Thomas Kull

First Demonstration of Leukemia Imaging with the Proliferation Marker 18F-Fluorodeoxythymidine

Acute myeloid leukemia (AML) is a neoplasm of hematopoietic stem cells with partial or complete loss of the ability to differentiate but with preserved proliferation capacity. The aim of our study was to evaluate if the in vivo proliferation marker 3′-deoxy-3′-18F-fluorothymidine (FLT) is suitable for visualizing leukemia manifestation sites and if 18F-FLT is a surrogate marker for disease activity. Methods: In this pilot study, 10 patients with AML underwent pretherapeutic imaging with 18F-FLT PET or 18F-FLT PET/CT. The biodistribution of 18F-FLT was assessed 60 min after intravenous injection of the radiotracer. Standardized uptake values were calculated for reference segments of bone marrow, spleen, and normal organs. 18F-FLT PET in 10 patients with benign pulmonary nodules and the absence of malignant or inflammatory disease served as controls. Results: Retention of 18F-FLT was observed predominantly in bone marrow and spleen and was significantly higher in AML patients than in controls (mean 18F-FLT SUV in bone marrow, 11.5 and 6.6, P < 0.05; mean 18F-FLT SUV in spleen, 6.1 and 1.8, P < 0.05). Outside bone marrow, focal 18F-FLT uptake showed extramedullary manifestation sites of leukemia in 4 patients (meningeal disease, pericardial, abdominal, testicular, and lymph node), proven by other diagnostic procedures. Conclusion: This pilot study indicated that PET using 18F-FLT is able to visualize extramedullary manifestation sites of AML and reflects disease activity. Because 18F-FLT uptake in bone marrow is caused by a combination of both neoplastic and normal hematopoietic cells, the correlation of 18F-FLT uptake in bone marrow and leukemic blast infiltration did not reach statistical significance.

Radioimmunotherapy with Anti-CD66 Antibody: Improving the Biodistribution Using a Physiologically Based Pharmacokinetic Model

To improve radioimmunotherapy with anti-CD66 antibody, a physiologically based pharmacokinetic (PBPK) model was developed that was capable of describing the biodistribution and extrapolating between different doses of anti-CD66 antibody. Methods: The biodistribution of the 111In-labeled anti-CD66 antibody of 8 patients with acute leukemia was measured. The data were fitted to 2 PBPK models. Model A incorporated effective values for antibody binding, and model B explicitly described mono- and bivalent binding. The best model was selected using the corrected Akaike information criterion. The predictive power of the model was validated comparing simulations and 90Y-anti-CD66 serum measurements. The amount of antibody (range, 0.1–4 mg) leading to the most favorable therapeutic distribution was determined using simulations. Results: Model B was better supported by the data. The fits of the selected model were good (adjusted R2 > 0.91), and the estimated parameters were in a physiologically reasonable range. The median deviation of the predicted and measured 90Y-anti-CD66 serum concentration values and the residence times were 24% (range, 17%−31%) and 9% (range, 1%−64%), respectively. The validated model predicted considerably different biodistributions for dosimetry and therapeutic settings. The smallest (0.1 mg) simulated amount of antibody resulted in the most favorable therapeutic biodistribution. Conclusion: The developed model is capable of adequately describing the anti-CD66 antibody biodistribution and accurately predicting the time–activity serum curve of 90Y-anti-CD66 antibody and the therapeutic serum residence time. Simulations indicate that an improvement of radioimmunotherapy with anti-CD66 antibody is achievable by reducing the amount of administered antibody; for example, the residence time of the red marrow could be increased by a factor of 1.9 ± 0.3 using 0.27 mg of anti-CD66 antibody.

Internal radionuclide therapy: The ULMDOS software for treatment planning

Before therapy with unsealed radionuclides, a dosimetry assessment must be performed for each patient. We present the interactive software tool ULMDOS, which facilitates dosimetric calculations, enhances traceability, and adequate documentation. ULMDOS is developed in IDL 6.1 (Interactive Data Language) under Windows XP/2000. First the patient data, the radiotracer data, and optionally urine and serum data are entered. After loading planar gamma camera images and drawing regions of interest, the residence times can be calculated using fits of the time activity data to exponential functions. Data can be saved in ASCII format for retrospective examination and further processing. ULMDOS allows one to process the dosimetric calculations within a standardized environment, spares the time-consuming transfer of data between different software tools, enables the documentation of ROI and raw data, and reduces intraindividual variability. ULMDOS satisfies the required conditions for traceability and documentation as a prerequisite to routine use in clinical settings.

Comparing time activity curves using the Akaike information criterion

The comparison of curves is a common task in many fields of science. Simply comparing the sums of squares or R(2) is not sufficient, and frequently used tests have many disadvantages. The basic idea of the presented method is turning the problem of comparing curves into a problem of model selection using the corrected Akaike Information Criterion. Here, this straightforward approach is applied for comparing curves using the example of (111)In- and (90)Y-labelled anti-CD66 antibody serum time activity data. As a result it is shown that for the investigated (111)In- and (90)Y-labelled anti-CD66 antibodies, the biokinetics between dosimetry and therapy are different with respect to the contribution of the second, longer half-life component. We advocate the use of the presented method rather than employing less advanced approaches for curve comparison.

Optimal preloading in radioimmunotherapy with anti-CD45 antibody

PURPOSE Anti-CD45 antibody is predominantly used in the treatment of acute leukemia. CD45 is stably expressed on all leukocytes and their precursors, and therefore the liver and spleen constitute major antigen sinks. Thus, as the red marrow is the target organ, in radioimmunotherapy with anti-CD45 antibody, preloading with unlabeled antibody is a method to increase the absorbed dose to the target cells. In a previous study, a method to individually determine the optimal preload for five patients with acute leukemia was developed. Here, this method is examined and improved using two pretherapeutic measurement series and a refined pharmacokinetic model. METHODS To obtain the biodistribution of 111In-labeled anti-CD45 antibody under different saturation conditions, two measurement series one with and one without preloading were conducted in five patients. For each patient, two physiologically based pharmacokinetic models were fitted to the data and the corrected Akaike information criterion was used to identify the model, which was empirically most supported. The resultant parameter values were compared to values reported in the literature. To individually determine the optimal amount of unlabeled antibody for therapy, computer simulations for preloads ranging from 0 to 60 mg were performed based on the estimated parameters of each patient. The prediction power of the model was assessed by comparing the simulated therapeutic serum curves to the actual 90Y measurements. RESULTS Visual inspection showed good fits and the adjusted R2 was >0.90 for all patients. All parameters were in a physiologically reasonable range. The relative deviation of the predicted area under the therapeutic serum curve and the measured curve was 15%-33%. The optimal preloading increased the marrow-over-liver selectivity up to 3.9 fold compared to the simulated biodistribution using a standard dose (0.5 mg/kg). CONCLUSIONS The presented method can be used to individually determine the optimal preload and the corresponding residence times in radioimmunotherapy with anti-CD45 antibody.

Preferential tumor targeting and selective tumor cell cytotoxicity of 5-[131/125I]iodo-4'-thio-2'-deoxyuridine.

Purpose: Auger electron emitting radiopharmaceuticals are attractive for targeted nanoirradiation therapy, provided that DNA of malignant cells is selectively addressed. Here, we examine 5-[123/125/131I]iodo-4′-thio-2′-deoxyuridine (ITdU) for targeting DNA in tumor cells in a HL60 xenograft severe combined immunodeficient mouse model. Experimental Design: Thymidine kinase and phosphorylase assays were done to determine phosphorylation and glycosidic bond cleavage of ITdU, respectively. The biodistribution and DNA incorporation of ITdU were determined in severe combined immunodeficient mice bearing HL60 xenografts receiving pretreatment with 5-fluoro-2′-deoxyuridine (FdUrd). Organ tissues were dissected 0.5, 4, and 24 h after radioinjection and uptake of [131I]ITdU (%ID/g tissue) was determined. Cellular distribution of [125I]ITdU was imaged by microautoradiography. Apoptosis and expression of the proliferation marker Ki-67 were determined by immunohistologic staining using corresponding paraffin tissue sections. Results: ITdU is phosphorylated by thymidine kinase 1 and stable toward thymidylate phosphatase-mediated glycosidic bond cleavage. Thymidylate synthase-mediated deiodination of [123/125/131I]ITdU was inhibited with FdUrd. Pretreatment with FdUrd increased preferentially tumor uptake of ITdU resulting in favorable tumor-to-normal tissue ratios and tumor selectivity. ITdU was exclusively localized within the nucleus and incorporated into DNA. In FdUrd-pretreated animals, we found in more than 90% of tumor cells apoptosis induction 24 h postinjection of ITdU, indicating a highly radiotoxic effect in tumor cells but not in cells of major proliferating tissues. Conclusion: ITdU preferentially targets DNA in proliferating tumor cells and leads to apoptosis provided that the thymidylate synthase is inhibited.

Optimized Peptide Amount and Activity for 90Y-Labeled DOTATATE Therapy

In peptide receptor radionuclide therapy with 90Y-labeled DOTATATE, the kidney absorbed dose limits the maximum amount of total activity that can be safely administered in many patients. A higher tumor-to-kidney absorbed dose ratio might be achieved by optimizing the amount of injected peptide and activity, as recent studies have shown different degrees of receptor saturation for normal tissue and tumor. The aim of this work was to develop and implement a modeling method for treatment planning to determine the optimal combination of peptide amount and pertaining therapeutic activity for each patient. Methods: A whole-body physiologically based pharmacokinetic (PBPK) model was developed. General physiologic parameters were taken from the literature. Individual model parameters were fitted to a series (n = 12) of planar γ-camera and serum measurements (111In-DOTATATE) of patients with meningioma or neuroendocrine tumors (NETs). Using the PBPK model and the individually estimated parameters, we determined the tumor, liver, spleen, and red marrow biologically effective doses (BEDs) for a maximal kidney BED (20 Gy2.5) for different peptide amounts and activities. The optimal combination of peptide amount and activity for maximal tumor BED, considering the additional constraint of a red marrow BED less than 1 Gy15, was individually quantified. Results: The PBPK model describes the biokinetic data well considering the criteria of visual inspection, the coefficients of determination, the relative standard errors (<50%), and the correlation of the parameters (<0.8). All fitted parameters were in a physiologically reasonable range but varied considerably between patients, especially tumor perfusion (meningioma, 0.1–1 mL·g−1·min−1, and NETs, 0.02–1 mL·g−1·min−1) and receptor density (meningioma, 5–34 nmol·L−1, and NETs, 7–35 nmol·L−1). Using the proposed method, we identified the optimal amount and pertaining activity to be 76 ± 46 nmol (118 ± 71 μg) and 4.2 ± 1.8 GBq for meningioma and 87 ± 50 nmol (135 ± 78 μg) and 5.1 ± 2.8 GBq for NET patients. Conclusion: The presented work suggests that to achieve higher efficacy and safety for 90Y-DOATATE therapy, both the administered amount of peptide and the activity should be optimized in treatment planning using the proposed method. This approach could also be adapted for therapy with other peptides.

New Molecular Markers for Prostate Tumor Imaging: A Study on 2-Methylene Substituted Fatty Acids as New AMACR Inhibitors†

The development of prostate carcinoma is associated with alterations in fatty acid metabolism. α-Methylacyl-CoA racemase (AMACR) is a peroxisomal and mitochondrial enzyme that catalyses interconversion between the (S)/(R)-isomers of a range of α-methylacyl-CoA thioesters. AMACR is involved in the β-oxidation of the dietary branched-chain fatty acids and bile acid intermediates. It is highly expressed in prostate (more than 95 %), colon (92 %), and breast cancers (44 %) but not in the respective normal or hyperplastic tissues. Thus, targeting of AMACR could be a new strategy for molecular imaging and therapy of prostate and some other cancers. Unlabeled 2-methylenacyl-CoA thioesters (12 a-c) were designed as AMACR binding ligands. The thioesters were tested for their ability to inhibit the AMACR-mediated epimerization of (25R)-THC-CoA and were found to be strong AMACR inhibitors. Radioiodinated (E)-(131) I-13-iodo-2-methylentridec-12-enoic acid ((131) I-7 c) demonstrated preferential retention in AMACR-positive prostate tumor cells (LNCaP, LNCaP C4-2wt and DU145) compared with both AMACR-knockout LNCaP C4-2 AMACR-siRNA and benign BPH1 prostate cell lines. A significant protein-bound radioactive fraction with main bands at 47 (sum of molecular weights of AMACR plus 12 c), 70, and 75 kDa was detected in LNCaP C4-2 wt cells. In contrast, only negligible amounts of protein-bound radioactivity were found in LNCaP C4-2 AMACR-siRNA cells.

Synthesis and Biologic Study of IV-14, a New Ribonucleoside Radiotracer for Tumor Visualization

Uridine-cytidine kinase (UCK) 2, an enzyme normally expressed in human placenta and testis and highly overexpressed in many neoplasias of blood and solid tissues, catalyzes monophosphorylation of pyrimidine ribonucleosides with efficiency 15- to 20-fold higher than that of ubiquitously expressed isozyme UCK1. In this paper, we report the synthesis of 3′-(E)-(2-iodovinyl)uridine (IV-14) and its preclinical evaluation as a new radiotracer derived from a UCK2-selective antitumor agent, 3′-(ethynyl)uridine. Methods: Radioiodinated IV-14 was prepared from the respective stannyl precursor. 131I-IV-14 was studied in cellular uptake assays and tested for stability in serum as well as for stability to thymidine phosphorylase, liver-, and mucosa-specific murine uridine phosphorylases. UCK1 and UCK2 expression levels in different tumor cell lines were determined by Western blot. Cellular distribution of 131I-IV-14 was determined in HL60 cells. Biodistribution studies and γ-camera scintigraphy were performed on an HL60-xenografted severe combined immunodeficiency (SCID) mouse model. Results: 131I-IV-14 demonstrated excellent stability in serum. It was stable to human thymidine phosphorylase and to liver- and mucosa-specific murine uridine phosphorylases. Cellular uptake after 24 h of incubation with 131I-IV-14 was 4.27 ± 0.21, 3.66 ± 0.13, 2.69 ± 0.07, 2.24 ± 0.18, and 3.26 ± 0.18 percentage injected dose per 5 × 105 Mia-PaCa-2, CX-1, HL60, Capan-1, and Panc-1 cells, respectively. Uptake and retention of IV-14 were regulated by 2 factors: UCK2 expression level and intracellular transport mediated partially via human equilibrating nucleoside transporter 1. A biodistribution study of 131I-IV-14 in an HL60-xenografted SCID mouse model showed that at 4 h after injection the greatest amount of retained radioactivity was in tumor. The tissue-to-tumor ratio 4 h after injection was 1.0 ± 0.24 for tumor, 0.40 ± 0.18 for spleen, 0.25 ± 0.12 for colon, 0.14 ± 0.07 for small intestine, and less than 0.1 for other sites. Scintigraphy with 123I-IV-14 4 h after injection showed the tumor well. In addition, high accumulation of radioiodide in the stomach content was observed and was presumably due to metabolic degradation of IV-14. Conclusion: IV-14 is a UCK2-specific marker, allowing for in vivo addressing of tumors with high RNA synthesis independent of proliferation rate.

Determination of individual organ masses for 90Y-anti-CD66 radioimmunotherapy: Influence on therapy planning

Dosimetry is important in the development of radioactive pharmaceuticals, especially for optimizing radionuclide therapy with respect to risk-benefit analysis. To calculate the applied absorbed doses in the target and risk organs standard phantom masses are frequently used. However, deviations to the true organ mass can lead to suboptimal decisions in dose finding studies. To estimate the magnitude of deviations introduced when using standard phantom masses instead of individual organ masses, we investigated 10 patients treated with radioimmunotherapy using (90)Y labelled anti-CD66 antibody. The use of standard phantom masses instead of individually measured organ masses results in mean deviations for liver, spleen and kidneys of 2% (Min. -22%, Max. 34%), -3% (Min. -34, Max. 100%) und -8% (Min. -37, Max. 38%), respectively. For the administered therapeutic activity differences of -16% (Min. -45%, Max. 4%) were observed depending on the used organ mass. These results demonstrate that using standard phantom masses for dosimetry before radionuclide therapy is not adequate.