Homogeneity of antibody-drug conjugates critically impacts the therapeutic efficacy in brain tumors.

Glioblastoma multiforme (GBM) is the most aggressive and fatal disease of all brain tumor types. Most therapies rarely provide clinically meaningful outcomes in the treatment of GBM. Although antibody-drug conjugates (ADCs) are promising anticancer drugs, no ADCs have been clinically successful for GBM, primarily because of poor blood-brain barrier (BBB) penetration. Here, we report that ADC homogeneity and payload loading rate are critical parameters contributing to this discrepancy. Although both homogeneous and heterogeneous conjugates exhibit comparable in vitro potency and pharmacokinetic profiles, the former shows enhanced payload delivery to brain tumors. Our homogeneous ADCs provide improved antitumor effects and survival benefits in orthotopic brain tumor models. We also demonstrate that overly drug-loaded species in heterogeneous conjugates are particularly poor at crossing the BBB, leading to deteriorated overall brain tumor targeting. Our findings indicate the importance of homogeneous conjugation with optimal payload loading in generating effective ADCs for intractable brain tumors.

Homogeneous antibody-angiopep 2 conjugates for effective brain targeting.

Antibody-based therapy has shown great success in the treatment of many diseases, including cancers. While antibodies and antibody-drug conjugates (ADCs) have also been evaluated for central nervous system (CNS) disorders as well as brain tumors, their therapeutic efficacy can be substantially limited due to low permeability across the blood-brain barrier (BBB). Thus, improving BBB permeability of therapeutic antibodies is critical in establishing this drug class as a reliable clinical option for CNS diseases. Here, we report that, compared with a conventional heterogeneous conjugation, homogeneous conjugation of the synthetic BBB shuttle peptide angiopep-2 (Ang2) to a monoclonal antibody (mAb) provides improved binding affinity for brain microvascular endothelial cells in vitro and accumulation into normal brain tissues in vivo. In a mouse model, we also demonstrate that the homogeneous anti-EGFR mAb-Ang2 conjugate administered intravenously efficiently accumulates in intracranial tumors. These findings suggest that homogeneous conjugation of BBB shuttle peptides such as Ang2 is a promising approach to enhancing the therapeutic efficacy of antibody agents for CNS diseases.

Enhancing the accumulation level of 3-[18F]fluoro-L-α-methyltyrosine in tumors by preloading probenecid.

INTRODUCTION: 3-[18F]fluoro-α-methyl-L-tyrosine ([18F]FAMT) is a promising amino acid tracer targeting L-type amino acid transporter 1 (LAT1). One concern regarding the diagnosis using [18F]FAMT is the possibility of false-negative findings because of its relatively low accumulation level even in malignant tumors. Moreover, preloading probenecid, an organic anion transporter inhibitor, markedly increased the tumor accumulation level of radioiodine-labeled α-methyltyrosine. In this study, we evaluated the usefulness of preloading probenecid in improving the tumor-imaging capability of [18F]FAMT. METHODS: Three biodistribution studies of [18F]FAMT were conducted in normal mice to elucidate the usefulness of probenecid preloading. Later, a biodistribution study and positron emission tomography (PET) imaging of [18F]FAMT were conducted with or without probenecid injection in tumor-bearing mice. RESULTS: Probenecid preloading significantly delayed blood clearance and consequently enhanced the accumulation of [18F]FAMT in the pancreas, a LAT1-positive organ. The effects of probenecid preloading were independent of the administration route. Tumor accumulation level in the biodistribution study and the maximum standardized uptake value in tumors on PET imaging of the probenecid preloading group were significantly higher than those of the control (without probenecid injection) group in tumor-bearing mice. CONCLUSIONS: Preloading probenecid significantly delayed blood clearance and consequently enhanced the accumulation of [18F]FAMT in tumors. These results indicate that preloading probenecid could improve the diagnostic accuracy of [18F]FAMT.

Reduced Fatty Acid Use from CD36 Deficiency Deteriorates Streptozotocin-Induced Diabetic Cardiomyopathy in Mice.

Cardiac dysfunction is induced by multifactorial mechanisms in diabetes. Deranged fatty acid (FA) utilization, known as lipotoxicity, has long been postulated as one of the upstream events in the development of diabetic cardiomyopathy. CD36, a transmembrane glycoprotein, plays a major role in FA uptake in the heart. CD36 knockout (CD36KO) hearts exhibit reduced rates of FA transport with marked enhancement of glucose use. In this study, we explore whether reduced FA use by CD36 ablation suppresses the development of streptozotocin (STZ)-induced diabetic cardiomyopathy. We found that cardiac contractile dysfunction had deteriorated 16 weeks after STZ treatment in CD36KO mice. Although accelerated glucose uptake was not reduced in CD36KO-STZ hearts, the total energy supply, estimated by the pool size in the TCA cycle, was significantly reduced. The isotopomer analysis with 13C6-glucose revealed that accelerated glycolysis, estimated by enrichment of 13C2-citrate and 13C2-malate, was markedly suppressed in CD36KO-STZ hearts. Levels of ceramides, which are cardiotoxic lipids, were not elevated in CD36KO-STZ hearts compared to wild-type-STZ ones. Furthermore, increased energy demand by transverse aortic constriction resulted in synergistic exacerbation of contractile dysfunction in CD36KO-STZ mice. These findings suggest that CD36KO-STZ hearts are energetically compromised by reduced FA use and suppressed glycolysis; therefore, the limitation of FA utilization is detrimental to cardiac energetics in this model of diabetic cardiomyopathy.

Antibody-drug conjugates with dual payloads for combating breast tumor heterogeneity and drug resistance.

Breast tumors generally consist of a diverse population of cells with varying gene expression profiles. Breast tumor heterogeneity is a major factor contributing to drug resistance, recurrence, and metastasis after chemotherapy. Antibody-drug conjugates (ADCs) are emerging chemotherapeutic agents with striking clinical success, including T-DM1 for HER2-positive breast cancer. However, these ADCs often suffer from issues associated with intratumor heterogeneity. Here, we show that homogeneous ADCs containing two distinct payloads are a promising drug class for addressing this clinical challenge. Our conjugates show HER2-specific cell killing potency, desirable pharmacokinetic profiles, minimal inflammatory response, and marginal toxicity at therapeutic doses. Notably, a dual-drug ADC exerts greater treatment effect and survival benefit than does co-administration of two single-drug variants in xenograft mouse models representing intratumor HER2 heterogeneity and elevated drug resistance. Our findings highlight the therapeutic potential of the dual-drug ADC format for treating refractory breast cancer and perhaps other cancers.

Chemical generation of small molecule-based bispecific antibody-drug conjugates for broadening the target scope.

Antibody-drug conjugates (ADCs) hold great therapeutic promise for cancer indications; however, treating tumors with intratumor heterogeneity remains challenging. We hypothesized that ADCs that can simultaneously target two different cancer antigens could address this issue. Here, we report controlled production and evaluation of bispecific ADCs chemically functionalized with tumor-targeting small molecules. Enzyme-mediated conjugation of bi-functional branched linkers and following sequential orthogonal click reactions with payload and tumor targeting modules (folic acid or RGD peptide) afforded homogeneous bispecific ADCs with defined ligand/drug-to-antibody ratios ranging from 4 + 4 to 16 + 4 (ligand/payload). Most bispecific ADCs were stable under physiological conditions for 14 days. Functionalization with the cancer-specific ligands did not impair cathepsin B-mediated payload release from ADCs. Bispecific ADCs targeting the folate receptor (FR)/human epidermal growth factor receptor 2 (HER2) demonstrated specific binding and high cell killing potency only in cells expressing either antigen (FR or HER2). Integrin/HER2 bispecific ADCs equipped with RGD peptides also showed target-specific binding and cytotoxicity in integrin- or HER2-positive cells. These findings suggest that our small-molecule based bispecific ADCs have the potential to effectively treat tumors with heterogeneous antigen expression.

DPP-4 inhibitor induces FGF21 expression via sirtuin 1 signaling and improves myocardial energy metabolism.

Dipeptidyl peptidase-4 (DPP-4) inhibitors are widely used incretin-based therapy for the treatment of type 2 diabetes. We investigated the cardioprotective effect of a DPP-4 inhibitor, vildagliptin (vilda), on myocardial metabolism and cardiac performance under pressure overload. Mice were treated with either vehicle or vilda, followed by transverse aortic constriction (TAC). After 3 weeks of TAC, cardiac hypertrophy and impairment of systolic function were attenuated in vilda-treated mice. Pressure-volume analysis showed that vilda treatment significantly improved left-ventricular contractile efficiency in TAC heart. Myocardial energy substrate analysis showed that vilda treatment significantly increased glucose uptake as well as fatty acid uptake. Fibroblast growth factor 21 (FGF21), a peptide involved in the regulation of energy metabolism, increased in TAC heart and was further increased by vilda treatment. FGF21 was strongly expressed in cardiac fibroblasts than in cardiomyocytes in mouse heart after TAC with vilda treatment. Vilda treatment markedly induced FGF21 expression in human cardiac fibroblasts through a sirtuin (Sirt) 1-mediated pathway, suggesting that fibroblast-mediated FGF21 expression may regulate energy metabolism and exert vilda-mediated beneficial effects in stressed heart. Vilda induced a metabolic regulator, FGF21 expression in cardiac fibroblasts via Sirt1, and increased contractile efficiency in murine pressure-overloaded heart.

64Cu-ATSM and 99mTc(CO)3-DCM20 potential in the early detection of rheumatoid arthritis.

OBJECTIVES: Molecular imaging constitutes a promising technique for the early detection of rheumatoid arthritis (RA). Macrophages and hypoxia play significant roles in inflamed synovium. In the present study, we evaluated the efficacy of radiopharmaceuticals that target macrophage mannose receptors (99mTc-labeled mannosylated dextran or 99mTc(CO)3-DCM20) and hypoxia (copper(II) diacetyl-di(N4-methylthiosemicarbazone) or Cu-ATSM) for the early detection of RA in collagen-induced arthritis (CIA) mice models. METHODS: CIA model was developed in DBA/1 mice, and the clinical score for arthritis was visually assessed on a regular basis. Two biodistribution studies were performed in a paired-labeled format using 2-deoxy-2-18F-fluoro-D-glucose (18F-FDG) as a reference: (1) 99mTc(CO)3-DCM20 with 18F-FDG and (2) 67Cu-ATSM with 18F-FDG. RESULTS: The accumulation levels of 99mTc(CO)3-DCM20 and 67Cu-ATSM in forepaws, hindpaws, and knee joints of CIA mice were significantly higher than that of control mice. In contrast, 18F-FDG uptake in hindpaws and knee joints showed no significant difference between CIA and control mice. The radioactivity levels of 99mTc(CO)3-DCM20 and 67Cu-ATSM were significantly correlated with the clinical scores for the paws. CONCLUSION: These results suggest the potential usefulness of 99mTc(CO)3-DCM20 and radiolabeled Cu-ATSM for the imaging and early detection of RA.

Reduced fatty acid uptake aggravates cardiac contractile dysfunction in streptozotocin-induced diabetic cardiomyopathy.

Diabetes is an independent risk factor for the development of heart failure. Increased fatty acid (FA) uptake and deranged utilization leads to reduced cardiac efficiency and accumulation of cardiotoxic lipids, which is suggested to facilitate diabetic cardiomyopathy. We studied whether reduced FA uptake in the heart is protective against streptozotocin (STZ)-induced diabetic cardiomyopathy by using mice doubly deficient in fatty acid binding protein 4 (FABP4) and FABP5 (DKO mice). Cardiac contractile dysfunction was aggravated 8 weeks after STZ treatment in DKO mice. Although compensatory glucose uptake was not reduced in DKO-STZ hearts, total energy supply, estimated by the pool size in the TCA cycle, was significantly reduced. Tracer analysis with 13C6-glucose revealed that accelerated glycolysis in DKO hearts was strongly suppressed by STZ treatment. Levels of ceramides, cardiotoxic lipids, were similarly elevated by STZ treatment. These findings suggest that a reduction in total energy supply by reduced FA uptake and suppressed glycolysis could account for exacerbated contractile dysfunction in DKO-STZ hearts. Thus, enhanced FA uptake in diabetic hearts seems to be a compensatory response to reduced energy supply from glucose, and therefore, limited FA use could be detrimental to cardiac contractile dysfunction due to energy insufficiency.

Antibody Clicking as a Strategy to Modify Antibody Functionalities on the Surface of Targeted Cells.

We established a methodology for initiating cross-linking of antibodies selectively on the cell surface through intermolecular copper-free click reactions facilitated by increased effective concentrations of antibodies binding to target antigens. Upon cross-linking of tetrazine- and bicyclononyne-modified trastuzumab on the surface of HER2-overexpressing cells, increased antibody uptake and activation of intracellular signaling were observed. Our findings demonstrate that the cross-linking reaction can significantly alter the biophysical properties of proteins, activating their unique functionalities on targeted cells to realize an increased cargo delivery and synthetic manipulation of cellular signaling.

LILRB4-targeting Antibody-Drug Conjugates for the Treatment of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the most common and aggressive blood cancer in adults. In particular, significant unmet medical needs exist for effective treatment strategies for acute myelomonocytic leukemia (M4) and acute monocytic leukemia (M5) AML subtypes. Antibody-drug conjugates (ADC) are a promising drug class for AML therapy, as demonstrated by the FDA-approved anti-CD33 ADC, gemtuzumab ozogamicin (Mylotarg). However, CD33 is expressed in normal hematopoietic stem cells, highlighting the critical need to identify AML-specific targets to minimize the risk of potential adverse effects. We have demonstrated that the leukocyte immunoglobulin-like receptor subfamily B4 (LILRB4) is expressed at significantly higher levels on monocytic M4 and M5 AML cells than on normal counterparts. Here, we test whether LILRB4 is a promising ADC target to kill monocytic AML cells while sparing healthy counterparts. To this end, we generated ADCs from a humanized anti-LILRB4 mAb and the antimitotic payload, monomethyl auristatin F. The conjugates constructed were characterized and evaluated for LILRB4-specific cell killing potency, toxicity to progenitor cells, pharmacokinetics, and therapeutic efficacy. Our ADC linker technology platform efficiently generated homogeneous anti-LILRB4 ADCs with defined drug-to-antibody ratios. The homogeneous anti-LILRB4 ADCs demonstrated the capacity for LILRB4-mediated internalization, suitable physicochemical properties, and high cell killing potency against LILRB4-positive AML cells. Importantly, our data indicate that these ADCs spare normal progenitor cells. One of our homogeneous conjugates exerted a remarkable therapeutic effect and no significant toxicity in a xenograft mouse model of disseminated human AML. Our findings highlight the clinical potential of anti-LILRB4 ADCs in monocytic AML therapy.

Diagnosis of bladder cancer using 18F-labeled α-methyl-phenylalanine tracers in a mouse model.

OBJECTIVE: Although 2-18F-fluoro-2-deoxy-glucose (18F-FDG) has established roles in the diagnosis of a variety of cancers, it has limited value in the detection of primary/recurrent lesions in the bladder, mainly because of interference by the pooled radioactivity in the urine. Our previous study revealed promising properties of L- and D-2-18F-α-methyl-phenylalanine (2-18F-FAMP) as radiotracers; i.e., their rapid blood clearance and low renal accumulation. In the present study we evaluated the utility of L- and D-2-18F-FAMP for imaging bladder cancer in a mouse model. METHODS: We used the human bladder cancer cell line HT1376 to prepare a bladder cancer xenograft model in mice bearing both orthotopic and subcutaneous tumors. Biodistribution and PET imaging studies were performed at 1 and 3 h after injection of L-2-18F-FAMP or D-2-18F-FAMP. 18F-FDG was used as a control. RESULTS: At 1 h after injection, greater accumulations of both L-2-18F-FAMP and D-2-18F-FAMP were observed in the orthotopic tumors compared to 18F-FDG. The orthotopic tumor-to-muscle ratio of D-2-18F-FAMP was significantly higher than that of 18F-FDG (p < 0.01), because of the rapid blood clearance of D-2-18F-FAMP. L-2-18F-FAMP showed the highest subcutaneous tumor-to-muscle ratio (p < 0.01) due to its high subcutaneous tumor uptake. Compared to L-2-18F-FAMP, D-2-18F-FAMP exhibited faster clearance and lower kidney accumulation. In the PET imaging studies, L- and D-2-18F-FAMP both clearly visualized the orthotopic bladder tumors at 1 h after injection. CONCLUSION: Our study showed that L-2-18F-FAMP and D-2-18F-FAMP have the potential to detect bladder cancer.

Selective synthesis of L-2-[18 F]fluoro-alpha-methylphenylalanine via copper-mediated 18 F-fluorination of (mesityl)(aryl)iodonium salt.

L-2-[18 F]fluoro-alpha-methylphenylalanine (2-[18 F]FAMP) is a promising amino acid tracer for positron emission tomography (PET) imaging, yet the low production yield of direct electrophilic radiofluorination with [18 F]F2 necessitates further optimization of the radiolabeling process. This paper describes a two-step preparation method for L-2-[18 F]fluoro-alpha-methylphenylalanine (2-[18 F]FAMP) starting from [18 F]fluoride. The (Mesityl)(L-alpha-methylphenylalanine)-2-iodonium tetrafluoroborate precursors with various protecting groups were prepared. The copper-mediated 18 F-fluorination of the iodonium salt precursors successfully produced 2-[18 F]FAMP. The highest radio chemical conversion of 57.6% was noted with N-Piv-protected (mesityl)(aryl)iodonium salt in the presence of 5 equivalent of Cu (OTf)2 . Subsequent deprotection with 57% hydrogen iodide produced 2-[18 F]FAMP within 120 min in 21.4 ± 11.7% overall radiochemical yield with >95% radiochemical purity and an enantiomeric excess >99%. The obtained 2-[18 F]FAMP showed comparable biodistribution profiles in normal mice with that of the carrier-added 2-[18 F]FAMP. These results indicate that usefulness of copper mediated 18 F-fluorination for the production of 2-[18 F]FAMP, which would facilitate clinical translation of the promising tumor specific amino acid tracer. Individual facilities could adopt either production method based on radioactivity demand and equipment availability.

Immuno-PET imaging for non-invasive assessment of cetuximab accumulation in non-small cell lung cancer.

BACKGROUNDS: Overexpression of epidermal growth factor receptor (EGFR) has been established as a valid therapeutic target of non-small cell lung cancer (NSCLC). However, the clinical benefit of cetuximab as an EGFR-targeting drug is still controversial, partially due to the lack of effective means to identify suitable patients. This study aimed to investigate the potential of radiolabeled cetuximab as a non-invasive tool to predict cetuximab accumulation in NSCLC tumor xenografts with varying EGFR expression levels. METHODS: The NSCLC tumors in model mice were subjected to in vivo biodistribution study and positron emission tomography (PET) imaging 48 h after injection of either 111In- or 64Cu-labeled cetuximab. The EGFR expression levels of NSCLC tumors were determined by ex vivo immunoblotting. RESULTS: We found that tumors with high EGFR expression had significantly higher [111In]In-DOTA-cetuximab accumulation than tumors with moderate to low EGFR expression (P < 0.05). Strong correlations were found between [111In]In-DOTA-cetuximab tumor uptake and EGFR expression level (r = 0.893), and between [64Cu]Cu-DOTA-cetuximab tumor uptake with EGFR expression level (r = 0.915). PET imaging with [64Cu]Cu-DOTA-cetuximab allowed clear visualization of tumors. CONCLUSION: Our findings suggest that this immuno-PET imaging can be clinically translated as a tool to predict cetuximab accumulation in NSCLC cancer patients prior to cetuximab therapy.

Al18F-labeled alpha-melanocyte-stimulating hormone (α-MSH) peptide derivative for the early detection of melanoma.

OBJECTIVE: Early detection plays a role in the prognosis of melanoma, the most aggressive skin cancer. 64Cu- and 68Ga-labeled alpha-melanocyte-stimulating hormone (α-MSH) analogs targeting the melanocortin-1 receptor are promising positron emission tomography (PET) tracers for detecting melanoma, and the use of 18F-labeling will further contribute to the detectability and availability. However, the high radiochemistry demand related to the conventional 18F-labeling methods has restricted the development of 18F-labeled α-MSH analogs. A recently developed radiofluorination method using aluminum-fluoride (Al18F) offers a simple, efficient, and time-saving labeling procedure compared to the conventional 18F-labeling methods. Herein, we sought to establish a simple preparation method for an 18F-labeled α-MSH analog using Al18F, and we examined its potential for the early detection of melanoma. METHODS: A 1,4,7-triazacyclononane-N,N',N″-triacetic acid (NOTA)-conjugated α-MSH analog (NOTA-GGNle-CycMSHhex) was prepared by the Fmoc solid-phase strategy. NOTA-GGNle-CycMSHhex was labeled with Al18F by heating at 105 °C using a microwave synthesizer for 15 min. Biodistribution study was conducted on B16/F10-luc melanoma-bearing mice at 30 min, 1 h and 3 h after injection of Al18F-NOTA-GGNle-CycMSHhex. PET imaging was conducted on melanoma-bearing mice at 1 h post-injection. One day prior to the PET imaging, bioluminescence imaging was also performed. RESULTS: Al18F-NOTA-GGNle-CycMSHhex was readily prepared with a high radiochemical yield (94.0 ± 2.8%). The biodistribution study showed a high accumulation of Al18F-NOTA-GGNle-CycMSHhex in the tumor at 30 min and 1 h post-injection (6.69 ± 1.49 and 7.70 ± 1.71%ID/g, respectively). The tumor-to-blood ratio increased with time: 3.46 ± 0.89, 12.67 ± 1.29, and 35.27 ± 9.12 at 30 min, 1 h, and 3 h post-injection, respectively. In the PET imaging, Al18F-NOTA-GGNle-CycMSHhex clearly visualized the tumors and depicted very small tumors (< 3 mm). CONCLUSIONS: We successfully prepared Al18F-NOTA-GGNle-CycMSHhex in a simple and efficient manner. Al18F-NOTA-GGNle-CycMSHhex showed high tumor accumulation and clearly visualized very small tumors in melanoma-bearing mice. These findings suggest that Al18F-NOTA-GGNle-CycMSHhex will be a promising PET tracer for melanoma imaging at an earlier stage.

Novel 18F-Labeled α-Methyl-Phenylalanine Derivative with High Tumor Accumulation and Ideal Pharmacokinetics for Tumor-Specific Imaging.

Positron emission tomography (PET) imaging with 18F-labeled α-methyl-substituted amino acids exerts significant influence on differential diagnosis of malignant tumors and tumor-like lesions. Exclusive uptake via L-type amino acid transporter 1 (LAT1), a tumor-specific transporter, accounts for their excellent tumor specificity and low background accumulation. However, further refinement and optimization in their tumor accumulation and pharmacokinetics are sorely needed. To address these issues, we newly designed 18F-labeled α-methyl-phenylalanine (18F-FAMP) regioisomers (2-, 3-, or 4-18F-FAMP) and stereoisomers (L- or D-form), and we comprehensively evaluated their potential as tumor-imaging agents. 18F-FAMPs were prepared from α-methyl phenylalanine by electrophilic radiofluorination and purified by reversed-phase HPLC. In biodistribution studies on normal mice, L-2-18F-FAMP and the three D-18F-FAMPs showed faster blood clearance and lower renal accumulation than L-3-18F-FAMP or L-4-18F-FAMP. In LS180 human colorectal cancer cell line xenograft mice, L-2-18F-FAMP exhibited significantly higher tumor accumulation than the D-18F-FAMPs or a clinically relevant tracer, L-3-18F-α-methyl-tyrosine (18F-FAMT) (p < 0.05). The renal accumulation levels of L-2-18F-FAMP were significantly lower than that of 18F-FAMT (p < 0.01). LAT-1 specificity of L-2-18F-FAMP was validated in the cellular uptake studies. The PET imaging with L-2-18F-FAMP clearly visualized the tumor as early as 1 h after injection, and the high tumor accumulation level was retained for 3 h. These findings suggest that L-2-18F-FAMP constitutes a potential PET tracer for tumor-specific imaging.

Thin-Shelled PEGylated Perfluorooctyl Bromide Nanocapsules for Tumor-Targeted Ultrasound Contrast Agent.

Shell thickness determines the acoustic response of polymer-based perfluorooctyl bromide (PFOB) nanocapsule ultrasound contrast agents. PEGylation provides stealth property and arms for targeting moieties. We investigated a modulation in the polymer formulation of carboxy-terminated poly(d,l-lactide-co-glycolide) (PLGA) and poly(d,l-lactide-co-glycolide)-block-polyethylene glycol (PLGA-b-PEG) to produce thin-shelled PFOB nanocapsules while keeping its echogenicity, stealth property, and active targeting potential. Polymer formulation contains 40% PLGA-PEG that yields the PEGylated PFOB nanocapsules of approximately 150 nm size with average thickness-to-radius ratio down to 0.15, which adequately hindered phagocytosis. Functionalization with antibody enables in vitro tumor-specific targeting. Despite the acoustic response improvement, the in vivo tumor accumulation was inadequate to generate an observable acoustic response to the ultrasound power at the clinical level. The use of PLGA and PLGA-PEG polymer blend allows the production of thin-shelled PFOB nanocapsules with echogenicity improvement while maintaining its potential for specific targeting.

Circulating FABP4 is eliminated by the kidney via glomerular filtration followed by megalin-mediated reabsorption.

Circulating fatty acid binding protein 4 (FABP4), secreted from adipocytes, is a potential biomarker for metabolic and cardiovascular diseases. Circulating FABP4 levels are positively associated with adiposity and adrenergic stimulation, but negatively with renal function. In this study, we addressed the issue of how the kidney regulates clearance of circulating FABP4. Tracing study revealed remarkable accumulation of 125I-labeled FABP4 in the kidney. Exogenous FABP4 was exclusively detected in the apical membrane of proximal tubule epithelial cells (PTECs). Bilateral nephrectomy resulted in marked elevation of circulating FABP4 levels. Accelerated lipolysis by ß-3 adrenergic stimulation led to a marked elevation in circulating FABP4 in mice with severe renal dysfunction. Megalin, an endocytic receptor expressed in PTECs, plays a major role in reabsorption of proteins filtered through glomeruli. Quartz-crystal microbalance study revealed that FABP4 binds to megalin. In kidney-specific megalin knockout mice, a large amount of FABP4 was excreted in urine while circulating FABP4 levels were significantly reduced. Our data suggest that circulating FABP4 is processed by the kidney via the glomerular filtration followed by megalin-mediated reabsorption. Thus, it is likely that circulating FABP4 levels are determined mainly by balance between secretion rate of FABP4 from adipocytes and clearance rate of the kidney.

CD36 is indispensable for nutrient homeostasis and endurance exercise capacity during prolonged fasting.

During fasting, most tissues including skeletal muscle heavily rely on utilization of fatty acids (FA) and minimize glucose use. In contrast, skeletal muscle prefers carbohydrate use as exercise intensity increases. In mice deficient for CD36 (CD36-/- mice), FA uptake is markedly reduced with a compensatory increase in glucose uptake in skeletal muscle even during fasting. In this study, we questioned how exercise endurance is affected during prolonged fasting in CD36-/- mice where glucose utilization is constantly increased. With or without a 24-h fast, a single bout of treadmill exercise was started at the speed of 10 m/min, and the speed was progressively increased up to 30 m/min until mice were exhausted. Running distance of wild type (WT) and CD36-/- mice was comparable in the fed state whereas that of CD36-/- mice was significantly reduced after a 24-h fast. Glycogen levels in liver and skeletal muscle were depleted both in WT and CD36-/- mice after a 24-h fast. In CD36-/- mice, FA uptake by skeletal muscle continued to be reduced during fasting. Glucose utilization also continued to be enhanced in the heart and oxidative skeletal muscle and glucose supply relative to its demand was diminished, resulting in accelerated hypoglycemia. Consequently, available energy substrates from serum and in muscle for exercise performance were very limited in CD36-/- mice during prolonged fasting, which could cause a remarkable reduction in exercise endurance. In conclusion, our study underscores the importance of CD36 for nutrient homeostasis to maintain exercise performance of skeletal muscle when nutrient supply is limited.

Myocardial fatty acid uptake through CD36 is indispensable for sufficient bioenergetic metabolism to prevent progression of pressure overload-induced heart failure.

The energy metabolism of the failing heart is characterized by reduced fatty acid (FA) oxidation and an increase in glucose utilization. However, little is known about how energy metabolism-function relationship is relevant to pathophysiology of heart failure. Recent study showed that the genetic deletion of CD36 (CD36KO), which causes reduction in FA use with an increased reliance on glucose, accelerates the progression from compensated hypertrophy to heart failure. Here, we show the mechanisms by which CD36 deletion accelerates heart failure in response to pressure overload. CD36KO mice exhibited contractile dysfunction and death from heart failure with enhanced cardiac hypertrophy and interstitial fibrosis when they were subjected to transverse aortic constriction (TAC). The pool size in the TCA cycle and levels of high-energy phosphate were significantly reduced in CD36KO-TAC hearts despite an increase in glycolytic flux. De novo synthesis of non-essential amino acids was facilitated in CD36KO-TAC hearts, which could cause a further decline of the pool size. The ingestion of a diet enriched in medium-chain FA improved cardiac dysfunction in CD36KO-TAC hearts. These findings suggest that myocardial FA uptake through CD36 is indispensable for sufficient ATP production and for preventing an increased glycolytic flux-mediated structural remodeling during pressure overload-induced hypertrophy.