Official Title
Combination of Recombinant Bacterial ACE2 Receptors -Like Enzyme of B38-CAP and Isotretinoin Could be Promising Treatment for COVID-19 Infection- and Its Inflammatory Complications
Brief Summary

Combination of Recombinant Bacterial ACE2 receptors -like enzyme of B38-CAP and Isotretinoin could be promising treatment for COVID-19 infection- and Its inflammatory complications Mahmoud ELkazzaz1 1Department of chemistry and biochemistry, Faculty of Science, Damietta University, Egypt. _____________________________________________________________________________________________ ________________________________________________________________________ B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac dysfunction Angiotensin-converting enzyme 2 (ACE2) is critically involved in cardiovascular physiology and pathology, and is currently clinically evaluated to treat acute lung failure. Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is an ACE2-like enzyme to decrease angiotensin II levels in mice. In protein 3D structure analysis, B38-CAP homolog shares structural similarity to mammalian ACE2 with low sequence identity. A study demonstrated that the bacterial B38-CAP as an ACE2-like carboxypeptidase, indicating that evolution has shaped a bacterial carboxypeptidase to a human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein drugs for hypertension and heart failure. pretreatment of B38-CAP markedly down regulated a massive increase of plasma Ang II levels at 5 min after Ang II injection In addition to the currently used drugs to inhibit Ang II generation or signaling, such as ACE inhibitors or Angiotensin receptor blockers, direct down-modulation of Ang II levels by rhACE2 protein is one of the promising candidates for new therapeutic strategy in cardiovascular disease and other Ang II-related diseases, e.g. ARDS. On the other hand, although mass production of rhACE2 as a protein drug costs due to requirement of mammalian cell expression systems, B38-CAP is easily prepared with E. coli expression system and is cost effective. Therapeutic efficacy and less toxicity in mouse heart failure models would warrant further investigation of B38-CAP or other microbial carboxypeptidases in disease models. Finally the principal investigator expects that treatment with ACE2-like enzyme of bacteria B38-CAP expected to work efficiently Like human ACE2 and it will save the lung cells from COVID - 19 inhibitory effect and down regulation of ACE2 because COVID-19 binds to human ACE2 and down regulates it and this receptors is very important for lung cells survival and function So ,the principal investigator also expects that B38-CAP ACE2 like enzyme may be not recognized by COVID -19 spike protein because evolutionary it is too far away from human ace2 and human ACE2 is a real receptor of COVID -19 not ACE2 like enzyme but in the same time it will make the same function of human ACE2 In another study by Sinha et al who analyzed a publicly available Connectivity Map (CMAP) dataset of pre/post transcriptomic profiles for drug treatment in cell lines for over 20,000 small molecules, isotretinoin was the strongest down-regulator of ACE 2 receptors. On the other hand, they found 6 drugs in CMAP that are currently being investigated in clinical trials for treating COVID-19 (chloroquine, thalidomide, methylprednisolone, losartan, lopinavir and ritonavir, from clinicaltrials.gov), none of which was found to significantly alter ACE2 expression (P>0.1) Moreover, another study demonstrated that isotretinoin is a Potential papain like protease (PLpro) inhibitors which is a protein encoded by SARS-CoV-2 genes and considered one of the proteins that should be targeted in COVID-19 treatment by performing target-based virtual ligand screening . So, the principal investigator expects strong inhibition of COVID - 19 infection And rescuing the lung cells from its serious attack by treating with ACE2 like enzyme and Isotretinoin Keywords: COVID 2019 , Isotretinoin,B38-CAP , Bacterial ACE2 receptors -like enzyme , rhACE226.

Detailed Description

This is a small pilot study investigating whether there is any efficacy signal that warrants
a larger Phase 2B trial, or any harm that suggests that such a trial should not be done. It
is expected to produce statistically significant results in the major endpoints. The
investigator will examine all of the biologic, physiological, and clinical data to determine
whether a Phase 2B trial is warranted.

- Primary efficacy analysis will be carried only on patients receiving at least 4 doses of
active combination drug. Safety analysis will be carried out on all patients receiving
at least one dose of active drug. It is planned to enroll more than or equal to 24
subjects with COVID-19. It is expected to have at least 12 evaluable patients in each
group.

- Experimental group: 0.4 mg/kg IV BID for 7 days (unblinded) plus Aerosolized 13 cis
retinoic acid in gradual in 2 divided doses increases forms 0.2 mg/kg/day to 4 mg/kg/day
as inhaled Isotretinoin therapy for 14 days and standard of care Control group: standard
of care Intervention duration: up to 14 days of therapy No planned interim analysis.

Combination of Recombinant Bacterial ACE2 receptors -like enzyme of B38-CAP and Isotretinoin
could be promising treatment for COVID-19 infection- and Its inflammatory complications

Mahmoud ELkazzaz1

1Department of chemistry and biochemistry, Faculty of Science, Damietta University, GOEIC,
Egypt.

_____________________________________________________________________________________________
________________________________________________________________________

Introduction

Angiotensin converting enzyme-2 (ACE2) receptors mediate the entry into the cell of three
strains of coronavirus: SARS-CoV, NL63 and SARS-CoV-2. ACE2 receptors are ubiquitous and
widely expressed in the heart, vessels, gut, lung (particularly in type 2 pneumocytes and
macrophages), kidney, testis and brain. ACE2 is mostly bound to cell membranes and only
scarcely present in the circulation in a soluble form. An important salutary function of
membrane-bound and soluble ACE2 is the degradation of angiotensin II to angiotensin1-7.
Consequently, ACE2 receptors limit several detrimental effects resulting from binding of
angiotensin II to AT1 receptors, which include vasoconstriction, enhanced inflammation and
thrombosis. The increased generation of angiotensin1-7 also triggers counter-regulatory
protective effects through binding to G-protein coupled Mas receptors. Unfortunately, the
entry of SARS-CoV2 into the cells through membrane fusion markedly down-regulates ACE2
receptors, with loss of the catalytic effect of these receptors at the external site of the
membrane. Increased pulmonary inflammation and coagulation have been reported as unwanted
effects of enhanced and unopposed angiotensin II effects via the ACE→Angiotensin II→AT1
receptor axis. Clinical reports of patients infected with SARS-CoV-2 show that several
features associated with infection and severity of the disease (i.e., older age,
hypertension, diabetes, cardiovascular disease) share a variable degree of ACE2 deficiency.
We suggest that ACE2 down-regulation induced by viral invasion may be especially detrimental
in people with baseline ACE2 deficiency associated with the above conditions. The additional
ACE2 deficiency after viral invasion might amplify the dysregulation between the 'adverse'
ACE→Angiotensin II→AT1 receptor axis and the 'protective' ACE2→Angiotensin1-7→Mas receptor
axis. In the lungs, such dysregulation would favor the progression of inflammatory and
thrombotic processes triggered by local angiotensin II hyperactivity unopposed by
angiotensin1-7. In this setting, recombinant brACE2 could be promising therapeutic approaches
in patients with SARS-CoV-2 infection.

Rescuing The renin-angiotensin system (RAS) by B38-CAP ACE2 which is a bacteria-derived
ACE2-like enzyme

The expected benefits of B38-CAP derived ACE2-like enzyme depending on previous research data
show that B38-CAP derived ACE2-like enzyme will do the same function of human ACE2 and in the
same time it will be resistant to COVID- spike protein because evolutionary it is too far
away from human ace2 and human ACE2 is a real receptor of COVID -19 not ACE2 like enzyme and
this discussed as follow :

B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac
dysfunction Angiotensin-converting enzyme 2 (ACE2) is critically involved in cardiovascular
physiology and pathology, and is currently clinically evaluated to treat acute lung failure.
Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is an
ACE2-like enzyme to decrease angiotensin II levels in mice. In protein 3D structure analysis,
B38-CAP homolog shares structural similarity to mammalian ACE2 with low sequence identity. In
vitro, recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin
1-7, as well as other known ACE2 target peptides. Treatment with B38-CAP suppressed
angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. Moreover,
B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial fibrosis,
and cardiac dysfunction in mice. Our data identify the bacterial B38-CAP as an ACE2-like
carboxypeptidase, indicating that evolution has shaped a bacterial carboxypeptidase to a
human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein
drugs for hypertension and heart failure. On the contrary Treatment with recombinant human
ACE2 protein (rhACE2), which is devoid of its membrane-anchored domain thus soluble, has been
demonstrated to exhibit beneficial effects in various animal models including heart failure,
acute lung injury, and diabetic nephropathy, and so forth. rhACE2 is currently tested in the
clinic to treat ARDS and COVID-19 infected patients . Using cell cultures and organoids,
researchers from the Karolinska Institute in Sweden and the University of British Columbia
(UBC) in Canada, showed that by adding a genetically modified variant of ACE2, called human
recombinant soluble angiotensin-converting enzyme 2 (hrsACE2), COVID-19 was prevented from
entering cells. The paper, published in Cell, shows that hrsACE2 had a dose dependent effect
of viral growth of SARS-CoV-2 and was able to reduce it by a factor of 1,000 to 5,000 in cell
cultures. Despite its beneficial effects, rhACE2 is a glycosylated protein and thus its
preparation requires time- and cost-consuming protein expression system with mammalian or
insect cells, which may not be advantageous in drug development and medical economy Although
it had been reported that an immune response is associated with the chronic infusion of
rhACE2 resulting in the degradation of rhACE226, this was not observed for B38-CAP; there
were no antibodies against B38-CAP detectable in the serum of mice infused with B38-CAP for 2
weeks. Implantation of B38-CAP-filled osmotic mini-pumps significantly suppressed Ang
II-induced hypertension in conscious mice .without affecting the heart rate. These results
indicate that B38-CAP antagonizes the vasopressor effect of Ang II. So the principle
investigator expects and suggests that treating with cloned Bacterial ACE2 receptors -like
enzyme of B38-CAP could be promising COVID-19 infection- and lung injury preventing drug
better than recombinant human ACE2 in addition to brsACE2, expected to lure the virus to
attach itself to the copy instead of the actual cells… It distracts the virus from infecting
the cells to the same degree and should lead to a reduction in the growth of the virus in the
lungs and other organs. A study showed that recombinant B38-CAP protein downregulates Ang II
levels in mice and antagonizes Ang II-induced hypertension, pathological cardiac hypertrophy,
and myocardial fibrosis. A study showed beneficial effects of B38-CAP on the pathology of
pressure overload-induced heart failure in mice without overt toxicities and also
pretreatment of B38-CAP markedly downregulated a massive increase of plasma Ang II levels at
5 min after Ang II injection In addition to the currently used drugs to inhibit Ang II
generation or signaling, such as ACE inhibitors or Angiotensin receptor blockers, direct
down-modulation of Ang II levels by rhACE2 protein is one of the promising candidates for new
therapeutic strategy in cardiovascular disease and other Ang II-related diseases, e.g. ARDS.
On the other hand, although mass production of rhACE2 as a protein drug costs due to
requirement of mammalian cell expression systems, B38-CAP is easily prepared with E. coli
expression system and is cost effective. Therapeutic efficacy and less toxicity in mouse
heart failure models would warrant further investigation of B38-CAP or other microbial
carboxypeptidases in disease models. Furthermore, human ACE2-like enzyme in bacteria might
pave the way to a new strategy to engineer evolution of bacterial proteins for better
designing and preparations of recombinant protein drugs Finally the principal investigator
expect that treatment with ACE2-like enzyme in bacteria B38-CAP may be do the same mechanism
of rhACE2 in inhibiting COVID -19 and the other suggested mechanism is that ACE2-like enzyme
in bacteria B38-CAP injection in human body will do the same function of human ACE2 and in
the same time it will be resistant to COVID- spike protein because evolutionary it is too far
away from human ace2 and human ACE2 is a real receptor of COVID -19 not ACE2 like enzyme and
Also among three bacterial carboxypeptidases we tested, only B38-CAP showed dependence of
proteolytic activity on anion concentration, which is characteristic of ACE2 activity.
B38-CAP also showed pH optimum of 7.5 equivalent to rhACE2.In addition, IC50 of MLN-4760 was
also equivalent between rhACE2 and B38-CAP. Although B38-CAP exhibited quite similar
proteolytic activity to rhACE2, there seems a difference in substrate specificity between two
enzymes So , the principal investigator also expects that B38-CAP ACE2 like enzyme may be not
recognized by COVID -19 spike protein but in the same time it will make the same function of
human ACE2 in addition to down regulating of human ACE2 which is the real receptor of COVID
-19

Blocking of ACE2 receptor of COVID -19 which is the real receptors of it

Isotretinoin(13cis RA) may be able to inhibit COVID 2019 entry via down regulation of ACE2
via action like shRNA targeting ACE2 gene expression and this is discussed as follow :

The COVID-19 pandemic caused by SARS-COV-2 has infected over 2,000,000 people causing over
150,000 deaths. A key host cellular protein required for the virus entry is
angiotensin-converting enzyme 2 (ACE2) whose expression has been demonstrated in many tissues
including alveolar epithelial type II cells in lungs, oral mucosa and intestine, heart,
kidney, endothelium and skin. ACE2-expressing cells can act as home cells and are prone to
SARS-CoV-2 infection as ACE2 receptor facilitates cellular viral entry and replication. A
study demonstrated that patients with hypertension and diabetes mellitus may be at higher
risk of SARS-CoV-2 infection, as these patients are often treated with ACE inhibitors (ACEIs)
or angiotensin II type-I receptor blockers (ARBs), which have been previously suggested to
increase ACE2 expression. In another study by Sinha et al who analyzed a publicly available
Connectivity Map (CMAP) dataset of pre/post transcriptomic profiles for drug treatment in
cell lines for over 20,000 small molecules, isotretinoin was the strongest down-regulator of
ACE 2 receptors. On the other hand, they found 6 drugs in CMAP that are currently being
investigated in clinical trials for treating COVID-19 (chloroquine, thalidomide,
methylprednisolone, losartan, lopinavir and ritonavir, from clinicaltrials.gov), none of
which was found to significantly alter ACE2 expression (P>0.1) Moreover, another study
demonstrated that isotretinoin is a Potential papain like protease (PLpro) inhibitors which
is a protein encoded by SARS-CoV-2 genes and considered one of the proteins that should be
targeted in COVID-19 treatment by performing target-based virtual ligand screening as the
Principal Investigator discussed before that (13cRA) is the strongest down-regulator of ACE2.
and the principal investigator expects that 13cRA can inhibit and downregulat ACE2 expression
,Suggesting its therapeutic potential in preventing the entry of COVID 2019 to the host cell.

Unknown status
COVID

Combination Product: Recombinant Bacterial ACE2 receptors -like enzyme of B38-CAP (rbACE2) plus Aerosolized 13 cis retinoic acid

In this study, the experimental group will receive 0.4 mg/kg rbACE2 IV plus Aerosolized 13 cis retinoic acid in gradual in 2 divided doses increases froms 0.2 mg/kg/day to 4 mg/kg/day as inhaled 13 cis retinoic acid therapy for 14 days

Eligibility Criteria

Inclusion Criteria:

1.Laboratory diagnosis:

Adult SARI patients with 2019-ncov infection confirmed by PCR; Absolute value of
lymphocytes < 0. 6x 109/L; Severe respiratory failure within 48 hours and requires
admission to ICU. (severe respiratory failure was defined as PaO2/FiO2 < 200 mmHg and was
supported by positive pressure mechanical ventilation (including non-invasive and invasive
mechanical ventilation, PEEP>=5cmH2O))

Exclusion Criteria:

- Age <18 years; Age >80 years

- Pregnant or breast feeding woman

- Patient in other therapeutic clinical trial within 30 days before ICF

- Receive any other ACE inhibitors (ACEI), angiotensin-receptor blockers (ARB) treatment
within 7 days before ICF

- Chronic immunosuppression: current autoimmune diseases or patients who received
immunotherapy within 30 days before ICF

- Hematologic malignancy (lymphoma, leukemia, multiple myeloma)

- Other patient characteristics (not thought to be related to underlying COVID-19) that
portend a very poor prognosis (e.g, severe liver failure, and ect)

- Known allergy to study drug or its ingredients related to renin-angiotensin system
(RAS), or frequent and/or severe allergic reactions with multiple medications

- Other uncontrolled diseases, as judged by investigators

- Body weight ≥85 kg

- Hypercholesterolemia

- Hypertriglyceridemia

- Liver disease

- Renal disease

- Sjögren syndrome

- Pregnancy

- Lactation

- Depressive disorder

- Contraindications for hormonal contraception or intrauterine device.

- Autoimmune diseases A history of organ, bone marrow or hematopoietic stem cell
transplantation

- Patients receiving anti-hcv treatment

- Permanent blindness in one eye

- History of iritis, endophthalmitis, scleral inflammation or retinitis 15-90 days of
retinal detachment or eye surgery

- The competent physician considered it inappropriate to participate in the study

Eligibility Gender
All
Eligibility Age
Minimum: 18 Years ~ Maximum: 60 Years
Contacts

M.Sc.Mahmoud Elkazzaz, M.Sc.Biochemistry
00201090302015
mahmoudramadan2051@yahoo.com

M.Sc. Mahmoud Elkazzaz, M.Sc.Biochemistry, Principal Investigator
General Organization of Export and Import control system

Kafrelsheikh University
NCT Number
MeSH Terms
COVID-19
Tretinoin
Isotretinoin