Safety/Efficacy of Intranasally-Administered Bioactive Factors Produced by Autologous M2 Macrophages in Patients With Organic Brain Syndrome
Phase: PHASE1
Conditions: Organic Brain Syndrome, Nonpsychotic, Neurocognitive Disorders, Mental Disorder, Organic, Delirium, Dementia, Amnestic, Cognitive Disorders, Nonpsychotic Organic Brain Syndrome, Organic Mental Disorder, Encephalopathy, Post-Traumatic, Chronic, Encephalopathy, Ischemic, Brain Ischemia
Sponsor: Russian Academy of Medical Sciences

Study Identification 
 
Unique Protocol ID: IFCI-25/05/2015 
Brief Title: Intranasal Inhalations of Bioactive Factors Produced by M2 Macrophages in Patients 
With Organic Brain Syndrome  
Official Title: Safety/Efficacy of Intranasally-Administered Bioactive Factors Produced by 
Autologous M2 Macrophages in Patients With Organic Brain Syndrome 
 
The protocol was approved by the Ethics Committee of the Institute of Fundamental and Clinical 
Immunology (approval number: no 92, 11/10/2015) 
 
Study Protocol 
 
 
 
 
 
 
 
October 11, 2015 
 
 
Scientific background 
 
Studies over the last two decades have demonstrated that macrophages could cause not 
only neurodestructive but also neuroreparative effects, with macrophage activation being an 
essential prerequisite for the functional restoration of central nervous system (CNS) following 
damage [1, 2, 3, 4]. This dual impact is related to the heterogeneity of macrophages [5]. 
Classically activated М1-type macrophages with pro-inflammatory phenotype possess 
neurotoxic properties, while М2-type alternatively activated macrophages with anti-
inflammatory properties mediate neuroprotection, activate neuro- and angio-genesis, and play an 
essential role in neuronal plasticity and axon remodelling [4, 6, 7]. 
 
Macrophages within CNS comprise microglia (the dominant macrophage population) and 
monocyte-derived CNS-infiltrating macrophages. Upon recognition of danger molecules, short-
term or moderate signal directs microglia toward M2 regenerative phenotype, while intensive 
acute or chronic activation induces M1 neurotoxic phenotype [8]. In last case, neuroreparative 
deficiency is likely to be compensated by recruiting peripheral blood monocytes and their M2 
polarisation [9, 10]. In accordance with this notion, therapeutic approaches targeting macrophage 
polarisation towards M2 phenotype has been considered as a novel strategy for stimulating 
neuroreparative processes [11, 12]. 
 
We have previously developed an original protocol for obtaining M2-like macrophages 
based on macrophage interaction with apoptotic cells [13]. As compared to M1, M2 
macrophages generated were characterised by low antigen-presenting and pro-inflammatory 
activity, while possessing more pronounced regeneration potential due to high production of a 
variety of growth and neurotrophic factors [13, 14]. Pilot studies of M2 intrathecal 
administrations in ischemic stroke [15] and severe cerebral palsy [16, 17] demonstrated the 
safety and amelioration of motor and cognitive functions. However, the invasiveness of 
intrathecal injection and the possibility of M2→M1 repolarization in pathological 
microenvironment were serious factors limiting clinical translation of this treatment. In this 
connection, the utilization of M2-derived bioactive factors (M2-BFs) instead of cells and 
intranasal administration of M2-BFs that allows  for rapid delivery of various substances 
(including neurotrophic factors) in brain tissues [18] via olfactorial and trigeminal route 
bypassing blood-brain barrier appears to be a promising approach  [19, 20, 21]. A possibility for 
cytokines and hormones to be delivered into brain tissue upon intranasal route of delivery has 
been demonstrated in rodent and primate experimental models [22] as well as in human being 
[23, 24]. 

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Since conditioned M2 macrophage culture medium contains a wide spectrum of 
neurotrophic, pro-angiogenic and immunoregulatory cytokines, we have designed an innovative 
proof-of-concept trial designed to provide data as to whether the treatment/rehabilitation efficacy 
and functional outcome of patients with organic brain syndrome are improved with intranasal 
inhalations of bioactive factors, produced by autologous M2 macrophages (auto-M2-BFs). 
 
The aim of this study was to assess safety and clinical effectiveness of intranasal administration 
of bioactive factors, produced by autologous M2 macrophages in patients with organic brain 
syndrome. 
 
Study Design 
Study Type: Interventional 
Primary Purpose: Treatment 
Study Phase: Phase 1/Phase 2 
Intervention Model: Single Group Assignment 
Number of Arms: 1 
Masking: No masking 
Enrollment: 30  
 
Criteria:  
Inclusion Criteria: 
• Adults: age 18 - 80 
• Persistent neurological deficits (cognitive, mental, motor, vestibular/ataxic disorders 
as a result of trauma, cardiovascular, neurodegenerative and others cerebral 
injuries), confirmed clinically and by CT or MRI 
• A written informed consent of the patient or close relatives  
Exclusion Criteria: 
• 
Psychiatric disorders 
• 
Seizures 
• 
Severe dementia 
• 
Hepatic or renal dysfunctions 
• 
Hemodynamic or respiratory instability 
• 
HIV or uncontrolled bacterial, fungal, or viral infections 
• 
Pregnancy 
• 
Malignancy 
• 
Intolerance to gentamicin and / or multiple drug allergies 
• 
Participation in other clinical trials 
 
Outcome Measures 
Primary Outcome Measure: 
1. The Number of Patients With Severe Adverse Events and Adverse Reactions 
Time Frame: up to 6 months after treatment 
Measure Description: Occurrence of severe adverse events and adverse reactions (allergic, toxic, 
inflammatory reactions; neurological deterioration, convulsive syndrome) 
 

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Secondary Outcome Measure: 
2. Change in Subjective Assessment of Clinical Symptoms (SACS) 
Time Frame: Baseline and 6 months after treatment 
Measure Description: Subjective Assessment of Clinical Symptoms (SACS) is a 5-point rating 
scale with standardized criteria (0 – no; 1 – mild; 2 – moderate; 3 – severe; 4 – intensive) 
subjective assessment of the severity of fifteen clinical symptoms most characteristic of 
neurological disorders (headache, dizziness, gait disturbance, speech, visual impairment, tremor 
et al). Minimum SACS "total" score is 0, and maximum SACS "total" score is 60. Neurological 
improvements are assessed by SACS "total" score as > 6 points’ reduction from baseline. 
 
3. Change in Hospital Anxiety and Depression Scale (HADS) 
Time Frame: Baseline and 6 months after treatment 
Measure Description: Hospital Anxiety and Depression Scale (HADS) is used to diagnose 
anxiety/depression symptoms (absence – 0~7 points; subclinical form – 8~10 points; clinical 
form – 11 points or more). Minimum HADS "total" score (anxiety +depression subscale) is 0, 
and maximum HADS "total" score is 42. Improvements in patients with anxiety/depression 
symptoms are assessed by HADS "total" score as > 4 points reduction from baseline. 
 
4. Change in Functional Mobility Assessment (FMA) Scale 
Time Frame: Baseline and 6 months after treatment 
Measure Description: Functional Mobility Assessment (FMA) is cale is designed to evaluate 
parameters characterizing stability (0~24 points) and gait (0~16 points). The maximum FMA 
"total" score on stability and gait subscales is 39-40 and corresponds to the norm, minimum 
FMA "total" score is 0 and corresponds to the gross impairment. The degree of impairment of 
"total" score is divided into significant (0~20 points), moderate (21~33 points), and light (34~38 
points), whereas 39~40 points indicate no impairments. Improved mobility is assessed as FMA 
"total" score enhancement > 4 points from baseline. 
 
5. Change in Montreal Cognitive Assessment (МоСА) 
Time Frame: Baseline and 6 months after treatment 
Measure Description: Montreal Cognitive Assessment (MoCa) is used to assess cognitive 
functions. The maximum MoCa "total" score is 26-30 points and corresponds to the norm, 19-25 
points - mild cognitive disorder; 11-21 points - dementia. Improvements in patients with 
cognitive disorder are assessed as MoCA "total" score increase > 3 points from baseline. 
 
Methods 
M2-BFs preparation  
 
M2 macrophages are generated from adherent mononuclear cells (MNCs), as described 
previously [13, 14]. Briefly, MNCs are separated standardly from heparinized venous blood 
(150-200 ml) of a patient. The plastic-adherent MNCs are cultured in RPMI-1640 medium 
containing 0.05 mM 2-mercapethanol, 2 mM sodium pyruvate, 0.3 mg / ml L-glutamine, 1% 
solution of essential amino acids, 100 μg/ml gentamicin, 2% autoplasma, and recombinant 
human GM-CSF (rhGM-CSF, 50 ng/ml, R&D Systems) for 7 days. Conditioned medium of M2 
macrophages is subjected to sterilizing filtration and collected in sterile vials (2.0 ml/vial), which 
are labeled as M2-BFs and stored at -20° C. 

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Intranasal M2-BFs administration 
 
The conditioned medium of patient M2 macrophages (2.0 ml) is thawed at room 
temperature and used as a fine aerosol intranasally using a compressor inhaler (nebulizer). 
Patient will receive their first doses (n=2-3) of M2-BFs in clinic under the supervision of a 
physician and wait 2 hrs to determine any short-time adverse effects of inhaled dose. The 
subsequent course of intranasal inhalations (once a day up to 30 days) performed as outpatient 
treatment. 
 
Statistical Analysis 
 
Data will be analyzed using Statistica 6.0 software for Windows (StatSoft Inc. USA). The 
results of statistical analysis will be presented as median (Me) and interquartile range (IQR; LQ-
UQ). To check the normality of the distribution of data, the Kolmogorov-Smirnov test and the W 
Shapiro-Wilk test will be used. To assess the significance of differences, Fisher’s exact test (for 
discrete variables) and the non-parametric Mann-Whitney test (for continuous variables) will be 
used. Spearman’s rank correlation coefficient (RS) will be used to measure the statistical 
dependence between two continuous variables. Differences with p less than 0.05 will be 
considered statistically significant. 
 
Contacts 
Central Contact: Natalia M Starostina, PhD 
 
Telephone: +7 (383) 228-27-41 
 
Email: starostina48@bk.ru 
 
Central Contact Backup: Mariya N Davydova 
 
Telephone: +7 (383) 228-54-21 
 
Email: davydoff2002@mail.ru 
 
Study Chair: Elena R Chernykh, MD, PhD 
 
Telephone: +7 (383) 236-03-29 
 
Email: ct_lab@mail.ru 
 
Study Principal Investigator: Alexander A Ostanin, MD, PhD 
 
Telephone: +7 (383) 236-03-29 
 
Email: ostanin62@mail.ru 
 
Location  
Institute of Fundamental and Clinical Immunology 
Novosibirsk, Russian Federation, 630099 
 
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