Cardiac Uptake of the Adrenergic Imaging Agent meta-Iodobenzylguanidine (mIBG) Is Mediated by Organic Cation Transporter 3 (Oct3).

Heart failure (HF) is a chronic disease affecting 1%-2% of the global population.123I-labeled meta-iodobenzylguanidine (mIBG) is US Food and Drug Administration-approved for cardiac imaging and prognosis risk assessment in patients with HF. As a norepinephrine analog, mIBG is believed to be transported into adrenergic nerve terminals by the neuronal norepinephrine transporter (NET) and hence image sympathetic innervation of the myocardium. We previously showed that mIBG is an excellent substrate of organic cation transporter 3 (OCT3), an extraneuronal transporter expressed in cardiomyocytes. Here, we evaluated the in vivo impact of Oct3 on mIBG disposition and tissue distribution using Oct3 knockout mice. Oct3 +/+ and Oct3 -/- mice were administered with mIBG intravenously, and mIBG plasma pharmacokinetics and tissue exposures were determined. In Oct3 +/+ mice, mIBG exhibited extensive accumulation in multiple tissues (heart, salivary gland, liver, and adrenal gland). No difference was observed in overall plasma exposure between Oct3 +/+ and Oct3 -/- mice. Strikingly, cardiac mIBG was depleted in Oct3 -/- mice, resulting in 83% reduction in overall cardiac exposure (AUC0-24 h: 12.7 vs. 2.1 µg × h/g). mIBG tissue exposure (AUC0-24 h) was also reduced by 66%, 36%, and 31% in skeletal muscle, salivary gland, and lung, respectively, in Oct3 -/- mice. Our data demonstrated that Oct3 is the primary transporter responsible for cardiac mIBG uptake in vivo and suggested that cardiac mIBG imaging mainly measures OCT3 activity in cardiomyocytes but not NET-mediated uptake in adrenergic nerve endings. Our findings challenge the current paradigm in interpreting cardiac mIBG imaging results and suggest OCT3 as a potential genetic risk marker for HF prognosis. SIGNIFICANCE STATEMENT: 123I-labeled meta-iodobenzylguanidine is used for cardiac imaging and risk assessment in heart failure patients. Contrary to the current belief that meta-iodobenzylguanidine (mIBG) tracks cardiac sympathetic innervation due to its uptake by the neuronal norepinephrine transporter, the authors demonstrated that cardiac mIBG uptake is mediated by the extraneuronal transporter Oct3. Their findings warrant a re-evaluation of the scientific rationale behind cardiac mIBG scan and further suggest organic cation transporter 3 as a risk factor for disease progression in heart failure patients.

The Plasma Membrane Monoamine Transporter is Highly Expressed in Neuroblastoma and Functions as an mIBG Transporter.

Neuroblastoma (NB) is a pediatric cancer with low survival rates in high-risk patients. 131I-mIBG has emerged as a promising therapy for high-risk NB and kills tumor cells by radiation. Consequently, 131I-mIBG tumor uptake and retention are major determinants for its therapeutic efficacy. mIBG enters NB cells through the norepinephrine transporter (NET), and accumulates in mitochondria through unknown mechanisms. Here we evaluated the expression of monoamine and organic cation transporters in high-risk NB tumors and explored their relationship with MYCN amplification and patient survival. We found that NB mainly expresses NET, the plasma membrane monoamine transporter (PMAT), and the vesicular membrane monoamine transporter 1/2 (VMAT1/2), and that the expression of these transporters is significantly reduced in MYCN-amplified tumor samples. PMAT expression is the highest and correlates with overall survival in high-risk NB patients without MYCN amplification. Immunostaining showed that PMAT resides intracellularly in NB cells and co-localizes with mitochondria. Using cells expressing PMAT, mIBG was identified as a PMAT substrate. In mitochondria isolated from NB cell lines, mIBG uptake was reduced by ∼50% by a PMAT inhibitor. Together, our data suggest that PMAT is a previously unrecognized transporter highly expressed in NB and could impact intracellular transport and therapeutic response to 131I-mIBG. SIGNIFICANCE STATEMENT: This study identified that plasma membrane monoamine transporter (PMAT) is a novel transporter highly expressed in neuroblastoma and its expression level is associated with overall survival rate in high-risk patients without MYCN amplification. PMAT is expressed intracellularly in neuroblastoma cells, transports meta-iodobenzylguanidine (mIBG) and thus could impact tumor retention and response to 131I-mIBG therapy. These findings have important clinical implications as PMAT could represent a novel molecular marker to help inform disease prognosis and predict response to 131I-mIBG therapy.

Clinical Applications and the Roles of Transporters in Disposition, Tumor Targeting, and Tissue Toxicity of meta-Iodobenzylguanidine (mIBG).

Transporters on the plasma membrane of tumor cells are promising molecular "Trojan horses" to deliver drugs and imaging agents into cancer cells. Radioiodine-labeled meta-iodobenzylguanidine (mIBG) is used as a diagnostic agent (123I-mIBG) and a targeted radiotherapy (131I-mIBG) for neuroendocrine cancers. mIBG enters cancer cells through the norepinephrine transporter (NET) where the radioactive decay of 131I causes DNA damage, cell death, and tumor necrosis. mIBG is predominantly eliminated unchanged by the kidney. Despite its selective uptake by neuroendocrine tumors, mIBG accumulates in several normal tissues and leads to tissue-specific radiation toxicities. Emerging evidences suggest that the polyspecific organic cation transporters play important roles in systemic disposition and tissue-specific uptake of mIBG. In particular, human organic cation transporter 2 (hOCT2) and toxin extrusion proteins 1 and 2-K (hMATE1/2-K) likely mediate renal secretion of mIBG whereas hOCT1 and hOCT3 may contribute to mIBG uptake into normal tissues such as the liver, salivary glands, and heart. This mini-review focuses on the clinical applications of mIBG in neuroendocrine cancers and the differential roles of NET, OCT and MATE transporters in mIBG disposition, response and toxicity. Understanding the molecular mechanisms governing mIBG transport in cancer and normal cells is a critical step for developing strategies to optimize the efficacy of 131I-mIBG while minimizing toxicity in normal tissues. Significance Statement Radiolabeled mIBG has been used as a diagnostic tool and as radiotherapy for neuroendocrine cancers and other diseases. NET, OCT and MATE transporters play differential roles in mIBG tumor targeting, systemic elimination, and accumulation in normal tissues. The clinical use of mIBG as a radiopharmaceutical in cancer diagnosis and treatment can be further improved by taking a holistic approach considering mIBG transporters in both cancer and normal tissues.

Characterization of Meta-Iodobenzylguanidine (mIBG) Transport by Polyspecific Organic Cation Transporters: Implication for mIBG Therapy.

Radiolabeled meta-iodobenzylguanidine (mIBG) is an important radiopharmaceutical used in the diagnosis and treatment of neuroendocrine cancers. mIBG is known to enter tumor cells through the norepinephrine transporter. Whole-body scintigraphy has shown rapid mIBG elimination through the kidney and high accumulation in several normal tissues, but the underlying molecular mechanisms are unclear. Using transporter-expressing cell lines, we show that mIBG is an excellent substrate for human organic cation transporters 1-3 (hOCT1-3) and the multidrug and toxin extrusion proteins 1 and 2-K (hMATE1/2-K), but not for the renal organic anion transporter 1 and 3 (hOAT1/3). Kinetic analysis revealed that hOCT1, hOCT2, hOCT3, hMATE1, and hMATE2-K transport mIBG with similar apparent affinities (K m of 19.5 ± 6.9, 17.2 ± 2.8, 14.5 ± 7.1, 17.7 ± 10.9, 12.6 ± 5.6 µM, respectively). Transwell studies in hOCT2/hMATE1 double-transfected Madin-Darby canine kidney cells showed that mIBG transport in the basal (B)-to-apical (A) direction is much greater than in the A-to-B direction. Compared with control cells, the B-to-A permeability of mIBG increased by 20-fold in hOCT2/hMATE1 double-transfected cells. Screening of 23 drugs used in the treatment of neuroblastoma identified several drugs with the potential to inhibit hOCT- or hMATE-mediated mIBG uptake. Interestingly, irinotecan selectively inhibited hOCT1, whereas crizotinib potently inhibited hOCT3-mediated mIBG uptake. Our results suggest that mIBG undergoes renal tubular secretion mediated by hOCT2 and hMATE1/2-K, and hOCT1 and hOCT3 may play important roles in mIBG uptake into normal tissues. SIGNIFICANCE STATEMENT: mIBG is eliminated by the kidney and extensively accumulates in several tissues known to express hOCT1 and hOCT3. Our results suggest that hOCT2 and human multidrug and toxin extrusion proteins 1 and 2-K are involved in mIBG renal elimination, whereas hOCT1 and hOCT3 may play important roles in mIBG uptake into normal tissues. These findings may help to predict and prevent adverse drug interaction with therapeutic [131I]mIBG and develop clinical strategies to reduce [131I]mIBG accumulation and toxicity in normal tissues and organs.

Development and validation of a LC-MS/MS method for in vivo quantification of meta-iodobenzylguanidine (mIBG).

meta-iodobenzylguanidine (mIBG) is a radiopharmaceutical used for the diagnosis and treatment of neuroendocrine cancers. Previous quantification of mIBG in biodistribution and pharmacokinetic studies mainly relied on the use of radiolabeled mIBG, which involves the handling of highly radioactive materials. The goal of this study was to develop a nonradioactive analytical method for quantifying mIBG in mouse plasma and tissue homogenates using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Samples were prepared for analysis using a protein precipitation method. Mass spectrometry analysis was performed using 4-hydroxyphenformin as the internal standard, and the mass-to-charge transitions were 276.1 → 217.0 for mIBG and 222.1 → 121.0 for 4-hydroxyphenformin. The quantification limit of mIBG was 0.98 ng/mL, and the method was linear up to 500 ng/mL. The accuracy, inter-day and intra-day precision were 96-112%, 5.5-14.4%, and 3.7-14.1%, respectively, suggesting that the method was accurate and precise in quantifying mIBG at multiple concentrations in mouse plasma and liver homogenates. The extraction recovery was 96-106% and the matrix effect was 95-110%, indicating that the method was reproducible in quantifying mIBG with minimal impact from the biological matrices. In summary, we have developed and validated a fast, high-throughput quantification method of non-radiolabeled mIBG using LC-MS/MS. This method is reproducible, accurate, and precise, and can be used to quantify mIBG in plasma and tissue matrices to determine the pharmacokinetics and biodistribution of mIBG in preclinical animal models.