Stephan Maus

DNA Double Strand Breaks as Predictor of Efficacy of the Alpha-Particle Emitter Ac-225 and the Electron Emitter Lu-177 for Somatostatin Receptor Targeted Radiotherapy

Rationale Key biologic effects of the alpha-particle emitter Actinium-225 in comparison to the beta-particle emitter Lutetium-177 labeled somatostatin-analogue DOTATOC in vitro and in vivo were studied to evaluate the significance of γH2AX-foci formation. Methods To determine the relative biological effectiveness (RBE) between the two isotopes (as - biological consequence of different ionisation-densities along a particle-track), somatostatin expressing AR42J cells were incubated with Ac-225-DOTATOC and Lu-177-DOTATOC up to 48 h and viability was analyzed using the MTT assay. DNA double strand breaks (DSB) were quantified by immunofluorescence staining of γH2AX-foci. Cell cycle was analyzed by flow cytometry. In vivo uptake of both radiolabeled somatostatin-analogues into subcutaneously growing AR42J tumors and the number of cells displaying γH2AX-foci were measured. Therapeutic efficacy was assayed by monitoring tumor growth after treatment with activities estimated from in vitro cytotoxicity. Results Ac-225-DOTATOC resulted in ED50 values of 14 kBq/ml after 48 h, whereas Lu-177-DOTATOC displayed ED50 values of 10 MBq/ml. The number of DSB grew with increasing concentration of Ac-225-DOTATOC and similarly with Lu-177-DOTATOC when applying a factor of 700-fold higher activity compared to Ac-225. Already 24 h after incubation with 2.5–10 kBq/ml, Ac-225-DOTATOC cell-cycle studies showed up to a 60% increase in the percentage of tumor cells in G2/M phase. After 72 h an apoptotic subG1 peak was also detectable. Tumor uptake for both radio peptides at 48 h was identical (7.5%ID/g), though the overall number of cells with γH2AX-foci was higher in tumors treated with 48 kBq Ac-225-DOTATOC compared to tumors treated with 30 MBq Lu-177-DOTATOC (35% vs. 21%). Tumors with a volume of 0.34 ml reached delayed exponential tumor growth after 25 days (44 kBq Ac-225-DOTATOC) and after 21 days (34 MBq Lu-177-DOTATOC). Conclusion γH2AX-foci formation, triggered by beta- and alpha-irradiation, is an early key parameter in predicting response to internal radiotherapy.

Labelling of commercially available human serum albumin kits with 68Ga as surrogates for 99mTc-MAA microspheres

An alternative method of labelling different commercially available human serum albumin kits with generator produced (68)Ga as possible surrogate markers for the investigation of the biodistribution, and to exclude a possible lung shunt in patients with solid primary tumours and liver metastases by positron emission-tomography (PET) and effective substitute for (99m)Tc HSA as a PET perfusion tracer is described. The radioactive labelling with (68)Ga was based on the elution of a commercial (68)Ge/(68)Ga generator system, which uses a modified tin dioxide column to absorb (68)Ga. We obtained (68)Ga-MAA and (68)Ga-HSA in radiochemical yields of 79 ± 5% with less than 5% free (68)Ga. The whole procedure takes a total of less than 30 min from elution to the final product.

Predicting the in vivo release from a liposomal formulation by IVIVC and non-invasive positron emission tomography imaging.

This study aimed to predict the in vivo performance from the in vitro release of a low-molecular weight model compound, [(18)F]-2-fluoro-2-deoxy-d-glucose ([(18)F]FDG), from liposomes and by means of positron emission tomography (PET). Liposomes composed of hydrogenated phosphatidylcholine (HPC) were prepared by a freeze-thaw method. Particle size distribution was measured by dynamic light scattering (DLS). In vitro release was examined with a dispersion method detecting the radioactivity of [(18)F]FDG. In vivo release of [(18)F]FDG, following i.p. injection of the liposomes in rats, was determined by using a Micro-PET scanner. Convolution was performed to predict the in vivo profiles from the in vitro data and to establish an in vitro-in vivo correlation (IVIVC). The in vivo predictions slightly underestimated the experimentally determined values. The magnitude of the prediction errors (13% and 19%) displayed a satisfactory IVIV relationship leaving yet room for further improvement. This study demonstrated for the first time the use of PET in attaining an IVIVC for a parenterally administered modified release dosage form. It is therefore possible to predict target tissue concentrations, e.g., in the brain, from in vitro release experiments. IVIVC using non-invasive PET imaging could thus be a valuable tool in drug formulation development, resulting in reduced animal testing.

Evaluation of cannabinoid type 1 receptor expression in the rat brain using [18F]MK-9470 microPET

PurposeThe ligand [18F]MK-9470 is an inverse agonist binding with high affinity and specificity to the cannabinoid type 1 (CB1) receptor. In this study, a semiquantitative acquisition and analysis protocol for investigation of the CB1 receptor distribution in the rat brain was established.MethodsTwo C57BL/6N mice (one CB1−/− and one wild-type) and 19 Sprague Dawley rats were investigated using a Focus 120 microPET scanner. Seven rats were scanned twice for test–retest evaluation, six rats were scanned for blocking experiments using the inverse CB1 receptor agonist rimonabant, and 19 rats were scanned for baseline studies. Percentage injected dose per millilitre (%ID/ml) or uptake ratios (VOItarget/VOIwhole brain) were calculated. A Bland-Altman-plot was computed and mean values were compared using a two-sided paired t test.ResultsComparing the data from the CB1−/− mouse and the wild-type mouse, [18F]MK-9470 showed good specificity. Regarding the rat data, there was no relationship between the difference between the test and retest measurements or their mean value. The test and retest data showed a strong correlation (ρc = 0.846, p ≤ 0.01; rPearson = 0.857). Equivalence was not found for all regions and not even in the pons at baseline or under blocking condition. Only the baseline studies showed the highest levels of uptake in the caudate-putamen and thalamus, whereas moderate uptake was found in the hippocampus, hypothalamus and cerebellum, and the lowest uptake was observed in the cortex, amygdala and pons.ConclusionA reference region is not available; however, the proposed analysis method using the parameter uptake ratio is simple and delivers stable results allowing the discrimination of distinct brain regions.

Effects of tetrahydrocannabinol on glucose uptake in the rat brain

&NA; &Dgr;9‐Tetrahydrocannabinol (THC) is the psychoactive component of the plant Cannabis sativa and acts as a partial agonist at cannabinoid type 1 and type 2 receptors in the brain. The goal of this study was to assess the effect of THC on the cerebral glucose uptake in the rat brain. 21 male Sprague Dawley rats (12–13 w) were examined and received five different doses of THC ranging from 0.01 to 1 mg/kg. For data acquisition a Focus 120 small animal PET scanner was used and 24.1–28.0 MBq of [18F]‐fluoro‐2‐deoxy‐D‐glucose were injected. The data were acquired for 70 min and arterial blood samples were collected throughout the scan. THC, THC‐OH and THC‐COOH were determined at 55 min p.i. Nine volumes of interest were defined, and the cerebral glucose uptake was calculated for each brain region. Low blood THC levels of < 1 ng/ml (injected dose: ≤ 0.01 mg/kg) corresponded to an increased glucose uptake (6–30 %), particularly in the hypothalamus (p = 0.007), while blood THC levels > 10 ng/ml (injected dose: ≥ 0.05 mg/kg) coincided with a decreased glucose uptake (−2 to −22 %), especially in the cerebellar cortex (p = 0.008). The effective concentration in this region was estimated 2.4 ng/ml. This glucose PET study showed that stimulation of CB1 receptors by THC affects the glucose uptake in the rat brain, whereby the effect of THC is regionally different and dependent on dose – an effect that may be of relevance in behavioural studies. HighlightsTHC affects the glucose uptake primarily in limbic structures and the cerebellar cortex.An increase of the glucose uptake in the rat brain was observed at rather low THC blood concentrations <1 ng/ml.High THC blood concentrations >10 ng/ml yielded a decreased glucose uptake.

Pharmacokinetics of the cannabinoid receptor ligand [18F]MK‐9470 in the rat brain – Evaluation of models using microPET

PURPOSE The positron emission tomography ligand [18 F]MK-9470 is an inverse agonist that binds reversibly and with high affinity to the cannabinoid type 1 receptor. Due to its slow brain kinetics, care is required in the definition of its dissociation rates from the receptor. The goal of this study was to investigate pharmacokinetic analysis methods using an arterial input function. METHODS Five Sprague-Dawley rats received injections of 13 to 25 MBq of [18 F]MK-9470 and were scanned over a period of 90 min. Arterial blood samples were collected throughout the scan. Data were analyzed using four different compartmental models: a reversible one-tissue model, reversible two tissue models with and without parameter constraints and an irreversible two-tissue model. The outcome values were goodness of fit measures (Akaike information criterion; standard error), pharmacokinetic modeling parameters (volume of distribution; irreversible uptake constant) and intersubject variability. RESULTS Goodness of fit measures indicated that the experimental data are more adequately described by a two-tissue model than a one-tissue model. Differences in mean Akaike information criterion values between all two-tissue models were < 5%. Mean standard errors of model parameters were lowest for the irreversible model (range: 1% to 6%). The irreversible model delivered plausible results for all animals that were less variable compared to results of the other two-tissue models. CONCLUSIONS A reversible two-tissue model may not deliver stable results for all animals and regions within a 90-min microPET study protocol. Stable parameters for all animals and regions are obtained when an irreversible model is used. If the acquisition time of the experiment is limited, an irreversible model provides a consistent distribution of composite outcome parameters, suggesting its suitability for use in future studies.

Acute effect of intravenously applied alcohol in the human striatal and extrastriatal D2/D3 dopamine system

Investigations on the acute effects of alcohol in the human mesolimbic dopamine D2/D3 receptor system have yielded conflicting results. With respect to the effects of alcohol on extrastriatal D2/D3 dopamine receptors no investigations have been reported yet. Therefore we applied PET imaging using the postsynaptic dopamine D2/D3 receptor ligand [18F]fallypride addressing the question, whether intravenously applied alcohol stimulates the extrastriatal and striatal dopamine system. We measured subjective effects of alcohol and made correlation analyses with the striatal and extrastriatal D2/D3 binding potential. Twenty‐four healthy male μ‐opioid receptor (OPRM1)118G allele carriers underwent a standardized intravenous and placebo alcohol administration. The subjective effects of alcohol were measured with a visual analogue scale. For the evaluation of the dopamine response we calculated the binding potential (BPND) by using the simplified reference tissue model (SRTM). In addition, we calculated distribution volumes (target and reference regions) in 10 subjects for which metabolite corrected arterial samples were available. In the alcohol condition no significant dopamine response in terms of a reduction of BPND was observed in striatal and extrastriatal brain regions. We found a positive correlation for ‘liking’ alcohol and the BPND in extrastriatal brain regions (Inferior frontal cortex (IFC) (r = 0.533, p = 0.007), orbitofrontal cortex (OFC) (r = 0.416, p = 0.043) and prefrontal cortex (PFC) (r = 0.625, p = 0.001)). The acute alcohol effects on the D2/D3 dopamine receptor binding potential of the striatal and extrastriatal system in our experiment were insignificant. A positive correlation of the subjective effect of ‘liking’ alcohol with cortical D2/D3 receptors may hint at an addiction relevant trait.