D_SOW.pdf

STATEMENT OF WORK (SERVICES)

(SOW)

GENERAL INFORMATION

Title of Project:

Evaluation of Exenatide actions on L-DOPA induced dyskinesia in a progressive Parkinson’s disease mouse model

Statement of Need, Purpose, and/or Objective:

The National Institute of Health (NIH), National Institute on Aging (NIA), Intramural Research Program (IRP), Translational Gerontology (TG) Branch requires support services to analyze markers of L-DOPA induced dyskinesia in a progressive animal model of Parkinson’s disease (MitoPark mouse). The support services will evaluate whether the drug Exenatide provides a potential treatment strategy and evaluate the efficacy of an approved and well-tolerated type 2 diabetes mellitus drug, Exenatide, in mitigating L-DOPA-induced dyskinesia in a mouse model of Parkinson’s disease; the MitoPark mouse that has parallels to the human disorder. Brain regional tyrosine hydroxylase quantification are required together with quality control studies, and extensive statistical analyses with other behavioral/biochemical data and report generation.

Background Information:

Parkinson’s disease (PD) impacts 6 million people worldwide. As it is an age-related neurodegenerative disorder, the figure is estimated to increase to around 10 million by 2030 due to longevity increasing in most Western countries. The social and economic impact on patients and their caregivers is huge and is estimated at $US14.4 billion per year in the USA.

Presently available symptomatic treatments for PD are predominantly directed on augmenting of dopaminergic signaling and can provide symptomatic relief for a limited time but have little effect on nonmotor symptoms, and none have been shown to affect the progressive pathological and clinical decline. The most widely used symptomatic treatment for PD worldwide is levodopa (L- DOPA). The use of L-DOPA elevates dopamine (DA) synthesis in the substantia nigra and restores motor functions in PD patients. However, chronic administration of L-DOPA is often associated with abnormal involuntary movements (AIMs), also called levodopa-induced dyskinesia (LID) in PD patients. Early clinical studies have shown that 20–50% of PD patients developed dyskinesia within 5 years after the initiation of L-DOPA treatment (Rascol et al., 2000; Manson et al., 2012; Bjornestad et al., 2016). The severity of dyskinesia positively correlates with disease duration, Hoehn–Yahr stage, and duration of L-DOPA treatment (Nicoletti et al., 2016). Other studies also suggest that the disease severity and dose of L-DOPA are more important than the duration of L-DOPA treatment for the development of LID (Nutt et al., 2010; Espay et al., 2018).

LID has also been established in experimental animals. Chronic administration of L-DOPA to unilaterally 6-OHDA-lesioned rats has been widely used to examine AIMs (Lundblad et al., 2002). Similar to the PD patients, LID in the lesioned rats significantly correlates with the dose of L-DOPA and the magnitude of DA depletion (Putterman et al., 2007). LID can severely impact PD patients and new treatments to mitigate it are sorely needed.

NIA recently reported that systemic administration of PT320 (a sustained release formulation of the GLP-1 receptor agonist Exendin-4, given once every 2 weeks to unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats, provides sustained plasma exendin-4 levels (Chen https://pubmed.ncbi.nlm.nih.gov/10816186/ https://pubmed.ncbi.nlm.nih.gov/10816186/ https://pubmed.ncbi.nlm.nih.gov/23938226/ https://pubmed.ncbi.nlm.nih.gov/26585090/ https://pubmed.ncbi.nlm.nih.gov/26964541/ https://pubmed.ncbi.nlm.nih.gov/20220120/ https://pubmed.ncbi.nlm.nih.gov/20220120/ https://pubmed.ncbi.nlm.nih.gov/30357892/ https://pubmed.ncbi.nlm.nih.gov/11860512/ https://pubmed.ncbi.nlm.nih.gov/11860512/ https://pubmed.ncbi.nlm.nih.gov/17660384/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431885/#B9 et al., 2018) and had the ability to significantly mitigate L-DOPA/benserazide-mediated dyskinesia in a rat 6-OHDA model of Parkinsonism. Three doses of PT320 were administered over 3 weeks together with daily L-DOPA/benserazide (a peripherally acting aromatic L-amino acid decarboxylase inhibitor). NIA found that PT320 normalized DA turnover in the striatum and reduced LID behavior in these lesioned animals (Yu et al., 2020). This data supports the future clinical use of PT320 as a co-treatment with L-DOPA for PD. However, importantly, before such development and associated resources are committed, the efficacy of PT320 to mitigate LID requires to be cross-validated in a different preclinical animal model of PD associated LID.

Period of Performance:

The period of performance is twelve (12) months after receipt of the purchase order.

SCOPE OF WORK

General Requirements:

Independently and not as an agent of the Government, the Contractor shall furnish all the necessary services, qualified personnel, material, equipment, and facilities, not otherwise provided by the Government as needed to perform the Statement of Work below:

1. Cross-validate the prior demonstration of PT320 mitigation of LID in a progressive mouse model of PD; specifically, in the MitoPark mouse;

2. Preclinical evaluation of PT320 Exenatide in a MitoPark mouse model of PD associated LID, and

3. Outcome measures of:

• Neurochemical;

• Analyses correlating neurochemical data with (i) behavioral and (ii) biochemical data;

• Quality control, and

• Report writing

Specific Requirements:

Animals. MitoPark mice have been generated on a C57BL6 background, in which the DA transporter (DAT) promoter is used to drive cre-recombinase expression, and these mice have been crossed with mice in which the Tfam gene has been loxP-flanked. These mice have been appropriately genotyped and used in approved Animal protocols whereby the LID protocol was followed as in the prior successful study of Yu et al., 2020.

L-dopa/benserazide-induced dyskinesia (LID) was examined time-dependently by the abnormal involuntary movements (AIMs) test in the presence and absence of PT320 Exenatide (0.6 mg/kg each two weeks, subcutaneously). Animals were placed in clear Perspex boxes (22 cm × 34 cm × 20 cm). Each mouse was then observed for 1 min at 30 min intervals following L- DOPA/benserazide administration over a 3 h period. Three subtypes of AIMs were assessed: (i) limb – random uncontrollable movements of forelimb contralateral to the lesion; (ii) orolingual – excess chewing and jaw movements with protrusion of the tongue; and (iii) axial – dystonic postures or choreiform twisting of the neck and upper body toward the contralateral side. The ALO score is the sum of all AIMs (axial, limb, and orolingual) scores. The severity of each AIM was scored non-parametrically between 1 and 4, based upon the following criteria:

1 = present for less than 30 s https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431885/#B9 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431885/#B42

2 = present for more than 30 s 3 = present throughout a minute but suppressed by external stimuli 4 = present throughout a minute but not suppressible by external stimuli

Dopamine release data has been generated by fast scan cyclic voltammetry to determine DA release in striatum and nucleus accumbens following the protocol of Wang et al., 2021.

The brains of MitoPark mice have been removed and frozen to allow Western blotting and evaluation of tyrosine hydroxylase (TH) levels in line with Wang et al., 2021.

Required services to be performed include:

(i) Neurochemical/TH quantification shall be performed on all brain samples (20 brains) – both striatum and substantia nigra to quantify TH levels.

Specifically, proteins shall be electrophoresed on a 12% SDS-polyacrylamide gel and electro-transferred onto a polyvinyl difluoride membrane. After a 45 min incubation with blocking buffer (2% bovine serum albumin with tris-buffered saline with Tween 20 (TBST), membranes shall be incubated with primary antibodies against TH (1:1000, rabbit, ab75875, Abcam) and beta-actin (1:5000, rabbit, ab8227, Abcam, Cambridge, United Kingdom) overnight. After washing 3 times with TBST, the membranes shall be incubated with goat HRP-linked antirabbit IgG antibodies (1:20,000, ab6721, Abcam) for 1 h, developed with an enhanced chemiluminescence (ECL) detection kit (Amersham Life Sciences, Piscataway, NJ), and imaged with UVP ChemStudio Plus (Analytik Jena AG, Germany). Results shall be normalized to the levels of beta-actin used as a loading control, and the amount of immunoreactivity will be calculated relative to the expression with the corresponding controls.

Importantly, each animal shall be tracked separately to allow analyses with other measures recorded in the same animal

(ii) Quality control Quality control shall be undertaken in relation to samples available from Wang et al., 2021.

(iii) Data analyses Data analyses shall be performed to (a) evaluate the impact of treatment – with and without PT320 exenatide (0.6 mg/kg biweekly, subcutaneously). (b) Evaluate correlation coefficients in relation to animal TH levels with behavioral measures (i.e., AIM Scores – for limb, orolingual and axial measure. (c) Evaluate correlation analyses between TH and DA release.

Statistics Statistical evaluations shall be preformed (ANOVA) followed by appropriate post hoc tests to support multiple comparisons. All statistical tests shall be two-tailed and performed using appropriate software (GraphPad Prism 5.02, GraphPad Scientific, San Diego, CA, USA). A p-value <0.05 using a two-tailed test was considered significant.

Report https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8033754/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8033754/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8033754/

A detailed report shall be provided of the background, methods and results (including statistical analyses and graphs/tables of major findings.

Brain samples and behavioral AIM score/DA release shall be provided by the Government.

GOVERNMENT RESPONSIBILITIES

The Government will provide:

1. Brain samples for the services to be performed in the SOW, and

2. Data, re: AIM score and DA release to support analyses

DELIVERABLE

A report providing a written overview of the data (i.e., tables and/or graphs/figures), statistical analyses, and evaluation of key results – as an electronic file.

REPORTING REQUIREMENTS

One (1) 1 report is required – see deliverables, above.

OTHER CONSIDERATIONS

Travel:

Not applicable.

Key Personnel:

Experienced neuroscientist with a strong background in PD research and neurodegeneration focused on drug development with background in neuroscience/neurodegeneration (with M.D., Ph.D. or both) and knowledge of Mitopark mice and GLP-1 receptor agonist drugs

Information System Security Plan:

Not applicable.

Data Rights:

The National Institute on Aging shall have unlimited rights to and ownership of all deliverables provided under this contract including reports, recommendations, briefings, work plans and all other deliverables. This includes the deliverables provided under the basic contract and any optional task deliverables exercised by the contracting officer. In addition, it includes any additional deliverables required by contract change. The definition of “unlimited rights” is contained in Federal Acquisition Regulation (FAR) 27.401, “Definitions.” FAR clause 52.227-14, “Rights in Data-General, ” shall be incorporated by reference and made a part of this contract/order.

Section 508—Electronic and Information Technology Standards:

Not applicable.

Publications and Publicity:

The Contractor is not required to write or publish articles of data in relation to the SOW. Data from this service may be included in publications.

Confidentiality of Information:

The Contractor shall keep confidential any documents/data provided by the Government that are not in the public domain and that are marked as ‘confidential.