Charles Frederik Sio

Automated synthesis of [18F]gefitinib on a modular system.

In recent years, [(18)F]gefitinib PET has successfully been employed for a number of applications ranging from oncology to in vivo studies of drug transporter proteins. We here report a reliable, automated procedure for routine synthesis of this radiotracer on an Eckert and Ziegler modular system. The 3-step radiosynthesis followed by preparative HPLC-purification provided [(18)F]gefitinib in 17.2±3.3% (n=22) overall decay-corrected radiochemical yield with radiochemical purity >99% in a total synthesis time of about 2.5h.

PET-CT imaging with [18F]-gefitinib to measure Abcb1a/1b (P-gp) and Abcg2 (Bcrp1) mediated drug-drug interactions at the murine blood-brain barrier

INTRODUCTION The efflux transporters P-glycoprotein (P-gp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2) are expressed at the blood-brain barrier (BBB), and can limit the access of a wide range of drugs to the brain. In this study we developed a PET-CT imaging method for non-invasive, quantitative analysis of the effect of ABCB1 and ABCG2 on brain penetration of the anti-cancer drug gefitinib, and demonstrated the applicability of this method for identification and quantification of potential modulators of ABCB1 and ABCB2 using the dual inhibitor elacridar. METHODS In vitro cellular accumulation studies with [(14)C]-gefitinib were conducted in LLC-PK1, MDCKII, and the corresponding ABCB1/Abcb1a and ABCG2/Abcg2 overexpressing cell lines. Subsequently, in vivo brain penetration of [(18)F]-gefitinib was quantified by PET-CT imaging studies in wild-type, Abcg2(-/-), Abcb1a/1b(-/-), and Abcb1a/1b;Abcg2(-/-) mice. RESULTS In vitro studies showed that [(14)C]-gefitinib is a substrate of the human ABCB1 and ABCG2 transporters. After i.v. administration of [(18)F]-gefitinib (1mg/kg), PET-CT imaging showed 2.3-fold increased brain levels of [(18)F]-gefitinib in Abcb1a/1b;Abcg2(-/-) mice, compared to wild-type. Levels in single knockout animals were not different from wild-type, showing that Abcb1a/1b and Abcg2 together limit access of [(18)F]-gefitinib to the brain. Furthermore, enhanced brain accumulation of [(18)F]-gefitinib after administration of the ABCB1 and ABCG2 inhibitor elacridar (10 mg/kg) could be quantified with PET-CT imaging. CONCLUSIONS PET-CT imaging with [(18)F]-gefitinib is a powerful tool to non-invasively assess potential ABCB1- and ABCG2-mediated drug-drug interactions (DDIs) in vivo. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE This minimally-invasive, [(18)F]-based PET-CT imaging method shows the interplay of ABCB1 and ABCG2 at the BBB in vivo. The method may be applied in the future to assess ABCB1 and ABCG2 activity at the BBB in humans, and for personalized treatment with drugs that are substrates of ABCB1 and/or ABCG2.

Evaluation of a 99mTc-Labeled AnnexinA5 Variant for Non-invasive SPECT Imaging of Cell Death in Liver, Spleen and Prostate

PurposeWe investigate radio-labeling and pharmacokinetics of a new AnnexinA5 variant (HYNIC-cys-AnxA5) and then assess its utility for the non-invasive detection of cell death in liver, spleen and prostate.MethodsAnnexinA5 binds to phosphatidylserine expressed on the surface of apoptotic and necrotic cells. Contrary to other AnnexinA5 variants, the new cys-AnxA5 allows for site-specific conjugation of a hydrazinonicotinamide-maleimide moiety and subsequent radio-labeling with 99mTc at a position not involved in the AnxA5-phosphatidylserine interaction. Distribution of 99mTc-HYNIC-cys-AnxA5 was studied in rats, both invasively and via SPECT/CT. Cycloheximide was used to induce cell death in liver and spleen, whereas apoptosis in the prostate was induced by castration.ResultsHYNIC-cys-AnxA5 was efficiently and reproducibly labeled with 99mTc. Blood clearance of radioactivity after iv-injection was adequately described by a two-compartment model, the renal cortex representing the main site of accumulation. Cycloheximide treatment resulted in increased accumulation of intravenous-injected 99mTc-HYNIC-cys-AnxA5 in liver and spleen over controls, which correlated well with TUNEL staining for cell death in corresponding tissue sections. However, the increase in TUNEL-positive prostate epithelial cells observed following castration was not paralleled by greater 99mTc-HYNIC-cys-AnxA5 accumulation.Conclusion99mTc-HYNIC-cys-AnxA5 appears a suitable tracer for assessment of cell death in liver and spleen, but not prostate.

Validation of interventional fiber optic spectroscopy with MR spectroscopy, MAS-NMR spectroscopy, high performance thin layer chromatography and histopathology for accurate hepatic fat quantification

Objectives:To validate near-infrared (NIR)-based optical spectroscopy measurements of hepatic fat content using a minimally invasive needle-like probe with integrated optical fibers, enabling real-time feedback during percutaneous interventions. The results were compared with magnetic resonance spectroscopy (MRS) as validation and with histopathology, being the clinical gold standard. Additionally, ex vivo magic angle spinning nuclear magnetic resonance spectroscopy and high-performance thin-layer chromatography were performed for comparison. Materials and Methods:Ten mice were used for the study, of which half received a regular chow diet and the other half received a high-fat diet to induce obesity and hepatosteatosis. The mice were imaged with a clinical 3-Tesla MR to select a region of interest within the right and left lobes of the liver, where MRS measurements were acquired in vivo. Subsequently, optical spectra were measured ex vivo at the surface of the liver at 6 different positions immediately after resection. Additionally, hepatic fat was determined by magic angle spinning nuclear magnetic resonance spectroscopy and high-performance thin-layer chromatography. Histopathologic analyses were performed and used as the reference standard. Pearson correlation and linear regression analyses were performed to assess the correlation of the various techniques with NIR. A 1-way analysis of variance including post hoc Tukey multiple comparison tests was used to study the difference in fat estimation between the various techniques. Results:For both the mice groups, the estimated fat fractions by the various techniques were significantly similar (P = 0.072 and 0.627 for chow diet and high-fat diet group, respectively). The Pearson correlation value between NIR and the other techniques for fat determination showed the same strong linear correlation (P above 0.990; P < 0.001), whereas for histopathologic analyses, which is a rather qualitative measure, the Pearson correlation value was slightly lower (P = 0.925, P < 0.001) . Linear regression coefficient computed to compare NIR with the other techniques resulted in values close to unity with MRS having the narrowest confidence interval (r = 0.935, 95% confidence interval: 0.860–1.009), demonstrating highly correlating results between NIR and MRS. Conclusions:NIR spectroscopy measurements from a needle-like probe with integrated optical fibers for sensing at the tip of the needle can quickly and accurately determine hepatic fat content during an interventional procedure and might therefore be a promising novel diagnosing tool in the clinic.

Ultrasound and Microbubble Mediated Doxil Delivery in a Murine Breast Cancer Model: Therapeutic Efficacy Dependence on Tumor Growth Rate

The effect of tumor growth rate and treatment repeats are examined as parameters in pressure-mediated ultrasound treatments with microbubbles and Doxil in a murine breast cancer model. For this purpose, mice with a tumor doubling time of 8 and 13 days respectively received either a single or two ultrasound treatments (at 1 MHz/1 MPa) in conjunction with Definity microbubbles (1:1 dilution) and Doxil (3 mg/Kg dose) after the tumor size reached 150 mm3. The tumor model was generated using MDA-MB-231-luc cells implanted into the lower mammary fat pad of SCID beige mice. At 15 days post-treatment, tumor size was reduced by 3±18%, 8±14%, and 20±10% as compared to control for the Doxil only, ultrasound + microbubbles + Doxil (single treatment), and ultrasound + microbubbles + Doxil (2 treatments) groups, respectively, in the mice with the slower growing tumors. The mice with the faster growing tumor yielded tumor size reductions of 46±27%, 71±10%, and 61±26%, respectively, for the same groups. We hypothesize that treatment efficacy is dependent on the dynamics of the tumor itself, even within the same cell line.