68Ga-labeled oligonucleotides for in vivo imaging with PET.

The biologic evaluation in living rats of (68)Ga-labeled oligonucleotides as imaging agents for PET is reported. METHODS: (68)Ga, a positron-emitting radionuclide (half-life, 68 min), along with a macrocyclic chelating agent, 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), was used for labeling of antisense oligonucleotides targeting activated human K-ras oncogene. The biologic properties of 3 different forms of the oligonucleotides-that is, 2'-deoxyphosphodiester (PO), 2'-deoxyphosphorothioate (PS), and 2'-O-methyl phosphodiester (OMe)-were studied first. The biodistribution and biokinetics were evaluated in vivo in athymic rats, each bearing a tumor of A549 cells, containing K-ras point mutation in codon 12, and a tumor of BxPC-3 cells, containing wild-type K-ras. Dynamic PET imaging lasting up to 2 h was performed immediately after intravenous injection of (68)Ga-oligonucleotide. Blank studies were performed using (68)GaCl(3) or (68)Ga-DOTA alone without oligonucleotide. The (68)Ga-antisense oligonucleotide uptake in tumors was also compared with the (18)F-FDG and (68)Ga-sense oligonucleotide uptakes. In addition, oligonucleotide binding to human plasma proteins and to human albumin was examined by means of ultrafiltration. RESULTS: The oligonucleotides can be stably labeled with (68)Ga and DOTA chelate. Intravenously injected (68)Ga-oligonucleotides of 17-mer length revealed high-quality PET images, allowing quantification of the biokinetics in major organs and in tumors. The biodistribution and biokinetics of intravenously administered (68)Ga-oligonucleotide varied considerably with the nature of the oligonucleotide backbone. CONCLUSION: We conclude that (68)Ga labeling of oligonucleotides is a convenient approach for in vivo imaging and quantification of oligonucleotide biokinetics in living animals with PET.     

Evaluation of 68Ga-labeled tracers for PET imaging of myocardial perfusion in pigs

PurposeWe evaluated four potential gallium-68 (⁶⁸Ga)-labeled tracers for positron emission tomography (PET) imaging of myocardial perfusion in comparison with oxygen-15-labeled water ([¹⁵O]water) in healthy pigs. Four hexadentate salicylaldimine ligands derived from bis(3-aminopropyl)ethylenediamine (BAPEN) that showed promise in previous rat experiments were selected for this study.MethodsFollowing an evaluation of myocardial blood flow with [¹⁵O]water PET, the pigs (total n=14) underwent a dynamic 90-min PET study with one of four ⁶⁸Ga-labeled BAPEN derivatives (n=3–5 per tracer) either at rest or under adenosine stress. Serial arterial blood samples were collected during the imaging for the measurements of total radioactivity, radiometabolites, plasma protein binding and blood-to-plasma ratio for the ⁶⁸Ga chelates. Time–activity curves of the left ventricular blood pool and myocardium were derived from PET images, and metabolite-corrected arterial input function was used for kinetic modeling. Also, ex vivo biodistribution of ⁶⁸Ga radioactivity was analyzed.ResultsAll four ⁶⁸Ga tracers showed undesirably slow myocardial accumulation over time, but their in vivo stability, clearance from blood and the kinetics of the myocardium uptake varied. [⁶⁸Ga][Ga-(sal)₂BAPDMEN]¹⁺ showed the highest myocardial uptake in PET images and tissue samples (myocardium-to-blood ratio 7.63±1.89, myocardium-to-lung ratio 3.03±0.33 and myocardium-to-liver ratio 1.80±0.82). However, there was no correlation between the myocardial perfusion measured with [¹⁵O]water and the net uptake rates or K₁ values of the ⁶⁸Ga chelates.ConclusionOur results revealed that myocardial accumulation of the ⁶⁸Ga chelates proposed for myocardial perfusion imaging with PET was slow and not determined by myocardial perfusion in a large animal model. These findings suggest that the studied tracers are not suitable for clinical imaging of myocardial perfusion.     

Synthesis and preclinical evaluation of 68Ga-labeled collagelin analogs for imaging and quantification of fibrosis

ObjectivesFibrosis affecting functionality of vital organs such as liver, lung, heart, and kidney, is involved in many chronic diseases. Positron emission tomography (PET) would not only provide precise localization and extent of the affected tissue but also allow the accurate quantification of the fibrotic process for the subsequent prognosis.MethodsA cyclic peptide c[CPGRVMHGLHLGDDEGPC] conjugated either to 2-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)acetic acid (NOTA(tBu)₂) or 4-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazacyclononan-1-yl)-5-(tert-butoxy)-5-oxopentanoic acid (NODAGA(tBu)₃) via polyethylene glycol link (PEG₂) was synthesized and labeled with ⁶⁸Ga. Non-specific organ distribution, blood clearance, and excretion were investigated ex vivo in healthy rats. The binding specificity of the radioligands was assessed in vitro using autoradiography on cryosections of dog fibrotic heart tissue.ResultsThe yield of NOTA-PEG₂-c[CPGRVMHGLHLGDDEGPC] and NODAGA-PEG₂-c[CPGRVMHGLHLGDDEGPC] was 56% and 41%, respectively. Non-decay-corrected radiochemical yield was 80 ± 5% with radiochemical purity of 95 ± 4%. Pharmacokinetic studies in healthy male Sprague–Dawley rats showed fast blood clearance and renal excretion. Lower uptake in liver, spleen, and kidney was found for [[⁶⁸Ga]Ga-NOTA]^+ 1-PEG₂-c[CPGRVMHGLHLGDDEGPC] as compared to [[⁶⁸Ga]Ga-NODAGA]⁰-PEG₂-c[CPGRVMHGLHLGDDEGPC]. Histologic evaluation of the left ventricle (LV) myocardium from a dog with severe mitral regurgitation (MR), revealed mild to moderate perivascular and subendocardial, and mild diffuse interstitial fibrosis. The tracer binding to the cryosections of the tissue was specific with the equilibrium Kd of 2.3 ± 0.8 μM and 2.1 ± 0.9 μM, respectively for [⁶⁸Ga]Ga-NO2A-Col and [⁶⁸Ga]Ga-NODAGA-Col.ConclusionsTwo novel peptide based agents for the imaging of fibrosis by PET were developed. Moderation of the biodistribution could be achieved by variation of the charge on the complex moiety of the agents. The combination of the fast clearance from non-target organs as well as organs of interest such as lung, heart, and liver and binding specificity to the target tissue suggests the potential of the analogs for the imaging of fibrosis.     

含末端半胱氨酸人表皮生长因子受体2亲和体的68Ga标记和显像

目的 在含末端半胱氨酸的人表皮生长因子受体2(HER2)亲和体的羧基末端甘氨酸-甘氨酸-甘氨酸-半胱氨酸(GGGC)的半胱氨酸残基标记68Ga,探讨该标记物用于乳腺癌模型小鼠的HER2显像.方法 通过基因工程制备含末端半胱氨酸的HER2亲和体,应用1,4,7,10-四氮环十二烷-1,4,7-三乙酸-10-马来酰亚胺乙基乙酰胺(MMA-DOTA)在半胱氨酸巯基上耦联DOTA.新洗脱的68Ga液和DOTA-HER2亲和体在90℃下反应,完成标记.标记产物经过C18柱纯化、乙醇洗脱后用于细胞结合分析和microPET显像.细胞结合分析采用表达HER2的乳腺癌细胞MBA-MD-361,动物模型采用该乳腺癌细胞接种的BALB/c裸鼠模型.于4只荷乳腺癌小鼠尾静脉注射3.7 MBq68Ga标记亲和体,分别于注射后20、60 min进行micmPET显像,随即将动物处死,取出心脏、肿瘤、肌肉、骨骼测质量,并用γ计数仪测量放射性,计算肿瘤与肝脏、肌肉、骨骼、心脏的放射性比值.结果 HER2亲和体纯度在95％以上,68Ga标记亲和体的放化纯为97％.应用HER2阳性细胞测出亲和体的亲和力KD值为1.5 nmoL/L.MicroPET显像示,68Ga标记亲和体进入荷瘤鼠血液循环系统后,很快结合于HER2阳性肿瘤,主要从泌尿系统清除.注射68Ga标记亲和体后,肿瘤与肝脏、肌肉、骨骼、心脏的放射性比值分别为17.4±1.0、35.1±10.9、20.7±6.2和20.9±4.0.结论 68Ga亲和体标记成功,适用于HER2阳性肿瘤的PET显像.     

P-gp功能的PET显像剂的研究进展

肿瘤对化疗药物产生耐药性是肿瘤治疗失败的主要原因,引起肿瘤对化疗药物耐药的重要机制是ATP结合盒（ABC）转运蛋白的过度表达.P-糖蛋白（P-gp）是ABC转运蛋白超家族中研究最广、最深入的一类转运蛋白,目前检测P-gp主要依赖于组织活检或术后病理组织在体外进行定性、定量分析,这些检测方法受取材技术、肿瘤标本差异等因素的限制,因此,寻找一种无创性、可以动态检测肿瘤多药耐药的方法就显得尤为重要.目前许多^18F、^11C标记的P-gp底物及抑制剂的正电子显像剂已经进行基础研究,部分进入了临床试验阶段.该文对P-gp的PET显像剂现状进行综述.     

肿瘤血管生成的PET检测

肿瘤血管生成作为肿瘤的主要特征,在肿瘤生长和转移中起着重要作用,为肿瘤治疗提供了新策略.通过标记血管生成相关的受体、多肽、激酶或细胞外基质蛋白,形成高亲和力的分子探针,与肿瘤血管生成过程中产生的特异性靶分子结合,从而显示包括整合素、VEGF/VEGFR、基质金属蛋白酶(MMPs)等与血管生成关系密切的特征性血管生成因子,可从分子水平对肿瘤新生血管及血管靶向治疗疗效进行无创性检测.笔者就肿瘤血管生成PET影像学检测研究进展及未来发展作一综述.     

Infrared-based module for the synthesis of 68Ga-labeled radiotracers

INTRODUCTION: Generator-produced positron emission tomography tracers have gained much attention recently due to favorable imaging characteristics, accessibility and affordability. The focus of this study was to design and validate a semiautomated module for 68Ga-labeled chemistry utilizing infrared-based heating for rapid control of thermal cycle. METHODS: A prototype module was built and installed in our laboratory. DOTA (1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetra-acetic acid) was manually labeled (10-1000 nmol) with 68Ga to optimize synthesis conditions. For automation, 250 nmol of DOTA was labeled with 68Ga with reaction times of 5 min (n=5), 10 min (n=5) and 20 min (n=6). A dose calibrator and radio-thin-layer chromatography were used to access the product yield and quality of both manual and automated syntheses. RESULTS: A semiautomated 68Ga synthesis module was developed. The system showed that software control could be used to drive a multistep radiochemical synthesis and to produce 68Ga-DOTA with >95% radiochemical purity, similar to that in manual synthesis. The device also showed that for a short reaction time of 5 min, decay-corrected radioactive yields of >70% could be achieved. The total synthesis was as short as 22 min, including 6-8 min for HCl evaporation. The temperature and pressure profiles of the process were consistent. CONCLUSION: We demonstrated the use of a commercially available 68Ga/68Ge generator with a semiautomated module to successfully label the bifunctional chelator DOTA with 68Ga. Further investigation with different 68Ga-labeled bioconjugates is warranted to demonstrate the usefulness of the module as a tool for tracer development and imaging research.     

Synthesis of novel 68Ga-labeled amino acid derivatives for positron emission tomography of cancer cells

ObjectivesWe developed amino acid derivatives of 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A) and 1,4,7,10-tetraazacyclododecane-1,4,7,-triacetic acid (DO3A) that can be labeled with ⁶⁸Ga, and we investigated their basic biological properties.Materials and methodsAlanine derivatives of DO2A and DO3A were synthesized by regiospecific nucleophilic attack of DO2tBu and DO3tBu on the β-position of Boc-l-serine-β-lactone, followed by acid hydrolysis. Also, homoalanine derivatives were synthesized by reacting with the protected bromo derivative of homoalanine, which was synthesized from N-Cbz-l-homoserine lactone. Further catalytic reduction and acid cleavage of protected groups resulted in the required products. All derivatives were labeled with ⁶⁸Ga. Cell uptake assays were carried out in Hep3B (human hepatoma) and U87MG (human glioma) cell lines at 37°C. Positron emission tomography (PET) imaging studies were performed using balb/c mice xenografted with CT-26 (mouse colon cancer).ResultsAll compounds were labeled with >97% efficiency. According to in vitro studies, the labeled amino acid derivatives showed significantly greater uptakes than the control (⁶⁸Ga 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) in cancer cells. Small animal PET images for labeled compounds showed high tumor uptake, as well as kidney and bladder uptakes, at 30 min postinjection. ⁶⁸Ga-DO3A-homoalanine showed the highest standardized uptake value ratio (3.9±0.3), followed by ⁶⁸Ga-DO2A-alanine (3.1±0.2), ⁶⁸Ga-DO3A-alanine (2.8±0.2) and ⁶⁸Ga-DO2A-homoalanine (2.3±0.2).ConclusionThese derivatives were found to have high labeling efficiencies, high stabilities, high tumor cell uptakes, high tumor/nontumor xenograft uptakes and low nonspecific uptake in normal organs, except for the kidneys. However, the uptake mechanism of these derivatives remains unclear, and uptake via specific amino acid transporters needs to be demonstrated.     

Preparation of 66Ga- and 68Ga-labeled Ga(III)-deferoxamine-folate as potential folate-receptor-targeted PET radiopharmaceuticals

A folate-receptor-targeting radiopharmaceutical, Ga(III)-deferoxamine-folate (Ga-DF-Folate), was radiolabeled with two positron-emitting isotopes of gallium, cyclotron-produced (66)Ga (9.5 hour half-life) and generator-produced (68)Ga (68 minute half-life). The [(66)Ga]Ga-DF-Folate was administered to athymic mice with folate-receptor-positive human KB cell tumor xenografts to demonstrate that microPET mouse tumor imaging is feasible with (66)Ga, despite the relatively high positron energy of this radionuclide. Using the athymic mouse KB tumor xenograft model, dual-isotope autoradiography was also performed following i.v. co-administration of [(18)F]-FDG, a marker of regional metabolic activity, and folate-receptor-targeted [(111)In]In-DTPA-Folate. The autoradiographic images of 1 mm tumor sections demonstrate the gross heterogeneity of the KB cell tumor xenograft, as well as subtle disparity in the regional accumulation of the two radiotracers.     

Development and preclinical evaluation of new 124I-folate conjugates for PET imaging of folate receptor-positive tumors

In an attempt to develop new folate radiotracers with favorable biochemical properties for detecting folate receptor-positive cancers, we have synthesized [¹²⁴I]-SIB- and [¹²⁴I]-SIP-folate conjugates using a straightforward and two-step simple reactions. Radiochemical yields for [¹²⁴I]-SIB- and [¹²⁴I]-SIP-folate conjugates were greater than 90 and 60% respectively, with total synthesis time of 30–40 min. Radiochemical purities were always greater than 98% without HPLC purification. These synthetic approaches hold considerable promise as rapid and simple method for ¹²⁴I-folate conjugate preparation with high radiochemical yield in short synthesis time. In vitro tests on KB cell line showed that the significant amounts of the radioconjugates were associated with cell fractions. In vivo characterization in normal Balb/c mice revealed rapid blood clearance of these radioconjugates and favorable biodistribution profile for [¹²⁴I]-SIP-folate conjugate over [¹²⁴I]-SIB-folate conjugate. Biodistribution studies of [¹²⁴I]-SIP-folate conjugate in nude mice bearing human KB cell line xenografts, demonstrated significant tumor uptake. The uptake in the tumors was blocked by excess injection of folic acid, suggesting a receptor-mediated process. These results demonstrate that [¹²⁴I]-SIP-folate conjugate may be useful as a molecular probe for detecting and staging of folate receptor-positive cancers, such as ovarian cancer and their metastasis as well as monitoring tumor response to treatment.     

Assessment of cardiac sympathetic neuronal function using PET imaging

The autonomic nervous system plays a key role for regulation of cardiac performance, and the importance of alterations of innervation in the pathophysiology of various heart diseases has been increasingly emphasized. Nuclear imaging techniques have been established that allow for global and regional investigation of the myocardial nervous system. The guanethidine analog iodine 123 metaiodobenzylguanidine (MIBG) has been introduced for scintigraphic mapping of presynaptic sympathetic innervation and is available today for imaging on a broad clinical basis. Not much later than MIBG, positron emission tomography (PET) has also been established for characterizing the cardiac autonomic nervous system. Although PET is methodologically demanding and less widely available, it provides substantial advantages. High spatial and temporal resolution along with routinely available attenuation correction allows for detailed definition of tracer kinetics and makes noninvasive absolute quantification a reality. Furthermore, a series of different radiolabeled catecholamines, catecholamine analogs, and receptor ligands are available. Those are often more physiologic than MIBG and well understood with regard to their tracer physiologic properties. PET imaging of sympathetic neuronal function has been successfully applied to gain mechanistic insights into myocardial biology and pathology. Available tracers allow dissection of processes of presynaptic and postsynaptic innervation contributing to cardiovascular disease. This review summarizes characteristics of currently available PET tracers for cardiac neuroimaging along with the major findings derived from their application in health and disease.     

Development of 68Ga-labelled DTPA galactosyl human serum albumin for liver function imaging

Purpose

The hepatic asialoglycoprotein receptor is responsible for degradation of desialylated glycoproteins through receptor-mediated endocytosis. It has been shown that imaging of the receptor density using [^99mTc]diethylenetriamine pentaacetic acid (DTPA) galactosyl human serum albumin ([^99mTc]GSA) allows non-invasive determination of functional hepatocellular mass. Here we present the synthesis and evaluation of [⁶⁸Ga]GSA for the potential use with positron emission tomography (PET).

Methods

Labelling of GSA with ⁶⁸Ga was carried out using a fractionated elution protocol. For quality control thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and size exclusion chromatography (SEC) techniques were evaluated. Stability of [⁶⁸Ga]GSA was studied in phosphate-buffered saline (PBS) and human serum. For in vivo evaluation [⁶⁸Ga]GSA distribution in Lewis rats was compared with [^99mTc]GSA by using a dual isotope protocol. PET and planar imaging studies were performed using the same scaled molar dose of [⁶⁸Ga]GSA and [^99mTc]GSA. Time-activity curves (TAC) for heart and liver were generated and corresponding parameters calculated (t50, t90).

Results

[⁶⁸Ga]GSA can be produced with high radiochemical purity. The best TLC methods for determining potential free ⁶⁸Ga include 0.1 M sodium citrate as eluent. None of the TLC methods tested were able to determine potential colloids. This can be achieved by SEC. HPLC confirmed high radiochemical purity (>98 %). Stability after 120 min incubation at 37 °C was high in PBS (>95 % intact tracer) and low in human serum (～27 % intact tracer). Biodistribution studies simultaneously injecting both tracers showed comparable liver uptake, whereas activity concentration in blood was higher for [⁶⁸Ga]GSA compared to [^99mTc]GSA. The [^99mTc]GSA TACs exhibited a small degree of hepatic metabolism compared to the [⁶⁸Ga]GSA curves. The mean [⁶⁸Ga]GSA t90 was higher than the mean t90 for [^99mTc]GSA. The mean [⁶⁸Ga]GSA t50 was not significantly different from the mean t50 for [^99mTc]GSA.

Conclusion

This study provides a promising new ⁶⁸Ga-labelled compound based on a commercially used kit for imaging the functional hepatocellular mass.     

(68)Ga-labeled DOTA-peptides and (68)Ga-labeled radiopharmaceuticals for positron emission tomography: current status of research, clinical applications, and future perspectives

In this review we give an overview of current knowledge of (68)Ga-labeled pharmaceuticals, with focus on imaging receptor-mediated processes. A major advantage of a (68)Ge/(68)Ga generator is its continuous source of (68)Ga, independently from an on-site cyclotron. The increase in knowledge of purification and concentration of the eluate and the complex ligand chemistry has led to (68)Ga-labeled pharmaceuticals with major clinical impact. (68)Ga-labeled pharmaceuticals have the potential to cover all today's clinical options with (99m)Tc, with the concordant higher resolution of positron emission tomography (PET) in comparison with single photon emission computed tomography. (68)Ga-labeled analogs of octreotide, such as DOTATOC, DOTANOC, and DOTA-TATE, are in clinical application in nuclear medicine, and these analogs are now the most frequently applied of all (68)Ga-labeled pharmaceuticals. All the above-mentioned items in favor of successful application of (68)Ga-labeled radiopharmaceuticals for imaging in patients are strong arguments for the development of a (68)Ge/(68)Ga generator with Marketing Authorization and thus to provide pharmaceutical grade eluate. Moreover, now not one United States Food and Drug Administration-approved or European Medicines Agency-approved (68)Ga-radiopharmaceutical is available. As soon as these are achieved, a whole new radiopharmacy providing PET radiopharmaceuticals might develop.     

Procedure guidelines for PET/CT tumour imaging with 68Ga-DOTA-conjugated peptides: 68Ga-DOTA-TOC, 68Ga-DOTA-NOC, 68Ga-DOTA-TATE

The aim of these guidelines is to assist nuclear medicine physicians in recommending, performing, reporting and interpreting the results of somatostatin (SST) receptor PET/CT imaging using ⁶⁸Ga-DOTA-conjugated peptides, analogues of octreotide, that bind to SST receptors. This imaging modality should not be regarded as the only approach to visualizing tumours expressing SST receptors or as excluding other imaging modalities useful for obtaining comparable results. The corresponding guidelines of ¹¹¹In-pentetreotide scintigraphy imaging have been considered and partially integrated with this text. The same has been done with the relevant and recent literature in this field and the final result has been discussed by distinguished experts.