A Plasma-based Apolipoprotein ELISA Assay to detect Neuropsychiatric Disorders – Project Proposal (ISSN 2753-8176 (online))

1. Ana Pedro

1.Gwyntwr1386 Pharmacy, Regus Chester Business Park, Heronsway, Chester, CH49QR, UK. info@gwyntwr1386.com

Protocol title

A Plasma-based Apolipoprotein ELISA Assay to detect Neuropsychiatric Disorders

Protocol identifying number

Version 0.1

Date

14/07/2024

Name and address of the Sponsor and monitor

Name and title of the person(s) authorised to sign the protocol and the protocol amendment(s) for the Sponsor

Ana Pedro, MPHARM, PhD

Name and title of the investigator(s) who is (are) responsible for conducting the study, and the address and telephone number(s) of the study site(s).

Ana Pedro, MPHARM, PhD

Name(s) and address(es) of the clinical laboratory(ies) and other medical and/or technical department(s) and/or institutions involved in the study

Background Information

Neuropsychiatric disorders (NDs) are a diverse group of pathologies, including schizophrenia or bipolar disorders, that directly affect the mental and physical health of those who suffer from them, with an incidence that is increasing worldwide. In addition to diverse clinical presentations, these diseases are heterogeneous in their pathogenesis, brain regions affected, and clinical symptoms, making diagnosis difficult. Therefore, finding new biomarkers is essential for the detection, prognosis, response prediction, and development of new treatments for NDs. Among the most promising candidates are the apolipoproteins (APO), “any of various proteins that combine with a lipid to form a lipoprotein, such as HDL and LDL”, or “The protein components of lipoproteins, which remain after the lipids to which the proteins are bound have been removed”(1). Changes in APOD levels have been demonstrated in neuropsychiatric conditions like schizophrenia (SZ) or bipolar disorders (BPD), not only in some brain areas but in corporal fluids, i.e., blood or serum of patients (2). We found out in a recent publication (3) that apoliproteins E (APOE) (total) and L1 (APOL1) are present in the plasma of autistic children and their parents but not in the plasma of their normal siblings. APOE present on high-density lipoproteins primarily function is lipid transport and cholesterol homeostasis in the central nervous system (CNS). APOE has an high expression in the prefrontal cortex and falls by approximately 50% from neonatal to adult ages (5). Moreover, APOE methylation in pediatric patients with autistic spectrum disorders (ASD) is much higher than that in the healthy controls and APOE hypermethylation is inversely correlated with lower expression (4). Also, a genetic study suggest that APOE2 and APOE4 variants are associated with ASD (6). Cultured astrocytes and neurons expressing APOE4 show reduced cholesterol and phospholipid secretion, decreased lipid-binding capacity, and increased intracellular degradation. Domain interaction, in which arginine-61 interacts ionically with glutamic acid-255, and a less stable conformation than APOE2 are supposed to be responsible for this dysfunction. Blocking this domain interaction by gene targeting (replacing arginine-61 with threonine) or by small-molecule structure correctors can increase CNS APOE4 levels and its lipid-binding capacity and decreases its intracellular degradation. Furthermore, plasma levels of APOE vary with APOE genotype (APOE2>APOE4) (7,8). In healthy controls, APOE2 is more abundant in is more abundant in plasma then APO4 (9). The presence of total APOE and APOL1 in the plasma precipitates of all of the autistic children and their parents but not in the plasma of their normal sibling may underline a still yet unknown feature of CNS cholesterol transport and metabolism which might be important to understand the causes of ASD.

We propose to collect around 50 plasma samples from patients with NDs and 50 plasma samples from healthy controls and to develop a plasma-based ELISA assay based on apoliprotein detection to detect and differentiate among these different type of disorders. Patient and controls plasma samples will be collected at …. and will be analysed at Gwyntwr1386 Pharmacy.

The study will be conducted in compliance with the protocol, GCP and the applicable regulatory requirement(s).

References:

1. Elliot et al (2010).Apolipoproteins in the brain: implications for neurological and psychiatric disorders. Clin Lipidol. 2010 August 1; 51(4): 555–573. doi:10.2217/CLP.10.37.

2. Del Valle et al (2023). Apolipoprotein D as a Potential Biomarker in Neuropsychiatric Disorders. Int. J. Mol. Sci. 2023, 24, 15631

3. Pedro A (2022). The role of apoliproteins APOE and APOL1 in Autism DOI:10.13140/RG.2.2.23055.05286, ISSN 2753-8176 (online)

4.Hu et al. (2018).APOE hypermethylation is associated with autism spectrum disorder in a Chinese population. EXPERIMENTAL AND THERAPEUTIC MEDICINE 15: 4749-4754, 2018

5.Elliot et al (2010). Apolipoproteins in the brain: implications for neurological and psychiatric disorders. Clin Lipidol. 2010 August 1; 51(4): 555–573.

6. Giunco CT, de Oliveira AB, Carvalho-Salles AB, Souza DS, Silva AE, da Rocha SS and Fett-Conte AC: Association between APOE polymorphisms and predisposition for autism. Psychiatr Genet 19: 338, 2009.

7.Utermann G. Genetic polymorphism of apolipoprotein E – impact on plasma lipoprotein metabolism. In: Crepaldi G, Tiengo A, Baggio G, eds. Diabetes, Obesity and Hyperlipidemias – III. Amsterdam: Elsevier Science Publishers; 1985:1–28

8. Rasmussen KL, Tybjaerg-Hansen A, Nordestgaard BG, Frikke-Schmidt R. Plasma levels of apolipoprotein E and risk of dementia in the general population. Ann Neurol. 2015;77:301–311. doi: 10.1002/ana.24326.

9. Gupta VB, Wilson AC, Burnham S, et al; AIBL Research Group. Follow-up plasma apolipoprotein E levels in the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing (AIBL) cohort. Alzheimers Res Ther. 2015;7:16. doi: 10.1186/s13195-015-0105-6.

Study Objectives and Purpose

Finding new biomarkers for NDs which will help in the detection, prognosis, response prediction, and development of new treatments for NDs.

Study Design

Pilot study – 4 years

We propose to collect around 50 plasma samples from patients with NDs (vulnerable groups) and 50 plasma samples from controls and to develop a plasma-based ELISA assay based on apoliprotein detection to detect and differentiate among these different type of disorders. Patient and controls plasma samples will be collected at …. and will be analysed at Gwyntwr1386 Pharmacy.

1. Plasma samples from controls and patients will be collected at ….and stored at -80C at ….and a sample number such as “GP1” will be associated with each patient clinical history and diagnosis.

2. Plasma samples will be tested for the presence different of different apolipoproteins types by ELISA.

3. Each patient clinical history and diagnosis with be annotated in an Excel table. The patients will be named by “GP” numbers in the Excel table.

4. ELISA testing results will be compared with each patient diagnosis and clinical history and statistical analysis and interpretation of the results will be undertaken. We will look forward to develop a test to diagnose NDs and will take in consideration reliability, validity, precision and interpretability.

Statistics

Number of participants planned to be enrolled – 100 controls and patients plasma samples

Reason for choice of sample size, including reflections on (or calculations of) the power of the study and clinical justification.

The level of significance to be used.

Procedure for accounting for missing, unused, and spurious data.

Procedures for reporting any deviation(s) from the original statistical plan (any deviation(s) from the original statistical plan should be described and justified in protocol and/or in the final report, as appropriate).

The selection of participants to be included in the analyses - all eligible participants diagnosed with NDs

If the chosen outcome measures have to be adapted in some way (e.g. to a specific illness or population), or designed because no existing measures are appropriate for the aims of the study, assessments of concurrent validation should be incorporated into the analysis plan (discuss with study statistician).

Sample sizes between 24 and 50 have been recommended (Lancaster et al, 2004; Sim & Lewis, 2012; Julious, 2005)

PPI

We will recruit people for PPI through …..

Direct Access to Source Data/Documents

The Sponsor should ensure that it is specified in the protocol or other written agreement that the investigator(s)/institution(s) will permit study-related monitoring, audits, REC review, and regulatory inspection(s), providing direct access to source data/documents.

Quality Control and Quality Assurance

We will make sure all experiments will be recorded in lab notebooks with details of those were preformed.

Ethics

Description of ethical considerations relating to the study.

Data Handling and Record Keeping

Description of data management procedures (see DM02 Research Data Management).

Finance and Insurance

Financing and insurance details to be included, if not addressed in a separate agreement. Reference to the separate agreements should be included if possible.

Any testing kits resulting from this study will be developed and commercialized by Gwyntwr1386 Healthcare CIC

Publication Policy

Publication policy, if not addressed in a separate agreement. Reference to the separate agreements should be included if possible.