WO2018052325A1

WO2018052325A1 - A method for detecting panic disorder

Info

Publication number: WO2018052325A1
Application number: PCT/PL2017/050044
Authority: WO
Inventors: Marcin JABŁOŃSKI; Jolanta KUCHARSKA MAZUR; Jerzy SAMOCHOWIEC; Mariusz Ratajczak; Maciej TARNOWSKI; Barbara DOŁĘGOWSKA
Original assignee: Pomorski Uniwersytet Medyczny W Szczecinie
Priority date: 2016-09-14
Filing date: 2017-09-13
Publication date: 2018-03-22
Also published as: EP3513199A1; PL418690A1

Abstract

Disclosed is the use of plasma level of complement components, plasma level of sphingosine-1 -phosphate and stromal factor as markers of panic disorder, as well as the use of the number of HSCs (Lin-/CD45+/AC133+) as a marker of panic disorder.

Description

A method for detecting panic disorder

The invention consists in the use of plasma levels of complement components, sphingosine -1- phosphate and stromal factor, as well as the number of HSCs (Lin- / CD45 + / AC133 +) as markers of panic disorder. The invention can find application in medical diagnostics and be used to determine or confirm the diagnosis of panic disorder.

To date, there there have been very few reports regarding potential correlations between stem cell behaviour, factors responsible for their movement and the issue of mental disorders. One such report constituted a systematic evaluation of the problem of stem cell behaviour and factors responsible for their movement in first-episode psychosis (Kucharska-Mazur et al., 2014), further studies involved the effect of lithium administration on stem cells in bipolar disorder (Ferensztajn-Rochowiak et al., 2016). Reports on the importance of regeneration processes in the etiology of anxiety disorders are extremely scarce, despite the fact that stem cells are detected in the peripheral blood in various models of serious disorders or injuries associated with significant stress level, such as myocardial infarction, burns, inflammatory bowel disease, stroke (Wojakowski et al., 2009; Kucia et al., 2006; Paczkowska et al., 2009, Marlicz et.al 2012).

Subject invention aims to provide a relatively easy to implement and unambiguous diagnostic detection method of panic disorder. The sought-after diagnostic method based on this invention should allow faster and more accurate recognition of anxiety disorders, and also enable to effectively monitor the course of patient recovery.

The object of this invention is a method for detecting panic disorder that consists in collecting a biological sample from a patient and analysing plasma concentration levels of complement complex components, sphingosine- 1 -phosphate and stromal growth factor, wherein a finding of reduced concentrations of those molecules, in comparison to their average concentrations observed in healthy individuals, indicates detection of panic disorder in said patient.

Preferably, C3a, C5a and C5b are analysed as complement complex components.

Additionally, the level of stem cells in peripheral blood is preferably analysed directly, and a reduction in the mobilization of peripheral blood stem cells, particularly HSCs (Lin- / CD45 + / AC133 +), is a marker of panic disorder.

In the study that led to the present invention it was sought to answer the question whether anxiety disorders affect mobilization of stem cells into the peripheral blood, and thus whether the concentration of SCs and related humoral factors may become a novel diagnostic tool for anxiety disorders.

Surprisingly, it has been observed significantly lower concentration of the analysed factors responsible for stem cell movement in patients with panic disorder as compared to the control group, and that the administered pharmacotherapy did not cause significant changes to that concentration.

Results obtained in accordance with the invention indicate that in patients with panic disorder may be observed mechanisms in which stem cells are engaged either directly or through their paracrine activity and may be involved in the etiopathogenesis of the above- mentioned disorders as well as in the repair processes that take place during recovery.

Accordingly, as there are no simple and unambiguous methods for the diagnosis of panic disorder with panic attacks in the art, the diagnostic method based on the subject invention should play an important role in better understanding processes responsible for: occurrence of anxiety disorders, effective course of recovery and, also, should enable faster and more accurate diagnosis.

Example

Methodology for the assays

Morphology was evaluated using ABX Micros 60 haematological analyser (Horiba). Blood taken on K₃EDTA was centrifuged (250g; 10 minutes; 20°C). The obtained plasma was transferred into a fresh tube and frozen in -80°C until further analysis.

Determination of sphingosine-l-phosphate using RP HPLC (reverse-phase high performance liquid chromatography)

Plasma was thawed at room temperature, followed by addition of D-erythro-sphingosine- 1-phosphate (SIP C17 - internal standard), lMNaCl and methanol. Mixing, chloroform adding, then mixing again and centrifuging were performed. Bottom organic phase was transferred into a fresh tube. Chloroform was added into non-organic phase, followed by mixing and centrifuging. The two organic phases were combined and then dried in a vacuum centrifuge.

The dried residue was dissolved in methanol. Reaction mixture (o-phthaldialdehyde, methanol, mercaptoethanol and boric acid, pH 10.5) was added, and then incubation and centrifugation were performed. Clear supernatant was transferred into a fresh tube and analysed using Hewlett Packard Series 1200 chromatograph. Chromatographic data were processed using HP Chemstation software (Agilent, former Hewlett Packard).

Separation by reverse-phase HPLC (RP-HPLC) was performed using Cosmosil 5 μιη CI 8-ARII column (150 x 4.6) with 5 μιη C18-ARII pre-column (10 x 4.6) (Waters). The column temperature was 25°C. Isocratic method was used, wherein the mobile phase was 10 mM K₂HPO₄ (pH 5.5) and methanol (15:85; v/v). The flow rate was 1 ml/min. Samples of 50 fl volume were injected every 28 minutes. Derivatives of SIP were detected using 340 nm excitation wavelength and 455 nm emission wavelength. Calculations for SIP content were based on the peak area relative to the peak area for the internal standard. A Mixture of SIP C 17 and SIP CI 8 standards (Avanti Polar Lipids) was used for peak identification and calibration (Blogowski et al., 2013b; Caligan et al., 2000; Egom et al., 2013; Starzynska et al., 2013).

Determination of plasma level of selected proteins, based on the example of C3a assayed using Human C3a ELISA Kit (BD OptEIA)

Standards and diluted plasma were pipetted into the wells of the plate. The plate was incubated and washed. Substrate (TMB) was added and the plate was incubated again, Stop Solution was added and absorbance was measured (at 450 nm wavelength) using En Vision plate reader (Perkin-Elmer) (Blogowski et al., 2012; Blogowski et al., 2013a; Blogowski et al., 2013b; Starzynska et al., 2013).

ELISA technique was used analogically for the determination of plasma level of other proteins, i.e. selected from complement complex and stromal growth factor.

Blood analysis using flow cytometry

Peripheral blood samples were lysed twice using BD Pharm Lyse lysing buffer (BD Bioscience) at room temperature for 10 minutes and then were washed with phosphate-buffered saline (PBS) with an addition of 2% fetal bovine serum (FBS; Sigma) in order to obtain total nucleated cells (TNCs) pool. Nucleated cells were then stained with hematopoietic cell lines markers using antibodies conjugated with fluorescein isothiocyanate (FITC) against human: CD2 (clone RPA-2.10); CD3 (clone UCHTl); CD14 (clone M5E2); CD16 (clone 3G8); CD19 (clone HIB19); CD24 (clone ML5); CD56 (clone NC AMI 6.2); CD66b (clone G10F5); and CD235a (clone GA-R2) (all from BD Bioscience). The cells were simultaneously stained for the panleukocytic marker, CD45, with antibodies conjugated with phycoerythrin (PE) (clone HI30; BD Biosciences) and for one of the following antigens: CD 34 - with antibodies conjugated with allophycocyanin (APC) (clone 581; BD Bioscience) or CD 133 (CD133/1) - with antibodies conjugated with APC; Miltenyi Biotec). Additionally, the following isotype controls were used: antibodies conjugated with FITC - mouse IgGl, I (clone MOPC-21), mouse IgG2a, I (clone G155-178), mouse IgG2b, I (clone 27-35); conjugated with PE - mouse IgGl, I (clone MOPC-21) and conjugated with APC - mouse IgGl, I (clone MOPC-21) (all from BD Bioscience). In addition, APC-conjugated mouse IgGl antibodies (clone IS5-21F5; Miltenyi Biotec) were used as isotype controls. Staining was performed in buffered saline (PBS) with 2 % FBS, on ice, for 30 minutes. Next, the cells were washed, resuspended in liquid and analysed using NAVIOS Flow Cytometer (Beckman Coulter). Et least 10⁶ events were acquired from each sample. The total number of VSELs and HSCs was calculated (individually for each patient) per 1 ml of peripheral blood based on the percentage content of these cells as detected by flow cytometry and the total number of white cells per 1 ml of peripheral blood. Kaluza software (Beckman Coulter) was used for analysis (Paczkowska et al., 2009; Zuba-Surma & Ratajczak 2010).

Study group

The patients were recruited in the West Pomeranian Voivodeship. All patients were examined at the Psychiatry Clinic of the Independent Public Clinical Hospital No. 1 (SPSK-1 PUM) of the Pomeranian Medical University in Szczecin. They received written information on the study and all their questions regarding the study were answered before they gave their written consent for participation.

The investigated group consisted of 30 unrelated patients diagnosed with panic disorder. Nosological diagnosis was made according to the International Classification of Mental and Behavioural Disorders (ICD-10):

• panic disorder (F 41.0) - 30 individuals, 12 of whom were additionally diagnosed with agoraphobia.

Study excluding criteria were:

• absence of written consent for participation in the study

• organic mental disorders or other Axis I disorders of the ICD-10 besides the above- mentioned diagnoses

• association of current mental disorders with the use of psychoactive substances, including alcohol

• serious somatic diseases, especially cases requiring administration of medicine

• glucose intolerance • currently active diseases of inflammatory etiology (patients were excluded based on the results of laboratory analyses and physical examination)

• use of psychotropic drugs due to any reason during the current episode and less than six months before the onset of the current episode.

A specialist psychiatrist examined individuals from the study group - medical interview, standard psychiatric examination, internal medicine examination and neurological examination were performed; additionally, accessible members of patient's family had medical interviews. Mental disorders other than anxiety disorders were excluded using MINI (Sheehan et al., 1998). The intensity of depressiveness was evaluated using MADRS scale (Montgomery et al., 1979); also, the intensity of anxiety disorders was evaluated using HAM-A scale (Bruss et al., 1994). Standardised medical history was used for collecting demographic data, family medical history and history of symptoms.

Control group

The control group consisted of 30 healthy individuals matched for gender, ethnics, age, socio- demographic factors and body mass index, without a history or current mental disorders of Axis I of the ICD-10. Those individuals were examined by a specialist psychiatrist analogically to the individuals from the study group. Also, standardised medical history was used for collecting demographic data, family medical history and history of symptoms.

Study procedures

In the study group, blood samples were collected twice - the first time in the morning, before the administration of any antidepressants, and the second time after observed clinical improvement following the treatment, wherein the clinical improvement was defined as disappearance or reduction of symptom intensity, or reduced score in the HAM-A scale.

The samples were immediately transferred for further analysis to the Laboratory of the Department of Physiology of the Pomeranian Medical University and to the Department of Medical Analytics of the Pomeranian Medical University. On blood collection days, psychiatric assessment, physical examination, psychometric evaluation and basic laboratory tests were also performed, and after blood collection, the following antidepressants were randomly administered:

• sertraline,

• paroxetine,

• venlafaxine, • amitriptyline,

• escitalopram,

• trazodone.

Doses were established depending on the tolerance of the drug by the patient.

Results and Conclusions

Table 1. Summary of plasma levels of complement components, sphingosine-1 -phosphate and stromal growth factor in the study and control group.

Key: PG - patient group, CG - control group, BT - before treatment, AT - after treatment, C3a, C5a, C5b - complement complex components, SDF-1 - stromal cell-derived factor 1, SIP - sphingosine- 1 -phosphate.

Moreover, statistically significant reduction in the number of HSCs ( Lin-/CD45+/AC133+) was found in those same patients suffering from panic disorder.

Based on the obtained results, the following conclusions were made:

- reduction in the concentration of the analysed components of complement complex ( C3a, C5a, C5b), as well as SDF-1 and SIP, is a significant marker of panic disorder.

- reduction in the level of mobilisation of HSCs ( Lin-/CD45+/AC133+) into peripheral blood is a marker of panic disorder. References:

Literature:

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Blogowski W, Dolegowska B, Budkowska M, Salata D, Domanski L, Starzynska T. Perioperative release of pro-regenerative biochemical signals from human renal allografts subjected to ischemia-reperfusion injury. Innate Immunity 2013 b Apr 22. [Epub ahead of print]

Blogowski W, Dolegowska B, Salata D, Budkowska M, Domanski L, Starzyhska T. Clinical analysis of perioperative complement activity during ischemia/reperfusion injury following renal transplantation. Clinical Journal of American Society of Nephrology 2012;7(11): 1843- 51.

Bruss, G.S., Gruenberg, A.M., Goldstein, R.D., Barber, J.P. (1994). Hamilton Anxiety Rating Scale Interview guide: joint interview and test-retest methods for interrater reliability. Psychiatry Research, 4, 53, 191-202.

Caligan TB, Peters K, Ou J, Wang E, Saba J, Merrill AH Jr. A high-performance liquid chromatographic method to measure sphingosine 1 -phosphate and related compounds from sphingosine kinase assays and other biological samples. Analytical Biochemistry 2000;281(l):36-44.

Egom EE, Mamas MA, Chacko S, Stringer SE, Charlton-Menys V, El-Omar M, Chirico D, Clarke B, Neyses L, Cruickshank JK, Lei M, Fath-Ordoubadi F. Serum sphingolipids level as a novel potential marker for early detection of human myocardial ischaemic injury. Frontiers in Physiology 2013;4: 130.

Ferensztajn-Rochowiak, E., Kucharska-Mazur, J., Samochowiec, J., Ratajczak, MZ., Michalak, M., Rybakowski, J.K. (2016). The effect of longterm lithium treatment of bipolar disorder on stem cells circulating in peripheral blood. The World Journal of Biological Psychiatry, Apr 13 : 1-25. [Epub ahead of print] Ferensztajn-Rochowiak, E., Rybakowski, J.K. (2016). The effect of lithium on hematopoetic, mesenchymal and neural stem cells. Pharmacological Reports, 68,224-30.

Kucia, M., Zhang, Y.P., Reca, R., et al. (2006). Cells enriched in markers of neural tissue- committed stem cells reside in the bone marrow and are mobilized into the peripheral blood following stroke. Leukemia, 20,18-28.

Marlicz, W., Zuba-Surma, E., Kucia, M., Blogowski, W., Starzynska, T., Ratajczak, M.Z. ( 2012). Various types of stem cells, including a population of very small embryonic-like stem cells, are mobilized into peripheral blood in patients with Crohn's disease. Inflammatory Bowel Diseases, 18,1711-22.

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Claims

1. A method for detection of panic disorder, characterised in that plasma level concentrations of complement complex components, sphingosine-1 -phosphate and stromal growth factor are analysed in the biological sample taken from the patient, wherein a finding of reduced concentrations of those molecules, in comparison to their average concentration observed in healthy individuals, indicates the detection of panic disorder in said patient.

2. A method, according to claim 1, characterised in that C3a, C5a and C5b are analysed as complement complex components.

1. A method, according to claim 1 , characterised in that additionally, the level of stem cells in peripheral blood is analysed directly, wherein reduced level of mobilisation of stem cells, particularly HSCs (Lin-/CD45+/AC133+), into peripheral blood is a marker for panic disorder.