Non Human Primates Using Vesicular Monoamine

Published Date: 02 Nov 2017

Yajing Liu1,2, Feng Yue1,2,6, Rongping Tang3, Guoxian Tao3, Xiaomei Pan3, Lin Zhu4, Hank F. Kung5, Piu Chan1,2,6

1Department of Neurobiology, Xuanwu Hospital of Capital Medical University, Beijing 100053, China（首都医科大学宣武医院神经生物室，北京市100053）

2Key Laboratory of Neurodegenerative Diseases for Ministry of Education, Xuanwu Hospital of Capital Medical University, Beijing 100053, China（首都医科大学宣武医院教育部神经变性病学重点实验室，北京市100053）

3Wincon Theracells Biotechnologies Inc., Nanning, Guangxi 530003, China（广西南宁灵康赛诺科生物科技有限公司，南宁市530003）

4Key Laboratory of Radiopharmaceuticals, Beijing Normal University, Ministry of Education, Beijing 100875, China（北京师范大学放射性药物教育部重点实验室，北京市100069）

5Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104, USA

6Beijing Key Laboratory on Parkinson’s Disease, Beijing 100053, China（帕金森病研究北京市重点实验室，北京100053）

*Correspondence: Piu Chan. M. D. Ph. D., Professor, Department of Neurobiology, Xuanwu Hospital of Capital Medical University, Beijing 100053, China. （首都医科大学宣武医院神经生物室，北京市100053） Tel.: +86 10 83198677; fax: +86 10 8316 1294. [email protected].

Yajing Liu and Feng Yue contributed equally to this work.

Abstract

The 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP)-induced Parkinson’s disease model, particularly in non-human primate (NHP), remains the gold standard for the study of pathogenesis and assessment of novel therapies for Parkinson’s disease (PD). However, whether or not the loss of dopamine neurons in this model is progressive remain controversial. This is mostly due to lacking of in vivo objective assessment of change in the integrity of dopamine neurons in life animals. In the present study, parkinsonism NHPs model was induced by intravenous administration of MPTP (0.2 mg/kg) for up to 15 days and stable parkinsonism was developed for at least 90 days until the symptoms were stable. Noninvasive neuroimaging of vesicular monoamine transporter 2 (VMAT2) with (2R,3R,11R)-9-(3-18F-fluoropropoxy) -3-isobutyl-10-methoxy-2,3,4,6,7,11-hexahydro-1H-pyrido[2,1-]isoquinolin-2-ol ([18F]AV-133) PET imaging was applied in NHPs before and 15 and 90 days after acute MPTP treatment. The PET imaging results indicated that a longitudinal and progressive loss of striatal uptake of [18F]AV-133 was evident. The dopaminergic denervation severity (DS) showed significant linear correlation with clinical rating scores and subscores of bradykinesia. These findings demonstrated that longitudinal [18F]AV-133 PET imaging could be a useful tool to noninvasively evaluate the evolution of monoaminergic terminals reductions in an acute MPTP-induced parkinsonism NHPs model.

Key words: Parkinson’s disease, Non-human primate, [18F]AV-133, VMAT2, PET

摘要：

1-甲基-4-苯基-1-1,2,3,4-四氢吡啶（MPTP）诱导的帕金森病非人灵长类模型是认识帕金森病及评价新的神经保护药物和治疗策略最为理想的动物模型。但是，由于缺乏活体检测多巴胺能神经元变化的手段，对于此模型中多巴胺能神经元是否为渐进性损失仍然存有争议。在本研究中，非人灵长类帕金森病模型通过静脉注射MPTP 2mg/kg，1次/d，连续15d得到。在给药前、给药15d和90d后进行[18F]AV-133 PET显像—一种以囊泡单胺转运体（VMAT2）为靶向的PET显像剂。PET结果表明，[18F]AV-133在纹状体的摄取渐行性减少。多巴胺能神经元损伤程度（DS）与临床总评分和临床症状运动迟缓评分呈线性正相关。这些研究结果表明：[18F]AV-133 PET显像是可以在体、无创评估急性MPTP诱导的帕金森病非人灵长类模型多巴胺能神经末梢完整性的有力工具。

关键词: 帕金森病；非人灵长类；[18F]AV-133；囊泡单胺转运体；正电子发射型计算机断层显像

1 Introduction

One of the most common neurodegenerative disorder is Parkinson’s disease (PD) which affects ~2% of the world’s population aged over 65 . The cardinal clinical symptoms of PD are bradykinesia, tremor, rigidity, and postural instability with pathological characteristic of evolutional nigrostriatal dopamine neurons degeneration. 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is of proven neuro toxin, and could induce parkinsonism in both humans and non-human primates (NHPs) with cognitive, biochemical, histologic and classic behaviora changes that present in PD . Therefore, MPTP-lesioned NHPs have been used to evaluate the efficacy of anti-parkinsonism therapy. However, whether acute MPTP-induced loss of dopamine neurons is a progressive process remains controversial, a biomarker which can non-invasively monitor this change longitudinally becomes critical.

Increasing evidence suggests that single photon emission computed tomography (SPECT) and positron emission tomography (PET) imaging could sensitively and objectively evaluate the integrity of nigrostriatal dopaminergic, and maybe a useful tool for providing diagnostic information of PD . There are numerous SPECT and PET imaging tracers for monitoring the integrity of dopamine neuron, for example 6-[18F]fluoro-DOPA ([18F]FDOPA) can be used to evaluate DA synthesis capacity , 123I-labeled 2β-carbomethoxy-3β- (4-[123I]iodophenyl) tropane ([123I]-β-CIT) to target membrane DA transporter (DAT) , and [11C]-raclopride specifically binds to DA receptors.

Vesicular monoamine transporter 2 (VMAT2), which is located on vesicle membranes in monoamine neurons, carries out the reuptake and package of monoamines into vesicles. The monoamine include dopamine, norepinephrine, and serotonin. Previous studies on immunochemical analysis of VMAT2 have founded that dopaminergic terminals is responsible for above 95% of VMAT2 expression . A multi-tracer PET imaging in NHPs of PD model induced by a chronic, low-dose of MPTP treatment has founded that the model has undergone the process of nigrostriatal dopamine neuron degeneration, which results in a reduced storage capacity of VMAT2 and decreased uptake of [11C]DTBZ in nigrostriatal . Imaging VMAT2 has thus been regarded as an effective tool to follow up degeneration of dopaminergic terminal. 9-[18F]fluoropropyl-(+)- dihydrotetrabenazine ([18F]AV-133) is a novel 18F-labeled tetrabenazine derivative which selectively binds with high affinity to VMAT2 . Previous [18F]AV-133 PET imaging studies clearly demonstrated that [18F]AV-133 could sensitively detecte VMAT2 reductions in PD patients, supporting that [18F]AV-133 may be a potential tool to identify presymptomatic patients with nigrostriatal movement disorders .

So far, there is no study has been done using [18F]AV-133 as a biomarker to evaluate parkinsonism in NHPs, particularly in acute intravenously infusion of MPTP model which has been extensively used for pre-clinical evaluation of anti-parkinsonian drugs. The current study is to investigate the utility of [18F]AV-133 as the biomarker for assessing the longitudinal loss of VMAT2 function and dopaminergic terminal degeneration in acute MPTP-induced parkinsonism NHPs.

2 Material and methods

2.1 Animals

Nine 10-15 year old cynomolgus monkeys (2 female and 7 male) with body weights ranging from 5.2-8.0 kg were used. All cynomolgus monkeys were sourced from Grandforest Co., a local NHP breeder base. Each individual was without physical impairments and healthy. And all included animals had detailed birth and quarantine certifications. MPTP treatment consisted of intravenous injection MPTP-HCl (Sigma Aldrich, USA) diluted in sterile saline at a concentration of 0.2mg/kg after anesthetized by a mixture of 3% isofluran and 97% oxygen. Low content of isofluran (1%) was continued for maintenance. Injection was performed daily for 15 days. In this study, there was no NHP euthanized.

2.2 Ethics statement

The animals were housed at the facility of Wincon TheraCells Biotechnologies Co., Ltd., which was certified by and met the guidelines of the council on accreditation of the Association for Assessment and Accreditation of Laboratory Animals Care (AAALAC, international), with 12h light and 12h dark alternately. The ambient temperature was 24 ± 2 °C, and humidity was 65 ± 4%. Uninterrupted IO water was available ad libitum. Fresh fruits and vegetabels were supplied twice daily. The Institutional Animal Care and Utilization Committee (IACUC) of Wincon TheraCells Biotechnologies facility approved this experiment (Permit Number: W00019).

2.3 PET Radiosynthesis of [18F]AV-133

Radiosynthesis of [18F]AV-133 was performed on a homemade automated radiosynthesis apparatus according to previously described methods . The mesylate precursor of AV-133 was nucleophilic-substituted by [18F] under the Kryptofix 222 catalyst. After [18F]fluorination, the crude [18F]AV-133 was purified via solid phase extraction column (Oasis HLB 3mL cartridge, Waters). The radiosynthesis yield of [18F]AV-133 was 45-50% (decay corrected) in the quantity of 350-500 mCi with radiochemical purity above 95%. The mean specific radioactivity of [18F]AV-133 was 1000 Ci/mmol.

2.4 PET acquisition and analysis

Each NHP underwent three [18F]AV-133 PET scans and clinical ratings, executed before (baseline), 15 days (day 15, acute phase) and 90 days (day 90, chronic parkinsonism phase) after the first MPTP infusion, respectively. The PET system used for NHPs brain imaging was Biograph Sensation 16HR (Siemens/CTI, Knoxville, TN, USA). The PET images were attenuation-corrected using low-dose helical CT. The scan protocol for CT was as follows: peak kilovoltage 120 kV, 420 mAs, pitch 6 mm and collimator 0.75 mm. Each individual was anesthetized with ketamine (15 mg/kg) via intramuscular injection, and then injected with ketamine (7.5 mg/kg) every 30 min. [18F]AV-133 in 1.0 mL sterile 10% ethanol saline was injected as a bolus into the femoral vein. The cannula was then immediately flushed with 10 mL saline. The total radioactivity injected was 0.2 mCi/kg. One 10-min frames from 30-40 min post-injection were used to estimate [18F]AV-133 binding. This was based on our preliminary study, which showed a transient equilibrium was achieved for [18F]AV-133 from 30min after i.v. injection .

The data were reconstructed using 3D iterative algorithm after randomness, scatter and attenuation corrections. Each NHP’s PET scans were analyzed simultaneously following the same protocol. Because of negligible density of VMAT2 in cerebellum region, cerebellum is a suitable region as nonspecific reference. Two irregular ROIs encompassing the whole right and left striatum were drawn on the four consecutive slices through striatum, and background were readily identified and delineated on the cerebellum guided by detailed CT images and with reference to the stereotaxic brain atlas. The results were expressed as specific uptake ratio (SUr), calculated by (mean striatal uptake-mean cerebellar uptake)/mean cerebellar uptake and dopaminergic denervation severity (DS) after MPTP treatment, calculated from SUr via (SUrbaseline-SUrlesioned)/SUrbaseline .

2.5 Clinical rating score

The behavioral changes were observed and quantified daily on a previously validated Parkinsonian monkey clinical rating scale . A videotape system was used to record animal behaviors in testing cages. The scale rated nine items: bradykinesia (0-5), posture (0-2), rigidity (0-2), gait (0-5), balance (0-2), resting tremor for each side (0-3 for each side), gross motor skill (0-4) for each upper limb, gross motor skill (0-4) for each lower limb, and defense reaction (0-2). Minimum score was 0, corresponding to normal, and the maximum total score achievable was 32, corresponding to severe disability. The scores were rated by both an experienced neurologist and technician who were blinded to the study protocol.

2.6 Statistical analysis

Comparisons of group characteristics were performed with paired sample t tests. Correlations between clinical data and PET imaging results were evaluated using a nonparametric Pearson analysis. A value of P < 0.05 was defined as statistical significance. All error bar were presented as mean ± SD.

3 Results

3.1 PET images

[18F]AV-133 PET imaging were performed three times on each NHPs, before (baseline), 15 (day 15, acute phase) and 90 days (day 90, chronic parkinsonism phase) after the first MPTP infusion, respectively. Representative images for each of the healthy and MPTP-lesioned status are shown in Figure 1, and revealed a progressive reduction of striatal [18F]AV-133 uptake. Before MPTP intoxication, [18F]AV-133 had symmetric uptake and retention in the bilateral striatum (ST), where there is a high density of VMAT2. In contrast, for the cerebellum (CB) region, which is nearly devoid of VMAT2, the uptake was low or not evident. The right and left striatal specific uptake ratios (SUr) were 1.59 ± 0.48 and 1.63 ± 0.48 (n = 9), respectively (Figure 2). Fifteen days after the first MPTP infusion when MPTP injection was stopped (day 15), decreased striatal uptake was detected. The right and left striatal SUrs were decreased to 1.01 ± 0.35 and 1.04 ± 0.33 (n = 9), respectively (Figure 2). MPTP toxicity lead to partial striatal denervation (right and left DS = 0.36 ± 0.30 and 0.34 ± 0.28, n =9/group, respectively) (Figure 3). Ninety days after the first MPTP infusion when parkinsonian symptoms were stable (day90), significant reduction of striatal uptake was observed. The right and left striatal SUrs were 0.26 ± 0.13 and 0.24 ± 0.16 (n = 9), respectively (Figure 2). It was found that compared with baseline, the right and left DSs were 0.84 ± 0.11 and 0.84 ± 0.12 (n = 9), respectively (Figure 3). The value of [18F]AV-133 uptake in the striatum did not overlap before and 90 days after MPTP injection (Figure 2). Statistical analyses indicated that there were also significant differences between before and 15 and 90 days after MPTP (before vs day 15: P < 0.05; before vs day 90: P < 0.05; day 15 vs day 90: P < 0.05).

Figure 1

Figure 1. Representative PET scans of [18F]AV-133 in the progressive PD model. A: before MPTP treatment (baseline), B: 15 days after the first MPTP treatment (day 15, acute phase), C: 90 days after the first MPTP treatment (day 90, chronic parkinsonism phase). Images are transverse slices at the level of the striatum.

Figure 2

Figure 2. Striatal specific uptake ratio (SUrs) of [18F]AV-133 in the progressive PD model. A: before MPTP treatment (baseline), B: 15 days after the first MPTP treatment (day 15, acute phase), C: 90 days after the first MPTP treatment (day 90, chronic parkinsonism phase). The values of striatal SUrs (mean ± SD) are 1.59 ± 0.48, 1.01 ± 0.35 and 0.26 ± 0.13 for healthy, day 15 group and day 90 group, respectively (n = 9).

Figure 3

Figure 3. Dopaminergic denervation severity (DS) of [18F]AV-133 in the progressive PD model. A: before MPTP treatment (baseline), B: 15 days after the first MPTP treatment (day 15, acute phase), C: 90 days after the first MPTP treatment (day 90, chronic parkinsonism phase). The values of DS (mean ± SD) are 0.00 ± 0.00, 0.34 ± 0.29 and 0.84 ± 0.11 for healthy, day 15 group and day 90 group, respectively (n = 9).

3.2 Correlation between [18F]AV-133 uptake and clinical rating scores

The correlations between the striatal SUr and DS of [18F]AV-133 and clinical rating scores were also analyzed. On day 15 after MPTP injection, the mean total clinical rating score was 14.56 ± 6.76. As shown in Figure 4, the increased DS in the striatum was accompanied by a significant increase in the total clinical rating scores and subscores for bradykinesia (R = 0.894, P < 0.05 and R = 0.902, P < 0.05, respectively), and a significant correlation was observed for the SUr for [18F]AV-133 (R = -0.684, P < 0.05, and R = -0.689, P < 0.05, respectively). However, no significant correlation was found when the subscores of rigidity were examined.

Ninety days after the first MPTP treatment, a state of stable experimental parkinsonism was obtained, and the mean total clinical rating scores reached 22.19 ± 5.34. As shown in Figure 5, the clinical rating scores and subscores for rigidity and bradykinesia positively correlated with DS (R = 0.894, P < 0.05, R = 0.823, P < 0.05, and R = 0.809, P < 0.05, respectively), and a significant trend was also observed for the SUr for [18F]AV-133 (R = -0.843, P < 0.05, R = -0.739, P < 0.05 and R = -0.759, P < 0.05, respectively).

Figure 4

Figure 4. Correlation of clinical rating results with [18F]AV-133 PET imaging results of day 15 group NHPs. Significant negative correlations were observed between SUr and clinical rating scores (R = -0.684, P < 0.05) (A), bradykinesia (R = -0.689, P < 0.05) (C) (n = 9). Significant positive correlations were observed between DS and clinical rating scores (R = 0.894, P < 0.05) (D), bradykinesia (R = 0.902, P < 0.05) (F) (n = 9). There was no correlation between rigidity and SUr (P > 0.05) (B) and DS (P > 0.05) (E) (n = 9).

Figure 5

Figure 5. Correlation of clinical rating results with [18F]AV-133 PET imaging results of day 90 group NHPs. Significant negative correlations were observed between SUr and clinical rating scores (R = -0.843, P < 0.05) (A), rigidity (-0.739, P < 0.05) (B), bradykinesia (R = -0.759, P < 0.05) (C) (n = 9). Significant positive correlations were observed between DS and clinical rating scores (R = 0.894, P < 0.05) (D), rigidity (R = 0.823, P < 0.05) (E), bradykinesia (R = 0.809, P < 0.05) (F) (n = 9).

4 Discussion

MPTP induced parkinsonism in NHPs provides opportunities to study the clinical efficacy of anti-parkinsonism therapy. [18F]AV-133 has successfully detected monoaminergic terminal reduction in PD patients. To assess the dopaminergic terminal degeneration with this PET tracer in acute MPTP-induced parkinsonism NHPs, it would likewise validate the use of the MPTP NHPs model to study in vivo effects of anti-parkinsonism therapy on dopaminergic function and constitute a useful tool for precinical research.

The presented data confirmed that [18F]AV-133 PET imaging could be a useful tool for assessing parkinsonism in NHPs, and found a progressive degeneration of dopamine neuron in an acute administration of MPTP model. Our PET study showed that [18F]AV-133 had high specific binding in the bilateral striatum in NHPs, and the PET imaging provided significant signal to noise ratio. The striatal [18F]AV-133 uptake observed here are consistent with previous studies on NHPs brain . MPTP administration could induce striatal dopaminergic denervation, and resulte in decreases in VMAT2 binding (Figure 2). After MPTP exposure for 15 days, the dopaminergic denervation severity (DS) was 34% higher as compared with baseline. When the NHPs achieved stable parkinsonism, demonstrated by maximum scores on the Parkinsonian monkey clinical rating scale, the PET imaging results measured by [18F]AV-133 in the striatum also showed significantly low VMAT2 densities, and the striatal specific uptake ratio (SUr) for [18F]AV-133 was only 16% of the baseline. Okamura et al. found that there was a greatest VMAT2 reductions in the posterior putamen (-81%) of PD patients. And the VMAT2 reduciton in anterior putamen and caudate nucleus was followed by (-70% and -48% respectively) . It appears that it is the same case for our MPTP-induced parkinsonian NHP models. In 2010, Obeso et al. evaluated [11C]DTBZ PET imaging in a model of progressive parkinsonism in NHPs, which was induced by small dose of MPTP administration over several months . Different from the previous study, our study used a model of acute infusion of MPTP (0.2 mg/kg every day for 15 day) which is the standard model for anti-parkinsonian drug study, and observed the progressive dopaminergic depletion in NHPs. In Obeso study, the comparisons were conducted between four groups, controls, asymptomatic, recovered and stable Parkinsonian. In the present study, changes of [18F]AV-133 uptake in striatum between the three stages were analyzed based on self-comparison. Because of individual differences in striatal uptake of [18F]AV-133 at healthy state, the self-comparison of SUrs were more reliable index for evaluating nigrostriatal dopamine neuron degeneration.

Bezard et al. reported that after receiving daily injection of MPTP (0.2 mg/kg, i.v.) for 15.5 ± 1.1 days, NHPs appeared to have parkinsonian symptoms, and reached a score over 8 on the clinical rating scale . Because of the different clinical rating scale, in our study the clinical rating score was 14.56 ± 6.76 after 15 MPTP treatments. In our study, there was a female cynomolgus monkey not sensitive to the MPTP. 90 days after the first MPTP injection, the clinical score was only 10, much less than the others in day 90 group. Accordingly, DS of this monkey was lower than others, confirmed that MPTP treatment produced less damage on dopaminergic neuron than others. Hence, the clinical extrapyramidal symptoms in this monkey was milder than others. Compared with the striatal SUr for [18F]AV-133, the DS in the striatum showed a strong linear correlation with the clinical rating scores at day 15 and day 90 (Figure 4A, D and Figure 5A, D). This indicated that the DS was a better index to evaluate VMAT2 loss than striatal SUr. Our PET study also showed that subscores of bradykinesia showed negative correlation with SUr for [18F]AV-133 and positive correlation with DS in MPTP-lesioned day 15 and day 90 monkeys (Figure 4C, F and Figure 5C, F). This is consistent with previous studies on human PD patients . On day 15 after MPTP injection, no significant correlation was observed between subscores of rigidity and SUr and DS, suggesting a weak association between rigidity symptom and [18F]AV-133 binding in mild parkinsonian NHPs (Figure 4B, E). However, PET studies on day 90 group showed a significant correlation of rigidity subscores with PET imaging results (Figure 5B, E). Additional studies with larger sample populations and high PET scan frequency are warranted to more fully examine the usefulness and application of [18F]AV-133 PET imaging.

In conclusion, a rapid and progressiveVMAT2 reductions were detected in the striatum from the longitudinal [18F]AV-133 PET imaging study in acute MPTP-lesioned NHPs. These observation indicated that PET with [18F]AV-133 is a sensitive marker to follow up the evolutional dopaminergic depletion in MPTP-induced acute parkinsonism NHPs models.

Acknowledgements: This study was supported by grants from the Ministry of Sciences and Technology of China (2012AA02A514, 2011CB504101), A Science Research and Technology Development Project from Bureau of Sciences and Technology of Nanning China (201001010A), China Postdoctoral Science Foundation (20110490452) and Beijing Postdoctoral Research Activity Foundation (2011ZZ-07), Open Fund of the Key Laboratory on Neurodegenerative Disease of Ministry of Education (2012SJBX01). We thank Avid radiopharmaceuticals, Inc. for kindly provided the mesylate precursor. We thank C. Qin, S. Huang and Y. Qi for their technical assistance with PET scans, and Q Li, Y Gan and M Yang for their valuable assistance in primate experiments.