Antita Adriaens

Analytical characterization and comparison of the blood–brain barrier permeability of eight opioid peptides

Opioid drugs, including the newly developed peptides, should penetrate the blood-brain barrier (BBB) for pain management activity. Although BBB transport is fragmentarily described for some mu-opioid peptides, a complete and comparative overview is currently lacking. In this study, the BBB transport of eight opioid peptides (EM-1, EM-2, CTAP, CTOP, DAMGO, dermorphin, TAPP and TAPS) is described and compared. In addition, the metabolic stability in plasma and brain was evaluated. The highest influx rate was obtained for dermorphin (K(in)=2.18 microl/(g x min)), followed by smaller rates for EM-1, EM-2 and TAPP (K(in)=1.06-1.14 microl/(g x min)). Negligible influx was observed for DAMGO, CTOP and TAPS (K(in)=0.18-0.40 microl/(g x min)) and no influx for CTAP. Capillary depletion revealed that all peptides reached brain parenchyma for over 75%. Efflux was shown for TAPP (t(1/2)=2.82 min) and to a lesser extent for EM-1, EM-2 and DAMGO (t(1/2)=10.66-21.98 min), while no significant efflux was observed for the other peptides. All peptides were stable in mouse plasma and brain, with generally higher stability in brain, except for EM-1 and EM-2 which showed plasma half-life stabilities of a few minutes only.

Desirability function combining metabolic stability and functionality of peptides

The evaluation of peptides as potential therapeutic or diagnostic agents requires the consideration of several criteria that are targeted around two axes: functionality and metabolic stability. Most often, a compromise has to be made between these mutually opposing characteristics.

The Influence of Morphine on Cerebral 5-HT2A Availability in Dogs: a SPECT Study

The opioid and serotonergic systems are closely involved in pain processing and mood disorders. The aim of this study was to assess the influence of systemic morphine on cerebral serotonin 2A receptor (5-HT2A) binding in dogs using SPECT with the 5-HT2A radioligand 123I-5I-R91150. Methods: 5-HT2A binding was estimated with and without morphine pretreatment in 8 dogs. The 5-HT2A binding indices in the frontal, parietal, temporal, and occipital cortex and in the subcortical region were obtained by semiquantification. Results: A significantly decreased 5-HT2A binding index was found in the morphine group for the right (morphine, 1.41 ± 0.06; control, 1.52 ± 0.10) and left (morphine, 1.44 ± 0.08; control, 1.55 ± 0.11) frontal cortices, with P = 0.012 and P = 0.040, respectively. No significant differences were noted for the other regions. Conclusion: Morphine decreased the frontocortical 5-HT2A availability, confirming an interaction between the 5-HTergic and the opioid systems. Whether this interaction is caused by decreased receptor density due to direct internalization or is the result of indirect actions, such as increased endogenous serotonin release, remains to be elucidated.

NORMAL REGIONAL DISTRIBUTION OF CEREBRAL BLOOD FLOW IN DOGS: COMPARISON BETWEEN 99mTc‐ETHYLCYSTEINATE DIMER AND 99mTc‐ HEXAMETHYLPROPYLENE AMINE OXIME SINGLE PHOTON EMISSION COMPUTED TOMOGRAPHY

Functional imaging provides important insights into canine brain pathologies such as behavioral problems. Two (99m) Tc-labeled single photon emission computed tomography (SPECT) cerebral blood flow tracers-ethylcysteinate dimer (ECD) and hexamethylpropylene amine oxime (HMPAO)-are commonly used in human medicine and have been used previously in dogs but intrasubject comparison of both tracers in dogs is lacking. Therefore, this study investigated whether regional distribution differences between both tracers occur in dogs as is reported in humans. Eight beagles underwent two SPECT examinations first with (99m) Tc-ECD and followed by (99m) Tc-HMPAO. SPECT scanning was performed with a triple head gamma camera equipped with ultrahigh resolution parallel hole collimators. Images were reconstructed using filtered backprojection with a Butterworth filter. Emission data were fitted to a template permitting semiquantification using predefined regions or volumes of interest (VOIs). For each VOI, perfusion indices were calculated by normalizing the regional counts per voxel to total brain counts per voxel. The obtained perfusion indices for each region for both tracers were compared with a paired Students T-test. Significant (P < 0.05) regional differences were seen in the subcortical region and the cerebellum. Both tracers can be used to visualize regional cerebral blood flow in dogs, however, due to the observed regional differences, they are not entirely interchangeable.

The effect of morphine on regional cerebral blood flow measured by 99mTc-ECD SPECT in dogs.

To gain insights into the working mechanism of morphine, regional cerebral blood flow (rCBF) patterns after morphine administration were assessed in dogs. In a randomized cross-over experimental study, rCBF was estimated with 99mTc-Ethylcysteinate Dimer single photon emission computed tomography in 8 dogs at baseline, at 30 minutes and at 120 minutes after a single bolus of morphine. Perfusion indices (PI) in the frontal, parietal, temporal and occipital cortex and in the subcortical and cerebellar region were calculated. PI was significantly decreased 30 min after morphine compared to baseline in the right frontal cortex. The left parietal cortex and subcortical region showed a significantly increased PI 30 min after morphine compared to baseline. No significant differences were noted for the other regions or at other time points. In conclusion, a single bolus of morphine generated a changing rCBF pattern at different time points.

The effect of prolonged exposure to morphine on canine cerebral 5-HT2A receptors measured with 123I-R91150 SPECT

Down-stream neuronal alterations, including changes in the 5-HT-2A receptor system, play an important role in the etiology and treatment of depression. The present study examined the effect of prolonged opioid treatment on cerebral 5-HT2A receptors. Cerebral 5-HT2A receptor availability was estimated in seven healthy five-year-old female neutered Beagle dogs pre and post 10-day morphine treatment (oral sustained release morphine 20mg twice daily for 10 days) with (123)I-R-91150, a 5-HT2A selective radioligand, and SPECT. 5-HT2A receptor binding indices (BI) for the frontal, parietal, temporal and occipital cortex and the subcortical region were calculated. Statistical analysis was performed using a linear mixed-effect model with treatment as fixed effect and dog as random effect. Morphine treatment significantly (P≤0.05) lowered 5-HT2A BIs in the right and left frontal cortex, the right and left temporal cortex, the right and left parietal cortex, and the subcortical region. The decreased cerebral 5-HT2A receptor availability following prolonged morphine exposure provides further evidence for an interaction between the opioid and serotonergic system.

Morphine decreases 5-HT2A receptor binding measured with SPECT in the canine frontal cortex

Objectives: paraneoplastic encephalitis is often linked to antibody induced neuronal decay. We describe an early detection of cerebellar affection using 18F‐FDG PET/CT while MRI and CT did not show significant changes at first. After time, specific cerebellar antibodies against rho‐GTPase‐activating protein 26 were identified and are suspected to be responsible for the symptoms. Methodology: sequential 18F‐FDG (N=6) every 6 months were done for whole body staging presuming paraneoplastic cause. In addition, five brain scans were done separately according to our paraneoplastic standard protocol. PET scans were analyzed using ROI comparison. Clinical progress was monitored using Klockgether Ataxy score and neurological examination, chemistry and sophisticated analytics of potential antibody targets were performed. In addition, MRI and CT brain were performed. Results: Decrease of cerebellar metabolism correlated with progression of clinical symptoms while neither MRI nor CT were able to show intraaxial pathologies at first. Meanwhile, MRI demonstrates a corresponding cerebellar atrophy. Statistical analysis regarding SUV changes in the cerebellum, antibody titer and Klockgether ataxia score are ongoing. Initial results demonstrate a close relation with progress of cerebellar hypometabolism. Up to date, no cause of a potential paraneoplastic origin was found. Conclusions: First results give evidence of an association of early changes in 18F‐FDG brain scan with newly identified antibody induced structural decay of the brain. In addition, 18F‐FDG PET/CT was able to show early changes in cerebellar metabolism compared to morphological imaging modalities. P280 Small animal PET imaging: Is strict standardization the key to more robust data? M. I. Kehl, M. Honer, P. A. Schubiger, S. M. Ametamey; ETH Zürich, Zürich, SWITZERLAND. Small animal PET imaging is a frequently used methodology to investigate the rodent brain in healthy and disease states. Non‐invasive imaging methods like PET allow longitudinal follow‐up studies, where animals can be used as their own controls. Such set‐ups are considered to give more reliable results than the traditional control group approach. In this study, the inherent variability of FDG and fallypride brain uptake within and between individual animals was assessed using a fixed protocol. Moreover, experimental factors were elicited, which might essentially impact the outcome of rodent PET studies and therefore require strict standardization. Finally, the potential benefit of standardization over heterogenization was evaluated for an experimental setting with varying parameters regarding age, gender and cage occupancy. Methods: Animals underwent fixed protocols of repeated FDG or fallypride scans using the dedicated small‐animal PET/CT scanner eXplore Vista. Each animal was measured three times with one week recovery phase between scans. SUVs determined by ROI analysis were compared between scans of the same animal (intra‐subject variability) and scans of different individuals (inter‐subject variability). Furthermore, several FDG scans were performed under variation of various experimental parameters, e.g. gender, age, cage occupancy, anaesthetic protocol, environmental temperature during uptake phase and 10% ethanol in tracer solution. Animals were sacrificed subsequent to the PET scan; organs were collected and measured by classical gamma counting. Results: No significant difference between inter‐ and intra‐animal FDG/fallypride uptake variability was identified (COV=14±7% vs. 21±10% for FDG). Significant differences in fallypride and FDG brain uptake were found between scans of different test days, suggesting false positive effects. Biodistribution was robust in terms of experimental parameters; only anaesthetic protocols proved to significantly impact brain uptake (27% less uptake for animals anaesthetized over 60min vs. 40min). In contrast to heterogenization, standardization of groups produced substantially more false positive effects in FDG organ distribution (false positive rate: 9% vs. 6%). Conclusion: Repeated measurements on the same animal did not reduce data variability of FDG/fallypride brain uptake compared to measurements on different animals. Strict standardization of experimental parameters is not advisable, as it produces high numbers of idiosyncratic results with no external validity. Except for anaesthetic protocols, all investigated parameters did not impact brain uptake significantly. Controlled heterogenization of test groups with regard to gender, age, cage occupancy and laboratory climate is therefore recommended, as it decreases the generation of false positive results and thus increases external validity of study