Introduction The 80kD glycoprotein transferrin (TF) and its related receptor (TFR1) play a major role in the recruitment by cancer cells of factors for their multiplication, adhesion, invasion and metastatic potential. Though primarily designed to bind iron and then be internalised into cells with its receptor, TF can also bind most transition metals such as Co, Cr, Mn, Zr, Ni, Cu, V, In & Ga. Under certain conditions TF binds Ti (IV) even more tightly than it does Fe and that this occurs at the N-lobe (as distinct from C) of apoTF. Further, under physiological conditions the species Fe(C)Ti(N)-TF may provide the route for Ti entry into cells via TFR1 (1). Thus, the radiometal PET reporter isotope 45Ti with an ‘intermediate’ (~hrs) half-life suited to tracking cell-focused biological mechanisms is an attractive option for elucidating cellular mechanisms involving TF binding and internalisation, at least in (preclinical) animal models. 45Ti (T½ = 3.08 hr; + branching ratio = 85 %; mean β+ energy = 439keV, no significant dose-conferring non-511keV γ-emissions) was produced using the reaction 45Sc(p,n)45Ti by irradiating (monoisotopic) scandium discs with an energy-degraded proton beam produced by an 18MeV isochronous medical cyclotron. Separation and purification was achieved with an hydroxylamine hydrochloride functionalised resin. Comparative microPET imaging was performed in an immunodeficient mouse model, measuring biodistributions of the radiolabels 45Ti-oxalate and 45Ti-human-TF (45Ti-h-TF), out to 6hr post-injection. Materials and Methods High purity 15mm diameter scandium disc foils (99.5%, Goodfellow, UK) each thickness 0.100 ± 0.005 mm (55 mg) were loaded into an in-house constructed solid-targetry system mounted on a 300mm external beam line utilising helium-gas and chilled water to cool the target body (2). The proton beam was degraded to 11.7 MeV using a graphite disc integrated into the graphite collimator. This energy abolishes the competing ‘contaminant’ reactions 45Sc(p,n+p)44Sc and 45Sc(p,2n)44Ti. Beam current was measured using the well documented 65Cu(p,n)65Zn reaction. Calculations showed that the chosen energy is close to the optimal primary energy (~12 MeV) for maximising the (thin-target) yield from a 0.100 mm thick target. For separation of Ti from the Sc target two methods were examined; (i) ion exchange column separation using 2000 mg AG 50W-X8 resin conditioned with 10mL 9M HCl. Disc is dissolved in 1 mL of 9M HCl, which at completion of reaction is pipetted into column. Successive 1 mL volumes of 9M HCl are added, and subsequent elutions collected. (ii) Following Gagnon et al., (3) a method employing hydroxylamine hydro-chloride functionalised resin (’hydroxamate method’) was applied, similar to its use in our hands for purification and separation of 89Zr (2) following its original description for 89Zr by Holland et al., (4). Disc dissolved in 2mL 6M HCl, then diluted to 2M. Elute through column to waste fraction 1 (w1 – see FIG. 1). Then elute 6 mL of 2M HCl through column to w2, followed by 6 mL of traceSELECT H2O to w3. Finally, elute Ti into successive 1 mL product fractions (p1, 2 etc.) using 5 mL of 1M oxalic acid. This procedure takes approximate 1 hr. 45Ti in elution vials was measured using γ-spectroscopy. Sc in the same vials was determined later using ICP-MS. Results A typical production run using a beam current of 40 μA for 60min on a 0.100mm-thick disc produced an activity of 1.83 GBq. Radionuclidic analysis of an irradiated disc using calibrated cryo-HPGe γ-spectroscopy revealed T½ = 2.97–3.19 hr (95% CI) for 45Ti, and with contaminant 44Sc < 0.19 %, with no other isotopes detected. Despite systematic adjustments to column conditions satisfactory chemical separation was not achieved using the ion exchange column method (i), despite previous reports of its success (5). Typical results of separation using the successful hydroxamate method (ii) are shown on the FIGURE 1. It is seen that significant portion of 45Ti is lost in the initial washing steps leading to waste collection. N = 4 replicate experiments showed a variation (SD) of 10 % of the mean in each elu-tion fraction. Subsequent ICP-MS of the same elutions for (cold) Sc showed approximately 80 % by mass appeared in w1 and 20 % in w2, with negligible total mass (total fraction ~1/6000) of Sc in product (p1–4) vials. However, the FIG. 1 shows that a total of only 30% of the original activity of 45Ti (corrected to EOB) is available in the product vials, with the vial of highest specific activity (p1) containing 14 %. However, using a stack of 2×0.100mm thick Sc discs as a target yields isotope of adequate specific activity with-out need for concentration, for subsequent labelling and small-animal imaging purposes. In a ‘proof-of-principle’ experiment, two groups of healthy Balb/c-nu/nu female adult mice were administered with 45Ti radiotracers. The first group (N = 3) received approximately 20 MBq IP of 45Ti-oxalate buffered to pH = 7.0, and under-went microPET/CT imaging (Super Argus PET, Sedecal, Spain) out to 6hr post-injection, plus biodistribution analysis of radioactivity by dis-section at sacrifice (6hr). The second group (N = 3) received approximately 20 MBq IP of 45Ti-h-TF and were also studied to 6hr post-injection, followed by radioactive analysis after dissection at sacrifice. Organ and tissue biodistributions of the two groups at 6hr were similar but with 45Ti-oxalate showing slightly greater affinity for bone. Biodistribution by dissection results broadly confirmed the findings from PET images. However, TLC results suggested that similarity of radiolabel biodistributions of the two groups may be due to contamination of the TF radiolabel with non-conjugated Ti at time of injection. An alternative explanation is dechelation in vivo of 45Ti from 45Ti-h-TF. Conclusion Despite significant loss of 45Ti to the waste fractions of the separation process (total 53 %, corrected to EOB), 45Ti of acceptable specific activity and high radionuclidic purity has been produced from the reaction 45Sc(p,n)45Ti, with separation and purification of the product by hydroxamate column chemistry, confirming an earlier report. Though microPET in vivo imaging using 45Ti-based radiolabels was shown to be feasible, the similarity in the results for the label 45Ti-h-TF compared with ‘raw’ 45Ti-oxalate suggests further investigations. These may include a direct comparison of in vivo 45Ti-h-TF small-animal imaging plus post-dissection biodistribution with the same procedures using 89Zr labelled h-apotransferrin (6).

Introduction 76Br is of interest for in vivo PET imaging applications. Its relatively long half-life (16.1 h) allows use not only on small molecules but also proteins which have slow excretion as carrier molecules. Irradiation using a low energy proton beam (~ 20 MeV) on an enriched Cu276Se target, followed by dry distillation with thermal chromatography, is one of the best methods to obtain sufficient amounts of 76Br for clinical applications1,2. However, the thermal chromatography is plagued by poor reproducibility and appears unsuitable for automation of its production, leading us to remove the thermal chroma-tography from the dry distillation. In this investigation we employed H2O solution to collect 76Br and optimized the distillation condition using a small amount of 77Br (57.0 h). We also produced large amount of 76Br under the optimized conditions to evaluate the dry distillation method. Material and Methods Target preparation and dry distillation were conducted based on the methods described in previous reports1,2. To produce 77Br, Cu2natSe target was irradiated with 20 MeV proton beams (5 µA) accelerated by AVF cyclotron in the Japan Atomic Energy Agency. The following two systems were used in the dry distillation optimization studies; (1) an initial system was composed of two furnaces, a main and an auxiliary furnace. Temperature of each furnace was set at 1050 °C (main) and 200 °C (auxiliary) respectively; (2) the second system was made of one large furnace composed of heating and cooling area. Temperature of the heating area was varied from 1050 to 1120 °C. In both systems PTFE tubing, leading to a H2O solution (15 mL), was inserted into the apparatus. The irradiated target was heated under streaming Ar gas (30 mL/min.). An enriched Cu276Se target (76Se enrichment: 99.67%) was used for 76Br production. Radioactivity was measured on a high-purity germanium (HPGe) detector coupled to a multichannel analyzer. TLC analyses were conducted on Al2O3 plates (Merck) using 7:1 acetone:H2O as the eluting solvent. Results and Conclusion Low efficiency (33 %) of 77Br recovery was ob-served in the initial system. Distribution of radioactivity inside the apparatus showed that 35 % was trapped in the PTFE tube and the quartz tube. The recovery yield was increased up to 54 % when the auxiliary furnace was turned off, indicating that the temperature gradient inside the quartz tube is suitable to carry 77Br effectively to the H2O trap. We initially used a quartz boat to place the irradiated target in the furnace, but found that using a reusable tungsten backing was better. However, we found that recovery yield was dramatically reduced to 18 %. The studies where the temperature was varied showed that releasing efficiency was increased up to 100 % at the temperature of 1120 °C. Good recovery yield (~ 77 %) was achieved after optimizing the temperature gradient (FIG. 1b). Using the optimized setup, 76Br production runs (n = 6) have been conducted, allowing us to recover up to 39.8 MBq/µAh (EOB) of 76Br. High specific activity (~4400 GBq/µmol) was obtained in the final solution. TLC analysis showed that chemical form obtained was bromide. We concluded that the dry distillation using H2O trap is capable of providing enough high purity 76Br for clinical applications.

Introduction Since its development by Al Wolf and colleagues in the 1970s1, [11C]cyanide has been a useful synthon for a wide variety of reactions, most notably those producing [1-11C]-labeled amino acids2. However, despite its position as rote gas-phase product, the catalytic synthesis is difficult to optimize and often only perfunctorily dis-cussed in the radiochemical literature. Recently, [11C]CN– has been used in the synthesis of indole-3-[1-11C]acetic acid ([11C]IAA), the principal phytohormone responsible for a wide variety of growth and development functions in plants3. The University of Wisconsin has expertise in cyclotron production and radiochemistry of 11C and previous experience in the PET imaging of plants4,5. In this abstract, we present work on optimizing [11C]CN– production for the synthesis of [11C]IAA and the PET imaging of auxin transport in living plants. Material and Methods [11C]CH4 was produced by irradiating 270 psi of 90% N2, 10% H2 with 30 µA of 16.1 MeV protons from a GE PETtrace cyclotron. After irradiation, the [11C]CH4 was converted to [11C]CN– by passing through a quartz tube containing 3.0 g of Pt wire and powder between quartz wool frits inside a 800–1000 ˚C Carbolite tube furnace. The constituents and flow rate of the [11C]CH4 carrier gas were varied in an effort to optimize the oven\'s catalytic production of [11C]CN– from CH4 and NH3. The following conditions were investigated: i. Directly flowing irradiated target gas versus trapping, purging and releasing [11C]CH4 from a −178 ˚C HayeSep D column in He through the Pt furnace. ii. Varying the amount of anhydrous NH3 (99.995%) mixed with the [11C]CH4 carrier gas prior to the Pt furnace. Amounts varied from zero to 35 % of gas flow. iii. Varying the purity of the added NH3 gas with the addition of a hydride gas purifier (Entegris model 35KF), reducing O2 and H2O impurities to < 12 ppb. iv. Varying the flow rate of He gas carrying trapped, purged and released [11C]CH4. After flowing through the Pt furnace, the gas stream was bubbled through 300 µL of DMSO containing IAA precursor gramine (1 mg), then passed through a 60×5 cm column containing ascarite to absorb [11C]CO2, followed by a −178˚C Porapak Q column to trap [11C]CH4 and [11C]CO. After bubbling, the DMSO/gramine vial was heated to 140 ˚C to react the gramine with [11C]CN–, forming the intermediate indole-3-[1-11C]acetonitrile ([11C]IAN), which was subsequently purified by solid phase extraction (SPE). The reaction mixture was diluted into 20 mL water and loaded onto a Waters Sep-Pak light C18 cartridge, followed by rinsing with 5 mL of 0.1% HCl : acetonitrile (99 : 1) and 10 mL of the same mixture in ratio 95 : 5, and finally eluted with 0.5 mL of diethyl ether. The ether was subsequently evaporated under argon flow, followed by the hydrolysis of [11C]IAN to [11C]IAA with the addition of 300 µL 1 M NaOH and heating to 140 ˚C for 5 minutes. After hydrolysis, the solution was neutralized with 300 µL 1 M HCl and purified using preparative high-performance liquid chromatography (HPLC) using a Phenomenex Luna C18 (10μ, 250×10mm) column with a mobile phase acetonitrile : 0.1% formic acid in H2O (35 : 65) at flow rate of 3 mL/min. The [11C]IAA peak, eluting at 12 minutes, was collected and rotary evaporated to dryness, then again after the addition of 5 mL acetonitrile, followed by its reconstitution in 50 µL of water. Analytical HPLC was performed on the [11C]IAA before and after this evaporation procedure using a Phenomenex Kinetex C18 (2.6μ, 75× 4.6 mm) column with a linear gradient elution over 20 minutes of 10 : 90–30 : 70 (acetonitrile : 0.1% formic acid) at a 1 mL/min flow rate, eluting at 7.6 minutes. The transport of [11C]IAA was monitored following administration through the severed petiole of rapid cycling Brassica oleracea (rcBo) using a Siemens microPET P4 scanner. Transport was compared following administration to the first true leaf versus the final fully formed leaf in plants with and without exposure to the polar auxin transport inhibitor naphthylphthalamic acid (NPA). Results and Conclusion Optimization of the [11C]CN– gas phase chemistry was performed using two key metrics for measuring conversion yield. First is the fraction of total produced radioactivity that trapped in the DMSO/gramine solution (denoted %DMSO), and second, the fraction of DMSO/gramine-trapped activity that was able to react with gramine to form [11C]IAN (denoted %CN–). Under certain conditions, the former of these metrics experienced significant losses due to unconverted [11C]CH4 or through combustion, forming [11C]CO2 or [11C]CO. The latter metric experienced losses due to production of incomplete oxidation products of the CH4-NH3 reaction, such as methylamine. Total [11C]CH4 to [11C]CN– con-version yields is reported by the product of the two metrics. It was initially hypothesized that the irradiation of a 90% N2, 10% H2 target gas would produce sufficient in-target-hot-atom-produced NH3 to convert [11C]CH4 to [11C]CN– in the Pt furnace. However, conversion yields were found to be low and highly variable, with 13 ± 8 % trapping in DMSO/gramine, 9 ± 9 % of which reacted as CN– (n = 15). While in disagreement with previous reports1, this is likely as a result the batch irradiation conditions resulting ammonia losses in the target chamber and along the tubing walls. Yields and reproducibility were improved when combining the target gas with a stream of anhydrous NH3 gas flow with conversion yields reported in TABLE 1. However, these yields remained undesirably low, potentially as a result of the 10% H2 carrier gas having an adverse effect on the oxidative conversion of [11C]CH4 to [11C]CN–. To remedy this, the irradiated target gas was trapped, purged, released in He and combined with NH3 gas before flowing through the Pt furnace. Initial experiments using 99.995% anhydrous NH3 gas resulted in very poor (< 0.1%) [11C]CN– yields as a result of nearly quantitative combustion forming [11C]CO2. Installation of a hydride gas purifier to reduce O2 and H2O impurities in NH3 improved yields for CH4 in He, but did not significantly affect those from [11C]CH4 in N2/H2 target gas. In disagreement with previous reports2, conversion yields were found to be highly sensitive to overall carrier gas flow rate, with lower flow rates giving the best yields, as shown in TABLE 1. Optimization experiments are continuing. The total decay-corrected yield for the 1 hour synthesis of [11C]IAA in 50 µL of water is 2.3 ± 0.7 %, based on the total produced [11C]CH4 with a specific activity ranging from 1–100 GBq/µmol. The principal radiochemical impurity was determined to be indole-3-carboxylic acid. The SPE procedure isolating the [11C]IAN intermediate product was optimized to minimize this impurity in the final sample. After a rapid distribution of the administered [11C]IAA through the cut petiole and throughout the rcBO plant, upward vascular transport of auxin and downward polar auxin transport was visualized through time-activity curves (TACs) of regions of interest along the shoot. Comparison of these TACS with and without exposure to NPA yields insight into the fundamental physiological process of polar auxin transport in plants. In conclusion, the Pt-catalyzed oxidative conversion of [11C]CH4 and NH3 to [11C]CN– is a challenging process to optimize and highly sensitive to carrier gas composition and flow rate. Optimization for our experimental conditions yielded several results which disagreed with previous reports. [11C]IAA produced using [11C]CN– is well suited for PET imaging of polar auxin transport in living plants.

Im Rahmen des vorliegenden Dissertationsprojektes wurde der Zusammenhang zwischen Ausprägung des regulativen Temperaments und SERT-Verfügbarkeit als Indikator für die Funktionalität des zentralen serotonergen Systems mittels Positronen-Emissions-Tomographie (PET) bei adipösen im Vergleich zu normalgewichtigen Personen untersucht.

Introduction 76Br is of interest for in vivo PET imaging applications. Its relatively long half-life (16.1 h) allows use not only on small molecules but also proteins which have slow excretion as carrier molecules. Irradiation using a low energy proton beam (~ 20 MeV) on an enriched Cu276Se target, followed by dry distillation with thermal chromatography, is one of the best methods to obtain sufficient amounts of 76Br for clinical applications1,2. However, the thermal chromatography is plagued by poor reproducibility and appears unsuitable for automation of its production, leading us to remove the thermal chroma-tography from the dry distillation. In this investigation we employed H2O solution to collect 76Br and optimized the distillation condition using a small amount of 77Br (57.0 h). We also produced large amount of 76Br under the optimized conditions to evaluate the dry distillation method. Material and Methods Target preparation and dry distillation were conducted based on the methods described in previous reports1,2. To produce 77Br, Cu2natSe target was irradiated with 20 MeV proton beams (5 µA) accelerated by AVF cyclotron in the Japan Atomic Energy Agency. The following two systems were used in the dry distillation optimization studies; (1) an initial system was composed of two furnaces, a main and an auxiliary furnace. Temperature of each furnace was set at 1050 °C (main) and 200 °C (auxiliary) respectively; (2) the second system was made of one large furnace composed of heating and cooling area. Temperature of the heating area was varied from 1050 to 1120 °C. In both systems PTFE tubing, leading to a H2O solution (15 mL), was inserted into the apparatus. The irradiated target was heated under streaming Ar gas (30 mL/min.). An enriched Cu276Se target (76Se enrichment: 99.67%) was used for 76Br production. Radioactivity was measured on a high-purity germanium (HPGe) detector coupled to a multichannel analyzer. TLC analyses were conducted on Al2O3 plates (Merck) using 7:1 acetone:H2O as the eluting solvent. Results and Conclusion Low efficiency (33 %) of 77Br recovery was ob-served in the initial system. Distribution of radioactivity inside the apparatus showed that 35 % was trapped in the PTFE tube and the quartz tube. The recovery yield was increased up to 54 % when the auxiliary furnace was turned off, indicating that the temperature gradient inside the quartz tube is suitable to carry 77Br effectively to the H2O trap. We initially used a quartz boat to place the irradiated target in the furnace, but found that using a reusable tungsten backing was better. However, we found that recovery yield was dramatically reduced to 18 %. The studies where the temperature was varied showed that releasing efficiency was increased up to 100 % at the temperature of 1120 °C. Good recovery yield (~ 77 %) was achieved after optimizing the temperature gradient (FIG. 1b). Using the optimized setup, 76Br production runs (n = 6) have been conducted, allowing us to recover up to 39.8 MBq/µAh (EOB) of 76Br. High specific activity (~4400 GBq/µmol) was obtained in the final solution. TLC analysis showed that chemical form obtained was bromide. We concluded that the dry distillation using H2O trap is capable of providing enough high purity 76Br for clinical applications.

Introduction The 80kD glycoprotein transferrin (TF) and its related receptor (TFR1) play a major role in the recruitment by cancer cells of factors for their multiplication, adhesion, invasion and metastatic potential. Though primarily designed to bind iron and then be internalised into cells with its receptor, TF can also bind most transition metals such as Co, Cr, Mn, Zr, Ni, Cu, V, In & Ga. Under certain conditions TF binds Ti (IV) even more tightly than it does Fe and that this occurs at the N-lobe (as distinct from C) of apoTF. Further, under physiological conditions the species Fe(C)Ti(N)-TF may provide the route for Ti entry into cells via TFR1 (1). Thus, the radiometal PET reporter isotope 45Ti with an ‘intermediate’ (~hrs) half-life suited to tracking cell-focused biological mechanisms is an attractive option for elucidating cellular mechanisms involving TF binding and internalisation, at least in (preclinical) animal models. 45Ti (T½ = 3.08 hr; + branching ratio = 85 %; mean β+ energy = 439keV, no significant dose-conferring non-511keV γ-emissions) was produced using the reaction 45Sc(p,n)45Ti by irradiating (monoisotopic) scandium discs with an energy-degraded proton beam produced by an 18MeV isochronous medical cyclotron. Separation and purification was achieved with an hydroxylamine hydrochloride functionalised resin. Comparative microPET imaging was performed in an immunodeficient mouse model, measuring biodistributions of the radiolabels 45Ti-oxalate and 45Ti-human-TF (45Ti-h-TF), out to 6hr post-injection. Materials and Methods High purity 15mm diameter scandium disc foils (99.5%, Goodfellow, UK) each thickness 0.100 ± 0.005 mm (55 mg) were loaded into an in-house constructed solid-targetry system mounted on a 300mm external beam line utilising helium-gas and chilled water to cool the target body (2). The proton beam was degraded to 11.7 MeV using a graphite disc integrated into the graphite collimator. This energy abolishes the competing ‘contaminant’ reactions 45Sc(p,n+p)44Sc and 45Sc(p,2n)44Ti. Beam current was measured using the well documented 65Cu(p,n)65Zn reaction. Calculations showed that the chosen energy is close to the optimal primary energy (~12 MeV) for maximising the (thin-target) yield from a 0.100 mm thick target. For separation of Ti from the Sc target two methods were examined; (i) ion exchange column separation using 2000 mg AG 50W-X8 resin conditioned with 10mL 9M HCl. Disc is dissolved in 1 mL of 9M HCl, which at completion of reaction is pipetted into column. Successive 1 mL volumes of 9M HCl are added, and subsequent elutions collected. (ii) Following Gagnon et al., (3) a method employing hydroxylamine hydro-chloride functionalised resin (’hydroxamate method’) was applied, similar to its use in our hands for purification and separation of 89Zr (2) following its original description for 89Zr by Holland et al., (4). Disc dissolved in 2mL 6M HCl, then diluted to 2M. Elute through column to waste fraction 1 (w1 – see FIG. 1). Then elute 6 mL of 2M HCl through column to w2, followed by 6 mL of traceSELECT H2O to w3. Finally, elute Ti into successive 1 mL product fractions (p1, 2 etc.) using 5 mL of 1M oxalic acid. This procedure takes approximate 1 hr. 45Ti in elution vials was measured using γ-spectroscopy. Sc in the same vials was determined later using ICP-MS. Results A typical production run using a beam current of 40 μA for 60min on a 0.100mm-thick disc produced an activity of 1.83 GBq. Radionuclidic analysis of an irradiated disc using calibrated cryo-HPGe γ-spectroscopy revealed T½ = 2.97–3.19 hr (95% CI) for 45Ti, and with contaminant 44Sc < 0.19 %, with no other isotopes detected. Despite systematic adjustments to column conditions satisfactory chemical separation was not achieved using the ion exchange column method (i), despite previous reports of its success (5). Typical results of separation using the successful hydroxamate method (ii) are shown on the FIGURE 1. It is seen that significant portion of 45Ti is lost in the initial washing steps leading to waste collection. N = 4 replicate experiments showed a variation (SD) of 10 % of the mean in each elu-tion fraction. Subsequent ICP-MS of the same elutions for (cold) Sc showed approximately 80 % by mass appeared in w1 and 20 % in w2, with negligible total mass (total fraction ~1/6000) of Sc in product (p1–4) vials. However, the FIG. 1 shows that a total of only 30% of the original activity of 45Ti (corrected to EOB) is available in the product vials, with the vial of highest specific activity (p1) containing 14 %. However, using a stack of 2×0.100mm thick Sc discs as a target yields isotope of adequate specific activity with-out need for concentration, for subsequent labelling and small-animal imaging purposes. In a ‘proof-of-principle’ experiment, two groups of healthy Balb/c-nu/nu female adult mice were administered with 45Ti radiotracers. The first group (N = 3) received approximately 20 MBq IP of 45Ti-oxalate buffered to pH = 7.0, and under-went microPET/CT imaging (Super Argus PET, Sedecal, Spain) out to 6hr post-injection, plus biodistribution analysis of radioactivity by dis-section at sacrifice (6hr). The second group (N = 3) received approximately 20 MBq IP of 45Ti-h-TF and were also studied to 6hr post-injection, followed by radioactive analysis after dissection at sacrifice. Organ and tissue biodistributions of the two groups at 6hr were similar but with 45Ti-oxalate showing slightly greater affinity for bone. Biodistribution by dissection results broadly confirmed the findings from PET images. However, TLC results suggested that similarity of radiolabel biodistributions of the two groups may be due to contamination of the TF radiolabel with non-conjugated Ti at time of injection. An alternative explanation is dechelation in vivo of 45Ti from 45Ti-h-TF. Conclusion Despite significant loss of 45Ti to the waste fractions of the separation process (total 53 %, corrected to EOB), 45Ti of acceptable specific activity and high radionuclidic purity has been produced from the reaction 45Sc(p,n)45Ti, with separation and purification of the product by hydroxamate column chemistry, confirming an earlier report. Though microPET in vivo imaging using 45Ti-based radiolabels was shown to be feasible, the similarity in the results for the label 45Ti-h-TF compared with ‘raw’ 45Ti-oxalate suggests further investigations. These may include a direct comparison of in vivo 45Ti-h-TF small-animal imaging plus post-dissection biodistribution with the same procedures using 89Zr labelled h-apotransferrin (6).

Introduction Since its development by Al Wolf and colleagues in the 1970s1, [11C]cyanide has been a useful synthon for a wide variety of reactions, most notably those producing [1-11C]-labeled amino acids2. However, despite its position as rote gas-phase product, the catalytic synthesis is difficult to optimize and often only perfunctorily dis-cussed in the radiochemical literature. Recently, [11C]CN– has been used in the synthesis of indole-3-[1-11C]acetic acid ([11C]IAA), the principal phytohormone responsible for a wide variety of growth and development functions in plants3. The University of Wisconsin has expertise in cyclotron production and radiochemistry of 11C and previous experience in the PET imaging of plants4,5. In this abstract, we present work on optimizing [11C]CN– production for the synthesis of [11C]IAA and the PET imaging of auxin transport in living plants. Material and Methods [11C]CH4 was produced by irradiating 270 psi of 90% N2, 10% H2 with 30 µA of 16.1 MeV protons from a GE PETtrace cyclotron. After irradiation, the [11C]CH4 was converted to [11C]CN– by passing through a quartz tube containing 3.0 g of Pt wire and powder between quartz wool frits inside a 800–1000 ˚C Carbolite tube furnace. The constituents and flow rate of the [11C]CH4 carrier gas were varied in an effort to optimize the oven\'s catalytic production of [11C]CN– from CH4 and NH3. The following conditions were investigated: i. Directly flowing irradiated target gas versus trapping, purging and releasing [11C]CH4 from a −178 ˚C HayeSep D column in He through the Pt furnace. ii. Varying the amount of anhydrous NH3 (99.995%) mixed with the [11C]CH4 carrier gas prior to the Pt furnace. Amounts varied from zero to 35 % of gas flow. iii. Varying the purity of the added NH3 gas with the addition of a hydride gas purifier (Entegris model 35KF), reducing O2 and H2O impurities to < 12 ppb. iv. Varying the flow rate of He gas carrying trapped, purged and released [11C]CH4. After flowing through the Pt furnace, the gas stream was bubbled through 300 µL of DMSO containing IAA precursor gramine (1 mg), then passed through a 60×5 cm column containing ascarite to absorb [11C]CO2, followed by a −178˚C Porapak Q column to trap [11C]CH4 and [11C]CO. After bubbling, the DMSO/gramine vial was heated to 140 ˚C to react the gramine with [11C]CN–, forming the intermediate indole-3-[1-11C]acetonitrile ([11C]IAN), which was subsequently purified by solid phase extraction (SPE). The reaction mixture was diluted into 20 mL water and loaded onto a Waters Sep-Pak light C18 cartridge, followed by rinsing with 5 mL of 0.1% HCl : acetonitrile (99 : 1) and 10 mL of the same mixture in ratio 95 : 5, and finally eluted with 0.5 mL of diethyl ether. The ether was subsequently evaporated under argon flow, followed by the hydrolysis of [11C]IAN to [11C]IAA with the addition of 300 µL 1 M NaOH and heating to 140 ˚C for 5 minutes. After hydrolysis, the solution was neutralized with 300 µL 1 M HCl and purified using preparative high-performance liquid chromatography (HPLC) using a Phenomenex Luna C18 (10μ, 250×10mm) column with a mobile phase acetonitrile : 0.1% formic acid in H2O (35 : 65) at flow rate of 3 mL/min. The [11C]IAA peak, eluting at 12 minutes, was collected and rotary evaporated to dryness, then again after the addition of 5 mL acetonitrile, followed by its reconstitution in 50 µL of water. Analytical HPLC was performed on the [11C]IAA before and after this evaporation procedure using a Phenomenex Kinetex C18 (2.6μ, 75× 4.6 mm) column with a linear gradient elution over 20 minutes of 10 : 90–30 : 70 (acetonitrile : 0.1% formic acid) at a 1 mL/min flow rate, eluting at 7.6 minutes. The transport of [11C]IAA was monitored following administration through the severed petiole of rapid cycling Brassica oleracea (rcBo) using a Siemens microPET P4 scanner. Transport was compared following administration to the first true leaf versus the final fully formed leaf in plants with and without exposure to the polar auxin transport inhibitor naphthylphthalamic acid (NPA). Results and Conclusion Optimization of the [11C]CN– gas phase chemistry was performed using two key metrics for measuring conversion yield. First is the fraction of total produced radioactivity that trapped in the DMSO/gramine solution (denoted %DMSO), and second, the fraction of DMSO/gramine-trapped activity that was able to react with gramine to form [11C]IAN (denoted %CN–). Under certain conditions, the former of these metrics experienced significant losses due to unconverted [11C]CH4 or through combustion, forming [11C]CO2 or [11C]CO. The latter metric experienced losses due to production of incomplete oxidation products of the CH4-NH3 reaction, such as methylamine. Total [11C]CH4 to [11C]CN– con-version yields is reported by the product of the two metrics. It was initially hypothesized that the irradiation of a 90% N2, 10% H2 target gas would produce sufficient in-target-hot-atom-produced NH3 to convert [11C]CH4 to [11C]CN– in the Pt furnace. However, conversion yields were found to be low and highly variable, with 13 ± 8 % trapping in DMSO/gramine, 9 ± 9 % of which reacted as CN– (n = 15). While in disagreement with previous reports1, this is likely as a result the batch irradiation conditions resulting ammonia losses in the target chamber and along the tubing walls. Yields and reproducibility were improved when combining the target gas with a stream of anhydrous NH3 gas flow with conversion yields reported in TABLE 1. However, these yields remained undesirably low, potentially as a result of the 10% H2 carrier gas having an adverse effect on the oxidative conversion of [11C]CH4 to [11C]CN–. To remedy this, the irradiated target gas was trapped, purged, released in He and combined with NH3 gas before flowing through the Pt furnace. Initial experiments using 99.995% anhydrous NH3 gas resulted in very poor (< 0.1%) [11C]CN– yields as a result of nearly quantitative combustion forming [11C]CO2. Installation of a hydride gas purifier to reduce O2 and H2O impurities in NH3 improved yields for CH4 in He, but did not significantly affect those from [11C]CH4 in N2/H2 target gas. In disagreement with previous reports2, conversion yields were found to be highly sensitive to overall carrier gas flow rate, with lower flow rates giving the best yields, as shown in TABLE 1. Optimization experiments are continuing. The total decay-corrected yield for the 1 hour synthesis of [11C]IAA in 50 µL of water is 2.3 ± 0.7 %, based on the total produced [11C]CH4 with a specific activity ranging from 1–100 GBq/µmol. The principal radiochemical impurity was determined to be indole-3-carboxylic acid. The SPE procedure isolating the [11C]IAN intermediate product was optimized to minimize this impurity in the final sample. After a rapid distribution of the administered [11C]IAA through the cut petiole and throughout the rcBO plant, upward vascular transport of auxin and downward polar auxin transport was visualized through time-activity curves (TACs) of regions of interest along the shoot. Comparison of these TACS with and without exposure to NPA yields insight into the fundamental physiological process of polar auxin transport in plants. In conclusion, the Pt-catalyzed oxidative conversion of [11C]CH4 and NH3 to [11C]CN– is a challenging process to optimize and highly sensitive to carrier gas composition and flow rate. Optimization for our experimental conditions yielded several results which disagreed with previous reports. [11C]IAA produced using [11C]CN– is well suited for PET imaging of polar auxin transport in living plants.

Der Programmed Death-Ligand 1 (PD-L1) wird von verschiedenen Tumorentitäten überexprimiert und hemmt durch die Bindung an das auf T-Zellen vorkommende Programmed Cell Death Protein (PD-1) die Immunantwort. Immuncheckpoint-Inhibitoren sind in der Lage, diese Blockade aufzubrechen und die Immunantwort zu reaktivieren. Auf eine solche Monotherapie sprechen aber nur etwa 30 % der Patienten an, sodass klinischer Bedarf an nichtinvasiven, bildgebenden Diagnosemethoden besteht. Eine Möglichkeit stellen Radioliganden für die nichtinvasive Bildgebung mittels Positronen-Emissions-Tomographie (PET) oder Einzelphotonen-Emissionscomputertomographie (SPECT) dar, welche einen Therapieerfolg prognostizieren könnten. Die vorliegende Dissertationsschrift beschäftigt sich mit der Entwicklung und Synthese einer neuen Klasse von PD-L1-adressierenden Radioliganden sowie deren biologischer Evaluierung in Zellen (in vitro) und in Kleintieren (in vivo). Nach der Identifikation von drei hochaffinen PD-L1-Inhibitoren in der Patentliteratur, wurden zunächst diese als Referenzverbindungen in einer neuen, konvergenten Synthesestrategie dargestellt. Diese ermöglichte nicht nur eine höhere Gesamtausbeute der Substanzen, sondern erlaubte auch dank des modularen Aufbaus den einfachen Zugang zu einer großen Substanzbibliothek an PD-L1-Radioliganden. Mithilfe einer Kokristallstruktur aus PD-L1 und einem Inhibitor wurden Modifikationen des Inhibitors für eine Transformation in einen Radioliganden geplant. Zur Erhöhung der Hydrophilie der Substanzen wurden löslichkeitsvermittelnde Gruppen (Sulfon-/Phosphonsäuren) in dem Molekülteil eingeführt, welcher in der „Solvent-Exposed-Region“ des Proteins lokalisiert ist. Funktionalisierung des Bindungsmotivs mit einem Alkin erlaubte die Einführung eines Linker-Chelator-Konstruktes mittels kupferkatalysierter Azid-Alkin-Cycloaddition (CuAAC) zur Komplexierung des Radiometalls. In der ersten Generation an PD-L1-Radioliganden wurden zwei Bindungsmotive und drei verschiedenen Linker erprobt, sodass nach Konjugation mit dem DOTA-Chelator sechs PD-L1-Radioliganden zur Verfügung standen. Nach erfolgreicher Radiomarkierung mit 64Cu, 68Ga und 177Lu erfolgten mit den 64Cu-markierten Radioliganden Bindungsaffinitätsbestimmungen in einem Sättigungsbindungsassay (Zugabe von 2,5 % BSA im Medium) an transduzierten PC3 PD-L1-Zellen. Dabei erwies sich [64Cu]Cu-62 mit Dimethylbiphenyl als Bindungsmotiv und Piperazinpropyl als Linker mit einem KD-Wert von 60 nM als der vielversprechendste Radiotracer, sodass dieses Bindungsmotiv als Grundlage für die folgenden Radioliganden dienen sollte. Nach bestätigter In-vitro-Stabilität der Radioliganden in verschiedenen Medien (PBS, Humanserum, gegenüber Lebermikrosomen), wurden für alle sechs Verbindungen aus der ersten Generation µPET-Aufnahmen in tumortragenden Mäusen durchgeführt. Die Aufnahmen zeigten eine geringe Akkumulation im PD-L1-positiven Tumor, eine hepatobiliäre Ausscheidung trotz niedriger log D7,4-Werten von –2,73 bis –3,50 und für kleine Moleküle ungewöhnlich lange Zirkulationszeiten. Diese konnten mit Gelelektrophorese und Größenausschlusschromatographie auf eine ausgeprägte Albuminbindung der Radioliganden zurückgeführt werden. In Echtzeit-Radioliganden-Bindungsversuchen mittels LigandTracer® konnten für die meisten Radioliganden einstellige, nanomolare KD-Werte erzielt werden. In Abwesenheit von Albumin zeigen diese Bindungsaffinitäten mit hoher Wahrscheinlichkeit die reine Bindung an PD-L1. Um die Tumoraufnahme zu erhöhen, wurden für die PD-L1-Radioliganden der zweiten Generation die funktionelle Gruppe am Pyridinring und das Halogen am zentralen Aromaten ausgetauscht. Trotz höherer KD-Werte in Sättigungsbindungsversuchen zeigten die µPET-Aufnahmen eine erhöhte Tumoraufnahme für das Brom- ([64Cu]Cu-115) und Iodderivat ([64Cu]Cu-116), vermutlich aufgrund höherer Bmax-Werte. Um eine Transchelatierung zu vermeiden, wurde für die PD-L1-Radioliganden der dritten Generation DOTA gegen NODA-GA als Chelator ausgetauscht. Außerdem wurde auf eine Verbesserung des pharmakokinetischen Profils abgezielt: Mittels Einführung von weiteren Sulfonsäuren und deren partiellen Austausch durch Phosphonsäuren sollte zum einen die Albuminbindung verringert und zum anderen die renale Ausscheidung begünstigt werden. Die sechs Radioliganden mit bis zu vier Sulfon-/Phosphonsäuren zeigten, abhängig von der Art und Anzahl der löslichkeitsvermittelnden Gruppe, stark unterschiedliche pharmakokinetische Profile. Dabei überzeugte der Radioligand [64Cu]Cu-148 mit einem KD-Wert von 94 nM im Sättigungsbindungsversuch und einem log D7,4-Wert von –3,80 insbesondere in der µPET-Bildgebung: Größtenteils renale Ausscheidung, moderate Tumoraufnahme (SUVmax = 3,5) und ausgeprägtes Signal-zu-Hintergrund-Verhältnis zum PD-L1-negativen Tumor. Die vierte Generation sollte das wünschenswerte pharmakokinetische Profil des Radioliganden [64Cu]Cu-148 mit den höheren Bmax-Werten der Radioliganden der zweiten Generation kombinieren. Die µPET-Aufnahmen zeigten für alle vier synthetisierten Radioliganden sehr geringe Tumoraufnahmen, dafür ein unerwartetes pharmakokinetisches Profil mit hoher Leberaufnahme – trotz niedriger log D7,4-Werte – und eine Akkumulation in Knochen/Knochenmark und Gelenken. Es wird vermutet, dass es trotz des räumlichen Abstandes der beiden Phosphonsäuren zu einer Ca2+-vermittelten Knochenanreicherung und/oder einer Bindung an anderen biologischen Strukturen wie Makrophagen kommt. Dank des modularen Aufbaus und der Einführung einer Alkin-Funktionalität wird über 18F-prosthetische Gruppen ein einfacher Zugang zu 18F-Radioliganden mittels CuAACReaktionen eröffnet, für welche bereits 19F-Referenzverbindungen und Markierungsvorläufer synthetisiert wurden. Nach weiteren Optimierungen des pharmakokinetischen Profils und damit kürzeren Zirkulationszeiten soll dies zukünftig den Zugang zu vielversprechenden 18F-Radiotracern ohne Chelator-Linker-Einheit ermöglichen.

Introduction: Prostate-specific membrane antigen (PSMA)-based imaging and theranostics have played an important ole in the diagnosis, staging, and treatment of prostate cancer (PCa). We aimed to evaluate the acceptance and use of PSMA theranostics among German urologists.- Methods: An anonymous online questionnaire was sent via survio.com to the members of the German Society of Urology (DGU). - Results: Seventy-two percent of participants performed PSMA positron emission tomography (PET) imaging regularly in biochemically recurrent PCa. Overall, 61% of participants considered PSMA-radioligand therapy to be very useful or extremely useful. PSMA PET imaging in high-risk PCa is more often considered by urologists working in a university setting than in nonuniversity settings or medical practices (51% vs. 25%, p < 0.001). Most perform PSMA-radioligand therapy as an option after all approved systemic treatments for metastatic metastatic castration-resistant PCa (56%) or after cabazitaxel (14%). A total of 93.9% and 70.3% of respondents consider the lack of reimbursement by health insurance to be the main obstacle to using PSMA PET imaging or radioligand therapy, respectively. - Discussion/Conclusion: PSMA-based maging/theranostics are already widely applied but would find even more widespread use if reimbursement is clearly regulated by health insurance in Germany.