Large Amino Acid Transporter Research Articles

Expression of the large amino acid transporter SLC7A5/LAT1 on immune cells is enhanced in primary sclerosing cholangitis-associated cholangiocarcinoma and correlates with poor prognosis in cholangiocarcinoma

Biliary tract cancers (BTC) are rare lethal malignancies arising along the biliary tree. Unfortunately, effective therapeutics are lacking and the prognosis remains dismal even for patients eligible for surgical resection. Therefore, novel therapeutic approaches along with early detection strategies and prognostic markers are urgently needed. Primary sclerosing cholangitis (PSC) is a chronic disease of the bile ducts leading to fibrosis and ultimately cirrhosis. Patients with PSC have a 5–20% lifetime risk of developing BTC; yet the molecular mechanisms that underpin the development of PSC- associated biliary tract cancer (PSC-BTC) have not been fully elucidated. SLC7A5/LAT1, a large amino acid transporter, has been shown to modulate cell growth and proliferation as well as other intracellular processes in solid tumors. In this study, we evaluated SLC7A5 expression in PSC-BTC and in sporadic BTC (sBTC) and its role as a prognostic factor. Analysis of the TGCA cohort showed a significantly higher expression of SLC7A5 in tumor tissue compared with adjacent normal tissue (p = 0.0002) in BTC. In our cohort (comprised of 69 BTC patients including 16 PSC-BTC), SLC7A5/LAT1 expression was observed in both tumor and intratumoral immune cells. A significantly higher percentage of SLC7A5/LAT1 positive intratumoral immune cells was observed in PSC-BTC compared with sBTC (p = 0.004). Multiplex immunofluorescence co-detection by indexing (CODEX) analysis identified CD4+ regulatory T lymphocytes and CD68+ macrophages as the largest immune cell populations expressing LAT1.SLC7A5/LAT1 expression as well as a higher intratumoral infiltration of SLC7A5/LAT1-positive immune cells (≥2%) were associated with a shorter overall survival in our cohort (LogRank test, p = 0.04 and p = 0.008; respectively). SLC7A5/LAT1 expressing tumors are higher staged tumors (pT3/4 versus pT1/2, p = 0.048).These results underline the potential use of SLC7A5/LAT1 as a prognostic marker in BTC. Furthermore, the higher frequency of SLC7A5/LAT1 positive immune cells in PSC-BTC compared to sBTC may hint at the potential role of SLC7A5/LAT1 in inflammation-driven carcinogenesis.

The Development of LAT1 Efflux Agonists as Mechanistic Probes of Cellular Amino Acid Stress.

Amino acid restriction induces cellular stress and cells often respond via the induction of autophagy. Autophagy or 'self-eating' enables the recycling of proteins and provides the essential amino acids needed for cell survival. Of the naturally occurring amino acids, methionine restriction has pleiotropic effects on cells because methionine also contributes to the intracellular methyl pools required for epigenetic controls as well as polyamine biosynthesis. In this report, we describe the chemical synthesis of four diastereomers of a methionine depletion agent and demonstrate how controlled methionine efflux from cells significantly reduces intracellular methionine, S-adenosylmethionine (SAM), S-adenosyl homocysteine (SAH), and polyamine levels. We also demonstrate that human pancreatic cancer cells respond via a lipid signaling pathway to induce autophagy. The methionine depletion agent causes the large amino acid transporter 1 (LAT1) to preferentially work in reverse and export the cell's methionine (and leucine) stores. The four diastereomers of the lead methionine/leucine depletion agent were synthesized and evaluated for their ability to (a) efflux 3H-leucine from cells, (b) dock to LAT1 in silico, (c) modulate intracellular SAM, SAH, and phosphatidylethanolamine (PE) pools, and (d) induce the formation of the autophagy-associated LC3-II marker. The ability to modulate the intracellular concentration of methionine regardless of exogenous methionine supply provides new molecular tools to better understand cancer response pathways. This information can then be used to design improved therapeutics that target downstream methionine-dependent processes like polyamines.

Prognostic value of large amino acid transporter type 1 (LAT1) expression in pulmonary adenocarcinoma: A tissue microarray study

BackgroundLarge amino acid transporter type 1 (LAT1) provides cancer cells with essential amino acids for both protein synthesis and cell growth and may predict patient prognosis. Additionally, LAT1 inhibition can be a therapeutic target. This study aimed to examine the prognostic significance of LAT1 expression in lung cancer, paying special attention to adenocarcinoma subtypes. MethodsTissue microarrays (TMA) of 1,560 total cores obtained from surgically resected lung cancer specimens between 1995 and 2008 at our hospital were used. Overall, 795 cases of adenocarcinoma were identified, and 717 underwent further evaluation. Immunohistochemical staining of whole slides and TMA cores were assessed to set H-score cutoff value.. Immunohistochemical expression of LAT1 was examined based on the subtypes of adenocarcinoma. Statistical analyses explored the prognostic significance of LAT1. ResultsAdenocarcinoma accounted for 71.8% of all cases (n = 795), and 216 cases (27.1%) expressed LAT1. The 795 cases were categorized into five subtypes: lepidic (n = 29, 3.6%), papillary (n = 601, 75.6%), acinar (n = 58, 7.3%), and solid (n = 9, 1.1%); 717 of the 795 cases were further assessed according to the exclusion criteria. The LAT1-positive ratio increased as the architectural grade increased. Notably, in papillary adenocarcinoma, the LAT1-positive group had significantly lower overall survival compared to the negative group (10-year survival: 45.6% vs. 60.8%, p < 0.001). ConclusionLAT1 expression was higher in high-grade subtypes of pulmonary adenocarcinoma. Moreover, LAT1 expression is useful for predicting prognosis, particularly in papillary adenocarcinoma, facilitating prognostic stratification of papillary adenocarcinoma.

Amino Acid-Based Boron Carriers in Boron Neutron Capture Therapy (BNCT).

Interest in the design of boronated amino acids has emerged, partly due to the utilization of boronophenylalanine (BPA), one of the two agents employed in clinical Boron Neutron Capture Therapy (BNCT). The boronated amino acids synthesized thus far for BNCT investigations can be classified into two categories based on the source of boron: boronic acids or carboranes. Amino acid-based boron carriers, employed in the context of BNCT treatment, demonstrate significant potential in the treatment of challenging tumors, such as those located in the brain. This review aims to shed light on the developmental journey and challenges encountered over the years in the field of amino acid-based boron delivery compound development. The primary focus centers on the utilization of the large amino acid transporter 1 (LAT1) as a target for boron carriers in BNCT. The development of efficient carriers remains a critical objective, addressing challenges related to tumor specificity, effective boron delivery, and rapid clearance from normal tissue and blood. LAT1 presents an intriguing and promising target for boron delivery, given its numerous characteristics that make it well suited for drug delivery into tumor tissues, particularly in the case of brain tumors.

Compensatory Processes in Striatal Neurons Expressing the Tyrosine Hydroxylase Gene in Transgenic Mice in a Model of Parkinson's Disease.

The mammalian striatum is known to contain non-dopaminergic neurons that express dopamine (DA)-synthesizing enzymes and produce DA, responsible for the regulation of motor function. This study assessed the expression of DA-synthesizing enzymes in striatal neurons and their role in DA synthesis in transgenic mice expressing the green fluorescent protein (GFP) gene under the tyrosine hydroxylase (TH) gene promoter in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease (PD). We showed that, in Parkinsonian animals, the number of neurons expressing the TH gene increased by 1.9 times compared with the control (0.9% NaCl), which indicates a compensatory response to the DAergic denervation of the striatum. This assumption is supported by a 2.5-fold increase in the expression of genes for TH and transcription factor Nurr1 and a 1.45-fold increase in the expression of the large amino acid transporter 1 gene. It is noteworthy that, in Parkinsonian mice, in contrast to the controls, DA-synthesizing enzymes were found not only in nerve fibers but also in neuronal cell bodies. Indeed, TH or TH and aromatic L-amino acid decarboxylase (AADC) were detected in GFP-positive neurons, and AADC was detected in GFP-negative neurons. These neurons were shown to synthesize DA, and this synthesis is compensatorily increased in Parkinsonian mice. The above data open the prospect of improving the treatment of PD by maintaining DA homeostasis in the striatum.

Valine conjugated polymeric nanocarriers for targeted co-delivery of rivastigmine and quercetin in rat model of Alzheimer disease

Multifunctional nanocarriers are increasingly promising for disease treatment aimed at finding effective therapy and overcoming barriers in drug delivery. Herein, valine conjugated chitosan (VLCS) was used for surface modification of nanocarriers (NCs) based on Poly (ε-caprolactone)-Poly (ethylene glycol)-Poly (ε-caprolactone) (PCL-PEG-PCL) triblock copolymers (NCs@VLCS). The nanocarriers were co-loaded with rivastigmine (RV) and quercetin (QT) to yield the final RV/QT-NCs@VLCS as a multifunctional nanocarrier for Alzheimer’s disease (AD) treatment. The large amino acid transporter 1 (LAT-1) was selected for the direction of the NCs to the brain. The biocompatibility of the nanocarrier was studied in HEK-293 and SH-SY5Y cells and rats. The Morris water maze test demonstrated a faster regain of memory loss with RV/QT-NCs@VLCS compared to the other groups. Furthermore, RV/QT-NCs@VLCS and RV/QT-NCs improved GSH depletion induced by scopolamine (SCO), with RV/QT-NCs@VLCS having a superior effect. The real-time PCR analysis revealed that co-delivery of RV and QT by NCs@VLCS showed significantly higher efficacy than sole delivery of RV. RV/QT-NCs@VLCS treatment also modulated the expression of BDNF, ACHE, and TNF-α. The findings revealed that NCs@VLCS co-loaded with RV and QT, significantly increased efficacy relative to the single use of RV and could be considered a potent multifunctional drug delivery system for Alzheimer’s treatment.

Insights into the Transport Cycle of LAT1 and Interaction with the Inhibitor JPH203.

The large Amino Acid Transporter 1 (LAT1) is an interesting target in drug discovery since this transporter is overexpressed in several human cancers. Furthermore, due to its location in the blood-brain barrier (BBB), LAT1 is interesting for delivering pro-drugs to the brain. In this work, we focused on defining the transport cycle of LAT1 using an in silico approach. So far, studies of the interaction of LAT1 with substrates and inhibitors have not considered that the transporter must undergo at least four different conformations to complete the transport cycle. We built outward-open and inward-occluded conformations of LAT1 using an optimized homology modelling procedure. We used these 3D models and the cryo-EM structures in outward-occluded and inward-open conformations to define the substrate/protein interaction during the transport cycle. We found that the binding scores for the substrate depend on the conformation, with the occluded states as the crucial steps affecting the substrate affinity. Finally, we analyzed the interaction of JPH203, a high-affinity inhibitor of LAT1. The results indicate that conformational states must be considered for in silico analyses and early-stage drug discovery. The two built models, together with the available cryo-EM 3D structures, provide important information on the LAT1 transport cycle, which could be used to speed up the identification of potential inhibitors through in silico screening.

Construction of Bio-conjugated nano-vesicles using non-ionic surfactants for targeted drug delivery: A computational supported experimental study

IntroductionTargeted delivery of drugs to the brain is a complicated activity. This complication is caused by the blood–brain barrier (BBB). Interestingly, there are various transporters on the surface of the BBB that are naturally used for the transportation of specific compounds. A practical approach for brain drug delivery is targeting these transporters with appropriate biomolecules. Large Amino Acid Transporter 1 (LAT1) and Glucose transporter 1 (GLUT1) are two essential transporters on the surface of the BBB that transport amino acids and carbohydrates, respectively. MethodsIn this study, we evaluated the binding affinity of various biomolecules such as amino acids and carbohydrates against their related transporters by conducting molecular docking study. The biomolecules with the highest binding affinity were selected for conjugation to the niosomes. After the conjugation of biomolecules to cetyl alcohol, the bio-conjugated vesicles were constructed with a non-ionic surfactant (Span60) and cholesterol by the thin-film hydration method. The size distribution of formulations was measured with Dynamic Light Scattering. The niosomal formulations were treated on the HUVEC cells and the cellular uptake of the niosomes was assessed by fluorescent microscopy. ResultsTrehalose and glucose showed the highest binding affinity to GLUT1 (-7.787 and −5.715 kcal/mol, respectively), and threonine and glutamine showed the highest binding affinity to LAT1 (-4.532 and −5.946, respectively). Trehalose-conjugated niosomes with a size distribution of 60 nm represent the highest cellular uptake in HUVEC cells. ConclusionComputational approaches are practical methods for selecting appropriate targeting agents. In addition, trehalose is a potential agent for bio-conjugation and can improve the transport of the niosomes through the BBB.

Chemical Approaches for Studying the Biology and Pharmacology of Membrane Transporters: The Histidine/Large Amino Acid Transporter SLC7A5 as a Benchmark.

The localization of membrane transporters at the forefront of natural barriers makes these proteins very interesting due to their involvement in the absorption and distribution of nutrients and xenobiotics, including drugs. Over the years, structure/function relationship studies have been performed employing several strategies, including chemical modification of exposed amino acid residues. These approaches are very meaningful when applied to membrane transporters, given that these proteins are characterized by both hydrophobic and hydrophilic domains with a different degree of accessibility to employed chemicals. Besides basic features, the chemical targeting approaches can disclose information useful for pharmacological applications as well. An eminent example of this picture is the histidine/large amino acid transporter SLC7A5, known as LAT1 (Large Amino Acid Transporter 1). This protein is crucial in cell life because it is responsible for mediating the absorption and distribution of essential amino acids in peculiar body districts, such as the blood brain barrier and placenta. Furthermore, LAT1 can recognize a large variety of molecules of pharmacological interest and is also considered a hot target for drugs due to its over-expression in virtually all human cancers. Therefore, it is not surprising that the chemical targeting approach, coupled with bioinformatics, site-directed mutagenesis and transport assays, proved fundamental in describing features of LAT1 such as the substrate binding site, regulatory domains and interactions with drugs that will be discussed in this review. The results on LAT1 can be considered to have general applicability to other transporters linked with human diseases.

Synthesis of L-DOPA conjugated doxorubicin-polyethylenimine nanocarrier and evaluation of its cytotoxicity on A375 and HepG2 cell lines

Objective(s): Polyethylenimine (PEI) is one of the most-extensively investigated cationic polymers for gene and drug delivery. Recently, great attention has been directed to design of carriers for co-delivery of nucleic acids and small molecules. These delivery systems are able to overcome the limitations of gene or drug delivery alone. The aim of this study is to prepare a targeted nano-carrier for co-delivery of doxorubicin (Dox) and gene using polyethylenimine. Materials and Methods: In order to prepare the ligand-containing polymer conjugates, succinic anhydride was conjugated onto the hydroxyl group of Dox through an ester bond following the protection of Dox amines by Fmoc. Drug-polymer conjugates were then coupled with L-DOPA in order to prepare the targeted nanocarriers to the cells through Large Amino Acid Transporter-1 (LAT-1). The PEI derivatives were characterized using 1H-NMR. The toxicity of conjugated polymer, Dox and PEI was assessed on HepG2 and A375 cell lines with different expression level of LAT-1 transporters using MTT assay. Results: The chemical structure of PEI conjugate was confirmed by 1H-NMR. The cytotoxicity measurement demonstrated a cell line-dependent toxicity profile at the concentrations tested in this study. It was shown that there was no significant difference in cell-induced toxicity between conjugated polymer and its parent form in A375 cell line while the cytotoxicity of conjugated polymer was significantly lower than the parent PEI in HepG2 cells.Conclusion: These results provide promising evidence for further evaluation of PEI conjugate for co-delivery of drug and gene via LAT-1 transporters.

A Novel Blood-Brain Barrier-Permeable Chemotherapeutic Agent for the Treatment of Glioblastoma.

IntroductionThe standard treatment for glioblastoma (GBM) patients is surgical tumor resection, followed by radiation and chemotherapy with temozolomide (TMZ). Unfortunately, 60% of newly diagnosed GBM patients express high levels of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) and are TMZ-resistant, and all patients eventually become refractory to treatment. The blood-brain barrier (BBB) is an obstacle to the delivery of chemotherapeutic agents to GBM, and BBB-permeable agents that are efficacious in TMZ-resistant and refractory patients are needed. The large amino acid transporter 1 (LAT1) is expressed on the BBB and in GBM and is detected at much lower levels in normal brain tissue. A LAT1-selective therapeutic would potentially target brain tumors while avoiding uptake by healthy tissue.MethodsWe report a novel chemical entity (QBS10072S) that combines a potent cytotoxic chemotherapeutic domain (tertiary N-bis(2-chloroethyl)amine) with the structural features of a selective LAT1 substrate and tested it against GBM models in vitro and in vivo. For in vitro studies, DNA damage was assessed with a gamma H2A.X antibody and cell viability was assessed by WST-1 assay and/or CellTiter-Glo assay. For in vivo studies, QBS10072S (with or without radiation) was tested in orthotopic glioblastoma xenograft models, using overall survival and tumor size (as measured by bioluminescence), as endpoints.ResultsQBS10072S is 50-fold more selective for LAT1 vs. LAT2 in transport assays and demonstrates significant growth suppression in vitro of LAT1-expressing GBM cell lines. Unlike TMZ, QBS10072S is cytotoxic to cells with both high and low levels of MGMT expression. In orthotopic GBM xenografts, QBS10072S treatment significantly delayed tumorigenesis and prolonged animal survival compared to the vehicle without adverse effects.ConclusionQBS10072S is a novel BBB-permeable chemotherapeutic agent with the potential to treat TMZ-resistant and recurrent GBM as monotherapy or in combination with radiation treatment.

Highly Specific L-Type Amino Acid Transporter 1 Inhibition by JPH203 as a Potential Pan-Cancer Treatment

Accelerated cancer cell growth requires a massive intake of amino acids. Overexpression of L-type (large) amino acid transporter 1 (LAT1) on the cancer cell membrane facilitates such a demand, which is limited in normal organs. Therefore, LAT1 overexpression is ideal as a molecular cancer therapeutic target. JPH203, a LAT1-selective non-transportable blocker, had demonstrated LAT1 inhibition in &lt;10 µM IC50 values and effectively suppressed cancer cell growth in studies involving several types of cancer cell lines and tumor xenograft models. A limited phase I clinical trial was performed on five different solid tumors and showed that JPH203 is well-tolerated and has a promising activity for the treatment of bile duct cancer. This review details the development and prospect of JPH203 as a LAT1-targeting cancer therapy.

Butyrate Rescues Oxidative Stress-Induced Transport Deficits of Tryptophan: Potential Implication in Affective or Gut-Brain Axis Disorders

Introduction: Butyrate is a short-chain fatty acid metabolite produced by microbiota in the colon. With its antioxidant properties, butyrate has also been shown to alter the neurological functions in affective disorder models, suggesting it as a key mediator in gut-brain interactions. Objective: Here, we evaluated the negative effect of oxidative stress on the transport of the serotonin precursor tryptophan as present in affective disorders. Butyrate was hypothesized to be able to rescue these deficits due to its antioxidative capacities and its effect on transmembrane transport of tryptophan. Human skin-derived fibroblasts were used as cellular models to address these objectives. Methods: Human fibroblasts were treated with hydrogen peroxide to induce oxidative stress. Stressed as well as control cells were treated with different concentrations of butyrate. Tryptophan (3H) was used as a tracer to measure the transport of tryptophan across the cell membranes (n = 6). Furthermore, gene expression profiles of different amino acid transporters were analyzed (n = 2). Results: As hypothesized,oxidative stress significantly decreased the uptake of tryptophan in fibroblast cells, while butyrate counteracted this effect. Oxidative stress did not alter the gene expression profile of amino acid transporters. However, treatment of stressed and control cells with different concentrations of butyrate differentially regulated the gene expression of large amino acid transporters 1 and 2, which are the major transporters of tryptophan. Conclusions:Gut microbiota-derived butyrate may have therapeutic potential in affective disorders characterized by either aberrant serotonergic activity or neuroinflammation due to its role in rescuing the oxidative stress-induced perturbations of tryptophan transport.

Abnormalities in the genes that encode Large Amino Acid Transporters increase the risk of Autism Spectrum Disorder.

BackgroundAutism spectrum disorder (ASD) is a common neurodevelopmental disorder whose molecular mechanisms are largely unknown. Several studies have shown an association between ASD and abnormalities in the metabolism of amino acids, specifically tryptophan and branched‐chain amino acids (BCAAs).MethodsNinety‐seven patients with ASD were screened by Sanger sequencing the genes encoding the heavy (SLC3A2) and light subunits (SLC7A5 and SLC7A8) of the large amino acid transporters (LAT) 1 and 2. LAT1 and 2 are responsible for the transportation of tryptophan and BCAA across the blood–brain barrier and are expressed both in blood and brain. Functional studies were performed employing the Biolog Phenotype Microarray Mammalian (PM‐M) technology to investigate the metabolic profiling in lymphoblastoid cell lines from 43 patients with ASD and 50 controls with particular focus on the amino acid substrates of LATs.ResultsWe detected nine likely pathogenic variants in 11 of 97 patients (11.3%): three in SLC3A2, three in SLC7A5, and three in SLC7A8. Six variants of unknown significance were detected in eight patients, two of which also carrying a likely pathogenic variant.The functional studies showed a consistently reduced utilization of tryptophan, accompanied by evidence of reduced utilization of other large aromatic amino acids (LAAs), either alone or as part of a dipeptide.ConclusionCoding variants in the LAT genes were detected in 17 of 97 patients with ASD (17.5%). Metabolic assays indicate that such abnormalities affect the utilization of certain amino acids, particularly tryptophan and other LAAs, with potential consequences on their transport across the blood barrier and their availability during brain development. Therefore, abnormalities in the LAT1 and two transporters are likely associated with an increased risk of developing ASD.

Large amino acid transporter 1 mediated glutamate modified mesoporous silica nanoparticles for chemophotothermal therapy

Glutamic acid modified Pluronic P123 (P123-G) was synthesized to coat hydrophobic mesoporous silica nanoparticles (PGMSN) aiming to target large amino acid transporter 1 (LAT1) overexpressed cancer cells. We discovered that, once internalized, PGMSN could be transported out of the cells with their cargo, and the exported nanoparticles were then taken up by neighboring cells. This intercellular delivery of particles led to a deeper tumor penetration, presumably through tandem cycles of LAT1 mediated endocytosis and exocytosis. Taking advantage of high drug loading capability of MSN, a kind of cyanine dye Cypate acting as a photothermal converting agent was loaded into MSN along with the chemotherapeutic drug doxorubicin hydrochloride (DOX) to improve the anti-cancer effect by the combination of chemotherapy and photothermal therapy (PTT). Both DOX and Cypate stably dispersed in the pores of MSN, and the coating of P123-G serving as diffusion barrier blocked drug preleakage. The system possessed pH and near-infrared (NIR) light dual-responsive DOX release property. Additionally, PGMSN exhibited targeted PTT effect which strengthened cytotoxic activity of the system due to the enhanced endocytosis. So the deep tumor penetration and targeted PTT effect made PGMSN especially suitable for cancers overexpressing LAT1 receptors.

Abstract B125: A novel BBB-permeable chemotherapeutic agent for the treatment of CNS tumors

Abstract Standard of care for glioblastoma multiforme (GBM) patients is surgical tumor resection, followed by radiation and chemotherapy with temozolomide. Unfortunately, 60% of newly diagnosed GBM patients express high levels of the DNA repair enzyme MGMT and are temozolomide-insensitive, and all patients eventually become refractory to treatment. The blood-brain barrier (BBB) remains an obstacle to adequate delivery of chemotherapeutic agents to brain tumors. Lipophilic drugs such as temozolomide and lomustine are able to cross the BBB based on passive diffusion; however, their tissue distribution also causes toxicity to critical systemic organs, such as the bone marrow, leading to dose-limiting toxicity. Therefore, BBB-permeable chemotherapeutic agents that are efficacious in temozolomide-insensitive and refractory patients are needed. The large amino acid transporter 1 (LAT1) is highly expressed on the BBB and in GBM and other malignant brain tumors such as diffuse intrinsic pontine glioma (DIPG), where it is associated with poor prognosis, but is undetectable in normal brain tissue. Targeting LAT1 transport, while avoiding interaction with other closely related transporters expressed on normal tissue such as LAT2, will target brain tumors and avoid uptake into healthy tissue. We report the generation of a novel molecule (QBS10072S) that combines a potent cytotoxic domain with the structural features of a selective LAT1 substrate. QBS10072S is 50-fold more selective for LAT1 vs. LAT2 in transport assays, and demonstrates significant in vitro cytotoxicity to LAT1-expressing GBM cell lines and minimal cytotoxicity to normal human astrocytes (LAT1-negative). Unlike temozolomide, QBS10072S is cytotoxic to cells with both high and low levels of MGMT expression. In orthotopic glioblastoma xenografts, QBS10072S causes significant delay in tumor growth and increase in mouse survival compared to vehicle. QBS10072S is well tolerated, with no toxicity seen in normal brain or the BBB. In summary, QBS10072S is a novel BBB-permeable chemotherapeutic agent with the potential to treat temozolomide-insensitive and recurrent GBM. Citation Format: Theodore P. Nicolaides, Tomoko Ozawa, Tsun Wen Yao, Michael Prados, Wolf-Nicolas Fischer, Bernd Jandeleit, Kerry Koller. A novel BBB-permeable chemotherapeutic agent for the treatment of CNS tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B125.

Large Amino Acid Transporter 1 Selective Liposomes of l-DOPA Functionalized Amphiphile for Combating Glioblastoma.

Despite significant progress in neurosurgery and radiation therapy during the past decade, overall survivability (OS) of glioblastoma patients continues to be less than 2 years. The scope of systemic chemotherapy is greatly limited by poor drug transport across the blood brain barrier (BBB) and, thereby, suboptimal drug accumulation in glioma tissue. To this end, use of large amino acid transporter-1 (LAT1) overexpressed both on brain capillary endothelial cells (BCECs) and glioma cells has begun. Prior reports on the use of LAT1 mediated delivery of model drugs showed their brain accumulations. However, in depth in vivo glioblastoma regression studies aimed at examining the therapeutic potential of LAT1 mediated delivery of potent chemotherapeutics to brain tumor tissues have not yet been undertaken. Herein, we report on the development of a nanometric (100-135 nm) promising LAT1 selective liposomal drug carrier prepared from a novel l-3,4-dihydroxyphenylalanine (l-DOPA) functionalized amphiphile (Amphi-DOPA). In vitro studies using Rh-PE labeled liposomes of Amphi-DOPA both in untreated glioma (GL261) cells and in GL261cells preincubated with LAT1 antibody revealed LAT1 mediated cellular uptake. Intravenously administered NIR-dye labeled liposomes of Amphi-DOPA in glioblastoma-bearing mice showed preferential accumulation of the dye in brain tissue. Notably iv administration of WP1066-loaded liposomes of Amphi-DOPA enhanced the overall survivability of C57BL/6J mice bearing orthotopically established mouse glioblastoma by ∼60% compared to that for the untreated mouse group. Furthermore, we show that the OS of established glioblastoma-bearing mice can be significantly enhanced (by >300% compared to that for the untreated mouse group) when the presently described LAT1 mediated targeted chemotherapy with WP1066-loaded liposomes of Amphi-DOPA is combined with in vivo DC-targeted DNA vaccination using a survivin (a glioblastoma antigen) encoded DNA vaccine. The present findings open a new door for LAT1 mediated systemic chemotherapy of glioblastoma.

Blood brain barrier permeable gold nanocluster for targeted brain imaging and therapy: an in vitro and in vivo study.

Blood brain barrier (BBB) is a dynamic interface, comprising polarized endothelial cells, that separates the brain from the circulatory system. The highly protective nature of this tight junction impairs diagnosis and treatment of brain disorders. In this study, we designed a sub atomic size, near infrared emitting, dual function glutathione gold cluster with high fluorescence yield to facilitate permeability of BBB, for imaging applications and drug delivery. The gold cluster was then modified with Levodopa (l-dopa), to utilize the large amino acid transporter 1 (LAT1) pathways to enhance brain entry. Uptake and permeability of the nanoprobes were demonstrated using an established model of BBB, comprising brain endothelial cells (bEnd.3). The uptake and the clearance of l-dopa modified cluster was faster than the glutathione cluster. l-Dopa modified cluster supports the slow and sustained delivery of a model drug, pilocarpine, to the brain. Results of in vivo imaging and drug release in normal mice hold promise for considering the probe for early diagnosis of brain diseases, when the barrier is not disrupted, and for subsequent drug treatment.

Targeting tumor highly-expressed LAT1 transporter with amino acid-modified nanoparticles: Toward a novel active targeting strategy in breast cancer therapy

Designing active targeting nanocarriers with increased cellular accumulation of chemotherapeutic agents is a promising strategy in cancer therapy. Herein, we report a novel active targeting strategy based on the large amino acid transporter 1 (LAT1) overexpressed in a variety of cancers. Glutamate was conjugated to polyoxyethylene stearate as a targeting ligand to achieve LAT1-targeting PLGA nanoparticles. The targeting efficiency of nanoparticles was investigated in HeLa and MCF-7 cells. Significant increase in cellular uptake and cytotoxicity was observed in LAT1-targeting nanoparticles compared to the unmodified ones. More interestingly, the internalized LAT1 together with targeting nanoparticles could recycle back to the cell membrane within 3 h, guaranteeing sufficient transporters on cell membrane for continuous cellular uptake. The LAT1 targeting nanoparticles exhibited better tumor accumulation and antitumor effects. These results suggested that the overexpressed LAT1 on cancer cells holds a great potential to be a high-efficiency target for the rational design of active-targeting nanosystems.

Amino Acid Metabolism Abnormity and Microenvironment Variation Mediated Targeting and Controlled Glioma Chemotherapy.

Energy metabolism abnormity is one of the most significant hallmarks of cancer. As a result, large amino acid transporter 1 (LAT1) is remarkably overexpressed in both blood-brain-barrier and glioma tumor cells, leading a rapid and sufficient substrate transportation. 3CDIT and 4CDIT are originally synthesized by modifying the existing most potent LAT1 substrate. 3CDIT is selected as its higher glioma-targeting ability. Since the microenvironment variation in tumor cells is another important feature of cancer, a great disparity in adenosine-5'-triphosphate (ATP) and glutathione (GSH) levels between extracellular and intracellular milieu can provide good possibilities for dual-responsive drug release in tumor cells. Doxorubicin (DOX) is successfully intercalated into the ATP aptamer DNA scaffolds, compressed by GSH-responsive polymer pOEI, and modified with 3CDIT to obtain 3CDIT-targeting pOEI/DOX/ATP aptamer nanoparticles (NPs). Enhanced NP accumulation and rapid GSH & ATP dual-responsive DOX release in glioma are demonstrated both in vitro and in vivo. More efficient therapeutic effects are shown with 3CDIT-targeting pOEI/DOX/ATP aptamer NPs than free DOX and no systemic toxicity is observed. Therefore, glioma-targeting delivery and GSH & ATP dual-responsive release guarantee an adequate DOX accumulation within tumor cells and ensure a safe and efficient chemotherapy for glioma.