Altering distribution profile of palbociclib by its prodrugs.

Palbociclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor, is currently used clinically for treating hormone receptor-positive and human epidermal growth factor receptor 2 negative breast cancer. Additionally, it has the potential to be utilized in the treatment of various tumors, including malignant glioblastoma. Previous research has indicated that palbociclib is a substrate for two efflux transporters, P-glycoprotein (P-gp; MDR1) and breast cancer-resistant protein (BCRP), which restrict the brain exposure of palbociclib. In the present study, our objective was to alter the brain distribution pattern of palbociclib by creating and assessing two novel prodrugs through in vitro, in situ, and in vivo evaluations. To this end, we synthesized two prodrugs of palbociclib by attaching it to the tyrosine promoiety at the para- (PD1) and meta-(PD2) position via a carbamate bond. We hypothesized that the prodrugs could bypass efflux transporter-mediated drug resistance by leveraging the l-type amino acid transporter (LAT1) to facilitate their transport across the blood-brain barrier (BBB) and into cancer cells, such as glioma cells that express LAT1. The compounds PD1 and PD2 did not show selective binding and had limited inhibitory effects on LAT1 in three cell lines (MCF-7, U87-MG, HEK-hLAT1). However, PD1 and PD2 demonstrated the ability to evade efflux mechanisms, and their in vitro uptake profiles were comparable to that of palbociclib, indicating their potential for effective cellular transport. In in situ and in vivo studies, brain uptake was not significantly improved compared to palbociclib, but the pharmacokinetic profiles showed encouraging enhancements. PD1 exhibited a higher AUCbrain/plasma ratio, suggesting safer dosing, while PD2 showed favorable long-acting pharmacokinetics. Although our prodrug design did not significantly improve palbociclib brain delivery due to the potential size limitation of the prodrugs, the study provides valuable insights for future prodrug development and drug delivery strategies targeting specific transporters.

Amino acid derivative of probenecid potentiates apoptosis-inducing effects of vinblastine by increasing oxidative stress in a cancer cell-specific manner.

Many chemotherapeutic drugs suffer from multidrug resistance (MDR). Efflux transporters, namely ATP-binding cassettes (ABCs), that pump the drugs out of the cancer cells comprise one major reason behind MDR. Therefore, ABC inhibitors have been under development for ages, but unfortunately, without clinical success. In the present study, an l-type amino acid transporter 1 (LAT1)-utilizing derivative of probenecid (PRB) was developed as a cancer cell-targeted efflux inhibitor for P-glycoprotein (P-gp), breast cancer resistant protein (BCRP) and/or several multidrug resistant proteins (MRPs), and its ability to increase vinblastine (VBL) cellular accumulation and apoptosis-inducing effects were explored. The novel amino acid derivative of PRB (2) increased the VBL exposure in triple-negative human breast cancer cells (MDA-MB-231) and human glioma cells (U-87MG) by 10-68 -times and 2-5-times, respectively, but not in estrogen receptor-positive human breast cancer cells (MCF-7). However, the combination therapy had greater cytotoxic effects in MCF-7 compared to MDA-MB-231 cells due to the increased oxidative stress recorded in MCF-7 cells. The metabolomic study also revealed that compound 2, together with VBL, decreased the transport of those amino acids essential for the biosynthesis of endogenous anti-oxidant glutathione (GSH). Moreover, the metabolic differences between the outcomes of the studied breast cancer cell lines were explained by the distinct expression profiles of solute carriers (SLCs) that can be concomitantly inhibited. Therefore, attacking several SLCs simultaneously to change the nutrient environment of cancer cells can serve as an adjuvant therapy to other chemotherapeutics, offering an alternative to ABC inhibitors.

Tetrazine Glycoconjugate for Pretargeted Positron Emission Tomography Imaging of trans-Cyclooctene-Functionalized Molecular Spherical Nucleic Acids.

Pretargeted concept in positron emission tomography (PET) together with bioorthogonal chemistry is an elegant solution to study processes with slow pharmacokinetics by utilizing radiotracers labeled with short-lived radionuclides. Namely, radiotracers based on tetrazine ligation with trans-cyclooctene (TCO) via the inverse electron demand Diels-Alder (IEDDA) reaction have become a state-of-the-art for the pretargeted PET imaging. For radiolabeling of tetrazine scaffolds, indirect radiofluorination methods are often preferred, as tetrazines are vulnerable to harsh conditions typically necessary for the direct radiofluorination. 18F-Fluoroglycosylation is an indirect radiofluorination method, which allows the introduction of a widely accessible glucose analog 2-[18F]fluoro-2-deoxy-d-glucose ([18F]FDG) to aminooxy-functionalized precursors via oxime formation. Here, we report the biological evaluation of [18F]FDG-Tz as a tracer for pretargeted PET imaging of TCO-functionalized molecular spherical nucleic acids (MSNA) against human epidermal growth factor receptor 2 (HER2) mRNA. The oxime ether formation between [18F]FDG and tetrazine oxyamine resulted in [18F]FDG-Tz with high radiochemical purity (>99%) and moderate yields (6.5 ± 3.6%, n = 5). Biological evaluation of [18F]FDG-Tz in healthy mice indicated favorable pharmacokinetics with quick blood clearance, urinary excretion as the main elimination route, and the absence of GLUT1 transportation. The successful pretargeted experiments with TCO-functionalized MSNA revealed higher tumor uptake compared to preclicked MSNA in HER2-expressing human breast cancer xenograft-bearing mice.

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.

Structural Features Affecting the Interactions and Transportability of LAT1-Targeted Phenylalanine Drug Conjugates.

L-type amino acid transporter 1 (LAT1) transfers essential amino acids across cell membranes. Owing to its predominant expression in the blood-brain barrier and tumor cells, LAT1 has been exploited for drug delivery and targeting to the central nervous system (CNS) and various cancers. Although the interactions of amino acids and their mimicking compounds with LAT1 have been extensively investigated, the specific structural features for an optimal drug scaffold have not yet been determined. Here, we evaluated a series of LAT1-targeted drug-phenylalanine conjugates (ligands) by determining their uptake rates by in vitro studies and investigating their interaction with LAT1 via induced-fit docking. Combining the experimental and computational data, we concluded that although LAT1 can accommodate various types of structures, smaller compounds are preferred. As the ligand size increased, its flexibility became more crucial in determining the compound's transportability and interactions. Compounds with linear or planar structures exhibited reduced uptake; those with rigid lipophilic structures lacked interactions and likely utilized other transport mechanisms for cellular entry. Introducing polar groups between aromatic structures enhanced interactions. Interestingly, compounds with a carbamate bond in the aromatic ring's para-position displayed very good transport efficiencies for the larger compounds. Compared to the ester bond, the corresponding amide bond had superior hydrogen bond acceptor properties and increased interactions. A reverse amide bond was less favorable than a direct amide bond for interactions with LAT1. The present information can be applied broadly to design appropriate CNS or antineoplastic drug candidates with a prodrug strategy and to discover novel LAT1 inhibitors used either as direct or adjuvant cancer therapy.

Synthesis and In Vitro Evaluation of a Set of 6-Deoxy-6-thio-carboranyl d-Glucoconjugates Shed Light on the Substrate Specificity of the GLUT1 Transporter.

Glucose- and sodium-dependent glucose transporters (GLUTs and SGLTs) play vital roles in human biology. Of the 14 GLUTs and 12 SGLTs, the GLUT1 transporter has gained the most widespread recognition because GLUT1 is overexpressed in several cancers and is a clinically valid therapeutic target. We have been pursuing a GLUT1-targeting approach in boron neutron capture therapy (BNCT). Here, we report on surprising findings encountered with a set of 6-deoxy-6-thio-carboranyl d-glucoconjugates. In more detail, we show that even subtle structural changes in the carborane cluster, and the linker, may significantly reduce the delivery capacity of GLUT1-based boron carriers. In addition to providing new insights on the substrate specificity of this important transporter, we reach a fresh perspective on the boundaries within which a GLUT1-targeting approach in BNCT can be further refined.

Development of Sesamol Carbamate-L-Phenylalanine Prodrug Targeting L-Type Amino Acid Transporter1 (LAT1) as a Potential Antiproliferative Agent against Melanoma.

Sesamol is a compound reported to have anti-melanogenesis and anti-melanoma actions. Sesamol, however, has low intracellular drug concentration and fast excretion, which can limit its benefits in the clinic. To overcome this drawback and increase intracellular delivery of sesamol into the target melanoma, research has focused on L-type amino acid transporter 1 (LAT1)-mediated prodrug delivery into melanoma cells. The sesamol prodrug was designed by conjugating sesamol with L-phenylalanine at the para position with a carbamate bond. LAT1 targeting was evaluated vis-à-vis a competitive [14C]-leucine uptake inhibition. The sesamol prodrug has a higher [14C]-leucine uptake inhibition than sesamol in human LAT1-transfected HEK293 cells. Moreover, the sesamol prodrug was taken up by LAT1-mediated transport into SK-MEL-2 cells more effectively than sesamol. The sesamol prodrug underwent complete hydrolysis, releasing the active sesamol at 72 h, which significantly exerted its cytotoxicity (IC50 of 29.3 µM) against SK-MEL-cells more than sesamol alone. Taken together, the strategy for LAT1-mediated prodrug delivery has utility for the selective uptake of sesamol, thereby increasing its intracellular concentration and antiproliferation activity, targeting melanoma SK-MEL-2 cells that overexpress the LAT1 protein. The sesamol prodrug thus warrants further evaluation in an in vivo model.

Exploring the Biochemical Foundations of a Successful GLUT1-Targeting Strategy to BNCT: Chemical Synthesis and In Vitro Evaluation of the Entire Positional Isomer Library of ortho-Carboranylmethyl-Bearing Glucoconjugates.

Boron neutron capture therapy (BNCT) is a noninvasive binary therapeutic modality applicable to the treatment of cancers. While BNCT offers a tumor-targeting selectivity that is difficult to match by other means, the last obstacles preventing the full harness of this potential come in the form of the suboptimal boron delivery strategies presently used in the clinics. To address these challenges, we have developed delivery agents that target the glucose transporter GLUT1. Here, we present the chemical synthesis of a number of ortho-carboranylmethyl-substituted glucoconjugates and the biological assessment of all positional isomers. Altogether, the study provides protocols for the synthesis and structural characterization of such glucoconjugates and insights into their essential properties, for example, cytotoxicity, GLUT1-affinity, metabolism, and boron delivery capacity. In addition to solidifying the biochemical foundations of a successful GLUT1-targeting approach to BNCT, we identify the most promising modification sites in d-glucose, which are critical in order to further develop this strategy toward clinical use.

L-Type Amino Acid Transporter 1 Enables the Efficient Brain Delivery of Small-Sized Prodrug across the Blood-Brain Barrier and into Human and Mouse Brain Parenchymal Cells.

Membrane transporters have long been utilized to improve the oral, hepatic, and renal (re)absorption. In the brain, however, the transporter-mediated drug delivery has not yet been fully achieved due to the complexity of the blood-brain barrier (BBB). Because L-type amino acid transporter 1 (LAT1) is a good candidate to improve the brain delivery, we developed here four novel LAT1-utilizing prodrugs of four nonsteroidal anti-inflammatory drugs. As a result, all the prodrugs were able to cross the BBB and localize into the brain cells. The brain uptake of salicylic acid (SA) was improved five times, not only across the mouse BBB but also into the cultured mouse and human brain cells. The naproxen prodrug was also transported efficiently into the mouse brain achieving less peripheral exposure, but the brain release of naproxen from the prodrug was not improved. Contrarily, the high plasma protein binding of the flurbiprofen prodrug and the premature bioconversion of the ibuprofen prodrug in the mouse blood hindered the efficient brain delivery. Thus, the structure of the parent drug affects the successful brain delivery of the LAT1-utilizing prodrugs, and the small-sized LAT1-utilizing prodrug of SA constituted a successful model to specifically deliver its parent drug across the mouse BBB and into the cultured mouse and human brain cells.

Addressing the Biochemical Foundations of a Glucose-Based "Trojan Horse"-Strategy to Boron Neutron Capture Therapy: From Chemical Synthesis to In Vitro Assessment.

Boron neutron capture therapy (BNCT) for cancer is on the rise worldwide due to recent developments of in-hospital neutron accelerators which are expected to revolutionize patient treatments. There is an urgent need for improved boron delivery agents, and herein we have focused on studying the biochemical foundations upon which a successful GLUT1-targeting strategy to BNCT could be based. By combining synthesis and molecular modeling with affinity and cytotoxicity studies, we unravel the mechanisms behind the considerable potential of appropriately designed glucoconjugates as boron delivery agents for BNCT. In addition to addressing the biochemical premises of the approach in detail, we report on a hit glucoconjugate which displays good cytocompatibility, aqueous solubility, high transporter affinity, and, crucially, an exceptional boron delivery capacity in the in vitro assessment thereby pointing toward the significant potential embedded in this approach.

Tyrosine-Chlorambucil Conjugates Facilitate Cellular Uptake through L-Type Amino Acid Transporter 1 (LAT1) in Human Breast Cancer Cell Line MCF-7.

l-type amino acid transporter 1 (LAT1) is an amino acid transporter that is overexpressed in several types of cancer and, thus, it can be a potential target for chemotherapy. The objectives of this study were to (a) synthesize LAT1-targeted chlorambucil derivatives and (b) evaluate their LAT1-mediated cellular uptake as well as antiproliferative activity in vitro in the human breast cancer MCF-7 cell line. Chlorambucil was conjugated to l-tyrosine-an endogenous LAT1 substrate-via either ester or amide linkage (compounds 1 and 2, respectively). While chlorambucil itself did not bind to LAT1, its derivatives 1 and 2 bound to LAT1 with a similar affinity as with l-tyrosine and their respective cellular uptake was significantly higher than that of chlorambucil in MCF-7. The results of our cellular uptake study are indicative of antiproliferative activity, as a higher intracellular uptake of chlorambucil derivatives resulted in greater cytotoxicity than chlorambucil by itself. LAT1 thus contributes to intracellular uptake of chlorambucil derivatives and, therefore, increases antiproliferative activity. The understanding gained from our research can be used in the development of LAT1-targeted anticancer drugs and prodrugs for site-selective and enhanced chemotherapeutic activity.