Review of Russian ezrin peptide treatment of acute viral respiratory disease and virus induced pneumonia; a potential treatment for covid-19

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COVID-19patients display a spectrum of disease severity.
About 80% haveAcute Viral Respiratory Disease (AVRI) withfever
around 38oC,dry cough and amild pneumonia. About 15% have
severe disease with lung inflammation leading to Acute Respiratory
DistressSyndrome (ARDS): including dyspnoea (shortness of
breath), and hypoxia (low blood oxygen). About 5% have critical
disease:
Acute Lung Injury (ALI)including respiratory failure,
shock, multi-organ dysfunctionand in about 0.5%to 2% cases,
death.11


Human Ezrin, Old-Age and COVID-19 fatality

In Chinese and Italian cohorts of COVID-19 virus infected
people, severe and critical disease was very common in people
over 65 years old. In contrast, symptomatic infection in children
with COVID-19 virusisrare and mild. In aChineseCDCreport,
less than2%of all symptomatic infectionswere in individuals
younger than 20 years old. In a small study of 10 childrenin China
who did develop symptoms, clinical illness was mild; 80 percent
had fever, which resolved within 24 hours, 60%had cough, 40%
had sore throat, and none required supplemental oxygen.12

There is clearly a human factor related to aging that is
interacting with COVID-19 virus.In 2012, it was discovered that
human ezrin, a submembrane protein that is involved in cell shape,
motility, receptor organisation and cell signalling, specificallybound
to the carboxy-terminus of the SARS coronavirus spike protein,
using its FERM domain. The coronavirus was dependent on using
a specific conformation of human ezrin to fuse with the epithelial
cells of the airways and successfully infect them. The fully active
conformation of ezrin, restrains coronavirus infection at the cell-
entry stage. 13

Increased expression of non-functional ezrin is associated
with organism age and senescence, which accumulates on the
interior surface of the cell membrane. 14In old rats, there is a
fourfold increase in membrane associated ezrin in old epithelial
cells, compared to young epithelial cells. 15Old mice also have
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defective CD4 T lymphocytes, which display age-related defects in
ezrin-mediated cytoskeletal signals16. The failure of Ezrin signalling
complexes over time with age, may in part explain the age-related
fatality observed with COVID-19 disease.


Ezrin Peptide Therapy

Ezrin peptides mimicking the trigger-hinge region of the alpha-
domain of human ezrin, are highly polar molecules, with alternating
negative and positive charges, which act locally on epithelial cells
and fibroblasts inmucus membranes.

The receptors for ezrin peptides are believed to be membrane
associated ezrin-protein signalling complexes. Ezrin peptides are
thought to have an allosteric effect, which results in changes of the
conformation of ezrin into various functional forms. These changes
can not only prevent viruses entering cells, but also can activate
specific signalling pathways. Ezrin-protein complexes are
associated with the regulation of the ras>raf>MEK>ERK and
PI3K>PKB>mTOR intracellular signalling pathways, andthey are
involved in the control of cytokine and interferon expression. Ezrin
peptides also act on fibroblasts to stimulate tissue repair
processes. 17

Clinical studies in Russia over twenty-five years have shown
that ezrin peptides can safely and effectively treat viral infections
caused by HIV, HCV, HPV, Herpes Simplex 1 & 2, and the
spectrum of viruses that cause Acute Viral Respiratory Infection
(AVRI). Clinical trials of ezrin peptide TEKKRRETVEREKE
[Gepon], determined that the ezrin-peptides possessanti-viral,
immuno-modulatingactivity andanti-inflammatory activity, and
could beused for successful prevention and treatment of a wide
range of infectious diseases caused by viruses, bacteria,
chlamydia, mycoplasmas, and candidafungi. 18192021222324252627282930

Generally, clinical studies have demonstrated that ezrin
peptides are safe, reduce virally induced inflammation, and lead to
faster recovery from Acute Viral Respiratory Infection (AVRI). Ezrin
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peptides have even successfullytreated viral pneumonia by
shutting down non-specific inflammation, and symptoms of viral
infection, probably by amplifying specific anti-viral responses
selected by the immune system.


CLINICAL STUDY ONE
100patient clinical study into intra-nasal Ezrin-peptide TEKKRRETVEREKE
[Gepon] solution, as a treatmentof Acute Viral Respiratory Infection[AVRI],
inflammatoryLaryngeal-Tracheal-Bronchitis with Stenosis [LTBS]and Recurrent
Croup[RC]


Introduction

In year 2000, V.F. Uchaikin, Member of The Russian
Academy of Medical Sciences, organizedapost-registration
clinical study offourteen amino-acidsyntheticEzrin-peptide
TEKKRRETVEREKE[Gepon], atthe MorozovMoscow Children’s
Hospital, in collaboration with The Russian Government Medical
University, Moscow.

The PrincipalClinical Investigators in the clinical study into the
safety and efficacy of Gepon in Recurrent Acute Respiratory
Diseases were: Kladova O.V., MD, Doctorof Medical Sciences,
Professor, Department of children’s infectious diseases of the
Russian State Medical University: Legkova T.P., Head of Moscow
Children’s Hospital No18 and Ovchinnikova G.S., Doctor of
Children’s Home No 5

The objective of the clinical study was to test the efficacy of
intra-nasal application of ezrin-peptide TEKKRRETVEREKE
[Gepon]31,in treatingofrecurrent Acute Viral Respiratory Infection
[AVRI], which results in inflammation,and laryngitis-tracheitis-
bronchitis complicated with laryngeal stenosis and/or croup
syndrome. 323334

The clinical research was approved by aDecision of the
Committee on Ethics (Minutes No: 6 of 06 December 2000), and a
Decision of the Pharmacology Committee (Minutes No: 14 of 21
December 2000).Permission to conduct clinical trialswasissued
by the Department of the Stateforcontrol of quality, efficiency and
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safety of the medical preparations and equipment (No: 183 of 31
January 2001).


Assessed Patient Population

125 child-patientsbetween the ages of 1 year to 14 years old,
sufferingAcute Respiratory Virus Infection [AVRI], Virus Induced
Inflammation, Laryngeal-Tracheal-Bronchitis with Stenosis [LTBS]
and Recurrent Croup [RC] syndrome,were assessed for the
clinical trial.

ARVI occurred in 85% of the children.50 children suffered
Laryngeal-Tracheal-Bronchitis with Laryngeal Stenosis (LTBS) and
75 children suffered Recurrent Croup(RC). The recurrent croup
was caused by a chronic virus-induced non-specific inflammation
of the upper respiratory tract.The frequency of recurring croup
(RC)in child-patients wasbetween 3 to 35 times per annum.
Recurrent croup syndrome was associated with 1stdegree stenosis
of the larynx in 54of the RC patients(72%), by 2nddegree stenosis
of the larynx in 21of the RC patients(28%).

10% of the croup cases had no fever, 60% of the croup cases
suffered sub-febrile fever, 30% of the croup cases suffered febrile
fever. Theaverageduration of fever was 3-4 days. Bacterial
complications were observed in 15%,including sore throats, acute
otitis media, eustachyitis, and pneumonia.Half of the child-patients
were under treatment in hospital,and half were treated as out-
patients at home.


Immune Statusof Assessed Patient Population

The 125 children of theassessed patient populationwere
offered immune status analysis, which was then compared to the
average status of healthy children.Generally, a profound non-
specificinflammatory response was being induced and maintained
by the viral infection. In contrast, specific anti-viral immunity had
beendisrupted.

Blood sampleswere assessedby flow-cytometry using
monoclonal antibody markers:CD3, CD4, CD8, CD16, CD20,
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CD1b, CD25, CD38, CD54, CD71, CD95, and HLA-DR,to
determinethe distribution oflymphocyte sub-populations. In
addition, co-expression of [CD4+, CD8+], [CD8, DR] and [CD16+,
CD8+]was also measured. The ex vivo phagocytic activities of
blood monocytes and neutrophils versus Staphylococcus Aureus
were alsodetermined.

The levels of inflammatory cytokinesIL-1, IL-6, IL-8 and
TNFwere analysed.The functional activity of Th1 and Th2 cells
were assessed andthe level of expression of the interleukins IL-2
and IL-4 were measured. Interferon status of the children was
assessed by the functional 1988 Yershov method and by
expression of Interferon-gamma.The concentrations of serum IgA
IgE, IgG and IgM immunoglobulins were also analysed.

The viral respiratory infection had induced a marked
imbalance in T cell and B cell immunity, and also significantly
impaired the normal functionality of blood monocytes, neutrophils
and lymphocytes. For example, lymphocyte adhesion and
apoptosis were4x to5x above normal levels.

The most consistent differencesbetweenthe assessedchild-
patients and healthy children, were the largesignificantincreases
inthe concentrations ofinflammatory cytokines: IL-1, IL-6, IL-8
and TNFIL-6 was significantly elevated 2.1x in vivoand 9x in
vitro, and hugely elevated 26x when induced in vitro. IL-8 and
TNFalso showed a similar pattern of massive elevation.(Table 1)

In the Assessed Patient Population,levels of T cell activating
IL-2 and IL-4,were significantly increased.The level of IL-2 in vivo
was increased 24x, in vitrospontaneous production of IL-2 was
increased 12x, and in vitroinduced production increased by 5.1x.
The level of IL-4 both in vivoand in vitroalso showed similar
elevations.

The level of mature T-lymphocytes in children was increased
by 1.3x over healthy children. Activated [CD71+, CD38+, CDA+,
ADD+] lymphocytes were significantly reduced. The assessed
patient population hadmarked changes in the immuno-regulatory
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population of cells. The Th1 cell population was 0.63x less than
normal, whereas the Th2 cellpopulation was 1.49x more than
normal. There was a significant reduction in the expression of IL-2
Receptors (IL2R+).

There was also a significant reduction in the proportion of
cytotoxic T cells [CD16+, CD8+, HLA-DR+] and activated [CD8+,
HLA-DR+] cells. Macrophage function was disrupted;phagocytosis
was reduced 1.6x, phagocytic-index was reduced 1.2x,but
absolute-phagocyte-indicator was 1.6x above normal.Neutrophils
were significantly increased.

Serum IgG and IgE were significantly increased, but IgA was
decreased, while IgM stayed normal. Interferon production was
alsodisrupted:interferon-interferon-and interferon-were also
significantly reduced.

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Table 1.

Immunological Status of Assessed Children

withAVRI, Virus Induced Inflammation,LTBS and RC,

vs healthy children

Less than healthy children:-, Same as healthy children: n, More than healthy children: +,


ImmunologicalVirus Induced Change

Parameter

Leucocytes++

Lymphocytes-

Mature T lymphocytes+

Th1Subpopulation--

Th2Subpopulation+

IRI++

Activated CD8+--

IL2R+ cells-

HLA DR+ cells--

NKcells-

CD16+CD8+--

Mature B lymphocytes++

IgG++

IgA--

IgMn

IgE++

Neutrophils++

Segmented nuclear+

Rod-like nuclearn/+

Phagocytes-

Phagocytic index--

Absolute phagocytic index+

Production of interferon--

IL-1 alpha (in vivo)++

IL-1 alpha (in vitro)++

IL-2 (in vitro)++

IL-2 (in vivo) ++

IL-4 (in vitro) ++

IL-4 (in vivo) ++

IL-6 (in vitro) ++

IL-6 (in vivo) ++

IL-8 (in vitro) ++

IL-8 (in vivo) ++

TNF alpha (in vitro) ++

TNF alpha (in vivo) ++
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Criteria for theGeponClinical Study

Child-patientsaged between 1 and 14 years, with a verified
diagnosis ofrecurrentrespiratory disease, with 5 or more incidents
per annumrecorded in theirmedical documentation,and suffering
from Laryngeal-Tracheal-Bronchitis with Stenosis and / or
Recurrent Croup, were included in the study.

Child-patients were excluded from the study: if the child-
patient refused to take part in the clinical trials; if they were below
1 year, or over 14 years of age; they had received any immuno-
modulator therapy with the previous 4 months; if otherdiseases
were present, such asinsulin-dependent diabetes, tuberculosis,
chronic kidneyand liver diseases, oncological diseasesor HIV-
infection. Patients were also excluded: if theirdoctor’s advice was
not followed, ifside effects appearedwhich might require special
treatment, and if thechild-patient’sdoctor decided that it was in the
interest ofthe child-patientto terminate participation in the Clinical
Study.


Entry of Child-Patients to the GeponClinical Study.

Of the 125 child-patients who had been assessed, 100 were invited
to join the clinical study of Gepon. Child-patient’svoluntary
participation in the clinical research,was subject to informed written
agreement by their parents or guardians. Participation in the clinical
research was voluntary,free of charge, and free of incentive
payment.

Each child-patient wasassessed for: body temperature, skin
condition, peripheral lymph nodes, fauces(the arched opening at
the back of the mouth leading to the pharynx), and tonsils; function
of the lungs, heart,nervoussystem, muscular system and other
evaluations.The time elapsed between receiving written
agreement and the start of theGepontherapy was between 1 and