Pulmonary surfactant in COVID-19; A role in etiology and treatment Myasar Alkotaji 1, Musab M. Khalaf 2 College of pharmacy, University of Mosul, Mosul-Iraq 1 Corresponding author: [email protected]. Received accepted Abstract 15.11.2020 30.11.2020 9 Introduction: Today, SARS-COV-2 represents a global threat. Dealing with this viral infection necessitates a comprehensive understanding of pathophysiology of the disease to reach to the suitable treatment. Treatment of this disease should not be restricted to the usual antiviral treatments, which suffered from several limitations including low effectiveness, development of virus- resistant mutations and the unwanted side effects.

Knowing that SARS-CoV-2 attack the machinery unit of production of surfactant in the lung, the alveolar type II cells, manifested the importance of this review article on the role of pulmonary surfactant in this disease and the possible role of pulmonary surfactant that can play in treating of COVID-19 patients.

Objective: This work tried to clarify the important role of pulmonary surfactant in lung physiology and possible immune-modulatory effect. In addition, the constituents of pulmonary surfactant and their roles against COVID-19 complications is highlighted. This article suggested that surfactant therapy may have a role in COVID-19 therapy and this can be in a form of exogenous (synthetic) surfactant administrated through endotracheal tube or through aerosolization. The pros and cons of these methods of administration have been discussed. Moreover, a possible way of stimulation of endogenous surfactant by administrating a drug that stimulates the synthesis of surfactant has been suggested.

Key words: Pulmonary surfactant, COVID-19, synthetic surfactant, alveolar type II cells

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العوامل السطحية الرئوية في مرض كوفيد-19, الدور المحتمل في المرض والعالج

الخالصة

تمثل اإلصابات بفايروس كرورنا المستجد تهديدا عالميا. ان التعامل مع هذه االصابات الفيروسية يتطلب فهما شامال للفسيولوجيا المرضية لهذا المرض كي نصل الى العالج المناسب. ان عالج هذا المرض يجب أن ال ينحصر بمضادات الفايروسات االعتيادية والتي تعاني من العديد من القصور كقلة الفعالية باإلضافة الى الطفرات المقاومة للدواء واألعراض الجانبية غير المرغوب بها. ان اإلحاطة علما بأن فايروس كورونا المستجد )SARS-CoV-2( يهاجم وحدة أنتاج العوامل السطحية )surfactant(في الرئة والمتمثلة بالخاليا الحويصلية من النوع الثاني يبين بجالء أهمية هذا البحث حول دور العوامل السطحية الرئوية في هذا المرض والدور المحتمل للعوامل السطحية الرئوية الذي ممكن ان تلعبه في عالج مرضى كوفيد19- .

يحاول هذا البحث توضيح الدور المهم للعوامل السطحية الرئوية في تنظيم وظيفة الرئة والدور المناعي المرتقب. باإلضافة الى ذلك يهدف البحث الى تقديم اضاءات حول مكونات العوامل السطحية الرئوية ودورها المضاد لتعقيدات اإلصابة بكوفيد- 19.تخلص هذه الورقة العلمية الى االقتراح بأن العالج بالعوامل السطحية الرئوية يمكن أن يلعب دورا في عالج كوفيد19- والذي يتمثل بالعوامل السطحية الرئوية الخارجية أو المصنعة والتي يمكن اعطاءها عن طريق الضخ المباشر عبر أنبوب من خالل القصبة الهوائية أو من خالل طريقة التبخير. باإلضافة لذلك تم مناقشة ايجابيات وسلبيات طرق اإلعطاء هذه. عالوة على ذلك تقدم هذه الورقة العلمية اقتراح تحفيز تصنيع المزيد من العوامل السطحية الرئوية في الرئة وذلك بإعطاء دواء ذو قابلية لتحفيز عملية تصنيع هذه العوامل في داخل الرئة.

Introduction reported in Saudi Arabia in 2012. Both of these outbreaks were ne of the many pathogens considered previously as a great threat that infect the respiratory to public health; however, they were O system of animals and not considered a pandemic. In humans is a coronavirus. This family December 2019, a varying number of of viruses was the cause of two patients have admitted to hospitals former outbreaks including the severe with the initial diagnosis was acute respiratory syndrome (SARS- pneumonia of mysterious reason 1,2. CoV) in China in 2003, and the Later, these patients were found to be Middle East Respiratory Syndrome infected with a novel series of (MERS-CoV) which was first

2 coronavirus defined as "SARS-CoV- ACE2 receptors on the cell membrane 2". This new disease caused by 5. coronavirus was named by WHO After some stages of progression of COVID-19 3. Later on, the WHO the disease, the virus reaches the considered this disease a pandemic. alveoli (the gas exchange units of the The source of SARS-CoV-2 is lung) and infects alveolar type II cells. unknown, however, some genetic Both SARS-CoV and SARS-CoV-2 studies assumed that SARS-CoV-2 infect, specifically, the alveolar type might be a result of a combination of II cells more than alveolar type I cells two viruses including a bat SARS-like 6,7. SARS-CoV proliferates inside CoV and a coronavirus of unknown type II cells and this leads to the origin 4. production and releases a lot of viral Pathophysiology of SARS-CoV-2 particles which damage the cells and infection may lead to cellular death 8. The viral replication will continue and extend to The initial step of viral infection is the adjacent cells and infect type II cells binding of the virus to the host surface in the invading areas. When this receptors and then the virus will fuse occurs, a large number of type II cells with the cytoplasmic membrane. It is will be lost 9,10. Knowing that alveolar well known that the main target of the type II cells are a precursor for type I virus in epithelial cells of the lung. cells explains the huge effect of the The sequence of binding of SARS- virus on the physiological role of CoV-2 spikes is similar to what is alveoli and the consequent respiratory happened with SARS-CoV where the complication. entry of the virus into the host cells occurs after binding of the virus to the The pathologic consequence of SARS and SARS-CoV-2 infection is

3 widespread damage of the alveoli, however, in some cases ( 5.37% of where the hyaline membranes become those aged under 60 years compared rich in fibrin with the presence of a to 16.91% of those above 60) the few multinucleated giant cells 11,12. disease progressed into what is known The irregular healing of the wound as Acute respiratory distress may lead to severe scarring and syndrome (ARDS) 15. Acute fibrosis. Retrieval of lung function respiratory distress syndrome and will require epithelial regeneration acute lung injury (ALI) are popular and intense acquired and innate diseases, required highly expensive immune response. It was found that intervention and they are potentially administrating epithelial growth fatal diseases with mortality rates of factors like keratinocyte growth factor about 40%. They are characterized by to COVID-19 patients might be acute respiratory failure with harmful and might increase the viral widespread, bilateral injury of the load by producing more ACE2 lung and severe hypoxemia. There are expressing cells 13. The risk of many clinical conditions associated COVID-19 is particularly high for with the development of ARDS, such elderly individuals where the immune as pneumonia, sepsis, aspiration of response is diminished and there is a gastric contents and major trauma 16. decline in the ability to reform the Unfortunately, this pulmonary damaged epithelium. besides, in damage may increase alveolar elderly there is a reduction in the endothelial-epithelial permeability, mucociliary clearance and this may leading to flooding of the alveoli, permit the virus to propagate in the decrease lung function, and deprive alveoli of the lung more readily 14. the lungs of adequate quantities of Although that most COVID-19 cases surfactant 17. are mild and required no treatment,

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Pulmonary surfactant lung. Surfactants have a role in mucociliary clearance since Pulmonary surfactant is a lipid-rotein surfactants are responsible for the complex 18, that is secreted by special displacement of particles into the cells of the alveolar epithelium. liquid phase, which is followed by Pulmonary surfactant is assembled subsequent processes of defense and secreted onto the respiratory mechanisms including the movement surface to play a dual function in of particles out of the lungs or ending innate defense mechanism and the particles into lymphatic glands biophysical stability of the membrane 19,21. of alveoli. Alveolar epithelial type II cells are the cells that are responsible Composition of pulmonary for the production of pulmonary surfactant surfactant. After production, a Pulmonary surfactant composition surfactant is transported to what is includes about 80 % phospholipid, 5– called lamellar bodies where the lipids 10% neutral lipids, which is mainly and proteins are combined and stored cholesterol, 8–10% proteins, and 5– before it can be secreted 19. 6% specific surfactant proteins 22. It is It is well known that pulmonary worthy to mention that the surfactant is responsible for reducing phospholipid, dipalmitoyl the surface tension, at the air-water phosphatidylcholine (DPPC) interface, of the intraluminal alveoli represents about 30-65% of the and prevent the collapse of the alveoli pulmonary surfactant. Alao, surfactant 20. However, pulmonary surfactant contains other saturated phosphatidyl represents a primary defense cholines (PC), such as mechanism against the entry of palmitoylmirystoyl phosphatidyl pathogenic microorganisms into the choline (PMPC) and unsaturated PCs,

5 such as palmitoyl oleoyl-PC (POPC) 42°C, which is above the body 23. temperature. At body temperature (37°C), DPPC exists as a rigid gel One of the important key constituents phase, which plays an important role of pulmonary surfactant is cholesterol, in reducing the surface tension of the which accounts for about 3–8% of the alveolar interface to very low values total mass under normal physiological during dynamic compression 26, 27. conditions 24. The proportion of cholesterol in surfactant might affect Membrane-associated surfactant its function. A study in 2016 proteins demonstrated that the presence of a Four surfactant proteins are involved high concentration of cholesterol, in pulmonary surfactant-associated above the physiological levels of functions including SP-A, SP-B, SP- cholesterol, might lead to the C, and SP-D 28. The first three impairment of surface tension proteins are called apolipoproteins reduction from injured lungs 25. because they are associated with Indeed, it is not only the percentage of surfactant phospholipids. SP-D is not different compositions that is considered apolipoprotein although it important in the effective functioning interacts with phospholipids under of the surfactant, but the orientation of specific conditions. Both SP-A and phospholipid is also important. SP-D proteins play a role in the Phospholipids of surfactant are intrinsic immune defense system oriented in a state of matter called while the hydrophobic proteins (SP-B liquid crystal (LC). Dipalmitoyl and SP-C) are involved in the surface phosphatidyl choline, the main activity of surfactant. However, the constituent of the lung surfactant, has importance of the contribution of an LC transition temperature of 41- these proteins in surfactant ability is

6 varied. It is known that SP-B is the innate immune system that modulates most important protein in pulmonary the inflammatory response and aids in surfactant for maintaining respiratory removing microbes from the function. Experiments showed that respiratory epithelial surfaces. In deactivation of the SP-B gene addition, SP-A and SP-D showed expression results in respiratory intrinsic antimicrobial activity 30. The failure at birth. This is due to the surfactant immune mechanism is not impossibility of maintaining the implemented against bacterial physiological function of the lungs 28. infection only. Fungal and viral In addition, the net positive charge of are implemented as well. SP-B might increase its interaction The surfactant will act through with anionic phospholipids. facilitating the uptake of these foreign Moreover, the sort of orientation of molecules and enhance the some proteins in the membrane phagocytosis process leading to the surface may enhance the binding with elimination of these invading A phospholipid, for example, the microorganisms. The surfactant parallel orientation to the surface of proteins SP-A and SP-D have the the membrane will maintain a sort of abilities to attach to both types of hydrophobic interactions between the bacteria, the Gram-negative and helical sectors (the amphipathic Gram-positive bacteria 31,32. The co- sectors) and the surface of the existence of SP-A and SP-D on the membrane 29. mucous membrane of the respiratory tract and alveoli aids in the In contrast to SP-B and SP-C, both aggregation of influenza A virus SP-A and SP-D proteins have a particles and enhances the neutrophil hydrophilic nature and can be attack on virus 33, 34. Moreover, the considered as components of the SP-D, which is available in lung and

7 respiratory fluid, can bind to the been noticed in infant respiratory envelope protein of the human distress syndrome (IRDS) and acute immune deficiency virus (HIV) and respiratory distress syndrome (ARDS) inhibits the in-vivo multiplication of 37,38,39. The main abnormalities that this virus 35. Very interestingly, a occur with a surfactant include an study published in 2007 demonstrated about 80% decline in the overall that the lung surfactant-associated phospholipid content, decrease in the protein (collectin SP-D) can bind to surfactant content of dipalmitoyl SARS-CoV spike-protein, phosphatidylcholine (DPPC) and specifically, to the glycoprotein of the phosphatidylglycerol and other spike of SARS-CoV 36. This fractions, and decrease of apoproteins highlights the possible role of contents 37,38. surfactant in the defense mechanism The surfactant lacks or deficiency due against coronavirus. to block of surfactant synthesis in Abnormalities of pulmonary type II alveolar cells might be the surfactant reason for the sudden deterioration of respiratory function in COVID-19 As mentioned before SARS-CoV-2 is patients. Therefore, treatment with a known to be capable of infecting type synthetic surfactant that may aid in II alveolar cells, the pneumocytes that renewal or replacement of the are responsible for the production of depleted endogenous surfactant surfactant. Abnormalities of lung storage may provide a strong biologic surfactant can produce intense lung and physiologic plausibility for the injury and they are associated with use of exogenous surfactant therapy in loss of stability of the alveoli, COVID-19–related ARDS. floating, and alveolar breakdown. These alveolar dysfunctions have Surfactant replacement therapy

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Exogenous-surfactant replacement causative condition that may lead to therapy has been tried with great adult respiratory distress syndrome 43. success in IRDS 39 while studies in For example, in the study conducted adult respiratory distress syndrome by Gregory et al., the patients who have had dissimilar results. Initial participated in the study had different reports about using surfactant in adult etiologies that lead to adult respiratory respiratory distress syndrome have distress syndrome whereas the shown that artificial exogenous Exosurf study was restricted to surfactant (Exosurf)(40) or bovine sepsis-induced adult respiratory surfactant (Survanta) 41 can improve distress syndrome. Therefore, the oxygenation and lung function. causative factor of adult respiratory distress syndrome may have to be In one study, the authors mentioned taken into consideration in future the results of a large, prospective, studies 41. multicenter trial involving 725 patients with adult respiratory distress The second important explanation for syndrome induced by sepsis and the variations in the exogenous treated with Exosurf or placebo. surfactant efficacy may be related to The obtained results demonstrated the type of surfactant preparation that that exogenous-surfactant was used. Exosurf is a type of supplementation did not cause an exogenous surfactant that does not amelioration in patients condition 42. contain apoproteins that normally present in natural surfactant and this There are many explanations for the can affect the onset of action of failure in the improvement of patients surfactant 43. with adult respiratory distress syndrome in the Exosurf  trial. The Whereas the study of Gregory et al. first explanation may be related to the used a bovine surfactant that contains

9 two apoproteins constituents, the the therapy failed to reach its intended synthetic preparation does not contain target in the adult lung. these apoproteins. These obtained Another way of administration of data indicated that any surfactant exogenous surfactant is direct preparation must contain apoproteins administration through a to be effective 44. bronchoscope. Gunther et al. 46 used Another important thing in surfactant natural bovine surfactant therapy is the mode of delivery of (Alveofact) in a dose of 300 mg/kg surfactant. In the Exosurf trial 42, of body weight administered through surfactant was administered by fibreoptic bronchoscope, which continuous aerosolization, the study delivered surfactant to each segment conducted by MacIntyre et al. 45 of lung. This study demonstrated that demonstrated that only a small using the fibreoptic bronchoscope to amount of aerosolized surfactant deliver surfactant had no harmful (about 4.5 %) was delivered to the effects on lung function or lungs. The problem with this route of hemodynamics. Within 12 hours of administration is that the small administration, these patients had a quantity of delivered surfactant has no substantial improvement in therapeutic effect because it oxygenation, although deterioration in undergoes inhibition by the similar gas exchange has been noted in seven inflammatory mediators that are patients; for these seven patients, the linked with ARDS. There are additional amount of surfactant was previous negative results with the use administered within the first 24 h. of surfactant in ARDS, where patients There was a substantial decline in failed to express noticeable benefit as mortality in these patients. This study provides additional supports for the

10 idea that larger amounts of surfactant syndrome in COVID-19 was is necessary to be administered to registered on produce a therapeutic effect. clinicaltrial.gov/ct2/show/M. In this controlled study, twenty COVID-19- Other researchers have suggested that induced respiratory failure patients administration of surfactant by will be randomized to receive either bronchoscopic installation provides the Bovine lipid extract surfactant two advantages including the delivery with standard care or the standard of a large amount of surfactant and care alone. The product will be allows the clinician to perform a administered through the endotracheal bronchoalveolar lavage which aid in tube directly to the lung and the removing inflammatory mediators 47. treatment will be repeated for three Recently, a new product named KL4 days. The anticipated results of this was introduced by Windtree study will be available at the Therapeutics. Aerosolized KL4 is a beginning of 2021. synthetic surfactant with an In the meantime, we are suggesting a amphipathic peptide and it was used better alternative, a drug with the successfully in RDS in premature ability to enhance the synthesis and infants. Windtree Therapeutics secretion of endogenous surfactant company announced to conduct a from its source (the alveolar type II clinical trial on KL4 as a potential cells). However, this is recommended therapeutic for ARDS in patients with in patients with mild to moderate COVID-19, however, no results is cases of COVID-19, where the published yet 48. alveolar type II cells are not Interestingly and very recently, the completely distressed. This article first clinical trial on using of synthetic suggests early treatment of COVID- surfactant in adult respiratory distress

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19 patients with a very high dose of CoV-2 in Vero cell line 51. This result Ambroxol. Ambroxol is a mucolytic is in accordance of a newly published agent that stimulates the secretion of article that suggested Ambroxol as a surfactant which will aid in potential therapy for SARS-CoV-2 mucociliary clearance of the virus in and assumed that it works in a similar addition to the surfactant role in mechanism to azithromycin 52. preventing the collapse of alveoli and Altogether there is a need for further prevention of the subsequent experimental work on Ambroxol to complication. Ambroxol is an confirm the proposed action of approved drug in many European Ambroxol in COVID-19 patients. countries and its unique action on Conclusion stimulating the secretion of surfactant from alveolar type II cells is well The respiratory complications documented 49. Also, Ambroxol has encountered with the severe cases of been used in the treatment of neonatal COVID-19 respiratory illness ARDS and it demonstrated the anti- motivates the sense about the inflammatory effect that might be possibilities of using synthetic useful in COVID-19 patients. In surfactant in treating COVID-19 addition, Ambroxol manifested an in- ARDS patients. This article vitro inhibitory action on rhinovirus highlighted the structure and infection of the human tracheal cell physiology of surfactant, the role of line, which is one of the common surfactant in respiratory distress in respiratory tract infections 50. Very general and surfactant role in the recently, a non-peer-reviewed work etiology of COVID-19. demonstrated the in-vitro The history of long years of effectiveness of clinically-relevant successful management of RDS in concentration of Ambroxol on SARS-

12 premature infants with surfactant, addition, the method of delivery of especially the natural surfactant, is surfactant is critical for achieving encouraging. However, although the adequate concentration of the product feasibility, and safety encountered in all lung segments. Moreover, the with the use of surfactant in ARDS in surfactant should be administered in a infants is very clear, there is no large amount to achieve a perfect obvious benefit from using of therapeutic response. surfactant in ARDS in adults. , The The potential role of surfactant in real problem with a surfactant therapy COVID-19 needs more investigations is in the critical components of the and this should include surfactant. interdisciplinary teams working The natural surfactant of animal together to reach the suitable synthetic origin is closely similar to human surfactant with a suitable dosage form surfactant; however, they lack certain to be administered at suitable time of essential proteins or certain the infection. Alternatively, the components that they lose during the suggested drug, Ambroxol, may extraction process. The real challenge achieve the goal by enhancing the with the synthetic surfactant is with production of endogenous surfactant. obtaining optimum composition, However, further investigation of the which resembles the natural surfactant Ambroxol effect in COVID-19 is constituents, especially the required in order to prove this apoproteins constituents. supposed role in COVID-19 treatments. To conclude, optimum surfactant composition is very important to achieve an ideal therapeutic response and must contain apoproteins. In

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Acknowledgment: No fund or grant has been received in this work at all. The authors would like to thank the College of pharmacy-University of Conflict of interest: There is no Mosul. conflict of interest.

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