Labeling and distribution of linear peptides identified using in vivo phage display selection for tumors

To develop targeting molecules to be used for vascular targeting of short half-lived -emitters for radioimmunotherapy, linear peptide phage display libraries were selected in vivo for binding to IC-12 rat tracheal tumors growing in severe combined immune deficient mice. After three rounds of selection, 15 phage clones were analyzed for DNA sequence, and the deduced translation products of cDNA inserts were compared. Three consensus sequences were chosen from three separate experimental selection series and peptides of these sequences with added -gly-gly-tyr were obtained. Peptides were radiolabeled on tyrosine with ¹²⁵I and the biodistribution in tumor-bearing mice was determined. The radioiodinated peptides were stable in vitro and when injected in tumor-bearing mice 3.0 %ID/g accumulated in the tumor; however, much of the ¹²⁵I was found in the gastrointestinal tract and thyroid, indicative of dehalogenation of the labeled peptide. Radiolabeling peptide 2 with N-succinimidyl-3-¹²⁵I-iodobenzoate resulted in faster excretion, which in turn resulted in lower levels in tumor and other organs, especially thyroid and gastrointestinal tract. Peptide 2 was derivatized with the bifunctional isothiocyanates of cyclohexyl-B diethylenetriaminepentaacetic acid (DTPA) or CHX-A″ DTPA by direct conjugation or with a hydroxylamine derivative of 1B4M-DTPA (2-(p-[O-(carboxamylmethyl)hydroxylamine]benzyl)-6-methyl-diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid ) coupled at the N-terminus. The primary molecular species in the conjugated products were shown by mass spectrometry to have one DTPA per peptide. Peptide chelate conjugates were radiolabeled with ²¹³Bi and the products tested for biodistribution in tumor-bearing mice. The data show that chelation of ²¹³Bi to peptides was accomplished by both the direct method of DTPA attachment and by the method using the linker at the N-terminus. Only small amounts of peptide accumulated at tumor sites. We conclude that phage display is a powerful tool to select peptides with restricted binding specificity; however, the peptides isolated to date do not bind with high retention to tumor sites in vivo.     

miR-424*在X射线照射后的A549细胞体内、外及非小细胞肺癌组织和血清中的表达

目的 研究2、4 Gy X射线照射后人肺腺癌细胞miR-424^*体内、外表达以及非小细胞肺癌患者肺组织及血清中miR-424^*的表达变化及其意义。方法 2、4 Gy X射线分别照射体外培养的A549细胞,以实时定量PCR（RT-qPCR）法检测A549细胞中miR-424^*表达水平;用照射后的A549细胞制备裸鼠肺转移动物模型,检测裸鼠肺组织及血清中miR-424^*的表达水平;收集肺癌患者肺组织及血清样本,检测miR-424^*表达水平。结果 2、4 Gy X射线照射后1、2、12、24及48 h,miR-424^*表达均显著升高（2 Gy：t=-45.886~-6.709,P<0.05;4 Gy：t=-29.087~-7.833,P<0.05）;0、2、4 Gy照射后,miR-424^*在裸鼠肺及血清中表达水平分别为空白对照组的9.72、8.58及4.7与11.93、9.22及8.99倍（t=-13.243~-3.052,P<0.05）。6/11例（54.5%）患者肺癌组织中高表达miR-424^*,腺癌、鳞癌病理类型间检出率差异无统计学意义（P>0.05）;43/84例（51.20%）肺癌患者与健康志愿者相比,血清miR-424^*表达升高 1.97~17.71倍,其中腺癌患者血清检出率为39.1%（18/46）,鳞癌患者血清检出率为65.8%（25/38）,两种病理类型检出率差异有统计学意义（t=5.919,P<0.05）;此外,84例肺癌患者中,miR-424^*[JP3]在未接受放疗的肺癌患者血清中的阳性检出率为41.5%（22/53）,显著低于接受放疗的肺癌患者血清的阳性检出率67.7%（21/31）[JP]（t=5.387,P<0.05）。结论 2、4 Gy X射线照射可增加A549细胞miRNA-424^*的体内、外表达水平,可能与增强A549细胞体内、外侵袭转移能力有关。肺癌患者中50%以上的肺癌组织及肺癌患者血清中miR-424^*表达水平显著升高,可能与肺癌的病理类型及放疗相关。     

Endocavitary in vivo Dosimetry for IMRT Treatments of Gynecologic Tumors

The accuracy and reproducibility of endometrial carcinoma treatment with intensity-modulated radiotherapy (IMRT) was assessed by means of in vivo dosimetry. Six patients who had previously undergone radical hysterectomy for endometrial carcinoma were treated with IMRT using a vaginal applicator with radio-opaque fiducial markers. An ion-chamber inserted into the applicator supplied an endocavitary in vivo dosimetry for quality assurance purposes. The ratio R = D/D_TPS between the in vivo measured dose D and the predicted dose by the treatment planning system D_TPS was determined for every fraction of the treatment. Results showed that 90% and 100% of the ratios resulted equal to 1 within 5% and 10%, respectively. The mean value of the ratios distribution for the 6 patients was R = 0.995 and the SD = 0.034. The ratio R* between the measured and predicted total doses for each patient was near to 1, within 2%. The dosimetric results suggest that the use of a vaginal applicator in an image-guided approach could make the interfractions target position stable and reproducible, allowing a safe use of the IMRT technique in the treatment of postoperative vaginal vault. In vivo dosimetry may supply useful information about the discrimination of random vs. systematic errors. The workload is minimum and this in vivo dosimetry can be applied also in the clinical routine.     

Synthesis and in vivo evaluation of [C]methyl-Ro 64-6198 as an ORL1 receptor imaging agent

(1S,3aS)-8-(2,3,3a,4,5,6-Hexahydro-1H-phenalen-1-yl)-3-N-[¹¹C]methyl-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one ([¹¹C]methyl-Ro 64-6198), a N-methylated analog of Ro 64-6198, was synthesized and evaluated as a potential radiopharmaceutical for investigating brain nociceptin/orphanin FQ receptors (ORL1 receptors) by positron emission tomography. A racemate of methyl-Ro 64-6198, Ro 66-7931, showed a high affinity and selectivity for the ORL1 receptor in vitro. An in vivo distribution study in mice demonstrated moderate brain uptake, however, only slight difference was observed among brain regions. Furthermore, pretreating with nociceptin or Ro 66-7931 did not affect the accumulation. Therefore, despite its high affinity, [¹¹C]methyl-Ro 64-6198 does not appear to be a suitable tracer for in vivo ORL1 receptor imaging studies.     

Non-invasive visualisation of the development of peritoneal carcinomatosis and tumour regression after <Superscript>213</Superscript>Bi-radioimmunotherapy using bioluminescence imaging

Purpose Non-invasive imaging of tumour development remains a challenge, especially for tumours in the intraperitoneal cavity. Therefore, the aim of this study was the visualisation of both the development of peritoneal carcinomatosis and tumour regression after radioimmunotherapy with tumour-specific ²¹³Bi-Immunoconjugates, via in vivo bioluminescence imaging of firefly luciferase-transfected cells. Methods Human diffuse-type gastric cancer cells expressing mutant d9-E-cadherin were stably transfected with firefly luciferase (HSC45-M2-luc). For bioluminescence imaging, nude mice were inoculated intraperitoneally with 1 × 10⁷ HSC45-M2-luc cells. On days 4 and 8 after tumour cell inoculation, imaging was performed following D-luciferin injection using a cooled CCD camera with an image intensifier unit. For therapy, mice were injected with 2.7 MBq ²¹³Bi-d9MAb targeting d9-E-cadherin on day 8 after tumour cell inoculation. Bioluminescence images were taken every 4 days to monitor tumour development. Results After i.p. inoculation of HSC45-M2-luc cells into nude mice, development as well as localisation of peritoneal carcinomatosis could be visualised using bioluminescence imaging. Following ²¹³Bi-d9MAb therapy on day 8 after intraperitoneal inoculation of HSC45-M2-luc cells, small tumour nodules were totally eliminated and larger nodules showed a clear reduction in size on day 12 after tumour cell inoculation. Subsequently a recurrence of tumour mass was observed, starting from the remaining tumour spots. By measuring the mean grey level intensity, tumour development over time could be demonstrated. Conclusion Non-invasive bioluminescence imaging permits visualisation of the development of peritoneal carcinomatosis, localisation of tumour in the intraperitoneal cavity and evaluation of therapeutic success after ²¹³Bi-d9MAb treatment.     

基于EPID在体剂量验证在肺癌和食管癌动态调强放疗中的应用研究

目的对肺癌和食管癌患者在放疗过程中行基于电子射野影像装置(EPID)的在体剂量验证,探讨影响在体剂量验证结果准确性的因素,并推荐应用在体剂量验证的流程及规范。方法单纯随机抽样法选取2022年5月至2022年8月在金华市中心医院放疗科行食管癌和肺癌放疗的患者32例(其中,肺癌14例,食管癌18例),在uRT-TPOIS计划系统上制作动态调强放疗(dIMRT)及EPID在体剂量验证(In vivo EPID)计划,使用uRT-linac 506c直线加速器进行治疗。治疗过程中行在体剂量验证,其中,肺癌病例行In vivo EPID的分次共238次,执行图像引导放疗(IGRT)共80次,食管癌病例行In vivo EPID的分次共414次,执行IGRT共105次。设置阈值并获取每个射野的2Dγ通过率,分析低于阈值的分次射野,并结合在线CT影像及三维重建剂量结果,进一步分析影响γ通过率下降的主要因素。结果肺癌、食管癌3 mm/5%γ通过率均值分别为95.1%±5.7%、96.5%±4.5%;3 mm/3%γ通过率均值为91.5%±8.4%、92.2%±4.9%;2 mm/2%γ通过率均值分别为:79.1%±14.1%、83.7.2%±8.2%,病种之间通过率差异无统计学意义(P>0.05)。靠近0°/180°射野组(A组)的γ通过率高于靠近90°/270°射野组(B组)(3 mm/5%,Z=-25.4,P<0.05;3 mm/3%,Z=-26.8,P<0.05)。通过IGRT纠正摆位误差,可显著提高γ通过率(IGRT和非IGRT下,3 mm/5%γ通过率均值为96.3%±5.1%、96.0%±4.9%,Z=-5.50,P<0.05;3 mm/3%γ通过率均值92.3%±8.0%、91.3%±7.7%,Z=-9.54,P<0.05)。肿瘤及正常组织体积变化和运动变化、定位和治疗前准备充分与否等都会影响在体剂量验证的结果。结论放疗过程中进行EPID在体剂量验证能避免错误照射,但需规范EPID在体剂量验证流程,以避免人为因素等导致的通过率降低。     

Evaluation of BPA uptake in clear cell sarcoma (CCS) in vitro and development of an in vivo model of CCS for BNCT studies

Clear cell sarcoma (CCS), a rare malignant tumor with a predilection for young adults, is of poor prognosis. Recently however, boron neutron capture therapy (BNCT) with the use of p-borono‐L‐phenylalanine (BPA) for malignant melanoma has provided good results. CCS also produces melanin; therefore, the uptake of BPA is the key to the application of BNCT to CCS. We describe, for the first time, the high accumulation of boron in CCS and the CCS tumor-bearing animal model generated for BNCT studies.     

Synthesis and biological evaluation of 1-(4-[f]fluorobenzyl)-4-[(5,6-dimethoxy-1-oxoindan-2-yl)methyl]piperidine for in vivo studies of acetylcholinesterase

We synthesized and evaluated 1-(4-fluorobenzyl)-4-[(5,6-dimethoxy-1-oxoindan-2-yl)methyl]piperidine (4-FDP), which is an analog of donepezil. The 4-[¹⁸F]FDP was prepared by reductive alkylation of debenzylated donepezil with 4-[¹⁸F]fluorobenzaldehyde in high radiochemical yield (decay-corrected, 40–52%) and with high effective specific activity (30–38 GBq/μmol). Tissue distribution studies in mice demonstrated nonspecific distribution of the 4-[¹⁸F]FDP in brain regions, suggesting that this radioligand may not be a suitable agent for in vivo studies of acetylcholinesterase (AChE), despite its potent in vitro biological activity.     

基于电子射野影像系统与加速器日志文件重建模体内剂量的初步比较

目的 研究基于电子射野影像系统（EPID）与加速器日志文件（dynalogs file）重建模体内剂量的差异性。方法 收集12例盆腔患者的容积旋转调强（VMAT）计划,将计划信息复制到“Cheese”模体上重新计算剂量,而后在瓦里安加速器（RapidArc）上执行,“Cheese”模体置于等中心处获取射野影像（EPI）,将EPI传入EPIgray软件中重建剂量。同时利用Mobius软件调用加速器日志文件,实现对模体计划剂量的重建。以A1SL型号的电离室和配套的剂量仪测量整个计划执行结束后射野等中心（电离室中心）处剂量值,在计划系统（TPS）中读取电离室敏感体积体内的平均剂量值（设置电离室中心与等中心重合）。结果 电离室测量值与TPS中读取的等中心处剂量值相比,两者偏差为1.31%。两种方式重建的射野等中心的剂量分别与电离室测量数值相比,差异均无统计学意义（P>0.05）。结论 两种重建体内剂量的方法均能为VMAT在体剂量验证提供参考。     

2β-carbomethoxy-3β-(4- and 2-[F]fluoromethylphenyl)tropanes: specific probes for in vivo quantification of central dopamine transporter sites

Dopamine reuptake transporter binding kinetics of 2β-carbomethoxy-3β-(4-[¹⁸F]fluoromethylphenyl)tropane (p-FWIN) and 2β-carbomethoxy-3β-(2-[¹⁸F]fluoromethylphenyl)tropane (o-FWIN) were determined in vervet monkeys using positron emission tomography (PET). Ligand localization was rapid and specific to the striatum with kinetic estimates comparable with those of ¹¹C-labeled WIN 35,428 (CWIN). Binding was more specific with p-FWIN than with CWIN or o-FWIN. The relatively longer half-life of the ¹⁸F radiolabel enabled longer acquisition times with p-FWIN, resulting in less variability in the kinetic estimates.     

252Cf裂变中子源在组织等效模体中的中子和γ辐射剂量分布的计算

目的：计算^252Cf裂变中子源的的中子和γ辐射在组织等模体内的剂量分布,为使用^252Cf裂变中子源进行中子放疗提供有用的剂量学参数。方法：建立^252Cf源和组织等效模体的三维几何计算模型,利用蒙特卡罗方法进行中子和γ辐射联合输运计算。结果：计算了两种医用^252Cf裂变中子源在水、血液、肌肉、皮肤、骨骼和肺组织等效材料构成的模体中距源不同距离点处的中子和γ辐射吸收剂量。结论：蒙特卡罗计算结果与文献数据以及使用双电离室实验测量的结果符合得较好。对^252Cf裂变中子源在5种组织材料构成的模体中中子和γ辐射的剂量分布进行了比较,使用水作为组织等效材料对^252Cf裂变中子源在在以肌肉、血液和皮肤构成的局部组织内的剂量分布进行模拟计算,可取得较可靠的结果。     

Development and validation of the 57Co assay for determining the ligand to antibody ratio in bifunctional chelate/antibody conjugates for use in radioimmunotherapy

Introduction

The ligand to antibody ratio is an important characteristic of a chelate/antibody conjugate. It has been widely reported that if the ratio is too high, there will be detrimental effects on immunoreactivity and biodistribution; conversely, if the ratio is too low, the radionuclide may not bind efficiently, and the stability and the specific activity will be reduced. There are little published data on the accuracy or precision of the ⁵⁷Co assay. The UK Clinical Trials Regulations state that “systems with procedures that assure the quality of every aspect of the trial should be implemented”. The aims of this study were to assess the reliability and accuracy of the ⁵⁷Co binding assay and validate it against defined criteria.

Method

Thirty-two serial assays were assessed for reliability. Two batches of conjugated antibody were also analysed by matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry (MS) to allow the comparison of the functional test with a physical method.

Results

Reliability: The coefficient of variation was 0.13. Accuracy: There was 9% variation between the ⁵⁷Co binding assay and MALDI-TOF MS results.

Conclusion

A detailed method for the ⁵⁷Co ligand to antibody test is described that allows a discrete value to be obtained. The assay was validated as fit for purpose against target values of coefficient of variation <0.20, accuracy±10%, over a permissive range of 0.5–3.0 ligand to antibody ratio.     

Determination of 5-hydroxy-l-[β-C]tryptophan and its in vivo-formed radiolabeled metabolites in brain tissue using high performance liquid chromatography: A study supporting radiotracer kinetics obtained with positron emission tomography

A high performance liquid chromatographic system was developed for separation of ¹¹C-labeled 5-hydroxy- -tryptophan ([¹¹C]HTP) and in vivo formed radiolabeled metabolites in rat brain tissue. Analysis of brain homogenate revealed that the main part of the radioactivity was associated with ¹¹C-labeled 5-hydroxyindole-3-acetic acid after intravenous injection of [¹¹C]HTP to the rat. The serotonin synthesis rate in the brain was calculated and closely correlated to the serotonin synthesis rate in monkey and human measured using positron emission tomography.     

六道管式炉氧化燃烧联合分离海产品中有机结合氚与14 C的影响因素研究与不确定度评定

目的 建立海产品中有机结合氚(OBT)与¹⁴C的联合处理检测方法,分析测量过程中不确定度的来源并进行评定,实现海鱼、海虾、海蟹、海贝和海藻等不同种类海产品中的OBT和¹⁴C的快速、准确测定。方法 采用六道管式燃烧装置结合自制六通玻璃管,研究联合氧化燃烧与收集海产品中OBT和¹⁴C的方法;分析评定元素分析、氧化燃烧、液体闪烁计数测量等过程中引入的不确定度。结果 建立了5管合并收集OBT、单管收集¹⁴C的联合分离方法,-110℃ 冷阱收集条件下氢的燃烧回收率得到有效提升,样品转移时的损耗更少,收集碳所需氢氧化钠、氯化铵和氯化钙等试剂的用量大大降低。联合分离5.0 g样品中氢和碳(包含所有氢和碳的同位素),葡萄糖中氢和碳的燃烧回收率分别为98.7%和93.1%,5种海产品中氢和碳的燃烧回收率分别为92.5% ~ 97.3%和82.7%~96.3%,3次平行性实验相对标准偏差均<5%,表明该方法具有良好的准确性和精密度。样品计数是结果不确定度的主要来源,其次为元素含量分析和计数效率。结论 本研究建立的海产品中OBT和¹⁴C 联合分离体系燃烧回收率高、处理时间短、分离效果好、获得量足,可有效减少样品使用量,能满足海产品放射性污染健康风险评估要求。     

Usefulness of rate of increase in SPECT counts in one-day method of <Emphasis Type="Italic">N</Emphasis>-isopropyl-4-iodoamphetamine [<Superscript>123</Superscript>I] SPECT studies at rest and after acetazolamide challenge using a method for estimating time-dependent distribution at rest

OBJECTIVE: When N-isopropyl-4-iodoamphetamine ((123)I-IMP) single-photon emission computed tomography (SPECT) studies at rest and after acetazolamide (ACZ) challenge are conducted in a day, the time-dependent change in IMP in the brain at rest should be estimated accurately. We devised the method and investigated whether our one-day method for measuring the rate of increase in SPECT counts allowed reduction in the acquisition time. METHODS: Sequential, 5-min SPECT scans were performed. We estimated the time-dependent change in the brain using the change in slopes of two linear equations derived from the first three SPECT counts. For the one-day method, ACZ was administered 15 min or 20 min after IMP administration. The second IMP was administered 10 min after ACZ administration. RESULTS: Time-dependent changes in the brain were classified into 13 patterns when estimation was started at 5 min after IMP administration and 6 patterns when estimation was started at 10 min, and fitting coefficients were determined. The correlation between actual measurements at 37.5 min and estimates was high with a correlation coefficient of 0.99 or greater. Rates of increase obtained from 20-min data were highly correlated with those obtained from 15-min or 10-min data (r = 0.97 or greater). In patients with unilateral cerebrovascular disease, the rate of increase on the unaffected side was 44.4 +/- 10.9% when ACZ was administered 15 min later and 48.0 +/- 16.0% when ACZ was administered 20 min later, and the rates of increase with different timings of administration were not significantly different. CONCLUSIONS: The examination time may be reduced from 50 min to 45 min or 40 min as needed. The rate of increase was not influenced by the time frame for determination or the timing of ACZ administration. These findings suggest that our estimation method is accurate and versatile.