Monocarboxylate Transporter Inhibitor Research Articles

In Vitro and In Vivo Efficacy of the Monocarboxylate Transporter 1 Inhibitor AR-C155858 in the Murine 4T1 Breast Cancer Tumor Model.

Monocarboxylate transporter 1 (MCT1), also known as a L-lactate transporter, is a potential therapeutic target in cancer. The objectives of this study were to evaluate efficacy and assess concentration-effect relationships of AR-C155858 (a selective and potent MCT1 inhibitor) in murine 4T1 breast cancer cells and in the 4T1 tumor xenograft model. Western blotting of 4T1 cells demonstrated triple negative breast cancer (TNBC) characteristics and overexpression of MCT1 and CD147 (a MCT1 accessory protein), but absence of MCT4 expression. AR-C155858 inhibited the cellular L-lactate uptake and cellular proliferation at low nanomolar potencies (IC50 values of 25.0 ± 4.2 and 20.2 ± 0.2nM, respectively). In the xenograft 4T1 mouse model of immunocompetent animals, AR-C155858 (10mg/kgi.p. once daily) had no effect on tumor volume and weight. Treatment with AR-C155858 resulted in slightly increased tumor lactate concentrations; however, the changes were not statistically significant. AR-C155858 was well tolerated, as demonstrated by the unchanged body weight and blood lactate concentrations. Average blood and tumor AR-C155858 concentrations (110 ± 22 and 574 ± 245nM, respectively), 24h after the last dose, were well above the IC50 values. These data indicate that AR-C155858 penetrated 4T1 xenograft tumors and was present at high concentrations but was ineffective in decreasing tumor growth. Evaluations of AR-C155858 in other preclinical models of breast cancer are needed to further assess its efficacy.

Lactate Transporter Monocarboxylate Transporter 4 Induces Bone Pain in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) poses a significant challenge clinically, as it can invade facial bones and cause bone pain that is undertreated and poorly understood. Here we studied HNSCC bone pain (HNSCC-BP) in an intratibial mouse xenograft model that uses a human HNSCC cell line (SAS cells). These mice develop HNSCC-BP associated with an upregulation of phosphorylated ERK1/2 (pERK1/2), which is a molecular indicator of neuron excitation in the dorsal root ganglia (DRGs) of sensory nerve cell bodies. Our experiments demonstrated that the inhibition of monocarboxylate transporter 4 (MCT4) by short hairpin (shRNA) transduction suppressed the HNSCC-BP, the lactate level in bone marrow, and the pERK1/2 expression in DRG. The sensory nerves also expressed increased levels of the acid-sensing receptor TRPV1. DRG neurons co-cultured with SAS cells showed increased neurite outgrowth, and were inhibited by MCT4 silencing with shRNA. Collectively, our results show that HNSCC induced an acidic bone microenvironment that evokes HNSCC-BP via MCT4 expression.

The Monocarboxylate transporter inhibitor Quercetin induces intracellular acidification in a mouse model of Glioblastoma Multiforme: in-vivo detection using magnetic resonance imaging.

The response of tumor intracellular pH to a pharmacological challenge could help identify aggressive cancer. Chemical exchange saturation transfer (CEST) is an MRI contrast mechanism that is dependent on intracellular pH (pHi). pHi is important in the maintenance of normal cell function and is normally maintained within a narrow range by the activity of transporters located at the plasma membrane. In cancer, changes in pHi have been correlated with both cell proliferation and cell death. Quercetin is a bioflavonoid and monocarboxylate transporter (MCT) inhibitor. Since MCTs plays a significant role in maintaining pH balance in the tumor microenvironment, we hypothesized that systemically administered quercetin could selectively acidify brain tumors. The goals of the current study were to determine whether CEST MRI measurements sensitive to tumor pH could detect acidification after quercetin injection and to measure the magnitude of the pH change (ΔpH). Using a 9.4T MRI, amine and amide concentration independent detection (AACID) CEST spectra were acquired in six mice approximately 15 ± 1days after implanting 105U87 human glioblastoma multiforme cells in the brain, before and after administration of quercetin (dose: 200mg/kg) by intraperitoneal injection. Three additional mice were studied as controls and received only vehicle dimethyl sulfoxide (DMSO) injection. Repeated measures t-test was used to compare AACID changes in tumor and contralateral tissue regions of interest. Two hours after quercetin injection there was a significant increase in tumor AACID by 0.07 ± 0.03 corresponding to a 0.27 decrease in pHi, and no change in AACID in contralateral tissue. There was also a small average increase in AACID in tumors within the three mice injected with DMSO only. The use of the natural compound quercetin in combination with pH weighted MRI represents a unique approach to cancer detection that does not require injection of an imaging contrast agent.

Exploring the functionalisation of the thieno[2,3-d]pyrimidinedione core: Late stage access to highly substituted 5-carboxamide-6-aryl scaffolds

The thieno[2,3-d]pyrimidinedione core is found as a component in a range of pharmaceutically active compounds, however, synthetic approaches to these scaffolds rely on access to functionalised, highly substituted thiophenes. Here we describe a new approach for the preparation of 5-carboxamide-6-aryl analogues that involves a two-step synthesis of the thieno[2,3-d]pyrimidinedione core from a readily available mercaptouracil derivative. Thio-alkylation with ethyl 3-bromopyruvate, followed by cyclisation and dehydration mediated by polyphosphoric acid allowed the scalable synthesis of the thieno[2,3-d]pyrimidinedione unit. The late-stage functionalisation of this core motif via bromination of the thiophene ring and a subsequent Suzuki-Miyaura reaction as the key steps permitted access to a novel library of 5-carboxamide-6-aryl analogues. The physicochemical properties of these compounds were determined, generating an insight into the potential bioavailability of these scaffolds. Based on these results, a selection of the novel 5-carboxamide-6-aryl analogues were tested as lactate uptake inhibitors of monocarboxylate transporters 1, 2 and 4 in Xenopus oocytes.

PO-437 Ultrasound-triggered delivery of halogenated pyruvate analogues to breast cancer cells

Introduction Targeting the altered cancer metabolism has opened new therapeutic opportunities. Upregulation of the expression of the monocarboxylate transporter 1 (MCT1) has been shown in various cancers, including breast tumours. MCT1 is responsible for the transport of lactate and pyruvate. Lactate/pyruvate analogues, such as 3-bromopyruvate, have been shown to be toxic to cells overexpressing MCT1. However, in order to successfully treat the tumour, the therapeutic must be shielded from the immune system, target the tumour and penetrate it. To overcome these challenges we have encapsulated lactate/pyruvate analogues inside ultrasound labile liposomes. These will protect the therapeutic and extend its circulation time, enabling delivery and triggered release at the tumour site using externally applied ultrasound, whereas MCT1 expression offers selective uptake of the compounds. Material and methods Western blotting was used to identify cell lines with high and low expression of MCT1. Cell viability after treatment was tested with an MTT assay. Chromatography techniques were used to measure encapsulation efficiency into the liposomes. A novel iodinated lactate analogue, 3-iodolactate (3IL), was synthesised via Finkelstein reaction and characterised by NMR spectroscopy and mass spectrometry. Ultrasound-labile liposomes were synthesised by dispersion of a lyophilised lipid film (DOPE, cholesterol and DSPC-PEG(2000) in 65:25:10 molar ratio) in an aqueous solution of the compound, followed by extrusion and size exclusion chromatography. Results and discussions Breast cancer cell lines with low, intermediate and high expression of MCT1 have been identified (MDA-MB-231, MDA-MB-468 and BT20, respectively). 3 BP toxicity correlated with MCT1 expression. BT20 had an LD50 of 87.10±0.03 µM, while MDA-MB-231, were 90% viable even up to concentrations of 300 µM 3 BP. Toxicity is reversed with MCT1 inhibition. 3IL has been synthesised and purified. Ultrasound labile liposomes with an average hydrodynamic diameter of 120 nm have been synthesised and encapsulation of 3 BP has been confirmed and tested with cells with and without ultrasound. Conclusion Cell toxicity of halogenated lactate/pyruvate analogues has been confirmed. A novel iodinated analogue has been synthesised. These molecules have been encapsulated within ultrasound labile liposomes. A novel drug delivery system has been engineered for the delivery of metabolic inhibitors targeting the lactate/pyruvate transporter (MCT1) for targeted cancer therapy.

Effects of a Series of Acidic Drugs on L-Lactic Acid Transport by the Monocarboxylate Transporters MCT1 and MCT4.

Drug-induced myopathy is a serious side effect that often requires removal of a medication from a drug regimen. For most drugs, the underlying mechanism of drug-induced myopathy remains unclear. Monocarboxylate transporters (MCTs) mediate L-lactic acid transport, and inhibition of MCTs may potentially lead to perturbation of L-lactic acid accumulation and muscular disorders. Therefore, we hypothesized that L-lactic acid transport may be involved in the development of drug-induced myopathy. The aim of this study was to assess the inhibitory potential of 24 acidic drugs on L-lactic acid transport using breast cancer cell lines Hs578T and MDA-MB-231, which selectively express MCT1 and MCT4, respectively. The influx transport of L-lactic acid was minimally inhibited by all drugs tested. The efflux transport was next examined: loratadine (IC50: 10 and 61 µM) and atorvastatin (IC50: 78 and 41 µM) demonstrated the greatest potency for inhibition of L-lactic acid efflux by MCT1 and MCT4, respectively. Acidic drugs including fluvastatin, cerivastatin, simvastatin acid, lovastatin acid, irbesartan and losartan exhibited weak inhibitory potency on L-lactic acid efflux. Our results suggest that some acidic drugs, such as loratadine and atorvastatin, can inhibit the efflux transport of L-lactic acid. This inhibition may cause an accumulation of intracellular L-lactic acid leading to acidification and muscular disorders.

Valproic acid transport in the choriocarcinoma placenta cell line JEG-3 proceeds independently of the proton-dependent transporters MCT1 and MCT4

Medication therapy is the first line of treatment in the management of epilepsy. Fetal exposure to valproic acid (VPA), an antiepileptic drug, poses an elevated risk of teratogenicity in early pregnancy. Some studies have reported that monocarboxylate transporters (MCTs) may be involved in the placental transport of VPA. However, it has not been determined which MCTs contribute to VPA transport into the placenta. Therefore, the aim of this study was to determine how MCTs contribute to VPA transport into the placenta using the human placenta choriocarcinoma cell line JEG-3. VPA uptake was investigated using JEG-3 cells and radiolabeled VPA. MCT expression in JEG-3 cells was detected using RT-PCR and western blotting. Knockdown of MCTs was carried out using siRNAs. VPA uptake into JEG-3 cells was pH- and concentration-dependent, and described by using the Michaelis–Menten equation (Km = 0.95 ± 0.17 mM; Vmax = 19.3 ± 1.21 nmol/mg protein/15 s). MCT1 and MCT4 expression was found in JEG-3 cells, and typical MCT inhibitors significantly inhibited VPA uptake into JEG-3 cells. However, knockdown of MCT1 and MCT4 did not alter VPA uptake. In conclusion, VPA transport is mediated by a proton-dependent transporter in JEG-3 cells, but not by MCT1 and MCT4.

Development and validation of a liquid chromatography tandem mass spectrometry assay for AZD3965 in mouse plasma and tumor tissue: Application to pharmacokinetic and breast tumor xenograft studies

AZD3965, a pyrole pyrimidine derivative, is a potent and orally bioavailable inhibitor of monocarboxylate transporter 1 (MCT1), currently in a Phase I clinical trial in UK for lymphomas and solid tumors. There is currently no published assay for AZD3965. The objectives of this study were to develop and validate a LC/MS/MS assay for quantifying AZD3965 in mouse plasma and tumor tissue. Protein precipitation with 0.1% formic acid in acetonitrile was used for sample preparation. Chromatographic separation was achieved on a C18 column followed by tandem mass spectrometry detection in multiple reaction monitoring mode with utilizing Atmospheric Pressure Chemical Ionization. AR-C155858 was used as the internal standard. The inter-day and intra-day precision and accuracy of quality control samples evaluated in plasma and tumor tissue were less than ±7% of the nominal concentrations. The extraction recovery, matrix effect and stability values were all within acceptable levels. Sample dilution integrity, accessed by diluting plasma spiked with AZD3965 10-fold with blank plasma, was 101%. The lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) were 0.15 ng/mL and 12 μg/mL, respectively, in plasma. The assay of AZD3965 in tumor tissue was also validated with good precision and accuracy. The LLOQ was 0.15 ng/mL in tumor tissue. This assay was successfully applied to pharmacokinetic and murine 4T1 breast tumor xenograft studies of AZD3965 in mice.

Glucose and lactate as metabolic constraints on presynaptic transmission at an excitatory synapse.

Synapses have high energy demands which increase during intense activity. We show that presynaptic terminals can utilise extracellular glucose or lactate to generate energy to maintain synaptic transmission. Reducing energy substrates induces a metabolic stress: presynaptic ATP depletion impaired synaptic transmission through a reduction in the number of functional synaptic vesicle release sites and a slowing of vesicle pool replenishment, without a consistent change in release probability. Metabolic function is compromised in many pathological conditions (e.g. stroke, traumatic brain injury and neurodegeneration). Knowledge of how synaptic transmission is constrained by metabolic stress, especially during intense brain activity, will provide insights to improve cognition following pathological insults. The synapse has high energy demands, which increase during intense activity. Presynaptic ATP production depends on substrate availability and usage will increase during activity, which in turn could influence transmitter release and information transmission. We investigated transmitter release at the mouse calyx of Held synapse using glucose or lactate (10, 1 or 0mm) as the extracellular substrates while inducing metabolic stress. High-frequency stimulation (HFS) and recovery paradigms evoked trains of EPSCs monitored under voltage-clamp. Whilst postsynaptic intracellular ATP was stabilised by diffusion from the patch pipette, depletion of glucose increased EPSC depression during HFS and impaired subsequent recovery. Computational modelling of these data demonstrated a reduction in the number of functional release sites and slowed vesicle pool replenishment during metabolic stress, with little change in release probability. Directly depleting presynaptic terminal ATP impaired transmitter release in an analogous manner to glucose depletion. In the absence of glucose, presynaptic terminal metabolism could utilise lactate from the aCSF and this was blocked by inhibition of monocarboxylate transporters (MCTs). MCT inhibitors significantly suppressed transmission in low glucose, implying that lactate is a presynaptic substrate. Additionally, block of glycogenolysis accelerated synaptic transmission failure in the absence of extracellular glucose, consistent with supplemental supply of lactate by local astrocytes. We conclude that both glucose and lactate support presynaptic metabolism and that limited availability, exacerbated by high-intensity firing, constrains presynaptic ATP, impeding transmission through a reduction in functional presynaptic release sites as vesicle recycling slows when ATP levels are low.

Interruption of lactate uptake by inhibiting mitochondrial pyruvate transport unravels direct antitumor and radiosensitizing effects

Lactate exchange between glycolytic and oxidative cancer cells is proposed to optimize tumor growth. Blocking lactate uptake through monocarboxylate transporter 1 (MCT1) represents an attractive therapeutic strategy but may stimulate glucose consumption by oxidative cancer cells. We report here that inhibition of mitochondrial pyruvate carrier (MPC) activity fulfils the tasks of blocking lactate use while preventing glucose oxidative metabolism. Using in vitro 13C-glucose and in vivo hyperpolarized 13C-pyruvate, we identify 7ACC2 as a potent inhibitor of mitochondrial pyruvate transport which consecutively blocks extracellular lactate uptake by promoting intracellular pyruvate accumulation. Also, while in spheroids MCT1 inhibition leads to cytostatic effects, MPC activity inhibition induces cytotoxic effects together with glycolysis stimulation and uncompensated inhibition of mitochondrial respiration. Hypoxia reduction obtained with 7ACC2 is further shown to sensitize tumor xenografts to radiotherapy. This study positions MPC as a control point for lactate metabolism and expands on the anticancer potential of MPC inhibition.

Regulation of Acetate Utilization by Monocarboxylate Transporter 1 (MCT1) in Hepatocellular Carcinoma (HCC).

Altered energy metabolism is a biochemical fingerprint of cancer cells. Hepatocellular carcinoma (HCC) shows reciprocal [18F]fluorodeoxyglucose (FDG) and [11C]acetate uptake, as revealed by positron emission tomography/computed tomography (PET/CT). Previous studies have focused on the role of FDG uptake in cancer cells. In this study, we evaluated the mechanism and roles of [11C]acetate uptake in human HCCs and cell lines. The expression of monocarboxylate transporters (MCTs) was assessed to determine the transporters of [11C]acetate uptake in HCC cell lines and human HCCs with different [11C]acetate uptake. Using two representative cell lines with widely different [11C]acetate uptake (HepG2 for high uptake and Hep3B for low uptake), changes in [11C]acetate uptake were measured after treatment with an MCT1 inhibitor or MCT1-targeted siRNA. To verify the roles of MCT1 in cells, oxygen consumption rate and the amount of lipid synthesis were measured. HepG2 cells with high [11C]acetate uptake showed higher MCT1 expression than other HCC cell lines with low [11C]acetate uptake. MCT1 expression was elevated in human HCCs with high [11C]acetate uptake compared to those with low [11C]acetate uptake. After blocking MCT1 with AR-C155858 or MCT1 knockdown, [11C]acetate uptake in HepG2 cells was significantly reduced. Additionally, inhibition of MCT1 suppressed mitochondrial oxidative phosphorylation, lipid synthesis, and cellular proliferation in HCC cells with high [11C]acetate uptake. MCT1 may be a new therapeutic target for acetate-dependent HCCs with high [11C]acetate uptake, which can be selected by [11C]acetate PET/CT imaging in clinical practice.

The effect of Monocarboxylate Transporter (MCT1) inhibitor, AR-C117977 on accelerated rejection of cardiac grafts in pre-sensitised rats and concordant xenotransplantation

The effect of Monocarboxylate Transporter (MCT1) inhibitor, AR-C117977 on accelerated rejection of cardiac grafts in pre-sensitised rats and concordant xenotransplantation

Glucose-dependent growth arrest of leukemia cells by MCT1 inhibition: Feeding Warburg’s sweet tooth and blocking acid export as an anticancer strategy

This study aims to investigate the utilization of The Warburg Effect, cancer’s “sweet tooth” and natural greed for glucose to enhance the effect of monocarboxylate transporter inhibition on cellular acidification. By simulating hyperglycemia with high glucose we may increase the effectiveness of inhibition of lactate and proton export on the dysregulation of cell pH homeostasis causing cell death or disruption of growth in cancer cells. MCT1 and MCT4 expression was determined in MCF7 and K562 cell lines using RT-PCR. Cell viability, growth, intracellular pH and cell cycle analysis was measured in the cell lines grown in 5 mM and 25 mM glucose containing media in the presence and absence of the MCT1 inhibitor AR-C155858 (1 μM) and the NHE1 inhibitor cariporide (10 μM). The MCT1 inhibitor, AR-C155858 had minimal effect on the viability, growth and intracellular pH of MCT4 expressing MCF7 cells. AR-C155858 had no effect on the viability of the MCT1 expressing K562 cells, but decreased intracellular pH and cell proliferation, by a glucose-dependent mechanism. Inhibition of NHE1 on its own had a no effect on cell growth, but together with AR-C155858 showed an additive effect on inhibition of cell growth. In cancer cells that only express MCT1, increased glucose concentrations in the presence of an MCT1 inhibitor decreased intracellular pH and reduced cell growth by G1 phase cell-cycle arrest. Thus we propose a transient hyperglycemic-clamp in combination with proton export inhibitors be evaluated as an adjunct to cancer treatment in clinical studies.

Short-Chain Fatty Acids Suppress Inflammatory Reactions in Caco-2 Cells and Mouse Colons.

Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, play an important role in the maintenance of intestinal homeostasis. In the present study, anti-inflammatory effects of SCFAs were examined in human intestinal Caco-2 cells and mouse colonic cultures. Stimulation of Caco-2 cells with tumor necrosis factor (TNF)-α induced interleukin (IL)-8 (TNF-α, 17.1 ± 7.2 vs Control, 1.00 ± 0.26, P < 0.01) and IL-6 expression (TNF-α, 21.7 ± 10.0 vs Control, 1.00 ± 0.28, P < 0.01) through the activation of nuclear factor κB p65, spleen tyrosine kinase, and mitogen-activated protein kinase pathways. Pretreatment of cells with acetate (5 mM, IL-8 1.23 ± 0.40, IL-6 2.19 ± 0.92, P < 0.01 ), propionate (2.5 mM, IL-8 2.45 ± 2.10, IL-6 2.19 ± 0.92, P < 0.01), or butyrate (0.625 mM, IL-8 1.44 ± 0.70, IL-6 2.31 ± 0.32, P < 0.01) suppressed inflammatory responses induced by TNF-α. Pharmacological inhibition of monocarboxylate transporter (MCT)-1 attenuated the suppression of inflammatory signals by SCFAs. High expression levels of CXC motif chemokine ligand 2 (CXCL2, an IL-8 homologue, DSS, 31.7 ± 9.8 vs Control, 1.00 ± 0.70, P < 0.01) and IL-6 (DSS, 17.5 ± 7.2 vs Control, 1.00 ± 0.68, P < 0.01) were observed in BALB/c mouse colonic cultures exposed to dextran sodium sulfate, whereas treatments with mixtures of SCFAs decreased these elevated expression levels (CXCL2 4.14 ± 2.88, IL-6 0.58 ± 0.28, P < 0.01). Our results suggest that SCFAs transported by MCT-1 suppress TNF-α-induced inflammatory signaling in intestinal cells.

Monocarboxylate Transporter 1 (MCT1) is an independent prognostic biomarker in endometrial cancer

BackgroundEndometrial cancer (EC) is a major health concern due to its rising incidence. Whilst early stage disease is generally cured by surgery, advanced EC has a poor prognosis with limited treatment options. Altered energy metabolism is a hallmark of malignancy. Cancer cells drive tumour growth through aerobic glycolysis and must export lactate to maintain intracellular pH. The aim of this study was to evaluate the expression of the lactate/proton monocarboxylate transporters MCT1 and MCT4 and their chaperone CD147 in EC, with the ultimate aim of directing future drug development.MethodsMCT1, MCT4 and CD147 expression was examined using immunohistochemical analysis in 90 endometrial tumours and correlated with clinico-pathological characteristics and survival outcomes.ResultsMCT1 and MCT4 expression was observed in the cytoplasm, the plasma membrane or both locations. CD147 was detected in the plasma membrane and associated with MCT1 (p = 0.003) but not with MCT4 (p = 0.207) expression. High MCT1 expression was associated with reduced overall survival (p = 0.029) and remained statistically significant after adjustment for survival covariates (p = 0.017).ConclusionOur data suggest that MCT1 expression is an important marker of poor prognosis in EC. MCT1 inhibition may have potential as a treatment for advanced or recurrent EC.

Tempo-phosphate as an ESR tool to study phosphate transport

TEMPO-phosphate has been introduced as a phosphate analogue to study phosphate transport in erythrocytes. The nitroxide is reduced intracellularly upon entering the cells, the membrane transport being the rate-limiting step of the loss of ESR signal. The use of TEMPO-phosphate is convenient and avoids the hazard of radioactivity. We studied the inhibition of TEMPO-phosphate transport to human erythrocytes by various compounds. DIDS and SITS, inhibitors of Band 3, inhibited the TEMPO-phosphate transport. 1-cyano-4-hydroxycinnamic acid, inhibitor of monocarboxylate transporters, did not affect the permeation of TEMPO-phosphate. The transport of TEMPO-phosphate was inhibited by various polyphenols, especially curcumin, naringin, quercetin, luteolin and kaempferol. Interestingly, 3-bromopyruvic acid, an alkylating agent and potential anticancer agent, induced an apparent enhancement of TEMPO-phosphate transport into erythrocytes.

Direct Neuronal Glucose Uptake Is Required for Contextual Fear Acquisition in the Dorsal Hippocampus.

The metabolism of glucose is a nearly exclusive source of energy for maintaining neuronal survival, synaptic transmission and information processing in the brain. Two glucose metabolism pathways have been reported, direct neuronal glucose uptake and the astrocyte-neuron lactate shuttle (ANLS), which can be involved in these functions simultaneously or separately. Although ANLS in the dorsal hippocampus (DH) has been proved to be required for memory consolidation, the specific metabolic pathway involved during memory acquisition remains unclear. The DH and amygdala are two key brain regions for acquisition of contextual fear conditioning (CFC). In 2-NBDG experiments, we observed that 2-NBDG-positive neurons were significantly increased during the acquisition of CFC in the DH. However, in the amygdala and cerebellum, 2-NBDG-positive neurons were not changed during CFC training. Strikingly, microinjection of a glucose transporter (GLUT) inhibitor into the DH decreased freezing values during CFC training and 1 h later, while injection of a monocarboxylate transporter (MCT) inhibitor into the amygdala also reduced freezing values. Therefore, we demonstrated that direct neuronal glucose uptake was the primary means of energy supply in the DH, while ANLS might supply energy in the amygdala during acquisition. Furthermore, knockdown of GLUT3 by a lentivirus in the DH impaired the acquisition of CFC. Taken together, the results indicated that there were two different glucose metabolism pathways in the DH and amygdala during acquisition of contextual fear memory and that direct neuronal glucose uptake in the DH may be regulated by GLUT3.

Study of Statin- and Loratadine-Induced Muscle Pain Mechanisms Using Human Skeletal Muscle Cells.

Many drugs can cause unexpected muscle disorders, often necessitating the cessation of an effective medication. Inhibition of monocarboxylate transporters (MCTs) may potentially lead to perturbation of l-lactic acid homeostasis and muscular toxicity. Previous studies have shown that statins and loratadine have the potential to inhibit l-lactic acid efflux by MCTs (MCT1 and 4). The main objective of this study was to confirm the inhibitory potentials of atorvastatin, simvastatin (acid and lactone forms), rosuvastatin, and loratadine on l-lactic acid transport using primary human skeletal muscle cells (SkMC). Loratadine (IC50 31 and 15 µM) and atorvastatin (IC50 ~130 and 210 µM) demonstrated the greatest potency for inhibition of l-lactic acid efflux at pH 7.0 and 7.4, respectively (~2.5-fold l-lactic acid intracellular accumulation). Simvastatin acid exhibited weak inhibitory potency on l-lactic acid efflux with an intracellular lactic acid increase of 25–35%. No l-lactic acid efflux inhibition was observed for simvastatin lactone or rosuvastatin. Pretreatment studies showed no change in inhibitory potential and did not affect lactic acid transport for all tested drugs. In conclusion, we have demonstrated that loratadine and atorvastatin can inhibit the efflux transport of l-lactic acid in SkMC. Inhibition of l-lactic acid efflux may cause an accumulation of intracellular l-lactic acid leading to the reported drug-induced myotoxicity.

Identification of a selective inhibitor of human monocarboxylate transporter 4

The human monocarboxylate transporters (hMCTs/SLC16As) mediate the uptake of various monocarboxylates. Several isoforms of hMCTs are expressed in cancerous tissue as well as in normal tissue. In cancerous tissue, hypoxia induces the expression of hMCT4, which transports the energetic metabolite l-lactate across the plasma membrane. Since hMCT4 is involved in pH regulation and the transport of l-lactate in cancer cells, an hMCT4 inhibitor could function as an anticancer agent. Although several non specific hMCT inhibitors have been developed, a selective hMCT4 inhibitor has not yet been identified. The aim of this study was therefore to identify a selective hMCT4 inhibitor for use as a pharmacological tool for studying hMCT4. The heterologous expression system of the Xenopus oocyte was used to assess the effects of test compounds on hMCT4, whereupon isobutyrate derivatives, fibrates, and bindarit (2-[(1-benzyl-1H-indazol-3-yl)methoxy]-2-methylpropanoic acid) were demonstrated to exhibit selective inhibitory effects against this transporter. It is suggested that the structure formed from the joining of an isobutyrate moiety and two aromatic rings by appropriate linkers is important for acquiring the selective hMCT4-inhibiting activity. These findings provide novel insights into the ligand recognition of hMCT4, and contribute to the development of novel anticancer agents.

Synthesis and evaluation of 11C-labeled coumarin analog as an imaging probe for detecting monocarboxylate transporters expression

Upregulated monocarboxylate transporters (MCTs) in tumors are considered diagnostic imaging targets. Herein, we synthesized the positron emission tomography probe candidates coumarin analogs 2 and 3, and showed 55 times higher affinity of 2 for MCTs than a representative MCT inhibitor. Whereas [11C]2 showed low tumor accumulation, probably due to adduct formation with plasma proteins, [11C]2 showed high initial brain uptake, suggesting that the scaffold of 2 has properties that are preferable in imaging probes for the astrocyte–neuron lactate shuttle. Although further optimization of 2 is required, our findings can be used to inform the development of MCT-targeted imaging agents.