Altered protein expression of membrane transporters in isolated cerebral microvessels and brain cortex of a rat Alzheimer's disease model.

There is growing evidence that membrane transporters expressed at the blood-brain barrier (BBB) and brain parenchymal cells play an important role in Alzheimer's disease (AD) development and progression. However, quantitative information about changes in transporter protein expression at neurovascular unit cells in AD is limited. Here, we studied the changes in the absolute protein expression of five ATP-binding cassette (ABC) and thirteen solute carrier (SLC) transporters in the isolated brain microvessels and brain cortical tissue of TgF344-AD rats compared to age-matched wild-type (WT) animals using liquid chromatography tandem mass spectrometry based quantitative targeted absolute proteomic analysis. Moreover, sex-specific alterations in transporter expression in the brain cortical tissue of this model were examined. Protein expressions of Abcg2, Abcc1 and FATP1 (encoded by Slc27a1) in the isolated brain microvessels of TgF344-AD rats were 3.1-, 2.0-, 4.3-fold higher compared to WT controls, respectively (p < 0.05). Abcc1 and 4F2hc (encoded by Slc3a2) protein expression was significantly up-regulated in the brain cortical tissue of male TgF344-AD rats compared to male WT rats (p < 0.05). The study provides novel information for the elucidation of molecular mechanisms underlying AD and valuable knowledge about the optimal use of the TgF344-AD rat model in AD drug development and drug delivery research.

Targeting Transporters for Drug Delivery to the Brain: Can We Do Better?

Limited drug delivery to the brain is one of the major reasons for high failure rates of central nervous system (CNS) drug candidates. The blood-brain barrier (BBB) with its tight junctions, membrane transporters, receptors and metabolizing enzymes is a main player in drug delivery to the brain, restricting the entrance of the drugs and other xenobiotics. Current knowledge about the uptake transporters expressed at the BBB and brain parenchymal cells has been used for delivery of CNS drugs to the brain via targeting transporters. Although many transporter-utilizing (pro)drugs and nanocarriers have been developed to improve the uptake of drugs to the brain, their success rate of translation from preclinical development to humans is negligible. In the present review, we provide a systematic summary of the current progress in development of transporter-utilizing (pro)drugs and nanocarriers for delivery of drugs to the brain. In addition, we applied CNS pharmacokinetic concepts for evaluation of the limitations and gaps in investigation of the developed transporter-utilizing (pro)drugs and nanocarriers. Finally, we give recommendations for a rational development of transporter-utilizing drug delivery systems targeting the brain based on CNS pharmacokinetic principles.

The Effect of Single Nucleotide Variations in the Transmembrane Domain of OATP1B1 on in vitro Functionality.

PURPOSE: Organic Anion Transporting Polypeptide 1B1 (OATP1B1) mediates hepatic influx and clearance of many drugs, including statins. The SLCO1B1 gene is highly polymorphic and its function-impairing variants can predispose patients to adverse effects. The effects of rare genetic variants of SLCO1B1 are mainly unexplored. We examined the impact of eight naturally occurring rare variants and the well-known SLCO1B1 c.521C > T (V174A) variant on in vitro transport activity, cellular localization and abundance. METHODS: Transport of rosuvastatin and 2,7-dichlorofluorescein (DCF) in OATP1B1 expressing HEK293 cells was measured to assess changes in activity of the variants. Immunofluorescence and confocal microscopy determined the cellular localization of OATP1B1 and LC-MS/MS based quantitative targeted absolute proteomics analysis quantified the amount of OATP1B1 in crude membrane fractions. RESULTS: All studied variants, with the exception of P336R, reduced protein abundance to varying degree. V174A reduced protein abundance the most, over 90% compared to wild type. Transport function was lost in G76E, V174A, L193R and R580Q variants. R181C decreased activity significantly, while T345M and L543W retained most of wild type OATP1B1 activity. P336R showed increased activity and H575L decreased the transport of DCF significantly, but not of rosuvastatin. Decreased activity was interrelated with lower absolute protein abundance in the studied variants. CONCLUSIONS: Transmembrane helices 2, 4 and 11 appear to be crucial for proper membrane localization and function of OATP1B1. Four of the studied variants were identified as loss-of-function variants and as such could make the individual harboring these variants susceptible to altered pharmacokinetics and adverse effects of substrate drugs.

Molecular characteristics supporting l-Type amino acid transporter 1 (LAT1)-mediated translocation.

l-Type amino acid transporter 1 (LAT1) is an interesting protein due to its peculiar expression profile. It can be utilized not only as a carrier for improved or targeted drug delivery, e.g., into the brain but also as a target protein by which amino acid supply can be restricted, e.g., from the cancer cells. The recognition and binding processes of LAT1-ligands, such as amino acids and clinically used small molecules, including l-dopa, gabapentin, and melphalan, are today well-known. Binding to LAT1 is crucial, particularly when designing the LAT1-inhibitors. However, it will not guarantee effective translocation across the cell membrane via LAT1, which is a definite requirement for LAT1-substrates, such as drugs that elicit their pharmacological effects inside the cells. Therefore, in the present study, the accumulation of known LAT1-utilizing compounds into the selected LAT1-expressing cancer cells (MCF-7) was explored experimentally over a time period. The differences found among the transport efficiency and affinity of the studied compounds for LAT1 were subsequently explained by docking the ligands into the human LAT1 model (based on the recent cryo-electron microscopy structure). Thus, the findings of this study clarify the favorable structural requirements of the size, shape, and polarity of the ligands that support the translocation and effective transport across the cell membrane via LAT1. This knowledge can be applied in future drug design to attain improved or targeted drug delivery and hence, successful LAT1-utilizing drugs with increased therapeutic effects.

L-Type amino acid transporter 1 as a target for drug delivery.

Our growing understanding of membrane transporters and their substrate specificity has opened a new avenue in the field of targeted drug delivery. The L-type amino acid transporter 1 (LAT1) has been one of the most extensively investigated transporters for delivering drugs across biological barriers. The transporter is predominantly expressed in cerebral cortex, blood-brain barrier, blood-retina barrier, testis, placenta, bone marrow and several types of cancer. Its physiological function is to mediate Na+ and pH independent exchange of essential amino acids: leucine, phenylalanine, etc. Several drugs and prodrugs designed as LAT1 substrates have been developed to improve targeted delivery into the brain and cancer cells. Thus, the anti-parkinsonian drug, L-Dopa, the anti-cancer drug, melphalan and the anti-epileptic drug gabapentin, all used in clinical practice, utilize LAT1 to reach their target site. These examples provide supporting evidence for the utility of the LAT1-mediated targeted delivery of the (pro)drug. This review comprehensively summarizes recent advances in LAT1-mediated targeted drug delivery. In addition, the use of LAT1 is critically evaluated and limitations of the approach are discussed.

Mechanistic Study on the Use of the l-Type Amino Acid Transporter 1 for Brain Intracellular Delivery of Ketoprofen via Prodrug: A Novel Approach Supporting the Development of Prodrugs for Intracellular Targets.

l-Type amino acid transporter 1 (LAT1), selectively expressed at the blood-brain barrier (BBB) and brain parenchymal cells, mediates brain delivery of drugs and prodrugs such as l-dopa and gabapentin. Although knowledge about BBB transport of LAT1-utilizing prodrugs is available, there is a lack of quantitative information about brain intracellular delivery and influence of prodrugs on the transporter's physiological state. We studied the LAT1-mediated intrabrain distribution of a recently developed prodrug of the cyclooxygenase inhibitor ketoprofen as well as its impact on transporter protein expression and function (i.e., amino acid exchange) using brain slice method in mice and rats. The intrabrain distribution of the prodrug was 16 times higher than that of ketoprofen. LAT1 involvement in brain cellular barrier uptake of the prodrug was confirmed, reflected by a higher unbound brain intracellular compared to brain extracellular fluid concentration. The prodrug did not alter LAT1 protein expression and amino acid exchange. Integration of derived parameters with previously performed in vivo pharmacokinetic study using the Combinatory Mapping Approach allowed to estimate the brain extra- and intracellular levels of unbound ketoprofen, prodrug, and released parent drug. The overall efficiency of plasma to brain intracellular delivery of prodrug-released ketoprofen was 11 times higher than after ketoprofen dosing. In summary, this study provides quantitative information supporting the use of the LAT1-mediated prodrug approach for enhanced brain delivery of drugs with intracellular targets.

Alzheimer's Disease Phenotype or Inflammatory Insult Does Not Alter Function of L-Type Amino Acid Transporter 1 in Mouse Blood-Brain Barrier and Primary Astrocytes.

PURPOSE: The study aim was to evaluate the effect of Alzheimer's disease (AD) and inflammatory insult on the function of L-type amino acid transporter 1 (Lat1) at the mouse blood-brain barrier (BBB) as well as Lat1 function and expression in mouse primary astrocytes. METHODS: The Lat1 function and expression was determined in wildtype astrocytes with and without lipopolysaccharide (LPS)-induced inflammation and in LPS treated AD APP/PS1 transgenic astrocytes. The function of Lat1 at the BBB was evaluated in wildtype mice with and without LPS-induced neuroinflammation and APP/PS1 transgenic mice by in situ brain perfusion. RESULTS: There were 2.1 and 1.6 -fold decreases in Lat1 mRNA and protein expression in LPS-treated wildtype astrocytes compared to vehicle-treated astrocytes. In contrast, Lat1 mRNA and protein expression were increased by 1.7 and 1.2 -fold (not statistically significant) in the transgenic cells. A similar trend was observed in the cell uptake of [14C]-L-leucine. There were no statistically significant differences in [14C]-L-leucine BBB permeation between the groups. CONCLUSIONS: The results showed that neither LPS-induced inflammation or the presence of APP/PS1 mutations alters Lat1 function at the mouse BBB as well as Lat1 protein expression and function in mouse primary astrocytes.

Effects of Dimeric PSD-95 Inhibition on Excitotoxic Cell Death and Outcome After Controlled Cortical Impact in Rats.

Therapeutic effects of PSD-95 inhibition have been demonstrated in numerous studies of stroke; however only few studies have assessed the effects of PSD-95 inhibitors in traumatic brain injury (TBI). As the pathophysiology of TBI partially overlaps with that of stroke, PSD-95 inhibition may also be an effective therapeutic strategy in TBI. The objectives of the present study were to assess the effects of a dimeric inhibitor of PSD-95, UCCB01-144, on excitotoxic cell death in vitro and outcome after experimental TBI in rats in vivo. In addition, the pharmacokinetic parameters of UCCB01-144 were investigated in order to assess uptake of the drug into the central nervous system of rats. After a controlled cortical impact rats were randomized to receive a single injection of either saline or two different doses of UCCB01-144 (10 or 20 mg/kg IV) immediately after injury. Spatial learning and memory were assessed in a water maze at 2 weeks post-trauma, and at 4 weeks lesion volumes were estimated. Overall, UCCB01-144 did not protect against NMDA-toxicity in neuronal cultures or experimental TBI in rats. Important factors that should be investigated further in future studies assessing the effects of PSD-95 inhibitors in TBI are discussed.

Disease-Induced Alterations in Brain Drug Transporters in Animal Models of Alzheimer's Disease : Theme: Drug Discovery, Development and Delivery in Alzheimer's Disease Guest Editor: Davide Brambilla.

PURPOSE: Alzheimer's disease (AD) may disturb functions of the blood-brain barrier and change the disposition of drugs to the brain. This study assessed the disease-induced changes in drug transporters in the brain capillaries of transgenic AD mice. METHODS: Eighteen drug transporters and four tight junction-associated proteins were analyzed by RT-qPCR in cortex, hippocampus and cerebellum tissue samples of 12-16-month-old APdE9, Tg2576 and APP/PS1 transgenic mice and their healthy age-matched controls. In addition, microvessel fractions enriched from 1-3-month-old APdE9 mice were analyzed using RT-qPCR and Western blotting. Brain transport of methotrexate in APdE9 mice was assessed by in vivo microdialysis. RESULTS: The expression profiles of studied genes were similar in brain tissues of AD and control mice. Instead, in the microvessel fraction in APdE9 mice, >2-fold alterations were detected in the expressions of 11 genes but none at the protein level. In control mice strains, >5-fold changes between different brain regions were identified for Slc15a2, Slc22a3 and occludin. Methotrexate distribution into hippocampus of APdE9 mice was faster than in controls. CONCLUSIONS: The expression profile of mice carrying presenilin and amyloid precursor protein mutations is comparable to controls, but clear regional differences exist in the expression of drug transporters in brain.

A liquid chromatography-tandem mass spectrometry analysis of nine cytochrome P450 probe drugs and their corresponding metabolites in human serum and urine.

Cocktail phenotyping using specific probe drugs for cytochrome P450 (CYP) enzymes provides information on the real-time activity of multiple CYPs. We investigated different sample preparation techniques and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with simple protein precipitation for the analysis of nine CYP probe drugs and their metabolites in human serum and urine. Specific CYP probe drugs (melatonin, CYP1A2; nicotine, CYP2A6; bupropion, CYP2B6; repaglinide, CYP2C8; losartan, CYP2C9; omeprazole, CYP2C19 and CYP3A4; dextromethorphan, CYP2D6; chlorzoxazone, CYP2E; midazolam, CYP3A4) and their main metabolites, with the exception of 3'-hydroxyrepaglinide, were quantified in human serum and urine using the developed LC-MS/MS method. The analytical method was fully validated showing high selectivity, linearity, acceptable accuracy (85-115 %) and precision (2-19 %) and applied to a pharmacokinetic study in four healthy volunteers after oral administration of drugs given as a cocktail. All probe drugs and their metabolites (totally 19 analytes) were detected and quantified from human serum and urine over the time range of 1 to 6 h after oral administration. Therefore, the proposed method is applicable for drug interaction and CYP phenotyping studies utilizing a cocktail approach. Graphical Abstract Workflow overwiew of cocktail CYP-phenotyping study.

In Vivo Characterization of the Ultrapotent Monoacylglycerol Lipase Inhibitor {4-[bis-(benzo[d][1,3]dioxol-5-yl)methyl]-piperidin-1-yl}(1H-1,2,4-triazol-1-yl)methanone (JJKK-048).

Monoacylglycerol lipase (MAGL) is a serine hydrolase that acts as a principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). In addition to terminating the signaling function of 2-AG, MAGL liberates arachidonic acid to be used as a primary source for neuroinflammatory prostaglandin synthesis in the brain. MAGL activity also contributes to cancer pathogenicity by producing precursors for tumor-promoting bioactive lipids. Pharmacological inhibitors of MAGL provide valuable tools for characterization of MAGL and 2-AG signaling pathways. They also hold great therapeutic potential to treat several pathophysiological conditions, such as pain, neurodegenerative disorders, and cancer. We have previously reported piperidine triazole urea, {4-[bis-(benzo[d][1,3]dioxol-5-yl)methyl]-piperidin-1-yl}(1H-1,2,4-triazol-1-yl)methanone (JJKK-048), to be an ultrapotent and highly selective inhibitor of MAGL in vitro. Here, we characterize in vivo effects of JJKK-048. Acute in vivo administration of JJKK-048 induced a massive increase in mouse brain 2-AG levels without affecting brain anandamide levels. JJKK-048 appeared to be extremely potent in vivo. Activity-based protein profiling revealed that JJKK-048 maintains good selectivity toward MAGL over other serine hydrolases. Our results are also the first to show that JJKK-048 promoted significant analgesia in a writhing test with a low dose that did not cause cannabimimetic side effects. At a high dose, JJKK-048 induced analgesia both in the writhing test and in the tail-immersion test, as well as hypomotility and hyperthermia, but not catalepsy.

Amino Acid Promoieties Alter Valproic Acid Pharmacokinetics and Enable Extended Brain Exposure.

Valproic acid (VPA) has been used to treat epileptic seizures for decades, but it may also possess therapeutic potential in other nervous system diseases. However, VPA is extensively bound to plasma proteins, asymmetrically transported across the blood-brain barrier and metabolized to toxic species in the liver, which all contribute to its severe off-target adverse effects and possible drug-drug interactions. In this study, we evaluated seven amino acid prodrugs of VPA that were targeted to utilize L-type amino acid transporter 1 (LAT1), if they could alter the brain uptake mechanism and systemic pharmacokinetics of VPA. All prodrugs had affinity for LAT1 studied as competitive inhibition of [14C]-L-leucine in human breast cancer (MCF-7) cell line. However, since the ester prodrugs were unstable they were not studied further, instead the corresponding amide prodrugs were used to evaluate their systemic pharmacokinetics in rats and the uptake mechanism via LAT1 into the rat brain. All amide prodrugs were bound to a lesser extent to plasma proteins than VPA and this being independent of the prodrug concentration. Amide prodrugs were also delivered into the brain after intravenous bolus injection. One of the prodrug showed greater brain uptake and high selectivity for LAT1 and it was able to release VPA slowly within the brain. Therefore, it was concluded that the VPA brain concentrations can be stabilized as well as the problematic pharmacokinetic profile can be altered by a LAT1-selective prodrug.

Systemic and Brain Pharmacokinetics of Perforin Inhibitor Prodrugs.

We have recently reported that by converting a perforin inhibitor into an l-type amino acid transporter 1 (LAT1)-utilizing prodrug its cellular uptake can be greatly increased. The aim of the present study was to determine the in vivo and brain pharmacokinetics of two perforin inhibitors and their LAT1-utilizing prodrugs 1 and 2. In addition, the brain uptake mechanism and entry into primary mouse cortical neurons and astrocytes were evaluated. After 23 µmol/kg i.p. bolus injection, the prodrugs' unbound area under the concentration curve in brain was 0.3 nmol/g × min, whereas the parent drugs could not reach the brain. The unbound brain concentrations of the prodrugs after 100 µM in situ mouse brain perfusion were 521.4 ± 46.9 and 126.9 ± 19.9 pmol/g for prodrugs 1 and 2, respectively. The combination of competing transporter substrates for LAT1, l-tryptophan, and for organic anion transporting polypeptides, probenecid, decreased the brain concentrations to 352.4 ± 44.5 and 70.9 ± 7.0 pmol/g, respectively. In addition, in vitro uptake studies showed that at 100 µM prodrug 1 had 3.4-fold and 4.5-fold higher uptake rate into neurons and astrocytes, respectively, compared to its parent drug. Thus, the prodrugs enhance significantly the therapeutic potential of the parent drugs for the treatment of disorders of central nervous system in which neuroinflammation is involved.

Brain pharmacokinetics of ganciclovir in rats with orthotopic BT4C glioma.

Ganciclovir (GCV) is an essential part of the Herpes simplex virus thymidine kinase (HSV-tk) gene therapy of malignant gliomas. The purpose of this study was to investigate the brain pharmacokinetics and tumor uptake of GCV in the BT4C rat glioma model. GCV's brain and tumor uptakes were investigated by in vivo microdialysis in rats with orthotopic BT4C glioma. In addition, the ability of GCV to cross the blood-brain barrier and tumor vasculature was assessed with in situ rat brain perfusion. Finally, the extent to which GCV could permeate across the BT4C glioma cell membrane was assessed in vitro. The areas under the concentration curve of unbound GCV in blood, brain extracellular fluid (ECF), and tumor ECF were 6157, 1658, and 4834 µM⋅min, respectively. The apparent maximum unbound concentrations achieved within 60 minutes were 46.9, 11.8, and 25.8 µM in blood, brain, and tumor, respectively. The unbound GCV concentrations in brain and tumor after in situ rat brain perfusion were 0.41 and 1.39 nmol/g, respectively. The highly polar GCV likely crosses the fenestrated tumor vasculature by paracellular diffusion. Thus, GCV is able to reach the extracellular space around the tumor at higher concentrations than that in healthy brain. However, GCV uptake into BT4C cells at 100 µM was only 2.1 pmol/mg of protein, and no active transporter-mediated disposition of GCV could be detected in vitro. In conclusion, the limited efficacy of HSV-tk/GCV gene therapy may be due to the poor cellular uptake and rapid elimination of GCV.

Blood-brain barrier permeability and brain uptake mechanism of kainic acid and dihydrokainic acid.

The glutamatergic neurotransmitter system is involved in important neurophysiological processes and thus constitutes a promising target for the treatment of neurological diseases. The two ionotropic glutamate receptor agonists kainic acid (KA) and dihydrokainic acid (DHK) have been used as research tools in various in vivo central nervous system disease models in rodents, as well as being templates in the design of novel ligands affecting the glutamatergic system. Both molecules are highly polar but yet capable of crossing the blood-brain barrier (BBB). We used an in situ rat brain perfusion technique to determine the brain uptake mechanism and permeability across the BBB. To determine KA and DHK concentrations in the rat brain, simple and rapid sample preparation and liquid chromatography mass spectrometer methods were developed. According to our results the BBB permeability of KA and DHK is low, 0.25 × 10(-6) and 0.28 × 10(-6) cm/s for KA and DHK, respectively. In addition, the brain uptake is mediated by passive diffusion, and not by active transport. Furthermore, the non-specific plasma and brain protein binding of KA and DHK was determined to be low, which means that the unbound drug volume of distribution in brain is also low. Therefore, even though the total KA and DHK concentrations in the brain are low after systemic dosing, the concentrations in the vicinity of the glutamate receptors are sufficient for their activation and thus the observed efficacy.

Sex-specific changes in protein expression of membrane transporters in the brain cortex of 5xFAD mouse model of Alzheimer's disease.

Membrane transporters playing an important role in the passage of drugs, metabolites and nutrients across the membranes of the brain cells have been shown to be involved in pathogenesis of Alzheimer's disease (AD). However, little is known about sex-specific changes in transporter protein expression at the brain in AD. Here, we investigated sex-specific alterations in protein expression of three ATP-binding cassette (ABC) and five solute carriers (SLC) transporters in the prefrontal cortex of a commonly used model of familial AD (FAD), 5xFAD mice. Sensitive liquid chromatography tandem mass spectrometry-based quantitative targeted absolute proteomic analysis was applied for absolute quantification of transporter protein expression. We compared the changes in transporter protein expressions in 7-month-old male and female 5xFAD mice versus sex-matched wild-type mice. The study revealed a significant sex-specific increase in protein expression of ABCC1 (p = 0.007) only in male 5xFAD mice as compared to sex-matched wild-type animals. In addition, the increased protein expression of glucose transporter 1 (p = 0.01), 4F2 cell-surface antigen heavy chain (p = 0.01) and long-chain fatty acid transport protein 1 (p = 0.02) were found only in female 5xFAD mice as compared to sex-matched wild-type animals. Finally, protein expression of alanine/serine/cysteine/threonine transporter 1 was upregulated in both male (p = 0.02) and female (p = 0.002) 5xFAD mice. The study provides important information about sex-specific changes in brain cortical transporter expression in 5xFAD mice, which will facilitate drug development of therapeutic strategies for AD targeting these transporters and drug delivery research.

Design, synthesis and brain uptake of LAT1-targeted amino acid prodrugs of dopamine.

PURPOSE: Drug delivery to the brain is impeded by the blood-brain barrier (BBB). Here, we attempted to enhance the brain uptake of cationic dopamine by utilizing the large amino acid transporter 1 (LAT1) at the BBB by prodrug approach. METHODS: Three amino acid prodrugs of dopamine were synthesized and their prodrug properties were examined in vitro. Their LAT1-binding and BBB-permeation were studied using the in situ rat brain perfusion technique. The brain uptake after intravenous administration and the dopamine-releasing ability in the rat striatum after intraperitoneal administration were also determined for the most promising prodrug. RESULTS: All prodrugs underwent adequate cleavage in rat tissue homogenates. The prodrug with phenylalanine derivative as the promoiety had both higher affinity for LAT1 and better brain uptake properties than those with an alkyl amino acid - mimicking promoiety. The phenylalanine prodrug was taken up into the brain after intravenous injection but after intraperitoneal injection the prodrug did not elevate striatal dopamine concentrations above those achieved by corresponding L-dopa treatment. CONCLUSIONS: These results indicate that attachment of phenylalanine to a cationic drug via an amide bond from the meta-position of its aromatic ring could be highly applicable in prodrug design for LAT1-mediated CNS-delivery of not only anionic but also cationic polar drugs.

The Role of Solute Carrier Transporters in Efficient Anticancer Drug Delivery and Therapy.

Transporter-mediated drug resistance is a major obstacle in anticancer drug delivery and a key reason for cancer drug therapy failure. Membrane solute carrier (SLC) transporters play a crucial role in the cellular uptake of drugs. The expression and function of the SLC transporters can be down-regulated in cancer cells, which limits the uptake of drugs into the tumor cells, resulting in the inefficiency of the drug therapy. In this review, we summarize the current understanding of low-SLC-transporter-expression-mediated drug resistance in different types of cancers. Recent advances in SLC-transporter-targeting strategies include the development of transporter-utilizing prodrugs and nanocarriers and the modulation of SLC transporter expression in cancer cells. These strategies will play an important role in the future development of anticancer drug therapies by enabling the efficient delivery of drugs into cancer cells.

Monoacylglycerol Lipase Inhibitor JJKK048 Ameliorates ABCG2 Transporter-Mediated Regorafenib Resistance Induced by Hypoxia in Triple Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is among the most aggressive and deadly cancer subtypes. Intra-tumoral hypoxia is associated with aggressiveness and drug resistance in TNBC. One of the underlying mechanisms of hypoxia-induced drug resistance is the elevated expression of efflux transporters such as breast cancer resistant protein (ABCG2). In the present study, we investigated the possibility of ameliorating ABCG2-mediated drug resistance in hypoxic TNBC cells by monoacylglycerol lipase (MAGL) inhibition and the consequent downregulation of ABCG2 expression. The effect of MAGL inhibition on ABCG2 expression, function, and efficacy of regorafenib, an ABCG2 substrate was investigated in cobalt dichloride (CoCl2) induced pseudohypoxic TNBC (MDA-MB-231) cells, using quantitative targeted absolute proteomics, qRT-PCR, anti-cancer drug accumulation in the cells, cell invasiveness and resazurin-based cell viability assays. Our results showed that hypoxia-induced ABCG2 expression led to low regorafenib intracellular concentrations, reduced the anti-invasiveness efficacy, and elevated half maximal inhibitory concentration (IC50) of regorafenib in vitro MDA-MB-231 cells. MAGL inhibitor, JJKK048, reduced ABCG2 expression, increased regorafenib cell accumulation, which led to higher regorafenib efficacy. In conclusion, hypoxia-induced regorafenib resistance due to ABCG2 over-expression in TNBC cells can be ameliorated by MAGL inhibition.

In vitro identification of decreased function phenotype ABCG2 variants.

Reduced activity of efflux transporter ABCG2, caused e.g., by inhibition or decreased function genetic variants, can increase drug absorption and plasma levels. ABCG2 has one clinically significant single nucleotide variant Q141K (c.421C>A), which leads to decreased protein levels and transport activity. In addition to Q141K, ABCG2 has over 500 rare (<1% minor allele frequency) nonsynonymous variants, but their functionality remains unknown. We studied the transport activity and abundance of 30 rare ABCG2 variants. The variants were transiently expressed in HEK293 cells. Transport activity and protein abundance were measured from inside-out crude membrane vesicles. Results were normalised to the reference ABCG2, while Q141K was used to categorise variants into decreased and normal function phenotypes based on their apparent transport activity. Fourteen variants (G80E, D128V, T434M, Q437R, C438R, C438W, C438Y, L479S, P480L, S486N, T512N, S519P, G553D and K647E) had similar or lower apparent transport activity than Q141K and thus were categorised as having a decreased function phenotype. Protein abundance could not explain all of the observed changes in transport activity: Only six variants (D128V, Q437R, C438R, S519P, G553D, and K647E) had similar or lower abundance compared to Q141K. The decreased function variants may increase systemic drug exposure and therefore cause interindividual variability in pharmacokinetics. In the future, in vitro phenotype classification may help to design personalised drug treatments.