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Ефективність комбінованої терапії препаратами Ксаврон, Тіворель і Реосорбілакт у пацієнтів з пневмонією, спричиненою COVID-19: клінічні випадки

У публікації подано серію клінічних випадків пацієнтів із пневмоніями, спричиненими COVID-19, до схеми лікування яких було включено препарати Ксаврон, Тіворель і Реосорбілакт, що дозволило швидко досягти покращання стану пацієнтів і сприяло полегшенню перебігу захворювання.

В умовах, коли етіотропні препарати, які діють безпосередньо на збудник COVID-19, ще не розроблені, надзвичайно важливим є комплексний синдромно-патогенетичний підхід до лікування пацієнтів з тяжкими формами COVID-19, при якому будуть враховані індивідуальні особливості й забезпечена максимально ефективна підтримка організму. У цьому контексті застосування комбінованої терапії препаратами Ксаврон, Тіворель і Реосорбілакт є надзвичайно перспективним, оскільки дозволяє покращити стан пацієнтів з COVID-19-асоційованими пневмоніями, запобігає інвалідизації і зниженню якості життя пацієнтів після перенесеної хвороби.

Authors: Мороз Л.В., Ходош Е.М., Кульчак О.І., Чишкевич І.В.
Posted: Новини медицини та фармації, № 1,2, 2021.
Нейроінвазія при коронавірусній інфекції: патогенез та перспективи лікування

Вірус SARS-CoV-2 має тропізм до клітин ЦНС і може викликати розвиток неврологічної симптоматики в інфікованих пацієнтів. У цьому контексті введення до схеми інтенсивної терапії едаравону (препарату Ксаврон®) може бути доцільним з огляду на його здатність зменшувати прояви не тільки оксидативного стресу, але й явищ «цитокінового шторму», а саме значущого зменшення вмісту ІЛ-6. Таким чином, у цих хворих вдається купірувати як неврологічні симптоми, так і рівень системно-запальної відповіді.

Authors: О.А. Лоскутов1, Д.О. Дзюба1, Н.В. Коротчук2, К.Р. Марушко 3, Ю.Г. Вакуленко3, Д.О. Лоскутов
Posted: Спецвипуск «Інсульт», 2021 р. Здоров'я України
Проміжні результати дослідження СТІКс (Супутня Терапія Інсульту Ксавроном) — відкритого багатоцентрового дослідження «випадок — контроль»

У статті наведено результати першого етапу дослідження СТІКс — відкритого багатоцентрового дослідження «випадок — контроль». Проміжний аналіз результатів (interim analysis) дає можливість переконатись у тенденціях на користь робочої гіпотези, дозволяє вчасно оцінити недоліки створеного протоколу та відповідає вимогам академічної та медичної доброчесності — відкритості в оприлюдненні результатів.

Усього за період із березня до вересня 2020 року (дані від Вінницького центру отримані за більший період — серпень 2019 — серпень 2020) отримані для аналізу 570 анкет пацієнтів основної групи (з додаванням до лікування Ксаврону згідно з інструкцією до препарату) та 430 анкет пацієнтів групи контролю (конвенційне лікування згідно з локальними протоколами в окремих клініках та центрах).

Проведений аналіз першого етапу у дослідженні за протоколом СТІКс дає підстави говорити про доцільність застосування Ксаврону як додаткової терапії гострого періоду ішемічного інсульту. Його ефективність підтверджують показники перебігу захворювання — менша ймовірність ускладнень, швидша клінічна стабілізація пацієнтів, швидше відновлення порушень свідомості, що приводить до більш ранньої активізації пацієнта, початку його реабілітації тощо.

Authors: С.П. Московко Вінницький національний медичний університет імені М.І. Пирогова, перелік співавторів надано у публікації.
Posted: THE JOURNAL OF NEUROSCIENCE of B.M. Mankovskyi’ 2021, VOLUME 9, № 1
The importance of non-motor symptoms in the clinical picture of motor neuron disease

Non-motor symptoms are an obligate sign of motor neuron disease and can often serve as a rather sensitive indicator of the general functional state of ALS patients. Gastrointestinal disorders, weight loss, pain, sweating and anxiety prevail in the structure of non-motor symptoms in ALS patients

The presence of non-motor symptoms in patients indicates the multisystemic nature of this fatal disease. The use of Non-Motor Symptom Scale (NMS) in patients with ALS in the early stages will allow neurologists to detect non-motor symptoms timely.

Authors: V. V. KOSTENKO, Y. I. GOLOVCHENKOP. L. Shupyk National Medical Academy оf Postgraduate Education Kyiv
Posted: Ukrainian Neurological Journal 2020, №3
At the forefront of the fight against COVID-19 – the experience of Ukrainian specialists (Video)

Doctors all over the world are still looking for and cannot find effective means to combat COVID-19. Therefore, every practical experience is invaluable. Let’s find out how Ukrainian doctors save the lives of the population.

The latest experience was shared with leading domestic specialists in the framework of the reports of the IV International Congress on Infusion Therapy.

We bring to your attention a brief overview of the section that attracted the most attention among the audience.

Posted: IV International Congress on Infusion Therapy (October 12-13, 2020)
Застосування препаратів едаравону, цитиколіну й електролітів та L-аргініну в пацієнтів із гострим порушенням мозкового кровообігу

У статті представлено результати застосування цитопротекторної терапії, застосованої на додаток до традиційного лікування пацієнтів із гострим порушенням мозкового кровообігу.

Актуальним напрямом у лікуванні пацієнтів із ГПМК є застосування цитопротекторів, які можуть бути ефективним доповненням до тромболізису. Ці засоби захищають ішемізовані нейрони та нейросудинні одиниці від незворотних ушкоджень, посилюють нейропластичність і сприяють процесу відновлення нервової тканини.

Наведено дані щодо 30 клінічних випадків гострого порушення мозкового кровообігу у пацієнтів, яких госпіталізували до спеціалізованих відділень медзакладів України. Отримані дані свідчать, що застосування препаратів едаравону, цитиколіну й електролітів та L-аргініну у пацієнтів на додаток до традиційної схеми лікування допомагає значно знизити неврологічний дефіцит, підвищити функціональну незалежність і поліпшити загальний прогноз.

Authors: Т. І. Негрич, Львівський національний медичний університет імені Данила Галицького
Posted: Нейроnews 9(120), 2020
Edaravone: A Free Radical Scavenger with Multiple Pleotropic Actions can be a Potential Game Changer Agent in Prevention and Alleviation of COVID-19 – Induced Cytokine Storm

Normal physiological process, such as cellular respiration generate small amount of oxidizing reactive oxygen species (ROS) and reactive nitrogen species (RNS). These free radicals play crucial role in activation of signalling pathways in animal and plant cells. These are chemically highly reactive molecules that can damage cell structures. However, there are endogenous defences (scavenger) or antioxidant systems to protect tissues from ROS and RNS induced injury. These antioxidants can be divided into enzymatic and non-enzymatic groups. The enzymatic antioxidants include superoxide dismutase (SOD), catalase and glutathione peroxidase. Their cofactors are selenium, zinc, copper, and manganese for SOD and iron for catalase. The non-enzymatic antioxidants include vitamin E, C, A or β-carotene etc. Vitamin E can directly quench ROS including O2, HO- and O2. Vitamin C in higher doses broadly scavenges water soluble ROS including the major neutrophils oxidants HO-, H2 O2 and hypochlorous acid. Vitamin C also is a pro-oxidant when combined with iron.1 Most of ROS are produced in cell through the mitochondrial respiratory chain reaction during endogenous aerobic metabolism, while in hypoxic situation generates nitric oxide (NO), which can produce other RNS. Free radicals have odd number of electrons, this make them short lived,highly reactive. Consequently it reacts quickly with other closest stable molecules/substances ‘stealing’ its required electron to obtain stability. Meanwhile, the attacked molecule become a free radical by losing its electron and start a chain reaction cascade causing damage to the living cells.2,3 The enzymatic and non-enzymatic antioxidants system are intimately interlinked to one another. Both vitamin C and glutathione implicated in recycling of α- tocopherol. The complex interactions of these different antioxidants systems with narrow spectrum of activity may imply that successful therapeutic strategies will depend on the use of a combination of various antioxidants rather than a single agent.4,5

Redox/oxidation status in the lung:

The redox environment in the lung lining fluid is important factor in determining the lung’s innate
and adaptive immune system. Normal lung have extremely high levels of extracellular antioxidants
function to maintain extracellular space in a highly reduced state and facilitate the maintenance of
immune response. Balance between antioxidants and oxidants are sufficient in normal lung. In oxidized states there is presence of hyperresponsive immune system. Increase in oxidants or decrease in antioxidants can disrupt this balance, referred to as “oxidative state” and associated with diverse lung pathologies and facilitate the binding of pathogens or antigens to effectors cells leading to a hyperresponsive innate immune system leading to enhanced release of oxidants such as superoxide and NO causing activation of nuclear factor-kappa β (NK-kβ) and enhance production of cytokines, including TNF-α, interleukin-1β, (IL-1β), IL-12. Creation of markedly reduced environment by addition of antioxidants blunts all of the above primary response of innate immune system.6

Redox/oxidation status in critical illness:

Oxidative stress has been implicated in the manifestation of critical illness, including ischemia and reperfusion injury and systemic inflammatory states. Excessive ROS production may leads to indiscriminate bystander injury in the host. ROS causes direct cellular injury to cellular proteins and nucleic acids and by inducing lipid peroxidation, leading to destruction of the cell membrane.4 ROS also play a role as second messenger in the intracellular signalling pathways of inflammatory cells. In particular, the activation of the NF-kβ induced by hydrogen peroxide .NF-kβ resides in the cytoplasm as an inactive complex bound to its inhibitor I-kβ. Upon stimulation with various agents including cytokines, viruses, ROS, mitogens causes dissociation of NF-kβ- Ik-β complex and translocation of NF-kβ to the nucleus with its high affinity of binding to specific sites in the promoter region of target genes stimulating their transcription.7 NF-kβ is involved in the regulation of numerous pro-inflammatory genes, including many cytokines (TNF-α,IL-1,IL-6,IL-8,IL-2), hematopoietic growth factors such as granulocytemacrophage-colony stimulating factor(GMCSF), cell adhesion molecules-1(CAM-1) i.e. intercellular adhesion molecule-1 (ICAM-1), endothelialleukocyte adhesion molecule-1(ELAM-1),vascular CAM-1(VCAM-1), and nitric oxide syntheses (iNO). A second messenger transcription factor activator -1(APC-1), which also seems to be regulated by changes in redox states of the cell may contribute to the ongoing inflammatory cytokine production and progressi on of systemic inflammation leading to end organs injury manifested by the development of ALI/ARDS or multiple organ failure syndrome. In addition ischemia/reperfusion can leads to significant production of ROS by the increased activity of xanthineoxidase and increased production of hypoxanthine due to reintroduction of both.6

Lungs in Systemic Inflammatory Response Syndrome (SIRS):

Any initiating severe events (sepsis, shock, trauma, acute pancreatitis ect.) can leads to activation of acute inflammatory response on a systemic level. One of the earliest manifestations is activation of
pulmonary endothelium and macrophages (alveolar and interstitial), upregulation of adhesion molecules and production of cytokines and chemokines that induce a massive sequestration of neutrophils within pulmonary microvasculature. These cells transmigrate across all endothelium and epithelium into the alveolar space and release varieties of cytokines and pro-inflammatory compounds including proteolytic enzymes, ROS, nitrogen species, cationic proteins, lipid mediators and additional inflammatory cytokines. This perpetuates a vicious circle by recruiting additional inflammatory cells that in turn produce more cytotoxic mediators leading to profound injury to
the alveolo-capillary membrane leading to respiratory failure i.e. ALI/ARDS. The source of ROS/RNS in this context also includes itinerant and resident leukocytes (neutrophils, monocytes, and macrophages), parenchymal cells (endothelial and epithelial cells, fibroblast) and circulating oxidants generating enzyme xanthine oxidase. Leukocytes expressed two enzyme systems – (i) the NADPH oxidase, (ii) Nitric oxide syntheses (NOS) that generate substantial amount of ROS. Finally inhaled gas oxidant like high oxygen concentration used for mechanical ventilation causes ROS production.8

Immunoscenesence:

Age associated dysregulation of immune response contribute to higher incidence of infectious diseases in the elderly people due to imbalance of Th1/Th2 cytokine production and increased production of pro-inflammatory cytokines i.e. IL-6. Syntheses of many cytokines are implicated by changes in the cellular oxidant/antioxidant balance. Influenza A virus infection can cause early increase in IL-1, IL-6, and TNF-α, GMCSF, interferon-у(IFN-у).But IL-6 remains elevated throughout the infection. The alveolar macrophage plays a prominent role in the initiation of an immune response during early stage of infection. The lung epithelial cells and peripheral blood mononuclear cells (PBMCs) increases production of IFN-α. In elderly Th1 activity is decreased with decreased production of IL-2 and IFN-α, while Th2 activity is increased with increased production of IL-6,IL-1β,TNF-α and IL- 1Ra. The persistent elevated levels of IL-6 may indicate presence of age-associated diseases like hypertension, diabetes, CVA, CAD, heart failure, COPD, CKD etc.9 Antioxidants such as vitamin E, C, β-carotene and glutathione enhance IFN production. Glutathione supplementation maintains intracellular redox balance; cellular defence against oxidative stress significantly increases IL-2 production and decrease TNF-α. Vitamin E supplementation increases IL-2 production and decreases IL-6 in animal and humans.10

Antioxidant regulates the production of cytokines:

There are two possible mechanism, first through effects on transcription factor NF-kβ and APC-1, that regulated by redox status. Many cytokines (IL-2, IL-1, IL-6 and TNF-α) contain NF-kβ and APC-1 binding sites in the promoter and enhancer region of the genes encoding them. Reduction /oxidation can either up or down regulate DNA binding and transactivation activities (or both) in transcriptional activator-dependent and cell type dependent manner. Vitamin E or its derivatives inhibit NF-kβ in human Jurkat T cells and also by other antioxidants differently in different cells. Thus, antioxidants have immunomodulatory role.11 The second possible mechanism is through PGE2 synthesis: PGE2 inhibit early stage of T cell activation and decreases IL-2 production, modulates Th1/Th2 cytokine secretion through its effects on IL-12, which increases Th2 response promoting differentiation of naïve T cell into a population of Th1 cells capable of production of large amount of IFN-у. Vitamin E inhibits cyclooxygenase activity and decreases PGE2 production in age associated increased PGE2 synthesis and COX activity in old mice.12 ROS are also generated by phagocytes following viral infection as the virus itself can change redox status of cells involved in generation of ROS directly and decreases total concentration of antioxidants such as glutathione, vitamin C and E from the lung in the early stage of infection attributed to increased production of oxidants. Antioxidant vitamin E supplementation in high doses increases NKactivity (3 fold) and decreases viral loads and lower pulmonary IL-6 and TNF-α levels and increase production of Th1 cytokines IL-2 and IFN-у in old mice.13

Pathogenesis of COVID-19-induced cytokine storm and ALI/ARDS:

After coronavirus get inhaled and infects the respiratory epithelial cells through its ACE2 receptor, dendrite cell phagocytes the virus infected cells and present the antigens to T cells. The effectors T cells function by killing the infected cells and cytotoxic CD. T cells produce and release pro-inflammatory cytokines which induces cell apoptosis. Both pathogens and infected cell apoptosis trigger and amplify immune response. There is exacerbation of cytokine production and excessive recruitment of immune cells into the lung and the uncontrollable epithelial cell damage generates a vicious circle for infection related to ALI/ARDS.14 In the early stage of viral infection dendrite cells and epithelial cells are activated and express a cluster of pro-inflammatory cytokines including IL-1β,IL-2,IL-6 INF-α/β, TNF-α, and CC motif chemokines (CCL-3,CCL-5,CCL-2) and interferon inducible protein-10(IP-10) etc. Overproduction of these cytokines, chemokines and ROS contribute to the development of disease complications. Some patients have high levels of IL-10 secreted by the helper Th2 cells a marker of counter anti-inflammatory response associated with down regulation of neutrophils and macrophages function referred as “immune paralysis” and there is repressed immune response with hypoalbuminemia, lymphopenia, neutropenia and decreased percentage of CD. T cells indicating immune suppression and patients may die after cytokine storm. The amplification of the inflammatory response promote cellular apoptosis or necrosis of the affected cells, which further fuel inflammation, followed by increase permeability of blood vessels and aberrant accumulation of inflammatory monocytes, macrophages and neutrophils in the lung’s alveoli. This vicious circle intensifies the situation as the regulation of immune response is lost and cytokine storm is further activated resulting dire consequences. Thus, there is excessive inflammation with depressed immune system and activated cytokine storm substantially contribute to the pathogenesis of COVID-19 infection.15 There also high levels of circulating serum ferritin in cytokine storm may reflect as an acute phase response and play a critical role in inflammation and ROS generation. 16Ferritin a intracellular iron storage constituting two subunit H and L and their ratio may differ depends on tissue type and physiologic status of the cell. H ferritin seems to be immunomodulatory function and have proinflammatory activity with induction of expression of different inflammatory mediators including IL- 1β. 17 Serum ferritin levels correlates with disease severity in COVID-19 infection.18 Once ferritin released it loses part of the inner iron content giving rise to extremely high serum levels of ‘free iron’ and the excess free iron induce a marked procoagulalant state able to favour the production of hydroxyl radicals. Oxidative stress on RBCs and fibrin can induce production of dense clots responsible for stroke development. Iron chelating agent can taper the inflammatory response through a reduction of ROS production.19

COVID-19 infection induce cytokine storm have raised levels of Angiotensin-II (Ang-II) associated with oxidative stress and correlated to severity of infection:

As the ACE2 is primary receptor for COVID-19 infection, ACE2 expression is reduced leading to increased Ang-II levels and AT1R activation by Ang-II leading to hypertension, hypertrophy, fibrosis and oxidative stress causing lung injury. Whereas Mas receptor (MasR) activation by Ang-1- 7 (product of ACE2 cleaving of Ang-II) results in effects opposite of AT1R activation i.e. vasodilatation, growth inhibition, antioxidant and anti-fibrotic actions and prevent lung injury. Reduced activity of ACE2 within the cell internalization and degradation of ACE2 are inhibited by AT1R blockers (ARBs) Losartan and ACEI have protective against lung injury.20

NAD+ Depletion:

Both viral infection and RAAS activation produce ROS in a reproductive manner resulting to oxidative burst. Increased ROS levels have destructive effects on cellular macromolecules such as lipids, proteins especially nucleic acids. Oxidative stress mediated DNA damage is repaired primarily via base excision repair (BER) pathway. Normally poly-ADP-ribose polymerase-1 (PARP-1), a DNA base excision repair enzyme activated by DNA breaks and contribute to BER pathway for maintenance of genome stability. Upon activation of PARP-1, rapidly uses the substrate NAD+ to transfer poly- ADP-ribose (PAR) to itself and nuclear receptor proteins and damaged DNA. PARP-1 has ADPribose transferase activity and function as an antiviral agent through ADP-ribosylation of viral genome (RNA) and inhibition of viral transcription translation. Excessive activation of PARP-1 occurs to compensate ADP-ribose hydrolyzation of viral macrodomain (NSP3) poly (ADP-ribose) glycohydrolase (PARG) which is associated with catalytic consumption of NAD+ followed by ATP reduction leading to depletion of energy and cell death.21


Endothelial dysfunction due to reduction of Nitric Oxide (NO) Production:

Decreased NO production is prominent in COVID-19 infection as NO degradation caused by ROS. The reduced NO bioavailability results in proliferative, pro-oxidant, pro-inflammatory and pro thrombotic response. In hypoxic situation ROS generation and HIF-1α activation occurs, which consequently induce expression of furin enzyme and viral activation and invasion to other non-ACE2 expressing cells. Decreased NO attributed to inflammation and endothelial dysfunction, induction of vascular smooth muscle cells (VSMCs) proliferation, LDL oxidation and vascular cell adhesion molecule-1 and monocyte chemoattractant protei-1(MCP-1) expression. 21

Edaravone

In 2001, edaravone was approved in Japan to treat acute-phase cerebral infarction. In 2017, the U.S.FDA approved for treatment of amyotrophic lateral sclerosis (ALS). Edaravone a member of substituted 2-pyrazolin-5-one class has the chemical name 3-methyl-1-phenyl-2-pyrazolin-5-one. In India available as clear colourless solution in glass ampoule containing 30mg/20 ml of water for
injection, given IV infusion diluted with 100ml of isotonic saline in 30 minutes, twice daily for treatment of cerebral ischemic stroke.


Pharmacokinetics and Metabolism:

The maximum plasma concentration (Cmax) of edaravone at the end of IV infusion during clinical trials was a trend of a greater than dose proportional increase in concentration of area under the curve (AUC) and Cmax of edaravone, but no plasma drug accumulation was observed with multiple dose administration. The drug is 92% protein bound primarily to albumin and in the range of 0.1 to 50 micromole/L yields no concentration dependence. The mean terminal half-lives of edaravone and its metabolites were 4.5-6 and 2.0-2.8 hours respectively. The drug is metabolize to inactive sulfate and glucoronide conjugated in the liver and kidney. It is excreted mainly in the urine as its glucoronide conjugate form after 70% to 90% of the dose was recovered in the urine as sulfate conjugate.22 In water at pH 7.4 the percentage of the neutral and anionic forms of edaravone have been calculated to be 28.5% and 71.5% respectively. Approximately a half of edaravone exists as anionic form at physiological pH is the active form of edaravone. As pKa of edaravone is 7.0 edaravone able to enter lipid environment such as cell membrane and scavenge peroxyl radicals generated from lipids in radical chain reactions. (Oxidation of lipids proceeds as a chain reaction in which a single radical can generate thousands of molecules of lipid hydroperoxide causing damage to biological membrane). Edaravone scavenges both lipid and water soluble peroxyl radicals exerts broad spectrum antioxidant activity acting in concert with α-tochopherol and other antioxidant present in the body. 23

Effective doses and blood levels in treatment of stroke:

The plasma concentration of edaravone after its infusion at doses of 1.5 or 3.0mg/kg body weight in rats cerebral ischemic model were 5.7 and 9.9μM/L. The effective concentration in vitro studies were between 1 and 10μM/L and had no detrimental effects. Thus, 3mg/kg IV dose of edaravone either bolus or infusion in 30 minute is effective therapeutic dose in cerebral ischemia and other diseases.24 Doses adjustment not required in patients with renal or mild to moderate hepatic impairment. No drug interaction known and not inhibit cytochrome p-450 enzyme, CYP2A2, CYP3B6 and CYP3A4.23

Anti-inflammatory effects in strokes:

Microglial cells are the resident immune cells mediate neuroinflammation in degenerative CNS diseases and strokes, activated to engage in different functions such as phagocytising the toxic cellular debris, producing pro-inflammatory cytokines and enhancing neuronal survival by release of tropic factors. Activation of microglial cells release excessive pro-inflammatory cytokines or cytotoxic factors such as NO, TNF-α, IL-1β and ROS.

Edaravone reduces infarct size, improve neurological scores by decreasing ROS generation, inhibiting production of pro-inflammatory mediators (TNF-α, IL-1β, NO, ROS) and prevent oxidative stress damage. Edaravone drastically decreases the number of activated microglial cells and suppress the upregulation of Toll- like receptor- 2 (TLR2) expression in microglial cells in acute strokes.25 The neuroprotective effects of edaravone includes: 1- quenching of hydroxyl radical(H0), 2- inhibit OH-dependent and OH-induced peroxidation system, 4-inhibition of both non-enzymatic lipid peroxidation and lipo-oxygenase pathway. Thus, edaravone can improve ROS-induced neurological impairment in treatment of subarachnoid haemorrhage, acute ischemic stroke and intracerebral haemorrhage.26 In post ischemic inflammation state inhibit neutrophils activations as well as inducible NOS (i NOS) and neuronal NOS (nNOS) expression. The anti-inflammatory effects of edaravone have been confirmed in non-ischemic models of inflammation in extracerebral organs. Edaravone inhibit the oxidative modification of low-density lipoproteins (LDL), but also reverses oxidized LDL-induced reduction in expression of endothelial nitric oxide synthease (eNOS).27,28,29

Membrane stabilizing effect:

Edaravone present in the cell surface and/or near the surface of liposomal membrane thereby scavenges peroxyl radicals in both the aqueous and lipid phase. It suppresses the generation of ROS such as hydrogen peroxide and hydroxyl radicals of human neutrophils by its quenching action rather than by inhibiting neutrophils function.30

Effects on non-neurological diseases (pleotropic effects):

In addition to anti-stroke effects, edaravone prevent oxidative damages to various extracerebral diseases. Edaravone scavenges free radicals and has antiapoptotic, anti-cytokine effects in various diseases as it can diffuse into many disease affected organs and shows protective effects in the heart, lungs, intestine, liver, pancreas, kidney, bladder and testes. Thus, edaravone should be considered for treatment of diseases other than stroke focusing on its pleotropic effects.31, 32, 33, 34, 35

Anti-cytokine effects:

Edaravone suppresses plasma monocyte chemoattractant protein-1(MCP-1), improve LV ejection fraction and reduce hospitalization due to heart failure with AMI. Decreases TNF-α production induced by I/R-induce injury, protect cardiac function and reduce infarct size in rats. In LPS-induced lung injury of mouse model ,prevent lung injury and attenuates inflammatory cell and pro-inflammatory cytokines production such as IL- 6,TNF-α, kerationocyte derived chemokines and macrophage inflammatory protein-2 (MIP-2) in BALF.36 Edaravone inhibit activation of phospholipase A2 and production of plateletactivating factors and ameliorates lung edema and leukocyte extravasation.24, 32 After I/R-injury of rats liver edaravone suppress the expression of cytokines and chemokines such as TNF-α,IL-1,IL-8, monocyte chemoattractant protein -2( MCP-2), macrophage inflammatory protein(MIP1α,MIP-1β) and reduces intracellular adhesion molecule- 1(ICAM-1) mRNA expression. Also suppresses endotoxin-induced liver damage by inhibiting expression of inflammatory cytokines and chemokines as well as inflammatory cell recruitment. Edaravone shows protective effects on ischemic insults and inflammation in heart, vessels and brain in experimental studies.37

Effects on I/R-induced acute Pancreatitis (AP):

Edaravone reduces pancreatic and intestinal injury after AP in mice. There was reduction in histological score, apoptosis, IL-6, IL-1β and TNF- α along with obstructing activation of TLR-4 and NF-Kβ activation .38 It suppresses NF-kβ activation in LPS-induced lung injury in mice.39 TLR-4 can upregulate expression of inflammatory mediators via receiving ligand signal and protein molecules of necrotic cells and can enter extracellular fluid and recognized as endogenous ligands by TLR-4 to initiate immune response and sterile inflammatory response. Edaravone restrain TLR-4-NF-kβ pathway and repairs pancreatic and intestinal injury via regulation of TLR-4-NF-kβ signal pathway.40

Effects on ischemic preconditioning of kidney:

Edaravone significantly decreases serum creatinine and BUN concentration and ameliorates histological damage of renal tissue, decrease TUNEL-positive cells and Bax expression (marker of apoptosis). ROS are produced mainly by the tubular cells and cause lipid peroxidation, which is a free radical – generating system closely related to I/R-induced tissue damage. Edaravone prevent depletion of SOD in the tubular cells and prevent lipid peroxidation, upregulate anti-apoptotic protein Bcl-2 and downregulate pro-apoptotic protein Bax, thus prevent oxidative stress damage and apoptosis induced by I/R- injury.41

Conclusions
Edaravone a broad spectrum antioxidant scavenges ROS and RNS in both aqueous and lipid environments. Besides anti-stroke effects it has multiple pleotropic action includes antiinflammatory, anti-cytokine, immunomodulatory, anti-apoptotic, anti-necrotic, anti-fibrotic, membrane stabilizing, protect lung surfactant and protect multiple organs against I/R-induced injury. Thus, it appears to be most useful agent in prevention and treatment of COVID-19–induced cytokine storm, ALI/ARDS. However, prior to instituting antioxidant therapy, we must define the appropriate time points for intervention in each disease process. Antioxidant treatment become increasingly difficult as the inflammatory process and damage induced become irreversible with time.COVID-19 infection classically proceeds with initial phase of viral replication with high viral loads most likely benefit from effective antiviral drugs, where as the overlapping and third phase is manifested with hyperimmunity with high cytokines levels with low viral loads with aberrant immune response causing much of the host damage most likely benefit from anti-inflammatory drugs therapy i.e. steroid eg. Dexamethasone and anticytokine Tocilizumab etc. Edaravone started in the early phase and/or at the overlapping second phase may prevent progression to cytokine storm in high risk patients and ameliorates the manifestations of cytokine storm ARDS in third phase. It may decrease need for ventilation and/or duration of ventilation and mortality. Thus, it can be used as an adjunctive therapy for the treatment of severe COVID-19 infection. Effectiveness of edaravone is evident from treatment of Paraquat poisoning (a highly oxidative burst state caused by free radical damage leading to AKI,ARDS and/or multiorgan failure with similar pathophysiology of COVID-19– induced cytokine storm ARDS), started within 24 hour of poisoning ,before development of complications had very low morbidity and mortality in mild to moderate poisoning. (unpublished recent personal observations in 7 cases of mild to moderate poisoning ,did not developed any complications with early edaravone therapy in comparison to >98% developed complications with high morbidity and mortality in retrospective observations treated with combinations antioxidants including IV N-Acetylcysteine and steroids etc). Thus, we need to explore the broad spectrum antioxidant edaravone alone or with combination of antioxidants for synergistic effects rather than a single agent. Large double blind placebo control dose finding study of early edaravone therapy should be considered in treatment of high risk COVID-19 patients to know whether edaravone prevent cytokine storm, ARDS and/or decreases the need for ventilation, duration of ventilation and mortality.

Acknowledgement
We are very much thankful to our family members in the coronavirus pandemic lockdown period for their kind cooperation and encouragement for this work for the benefits of COVID-19 patients. We have no any conflict of interest.

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Authors: Dr Butungeshwar Pradhan, Dr Gourav Pradhan, Deblina Pradhan
Posted: Journal of Medical Science and Clinical Research Vol 08, Issue 07, Page 227-236, July
Нейроінвазія та неврологічні ускладнення при COVID-19 (ВІДЕО)

У відео на основі наявних публікацій систематизовані відомості про механізми нейроінвазії, основні форми ураження периферичної та центральної нервової систем та їх клінічні прояви при СOVID-19.

Обговорюються методи діагностики та лікувальна тактика при різних нозологічних формах.

Представлено пілотне дослідження вивчення впливу едаравону на маніфестацію IL-6 у пацієнтів з COVID-19.

Authors: Лоскутов О.А., проф., д.мед.н., завідувач кафедри анестезіології та інтенсивної терапії НМАПО ім. П.Л. Шупика
Posted: IV Міжнародний конгрес з інфузійної терапії (12-13 жовтня 2020 року)
Потенційні можливості лікування COVID-19: протидія надмірному запаленню, антиоксидантний вплив і цитопротекція

Зважаючи на небезпеку пандемії, що й досі не стихає, існує потреба в розробленні нових схем лікування коронавірусної хвороби (COVID‑19), у тому числі в переосмисленні вже знайомих, добре вивчених препаратів і застосуванні їх у новому контексті. Причиною мультиорганного пошкодження та смерті при тяжких випадках COVID‑19 є надмірне та некероване вивільнення певних прозапальних цитокінів і хемокінів (Huang C. et al., 2020), тому зменшення цієї агресивної запальної відповіді потенційно здатне знизити смертність і пошкодження органів.

На думку S.E. Reznik і співавт. (2020), препаратом, який може протидіяти запаленню при COVID‑19, є едаравон (як у вигляді монотерапії, так і в комбінації з одним або кількома противірусними препаратами). Відповідно до інструкції для медичного застосування, едаравон уводиться внутрішньовенно з метою лікування бічного аміотрофічного склерозу й інфаркту мозку в гострій фазі. Едаравон є ліпофільною речовиною, що легко проникає в низку тканин організму та має значну антиоксидантну та протизапальну активність (Watanabe T. et al., 2008). Системне призначення едаравону сприяє протекторній протизапальній дії в експериментальних моделях хімічного пошкодження легень, нирок, кишечнику, підшлункової залози, мозку та печінки (Kikuchi K. et al., 2012). Едаравон також здатен знижувати вміст певних цитокінів (інтерлейкінів (ІЛ) 2, 6, 1β, фактора некрозу пухлини), хемокінів (ІЛ‑8, моноцитарного хемоатрактантного білка‑1, макрофагального запального білка‑1α тощо), печінкових ферментів, активних форм кисню (АФК) й оксиду азоту – NO (Kikuchi K. et al., 2012).

Ефективність едаравону при COVID‑19 доцільно довести в подвійному сліпому рандомізованому плацебо-контрольованому клінічному дослідженні за участю дорослих пацієнтів із цим захворюванням, підтвердженим за допомогою полімеразної ланцюгової реакції (ПЛР), і тяжким перебігом, визначеним за критеріями G. Chen і співавт. (2020). Оптимальним режимом призначення едаравону в цьому випадку може бути введення 60 мг препарату внутрішньовенно 1 раз на добу протягом 10 днів. Первинними кінцевими точками мають виступати смертність і клінічне покращення (Grein J. et al., 2020). У разі успішного застосування едаравону для зменшення мультиорганного пошкодження, кількості ускладнень і смертності при тяжких випадках COVID‑19 доцільно вивчити його ефективність за умови інших інфекційних захворювань, які супроводжуються надмірною запальною відповіддю (Reznik S. et al., 2020).

Сьогодні українські науковці можуть поділитися власним досвідом застосування цього препарату в клінічній практиці. Зокрема, результати своїх досліджень і спостережень вони представили в рамках IV Міжнародного конгресу з інфузійної терапії, що відбувся в режимі онлайн 12-13 жовтня.

Досвідом застосування препарат у едаравон у лікуванні пневмоній коронавірусної етіології поділилася завідувачка пульмонологічного відділення Чернівецької обласної клінічної лікарні, лікар-пульмонолог вищої кваліфікаційної категорії, доктор медичних наук, професор Світлана Вікторівна Коваленко. З початком пандемії коронавіруної хвороби саме до очолюваного нею відділення почали надходити перші па цієнти з COVID‑19. У дослідження було включено пацієнтів (n=30 в основній групі та n=30 у  групі контролю), котрі за хворіли на  COVID‑19. Діагноз підтверджували за  допомогою КТ та  ПЛР‑тестування. Пацієнти мали приблизно однакову площу ураження легень за  даними КТ (від 50 до 70% площі легеневої тканини). Як контрольні точки були обрані кількість ліжко-днів, сатурація крові, температура тіла, рівень феритину, D‑димеру, С‑реактивного білка, загальний клінічний аналіз крові з визначенням кількості еритроцитів, лейкоцитів, тромбоцитів, ШОЕ, лейкоцитарної формули, вмісту гемоглобіну.

Пацієнти основної групи поряд зі стандартним лікуванням (антибіотики, антикоагулянти, протизапальні препарати) отримували препарат едаравону Ксаврон (з метою впливу на так званий цитокіновий шторм), Реосорбілакт (препарат, здатний зменшувати частоту легеневих ускладнень і тривалість перебування на ШВЛ), Тіворель (комбінація L‑аргініну та L‑карнітину для нормалізації вмісту NO в легеневих артеріях і пригнічення медіаторів запалення).

У пацієнтів основної групи спостерігалося більш швидке та виражене зниження рівня С‑реактивного білка, що свідчить про виражене пригнічення запального процесу в  легенях. Зменшення вмісту D‑димеру в  пацієнтів основної групи також свідчило про поліпшення їхнього загального стану порівняно з учасниками контрольної групи в динаміці лікування. Як відомо, високий рівень D‑димеру у хворих на COVID‑19 є прогностичним маркером тяжкого перебігу захворювання, а також збільшує ризик тромботичних ускладнень.

Терапія препаратами Ксаврон, Тіворель і Реосорбілакт сприяла більш вираженому насиченню крові киснем, супроводжувалася прискоренням нормалізації температури тіла. Загалом термін госпіталізації пацієнтів основної групи склав 13 ліжко-днів, натомість хворих контрольної групи – 15 ліжко-днів. Побічних ефектів лікування не спостерігалося.

Таким чином, застосування препаратів Ксаврон, Тіворель і Реосорбілакт у комплексному лікуванні пневмонії коронавірусної етіології дозволяє зменшити тяжкість перебігу захворювання та прискорює одужання.

Завідувачка кафедри інфекційних хвороб із  курсом епідеміології Вінницького національного медичного університету ім. М.І. Пирогова, доктор медичних наук, професор Лариса Василівна Мороз представила власний досвід ведення пацієнтів із COVID‑19 з акцентом на пневмонії. Доповідачка нагадала, що коронавіруси являють собою РНК‑віруси з  характерною оболонкою у формі корони, котрі мають тропність до рецепторів епітелію дихальної системи. Серед найвідоміших коронавірусів, здатних уражати організм людини, – SARSCoV (коронавірус тяжкого гострого респіраторного синдрому), MERS-CoV (коронавірус близькосхідного респіраторного синдрому) та новий SARS-CoV‑2.

Патогенез COVID‑19 передбачає розмноження вірусу в епітелії верхніх і нижніх дихальних шляхів із подальшим дифузним ураженням альвеолоцитів, що призводить до вірусної пневмонії чи гострого респіраторного дистрес-синдрому (ГРДС) із 40% летальністю. Основними симптомами є лихоманка (83-99%), втрата апетиту (40-84%), кашель (59-82%), підвищена втомлюваність (44-70%), аносмія (15-30%), біль у м’язах (11-35%). Окрім того, коронавірусна інфекція часто супроводжується коагулопатіями, що зумовлюють венозний тромбоз, інфаркт міокарда, дисеміноване внутрішньосудинне згортання крові. Факторами ризику коагулопатій виступають сепсис, хронічне обструктивне захворювання легень та ураження печінки, онкологічні захворювання, гіпертермія та гострий перебіг COVID‑19.

У 40% пацієнтів із коронавірусною інфекцією відзначається легкий перебіг, іще в 40% – середньотяжкий, у 15% – тяжкий і в 5% – критично тяжкий. У 94% пацієнтів, у яких це захворювання закінчилося летально, спостерігалася щонайменше одна з таких ознак: наявність злоякісної пухлини, патологічне ожиріння, цукровий діабет, хвороби серця та судин, нирок, легень, гіпоальбумінемія, вік понад 60 років.

У  діагностиці коронавірусного ураження легень провідне місце належить комп’ютерній томографії (КТ). Цей метод візуалізації необхідно застосовувати при первинному обстеженні чи госпіталізації, далі – повторно через 2-3 дні та що 2-3 дні за відсутності потрібного терапевтичного ефекту чи в разі клінічного погіршення з метою оцінки прогресування ураження легень, через 5-7 днів за відсутності позитивної динаміки симптомів. Ураження легень за даними КТ поділяється на 4 основні ступені (табл.).

Поки не  розроблена вакцина від COVID‑19, пріоритетною задачею клініцистів є зниження ризику фатальних ускладнень. Із цією метою може застосовуватися патогенетичне лікування, проте варто зауважити, що всі препарати патогенетичної дії застосовуються off-label, тобто поза межами показань відповідно до офіційних інструкцій. Так, едаравон (Ксаврон, ТОВ «Юрія-Фарм», Україна) являє собою низькомолекулярний антиоксидантний засіб, який має протизапальну дію за рахунок пригнічення цитокінового шторму (зменшення продукції фактора некрозу пухлини, ІЛ‑6), а також чинить інгібувальний вплив на проникність ендотеліоцитів мікроциркулярного русла легень. Едаравон швидко нейтралізує вільні радикали; гальмує перекисне окиснення ліпідів, захищаючи клітини від руйнування; активує ферменти антиоксидантного захисту (супероксиддисмутазу, каталазу, глутатіонпероксидазу). Ці властивості роблять доцільним застосування едаравону при ГРДС.

Таблиця. Ступені ураження легень відповідно до даних КТ

Порівняння едаравону з дексаметазоном виявило, що обидва препарати здатні запобігати розвитку підвищеної проникності ендотеліоцитів мікроциркуляторного русла легень, однак у зв’язку з меншою кількістю побічних явищ перевагу варто віддавати едаравону (Saitoa Y. et al., 2015).

Іншими діючими речовинами, вплив яких при COVID‑19 активно вивчається, є L‑аргінін і  L‑карнітин. Ці речовини є складниками вітчизняного препарату Тіворель (ТОВ «Юрія-Фарм»). L‑аргінін покращує мікроциркуляцію, забезпечуючи стійку вазодилатацію, зміцнює імунну систему й активує Т‑клітинний імунітет, чинить мембраностабілізувальну, цитопротекторну й антиоксидантну дії, збільшує вміст NO в легеневій тканині, зменшуючи спазм бронхів і легеневих артерій. Своєю чергою, L‑карнітин сприяє отриманню енергії із жирних кислот, чинить імуномодулювальну дію, пригнічуючи вивільнення прозапальних цитокінів, виступає прямим антиоксидантом, запобігає апоптозу клітин, є відомим кардіопротектором. За рахунок аргініну препарат Тіворель виступає донатором NO, основна функція котрого пов’язана з вазодилатацією та гальмуванням процесів агрегації й адгезії тромбоцитів. Цей препарат інгібує здатність коронавірусів прикріплятися до клітин, протидіє реплікації вірусів, сприяє зменшенню ендотеліальної дисфункції.

Для підтвердження наведених теоретичних даних професор Л.В. Мороз представила клінічний випадок 63-річного чоловіка з двобічною полісегментарною пневмонією, спричиненою вірусом SARSCoV‑2 (підтверджено за допомогою ПЛР і КТ). При надходженні пацієнт скаржився на загальну слабкість, малопродуктивний кашель, утруднений вдих, задишку під час незначного фізичного навантаження. Температура тіла становила 39,0 °C, сатурація артеріальної крові киснем – 88% (96% на тлі оксигенотерапії). Пацієнту було призначено меропенем, левофлоксацин, фраксипарин, інфузії парацетамолу (Інфулган, ТОВ «Юрія-Фарм»), а наступного дня додано Ксаврон (10 мл 2 рази на добу) та Тіворель. Через 7 днів лікування сатурація артеріальної крові киснем становила вже 96%, а на тлі оксигенотерапії – 99%. Позитивна динаміка також спостерігалася на КТ, а повторна ПЛР виявилася негативною. У зв’язку з клінічним і рентгенологічним покращенням було відмінено левофлоксацин і парацетамол.

У другому клінічному випадку, представленому доповідачкою, фігурував 56-річний чоловік, у якого також була діагностована двобічна полісегментарна пневмонія, спричинена вірусом SARS-CoV‑2. Скаргами цього хворого були загальна слабкість, пітливість, сухість у носі, малопродуктивний кашель, погіршення апетиту. Температура тіла становила 38,4 °C. Пацієнту призначили меропенем, моксифлоксацин, фраксипарин, Ксаврон і Тіворель у стандартних дозуваннях. Через тиждень сатурація крові киснем нормалізувалася, а повторна ПЛР була негативною.

Отже, теоретичні дані та клінічний досвід свідчать, що включення до схеми комплексного лікування пневмонії, спричиненої SARS-CoV‑2, препаратів Ксаврон і Тіворель чинить сприятливий вплив за рахунок протидії надмірній продукції цитокінів та окисним реакціям.

Завідувач кафедри внутрішньої медицини, фізичної реабілітації та спортивної медицини Буковинського державного медичного університету (м.  Чернівці), доктор медичних наук, професор Віктор Корнійович Тащук представив доповідь на тему «Органопротекція кардіологічного хворого в епоху COVID‑19».

Основними аспектами лікування хронічних коронарних синдромів є усунення гострого ішемічного болю (нітрати короткої дії); профілактика ішемічного болю та  симптоматичне лікування (нітрати тривалої дії, антагоністи кальцію, метаболічні препарати, реваскуляризація тощо); вплив на прогноз (ацетилсаліцилова кислота, статини, інгібітори ангіотензинперетворювального ферменту, β-блокатори).

Із 2005 р. для означення сукупності факторів, які впливають на здоров’я протягом життя, застосовують термін «експосом». Експосом включає особливості харчування, клімат, уживання ліків, вплив токсичних речовин, а з кінця 2019 р. – й епідемію COVID‑19. Це необхідно враховувати при веденні пацієнтів, передусім кардіоваскулярного профілю.

Не слід вважати, що кардіоцитопротекція є виключно прерогативою пострадянського простору. Проблемам надмірного вивільнення вільних радикалів, пошкодження мітохондріальної ДНК та зменшення вмісту аденозинтрифосфату (АТФ) присвячена значна кількість робіт зарубіжних науковців (Vilahur G., 2015; Koller A., 2016). За відомим висловом L. Opie (1999), серце – це більше, ніж помпа. Це також орган, який потребує енергії, отриманої внаслідок метаболізму. Метаболічна хвороба – ішемія – в ідеалі має підлягати метаболічному лікуванню.

Навіть короткий епізод ішемії сповільнює відновлення запасів АТФ, тому протидія виснаженню цих запасів здатна захистити кардіоміоцити від загибелі внаслідок «енергетичного голоду». Говорячи про хронічні коронарні синдроми, ми завжди стикаємося з питанням гіпоксії – невідповідності енергопродукції в системі мітохондріального окисного фосфорилювання енергетичним потребам клітини. Вплинути на цю ланку патогенезу, запобігаючи гіпоксії, можна саме за допомогою метаболічної терапії.

Авторське дослідження «Смарт-ЕКГ» передбачало аналіз варіабельності серцевого ритму, фази реполяризації та деяких інших показників (нахил сегмента ST, форма зубця Т) на електрокардіограмі, в тому числі на тлі застосування різних препаратів. Було зроблено висновок, що Тіворель активує парасимпатичний контур у разі стабільної стенокардії, зменшуючи ризик несприятливих подій.

Без сумніву, NO, донатором якого виступає Тіворель, є ключовою молекулою в лікуванні гіпоксії, а отже, і пневмоній, спричинених новим коронавірусом. Судинні, імуномодулювальні, антиоксидантні та  цитопротекторні властивості L‑карнітину й L‑аргініну обґрунтовують їх призначення при COVID‑19.

На  завершення виступу професор В.К. Тащук нагадав про пандемію іспанського грипу, що мала місце в 1918-1919 рр., і наголосив, що ми не маємо часу чекати, потрібно діяти негайно.

Виступ завідувача кафедри анестезіології та інтенсивної терапії Національної медичної академії ім. П.Л. Шупика (м. Київ), доктора медичних наук, професора Олега Анатолійовича Лоскутова стосувався нейроінвазії та неврологічних ускладнень COVID‑19.

Типові клінічні ознаки COVID‑19 нагадують такі тяжкого гострого респіраторного синдрому (SARS), але в міру накопиченнях нових клінічних даних науковці знаходять дедалі більше відмінностей. Зокрема, порівняння тропності, кінетики реплікації та профілювання ушкодження клітин виявило, що SARS-CoV‑2 властива набагато вища тропність до нейронів, аніж SARS-CoV (Chu H. et al., 2020). COVID‑19 уражає не лише легені, але й серце та центральну нервову систему, що значною мірою відображається на  клінічному перебігу захворювання. Патофізіологія неврологічних ускладнень COVID‑19 включає безпосереднє пошкодження рецепторів, цитокін-опосередковане ураження, вторинне пошкодження нервової системи внаслідок гіпоксії, ураження нейронів, зумовлене ретроградним транспортом вірусу вздовж нервових волокон. Уже через 3 дні після зараження M. Dube та співавт. (2020) виявляли вірусні антигени у зв’язаних із дендритами війках і клітинному тілі нюхових сенсорних нейронів епітелію порожнини носа. На думку авторів, нейроінвазія коронавірусів відбувається трансназальним шляхом. Згідно з результатами експерименту тих самих авторів, субепітеліальна ін’єкція вірусів у язик забезпечувала меншу інвазію вірусів у центральну нервову систему, ніж інтраназальний шлях уведення.

Відповідно до даних іноземних науковців, у 36,4% пацієнтів із COVID‑19 спостерігаються неврологічні прояви. У пацієнтів із тяжким перебігом частіше відзначаються цереброваскулярні порушення (5,4% проти 0,8% у осіб із легким перебігом), розлади свідомості (14,8% проти 2,4%), скелетно-м’язові симптоми (19,3% проти 4,8%). J. Helms і співавт. (2020) проаналізували підгрупу пацієнтів із тяжким перебігом COVID‑19 і з’ясували, що неврологічні симптоми (енцефалопатія, збудження, сплутаність свідомості, ураження кортикоспінального тракту) спостерігались у 84% хворих. Ризик тяжкого перебігу інфекції був у 2,5 раза вищим у пацієнтів з інсультом в анамнезі.

В опублікованій французькими науковцями послідовній серії клінічних випадків ГРДС, спричиненого SARS-CoV‑2, зміни свідомості було задокументовано в ≥2/3 випадків. У 67% пацієнтів також відзначалися кортикоспінальні симптоми, а у 20% – гіпоксична ішемічна енцефалопатія. Автори зауважили, що на тлі COVID‑19 навіть у молодих пацієнтів нерідко трапляється ішемічний інсульт, включаючи оклюзію великих судин.

Загалом неврологічні симптоми при COVID‑19 поділяють на  три категорії: з боку центральної нервової системи (головний біль, запаморочення, порушення свідомості, нудота та блювання, атаксія, гострі цереброваскулярні захворювання, епілепсія), з боку периферичної нервової системи (гіпогевзія, гіпосмія, невралгія) та скелетно-м’язові симптоми.

Відмінною рисою енцефалопатії в цьому випадку є порушення уваги та збудження, що супроводжується сплутаністю свідомості, в’ялістю, делірієм або комою. Метаболічні й ендокринні порушення ще вираженіше підвищують ризик енцефалопатії. Спричиняти це неврологічне ускладнення можуть також сепсис і надмірна запальна реакція.

Відомо, що більшість вірусних реакцій зумовлюють цитокіновий шторм, призводячи до загибелі клітин через викид інтерферон-індукованого білка ІР‑10, ІЛ‑6, інтерферону-β. Оскільки провідну роль у розвитку запалення на тлі цитокінового шторму відіграють АФК, найважливішими лікарськими засобами в цьому аспекті виступають антиоксиданти, найперше ті, що здатні проникати в мітохондрії (Zhang Z. et al., 2016). Японські клінічні рекомендації з лікування ГРДС перелічують кілька унікальних медикаментів, які можуть бути застосовані в таких пацієнтів, у тому числі едаравон як поглинач вільних радикалів.

В експерименті S. Ikeda та співавт. (2013) було оцінено вплив едаравону на індукований фотохімічним способом інфаркт головного мозку. Автори з’ясували, що вміст вільних радикалів у ділянках, які оточують вогнище ішемії, поступово зростає, в тому числі після реперфузії. Едаравон зменшував площу інфаркту та сприяв функціональному відновленню геміпарезу при церебральному тромбозі в моделі на щурах. Підґрунтям такої дії є здатність едаравону чинити протекторний вплив на мозок, усуваючи вільні радикали та протидіючи окисним пошкодженням.

Едаравон інгібує цитокін-індуковану гіперпроникність легеневих мікросудинних ендотеліальних клітин, причому ця дія є вираженішою, ніж у дексаметазону. На тлі застосування едаравону індекс проникності знижувався на 45%, а дексаметазону – лише на 35% (Saitoa Y. et al., 2015).

У власному пілотному дослідженні було з’ясовано, що едаравон (Ксаврон, ТОВ «Юрія-Фарм») зменшує вираженість запалення та смертність у пацієнтів із коронавірусною інфекцією. У контрольній групі рівень прозапального ІЛ‑6 перевищував верхню межу норми на 1652,40%, а в групі Ксаврону – лише на 269,97%. Летальність у групі контролю становила 14,3%, а в групі Ксаврону – 0%.

На жаль, вірусні захворювання характеризуються не лише гострими неврологічними ускладненнями, а й відтермінованими наслідками. Дані наукової роботи працівників лабораторії нейроімунології (Канада) свідчать, що респіраторні патогени можуть зберігатися в центральній нервовій системі людини й, можливо, така персистентна інфекція може стати фактором або кофактором патогенезу довготривалих неврологічних наслідків у генетично схильних осіб (Desforges M. et al., 2014). Це обґрунтовує важливість своєчасного багатокомпонентного лікування.

Доцент кафедри фтизіатрії, пульмонології та сімейної медицини Харківської медичної академії післядипломної освіти, доктор медичних наук Едуард Михайлович Ходош охарактеризував патогенетичну синдромну тактику ведення пацієнта з COVID‑19.

Основними ланками патогенезу коронавірусної пневмонії є пошкодження вірусом дрібних клітин бронхів та альвеол і потужна запальна відповідь, здатна уражати тканину легень. Як наслідок порушується стан сурфактанта, альвеоли та капіляри перестають забезпечувати повноцінне надходження кисню, через що розвивається ГРДС. Окрім того, виникають поліорганна гіпоксія та синдром дисемінованого внутрішньосудинного згортання, а надалі – пневмофіброз.

Одна з головних причин ураження органів при COVID‑19 – гіпоксія. Її супроводжують біохімічні та морфологічні розлади, в тому числі ослаблення аеробного гліколізу й активація анаеробного гліколізу, посилення окиснення жирних кислот у мітохондріях, накопичення недоокиснених продуктів, ацидоз (біохімічні порушення), порушення мікроциркуляції, набряки тканин, білкова та жирова дистрофія, неефективність дихання та кровопостачання, розвиток серцево-легеневої недостатності (морфологічні й функціональні).

Прозапальні цитокіни та макрофаги запускають процеси активації нейтрофілів і моноцитів, що призводить до зростання вмісту АФК приблизно вдесятеро, окисного пошкодження клітинних мембран, викиду лізосомальних протеаз. Таке патологічно активне запалення запускає хибне коло, ланками котрого є масивне пошкодження легеневих капілярів, утруднення мікроциркуляції, відкриття артеріовенозних шлюзів зі скидом крові справа наліво в обхід легеневих капілярів, що запускає першу фазу ГРДС із посиленням гіпоксії та подальшою активацією запальних процесів.

Основними індукторами мітохондріальної дисфункції й апоптозу виступають АФК, тому однією з найважливіших клінічних задач є вибір правильного антиоксиданту. Ефективність останніх залежить від здатності проникати та накопичуватися в зоні пошкодження без видалення сигнальних молекул. Отже, ключова вимога до антиоксидантів – жиророзчинність, тобто здатність проникати крізь мембрани клітин і мітохондрій, щоби блокувати сигнальні шляхи, котрими подається сигнал продукувати запальні цитокіни. Крім того, оптимальний антиоксидант має діяти синергічно з водорозчинними антиоксидантами (в цитозолі).

Х ара ктерис т ика м и циток і нового шторму є не лише активне запалення, а й периферична інсулінорезистентність, ацидоз, анаеробний метаболізм, зменшення чи припинення обміну речовин у тканинах на кшталт гібернації, що спочатку має адаптивний характер, а згодом – патологічний. Загалом патогенез ГРДС певною мірою споріднений із таким ішемічних і постішемічних явищ.

Едаравон (Ксаврон) здатен «гасити» цитокінову пожежу. Цей препарат швидко нейтралізує широкий спектр вільних радикалів, гальмує перекисне окиснення ліпідів та активує власні механізми антиоксидантного захисту. Важливо, що едаравон потрапляє в біологічні мембрани шляхом пасивної дифузії та проникає крізь гематоенцефалічний бар’єр.

Окрім Ксаврону, компонентами патогенетичної терапії COVID‑19 можуть виступати Тівортін і Тіворель (ТОВ «Юрія-Фарм»). Аргінін, який є компонентом обох цих засобів, виступає субстратом для синтезу NO, таким чином сприяючи вазодилатації, а також має імуностимулювальний, мембраностабілізувальний, цитопротекторний та антиоксидантний вплив. Тіворель, окрім аргініну, містить левокарнітин, роль якого полягає в доставці жирних кислот до мітохондріального матриксу («карнітиновий човник»), гальмуванні апоптозу, антиоксидантній дії, кардіопротекції.

NO є природною молекулою метаболізму людини, що відіграє важливу роль у формуванні імунної відповіді на вплив патогенної флори. Дослідження in vitro показали, що NO гальмує реплікацію SARS-CoV і покращує виживаність інфікованих клітин. Це обґрунтовує доцільність призначення Тівортіну/Тіворелю при COVID‑19.

Ще одним патогенетичним засобом, який традиційно застосовується при пневмоніях, є Реосорбілакт (ТОВ «Юрія-Фарм»). Цей препарат потенціює дію вазопресорів, зменшуючи потребу в них; запобігає набряку легень за рахунок виведення рідини з інтерстиційного простору; відновлює мікроциркуляцію завдяки дезагрегантній дії та зменшенню в’язкості крові.

На завершення Е.М. Ходош представив клінічний випадок пацієнта з пневмонією, спричиненою SARS-CoV‑2, в якого призначене лікування (азитроміцин, гідроксихлорохін, Тіворель, Ксаврон, Реосорбілакт, еноксапарин) сприяло усуненню клінічних проявів хвороби та зменшенню потреби в кисні, а також іще кілька клінічних випадків, у яких лікування схожими схемами фармакотерапії забезпечувало значне покращення.

Обираючи тактику лікування COVID-19, слід враховувати, що це захворювання може мати хвилеподібний перебіг. Перша хвиля характеризується досить легким перебігом, після чого приблизно у 80% пацієнтів відбуваються покращення й одужання. У 20% пацієнтів після першої хвилі може настати період тимчасового покращення, однак у подальшому настає друга хвиля розвитку симптомів (так звана легенева фаза), перебіг якої є тяжчим. І саме в цей час на додаток до протокольної терапії доцільним є застосування засобів, дія яких спрямована на комплексне пригнічення цитокінового шторму – препаратів Ксаврон, Тіворель і Реосорбілакт. Важливо призначити таке лікування на самому початку фази другої хвилі, що дасть можливість «зрізати» її подальше прогресування. Ця лікувальна стратегія дозволить полегшити перебіг захворювання, скоротити термін перебування пацієнтів у стаціонарі й уникнути тяжких ускладнень.

Authors: С.В. Коваленко, Л.В. Мороз, В.К. Тащук, О.А. Лоскутов, Е.М. Ходош
Posted: Медична газета «Здоров'я України», 2020
Edaravone: A potential treatment for the COVID-19-induced inflammatory syndrome?

We read with great interest the recent article by Zhong et al. titled “Efficacy and Safety of Current Therapeutic Options for COVID-19 – Lessons to Be Learnt from SARS and MERS Epidemic: A Systematic Review and Meta-analysis [1],” which is the most complete review of randomized control trials and cohort studies of potential pharmacotherapies for COVID-19 we have seen. However, after completing their thorough meta-analysis, the authors were not able to identify any particular drug or drug combination they could recommend for the treatment of COVID-19. This conclusion speaks to the need for venturing beyond the relatively short list of drugs that have been re-purposed for COVID-19 because of their efficacy in other viral infections.

In severe cases of COVID-19, an excessive and unregulated release of certain pro-inflammatory cytokines and chemokines can occur, producing an inflammatory syndrome that elicits significant systemic inflammation, multiple organ damage and failure and death [2]. It has been hypothesized that therapeutic interventions that significantly attenuate this severe, injurious inflammatory response could decrease the incidence of organ damage and mortality. Currently, there are no effective treatments for the COVID-19-induced inflammatory syndrome. We suggest that edaravone be considered for such use either alone or in combination with one or more anti-viral drugs, such as Lopinovir/Ritonavir.

Edaravone is given intravenously to treat amyotrophic lateral sclerosis and acute phase cerebral infarction. Edaravone is a lipophilic compound that penetrates readily into numerous tissues and organs and has significant anti-oxidant and anti-inflammatory efficacy [3]. The systemic administration of edaravone produces protective effects against inflammation and injury in animal models of chemical-induced injury to the lungs, kidneys, intestines, pancreas, brain and liver [4]. Edaravone also decreases the levels of 1) certain cytokines (IL-2, IL-6, IL-1β, TNF-α) and chemokines (IL-8, MCP-1, MIP-1a, 2,5); 2) reactive oxygen species and nitric oxide and 3) hepatic AST and ALT, in various animal models [4].

Edaravone’s efficacy could be determined by identifying individuals 18 years of age or older who have laboratory – confirmed (using real time RT-PCR) COVID-19 and are diagnosed with severe COVID-19 (based on the criteria of Chen et al. [5]), for a double-blind, randomized placebo-controlled trial. A 60 mg i.v dose of edaravone would be administered once daily in one hour for 10 days to individuals in the test group, while control participants would receive an i.v. placebo solution. All patients should receive the best supportive care. The primary efficacy point would be mortality and clinical improvement would be evaluated as previously described [6]. Edaravone can produce contusion, gait disturbance and headache and patients should be closely monitored for hypersensitivity reactions. Edaravone can be used in patients with mild to moderate hepatic impairment and renal impairment is not expected to significantly alter edaravone exposure. Finally, edaravone has not been reported to produce immunosuppression.

In conclusion, we hypothesize that edaravone, if given in a timely manner, would decrease organ damage, clinical complications and mortality in severe cases of COVID-19. If edaravone decreases mortality and produces significant clinical improvement, its efficacy should be tested in infectious diseases that produce an overexuberant inflammatory response, such as Ebola.

Declaration of Competing Interest

None of the authors has any declaration of interest.

References

[1] H. Zhong, Y. Wang, Z.-L. Zhang, Y.-X. Liu, K.-J. Le, M. Cui, Y.-T. Yu, Z.-C. Gu, Y. Gao, H.- W. Lin, Efficacy and safety of current therapeutic options for COVID-19 – lessons to be learnt from SARS and MERS epidemic: a systematic review and meta-analysis, Pharmacol. Res. 157 (2020) 104872.

[2] C. Huang, X. Wang, L. Li, L. Ren, J. Zhao, Y. Hu, L. Zhang, G. Fan, J. Xu, X. Gu, Z. Cheng, https://doi.org/10.1016/j.phrs.2020.105055 Received 26 June 2020 Pharmacological Research 160 (2020) 105055 Available online 30 June 2020 1043-6618/ © 2020 Elsevier Ltd. All rights reserved. T T. Yu, J. Xia, Y. Wei, W. Wu, X. Xie, W. Yin, H. Li, M. Liu, Y. Xiao, H. Gao, L. Guo, J. Xie, G. Wang, R. Jiang, Z. Gao, Q. Jin, J. Wang, B. Cao, Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China, Lancet 395 (10223) (2020) 497–506.

[3] T. Watanabe, M. Tahara, S. Todo, The novel antioxidant edaravone: from bench to bedside, Cardiovasc. Ther. 26 (2) (2008) 101–114.

[4] K. Kikuchi, N. Takeshige, N. Miura, Y. Morimoto, T. Ito, S. Tancharoen, K. Miyata, C. Kikuchi, N. Iida, H. Uchikado, N. Miyagi, N. Shiomi, T. Kuramoto, I. Maruyama, M. Morioka, K.-I. Kawahara, Beyond free radical scavenging: beneficial effects of edaravone (Radicut) in various diseases (Review), Exp. Ther. Med. 3 (1) (2012) 3–8.

[5] G. Chen, D. Wu, W. Guo, Y. Cao, D. Huang, H. Wang, T. Wang, X. Zhang, H. Chen, H. Yu, X. Zhang, M. Zhang, S. Wu, J. Song, T. Chen, M. Han, S. Li, X. Luo, J. Zhao, Q. Ning, Clinical and immunological features of severe and moderate coronavirus disease, J. Clin. Invest. 130 (5) (2020) 2620–2629.

[6] J. Grein, N. Ohmagari, D. Shin, G. Diaz, E. Asperges, A. Castagna, T. Feldt, G. Green, M.L. Green, F.-X. Lescure, E. Nicastri, R. Oda, K. Yo, E. Quiros-Roldan, A. Studemeister, J. Redinski, S. Ahmed, J. Bernett, D. Chelliah, D. Chen, S. Chihara, S.H. Cohen, J. Cunningham, A.D. Monforte, S. Ismail, H. Kato, G. Lapadula, E. L’Her, T. Maeno, S. Majumder, M. Massari, M. Mora-Rillo, Y. Mutoh, D. Nguyen, E. Verweij, A. Zoufaly, A.O. Osinusi, A. DeZure, Y. Zhao, L. Zhong, A. Chokkalingam, E. Elboudwarej, L. Telep, L. Timbs, I. Henne, S. Sellers, H. Cao, S.K. Tan, L. Winterbourne, P. Desai, R. Mera, A. Gaggar, R.P. Myers, D.M. Brainard, R. Childs, T. Flanigan, Compassionate use of remdesivir for patients with severe Covid-19, N. Eng. J. Med. 382 (24) (2020) 2327–2336.

Authors:

  • Sandra E. Reznik, Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, Queens, NY11439, United States Departments of Pathology and Obstetrics and Gynecology and Women’s Health, Montefiore Medical Center/Albert Einstein College of Medicine, Bronx, NY10461, United States
  • Amit K. Tiwari, Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, OH43614, United States
  • Charles R. Ashby Jr., Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, Queens, NY11439, United States. E-mail address: cnsratdoc@optonline.net
Authors: Sandra E. Reznik, Amit K. Tiwari, Charles R. Ashby Jr.
Posted: Pharmacological Research 160 (2020) 105055
Inhibitory Effects of Edaravone, a Free Radical Scavenger, on Cytokine-induced Hyperpermeability of Human Pulmonary Microvascular Endothelial Cells: A Comparison with Dexamethasone and Nitric Oxide Synthase Inhibitor

Lung hyperpermeability affects the development of acute respiratory distress syndrome (ARDS), but therapeutic strategies for the control of microvascular permeability have not been established. We examined the effects of edaravone, dexamethasone, and N-monomethyl-L-arginine (L-NMMA) on permeability changes in human pulmonary microvascular endothelial cells (PMVEC) under a hypercytokinemic state. Human PMVEC were seeded in a Boyden chamber. After monolayer confluence was achieved, the culture media were replaced respectively by culture media containing edaravone, dexamethasone, and L-NMMA. After 24-h incubation, the monolayer was stimulated with tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β). Fluorescein-labeled dextran was added. Then the transhuman PMVEC leak was measured. Expressions of vascular endothelial-cadherin (VE-cadherin) and zonula occludens-1 protein (ZO-1) were evaluated using real-time quantitative polymerase chain reaction and immunofluorescence microscopy. The results showed that TNF-α+IL-1βmarkedly increased pulmonary microvascular permeability. Pretreatment with edaravone, dexamethasone, or L-NMMA attenuated the hyperpermeability and inhibited the cytokine-induced reduction of VE-cadherin expression on immunofluorescence staining. Edaravone and dexamethasone increased the expression of ZO-1 at both the mRNA and protein levels. Edaravone and dexamethasone inhibited the permeability changes of human PMVEC, at least partly through an enhancement of VE-cadherin. Collectively, these results suggest a potential therapeutic approach for intervention in patients with ARDS.

Influenza virus infection causes severe pneumonia, especially in children and adults with underlying disease [1, 2]. Many patients need ventilator support because of severe complications such as acute respiratory distress syndrome (ARDS) [3]. Influenza A/H1N1pandemic viruses have been reported to show an affinity for type II alveolar epithelial cells and to invade the lung directly [4]. Although lung hyperpermeability is thought to contribute to ARDS development [5], no clinically available medication for the control of vascular permeability has been established.

Vascular endothelial cell-cell adhesions that control vascular permeability are organized mainly by adherence junctions and tight junctions [6, 7]. Adherence junctions are formed by members of the cadherin family. Vascular endothelial-cadherin (VE-cadherin) receives positive and negative control by various signals. It regulates endothelial cell adhesion and barrier function of blood vessels dynamically [6, 8]. Tight junctions include transmembrane molecules such as occludin, claudin, and junctional adhesion molecules. Zonula occludens-1 protein (ZO-1), a lining protein of tight junctions, connects transmembrane molecules with the actin cytoskeleton. In response to inflammatory cytokines, the vascular endothelium expresses cellular adhesion molecules including intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), and secretes chemokines such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) to upregulate leukocyte recruitment into inflamed tissues [9].

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), which is used to treat cerebral infarction as a free radical scavenger [10], reportedly protects various organs [11]. Several animal studies have demonstrated that edaravone prevents lung injury induced by various factors, including anticancer drugs, lipopolysaccharide, and acute pancreatitis via antioxidant and anti-inflammatory effects [12-15]. Nevertheless, little is known about whether edaravone has any protective effect for ARDS characterized by enhanced vascular permeability.

The 2009 pandemic influenza A virus infection caused severe pneumonia and ARDS related to lung hyperpermeability more often than seasonal influenza virus infection did [2, 3]. Although we initially tried to perform influenza virus infection experiments using human pulmonary microvascular endothelial cells (PMVEC), neither influenza A/H1N1pandemic nor A/H3N3Aichi was found to infect endothelial cells or change the permeability parameters (unpublished data). Therefore, we specifically examined the permeability changes of endothelial cells by inflammatory cytokines that are produced during severe infectious diseases including influenza. For this study, we established a human PMVEC monolayer model to examine the barrier permeability changes that occur in endothelial cells in a hypercytokinemic state. Dexamethasone and nitric oxide synthase (NOS) inhibitor are reported to decrease pulmonary vascular hyperpermeability in ARDS [16, 17]. Therefore, we examined the therapeutic effects of edaravone on permeability changes and, in the same experimental setting, compared the effects of edaravone to those of dexamethasone and NOS inhibitor.

Cell culture. Human PMVEC were purchased from Lonza Group, Ltd. (Walkersville, MD, USA) and were maintained using a BulletKit (EGM-2MV; Lonza) as recommended by the manufacturer. The cells were cultured in 95オ air, 5オ CO2 at 37℃. All experiments were performed on cells in the 3rd to 6th passage. Permeability assay. A Boyden chamber was used for the permeability evaluation. Human PMVEC were seeded in Transwell Inserts (3.0µm pore; Becton, Dickinson and Co., Franklin Lakes, NJ, USA) at a concentration of 5×104 cells/well. They were cultured for 3 days. After monolayer confluence was achieved, the culture media in the upper chamber (150µl) and the lower chamber (750µl) were replaced respectively by culture media containing edaravone (100µM; Mitsubishi Tanabe Pharma Corp., Tokyo, Japan), dexamethasone (100 µM; Calbiochem Novabiochem Corp., La Jolla, CA, USA), and N-monomethyl-L-arginine (L-NMMA, 1 mM; Calbiochem Novabiochem Corp.). After 24h incubation, the culture medium was replaced with culture medium containing each chemical and cytokines. Tumor necrosis factor-α (TNF-α) (Peprotech Inc., Rocky Hill, NJ, USA) and interleukin-1β (IL-1β) (Peprotech Inc. ) were used as pro-inflammatory cytokines. In a preliminary set of experiments, human PMVEC were treated with TNF-α (100ng/ml) and IL-1β (100ng/ml) singly or in combination. Used alone, TNF-αand IL-1βeach tended to increase the endothelial permeability, but the permeability changes were not significant. Only the combination of TNF-α +IL-1β increased the permeability significantly (data not shown). In subsequent experiments, therefore, human PMVEC were treated with TNF-α+ 280 Saito et al. Acta Med. Okayama Vol. 69, No. 5 IL-1β at either of 2 concentrations (10ng/ml or 100ng/ml) for 24h.

Phosphate-buffered saline was used as a control buffer. To measure trans-human PMVEC dextran leak, 150µl assay medium containing fluorescein isothiocyanate-labeled dextran (FITC-Dx; MW 3,000) (100µg/ml) was added to each upper chamber; and 750µl assay medium was added to each lower chamber. After incubation for 2h at 37℃, the fluorescence intensity of medium from the lower chambers was measured at 485-538nm. The data were expressed as follows: permeability index (オ)=[(experimental clearance)-(spontaneous clearance)]×100/[(clearance of filter alone)-(spontaneous clearance)] [18]. The experiments were repeated 3 times, with each repetition consisting of 4 replicates.

Lactate dehydrogenase (LDH) release assay. Cell damage was assessed using the LDH release assay (LDH Cytotoxicity Detection Kit; Takara Bio Inc., Shiga, Japan). The percentage cytotoxicity was calculated as follows: cytotoxicity (オ)=[experimental LDH release (OD492)-spontaneous LDH release (OD492)]×100/[maximum LDH release (OD492)]- [spontaneous LDH release (OD492)]. The OD492 of spontaneous LDH release and the OD492 of maximum LDH release were obtained respectively from the supernatant of controls and the supernatant of the cells treated with 2オ Triton X-100. The experiments were repeated twice, with each repetition consisting of 4 replicates.

Immunofluorescence microscopy. For immunofluorescence experiments, the endothelial cells were grown on collagen I- coated slide chambers (Becton, Dickinson and Co.). They were subsequently treated for 24h with edaravone, dexamethasone, or L-NMMA followed by treatment with TNF-α+ IL-1β (10ng/ml) for 24h. The cells were then washed with phosphate-buffered saline and fixed in 2オ formaldehyde. After permeabilization with 0.1オ Triton X-100 and blocking, the cells were incubated with anti-VE-cadherin antibody (10µg/ml; R & D Systems, Minneapolis, MN, USA) and anti-ZO-1 antibody (5µg/ml; Genetex Inc., Irvine, CA, USA) for 60min. Alexa Fluor 488-conjugated antibody (Invitrogen Corp., Carlsbad, CA, USA) was used as a secondary antibody. The nucleus was stained with 4ʼ, 6-diamidino-2-phenylindole (DAPI) in mounting medium (Vectashield; Vector Laboratories Inc., Burlingame, CA, USA). Samples were evaluated under a fluorescence microscope (BZ-9000 generation II; Keyence Co., Osaka, Japan). The experiments were repeated 4 times.

Real-time quantitative polymerase chain reaction (real-time PCR). To examine the effects of cytokines and medications on VE-cadherin and ZO-1 mRNA expression in human PMVEC under the experimental conditions employed, real-time PCR was performed with total RNAs extracted from the cells using the specific primers presented in Table 1 [19]. After the cells reached confluence in 24-well plates, they were incubated for 24h with edaravone, dexamethasone, or L-NMMA, followed by treatment with TNF-α+IL-1β. After stimulation, the complementary DNA was extracted using FastLane® Cell cDNA (Qiagen Inc., Hilden, Germany). Real-time PCR was performed using a real-time PCR system (7500 Fast; Applied Biosystems, Foster City, CA, USA) with SYBR ®Premix Ex Taq (Takara Bio Inc.). The PCR mixture, which had a total volume of 50µl, consisted of 1×SYBR ®Premix Ex Taq, which included DNA polymerase, SYBR Green dye, dNTP mixture, and PCR buffer, 0.2µM each of the forward and reverse primers, and cDNA of the samples. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

Enzyme-linked immunosorbent assay (ELISA). Expressions of adhesion molecules in the culture supernatant were measured following Lehleʼs in vitro model [20-22]. The levels of soluble ICAM-1 (sICAM-1) and soluble VCAM-1 (sVCAM-1) were measured (Luminex 100/200 System; Luminex Corp., Austin, TX, USA) with a Human CVD Panel 1 96-well Plate Assay (Millipore Corp., Bedford, MA, USA). The levels of MCP-1 and IL-8 were also measured by ELISA (R & D Systems). The results were obtained under 2 experimental conditions, each consisting of 4 replicates.

Statistical analyses. All values were expressed as the mean ± SD. Differences between groups were examined for statistical significance using one-way analysis of variance with Tukeyʼs multiple comparison test. Statistical significance was inferred for P-values less than 0.05.

Permeability change of the human PMVEC monolayer. As presented in Fig. 1, the endothelial permeability was increased significantly by TNF-α+IL-1β at the concentrations of 10ng/ml and 100ng/ml compared to the control group. No significant difference was found between 10ng/ml and 100ng/ml. For subsequent experiments, the concentration of 10ng/ml was adopted. Pretreatment with edaravone (100µM) significantly decreased the permeability by 45オ compared with the group treated with TNF-α+IL-1βalone (Fig. 2A). Pretreatment with dexamethasone (100µM) significantly decreased the permeability by 35オ compared with the group treated with TNF-α+IL-1βalone (Fig. 2B). Pretreatment with L-NMMA (1mM) significantly decreased the permeability by 50オ compared with the group exposed to only TNF-α+IL-1β(Fig. 2C).

Cell cytotoxicity. Cell damage was assessed by using an LDH release assay. The LDH release was not altered significantly by the pretreatment of endothelial cells with edaravone, dexamethasone, or L-NMMA compared with TNF-α+IL-1β alone (Fig. 3). This result showed that the attenuation of hyperpermeability by pretreatment in this study was not caused by the inhibition of cell membrane damage or cell death. Effects of edaravone, dexamethasone, and L-NMMA on the expression of endothelial adherence junction protein VE-cadherin. The effects of edaravone, dexamethasone, and L-NMMA on the expression of VE-cadherin were examined to identify the relation between permeability changes and cell-cell adhesion. Immunofluorescence staining showed that staining for VE-cadherin was reduced after TNF-α+IL-1βstimulation. Pretreatment with edaravone, dexamethasone, or L-NMMA for 24h attenuated the effects of TNF-α+IL-1βstimulation (Figs. 4A-4E). Pretreatment with edaravone, dexamethasone, or L-NMMA for 24h followed by treatment with TNF-α+IL-1β for 3h did not alter the mRNA level of VE-cadherin (Fig. 4F).

Effects of edaravone, dexamethasone, and L-NMMA on the expression of endothelial tight junction protein ZO-1. No change of ZO-1 was shown by treatment with TNF-α+IL-1β for 24h. The near-circumferential expression of ZO-1 was increased by pretreatment with edaravone or dexamethasone for 24h compared with the other groups (Figs. 5A-5E). In human PMVEC, ZO-1 mRNA expression was unaffected by stimulation with TNF-α +IL-1β, but it was increased significantly by pretreatment with edaravone or dexamethasone (Fig. 5F). Effects of TNF-α+IL-1β, edaravone, dexamethasone, and L-NMMA on the generation of sICAM-1, sVCAM-1, MCP-1, and IL-8 in human PMVEC. Leukocyte recruitment into inflamed tissues engenders microvascular hyperpermeability. Therefore, the effects of TNF-α+IL-1β, edaravone, dexamethasone, and L-NMMA on the production of sICAM-1, sVCAM-1, MCP-1, and IL-8 in human PMVEC were examined. Treatment of human PMVEC with TNF-α+ IL-1β for longer than 6h caused a significant increase in the production of sICAM-1 and sVCAM-1 in the supernatant compared with the non-stimulated cells. The levels of MCP-1 and IL-8 were increased significantly after 3h and 6h, respectively, by the administration of TNF-α+IL-1β(Fig. 6). We examined the inhibitory effects of pretreatment with edaravone, dexamethasone, and L-NMMA for 24h on the production of adherence molecules and chemokines. The sICAM-1 and sVCAM-1 production tended to be decreased by these pretreatments, but the differences were not significant. Pretreatment with dexamethasone significantly inhibited the production of MCP-1 and IL-8 induced by TNF-α+IL-1β

Various diseases including viral pneumonia, sepsis, and acute pancreatitis can cause ARDS, a state of pulmonary edema caused by the influx of protein-rich edema fluid into the air spaces. ARDS can also occur as a consequence of endothelial and epithelial injury [5]. Microvascular endothelial cells respond to local changes in biological needs caused by inflammation. These cells play an important role in the control of exchange of leukocytes and fluids between the lung microvessels and alveoli [23, 24]. The cytokine profiles of suctioned pulmonary secretions from children infected with influenza A/H1N1pdm revealed that TNF-α, IL-1βand other inflammatory cytokine concentrations were markedly higher on the fifth day after hospitalization than on the first day [25]. It has been reported that influenza A/H1N1pdm viruses replicate efficiently in the lungs of infected mice, ferrets, and non-human primates. These viruses can cause remarkable pathology in the lungs [4]. Consequently, locally released inflammatory cytokines in the lung are increased in influenza A/H1N1pdm pneumonia, and are likely to contribute to lung microvascular hyperpermeability. Our experiments showed that stimulation with inflammatory cytokines (TNF-α+IL-1β, 10ng/ml in each) for 24h significantly increased dextran leakage across human PMVEC. Pretreatment with edaravone, dexamethasone, or L-NMMA significantly inhibited the increase of permeability caused by these cytokines in the endothelial cells to the same extent. Stimulation with inflammatory cytokines caused perturbation of VE-cadherin, a major component of the adherence

Discussion

junction [8]. The finding that pretreatment with edaravone, dexamethasone, or L-NMMA inhibited the cytokine-induced perturbation of VE-cadherin suggests that these medications have the effect of holding VE-cadherin. Differential results were observed in terms of the protein levels and mRNA expression of VE-cadherin: although no remarkable change of the mRNA expression was observed, stimulation with inflammatory cytokines led to a decrease in VE-cadherin protein. Pretreatment with edaravone, dexamethasone, or L-NMMA inhibited the effect. This discrepancy points to indirect effects on VE-cadherin expression rather than to direct transcriptional regulation of the VE-cadherin gene. The results also showed that ZO-1 mRNA and near-circumferential expression were increased by pretreatment with edaravone or dexamethasone. These results support the contention that edaravone and dexamethasone can reinforce a tight junction through the enhancement of ZO-1 expression [26, 27]. Pretreatment with edaravone or dexamethasone might contribute to enhancement of the tight junction, resulting in the inhibition of hyperpermeability in human PMVEC.

Several previous studies have revealed that edaravone increases transmonolayer electrical resistance by enhancing the expression of the adherence junction protein [28]. Edaravone prevents the increase of permeability under oxidative stress [29] in human umbilical vein endothelial cells. Dexamethasone increases VE-cadherin protein levels and rearranges the cytoskeleton [30]. The present study provided similar results that edaravone and dexamethasone prevented hyperpermeability in human PMVEC under cytokine stimulation. Our experiments also showed that stimulation with cytokines increased the mRNA expression and protein levels of ICAM-1, VCAM-1, MCP-1, and IL-8, similar to human dermal microvascular endothelial cells [31, 32]. Pretreatment with edaravone or dexamethasone showed a tendency to inhibit the expression of ICAM-1 and VCAM-1, although the effect was not significant. Pretreatment with dexamethasone reduced MCP-1 and IL-8 significantly. These results present the possibility that edaravone and dexamethasone prevent the increase of vascular permeability partly through the inhibition of leukocyte recruitment into inflamed tissues. Our recent report described that administration of TNF-α enhances the expression of MMP-9, which dissolves type IV collagen in the brain endothelial cells of mice, and results in disorders of the bloodbrain barrier [33]. However, administration of TNF-α+IL-1β induced neither mRNA expression nor secretion of MMP-9 in human PMVEC (data not shown). This discrepancy might have resulted from differences in the species, cell types, experimental conditions, or sensitivity of detection methods. In conclusion, our experiments show that edaravone and dexamethasone can prevent hyperpermeability in human PMVEC stimulated by inflammatory cytokines, possibly through enhancement of the adherence junction, and potentially through other mechanisms as well (Fig. 8). These results might provide

Several previous studies have revealed that edaravone increases transmonolayer electrical resistance by enhancing the expression of the adherence junction protein [28]. Edaravone prevents the increase of permeability under oxidative stress [29] in human umbilical vein endothelial cells. Dexamethasone increases VE-cadherin protein levels and rearranges the cytoskeleton [30]. The present study provided similar results that edaravone and dexamethasone prevented hyperpermeability in human PMVEC under cytokine stimulation. Our experiments also showed that stimulation with cytokines increased the mRNA expression and protein levels of ICAM-1, VCAM-1, MCP-1, and IL-8, similar to human dermal microvascular endothelial cells [31, 32]. Pretreatment with edaravone or dexamethasone showed a tendency to inhibit the expression of ICAM-1 and VCAM-1, although the effect was not significant. Pretreatment with dexamethasone reduced MCP-1 and IL-8 significantly. These results present the possibility that edaravone and dexamethasone prevent the increase of vascular permeability partly through the inhibition of leukocyte recruitment into inflamed tissues. Our recent report described that administration of TNF-α enhances the expression of MMP-9, which dissolves type IV collagen in the brain endothelial cells of mice, and results in disorders of the bloodbrain barrier [33]. However, administration of TNF-α+IL-1β induced neither mRNA expression nor secretion of MMP-9 in human PMVEC (data not shown). This discrepancy might have resulted from differences in the species, cell types, experimental conditions, or sensitivity of detection methods. In conclusion, our experiments show that edaravone and dexamethasone can prevent hyperpermeability in human PMVEC stimulated by inflammatory cytokines, possibly through enhancement of the adherence junction, and potentially through other mechanisms as well (Fig. 8). These results might provide

Several previous studies have revealed that edaravone increases transmonolayer electrical resistance by enhancing the expression of the adherence junction protein [28]. Edaravone prevents the increase of permeability under oxidative stress [29] in human umbilical vein endothelial cells. Dexamethasone increases VE-cadherin protein levels and rearranges the cytoskeleton [30]. The present study provided similar results that edaravone and dexamethasone prevented hyperpermeability in human PMVEC under cytokine stimulation. Our experiments also showed that stimulation with cytokines increased the mRNA expression and protein levels of ICAM-1, VCAM-1, MCP-1, and IL-8, similar to human dermal microvascular endothelial cells [31, 32]. Pretreatment with edaravone or dexamethasone showed a tendency to inhibit the expression of ICAM-1 and VCAM-1, although the effect was not significant. Pretreatment with dexamethasone reduced MCP-1 and IL-8 significantly. These results present the possibility that edaravone and dexamethasone prevent the increase of vascular permeability partly through the inhibition of leukocyte recruitment into inflamed tissues. Our recent report described that administration of TNF-α enhances the expression of MMP-9, which dissolves type IV collagen in the brain endothelial cells of mice, and results in disorders of the bloodbrain barrier [33]. However, administration of TNF-α+IL-1β induced neither mRNA expression nor secretion of MMP-9 in human PMVEC (data not shown). This discrepancy might have resulted from differences in the species, cell types, experimental conditions, or sensitivity of detection methods. In conclusion, our experiments show that edaravone and dexamethasone can prevent hyperpermeability in human PMVEC stimulated by inflammatory cytokines, possibly through enhancement of the adherence junction, and potentially through other mechanisms as well (Fig. 8). These results might provide

Several previous studies

Several previous studies have revealed that edaravone increases transmonolayer electrical resistance byenhancing the expression of the adherence junctionprotein [28]. Edaravone prevents the increase ofpermeability under oxidative stress [29] in humanumbilical vein endothelial cells. Dexamethasoneincreases VE-cadherin protein levels and rearrangesthe cytoskeleton [30]. The present study providedsimilar results that edaravone and dexamethasoneprevented hyperpermeability in human PMVEC undercytokine stimulation.Our experiments also showed that stimulation withcytokines increased the mRNA expression and proteinlevels of ICAM-1, VCAM-1, MCP-1, and IL-8,similar to human dermal microvascular endothelialcells [31, 32]. Pretreatment with edaravone or dexamethasone showed a tendency to inhibit the expression of ICAM-1 and VCAM-1, although the effect wasnot significant. Pretreatment with dexamethasonereduced MCP-1 and IL-8 significantly. These resultspresent the possibility that edaravone and dexamethasone prevent the increase of vascular permeabilitypartly through the inhibition of leukocyte recruitmentinto inflamed tissues.Our recent report described that administration ofTNF-α enhances the expression of MMP-9, whichdissolves type IV collagen in the brain endothelialcells of mice, and results in disorders of the bloodbrain barrier [33]. However, administration ofTNF-α+IL-1β induced neither mRNA expressionnor secretion of MMP-9 in human PMVEC (data notshown). This discrepancy might have resulted fromdifferences in the species, cell types, experimentalconditions, or sensitivity of detection methods.In conclusion, our experiments show that edaravone and dexamethasone can prevent hyperpermeability in human PMVEC stimulated by inflammatorycytokines, possibly through enhancement of the adherence junction, and potentially through other mechanisms as well (Fig. 8). These results might provide

Fig. 3 Effects of edaravone, dexamethasone, and N-monomethyl-L-arginine (L-NMMA) on cell cytotoxicity in human PMVEC stimulated with TNF-α+IL-1β. In evaluation of cell cytotoxicity as determined by the LDH release, no significant differences were found among groups.

expression was unaffected by stimulation with TNF-α +IL-1β, but it was increased significantly by pretreatment with edaravone or dexamethasone (Fig. 5F).

Effects of TNF-α+IL-1β, edaravone, dexamethasone, and L-NMMA on the generation of sICAM-1, sVCAM-1, MCP-1, and IL-8 in human PMVEC.

Treatment of human PMVEC with TNF-α+ IL-1β for longer than 6h caused a significant increase in the production of sICAM-1 and sVCAM-1 in the supernatant compared with the non-stimulated cells. The levels of MCP-1 and IL-8 were increased significantly after 3h and 6h, respectively, by the administration of TNF-α+IL-1β(Fig. 6).

Fig. 4 Effects of TNF-α+IL-1β, edaravone, dexamethasone, and N-monomethyl-L-arginine (L-NMMA) on the expression of vascular endothelial (VE)-cadherin in human PMVEC. For immunofluorescence analysis, human PMVEC were treated with culture medium alone (A), with TNF-α+IL-1β for 24h (B), pretreatment with edaravone (C), with dexamethasone (D), with L-NMMA (E) for 24h and treatment with TNF-α+IL-1β for 24 h. TNF-α+IL-1β reduced staining for VE-cadherin. Pretreatment with edaravone, dexamethasone, or L-NMMA apparently inhibited this effect. VE-cadherin mRNA (F) was not altered significantly by these chemicals. Representative immunofluorescence studies are shown.

Fig. 5 Effects of TNF-α+IL-1β, edaravone, dexamethasone, and N-monomethyl-L-arginine (L-NMMA) on the expression of zonula occuldens-1 protein (ZO-1) in human PMVEC. For immunofluorescence analysis, human PMVEC were treated with culture medium alone (A), with TNF-α+IL-1β for 24h (B), pretreatment with edaravone (C), with dexamethasone (D), with L-NMMA (E) for 24h and treatment with TNF-α+IL-1β for 24 h. No change of ZO-1 expression was found from TNF-α+IL-1β treatment. Pretreatment with edaravone or dexamethasone increased ZO-1 expression compared with control or cytokine group. Representative immunofluorescence studies are shown. ZO-1 mRNA (F) was enhanced by pretreatment with edaravone or dexamethasone compared with control and TNF-α+IL-1β group. Data are presented as mean ± SD (n=4 in each group). *p<0.05 in comparison with TNF-α+IL-1β group. **p<0.01 in comparison with TNF-α+IL-1βgroup.

Fig. 6 Effects of TNF-α+IL-1βon the secretion of sICAM-1, sVCAM-1, MCP-1, and IL-8 in human PMVEC. Treatment with TNFα+IL-1β(10ng/ml) for 6h significantly increased the secretion of sICAM-1 (A), sVCAM-1 (B), and IL-8 (D). The secretion of MCP-1 (C) was increased by stimulation for 3h. Data are presented as mean ± SD (n=4 in each group). *p<0.05 in comparison with 0 h in each group. sICAM-1, soluble intercellular adhesion molecule-1; sVCAM-1, soluble vascular cell adhesion molecule-1; MCP-1, monocyte chemoattractant protein-1; IL-8, interleukin-8.

Discussion

Various diseases including viral pneumonia, sepsis, and acute pancreatitis can cause ARDS, a state of pulmonary edema caused by the influx of protein-rich edema fluid into the air spaces. ARDS can also occur as a consequence of endothelial and epithelial injury [5]. Microvascular endothelial cells respond to local changes in biological needs caused by inflammation. These cells play an important role in the control of exchange of leukocytes and fluids between the lung microvessels and alveoli [23, 24].

Fig. 5 Effects of TNF-α+IL-1β, edaravone, dexamethasone, and N-monomethyl-L-arginine (L-NMMA) on the expression of zonula occuldens-1 protein (ZO-1) in human PMVEC. For immunofluorescence analysis, human PMVEC were treated with culture medium alone (A), with TNF-α+IL-1β for 24h (B), pretreatment with edaravone (C), with dexamethasone (D), with L-NMMA (E) for 24h and treatment with TNF-α+IL-1β for 24 h. No change of ZO-1 expression was found from TNF-α+IL-1β treatment. Pretreatment with edaravone or dexamethasone increased ZO-1 expression compared with control or cytokine group. Representative immunofluorescence studies are shown. ZO-1 mRNA (F) was enhanced by pretreatment with edaravone or dexamethasone compared with control and TNF-α+IL-1β group. Data are presented as mean ± SD (n=4 in each group). *p<0.05 in comparison with TNF-α+IL-1β group. **p<0.01 in comparison with TNF-α+IL-1βgroup.

Fig. 7 Effects of edaravone, dexamethasone, and N-monomethyl-L-arginine (L-NMMA) on the secretion of sICAM-1, sVCAM-1, MCP-1, and IL-8 in the human PMVEC stimulated with TNF-α+IL-1β(10ng/ml in both). Up-regulation of sICAM-1 and sVCAM-1 were not suppressed by these chemicals. MCP-1 and IL-8 were suppressed significantly by pretreatment with dexamethasone. Data are presented as mean ± SD (n=4 in each group). *p<0.05, **p<0.01. sICAM-1, soluble intercellular adhesion molecule-1; sVCAM-1, soluble vascular cell adhesion molecule-1; MCP-1, monocyte chemoattractant protein-1; IL-8, interleukin-8.

It has been reported that influenza A/H1N1pdm viruses replicate efficiently in the lungs of infected mice, ferrets, and non-human primates. These viruses can cause remarkable pathology in the lungs [4]. Consequently, locally released inflammatory cytokines in the lung are increased in influenza A/H1N1pdm pneumonia, and are likely to contribute to lung microvascular hyperpermeability.

These results might provide 288 Saito et al. Acta Med. Okayama Vol. 69, No. 5 endothelial cell ZO-1 tight junction permeability edaravone, dexamethasone VE-cadherin adherence junction permeability TNF-α IL-1β alveolar cell Fig. 8 Schematic diagram showing TNF-α+IL-1β-induced lung hyperpermeability and the inhibitory effects of edaravone and dexamethasone. cellular- and molecular-level insight into treatment strategies for patients with ARDS and highly pathogenic avian influenza pneumonia, which strongly impair lung function. Edaravone and dexamethasone showed the effect to the same extent. In addition, because edaravone has fewer side effects than dexamethasone [11], edaravone alone or in combination with dexamethasone is expected to be effective for therapy for ARDS in a clinical setting.

Edaravone and dexamethasone showed the effect to the same extent. In addition, because edaravone has fewer side effects than dexamethasone [11], edaravone alone or in combination with dexamethasone is expected to be effective for therapy for ARDS in a clinical setting.

Fig. 8 Schematic diagram showing TNF-α+IL-1β-induced lung hyperpermeability and the inhibitory effects of edaravone and dexamethasone.

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Departments of Pediatrics and Virology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan

Authors: Yukie Saitoa, Yousuke Fujii, Masato Yashiroa, Mitsuru Tsugea, Nobuyuki Nosakaa, Nobuko, Yamashitab, Mutsuko Yamadaa, Hirokazu Tsukaharaa, Tsuneo Morishimaa
Experience of using methods for syndromic and pathogenetic therapy in pneumonia caused by COVID-19 at the pulmonary department

The treatment of pneumonia caused by SARS-CoV-2 virus is extremely important today. Coronavirus disease (COVID-19) is a new infection, against which there are no specific drugs or vaccines. In many ways, its complications became unexpected. Leading experts believe that vaccines and/or special drugs for the treatment of SARS-CoV-2 infection will be developed in the nearest future. While this search continues, physicians around the world are experimenting with a variety of therapeutic modalities and existing drugs to treat patients with COVID-19.

During the pandemic period, physicians from many countries have gained extensive experience considering the course of coronavirus-associated pneumonia, its complications and causes of death. Unfortunately, there are currently no well-managed protocols for the treatment of pneumonia caused by the new coronavirus, so often doctors supplement previously established treatment regimens for viral and bacterial pneumonia with drugs that do not yet have sufficient evidential effect for the treatment of pneumonia specifically, but which may be useful due to their known pharmacological mechanisms of influence on certain links of the COVID 19 pathogenesis.

A pandemic of a new coronavirus infection that develops as a result of SARSCoV2 infection and is severely manifested by acute respiratory distress syndrome (ARDS) has caused a global crisis that is the biggest challenge to the global health system for the last 100 years.  Approximately 15-20% of patients, especially the elderly ones and those suffering from serious comorbidities, have a severe form of the disease which is characterized by the 4% risk of death [7].

Penetration of SARS-CoV-2 into the cell and development of the immune response

The main target of the virus is lung epitheliocytes. It has been shown that SARSCoV2 can use the angiotensin-converting type 2 enzyme (ACE2) receptor to penetrate cells; this receptor is the same that facilitates infection of the respiratory epithelium and alveolocytes with type 2 SARSCoV virus. After binding with the SARSCoV2 virus, the latter enters the cells, followed by internalization, replication, and release of new virions from the infected cell. They affect the target organs and induce the development of local and systemic inflammatory response.

The main target of the virus is lung epitheliocytes. It has been shown that SARSCoV2 can use the angiotensin-converting type 2 enzyme (ACE2) receptor to penetrate cells; this receptor is the same that facilitates infection of the respiratory epithelium and alveolocytes with type 2 SARSCoV virus. After binding with the SARSCoV2 virus, the latter enters the cells, followed by internalization, replication, and release of new virions from the infected cell. They affect the target organs and induce the development of local and systemic inflammatory response.

It is not yet clear how the virus can avoid an immune response and stimulate pathogenesis. To overcome antiviral activity, SARSCoV encodes viral antagonists that modulate the induction of interferon (IFN) and cytokines; such mechanism allows to evade the effector function of serum immunoglobulins [8, 9].

In the human respiratory tract, SARSCoV2 causes suppression of mucociliary clearance and epithelial cell death, penetrates through the mucous membrane of the nose, larynx and bronchial tree into the peripheral blood and affects such organs as lungs, digestive tract, heart, kidneys. There are two phases of SARSCoV2 infection: early and late.

The early phase is usually manifested by a mild severity of the disease, and the main role in it is played by non-specific defense mechanisms and a specific adaptive immune response, which allow to eliminate the virus from the body. At this stage, it is recommended to carry out therapeutic measures aimed at strengthening the immune response [10]. Like many viruses, SARSCoV2 encodes proteins that counteract innate immune protection, particularly by inhibiting the activity of IFN type 1 [11].

The response of the innate immune system among infected patients is currently insufficiently studied. A key manifestation of innate immune activation during COVID19 is an increase of neutrophils amount, as well as an increase in the serum concentration of interleukin 6 (IL6) and C-reactive protein (CRP) [12]. Lymphocytopenia is developing during severe forms of COVID19 [13].

Damaged ACE2-expressing cells produce proinflammatory cytokines that recruit effector cells (macrophages, neutrophils) and release alarmines that induce inflammasome activity. Inflammasomes functioning is accompanied by the release of a significant amount of pro-inflammatory cytokines, which, in turn, enhance the recruitment of macrophages and neutrophils, and the development of the so-called cytokine storm, that causes an extraordinary level of inflammation in the lungs.

“Cytokine Storm”

“Cytokine storm” is characterized by a surge in the production of proinflammatory cytokines: IFNα and γ, IL1β, 6, 12, 18, 33, tumor necrosis factor, granulocyte-macrophage colony-stimulating factor (GMCSF), chemokines (CCL2, CCLC, CCL5, CCLC, etc.). They recruit effector immunocytes, leading to the development of a local inflammatory response. “Cytokine storm” causes ARDS development, which is accompanied by oxidative damage of cytomembranes lipids, an increase in the content of reactive oxygen species (ROS) by 10 times, and the release of lysosomal proteases. 

Apoptosis of pulmonary epithelial and endothelial cells develops, cell barrier is getting damaged, vascular permeability increases, resulting in edema and hypoxia. All this leads to multiple organ failure, which can be fatal. It was found that the risk of lethal outcome of the disease is associated with high levels of IL6 in the serum [4, 14].

The consequences of high viral replication and “cytokine storm” are massive lesions of target body tissues [10]:

  • vascular endothelium, which causes systemic inflammation, alteration of coagulation homeostasis, thromboembolism;
  • lungs, associated with the development of pneumonia and ARDS;
  • cardiovascular system, which leads to heart attacks, myocardites and death;
  • intestines accompanied by diarrhea;
  • kidney, associated with the development of acute renal failure;
  • other organs and systems.

The release of significant amounts of cytokines is closely related to the development of known clinical symptoms of COVID19 [15]:

  • IFN-γ causes fever, chills, headache, dizziness and fatigue (endogenous intoxication syndrome);
  • TNF can cause flu-like symptoms (fever, malaise, weakness) and increased vascular permeability, cardiomyopathy, lung damage and acute phase protein synthesis;
  • IL-6 can сause increased vascular permeability, activation of the complement system and coagulation cascade, which is associated with the specific symptoms of severe cytokine release syndrome (CRS), including disseminated intravascular coagulation (DIC); in addition, IL6 is likely to cause cardiomyopathy, followed by myocardial dysfunction, which is common among patients with CRS;
  • activation of endothelial cells may also be a sign of severe CRS, and endothelial dysfunction may lead to the development of capillary leakage, hypotension and coagulopathy.

Endothelial dysfunction

Among patients with a high risk of cardiovascular disease development, decrease of ACE receptor expression in the vessel wall as a result of SARSCoV2 receptor internalisation exacerbates signs of cardiovascular pathology, endothelial dysfunction, and inflammation, especially in the presence of atherosclerosis and diabetes [16].

Endothelial dysfunction, in turn, causes the development of inflammatory processes and thrombosis, accompanied by impaired coagulation, increased fibrinogen levels, decreased fibrinolysis and anticoagulation. The production of nitric oxide (NO) is significantly reduced due to its increased destruction by the free radicals, and due to the reduced availability of the L-arginine as NO-precursor, which leads to a predominance of vasoconstrictors and increased platelet adhesion. Therefore, exogenous intake of L-arginine as a substrate for NO synthesis is pathogenetically justified by the possibility to level out endothelial dysfunction [17].

Lesions of the respiratory system

Lung damage is a major cause of both severe course and mortality due to COVID19 [18].

At the first stage of lung damage development, alveolar macrophages, having recognized SARSCoV2, begin to produce proinflammatory interleukins and chemokines, which recruit effector T-lymphocytes. In the late period of the disease development, extremely high levels of proinflammatory cytokines (IL6, 1β, TNF, etc.) production provide an influx of large numbers of monocytes and neutrophils, which exacerbate inflammation and cause pulmonary edema [10].

IL1β and TNF induce the activity of the hyaluronan synthase 2 enzyme in endothelial CD31+ cells, alveolar epithelial EpCAM+ cells of the lungs and fibroblasts, which leads to excessive production of hyaluronic acid and accumulation of fluid in the alveolar space, which in turn plays a key role in the development of inflammation and edema [19, 20].

Risk factors associated with the development of ARDS and its progression to the lethal outcome include old age, elevated neutrophil counts, organ dysfunction, and coagulopathy (e.g., higher lactate dehydrogenase activity and D-dimer content). The severity of lung damage correlates with significant pulmonary infiltration by neutrophils and macrophages and a higher number of these cells in the peripheral blood.

Neutrophils are the main source of chemokines and cytokines. They are recruited into the lungs by cytokines, which in turn become activated and release toxic mediators, accompanied by the formation of free radicals and ROS. The latter inhibit endogenous antioxidants, which leads to oxidative damage of the lung tissue cells [21].

Patients with pneumonia caused by coronavirus infection who have developed ARDS have more neutrophils than patients without ARDS. This may be due to the activation of neutrophils for the realization of the immune response against the virus, but at the same time it leads to the so-called cytokine storm [8].

The virus is thought to start a second attack, causing deterioration approximately 7-14 days after the onset of the disease. On average, 8 days pass from the appearance of the first symptoms of COVID19 to the development of ARDS [22].

The development of infection associated with the SARSCoV2 virus is accompanied by excessive activation of cellular immunity. Also patients with COVID19 have a high content of proinflammatory CCR6 + Th17 cells.

Excessive activation of Th17 cells and extremely high levels of cytotoxicity of CD8 + T cells may be responsible for the severity of immune damage within lung tissue. Moreover, patients demonstrate a depletion of the T-reg cells pool, which causes unlimited activation of inflammatory mechanisms and delays the resolution of the inflammatory process [18].

Cardiovascular system lesions

Infection with SARSCoV2 virus can inhibit ACP2 expression activity, leading to toxic over-accumulation of angiotensin II, which causes ARDS and myocarditis [23].

Myocardial damage associated with SARSCoV2 virus infection, accompanied by a sharp increase in troponin I concentration (> 28 pg/ml), was observed in 5 of the first 41 patients diagnosed with COVID19 in Wuhan, China [24].

More than 60% of patients with lethal outcome because of COVID19, had hypertension, cardiovascular pathologies or diabetes in the anamnesis [25].

It has been suggested that blockade of the renin-angiotensin system increases ACE 2 expression, leading to internalization of SARSCoV2 into lung and heart cells, which cause ARDS, myocarditis, and death [26].

Other possible mechanisms of myocardial damage include a “cytokine storm” caused by an imbalance of the Th1 and T-reg cells response, and hypoxemia caused by COVID19 [27].

Hypercoagulation and Thrombosis

Most critical patients have severe systemic disease and multiple organ failure in addition to respiratory symptoms (pneumonia and ARDS). This is why many patients with COVID19 do not respond adequately to provided respiratory support.

One of the most significant manifestations associated with poor prognosis is the development of coagulopathy and endothelial dysfunction with diffuse micro- and macrothrombosis. The lungs are the most frequently affected organ due to the highest level of inflammation.

This justifies the idea that patients should not be transferred to artificial lung ventilation (ALV) too early, because without adequate blood flow inside the lungs such approach is useless. The main idea of ​​ventilation is to improve oxygenation, which requires an optimal ratio of ventilation and perfusion, as well as maintaining the mechanism of venous hypoxic vasoconstriction. In fact, 9 out of 10 patients die because of cardiovascular rather than respiratory causes, since venous microthrombosis, and not pneumonia, determines mortality, and thrombosis and endothelial dysfunction are the triggers of respiratory failure among a significant amount of patients [8].

A number of reports indicate a high incidence of both arterial and venous thromboembolic complications among patients with COVID19, suggesting the need for the use of simple and accessible laboratory markers.  The incidence of venous thromboembolism can be as high as 25%, sometimes resulting in lethal outcome. Patients have an increased incidence of heart attacks, strokes and other thrombotic diseases.

Among hematological changes, an increase in the content of D dimer, fibrinogen and other inflammatory markers is noted. In contrast to classical DIC, these patients have a lower degree of increase in activated partial thromboplastin time in comparison to prolongation of prothrombin time (probably due to elevated levels of VIII factor).  DIC may develop in later stages of the disease, causing a prognosis worsening. Anti-inflammatory drugs should be used due to the critical role of thromboinflammation and endothelial dysfunction during the conditions of “cytokine storm” in the early stages of the disease [28].

Treatment approaches of COVID19 patients and its complications

Since generally accepted approved treatment method for a new coronavirus infection is absent, specialists are forced to use drugs that are not registered as medicinal products for the treatment of COVID19 to alleviate the patient symptoms.

Thus, a situation has occurred, in which practical clinical experience becomes more important than the principles of evidence-based medicine [29].

Due to the fact that there is currently no etiotropic therapy with proven efficacy, treatment approaches of COVID19 patients should include pathogenetic, symptomatic and replacement therapy.

The World Health Organization’s guideline from March 13, 2020 states that treatment of mild COVID19 form should include symptomatic therapy and monitoring; the severe form of pathology requires oxygen therapy, monitoring and treatment of coinfection. Critically ill patients require additional treatment measures for ARDS and septic shock and the prevention of complications [1].

About 15% of adults infected by SARSCoV2 suffer from severe pneumonia, which requires additional oxygen insufflation. In 5% of patients, the course of pneumonia progresses to a critical level with the development of hypoxemic respiratory failure, ARDS and multiple organ failure, requiring respiratory support often during a few weeks [30].

Pneumonia caused by the COVID19 is characterized by an increase of effector T-cells, inflammatory cytokines and D-dimer, activation of the coagulation cascade, increased levels of fibrosis and microembolism, which must be taken into account when choosing a therapeutic strategy.

As life-threatening systemic and pulmonary inflammation develops rapidly, early detection of clinical and humoral markers with reactive hyperimmune response becomes important. Therefore, early initiation of treatment can dramatically affect the therapy effectiveness and minimize the possible impact of viral replication [31, 32].

Syndrome-pathogenetic approach

When choosing a treatment regimen for patients with COVID19, it is important to keep in mind the need of mitigation of consequences in order to reduce the frequency of disablement status development and avoid a decline in quality of life for those who have suffered from this serious disease. Among the possible consequences, the following should be noted:

  • primarily somatic disorders: microthromboendotheliitis, which is a systemic vascular damage with the formation of microthrombi, resulting in the respiratory, excretory, cardiovascular, endocrine, gastrointestinal systems alterations; fibrous changes in lung tissue, pneumosclerosis. Lung patency restoration could be obtained by medication supply (broncho- and mucolytics, steroids), intrapulmonary percussion ventilation. The therapy of patients with diabetes mellitus, whose vessels, especially small ones, have already been affected, should be adjusted with sugar-lowering drugs;
  • consequences of treatment with the use of dangerous drugs: gastrointestinal complications associated with COVID19 are mainly presented by hepatic insufficiency, and largely related to drug exposure;
  • exacerbation of chronic diseases, changes in the nature of their course, which requires correction;
  • psychological problems, depression development. The formation of the rehabilitation measures complex should be based on the specific clinical situation. The application of the syndrome-pathogenetic approach which consists of specific manifestations and syndromes treatment, instead of diagnosis treatment, acquires extreme urgency.

For example, when the endothelium of the lungs is getting affected, the plasma is moved into the interstitial space, then hyaline membranes are formed, and this is how fibrosis process is developing. However, even if fibrotic changes persist for a long time, a syndromic approach should be followed: oxygen support and non-invasive lung ventilation should be provided only in case of respiratory failure. Moreover, the use of lung ventilators in the case of COVID19 is associated with high (up to 90%) mortality, ventilator-associated pneumonia and other complications [33].

Methods of COVID19 pharmacotherapy

Among the drugs used for the COVID19 control there are antiviral drugs (both for monotherapy and for various combinations, including interferons). For example, nelfinavir developed for the combination treatment of HIV patients, is included in the list of potentially effective drugs for the COVID19 treatment [34].

There are also data on the effectiveness of remdesivir, which acts directly on the causative agent of coronavirus infection: its use helped to improve the condition of 68% of patients, reducing the length of hospital stay from 15 to 11 days [35].

Favipiravir, which was previously used for influenza treatment, and is now successfully implemented as an experimental COVID19 treatment, acts on one of the viral RNA replication units, halving the incidence of fatalities and reducing the incidence of serious complications in 4 times [36, 37].

Data considering the use of chloroquine-based drugs (sometimes in combination with the antibiotic azithromycin) among patients with COVID19 is contradictory: these agents provide antiviral effects in high concentrations, leading to toxical influence; in addition, cardiac arrhythmia is a serious side effect of their use [38].

The use of recombinant human monoclonal antibodies to IL6 receptors, such as levilimab and tocilizumab, as well as other immunosuppressive agents that prevent an overactive immune response that could cause worsening of the clinical condition is of particular interest. Thus, tocilizumab blocks IL6 receptors, which contributes to a positive therapeutic effect during many inflammatory diseases, including COVID19 [39].

A retrospective study of the tocilizumab efficacy during patients treatment with severe or critical COVID19 condition has shown that this drug improved oxygenation rate among 75% of the patients and normalized peripheral lymphocyte counts among 52,6% of patients [40].

Patients with weakened immune system may be more prone to complications of COVID19 due to the lack of a rapid immunological response that helps to overcome the virus. However, it has now been suggested that immunosuppression may also play a protective role during COVID19 infection, preventing an overactive immune response, which sometimes worsens or weakens the clinical condition of the patient [41].

In particular, a case of B-cell depletion was described, during which COVID19 developed without serious complications in patient suffering from multiple sclerosis, bronchial asthma and peptic ulcer disease.  The authors of the publication suggested that the persistence of B-cells in secondary lymphoid organs, associated with a moderately reduced immune response due to the absence of peripheral cells, may have played a favorable role for the clinical outcome. The fact that the patient did not show significant increase of IL6 amount (which may be released by peripheral B-cells) probably supports this hypothesis. If such hypothesis will be confirmed in more cases, it can be assumed that patients who experience B-cell depletion are protected from serious complications of COVID19. This would favor the use of selective immunosuppressants during severe COVID19 cases [42].

Since the activity of the hyaluronan synthase 2 enzyme in the lung tissues leads to an excess of hyaluronic acid production, it is thought that inhibition of its production will increase the area of gas exchange surface in the alveoli and support the recovery of COVID19 patients.

In particular, it is proposed to use for such purpose hymecromone as an approved drug for the treatment of gallbladder dysfunction, which is an inhibitor of hyaluronan synthase 2 [13].

According to recent clinical reports, the therapeutic range during the COVID19 is significantly longer than 14 days, but prolonged exposure to the virus can lead to the development of sudden intense immunological reactions, “cytokine storm” and immune cells infiltration. Some immunocytes, especially macrophages and neutrophils, are able to produce numerous ROS [43, 44].

A certain level of ROS is important for the regulation of immune responses and for clearance from viruses, but their excessive content leads to oxidation of cellular proteins and membrane lipids, as well as to rapid destruction of not only virus-infected but also normal lung cells and even heart cells, as a result of which multiorgan failure develops.

Therefore, antioxidant therapy can be proposed as a potential therapeutic approach for COVID19-induced lung and cardiovascular tissue damage reduction.  Potential drugs include antioxidants such as vitamin C (ascorbic acid) and vitamin E, since their reducing hydrogen atoms can react with ROS and neutralize them without the formation of toxic substances. Also molecules of plant origin (used in ancient Chinese medicine), such as curcumin and baikalin should be mentioned [45].

It is suggested that an adequate dose of antioxidants may help to reduce heart and lung damage in patients with severe COVID19 form, but scientists do not rule out that this approach may be also useful for patients with mild symptoms of disease [46].

Possibilities of edaravon, L-arginine, L-carnitine and Reosorbilact use in complex treatment of patients with COVID-19

The potential benefits of antioxidant therapy depend on the right choice of drugs. The key requirements for them are the ability to affect mitochondrial permeability, block the signaling pathways of inflammation (IL1, 6, 18) and act in synergy with water-soluble antioxidants (in the cytoplasm).

Edaravone

Edaravon is a low-molecular-weight antioxidant drug that demonstrates targeted interaction with peroxyl radicals.  Due to its amphiphilicity, it absorbs both fat and water-soluble peroxyl radicals by transferring an electron to the radical; inhibits the oxidation of lipids by absorbing water-soluble peroxyl radicals that initiate chain chemical reactions, as well as fat-soluble peroxyl radicals that support this chain [47].

In 2001, this drug was approved in Japan as a drug for the treatment of acute ischemic stroke, and in 2015 it was approved for the treatment of amyotrophic lateral sclerosis. Recommendations of the Japanese societies considering the use of respiratory support and intensive care include edaravon as a drug that should be used for the treatment of patients with ARDS within intensive care units [52]. Edaravon is able to quickly neutralize a wide range of free radicals. It activates eNOS and can improve blood circulation, blocks inflammatory iNOS; effectively inhibits lipid peroxidation (LPO), and protects cells from destruction by suppressing the chain reaction of LPO by absorbing peroxyl radicals.

Edaravon activates the enzymes of antioxidant protection, such as SOD (superoxide dismutase), CAT (catalase) and GSHRx (glutathione peroxidase).

The drug easily penetrates the blood-brain barrier, unlike other free radical acceptors, which explains its pronounced therapeutic effect; protects the endothelium of the brain from damage due to the protective effect on microvessels, which determines its possible therapeutic effect during disorders and diseases associated with carbonyl stress [48]. Edaravon reduces damage of the blood-brain barrier and inhibits the development of cerebral edema, directly and indirectly reduces the production of such pro-inflammatory cytokines, as IL6, iNOS, TNF and metalloproteinases.

Edaravon has been used for almost 20 years for treatment of the stroke patients (for the first 24 hours), but due to its free radical scavenger properties, it has a protective effect on the heart, lungs, intestines, liver, pancreas, kidneys and other organs.  Edaravon has recently been shown to be useful in patients with acute myocardial infarction. The drug also helped to increase the fraction of left ventricular ejection and reduce the frequency of re-hospitalizations among patients with acute myocardial infarction. Animal studies have shown that edaravon reduced the number of IL1β-positive myocardial cells among rats with experimental autoimmune myocarditis; protected cardiac function and reduced the size of the heart attack by decreasing the production of TNF in the myocardium [49].

Experimental studies have shown that edaravon is able to prevent the development of increased permeability of endothelial cells in the pulmonary microcirculatory tract caused by proinflammatory cytokines, probably due to increased adhesive contacts, as well as due to other mechanisms of action. Thus, the drug may be useful for the treatment of patients with ARDS and pneumonia in clinical practice. The safety of edaravon has been proven in a number of studies [50, 51].

Thus, the use of edaravon, which is capable of effectively “extinguishing a cytokine fire,” can significantly improve the condition of patients with COVID19, the pathogenesis of which is associated with the development of ARDS and a sharp increase of cytokine levels.

L-arginine

L-arginine is a conditionally essential amino acid that is an active cellular regulator of many vital functions of the body, revealing protective effects that are important in a critical condition. Arginine has antihypoxic, membrane stabilizing, cytoprotective, antioxidant, antiradical and detoxifying effects.

This amino acid is a substrate for the synthesis of NO, and because of that it improves microcirculation, promotes strong vasodilation, prevents activation and adhesion of lymphocytes and platelets.

The physiological action of NO varies from the modulation of the vascular system to the regulation of immune processes (cell-mediated immunity, the effect of neutrophils on pathogenic microorganisms, nonspecific immune protection) and the control of neuronal functions.

L-arginine restores the content of NO in the lung tissue, which reduces the spasm of the unstriated muscles of the bronchi and improves the vasomotor function of the endothelium among the pulmonary arteries [53]. L-arginine is effective for blood pressure decrease among patients with hypertension [54]. With COVID19 vaccine under development and no universal effective treatment available, the next question arises: how can the avoidance of the pathology be ensured? It is especially relevant for the elderly and medically compromised patients.

Based on experts opinion, the use of drugs that contribute to the overall strengthening of the body and improve the protection function of the immune system is promising. Such substances, of course, include L-arginine [55].

The usefulness of this approach is confirmed by a clinical trial initiated by the Medical Center of Ohio State University, USA, dedicated to the use of inhaled NO for the treatment of patients diagnosed with COVID19. The goal is to avoid intensive care [56].

Therefore, increasing the NO content is a promising perspective of ​​treatment for patients with coronavirus infection, and L-arginine can be successfully used to achieve this goal.

L-carnitine

L-carnitine is a natural substance involved in energy metabolism as well as the metabolism of ketone bodies. It is necessary for the transportation of long-chain fatty acids into the mitochondria (which are used as an energy substrate by all tissues except the brain) for their further oxidation and energy production.

This substance plays an important role in cardiac metabolism, as the oxidation of fatty acids depends on the availability of its sufficient amounts. The positive effect of L-carnitine has been proved during acute and chronic ischemia, decompensation of cardiac activity, heart failure caused by myocarditis and drug cardiotoxicity.

The immunomodulatory effect of L-carnitine is based on the inhibition of proinflammatory cytokines TNF, IL6 and IL1 under the conditions of “cytokine storm”. The drug is a direct antioxidant that prevents cell apoptosis and has a cardioprotective effect.

The results of two major meta-analyzes provided by DiNicolantonio (2013) and Askarpour et al. (2019) confirmed the effectiveness of L-carnitine therapy in patients with cardiovascular disease by reducing mortality, improving quality of life, lowering cholesterol level, normalizing heart rate and reducing the need for nitrates intake [57, 58].

L-carnitine may be beneficial for patients with COVID19 due to its immunomodulatory effects and inhibition of inflammatory mediators, including CRP, TNF and IL6, which may help to reduce the “cytokine storm” [59].

Reosorbilact

Reosorbilact is a unique hyperosmolar crystalloid solution that does not contain excess of chlorine, and therefore helps to avoid the main disadvantage of such solutions, such as an increased risk of hyperchloremia.

An important feature of Reosorbilact is the combination of both hyperosmolar properties and the properties of balanced crystalloids (a set of necessary Ca2 +, K +, Na +, Mg2 + ions in isoplasmic concentration). Due to hyperosmolarity, Reosorbilact induces fluid transfer from the intercellular sector to the vascular bed, which improves microcirculation and tissue perfusion due to the potent osmodiuretic effect of sorbitol, which is associated with the lack of natural human mechanisms of reabsorption for polyhydric alcohols in renal proximal tubules.  Reosorbilact has a pronounced diuretic and anti-edematous effect [60].

The main pharmacologically active ingredients of the drug are sorbitol and sodium lactate. Sorbitol improves microcirculation and tissue perfusion, and has a disaggregating effect.

Sodium lactate helps to correct metabolic acidosis; sodium chloride has a rehydrating effect, and replenishes the deficiency of sodium and chlorine ions in various pathological conditions; calcium chloride restores the content of calcium ions necessary for the transmission of nerve impulses, contraction of skeletal and non-striated muscles, myocardial activity, bone formation, blood clotting, and prevents the development of inflammatory reactions, increases the body’s resistance to infections; potassium chloride restores water-electrolyte balance, has a negative chronotropic and bathmotropic effect, and moderate diuretic effect. Several studies have shown that the use of hypertonic saline in patients with hemorrhagic shock due to traumatic injury reduces the length of stay on mechanical ventilation

It has also been found that the use of hyperosmolar solutions during the treatment of patients with severe sepsis can reduce the volume of infusion. Such positive effects as reduction of pulmonary complications, duration of mechanical ventilation and volume of infusion are important and promising during the use of hyperosmolar solutions for patients with pneumonia, particularly caused by COVID19, when it is important to adhere to a restrictive infusion [61, 62].

Study objective

In view of the foregoing, we conducted a study on the effectiveness of the syndrome-pathogenetic treatment approach. The objective was to evaluate the effect of combination including edaravon (Xavron), L-arginine hydrochloride + L-carnitine (Tivorel) and Reosorbilact (sorbitol, sodium lactate, sodium chloride, calcium chloride, potassium chloride, magnesium chloride) on clinical course of moderate severity pneumonia, caused by COVID19, as adjunctive therapy to basical treatment under conditions of a pulmonary department.

Design of the study

The study included two groups of 30 patients each with diagnosed moderate-grade pneumonia caused by COVID19. The main group received a combination of edaravon (Xavron) 30 mg 2 times per day intravenously, Larginine hydrochloride + L-carnitine (Tivorel) 1 vial / day intravenously and Reosorbilact 200 ml per day in addition to basic therapy. The control group received basic therapy.

Control points

The studied indicators were: the number of days spent in the hospital, saturation (oxygen saturation of the blood), body temperature, the levels of ferritin, D-dimer, CRP, procalcitonin; general clinical blood test to determine the number of erythrocytes, leukocytes, platelets, ESR, leukocyte formula and hemoglobin content. Patients also underwent computed tomography of the chest area.

Biochemical analysis of blood does not provide specific information, but the detected abnormalities may indicate the presence of organ dysfunction, decompensation of comorbidities and the development of complications; so such changes have some prognostic value, indicating the effectiveness of the drugs choice.

Ferritin content.  Hyperferritinemia is called a marker of severe coronavirus infection associated with a high risk of “cytokine storm” development. It is noted that researchers around the world are looking for a way to quickly reduce the level of ferritin in the blood of such patients.

The content of D-dimer, which is 3-4 times higher compared to the age norm, has clinical significance.

The content of CRP is the main laboratory marker for the activity of the process going on in the lungs; its increase correlates with the extent of lung tissue damage and is the basis for starting anti-inflammatory therapy.

Lymphocytopenia and thrombocytopenia. Leukocyte counts are normal in most patients with COVID-19; one third have leukopenia, and 83.2% have lymphopenia; thrombocytopenia is moderate, but is more common in patients with severe form of disease.

Laboratory signs of “cytokine storm” and ARDS may be following: a sudden increase in clinical manifestations after 12 weeks from the onset of the disease; febrile fever that increases or reappears; severe lymphopenia in complete blood count; reduction of the T- and B-lymphocytes number; a significant increase of the D-dimer level (> 1500) or its rapid increase; increase of CRP content> 75 mg/l.

The development of cardiovascular complications in the case of COVID19 is also accompanied by lymphocytopenia, thrombocytopenia and increased CRP level.

Results and Discussion Figure 1 demonstrates the mean values ​​of CRP (mg/l) before and after treatment among patients of the main and control groups. If before treatment the difference between the values was statistically insignificant (39.45 ± 9.7 vs. 46.26 ± 5.53 mg/ml, respectively), then after treatment patients of the main group demonstrated significant improvement compared to the patients in the control group (7.59 ± 1.71 vs. 12.5 ± 1.67 mg/ml, respectively; p = 0.04).

Figure 2 demonstrates the mean values ​​of D-dimer (ng/ml) before and after treatment among patients of the main and control groups. The difference between the D-dimer levels was statistically insignificant before treatment (1304.54 ± 230.32 vs. 1541.06 ± 477.79 ng/ml, respectively), but after treatment patients of the main group demonstrated significant improvement compared to the patients of the control group (1445.38 ± 106.03 vs. 1903.27 ± 129.68 ng/ml; p = 0.01).

Figure 3 demonstrates the mean values ​​of ferritin content (ng/ml) before and after treatment among patients of the main and control groups. The difference between the ferritin values was statistically insignificant before the treatment, (500.6 ± 89.14 vs. 598 ± 94.03 ng / ml, respectively), but after the treatment patients of the main group demonstrated significant improvements compared to the patients from the control group (393.72 ± 51, 73 vs. 540.11 ± 49.93 ng/ml, respectively; p = 0.05).

Figure 4 makes it possible to compare the average values ​​of treatment duration, body temperature and saturation among patients of the main and control groups. All indicators at the end of treatment were significantly better in patients of the main group (combination of basic therapy with additional treatment with Xavron, Tivorel and Reosorbilakt) compared to the patients in the control group: saturation levels were 93.31 ± 0.57 vs. 91.24 ± 0.77 (%oxygen saturation; p = 0.03), body temperature values were 37.86 ± 0.1 vs. 38.18 ± 0.12 ( oC, p = 0.04), the number of bed/days in the hospital was 13.1 ± 0.49 vs. 14.56 ± 0.42 (p = 0.03), respectively.

Conclusions

• The results of the study have shown that a number of indicators in the main group (patients whose basic treatment of pneumonia caused by COVID19 was combined with adjunctive therapy by Xavron, Tivorel and Reosorbilact) has significantly improved compared with the control group (patients who received only basic treatment). Adjunctive therapy helped to improve blood oxygen saturation, lower body temperature, and reduce hospital duration stays.

• At the end of treatment the main group demonstrated significantly decrease of CRP concentration, which is a marker of inflammatory activity in the lungs, and increase of which correlates with the extent of lung tissue damage and the severity of the disease.

• A decrease of the D-dimer content, which also belongs to the markers of the inflammatory process, among patients of the main group indicates an improvement considering their condition compared to the control group.

• Increase of acute phase ferritin protein concentration was noted in case of an unfavorable course of a disease and during so-called cytokine storm, so decrease of this specific indicator in the main group demonstrated an evidence of adjunctive therapy effectiveness using Xavron, Tivorel and Reosorbilact.

• Special attention is now being paid to the application of a syndrome-pathogenetic approach of coronavirus infection treatment, since etiotropic drugs that could affect directly COVID19 pathogen have not yet been developed.  Considering such conditions, it is extremely important to use a comprehensive approach during the treatment of patients with severe forms of disease, which takes into account individual characteristics and provide the most effective support for the body. Every effort should also be made to prevent disablement status development and reduction of patients quality of life after illness due to possible complications (damage of the respiratory system due to fibrosis, severe consequences for the cardiovascular system due to thrombosis, etc.). This will be facilitated by the additional use of Xavron, Tivorel and Reosorbilact.

Perspectives of further research

It was established that, in addition to pneumonia, COVID19 is accompanied by the development of respiratory failure, changes in rheological and fibrinolytic properties of blood, increased thrombosis, damage to the cardiovascular and nervous systems, and increased fibrosis in the lungs. The tendency of fibrosis manifestations decrease was noted in the experimental group during the research. Therefore, it would be useful to investigate the effect of our proposed combination of drugs on the fibrosis processes within the lungs after viral pneumonia, given the wide range of already known therapeutic properties of these drugs.

Author: S.V. Kovalenko, Doctor of Medical Sciences, Department of Internal Medicine, Clinical Pharmacology and Professional Diseases Bukovinian State Medical University; Pulmonary Department of Chernivtsi Regional Clinical Hospital.

Authors: S.V. Kovalenko
Posted: Medical newspaper "Health of Ukraine", № 13-14 (482-483), July 2020
Japanese Guidelines for the Management of Stroke 2015 [2017].

Edaravone, a drug with an expected neuroprotective effect, recommended as a medicine for patients with cerebral ischemic stroke (thromboembolism) (grade B).

Authors: The Japan Stroke Society 2017
Posted: Міжнародний неврологічний журнал, №1 (103), 2019
Догляд та лікування в гострому періоді інсульту: необхідне, достатнє та межі дозволеного

Актуальність роботи: публікація містить огляд основних положень для організації догляду за пацієнтами в гострому періоді інсульту, що ґрунтуються на поточних рекомендаціях і найкращому клінічному досвіді.

Мета публікації:  допомогти новим інсультним центрам в організації процесу ведення хворих, доповнивши документальний супровід (локальний протокол та маршрут пацієнта) ключовими положеннями з догляду для мінімізації ризиків ускладнень і збільшення шансів позитивного наслідку захворювання.

У ситуації обмеження фінансових спроможностей закладів, і, беручи до уваги концепцію мінімальних гарантій надання допомоги, а також право на вибір у пацієнта (чи його родичів – як на місце лікування, так і на його склад), варто роздивитись можливість партнерської участі сторони пацієнта стосовно обсягу лікувальних впливів. У світі розвивається і поширюється стратегія спільного прийняття рішення (sharing decision making), коли спеціальна модель спілкування, з висвітленням імовірних переваг і ризиків застосування того чи іншого засобу, приводить до прийняття консенсусного рішення і вважається за легітимну можливість відхилюватись в окремих випадках від наявних рекомендацій, протоколів, настанов.

Ключові слова: локальний протокол, маршрут пацієнта, гострий ішемічний інсульт, едаравон, мінімальні гарантії надання послуг, інсультний центр.

Authors: С.П. Московко д. мед. н. професор. Вінницький національний медичний університет імені М.І. Пирогова.
Кохранівська база даних систематичних оглядів. Едаравон для лікування гострого ішемічного інсульту.

Більшість випадків інсульту відбуваються, коли згусток крові блокує кровоносну судину, що веде до головного мозку. Без належного кровопостачання головний мозок швидко зазнає пошкодження, яке може бути постійним. Пошкодження, викликане інсультом, може спричинити слабкість рук або ніг, або проблеми з мовою чи зором. Дані експериментальних та клінічних досліджень показали, едаравон може бути корисним для людей з гострим ішемічним інсультом.

Ціль дослідження: за допомогою Кохранівського огляду оцінити ефективність та безпеку едаравону при гострому ішемічному інсульті.

Було ідентифіковано 1 788 посилань за допомогою електронного та ручного пошуку. З них ідентифікували 24 потенційно прийнятних випробування, з яких три були включені у дослідження.

У огляді надані висновки з ефективності та з безпеки, згідно яких едаравон може зменшити ступінь неврологічного порушення при лікуванні гострого ішемічного інсульту.

Authors: Feng S, Yang Q, Liu M, Li W, Yuan W, Zhang S, Wu B, Li J.
Posted: Кохранівська база даних систематичних оглядів 2011, випуск 12.
Алгоритми дій, мінімальні умови та складові надання якісної медичної допомоги при гострому інсульті

Актуальність роботи: лікування гострого інсульту потребує застосування процедури документального менеджменту, лікувальні заклади мають різні ресурси та умови надання медичної допомоги, у параметри реформи закладені «індикатори якості надання послуг» за принципом їхнього регулярного загального оприлюднення, згідно рекомендацій 10-го круглого столу академічної науки STAIR залишається нагальною потреба у цитопротекції та захисті нейроваскулярного юніту.

Ключова мета публікації – запропонувати основи створення внутрішнього керівного документа, що регламентує весь процес надання медичної допомоги при гострому інсульті в умовах реформи НСЗУ. Необхідні складові документу викладені з доступом до редагування, уточнення чи розширення відповідно до потреб та можливостей конкретного лікувального закладу.

Authors: С.П. Московко, д. мед. н., професор, Вінницький національний медичний університет імені М.І. Пирогова.
Протокол відкритого багатоцентрового дослідження «випадок – контроль» …

Протокол відкритого багатоцентрового дослідження «випадок – контроль» щодо безпеки та ефективності використання едаравону (Ксаврон®) у гострому періоді ішемічного інсульту в умовах реальної клінічної практики

На вітчизняному фармакологічному ринку з’явився препарат класу скавенджерів (прибиральників) активних радикалів та перекисних сполук. Це препарат Ксаврон виробництва «Юрія-Фарм», діючою речовиною якого є едаравон.

Едаравон був схвалений FDA для лікування БАС (боковий аміотрофічний склероз), його ефективність та безпеку у лікуванні гострого ішемічного інсульту доведено у низці рандомізованих плацебо-контрольованих досліджень. З 2009 р. Едаравон внесений в Японський гайдлайн по лікуванню гострого ішемічного інсульту.

В Україні розпочато вивчення  безпеки та ефективності Ксаврон в реальній клінічній практиці на основі відкритого багатоцентрового дослідження за принципом «випадок – ​контроль».

Наданий протокол дослідження: дизайн дослідження, популяція пацієнтів, первинні і вторинні кінцеві точки, організація дослідження.

Authors: С.П. Московко, О.В. Кириченко, Г.С. Руденко, Вінницький національний медичний університет імені М.І. Пирогова.
Posted: Медична газета «Здоров’я України». Спецвипуск «Інсульт». Додаток № 1 (52), 2020 р.
Japanese experience in the treatment of patients with acute ischemic stroke. Interview with Professor Yukito Shinohara

The Academy of Stroke, Annual Scientific and Educational Forum, traditionally dedicated to World Stroke Day was held in Kyiv October 31 – November 1, 2019.

Yukito Shinohara, Professor Emeritus of Tokai University School of Medicine, former president of the Japan Stroke Society and the Asia Pacific Stroke Organization, Chairman of the Japanese Stroke Guidelines in 2004 and 2009, has held a lecture in Kyiv during the Academy of Stroke 2019. Professor Yukito Shinohara has agreed to give interview to the readers of the International Neurological Journal.

Authors: Nataliia Kuprinenko
Posted: International Neurological Journal №8 (110), 2019
The use of the drug Xavron (Edaravon) in the complex treatment of patients with ischemic stroke in combination with thrombolytic therapy

Resume. The article describes the experience of the early use of Xavron (Edaravon) in patients with ischemic stroke in combination with thrombolytic therapy (TLT). These studies indicate greater efficacy of using Edaravon in combination with TLT in the treatment of ischemic stroke. When using the drug Xavron side effects of the drug was not observed.

Authors: O.B. Leskiv, T.F. Khabaznia
Posted: THE JOURNAL OF NEUROSCIENCE of B.M. Mankovskyi’ 2019, ТОМ 7, № 3-4
Usage of the new edaravone free radical scavenger (XAVRON) in the treatment of acute ischemic stroke

The purpose of the study is to evaluate the effectiveness of use of the drug edaravone for treatment of patients with acute cerebral infarction comparing to citicoline.The results of the treatment of ischemic stroke by intravenous administration of edaravone (Xavron) and citicoline indicated a more pronounced elimination of neurological deficit in the group edaravone.

Authors: I.S. Cuckoo; A.O. Volosovets, A.I. Cuckoo and others
Posted: МЛ №1-2 (157-158) 2019
Perspective Treatment Goals for Brain Protection in Case of Acute Ischemia

Abstract. Ischemic stroke initiates a cascade of biochemical reactions, among which the processes of free radicaloxidation occupy a key place. The most important place in management this disease takes reperfusion therapy, theconduct of which is closely related to the problem of neuroprotection. Use of antioxidants is a promising direction for the treatment of ischemic stroke. They have antioxidant effects and could prevent free radical processes. Edaravone is a new low molecular weight free radical scavenger, which inhibits the ischemic cascade. The article provides an overview of studies on the effectiveness and safety of edaravone in patients with ischemic stroke.

Authors: L. A. Dzyak, O. S. Tsurkalenko, V. M. Suk
Posted: Infusion & Chemotherapy № 2, 2019
Brain protection in acute ischemia

From 15 to 16 million people suffer a stroke every year, of which 100-110 thousand are residents of Ukraine. This vascular desiase ranks first among causes of disability and is one of the leading causes of death. One third of patients die during the first year after stroke, one out of four becomes disabled with a violation of basic functions, the daily activity of every second patient is limited, and only one out of five returns to their previous lives. The causes of the catastrophic consequences of a stroke, as well as the possibility of minimizing them, were discussed by the participants of the 11th scientific and practical conference “Neurosymposium”, which was held on September 12-12, 2019 in Odessa.

Authors: Dziak L.A., Chemer N.M., Khubetova I.V
Posted: Medical newspaper “Health of Ukraine. Neurology, Psychiatry, Psychotherapy”, No 3 (50), October 2019
Japan’s striking success in fighting stroke: review by Prof. Yukito Shinohara in Ukraine, 2019

Neurologist from Japan visits Ukraine for the first time. Dr. Yukito Shinohara is Immediate Past President of the Japan Stroke Society and the Asia Pacific Stroke Organization. He was Chairman of the Japanese Stroke Guidelines in 2004 and 2009. Professor Shinohara gave a presentation on theme “Present Stroke Situation in Japan and Japanese Stroke Guidelines” at the Scientific and Educational Forum “Academy of Stroke 2019”

First experience of using Xavron, a free radical scavenger, in patients with acute ischemic stroke

Abstract. Background. In acute ischemic stroke (AIS), more than 1,000 substances with known neuroprotective effects have been studied, but their effectiveness is considered insufficiently convincing. In 2018, Ukraine launched the release of a new free radical scavenger Xavron (active ingredient — edaravone), which since 2001 has been successfully prescribed in Japan for the treatment of AIS and is a part of the Japanese national guidelines for the treatment of AIS. The purpose of this work is to study the effectiveness of a new neuroprotective drug Xavron (edaravone) in the comprehensive therapy of patients with AIS. Materials and methods. A prospective, integrated clinical, neurological and laboratory examination was conducted in 28 patients (13 women and 15 men) with AIS. The patients were divided into two groups that did not differ in terms of main characteristics and treatment. However, patients in the first group (n = 18) received Xavron (30 mg edaravone) twice daily intravenously. In the control group (n = 10), the drugs with neuroprotective effect were not used. Results. The analysis of Glasgow Coma Scale scores showed a positive dynamics in the majority of patients in both groups without significant statistical difference (p > 0.05). However, the analysis of FOUR (Full Outline of UnResponsiveness) scores showed that in the group where Xavron was used for neuroprotection, since day 5 the level of consciousness was restored more quickly than in the control group. The difference between group 1 and controls became significant within 9–10 days of treatment (p< 0.05). On day 3 in the control group, the level of neuron-specific enolase (NSE) increased by 10 times (from 9.2 to 96.4 ng/ml). Subsequently, there was a rapid decrease in the NSE level, which in the main group of patients was normalized until day 10 of treatment, and in the control group, the NSE level did not reach the reference values within 10 days of therapy (p < 0.05). Conclusions. The use of Xavron (edaravone) in patients with AIS was significantly effective in terms of neurological status (level of consciousness on the FOUR scale) and the dynamics of neurological markers (NSE). Further research is needed to clarify the role of Xavron (edaravone) in the intensive care of AIS patients.

Authors: Emergency medicine № 3 (98), 2019
Posted: Emergency medicine № 3 (98), 2019
Effect of a Novel Free Radical Scavenger, Edaravone (MCI-186), on Acute Brain Infarction

Edaravone, a novel free radical scavenger, demonstrates neuroprotective effects by inhibiting vascular endothelial cell injury and ameliorating neuronal damage in ischemic brain models. The present study was undertaken to verify its therapeutic efficacy following acute ischemic stroke.

We performed a multicenter, randomized, placebo-controlled, double-blind study on acute ischemic stroke patients commencing within 72 h of onset. Edaravone was infused at a dose of 30 mg, twice a day, for 14 days. At discharge within 3 months or at 3 months after onset, the functional outcome was evaluated using the modified Rankin Scale. Two hundred and fifty-two patients were initially enrolled. Of these, 125 were allocated to the edaravone group and 125 to the placebo group for analysis. Two patients were excluded because of subarachnoid hemorrhage and disseminated intravascular coagulation.

A significant improvement in functional outcome was observed in the edaravone group as evaluated by the modified Rankin Scale (p = 0.0382).

Edaravone represents a neuroprotective agent which is potentially useful for treating acute ischemic stroke, since it can exert significant effects on functional outcome as compared with placebo.

Authors: Eiichi Otomo
Posted: Cerebrovasc Dis 2003
Acute ischemic stroke: revealing the maximum possibilities of pharmacotherapy

Materials of the XXI International Scientific and Practical Conference “Interdisciplinary Issues in Modern Neurology”, April 22-24, Truskavets, Ukraine.

Much attention was paid during the event to the problematic aspects of managing patients with acute ischemic stroke. Within the framework of intensive therapy in cardioneurology a separate scientific symposium discussed the possibilities of modern pharmacotherapy for acute stroke, which are available to doctors today and allow to improve the results of treatment and reduce the disability of patients with stroke.

Authors: Chemer N.M., Darii V.I., Oros M.M.
Posted: Medical newspaper "Health of Ukraine", № 12 (457), 2019
Acute Kidney Injury and Edaravone in Acute Ischemic Stroke: The Fukuoka Stroke Registry

Background: A free radical scavenger, edaravone, which has been used for the treatment of ischemic stroke, was reported to cause acute kidney injury (AKI) as a fatal adverse event. The aim of the present study was to clarify whether edaravone is associated with AKI in patients with acute ischemic stroke.Methods: From the Fukuoka Stroke Registry database, 5689 consecutive patients with acute ischemic stroke who were hospitalized within 24 hours of the onset of symptoms were included in this study. A logistic regression analysis for the Fukuoka Stroke Registry cohort was done to identify the predictors for AKI. A propensity score–matched nested case–control study was also performed to elucidate any association between AKI and edaravone.Results: Acute kidney injury occurred in 128 of 5689 patients (2.2%) with acute ischemic stroke. A multivariate analysis revealed that the stroke subtype, the basal serum creatinine level, and the presence of infectious complications on admission were each predictors of developing AKI. In contrast, a free radical scavenger, edaravone, reduced the risk of developing AKI (multivariate-adjusted odds ratio [OR] .45, 95% confidence interval [CI] .30-.67). Propensity score–matched case–control study confirmed that edaravone use was negatively associated with AKI (propensity score–adjusted OR .46, 95% CI .29-.74).Conclusions: Although AKI has a significant impact on the clinical outcome of hospital inpatients, edaravone has a protective effect against the development of AKI in patients with acute ischemic stroke.

Authors: Masahiro Kamouchi et al.
Posted: International Neurological Journal, №3 (105), 2019
The search for new methods and drugs to struggle with cerebral stroke and its complications

The scientific-practical conference “Opportunities and Achievements of Modern Pharmacotherapy in the Practice of a Neurologist” held in Kharkov on March 14-15, 2019. It was organized by Association of Neurologists, Psychiatrists and Narcologists of Ukraine, Institute of Neurology, Psychiatry and Narcology of the National Academy of Medical Sciences of Ukraine, Kharkiv National University named after V.N. Karazin.

The event was devoted to innovative methods of diagnosis, treatment and prevention of pathologies of central nervous system. Stroke remains a major central nervous system disease due to high prevalence, disability and mortality. You will find an overview of some of the reports voiced at the conference.

Authors: V.M. Mishchenko, V.I. Darii, I.V. Khubetova, T.S. Mishchenko
Posted: Health of Ukraine. Thematic issue "Neurology, Psychiatry, Psychotherapy" No. 2, 2019.
How is edaravone effective against acute ischemic stroke and amyotrophic lateral  sclerosis?

Abstract. Edaravone isa low-molecular-weight antioxidant drug targeting peroxyl radicals among many types of reactive oxygen species. Because of its amphiphilicity, it scavenges both lipid- and water-soluble peroxyl radicals by donating an electron to the radical. Thus, it inhibits the oxidation of lipids by scavenging chain-initiating water-soluble peroxyl radicals and chain-carrying lipid peroxyl radicals. In 2001, it was approved in Japan as a drug to treat acute-phase cerebral infarction, and then in 2015 it was approved for amyotrophic lateral sclerosis (ALS). In 2017, the U.S. Food and Drug Administration also approved edaravone for treatment of patients with ALS. Its mechanism of action was inferred to be scavenging of peroxynitrite. In this review, we focus on the radical-scavenging characteristics of edaravone in comparison with some other antioxidants that have been studied in clinical trials, and we summarize its pharmacological action and clinical efficacy in patients with acute cerebral infarction and ALS.

Authors: Kazutoshi Watanabe, Masahiko Tanaka, Satoshi Yuki, Manabu Hirai, Yorihiro Yamamoto
Posted: J. Clin. Biochem. Nutr. January 2018. Vol. 62, № 1. Р. 20-38
Ukrainian legislation on the prevention and treatment of rare (orphan) diseases

From 01 January 2015, amendments to the Fundamentals of Legislation of Ukraine on Health Care implemented by the Law of Ukraine No. 1213-VII dated 15 April 2014, have become effective. According to the implemented amendments to the legislation, patients with rare (orphan) diseases shall be provided, on a continuous and free basis, with medicinal products for the treatment of these diseases and appropriate food products for special dietary consumption in accordance with their list and volumes approved by the central executive body responsible for the formation of the national health care policy, in accordance with the procedure established by the Cabinet of Ministers of Ukraine.

At the same time, on 15 April 2015, the CMU Resolution No. 160 of 31 March 2015 “On approval of the procedure for providing citizens suffering from rare (orphan) diseases with medicines and appropriate food products for special dietary consumption” became effective. According to the specified Procedure, provision of the citizens suffering from rare (orphan) diseases with medicines and food products is performed by respective healthcare institutions according to the place of residence or treatment of such citizens.

References to laws:

Authors: Верховна Рада України; Кабінет міністрів України.
Posted: Відомості Верховної Ради (ВВР), 2014, № 26; Постанова Кабінету міністрів України №160 від 31.03.2015 р.