracellular uptake efficacy of EVs derived from PA-treated AML12 cells into activated LX-2 cells was significantly higher than those without PA treatment. Our study revealed that PA treatment induces hepatocytes to release EVs with aHSC-tropism. These findings may contribute to the development of an EV-based drug delivery system (DDS) for aHSC-targeted therapy and provide new insights into the role of steatotic hepatocyte-derived EVs in physiological or pathophysiological functions.

Comparing Variants of the Cell-Penetrating Peptide sC18 to Design Peptide-Drug Conjugates.

Herein, the design and synthesis of peptide-drug conjugates (PDCs) including different variants of the cell-penetrating peptide sC18 is presented. We first generated a series of novel sequence mutants of sC18 having either amino acid deletions and/or substitutions, and then tested their biological activity. The effects of histidine substituents were found to be not meaningful for sC18 uptake and cell selectivity. Moreover, building a nearly perfect amphipathic structure within a shortened sC18 derivative provided a peptide that was highly membrane-active, but also too cytotoxic. As a result, the most promising analog was sC18ΔE, which stands out due to its higher uptake efficacy compared to parent sC18. In the last set of experiments, we let the peptides react with the cytotoxic drug doxorubicin by Thiol–Michael addition to form novel PDCs. Our results indicate that sC18ΔE could be a more efficient drug carrier than parent sC18 for biomedical applications. However, cellular uptake using endocytosis and resulting entrapment of cargo inside vesicles is still a major critical step to overcome in CPP-containing peptide-drug development.

Multi-functionalized single-walled carbon nanotubes as delivery carriers: promote the targeting uptake and antitumor efficacy of doxorubicin

Multi-functionalized single-walled carbon nanotubes as delivery carriers: promote the targeting uptake and antitumor efficacy of doxorubicin

Triggered amplification of gene theranostics with high accuracy and efficacy using metallo-nanoassemblies

RNA-cleaving DNAzymes hold great promise for gene regulation, yet the low in vivo catalytic efficacy and the unavoidable nonspecific response in normal tissues hinder their practical applications. Herein, we develop a DNA prodrug-laden metallo-nanoassembly for sensitive orthogonal activation of DNAzymes to regulate disease gene with high precision and efficacy. With rich positive charges, metallo-nanoassemblies efficiently load DNA prodrugs (functional DNAzyme subunits), and thus improve drug stability and cellular uptake efficacy. Under tumor microenvironment, the cancer-specific biomarker triggers the amplified assembly of DNA prodrugs into active DNAzymes to efficiently cleave tumor-involved mRNA. Meanwhile, these glutathione-responsive metallo-nanoassemblies decompose and release metal ions as self-sufficient DNAzyme cofactors, which are accommodated into the afore-assembled DNAzymes to enhance the gene cleavage activity. Moreover, upon laser stimulus, these photothermal metallo-nanoassemblies can also remarkably promote the catalytic reaction of DNAzymes via the elevated local temperature. Such a smart delivery approach allows disease diagnosis via sensitive fluorescence and photothermal imaging, and achieves the precise and robust gene regulation with photothermal ablation as an auxiliary therapy. These results provide a new route to design programmable and activatable prodrugs for precision nanomedicine.

Construction of functionalized ruthenium-modified selenium coated with pH-responsive silk fibroin nanomaterials enhanced anticancer efficacy in hepatocellular cancer

Nanomaterials (NMs)-based cancer-targeted drug delivery systems have been promoted as a promising therapy. Using silk fibroin (SF), Ru(II)-modified selenium (Ru@Se), Nivolumab (NMB), and heptapeptide (HP), we designed a pH-response innovative Nanomaterials for the drug delivery framework. The constructed NMB@HP/SF-Ru@Se-NMs were nanospheres of ∼100 nm, contributing to enhanced permeability and retention (EPR) in tumor sites. The NMB was released from the pH-dependent nanocarriers, and in an acidic environment, the release of NMB was accelerated. NMB@HP/SF-Ru@Se-NMs demonstrated improved cellular uptake and anticancer efficacy for hepatocellular carcinoma. In addition, the flow cytometry results demonstrated that NMB@HP/SF-Ru@Se-NMs could efficiently destroy cancer cells. Further, histochemical investigations revealed that NMB@HP/SF-Ru@Se-NMs exhibited less toxicity to the primary organs, suggesting that the constructed NMs are biocompatible in vivo. Overall, the findings imply that the nanomaterials-based delivery framework (NMB@HP/SF-Ru@Se-NMs) is a novel treatment for hepatocellular carcinoma.

Responsive Peptide Nanofibers with Theranostic and Prognostic Capacity.

Photodynamic therapy (PDT) is a highly promising therapeutic modality for cancer treatment. The development of stimuli‐responsive photosensitizer nanomaterials overcomes certain limitations in clinical PDT. Herein, we report the rational design of a highly sensitive PEGylated photosensitizer‐peptide nanofiber (termed PHHPEG6 NF) that selectively aggregates in the acidic tumor and lysosomal microenvironment. These nanofibers exhibit acid‐induced enhanced singlet oxygen generation, cellular uptake, and PDT efficacy in vitro, as well as fast tumor accumulation, long‐term tumor imaging capacity and effective PDT in vivo. Moreover, based on the prolonged presence of the fluorescent signal at the tumor site, we demonstrate that PHHPEG6 NFs can also be applied for prognostic monitoring of the efficacy of PDT in vivo, which would potentially guide cancer treatment. Therefore, these multifunctional PHHPEG6 NFs allow control over the entire PDT process, from visualization of photosensitizer accumulation, via actual PDT to the assessment of the efficacy of the treatment.

Unraveling the Pivotal Role of Atropisomerism for Cellular Internalization.

The intrinsic challenge of large molecules to cross the cell membrane and reach intracellular targets is a major obstacle for the development of new medicines. We report how rotation along a single C–C bond, between atropisomers of a drug in clinical trials, improves cell uptake and therapeutic efficacy. The atropisomers of redaporfin (a fluorinated sulfonamide bacteriochlorin photosensitizer of 1135 Da) are separable and display orders of magnitude differences in photodynamic efficacy that are directly related to their differential cellular uptake. We show that redaporfin atropisomer uptake is passive and only marginally affected by ATP depletion, plasma proteins, or formulation in micelles. The α4 atropisomer, where meso-phenyl sulfonamide substituents are on the same side of the tetrapyrrole macrocycle, exhibits the highest cellular uptake and phototoxicity. This is the most amphipathic atropisomer with a conformation that optimizes hydrogen bonding (H-bonding) with polar head groups of membrane phospholipids. Consequently, α4 binds to the phospholipids on the surface of the membrane, flips into the membrane to adopt the orientation of a surfactant, and eventually diffuses to the interior of the cell (bind-flip mechanism). We observed increased α4 internalization by cells of the tumor microenvironment in vivo and correlated this to the response of photodynamic therapy when tumor illumination was performed 24 h after α4 administration. These results show that properly orientated aryl sulfonamide groups can be incorporated into drug design as efficient cell-penetrating motifs in vivo and reveal the unexpected biological consequences of atropisomerism.

Responsive Peptide Nanofibers with Theranostic and Prognostic Capacity

AbstractPhotodynamic therapy (PDT) is a highly promising therapeutic modality for cancer treatment. The development of stimuli‐responsive photosensitizer nanomaterials overcomes certain limitations in clinical PDT. Herein, we report the rational design of a highly sensitive PEGylated photosensitizer‐peptide nanofiber (termed PHHPEG6 NF) that selectively aggregates in the acidic tumor and lysosomal microenvironment. These nanofibers exhibit acid‐induced enhanced singlet oxygen generation, cellular uptake, and PDT efficacy in vitro, as well as fast tumor accumulation, long‐term tumor imaging capacity and effective PDT in vivo. Moreover, based on the prolonged presence of the fluorescent signal at the tumor site, we demonstrate that PHHPEG6 NFs can also be applied for prognostic monitoring of the efficacy of PDT in vivo, which would potentially guide cancer treatment. Therefore, these multifunctional PHHPEG6 NFs allow control over the entire PDT process, from visualization of photosensitizer accumulation, via actual PDT to the assessment of the efficacy of the treatment.

Recent Progress Toward Imaging Application of Multifunction Sonosensitizers in Sonodynamic Therapy.

Sonodynamic therapy (SDT) is a rapidly developing non-surgical therapy that initiates sensitizers’ catalytic reaction using ultrasound, showing great potential for cancer treatment due to its high safety and non-invasive nature. In addition, recent research has found that using different diagnostic and therapeutic methods in tandem can lead to better anticancer outcomes. Therefore, as essential components of SDT, sonosensitizers have been extensively explored to optimize their functions and integrate multiple medical fields. The review is based on five years of articles evaluating the combined use of SDT and imaging in treating cancer. By developing multifunctional sonosensitive particles that combine imaging and sonodynamic therapy, we have integrated diagnosis into the treatment of precision medicine applications, improving SDT cell uptake and antitumor efficacy utilizing different tumour models. This paper describes the imaging principle and the results of cellular and animal imaging of the multifunctional sonosensitizers. Efforts are made in this paper to provide data and design references for future SDT combined imaging research and clinical application development and to provide offer suggestions.

A dynamic rhizosphere interplay between tree roots and soil bacteria under drought stress.

Root exudates are thought to play an important role in plant-microbial interactions. In return for nutrition, soil bacteria can increase the bioavailability of soil nutrients. However, root exudates typically decrease in situations such as drought, calling into question the efficacy of solvation and bacteria-dependent mineral uptake in such stress. Here, we tested the hypothesis of exudate-driven microbial priming on Cupressus saplings grown in forest soil in custom-made rhizotron boxes. A 1-month imposed drought and concomitant inoculations with a mix of Bacillus subtilis and Pseudomonas stutzeri, bacteria species isolated from the forest soil, were applied using factorial design. Direct bacteria counts and visualization by confocal microscopy showed that both bacteria associated with Cupressus roots. Interestingly, root exudation rates increased 2.3-fold with bacteria under drought, as well as irrigation. Forty-four metabolites in exudates were significantly different in concentration between irrigated and drought trees, including phenolic acid compounds and quinate. When adding these metabolites as carbon and nitrogen sources to bacterial cultures of both bacterial species, eight of nine metabolites stimulated bacterial growth. Importantly, soil phosphorous bioavailability was maintained only in inoculated trees, mitigating drought-induced decrease in leaf phosphorus and iron. Our observations of increased root exudation rate when drought and inoculation regimes were combined support the idea of root recruitment of beneficial bacteria, especially under water stress.

Effect of Lugol's solution on 131I therapy efficacy in Graves' disease.

Lugol's solution could control thyroid function and suppress 131I uptake in hyperthyroidism. This study aimed to investigate the appropriate time to withdraw Lugol's solution before 131I therapy (RIT) in Graves' disease (GD) patients, and how this should influence 131I uptake and RIT outcome. Two groups (125 cases and 1805 cases) of GD patients received RIT, who were pre-treated with and without Lugol's solution (RI-CI group and RI group). The RI-CI group was further divided into the following sub-groups depending on the duration span between Lugol's solution withdrawal and RIT: sub-group A, 4-7 d (n = 49); sub-group B, 8-14 d (n = 41); and sub-group C, 15-30 d (n = 35). The highest radioactive iodine uptake rate (RAIUmax), effective half-life (Teff), TRAb, and free triiodothyronine (FT3) and free thyroxine (FT4) levels were compared, and therapeutic outcome was evaluated. There were no significant differences in RAIUmax, TRAb, and Teff among the four sub-groups (P > 0.05). Both FT3 and FT4 levels in sub-groups A and B were lower than those in group RI and sub-group C (P < 0.05). The outcome of non-hyperthyroidism (euthyroidism + hypothyroidism) in groups RI-CI and RI was significantly different at post-RIT month 1 and 3 (P < 0.05). However, intergroup differences at 6 and 12months were not significant (P > 0.05). Withdrawal of Lugol's solution 4-7 or 8-14 d before RIT does not influence 131I uptake and RIT efficacy in GD. Moreover, in order to avoid a rapid increase in thyroid hormone levels at the same time, Lugol's solution should be withdrawn 4-7 d before RIT.

Metal uptake efficacy and phytoremediation potential of plants grown around soil dumpsites in Anyigba, Kogi State, Nigeria

Plant’s accumulation and translocation potentials were investigated for phytoremediation purposes, recovery of metals, potential for edibles/vegetables to cause harm to humans. Plant and soil were collected, prepared, digested in mixturesof H2O2-HNO3 and Li2B4O7-LiBO2 for plants and soils respectively. The concentrations of Co, Cd, Cr, and As, accumulation and translocation were determined. Data were evaluated using bioconcentration (BCF), translocation factor (TF), bioaccumulation coefficient (BAC), metal uptake efficiency (ME%), multivariate analyses to establish hyperaccumulators, phytoextractors, phytostabilizers, source of metals and theirtoxicity. Analysis of variance (ANOVA) showed data were significant at p &lt;0.05. Correlation,factor and cluster analyses were employed to understand the accumulation and translocation of metals in soils and plants tissues.From this study, Colocasia asculenta, Corchorus aestuans and Laportea aestuans were hyperaccumulators of Co. Arsenic had only phytostabilizers. COA and LA were phytostabilizers and Sida acuta was phytoextractorof Cd respectively. Chromium, Co and Cd could be phytomined from some of the plants. Vegetables/edibles values in shoots and leaves were above permissible levels for Cr, Co and Cd. Only Sida acuta was not edible.Metal uptake efficacy (%) order were Co (28.99 to 89.08) &gt; Cd (21.74 to 50.96) &gt;Cr (22.90 to 49.06) &gt; and As (9.65 to 39.19).

Abstract 1438: Organic anion transporter(SLCO)genotype and prostate cancer progression: Prospective cohort study and meta-analysis

Abstract Background: Organic anion-transporting polypeptides, encoded by SLCO genes, have multiple substrates, including androgens. Genotype and protein levels moderate the uptake of androgens into prostate cells, a process competitively inhibited in vitro by statin medications. Single-nucleotide polymorphisms (SNPs) in SLCO2B1 influence SLCO1B2 expression and efficacy of androgen uptake in vitro. Methods: Among men of self-reported European ancestry diagnosed with prostate cancer during prospective follow-up of the Health Professionals Follow-up Study (HPFS, diagnosed 1986-2017) and the Physicians’ Health Study (PHS, 1982-2010) combined, we evaluated four SLCO2B1 SNPs identified in prior studies and time from diagnosis to lethal disease (metastases/prostate cancer-specific death), adjusting for age, calendar year of diagnosis, and principal components of genetic variation to account for local ancestry. We also conducted a systematic review and harmonized allele coding in prior studies for a two-stage dose-response meta-analysis with additive allele coding. Results: Of the 3208 men in the prospective cohort study, 96% were diagnosed with clinically localized (T1/T2) prostate cancer. Most were treated with radical prostatectomy (47%) or radiation (36%); 33% ever received androgen deprivation therapy (ADT). Over up to 32 years of follow-up (median, 14.4 years), 382 lethal events occurred. The intronic SNP rs1077858 had the strongest association with lethal disease (hazard ratio [HR] per minor allele 1.14, 95% confidence interval [CI] 0.98-1.33). Data were suggestive of potential differences for the four SNPs by use of statins and ADT but were not conclusive. The systematic review identified 2 additional population-based studies of primary disease and 4 hospital-based studies of advanced disease, none of which studied effect modification by statins or ADT. Among these 2377 men, 1088 events of recurrence, metastasis, or prostate cancer death occurred. In a meta-analysis of all 7 studies, the genotype of the exonic SNP rs12422149 (minor allele frequency 15%) that results in lower androgen uptake was associated with better outcomes (HR per minor allele 0.80, 95% CI 0.69-0.93), with little between-study heterogeneity (I2 0.27). For the intronic SNP rs1077858, the genotype with higher SLCO2B1 mRNA expression was associated with worse outcomes (HR per minor allele 1.14, 95% CI 0.99-1.31, 3 studies with I2 0.68). Conclusion: These results are compatible with the hypothesis that inherited genetic variation in SLCO2B1 influences androgen uptake and prostate cancer prognosis. These and other genetic variants, together with clinical features, may have potential utility as predictive biomarkers for identifying men with prostate cancer who might benefit from early therapy beyond ADT. Whether adjuvant statin therapy could mitigate the inherited risk requires further study. Citation Format: Konrad H. Stopsack, Harisha Rajanala, Anna Plym, Jane B. Vaselkiv, Konstantina Matsoukas, Ericka M. Ebot, Sarah C. Markt, Kathryn L. Penney, Gwo-Shu M. Lee, Lorelei A. Mucci, Philip W. Kantoff. Organic anion transporter(SLCO)genotype and prostate cancer progression: Prospective cohort study and meta-analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1438.

Abstract 3309: Self-assembling and disassembling (SADA) domain is critical to the binding, and anti-tumor efficacy of GD2-SADA

Abstract Introduction: GD2-SADA is a pre-targeted radioimmunotherapy drug-candidate designed to target radioactive lutetium-177 (in the form of 177Lu-DOTA) to GD2-expressing tumor cells (GD2-SADA:177Lu-DOTA Drug Complex). GD2-SADA is comprised of an anti-GD2 single-chain variable fragment (scFv), an anti-DOTA scFv that binds specifically to DOTA metal chelates, such as 177Lu-DOTA, and a SADA domain based on the human P53 tetramerization sequence, which can self-assemble from a monomeric 60-kDa polypeptide into 240-kDa tetrameric protein. We have previously demonstrated (1) that GD2-SADA has a unique clearance profile that allowed for the delivery of 177Lu-DOTA to tumors with minimal exposure to normal tissues like the bone marrow or kidneys and shrank established neuroblastoma in preclinical mouse models. We now provide additional mechanistic data demonstrating the importance of the SADA domain in providing this potent anti-tumor function. Methods: To study the role of the SADA domain, we designed a modified version of GD2-SADA that could not self-assemble into a tetrameric state by removing the entire SADA domain. The resulting P53(-/-)GD2-SADA protein retained binding to GD2 and Lu-DOTA but remained in a 60 kDa monomeric state in assays. P53(-/-)GD2-SADA and GD2-SADA were compared using SPR to measure antigen binding affinities, flow cytometry to evaluate cell binding, and in preclinical mouse models to evaluate tumor uptake and anti-tumor efficacy. Results: GD2-SADA demonstrated stronger binding affinity to GD2 antigen compared to P53(-/-)GD2-SADA, but comparable binding affinity to Lu-DOTA. In SPECT/CT imaging studies using tumor bearing mice, GD2-SADA demonstrated substantially higher uptake and persistence in GD2-expressing tumors compared with P53(-/-)GD2-SADA. Finally, treatment of tumor bearing mice with 3 cycles of GD2-SADA or P53(-/-)GD2-SADA and 177Lu-DOTA (once per week each for 3 weeks) resulted in potent anti-tumor responses from GD2-SADA, with only modest anti-tumor efficacy from P53(-/-)GD2-SADA at two different dose levels corresponding to equal molar doses (1 GD2-SADA to 1 P53(-/-)GD2-SADA) or equal mass doses (1 GD2-SADA to 4 P53(-/-)GD2-SADA i.e. same number of binding sites). Conclusions: The tetramerizing function of the SADA domain is critically important to the binding activity and anti-tumor efficacy of GD2 SADA. These data confirm that the SADA domain increases tumor antigen binding, uptake and persistence in tumor tissue, and markedly improves anti-tumor responses in preclinical models.