Clinical trial NCT02957123
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
Keywords: macrophages M2 type cytokines intranasal administration neuroprotection neuroregeneration organic brain syndrome neurocognitive disorders
Sponsor: Russian Academy of Medical Sciences
Start date: 2016-03-01; Completion date: 2020-09-01
Enrollment: 30 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The investigators 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 (BF), produced by autologous M2 macrophages (auto-M2-BFs). The rationale for this approach is the ability of central nervous system to repair and the important role of macrophages in the regulation of this process. It was found that type 2 macrophages have anti-inflammatory and reparative potential, whereas M1 cells possess pro-inflammatory and neurotoxic effects. Action of M2 macrophages is largely realized through the production a wide variety of bioactive factors (cytokines, chemokines, growth factors, neuropeptides, microvesicles etc) that inhibit inflammation, protect neurons from apoptosis, stimulate neurogenesis, the growth and remyelination of axons, the formation of new synapses and activate angiogenesis. This study uses auto-M2-BFs, as therapeutic agents and intranasal administration focusing on nose to brain transport, as a mode of delivery. Expected clinical effects in treated subjects: improvement of cognitive functions (memory, language, attention); correction of focal neurological deficit (paresis, spasticity, sensory disorders); reduction vestibular/ataxic disorders (vertigo, unsteadiness when walking); reduction of headaches; reduction of asthenia (weakness, fatigue); correction of emotional disorders (anxiety, depression).

Detailed description:
Following injury to the central nervous system (CNS), immune-mediated inflammation profoundly affects the ability of neural cells to survive and to regenerate. The role of inflammation comprises mostly of macrophages, is controversial, since macrophages can both induce neuronal and glial toxicity and promote tissue repair. The opposite effects of macrophages may be conditioned by their functional heterogeneity. Thus, classical pro-inflammatory macrophages (M1) are tissue-destructive, while anti-inflammatory (M2) macrophages mediate tissue repair. In addition, M2 macrophages predominantly induce the Th2 response, which is particularly beneficial in CNS repair. Using low serum conditions the investigators have generated M2-like macrophages and evaluated their phenotypic and functional features \[1, 2\]. Our data indicate that M2 macrophages, in contrast to pro-inflammatory M1 cells, produced significantly lower levels of pro-inflammatory cytokines (IL-1β, tumor necrosis factor-α, IL-6, IL-18, IL-12), chemokines (IL-8, monocyte chemoattractant protein 1-1) and Th1/Th2-cytokines (interferon-γ, IL-2, IL-4) coupled with a higher IL-10 level. M2 macrophages were capable of producing neurotrophic- (brain-derived neurotrophic factor,insulin-like growth factor-1), angiogenic- (vascular endothelial growth factor), and other growth factors (erythropoietin, granulocyte-colony stimulating factor , basic fibroblast growth factor, epidermal growth factor) with neuroprotective and regenerative activity.

Our pilot clinical trials have demonstrated the safety and clinical efficacy of intrathecal administration of M2 macrophages in children with severe cerebral palsy \[3\] and in non-acute stroke patients \[4\].

Since cell-free culture medium of M2 macrophages contains a wide variety of neurotrophic, immunoregulatory and pro-angiogenic factors, the investigators expect that intranasal administration of these auto-M2-BFs will improve the treatment/rehabilitation efficacy and functional outcome of patients with organic brain syndrome. Of note, intranasal administration of M2-macrophage soluble factors allow to delivery bioactive agents to brain through the olfactory and trigeminal ways across brain-blood barrier.

Arms: arm 1: Intranasally-Administered Bioactive Factors, Produced by Autologous M2 Macrophage (auto-M2-BFs). M2 macrophages were generated in vitro from peripheral blood of patients during 7 days.Cell-free culture medium, containing auto-M2-BFs, was collected and aliquots of 2 mL/vial were cryopreserved.

30 patients with organic brain syndrome will receive auto-M2-BFs with the aerosol inhaler device (nebulizer), 2.0 mL once a day up to 30 days.
Interventions: intervention 1: Intranasal auto-M2-BFs
Intervention descriptions: intervention 1: Delivery is performed with the aerosol inhaler device (nebulizer), 2.0 mL once a day up to 30 days.

Protocol documents:
SAP_002.pdf: 
Study Identification 
 
Project Title: Safety/Efficacy of Intranasally-Administered Bioactive Factors 
Produced by Autologous M2 Macrophages in Patients With Organic Brain 
Syndrome  
Brief Title: Intranasal Inhalations of Bioactive Factors Produced by 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 no 92, 11/10/2015). 
 
October 11, 2015 
 
Statistical Analysis Plan 
 
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.  

ICF_001.pdf: 
Study Identification 
 
Brief Title: Intranasal Inhalations of Bioactive Factors Produced by M2 Macrophages 
in Patients With Organic Brain Syndrome  
October 11, 2015 
 
 Informed Consent Form for Research Involving Human Subjects  
You are being invited to participate in a research study, which the Institute of Fundamental and Clinical 
Immunology (IFCI) Institutional Review Board (IRB) has reviewed and approved (approval no 92, 11/10/2015). 
This form is designed to provide you - as a human subject - with information about this study. The Investigator 
will describe this study to you and answer any of your questions. You are entitled to a copy of this form. If you 
have any questions or complaints about the informed consent process of this research study or your rights as a 
subject, please contact the IRB at the IFCI  Office of Institutional Research at (383) 228-54-21 or cke@ngs.ru. 
 
 Thank you for agreeing to participate in this research project.   
 
Project Title: Safety/Efficacy of Intranasally-Administered Bioactive Factors Produced by Autologous 
M2 Macrophages in Patients With Organic Brain Syndrome 
Principal Investigator: Alexander A Ostanin, PhD, MD, Head of Clinical Department, (383) 236-03-29 
 
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Prot_000.pdf: 
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 
 
References 
1. Donnelly DJ, Popovich PG. Inflammation and its role in neuroprotection, axonal regeneration 
and functional recovery after spinal cord injury. Exp Neurol. 2008, 209(2): 378–338. 
2. Hohlfeld R, Kerschensteiner M, Meinl E. Dual role of inflammation in CNS disease. 
Neurology. 2007, 68(22): S58–S63. 
3. Kerschensteiner M, Gallmeier E, Behrens L, et al. Activated human T cells, B cells, and 
monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: 
a neuroprotective role of inflammation. J Exp Med. 1999, 189(5): 865–870. 


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4. Yin Y, Cui Q, Li Y, et al. Macrophage-derived factors stimulate optic nerve regeneration. J 
Neurosci. 2003, 23(6): 2284–229. 
5. Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nat Rev Immunol. 2005, 
5(12): 953–964. 
6. Hu X, Leak RK, Shi J, et al. Microglial and macrophage polarization - new prospects for brain 
repair. Nat Rev Neurol. 2015, 11(1): 56–64. 
7. Kigerl KA, Gensel JC, Ankeny DP, et al. Identification of two distinct macrophage subsets 
with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal 
cord. J Neurosci. 2009; 29(43):13435–13444. 
8. Wake H, Moorhouse AJ, Nabekura J. Functions of microglia in the central nervous system--
beyond the immune response. Neuron Glia Biol. 2011, 7(1):47-53. 
9. Perego C, Fumagalli S, Zanier ER, et al. Macrophages are essential for maintaining a M2 
protective response early after ischemic brain injury. Neurobiol Dis. 2016, 96:284-293. 
10. London A, Cohen M, Schwartz M. Microglia and monocyte-derived macrophages: 
functionally distinct populations that act in concertin CNS plasticity and repair. Front Cell 
Neurosci. 2013, 7:34. 
11. Hu X, Li P, Guo Y, et al. Microglia/macrophage polarization dynamics reveal novel 
mechanism of injury expansion after focal cerebral ischemia. Stroke. 2012; 43(11):3063–3070. 
12. Sanberg PR, Park D-H, Kuzmin-Nichols N, et al. Monocyte transplantation for neural and 
cardiovascular ischemia repair. J Cell Mol Med. 2010, 14(3): 553–563. 
13. Chernykh ER, Shevela EY, Sakhno LV, et al. The generation and properties of human M2-
like macrophages: potential candidates for CNS repair? Cell Ther Transplant. 2010, 2(6): 
e.000080.01. 
14. Sakhno LV, Shevela EY, Tikhonova MA, et al. The phenotypic and functional features of 
human M2 macrophages generated under low serum conditions. Scand J Immunol. 2016, 83(2): 
151-159. 
15. Chernykh ER, Shevela EY, Starostina NM, et al. Safety and therapeutic potential of M2-
macrophages in stroke treatment. Cell Transplant. 2016, 25(8): 1461-1471. 
16. Chernykh ER, Kafanova MY, Shevela EY, et al. Clinical experience with autologous M2-
macrophages in children with severe cerebral palsy. Cell Transplant. 2014, 23(Suppl. 1): S97-
104. 
17. Chernykh ER, Shevela EY, Kafanova MY, et al. Monocyte-derived macrophages for 
treatment of cerebral palsy: A study of 57 cases. J Neurorestoratology. 2018, 6:41–47. 
18. Dhuria SV, Hanson LR, Frey WH. Intranasal delivery to the central nervous system: 
mechanisms and experimental considerations. J Pharm Sci. 2010, 99(4): 1654–1673. 
19. Galeano C, Qiu Z, Mishra A, et al. The route by which intranasally delivered stem cells enter 
the central nervous system. Cell Transplant. 2018, 27(3):501-514. 
20. Alcalá-Barraza SR, Lee MS, Hanson LR, et al. Intranasal delivery of neurotrophic factors 
BDNF, CNTF, EPO and NT-4 to the CNS. J Drug Target. 2010, 18(3): 179–190. 
21. Zhu J, Jiang Y, Xu G, Liu X. Intranasal administration: a potential solution for cross-BBB 
delivering neurotrophic factors. Histol Histopathol. 2012, 27(5): 537–548. 
22. Thorne RG, Pronk GJ, Padmanabhan V, Frey WH. Delivery of IGF-I to the rat brain and 
spinal cord along olfactory and trigeminal pathways following intranasal administration. 
Neuroscience. 2004, 127(2): 481–496. 


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23. Dewji NN, Azar MR, Hanson LR, et al. Pharmacokinetics in rat of p8, a peptide drug 
candidate for the treatment of alzheimer’s disease: Stability and delivery to the brain. J 
Alzheimers Dis Rep. 2018; 2(1): 169–179. 
24. Benedict C, Kern W, Schultes B, et al. Differential sensitivity of men and women to 
anorexigenic and memory-improving effects of intranasal insulin. J Clin Endocrinol Metab. 
2008, 93(4): 1339–1344.