INTRODUCTION:

During the COVID-19 pandemic, researchers begun to study literature extensively to explain the pathways that lead to development of disease and identify potential therapies^1,2, including target proteins such as angiotensin converting enzyme (ACE2), a membrane- bound protein that serves as a receptor for SARS-CoV-2³. The ACE2 role in initiation and spread of coronavirus infection was initially documented during the SARS outbreak, caused by SARS-CoV-1, in 2003. SARS-CoV-2 likewise uses the ACE2 receptor to invade human cells^4-5. Here, we review potential methods for blocking the binding of –SARS-CoV-2 to ACE2, using a soluble recombinant human ACE2- like peptide to bind virions outside of cells, neutralizing their infective capacity.

Association between ACE2 and coronaviruses:

ACE2 is expressed in the lungs, kidneys, and gastrointestinal tract⁶, with particularly high expression in type 2 pneumocytes⁷. It is the entry point receptor for SARS-CoV-1 and SARS-CoV-2^8-9. Genome sequencing has indicated four major structural proteins, including the spike (S) protein, nucleocapsid (N), membrane (M) and envelope (E). For coronaviruses, the ‘S’ protein is crucial for viral transmission and infection of host cells. Its composition includes a short intracellular tail, a transmembrane anchor, and large ectodomain consisting of a receptor- binding S1 subunit and a membrane- fusing S2 subunit. The SAR-CoV-2 ‘S’ protein genome shows 75 % similarity with that of SARS-CoV-1^10-11. Analysis of the receptor- binding motif in the ‘S’ protein has indicated that most of the amino acid residues essential for receptor binding are conserved between SARS-CoV-1 and SARS-CoV-2, explaining their affinity for the same host cell receptor. Mutations in the ‘S’ protein domain of SARS-CoV-2 virus allow it to bind to human ACE2 receptors with strong affinity, which may explain its transmissibility^10,12-13. Many reports also indicate that ACE2 expression increases with age and is elevated in individuals suffering with cardiovascular complications, which could account for the greater severity of COVID-19 in these subjects^14-15. SARS-CoV-2 also involves proteases enzymes that have key roles in promoting interaction of ACE2 and the ‘S’ protein. Blocking binding between ACE2 and SARS-CoV-2 or other related proteins could potentially serve as a treatment strategy against COVID-19¹⁴.

Role of Recombinant Human ACE2:

Many bacterial recombinant proteins are now being produced on a large scale or are under clinical trials. A human ACE2-like carboxypeptidase derived from bacterial B38-CAP. Several proteins produced by bacteria after DNA recombination are presently undergoing clinical trials while others are already being produced on a large scale¹⁶. Great emphasis is placed on the molecular biological aspects of this approach. Recombinant ACE2 has been shown to prevent acute lung injury in animal models of acute respiratory syndrome (ARDS)¹⁷, suggesting that recombinant ACE2 could treat acute lung failure in a number of diseases. ACE2 knockout mouse models exhibit more severe disease compared with control mice expressing ACE2 acid-aspiration-induced ARDS, endotoxin-induced ARDS, and peritoneal sepsis-induced ARDS¹¹. Loss of ACE2 expression in mutant mice also results in increased vascular permeability, increased pulmonary edema, neutrophil accumulation, and decreased lung function. Importantly, treatment with catalytically active, recombinant ACE2 protein improves recovery from acute lung injury in wild-type mice, as well as in ACE2 knockout mice¹⁷. As such, ACE2 can be characterized as playing a protective role in acute lung injury.

The soluble form of ACE2 lacks a membrane anchor and is present in small amounts in circulation¹¹. This form may act as a competitive interceptor of SARS-CoV-1 and other coronaviruses by preventing binding of the viral particle to the surface-bound, full-length ACE2. In the Vero-E6 monkey kidney cell line, SARS-CoV-1 replication can be blocked by a soluble form of ACE2^9-10. Mouse models carrying the human version of ACE2, with the original mouse ACE2 knocked out, are enabling novel research in this discipline^18-19. In this context, provision of soluble ACE2-like peptide could be beneficial as a novel biologic therapy to combat or limit infection progression caused by corona viruses that utilize ACE2 as a receptor.

The membrane-embedded ACE2 receptor itself may be used in target- specific therapy against SARS-CoV-2, such as masking it to binding; various mechanisms to this end are under investigation^3,20-22.

Recombinant human ACE2 (rhACE2) has recently been developed, and with clinical trials to treat ARDS conducted in 2014-2017. In 2019, human ACE2 was demonstrated to be a vital receptor for SARS-CoV-2 infection. As such, recombinant human ACE2 might be employed to block both viremia and prevent lung injury (Table: 1). Based on this hypothesis, rhACE2 was successfully administered as an intravenous infusion to 89 patients and volunteers in China²³. This shows that, therapies employing a soluble recombinant human ACE2- like peptide can decrease the rate of entry into cells and hinder viral replication^16,24 protecting against lung injury^25-27. Such therapeutic treatments that improve local or systemic immunity can be beneficial against the COVID-19 infection. Further, recombinant or soluble human ACE2, can be utilized that to inhibit transmission and reduce pulmonary complications.

Aerosolized pulmonary or nasal delivery of soluble recombinant human ACE2 Like peptide:

Hormones, vaccines, antibodies, and growth factors with specific activities can also serve as targeted therapy²⁸. Injectable protein drugs are commonly used, but can be painful, costly, carry risks of infection and low patient compliance²⁹. By contrast, drug delivery via a pulmonary route renders a number of advantages in comparison with oral or parenteral administration³⁰. Progression of influenza and respiratory syncytial virus (RSV) infections is due to their interaction with respiratory mucosa cells. Parenteral immunization may precipitate poor mucosal immunity insufficient to defend against pulmonary infection³¹. Conversely, noninvasive delivery of drugs by pulmonary and nasal routes, provide benefits in transporting macromolecules³².

Aerosols follow different mechanisms of interception, inertial impaction, gravitational settling, and Brownian diffusion through which the drugs are deposited in the airways^33-34. The main considerations for efficient delivery of aerosols are surface charge, size, shape, and density, as well as the pathophysiology of the lungs. Three widely known systems for aerosol drug delivery are nebulizers, metered dose inhalers (MDIs), and dry powder inhalers (DPIs). The choice of device depends on the active agent, its formulation characteristics, its target site, and pulmonary characteristics. The large vascularized membranous area of the lungs (100-140 m²) provides an efficient route for pulmonary delivery of macromolecules. Additionally, aerosol inhalation offers increased bioavailability due to quick onset of action, thereby reducing the required dose and possibility of adverse reactions^35,36.

Recent studies have demonstrated that SARS-CoV-2 is transmitted via an oral-nasal route, indicating that the nasal epithelium acts as a gateway for infection and further transmission^10,13,37.

ACE2 is present in the nasal epithelium, principally in goblet and ciliated cells³⁸. Since SARS-CoV-2 is an enveloped virus, it does not require rupture of the cell for its entry, and can make continuous use of secretary pathways in the nasal goblet cells prior to the appearance of symptoms. Viral transmission mainly occurs through infectious droplets¹⁰. Drug delivery through nasal is recognized as a perfect route for local effect. Standard procedures involve dosage regimen via nose for treating various conditions like sinusitis, rhinorrhea, nasal congestion and nasal infections^39-40. Notably, nasal drug administration has been studied as a surrogate for avoiding hepatic first pass effect. This route also offers bioavailability and smooth delivery of site-specific medications from the nose to other organs⁴¹. The lungs have specialized structures that allow penetration of macromolecules and can serve as a non-invasive route of administration of proteins^42-43. The developmental changes in biotechnology and pharmacology have enabled delivery of endogenous proteins and peptides through nasal route⁴⁴.

CONCLUSION:

Clinical trials are now underway to examine recombinant angiotensin converting enzyme 2 as therapy for COVID-19 (Table:1). The present review and available literature suggests the role of membrane-bound ACE2 in progression of COVID-19 leading to lung injury. Therefore, there is an urgent need to formulate an aerosol or a nasal drops containing soluble ACE2- like peptide that can be easily administered to patients with COVID-19, since a mechanism of binding the virus prior to its interaction with human cells could work prophylactically against COVID-19.

ACKNOWLEDGEMENT:

The authors thank Al-Hawash Private University and Dr. Farzat Ayoub University Hospital, Homs, Syria for providing necessary support for successful completion of this review on COVID 19.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 24.06.2020 Modified on 09.08.2020

Research J. Pharm. and Tech. 2021; 14(6):3433-3436.

DOI: 10.52711/0974-360X.2021.00597

Trial ID	Scientific title	Study Population	Indication
EUCTR2020-001172-15-DK	Recombinant human angiotensin-converting enzyme 2 (rhACE2) as a treatment for patients with COVID-19 - APN01-01-COVID19 (Denmark)	200	Severe COVID-19
NCT04335136	Recombinant Human Angiotensin-converting Enzyme 2 (rhACE2) as a Treatment for Patients With COVID-19 (Austria; Denmark; Germany; Austria; Denmark; Germany)	200	COVID-19