Promising Positron Emission Tomography Probe Research Articles

Quinazoline-2,4(1H,3H)-dione Scaffold for development of a novel PARP-targeting PET probe for tumor imaging.

Overexpression of Poly (ADP-ribose) polymerase (PARP) is associated with many diseases such as oncological diseases. Several PARP-targeting radiotracers have been developed to detect tumor in recent years. Two 18F labelled probes based on Olaparib and Rucaparib molecular scaffolds have been evaluated in clinical trials, but their slow hepatic clearance hinders their tumor imaging performance. Although a number of positron emission tomography (PET) probes with lower liver uptake have been designed, the tumor to background ratios remains to be low. Therefore, we designed a probe with low lipid-water partition coefficient to solve this problem. A pyridine-containing quinazoline-2,4(1H,3H)-dione PARP-targeting group was rationally designed and used to conjugate with the chelator 2,2',2'',2'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) to prepare the lead compound named as SMIC-2001 for radiolabeling. In vitro experiments, the lipid-water partition coefficient, stability, binding affinity, and cellular uptake of [68Ga]Ga-SMIC-2001 were determined. In vivo experiments, the U87MG xenograft models were used to evaluate its tumor imaging properties. [68Ga]Ga-SMIC-2001 showed a low Log D7.4 (-3.82 ± 0.06) and high affinity for PARP-1 (48.13 nM). In vivo study revealed that it exhibited a high tumor-to-background contrast in the U87MG xenograft models and mainly renal clearance. And the ratios of tumor to main organs were high except for the kidney (e.g. tumor to liver ratio reached 2.20 ± 0.51) at 60min p.i. In summary, pyridine-containing quinazoline-2,4(1H,3H)-dione is a novel PARP-targeting molecular scaffold for imaging probe development, and [68Ga]Ga-SMIC-2001 is a highly promising PET probe capable of imaging tumors with PARP overexpression.

PET Imaging of Acidic Tumor Environment With 89Zr-labeled pHLIP Probes.

Acidosis of the tumor microenvironment is a hallmark of tumor progression and has emerged as an essential biomarker for cancer diagnosis, prognosis, and evaluation of treatment response. A tool for quantitatively visualizing the acidic tumor environment could significantly advance our understanding of the behavior of aggressive tumors, improving patient management and outcomes. 89Zr-labeled pH-low insertion peptides (pHLIP) are a class of radiopharmaceutical imaging probes for the in vivo analysis of acidic tumor microenvironments via positron emission tomography (PET). Their unique structure allows them to sense and target acidic cancer cells. In contrast to traditional molecular imaging agents, pHLIP’s mechanism of action is pH-dependent and does not rely on the presence of tumor-specific molecular markers. In this study, one promising acidity-imaging PET probe ([89Zr]Zr-DFO-Cys-Var3) was identified as a candidate for clinical translation.

Development of High Brain-Penetrant and Reversible Monoacylglycerol Lipase PET Tracers for Neuroimaging.

Monoacylglycerol lipase (MAGL) is one of the key enzymes in the endocannabinoid system. Inhibition of MAGL has been proposed as an attractive approach for the treatment of various diseases. In this study, we designed and successfully synthesized two series of piperazinyl pyrrolidin-2-one derivatives as novel reversible MAGL inhibitors. (R)-[18F]13 was identified through the preliminary evaluation of two carbon-11-labeled racemic structures [11C]11 and [11C]16. In dynamic positron-emission tomography (PET) scans, (R)-[18F]13 showed a heterogeneous distribution and matched the MAGL expression pattern in the mouse brain. High brain uptake and brain-to-blood ratio were achieved by (R)-[18F]13 in comparison with previously reported reversible MAGL PET radiotracers. Target occupancy studies with a therapeutic MAGL inhibitor revealed a dose-dependent reduction of (R)-[18F]13 accumulation in the mouse brain. These findings indicate that (R)-[18F]13 ([18F]YH149) is a highly promising PET probe for visualizing MAGL non-invasively in vivo and holds great potential to support drug development.

Preclinical Evaluation of a Fluorine-18 Labeled Probe for the Detection of the Expression of PSMA Level in Cancer.

Prostate-specific membrane antigen (PSMA) is a prospect biomarker for the treatment of prostate cancer. Meanwhile, positron emission tomography (PET) is being developed as a significant imaging modality in cancer diagnosis. A new PET probe Glu-ureido-Lys-naphthylalanine-tranexamic acid-Gly(AMBF3)-triiodobenzoic acid (18F-GLNTGT) was radiosynthesized by a one-step 18F-labeled method. 18F-GLNTGT was obtained with a radioactivity yield (RCY) of 12.16 ± 6.4% and a good radiochemical purity (RCP > 96%). The cell uptakes of 18F-GLNTGT were determined to be 15.9 ± 0.43% ID and 9.47 ± 1.26% ID at 15 min in LNCaP cells and PC-3 cells, respectively. The cell internalization of 18F-GLNTGT was determined to be 12.89 ± 0.94% ID and 5.34 ± 0.15% ID at 15 min in LNCaP cells and PC-3 cells, respectively. It is suggested that the probe has good specificity targeting PSMA. From the results of 18F-GLNTGT binding affinity with PSMA, it has a higher affinity and a Ki value of 0.49 nM (95% confidence interval (CI): 0.35–0.67 nM). In PET imaging, 18F-GLNTGT showed the highest tumor uptake of 3.51 ± 0.15% ID/g at 45 min and the maximum tumor/muscle (T/Mmax) ratio of 3.68 ± 0.29 at 60 min post-injection (p.i.) in LNCaP tumors. The control probe 18F-AlF-NOTA-RGD2 presented the highest tumor uptake of 4.2 ± 0.54% ID/g at 7.5 min and the T/Mmax ratio of 2.72 ± 0.63 at 45 min p.i. in LNCaP tumors. The results showed that the probe has a higher tumor/muscle ratio compared with the control probe 18F-AlF-NOTA-RGD2. Although the probe 18F-GLNTGT has some limitations for CT signal detection both in cells and in vivo, it is still a promising PET probe for targeting PSMA membrane protein.

Development of a Novel Positron Emission Tomography (PET) Radiotracer Targeting Bromodomain and Extra-Terminal Domain (BET) Family Proteins.

Bromodomain and extra-terminal domain (BET) family proteins have become a hot research area because of their close relationship with a variety of human diseases. The non-invasive imaging technique, such as positron emission tomography (PET), provides a powerful tool to visualize and quantify the BET family proteins that accelerating the investigation of this domain. Herein, we describe the development of a promising PET probe, [11C]1, specifically targeting BET family proteins based on the potent BET inhibitor CF53. [11C]1 was successfully radio-synthesized with good yield and high purity after the optimization of radiolabeling conditions. The in vivo bio-activities evaluation of [11C]1 was performed using PET imaging in rodents. The results demonstrated that [11C]1 has favorable uptake in peripheral organs and moderate uptake in the brain. Further blocking studies indicated the high binding specificity and selectivity for BET proteins of this probe. Our findings suggest that [11C]1 is a promising BET PET probe for BET proteins as well as epigenetic imaging.

18F]-GE-179 positron emission tomography (PET) tracer for N-methyl-d-aspartate receptors: One-pot synthesis and preliminary micro-PET study in a rat model of MCAO

IntroductionThe objective of this study was to synthesize an N-methyl-d-aspartate receptor (NMDAR) radiotracer [18F]-GE-179 in one-pot and evaluate its in vivo binding for NMDAR activation after brain ischemia reperfusion injury. Methods[18F]-GE-179 was auto-synthesized using a quick one-pot method from a stable disulfide precursor and purified using semi-preparative high-performance liquid chromatography (HPLC) with ethanol/aqueous NaH2PO4 as the eluent. Dynamic micro-positron emission tomography (PET)/computed tomography (CT) study of [18F]-GE-179 was successfully performed using a rat model of middle cerebral artery occlusion (MCAO, induced by transient occlusion into the left MCA). A simplified reference tissue model method was used to calculate the [18F]-GE-179 non-displaceable binding potential (BPND) for intracranial NMDAR activation assessment. Immunofluorescence staining of NMDAR NR1 subunit in brain slices containing lesion was also performed. Results[18F]-GE-179 was successfully prepared in a yield of 23–30% in a formulation that could be injected directly after dilution. Localized radioactivity accumulation was observed in the animal model of MCAO. Significantly higher (p = 0.0003–0.0404) BPND relative to equivalent contralateral was found in the ipsilateral caudate putamen, Acb core/shell, cortex cingulate, amygdala, hypothalamus, and superior colliculus. Immunofluorescence staining showed elevated NMDAR expression in the affected hemisphere. Conclusions[18F]-GE-179 was synthesized using a one-pot process with a markedly improved yield. Preliminary in vivo micro-PET study visualized excessive NMDAR stimulation successfully in a rodent model of MCAO, which was consistent with results of in vitro immunofluorescence staining. The study demonstrates that [18F]-GE-179 is a promising PET probe for the detection of functional NMDAR alterations.

Discovery of 7-[18F]Fluorotryptophan as a Novel Positron Emission Tomography (PET) Probe for the Visualization of Tryptophan Metabolism in Vivo

Tryptophan and its metabolites are involved in different physiological and pathophysiological processes. Consequently, positron emission tomography (PET) tracers addressing tryptophan metabolic pathways should allow the detection of different pathologies like neurological disorders and cancer. Herein we report an efficient method for the preparation of fluorotryptophans labeled in different positions with 18F and their biological evaluation. 4-7-[18F]Fluorotryptophans ([18F]FTrps) were prepared according to a modified protocol of alcohol-enhanced Cu-mediated radiofluorination in 30-53% radiochemical yields. In vitro experiments demonstrated high cellular uptake of 4-7-[18F]FTrps in different tumor cell lines. 4, 5-, and 6-[18F]FTrps, although stable in vitro, suffered from rapid in vivo defluorination. In contrast, 7-[18F]FTrp demonstrated a high in vivo stability and enabled a clear delineation of serotonergic areas and melatonin-producing pineal gland in rat brains. Moreover 7-[18F]FTrp accumulated in different tumor xenografts in a chick embryo CAM model. Thus, 7-[18F]FTrp represents a highly promising PET probe for imaging of Trp metabolism.

Design, synthesis, in vitro characterization and preliminary imaging studies on fluorinated bile acid derivatives as PET tracers to study hepatic transporters

With the aim of identifying a fluorinated bile acid derivative that could be used as [18F]-labeled Positron Emission Tomography (PET) tracer for imaging the in vivo functioning of liver transporter proteins, and particularly of OATP1B1, three fluorinated bile acid triazole derivatives of cholic, deoxycholic and lithocholic acid (CATD, DCATD and LCATD 4a–c, respectively) were synthesized and labeled with tritium. In vitro transport properties were studied with cell-based assays to identify the best substrate for OATP1B1. In addition, the lead compound, LCATD (4c), was tested as a substrate of other liver uptake transporters OATP1B3, NTCP and efflux transporter BSEP to evaluate its specificity of liver transport. The results suggest that 4c is a good substrate of OATP1B1 and NTCP, whereas it is a poor substrate of OATP1B3. The efflux transporter BSEP also appears to be involved in the excretion of 4c from hepatocytes. The automated radiosynthesis of [18F]-4c was accomplished in a multi-GBq scale and a pilot imaging experiment in a wild type rat was performed after i.v. administration to assess the biodistribution and clearance of the tracer. PET imaging revealed that radioactivity was primarily located in the liver (tmax=75s) and cleared exclusively through the bile, thus allowing to image the hepatobiliary excretion of bile acids in the animal model. These findings suggest that [18F]-LCATD 4c is a promising PET probe for the evaluation of hepatic transporters OATP1B1, NTCP and BSEP activity with potential for studying drug-drug interactions and drug-induced toxicity involving these transporters.

PET imaging of insulin-like growth factor type 1 receptor expression with a 64Cu-labeled Affibody molecule.

The insulin-like growth factor 1 receptor (IGF-1R) serves as an attractive target for cancer molecular imaging and therapy. Previous single photon emission computerized tomography (SPECT) studies showed that the IGF-1R-targeting Affibody molecules (99m)Tc-ZIGF1R:4551-GGGC, [(99m)Tc(CO)3](+)-(HE)3-ZIGF1R:4551 and (111)In-DOTA-ZIGF1R:4551 can discriminate between high and low IGF-1R-expression tumors and have the potential for patient selection for IGF-1R-targeted therapy. Compared with SPECT, positron emission tomography (PET) may improve imaging of IGF-1R-expression, because of its high sensitivity, high spatial resolution, strong quantification ability. The aim of the present study was to develop the (64)Cu-labeled NOTA-conjugated Affibody molecule ZIGF-1R:4:40 as a PET probe for imaging of IGF-1R-positive tumor. An Affibody analogue (Ac-Cys-ZIGF-1R:4:40) binding to IGF-1R was site-specifically conjugated with NOTA and labeled with (64)Cu. Binding affinity and specificity of (64)Cu-NOTA-ZIGF-1R:4:40 to IGF-1R were evaluated using human glioblastoma U87MG cells. Small-animal PET, biodistribution, and metabolic stability studies were conducted on mice bearing U87MG xenografts after the injection of (64)Cu-NOTA-ZIGF-1R:4:40 with or without co-injection of unlabeled Affibody proteins. The radiosynthesis of (64)Cu-NOTA-ZIGF-1R:4:40 was completed successfully within 60min with a decay-corrected yield of 75%. (64)Cu-NOTA-ZIGF-1R:4:40 bound to IGF-1R with low nanomolar affinity (K D=28.55±3.95nM) in U87MG cells. (64)Cu-NOTA-ZIGF-1R:4:40 also displayed excellent in vitro and in vivo stability. In vivo biodistribution and PET studies demonstrated targeting of U87MG gliomas xenografts was IGF-1R specific. The tumor uptake was 5.08±1.07%ID/g, and the tumor to muscle ratio was 11.89±2.16 at 24h after injection. Small animal PET imaging studies revealed that (64)Cu-NOTA-ZIGF-1R:4:40 could clearly identify U87MG tumors with good contrast at 1-24h after injection. This study demonstrates that (64)Cu-NOTA-ZIGF-1R:4:40 is a promising PET probe for imaging IGF-1R positive tumor.

Radiofluorinated probe for PET imaging of fatty acid binding protein 4 in cancer

IntroductionCancer-associated adipocytes metabolically interact with adjacent cancer cells to promote tumor proliferation and metastasis. Fatty acid binding protein 4 (FABP4) participates in this interaction, and is gathering attention as a therapeutic and diagnostic target. Positron emission tomography (PET) is a useful diagnostic method that enables noninvasive in vivo quantitative imaging of biofunctional molecules with probes labeled with positron-emitting radioisotopes. Here a novel 18F labeled probe for PET FABP4 imaging developed through dedicated drug design from a radioiodinated probe we recently reported is evaluated in vitro and in vivo. MethodsWe designed the [18F]-labeled FTAP1 and FTAP3 probe, composed of a single or triple oxyethylene linker and a triazolopyrimidine scaffold derived from an FABP4 inhibitor. FABP4 binding affinities for chemically synthesized FTAP1 and FTAP3 were measured using FABP4 and 8-anilino-1-naphthalene sulfonic acid. Cell membrane permeability was measured using a commercially available plate assay system. After radiosynthesis, [18F]FTAP1 affinity and selectivity were evaluated using immobilized FABP3, FABP4, and FABP5. Cell uptake was investigated using differentiated adipocytes expressing FABP4 with inhibitor treatment. Following biodistribution studies in C6 glioblastoma-bearing mice, ex vivo autoradiography and immunohistochemistry were performed using thin sliced tumor sections. PET/CT imaging was then performed on C6 tumor bearing mice. ResultsFTAP1 showed high FABP4 affinity (Ki=68±8.9 nM) and adequate cell permeability. [18F]FTAP1 with ≥98% radiochemical purity was shown to selectively bind to FABP4 (16.3- and 9.3-fold higher than for FABP3 and FABP5, respectively). [18F]FTAP1 was taken up by FABP4 expressing cells, and this uptake could be blocked by an inhibitor, indicating very low non-specific cell binding. [18F]FTAP1 showed high tumor accumulation, which demonstrates its potential use for in vivo tumor PET imaging, and the intratumoral radioactivity distribution corresponded to the FABP4 expression profile. Conclusion[18F]FTAP1 is a promising PET probe to target FABP4.

Evaluation of 89Zr-Labeled Human Anti-CD147 Monoclonal Antibody as a Positron Emission Tomography Probe in a Mouse Model of Pancreatic Cancer

IntroductionPancreatic cancer is an aggressive cancer and its prognosis remains poor. Therefore, additional effective therapy is required to augment and/or complement current therapy. CD147, high expression in pancreatic cancer, is involved in the metastatic process and is considered a good candidate for targeted therapy. CD147-specfic imaging could be useful for selection of appropriate patients. Therefore, we evaluated the potential of a fully human anti-CD147 monoclonal antibody 059-053 as a new positron emission tomography (PET) probe for pancreatic cancer.MethodsCD147 expression was evaluated in four pancreatic cancer cell lines (MIA Paca-2, PANC-1, BxPC-3, and AsPC-1) and a mouse cell line A4 as a negative control. Cell binding, competitive inhibition and internalization assays were conducted with 125I-, 67Ga-, or 89Zr-labeled 059-053. In vivo biodistribution of 125I- or 89Zr-labeled 059-053 was conducted in mice bearing MIA Paca-2 and A4 tumors. PET imaging with [89Zr]059-053 was conducted in subcutaneous and orthotopic tumor mouse models.ResultsAmong four pancreatic cancer cell lines, MIA Paca-2 cells showed the highest expression of CD147, while A4 cells had no expression. Immunohistochemical staining showed that MIA Paca-2 xenografts also highly expressed CD147 in vivo. Radiolabeled 059-053 specifically bound to MIA Paca-2 cells with high affinity, but not to A4. [89Zr]059-053 uptake in MIA Paca-2 tumors increased with time from 11.0±1.3% injected dose per gram (ID/g) at day 1 to 16.9±3.2% ID/g at day 6, while [125I]059-053 uptake was relatively low and decreased with time, suggesting that 059-053 was internalized into tumor cells in vivo and 125I was released from the cells. PET with [89Zr]059-053 clearly visualized subcutaneous and orthotopic tumors.Conclusion[89Zr]059-053 is a promising PET probe for imaging CD147 expression in pancreatic cancer and has the potential to select appropriate patients with CD147-expressing tumors who could gain benefit from anti-CD147 therapy.

Development of 18F-Labeled Picolinamide Probes for PET Imaging of Malignant Melanoma

Melanoma is an aggressive skin cancer with worldwide increasing incidence. Development of positron emission tomography (PET) probes for early detection of melanoma is critical for improving the survival rate of melanoma patients. In this research, (18)F-picolinamide-based PET probes were prepared by direct radiofluorination of the bromopicolinamide precursors using no-carrier-added (18)F-fluoride. The resulting probes, (18)F-1, (18)F-2 and (18)F-3, were then evaluated in vivo by small animal PET imaging and biodistribution studies in C57BL/6 mice bearing B16F10 murine melanoma tumors. Noninvasive small animal PET studies demonstrated excellent tumor imaging contrasts for all probes, while (18)F-2 showed higher tumor to muscle ratios than (18)F-1 and (18)F-3. Furthermore, (18)F-2 demonstrated good in vivo stability as evidenced by the low bone uptake in biodistribution studies. Collectively, these findings suggest (18)F-2 as a highly promising PET probe for translation into clinical detection of melanoma.

Synthesis and in vivo evaluation of 18F-fluoroethyl GF120918 and XR9576 as positron emission tomography probes for assessing the function of drug efflux transporters

The purpose of this study was to synthesize two new positron emission tomography (PET) probes, N-(4-(2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl)phenyl)-9,10-dihydro-5-[¹⁸F]fluoroethoxy-9-oxo-4-acridine carboxamide ([¹⁸F]3) and quinoline-3-carboxylic acid [2-(4-{2-[7-(2-[¹⁸F]fluoroethoxy)-6-methoxy-3,4-dihydro-1H-isoquinolin-2-yl]ethyl}phenylcarbamoyl)-4,5-dimethoxyphenyl]amide ([¹⁸F]4), and to evaluate the potential of these PET probes for assessing the function of two major drug efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). [¹⁸F]3 and [¹⁸F]4 were synthesized by ¹⁸F-alkylation of each O-desmethyl precursor with [¹⁸F]2-fluoroethyl bromide for injection as PET probes. In vitro accumulation assay showed that treatment with P-gp/BCRP inhibitors (1 and 2) enhanced the intracellular accumulation capacity of P-gp- and BCRP-overexpressing MES-SA/Dx5 cells. In PET studies, the uptake (AUC(brain[0-)₆₀ (min])) of [¹⁸F]3 and [¹⁸F]4 in wild-type mice co-injected with 1 were approximately sevenfold higher than that in wild-type mice, and the uptake of [¹⁸F]3 and [¹⁸F]4 in P-gp/Bcrp knockout mice were eight- to ninefold higher than that in wild-type mice. The increased uptake of [¹⁸F]3 and [¹⁸F]4 was similar to that of parent compounds ([¹¹C]1 and [¹¹C]2) previously described, indicating that radioactivity levels in the brain after injection of [¹⁸F]3 and [¹⁸F]4 are related to the function of drug efflux transporters. Also, these results suggest that the structural difference between parent compounds ([¹¹C]1 and [¹¹C]2) and fluoroethyl analogs ([¹⁸F]3 and [¹⁸F]4) do not obviously affect the potency against drug efflux transporters. In metabolite analysis of mice, the unchanged form in the brain and plasma at 60 min after co-injection of [¹⁸F]4 plus 1 were higher (95% for brain; 81% for plasma) than that after co-injection of [¹⁸F]3 plus 1. [¹⁸F]4 is a promising PET probe to assess the function of drug efflux transporters.

Radiofluorinated Rhenium Cyclized α-MSH Analogues for PET Imaging of Melanocortin Receptor 1

In order to accomplish in vivo molecular imaging of melanoma biomarker melanocortin 1 receptor (MC1R), several α-melanocyte-stimulating hormone (α-MSH) analogues have been labeled with N-succinimidyl-4-¹⁸F-fluorobenzoate (¹⁸)F-SFB) and studied as positron emission tomography (PET) probes in our recent studies. To further pursue a radiofluorinated α-MSH peptide with high clinical translation potential, we utilized 4-nitrophenyl 2-¹⁸F-fluoropropionate (¹⁸F-NFP) to radiofluorinate the transition metal rhenium cyclized α-MSH metallopeptides for PET imaging of MC1R positive malignant melanoma. Metallopeptides Ac-d,Lys-ReCCMSH(Arg¹¹) (two isomers, namely RMSH-1 and RMSH-2) were synthesized using conventional solid phase peptide synthesis chemistry and rhenium cyclization reaction. The two isomers were then conjugated with ¹⁹F-NFP or ¹⁸F-NFP. The resulting cold or radiofluorinated metallopeptides, (¹⁸/¹⁹)F-FP-RMSH-1 and (¹⁸/¹⁹)F-FP-RMSH-2, were further evaluated for their in vitro receptor binding affinities, in vivo biodistribution, and small-animal PET imaging properties. The binding affinities of ¹⁹F-FP-RMSH-1 and ¹⁹F-FP-RMSH-2 were determined to be within low nanomolar range. In vivo studies revealed that both F-labeled metallopeptides possessed good tumor uptake in the B16F10 murine model with high MC1R expression, while possessing much lower uptake in A375M human melanoma xenografts. Moreover, ¹⁸F-FP-RMSH-1 displayed more favorable in vivo performance in terms of higher tumor uptake and much lower accumulation in the kidney and liver, when compared to that of ¹⁸F-FP-RMSH-2 at 2 h postinjection (p.i.). ¹⁸F-FP-RMSH-1 also displayed lower liver and lung uptake when compared with that of the same peptide labeled with ¹⁸F-SFB (named as ¹⁸F-FB-RMSH-1). Small animal PET imaging of ¹⁸F-FP-RMSH-1 in mice bearing B16F10 tumors at 1 and 2 h showed good tumor imaging quality. As expected, much lower tumor uptake and poorer tumor/normal organ contrast were observed for A375M model compared to those of the B16F10 model. ¹⁸F-FP-RMSH-1 also exhibited higher tumor uptake and better tumor retention when compared with ¹⁸F-FB-RMSH-1. ¹⁸F-FP-RMSH-1 demonstrates significant advantages over ¹⁸F-FB-RMSH-1 and ¹⁸F-FP-RMSH-2. It is a promising PET probe for imaging MC1R positive melanoma and MC1R expression in vivo.