The solute carrier organic anion transporting polypeptide 1A2 (OATP1A2, SLCO1A2) is implicated in the cellular influx of a number of drugs. We identified five novel single nucleotide polymorphisms (SNPs) in coding exons of the SLCO1A2 gene in a cohort of subjects: G550A, G553A, G673A, A775C, and G862A, that encoded the OATP1A2 variants E184K, D185N, V255I, T259P, and D288N, respectively. The function and expression of these variant transporters were assessed in HEK-293 cells. We found that the novel variants, E184K, D185N, T259P, and D288N, were associated with impaired estrone-3-sulfate, imatinib, and methotrexate transport (∼20-50% of wild-type control); function was retained by OATP1A2-V255I. From biotinylation assays, the decreased function of these variants was due, at least in part, to impaired plasma membrane expression. The four loss-of-function variants were studied further using mutagenesis to produce variants that encode residues with different charges or steric properties. From immunoblotting, the replacement of negatively charged residues at amino acid positions 184 and 185 impaired membrane expression, while either a positive or negative charge at residue 288 supported the correct membrane targeting of OATP1A2. Replacement of T259 with bulky residues disrupted transporter stability. From molecular models, E184, D185, and D288 were located near several charged residues such that intramolecular ionic interactions may stabilize the transporter structure. Individuals who carry these novel SNPs in the SLCO1A2 gene may be at risk from impaired efficacy or enhanced toxicity during treatment with drugs that are substrates for OATP1A2.

BackgroundVarious diseases derive from pathologically altered β-cells. Their function can be increased, leading to hyperinsulinism, or decreased, resulting in diabetes. Non-invasive imaging of the β-cell-specific glucagon-like peptide receptor-1 (GLP-1R) would allow the assessment of both β-cell mass and derived tumours, potentially improving the diagnosis of various conditions. We tested three new 67/68Ga-labelled derivatives of exendin-4, an agonist of GLP-1R, in vitro and in vivo. We determined the influence of the chelator NODAGA conjugated to resident lysines either at positions 12 and 27 or the C-terminally attached lysine at position 40 on the binding and kinetics of the peptide.MethodsBinding and internalisation of 67Ga-labelled Ex4NOD12, Ex4NOD27 and Ex4NOD40 were tested on Chinese hamster lung (CHL) cells stably transfected to express the GLP-1 receptor (GLP-1R). In vivo biodistribution of 68Ga-labelled peptides was investigated in CD1 nu/nu mice with subcutaneous CHL-GLP-1R positive tumours; the specificity of the binding to GLP-1R was determined by pre-injecting excess peptide.ResultsAll peptides showed good in vitro binding affinities to GLP-1R in the range of 29 to 54 nM. 67/68Ga-Ex4NOD40 and 67/68Ga-Ex4NOD12 show excellent internalisation (>30%) and high specific uptake in GLP-1R positive tissue, but high activity was also found in the kidneys.ConclusionsWe show that of the three peptides, Ga-Ex4NOD40 and Ga-Ex4NOD12 demonstrate the most favourable in vitro properties and in vivo binding to GLP-1R positive tissue. Therefore, we conclude that the lysines at positions 12 and 40 might preferentially be utilised for modifying exendin-4.

BackgroundCleavable linkers, which are specifically cleaved by defined conditions or enzymes, are powerful tools that can be used for various purposes. Amongst other things, they have been successfully used to deliver toxic payloads as prodrugs into target tissues. In this work novel linker sequences targeting meprin β, a metalloprotease expressed in the kidney brush-border membrane, were designed and included in the sequence of three radiolabelled exendin-4 derivatives. As radiolabelled exendin-4 derivatives strongly accumulate in the kidneys, we hypothesised that specific cleavage of the radiolabelled moiety at the kidney brush-border membrane would allow easier excretion of the activity into the urine and therefore improve the pharmacological properties of the peptide.ResultsThe insertion of a cleavable linker did not negatively influence the in vitro properties of the peptides. They showed a good affinity to the GLP-1 receptor expressed in CHL cells, a high internalisation and sufficiently high stability in fresh human blood plasma. In vitro digestion with recombinant meprin β rapidly metabolised the corresponding linker sequences. After 60 min the majority of the corresponding peptides were digested and at the same time the anticipated fragments were formed. The peptides were also quickly metabolised in CD1 nu/nu mouse kidney homogenates. Immunofluorescence staining of meprin β in kidney sections confirmed the expression of the protease in the kidney brush-border membrane. Biodistribution in GLP-1 receptor positive tumour-xenograft bearing mice revealed high specific uptake of the 111In-labelled tracers in receptor positive tissue. Accumulation in the kidneys, however, was still high and comparable to the lead compound 111In-Ex4NOD40.ConclusionIn conclusion, we show that the concept of cleavable linkers specific for meprin β is feasible, as the peptides are rapidly cleaved by the enzyme while retaining their biological properties.

β-cells, located in the islets of the pancreas, are responsible for production and secretion of insulin and play a crucial role in blood sugar regulation. Pathologic β-cells often cause serious medical conditions affecting blood glucose level, which severely impact life quality and are life-threatening if untreated. With 347 million patients, diabetes is one of the most prevalent diseases, and will continue to be one of the largest socioeconomic challenges in the future. The diagnosis still relies mainly on indirect methods like blood sugar measurements. A non-invasive diagnostic imaging modality would allow direct evaluation of β-cell mass and would be a huge step towards personalized medicine. Hyperinsulinism is another serious condition caused by β-cells that excessively secrete insulin, like for instance β-cell hyperplasia and insulinomas. Treatment options with drugs are normally not curative, whereas curative procedures usually consist of the resection of affected regions for which, however, an exact localization of the foci is necessary. In this review, we describe potential tracers under development for targeting β-cells with focus on radiotracers for PET and SPECT imaging, which allow the non-invasive visualization of β-cells. We discuss either the advantages or limitations for the various tracers and modalities. This article concludes with an outlook on future developments and discuss the potential of new imaging probes including dual probes that utilize functionalities for both a radioactive and optical moiety as well as for theranostic applications.

The glucagon-like peptide-1 receptor (GLP-1R) is highly and specifically expressed on the pancreatic β-cells. It plays an important role in glucose metabolism as well as in β-cell-derived diseases like diabetes, insulinoma, or congenital and adult hyperinsulinemic hypoglycemia. Radiolabeled exendin-4, a ligand of GLP-1R, has routinely been used in clinics to image insulinomas. However, its major drawback is the high kidney accumulation. Here, we show that the addition of an albumin-binding moiety (ABM) to radiolabeled exendin-4 results in a significant reduction of kidney uptake while retaining its high affinity and specificity to GLP-1R. The four tested peptides were shown to have high affinity to the GLP-1 receptor (IC50 of 3.7 ± 0.6 to 15.1 ± 0.8 nM). The radiolabeled derivatives were taken up into cells efficiently, internalizing between 39 ± 2 and 56 ± 2% after 2 h. Thus, the derivatives with ABM outperformed the reference peptide with its IC50 of 22.5 ± 2.9 nM and internalization of 41 ± 4%. Stability in human blood plasma was slightly enhanced by the addition of the albumin binder. In biodistribution studies, the radioligands exhibited an improved target-to-kidney ratio in comparison to the reference peptide of up to seven-fold. This was confirmed qualitatively in single-photon-emission computed tomography (SPECT)/CT imaging. This study demonstrated in vitro and in vivo that the addition of an ABM to radiolabeled exendin-4 strongly decreased kidney accumulation while retaining affinity to GLP-1R. Thus, exendin-4 derivatives with an albumin-binding moiety could present a viable class of diagnostic tracers for the detection of insulinomas and other GLP-1R-positive tissue in clinical application.