Ladislav Mrzljak

Development of a Series of Aryl Pyrimidine Kynurenine Monooxygenase Inhibitors as Potential Therapeutic Agents for the Treatment of Huntington's Disease

We report on the development of a series of pyrimidine carboxylic acids that are potent and selective inhibitors of kynurenine monooxygenase and competitive for kynurenine. We describe the SAR for this novel series and report on their inhibition of KMO activity in biochemical and cellular assays and their selectivity against other kynurenine pathway enzymes. We describe the optimization process that led to the identification of a program lead compound with a suitable ADME/PK profile for therapeutic development. We demonstrate that systemic inhibition of KMO in vivo with this lead compound provides pharmacodynamic evidence for modulation of kynurenine pathway metabolites both in the periphery and in the central nervous system.

The novel KMO inhibitor CHDI-340246 leads to a restoration of electrophysiological alterations in mouse models of Huntington's disease.

Dysregulation of the kynurenine (Kyn) pathway has been associated with the progression of Huntingtons disease (HD). In particular, elevated levels of the kynurenine metabolites 3-hydroxy kynurenine (3-OH-Kyn) and quinolinic acid (Quin), have been reported in the brains of HD patients as well as in rodent models of HD. The production of these metabolites is controlled by the activity of kynurenine mono-oxygenase (KMO), an enzyme which catalyzes the synthesis of 3-OH-Kyn from Kyn. In order to determine the role of KMO in the phenotype of mouse models of HD, we have developed a potent and selective KMO inhibitor termed CHDI-340246. We show that this compound, when administered orally to transgenic mouse models of HD, potently and dose-dependently modulates the Kyn pathway in peripheral tissues and in the central nervous system. The administration of CHDI-340246 leads to an inhibition of the formation of 3-OH-Kyn and Quin, and to an elevation of Kyn and Kynurenic acid (KynA) levels in brain tissues. We show that administration of CHDI-340246 or of Kyn and of KynA can restore several electrophysiological alterations in mouse models of HD, both acutely and after chronic administration. However, using a comprehensive panel of behavioral tests, we demonstrate that the chronic dosing of a selective KMO inhibitor does not significantly modify behavioral phenotypes or natural progression in mouse models of HD.

Patterns of age related changes for phosphodiesterase type-10A in comparison with dopamine D 2/3 receptors and sub-cortical volumes in the human basal ganglia: A PET study with 18 F-MNI-659 and 11 C-raclopride with correction for partial volume effect

ABSTRACT Phosphodiesterase 10A enzyme (PDE10A) is an important striatal target that has been shown to be affected in patients with neurodegenerative disorders, particularly Huntingtons disease (HD). PDE10A is expressed on striatal neurones in basal ganglia where other known molecular targets are enriched such as dopamine D2/3 receptors (D2/3 R). The aim of this study was to examine the availability of PDE10A enzyme in relation with age and gender and to compare those changes with those related to D2/3 R and volumes in different regions of the basal ganglia. As a secondary objective we examined the relative distribution of D2/3 R and PDE10A enzyme in the striatum and globus pallidus. Forty control subjects (20F/20M; age: 44±11y, age range 27–69) from an ongoing positron emission tomography (PET) study in HD gene expansion carriers were included. Subjects were examined with PET using the high‐resolution research tomograph (HRRT) and with 3T magnetic resonance imaging (MRI). The PDE10A radioligand 18F‐MNI‐659 and D2/3 R radioligand 11C‐raclopride were used. The outcome measure was the binding potential (BPND) estimated with the two‐tissue compartment model (18F‐MNI‐659) and the simplified reference tissue model (11C‐raclopride) using the cerebellum as reference region. The PET data were corrected for partial volume effects. In the striatum, PDE10A availability showed a significant age‐related decline that was larger compared to the age‐related decline of D2/3 R availability and to the age‐related decline of volumes measured with MRI. In the globus pallidus, a less pronounced decline of PDE10A availability was observed, whereas D2/3 R availability and volumes seemed to be rather stable with aging. The distribution of the PDE10A enzyme was different from the distribution of D2/3 R, with higher availability in the globus pallidus. These results indicate that aging is associated with a considerable physiological reduction of the availability of PDE10A enzyme in the striatum. Moreover as result of the analysis, in the striatum for both the molecular targets, we observed a gender effect with higher BPND the female group. Highlights18F‐MNI‐659 binding is a measure of Phosphodiestrease 10A enzyme availability.We examined the effect of age on 18F‐MNI‐659 binding in 40 healthy controls.Age related changes were also evaluated for 11C‐Raclopride and structural volumes.Patterns of molecular age related changes were evaluated with PVEc.We found an evident association between age and striatal 18F‐MNI‐659 binding.

Striatal molecular alterations in HD gene carriers: a systematic review and meta-analysis of PET studies

Background Over the past years, positron emission tomography (PET) imaging studies have investigated striatal molecular changes in premanifest and manifest Huntington’s disease (HD) gene expansion carriers (HDGECs), but they have yielded inconsistent results. Objective To systematically examine the evidence of striatal molecular alterations in manifest and premanifest HDGECs as measured by PET imaging studies. Methods MEDLINE, ISI Web of Science, Cochrane Library and Scopus databases were searched for articles published until 7 June 2017 that included PET studies in manifest and premanifest HDGECs. Meta-analyses were conducted with random effect models, and heterogeneity was addressed with I2 index, controlling for publication bias and quality of study. The primary outcome was the standardised mean difference (SMD) of PET uptakes in the whole striatum, caudate and putamen in manifest and premanifest HDGECs compared with healthy controls (HCs). Results Twenty-four out of 63 PET studies in premanifest (n=158) and manifest (n=191) HDGECs and HCs (n=333) were included in the meta-analysis. Premanifest and manifest HDGECs showed significant decreases in dopamine D2 receptors in caudate (SMD=−1.233, 95% CI −1.753 to −0.713, p<0.0001; SMD=−5.792, 95% CI −7.695 to −3.890, p<0.0001) and putamen (SMD=−1.479, 95% CI −1.965 to −0.992, p<0.0001; SMD=−5.053, 95% CI −6.558 to −3.549, p<0.0001), in glucose metabolism in caudate (SMD=−0.758, 95% CI −1.139 to −0.376, p<0.0001; SMD=−3.738, 95% CI −4.880 to −2.597, p<0.0001) and putamen (SMD=−2.462, 95% CI −4.208 to −0.717, p=0.006; SMD=−1.650, 95% CI −2.842 to −0.458, p<0.001) and in striatal PDE10A binding (SMD=−1.663, 95% CI −2.603 to −0.723, p=0.001; SMD=−2.445, 95% CI −3.371 to −1.519, p<0.001). Conclusions PET imaging has the potential to detect striatal molecular changes even at the early premanifest stage of HD, which are relevant to the neuropathological mechanisms underlying the development of the disease.

Noninvasive relative quantification of [11C]ABP688 PET imaging in mice versus an input function measured over an arteriovenous shunt

Impairment of the metabotropic glutamate receptor 5 (mGluR5) has been implicated with various neurologic disorders. Although mGluR5 density can be quantified with the PET radiotracer [11C]ABP688, the methods for reproducible quantification of [11C]ABP688 PET imaging in mice have not been thoroughly investigated yet. Thus, this study aimed to assess and validate cerebellum as reference region for simplified reference tissue model (SRTM), investigate the feasibility of a noninvasive cardiac image-derived input function (IDIF) for relative quantification, to validate the use of a PET template instead of an MRI template for spatial normalization, and to determine the reproducibility and within-subject variability of [11C]ABP688 PET imaging in mice. Blocking with the mGluR5 antagonist MPEP resulted in a reduction of [11C]ABP688 binding of 41% in striatum (p < 0.0001), while no significant effect could be found in cerebellum (−4.8%, p > 0.99) indicating cerebellum as suitable reference region for mice. DVR-1 calculated using a noninvasive IDIF and an arteriovenous input function correlated significantly when considering the cerebellum as the reference region (striatum: DVR-1, r = 0.978, p < 0.0001). Additionally, strong correlations between binding potential calculated from SRTM (BPND) with DVR-1 based on IDIF (striatum: r = 0.980, p < 0.0001) and AV shunt (striatum: r = 0.987, p < 0.0001). BPND displayed higher discrimination power than VT values in determining differences between wild-types and heterozygous Q175 mice, an animal model of Huntingtons disease. Furthermore, we showed high agreement between PET- and MRI-based spatial normalization approaches (striatum: r = 0.989, p < 0.0001). Finally, both spatial normalization approaches did not reveal any significant bias between test-retest scans, with a relative difference below 5%. This study indicates that noninvasive quantification of [11C]ABP688 PET imaging is reproducible and cerebellum can be used as reference region in mice.

Longitudinal microPET imaging of the zQ175 mouse model of Huntington's disease shows in vivo changes of molecular targets in the striatum and the cerebral cortex.

Since the discovery of the HTT gene in 1993, numerous animal models have been developed to study the progression of Huntington disease (HD) and to evaluate potential new therapeutics. In the present study, we used small-animal PET to characterize the expression of molecular targets in the recently reported HD animal model, the zQ175 mouse model. Methods: Male heterozygous zQ175 (Htttm1Mfc/190JChdi, CHDI-81003003) and wild-type (WT, C57BL/6J) animals were imaged with the dopamine D2 receptor radioligand 11C-raclopride, the PDE10A radioligand 18F-MNI-659, the dopamine D1 receptor radioligand 11C-NNC 112, and the 5-HT2A radioligand 11C-MDL 100907 at 6 and 9 mo of age. The outcome measure was the binding potential (BPND), using the cerebellum as the reference region. Selected regions of interest were the striatum for all radioligands and additionally the striatum, rostral cortex, caudal cortex, and hippocampus for 11C-NNC 112 and 11C-MDL 100907. Results: At 6 mo of age, the BPND in the striatum was lower in zQ175 than WT animals by 40% for 11C-raclopride, by 52% for 18F-MNI-659, by 28% for 11C-NNC, and by 11% for 11C-MDL 100907. In the rostral cortex, D1 receptor binding was 22% lower in zQ175 than WT animals. We found an overall reduction in D1 and 5-HT2A binding in the hippocampus of zQ175 compared with WT animals. The BPND of 11C-MDL 100907 in the caudal cortex was also lower in zQ175 WT animals. At 9 mo, there was a slight further reduction of D1, D2, and 5-HT2A BPND in the striatum, whereas PDE10A reached a plateau. Cortical markers were also slightly further decreased at 9 mo in zQ175 animals. Conclusion: Our study indicates a marked reduction of ligand binding to D1 and D2 and 5-HT2A receptors as well as loss of PDE10A enzyme in the striatum of zQ175 mice as compared with WT animals, in agreement with data obtained in clinical PET studies of patients with HD. The zQ175 mouse model recapitulates the expression pattern seen in humans with HD and may have value in further elucidating pathophysiologic events and therapeutic strategies.

Revisiting the Logan plot to account for non-negligible blood volume in brain tissue

BackgroundReference tissue-based quantification of brain PET data does not typically include correction for signal originating from blood vessels, which is known to result in biased outcome measures. The bias extent depends on the amount of radioactivity in the blood vessels. In this study, we seek to revisit the well-established Logan plot and derive alternative formulations that provide estimation of distribution volume ratios (DVRs) that are corrected for the signal originating from the vasculature.ResultsNew expressions for the Logan plot based on arterial input function and reference tissue were derived, which included explicit terms for whole blood radioactivity. The new methods were evaluated using PET data acquired using [11C]raclopride and [18F]MNI-659. The two-tissue compartment model (2TCM), with which signal originating from blood can be explicitly modeled, was used as a gold standard.DVR values obtained for [11C]raclopride using the either blood-based or reference tissue-based Logan plot were systematically underestimated compared to 2TCM, and for [18F]MNI-659, a proportionality bias was observed, i.e., the bias varied across regions. The biases disappeared when optimal blood-signal correction was used for respective tracer, although for the case of [18F]MNI-659 a small but systematic overestimation of DVR was still observed.ConclusionsThe new method appears to remove the bias introduced due to absence of correction for blood volume in regular graphical analysis and can be considered in clinical studies. Further studies are however required to derive a generic mapping between plasma and whole-blood radioactivity levels.

Longitudinal characterization of mGluR5 using11C-ABP688 PET imaging in the Q175 mouse model of Huntington's disease

Metabotropic glutamate receptor 5 (mGluR5) represents a potential therapeutic target for Huntington disease. Using 11C-ABP688 (3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-O-11C-methyl-oxime), a noncompetitive and highly selective antagonist for mGluR5, we aimed to longitudinally characterize in vivo changes in mGluR5 by means of PET imaging in the Q175 mouse model of Huntington disease. Methods: 11C-ABP688 PET imaging, followed by a CT scan, was performed on 18 heterozygous mice and 18 wild-type (WT) littermates at 3 different time points (6, 9, and 13 mo old). 11C-ABP688 nondisplaceable binding potential (BPND) was calculated for each time point in striatum and cortex using the cerebellum as the reference region. In addition, voxel-based statistical parametric mapping (SPM) analysis was performed on BPND images. Postmortem validation of mGluR5 level and neuronal density was performed on the mice at 6 mo old. Results: The 11C-ABP688 BPND of heterozygous animals was significantly reduced at all time points in the striatum (−13.1%, −13.5%, and −14.2% at 6, 9, and 13 mo, respectively; P < 0.001 for all) and in the cortex (−9.8%, −10.2%, and −10.6%, respectively; P < 0.01 for all), when compared with WT animals. Longitudinal changes in 11C-ABP688 BPND were also found in heterozygous mice, showing a reduction at 13 mo compared with 6 mo (−10.4%, P < 0.05). SPM analysis confirmed reduced BPND in heterozygous compared with WT mice, as well as a time-related decline in 11C-ABP688 binding in the striatum of heterozygous mice. Postmortem analysis confirmed a mGluR5 decrease in both striatum (−36.6%; P < 0.01) and cortex (−16.6%; P < 0.05) in heterozygous mice, whereas no difference in neuronal density was found. Conclusion: In vivo imaging of mGluR5 using 11C-ABP688 PET/CT revealed a marked reduction in ligand binding in the striatum and cortex of heterozygous mice, compared with WT mice, as well as a temporal decline. This study suggests that 11C-ABP688 PET imaging is a potential biomarker to monitor the progression of, and therapeutic strategies for, Huntington disease.

MR-based spatial normalization improves [18F]MNI-659 PET regional quantification and detectability of disease effect in the Q175 mouse model of Huntington’s disease

The positron emission tomography (PET) tracer [18F]MNI-659, selective for phosphodiesterase 10A (PDE10A), is a promising tool to assess an early biomarker for Huntington’s disease (HD). In this study we investigated [18F]MNI-659 uptake in the Q175 mouse model of HD. Given the focal striatal distribution of PDE10A as well as the striatal atrophy occurring in HD, the spatial normalization approach applied during the processing could sensibly affect the accuracy of the regional quantification. We compared the use of a magnetic resonance images (MRI) template based on individual MRI over a PET and CT templates for regional quantification and spatial normalization of [18F]MNI-659 PET images. We performed [18F]MNI-659 PET imaging in six months old heterozygous (HET) Q175 mice and wild-type (WT) littermates, followed by X-ray computed tomography (CT) scan. In the same week, individual T2-weighted MRI were acquired. Spatial normalization and regional quantification of the PET/CT images was performed on MRI, [18F]MNI-659 PET, or CT template and compared to binding potential (BPND) using volumes manually delineated on the individual MR images. Striatal volume was significantly reduced in HET mice (-7.7%, p<0.0001) compared to WT littermates. [18F]MNI-659 BPND in striatum of HET animals was significantly reduced (p<0.0001) when compared to WT littermates using all three templates. However, BPND values were significantly higher for HET mice using the PET template compared to the MRI and CT ones (p<0.0001), with an overestimation at lower activities. On the other hand, the CT template spatial normalization introduced larger variability reducing the effect size. The PET and CT template-based approaches resulted in a lower accuracy in BPND quantification with consequent decrease in the detectability of disease effect. This study demonstrates that for [18F]MNI-659 brain PET imaging in mice the use of an MRI-based spatial normalization is recommended to achieve accurate quantification and fully exploit the detectability of disease effect.