Background[11C]Cimbi-36 is a serotonin 2A receptor agonist positron emission tomography radioligand that has recently been examined in humans. The binding of agonist radioligand is expected to be more sensitive to endogenous neurotransmitter concentrations than antagonist radioligands. In the current study, we compared the effect of serotonin releaser fenfluramine on the binding of [11C]Cimbi-36, [11C]MDL 100907 (a serotonin 2A receptor antagonist radioligand), and [11C]AZ10419369 (a serotonin 1B receptor partial agonist radioligand with established serotonin sensitivity) in the monkey brain.MethodsEighteen positron emission tomography measurements, 6 for each radioligand, were performed in 3 rhesus monkeys before or after administration of 5.0 mg/kg fenfluramine. Binding potential values were determined with the simplified reference tissue model using cerebellum as the reference region.ResultsFenfluramine significantly decreased [11C]Cimbi-36 (26–62%) and [11C]AZ10419369 (35–58%) binding potential values in most regions (P < 0.05). Fenfluramine-induced decreases in [11C]MDL 100907 binding potential were 8% to 30% and statistically significant in 3 regions. Decreases in [11C]Cimbi-36 binding potential were larger than for [11C]AZ10419369 in neocortical and limbic regions (~35%) but smaller in striatum and thalamus (~40%). Decreases in [11C]Cimbi-36 binding potential were 0.9 to 2.8 times larger than for [11C]MDL 100907, and the fraction of serotonin 2A receptor in the high-affinity state was estimated as 54% in the neocortex.ConclusionsThe serotonin sensitivity of serotonin 2A receptor agonist radioligand [11C]Cimbi-36 was higher than for antagonist radioligand [11C]MDL 100907. The serotonin sensitivity of [11C]Cimbi-36 was similar to [11C]AZ10419369, which is one of the most sensitive radioligands. [11C]Cimbi-36 is a promising radioligand to examine serotonin release in the primate brain.

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Historically, the focus has been to use in vitro BBB models to optimize rate of drug delivery to the CNS, whereas total in vivo brain/plasma ratios have been used for optimizing extent. However, these two parameters do not necessarily show good correlations with receptor occupancy data or other pharmacological readouts. In line with the free drug hypothesis, the use of unbound brain concentrations (Cu,br) has been shown to provide the best correlations with pharmacological data. However, typically the determination of this parameter requires microdialysis, a technique not ideally suited for screening in early drug development. Alternative, and less resource-demanding methodologies to determine Cu,br employ either equilibrium dialysis of brain homogenates or incubations of brain slices in buffer to determine fraction unbound brain (fu,br), which is subsequently multiplied by the total brain concentration to yield Cu,br. To determine Cu,br/Cu,pl ratios this way, still requires both in vitro and in vivo experiments that are quite time consuming. The main objective of this study was to explore the possibility to directly generate Cu,br/Cu,pl ratios in a single in vitro model of the BBB, using a co-culture of brain capillary endothelial and glial cells in an attempt to mimick the in vivo situation, thereby greatly simplifying existing experimental procedures. Comparison to microdialysis brain concentration profiles demonstrates the possibility to estimate brain exposure over time in the BBB model. A stronger correlation was found between in vitro Cu,br/Cu,pl ratios and in vivo Cu,br/Cu,pl obtained using fu,br from brain slice than with fu,br from brain homogenate for a set of 30 drugs. Overall, Cu,br/Cu,pl ratios were successfully predicted in vitro for 88% of the 92 studied compounds. This result supports the possibility to use this methodology for identifying compounds with a desirable in vivo response in the CNS early on in the drug discovery process.

Neuropil deposition of beta-amyloid (Aβ) peptides is believed to be a key event in the neurodegenerative process of Alzheimer's disease (AD). An early and consistent clinical finding in AD is olfactory dysfunction with associated pathology. Interestingly, transgenic amyloid precursor protein (Tg2576) mice also show early amyloid pathology in olfactory regions. Moreover, a recent study indicates that axonal transport is compromised in the olfactory system of Tg2576 mice, as measured by manganese-enhanced magnetic resonance imaging (MEMRI). Here we tested whether the putative axonal transport deficit in the Tg2576 mouse model improves in response to a selective gamma-secretase inhibitor, N-[cis-4-[(4-chlorophenyl)-sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560). Tg2576 mice or wild-type (WT) littermates were treated daily with MRK-560 (30 μmol/kg) or vehicle for 4 (acute) or 29 days (chronic). The subsequent MEMRI analysis revealed a distinct axonal transport dysfunction in the Tg2576 mice compared with its littermate controls. Interestingly, the impairment of axonal transport could be fully reversed by chronic administration of MRK-560, in line with the significantly lowered levels of both soluble and insoluble forms of Aβ found in the brain and olfactory bulbs (OBs) following treatment. However, no improvement of axonal transport was observed after acute treatment with MRK-560, where soluble but not insoluble forms of Aβ were reduced in the brain and OBs. The present results show that axonal transport is impaired in Tg2576 mice compared with WT controls, as measured by MEMRI. Chronic treatment in vivo with a gamma-secretase inhibitor, MRK-560, significantly reduces soluble and insoluble forms of Aβ, and fully reverses the axonal transport dysfunction.

Purpose[11C]Lu AE92686 is a positron emission tomography (PET) radioligand that has recently been validated for examining phosphodiesterase 10A (PDE10A) in the human striatum. [11C]Lu AE92686 has high affinity for PDE10A (IC 50 = 0.39 nM) and may also be suitable for examination of the substantia nigra, a region with low density of PDE10A. Here, we report characterization of regional [11C]Lu AE92686 binding to PDE10A in the nonhuman primate (NHP) brain.MethodsA total of 11 PET measurements, seven baseline and four following pretreatment with unlabeled Lu AE92686 or the structurally unrelated PDE10A inhibitor MP-10, were performed in five NHPs using a high resolution research tomograph (HRRT). [11C]Lu AE92686 binding was quantified using a radiometabolite-corrected arterial input function and compartmental and graphical modeling approaches.ResultsRegional time-activity curves were best described with the two-tissue compartment model (2TCM). However, the distribution volume (V T) values for all regions were obtained by the Logan plot analysis, as reliable cerebellar V T values could not be derived by the 2TCM. For cerebellum, a proposed reference region, V T values increased by ∼30 % with increasing PET measurement duration from 63 to 123 min, while V T values in target regions remained stable. Both pretreatment drugs significantly decreased [11C]Lu AE92686 binding in target regions, while no significant effect on cerebellum was observed. Binding potential (BP ND) values, derived with the simplified reference tissue model (SRTM), were 13–17 in putamen and 3–5 in substantia nigra and correlated well to values from the Logan plot analysis.ConclusionsThe method proposed for quantification of [11C]Lu AE92686 binding in applied studies in NHP is based on 63 min PET data and SRTM with cerebellum as a reference region. The study supports that [11C]Lu AE92686 can be used for PET examinations of PDE10A binding also in substantia nigra.Electronic supplementary materialThe online version of this article (doi:10.1007/s00259-016-3544-9) contains supplementary material, which is available to authorized users.

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In drug discovery today, drug exposure is determined in preclinical efficacy and safety studies and drug effects are related to measured concentrations rather than to the administered dose. This leads to a strong increase in the number of bioanalytical samples, demanding the development of higher throughput methods to cope with the increased workload. Here, a combined approach is described for the high-throughput preparation and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of drug levels in plasma samples from the preclinical efficacy and safety studies, i.e. exposure studies. Appropriate pharmacokinetic (PK) compartmental models were fitted to data from PK screening studies in the rat, which were subsequently used to simulate the expected plasma concentrations of the respective exposure studies. Information on the estimated drug concentrations was used to dilute the samples to appropriate concentration levels. A Tecan Genesis RSP liquid handling system was utilized to perform automated plasma sample preparation including serial dilution of standard solutions, dilution of plasma samples, addition of internal standard solution and precipitation with acetonitrile. This robotic sample preparation process permitted two studies of 1-96 samples each to be run simultaneously. To ensure the performance of this method the accuracy and precision for diazepam were examined. Two novel drugs were used to illustrate the suggested approach. In conclusion, our method for sample preparation of exposure samples, based on the combined use of PK simulations, a liquid handling system and a fast LC/MS/MS method, increased the throughput more than three times and minimized the errors, while maintaining the required accuracy and precision.

The small sample volumes and flexibility in choice of liquid matrices using the capillary microsampling technique enable repeated sampling in mouse studies, as well as multi-matrix analysis if needed. Liquid microsampling is well suited for micro- and nano-liter scale immunoassays.