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\begin{document}

\title{Metformin and its analogues as a therapeutic tool for COVID-19: a
narrative review.}



\author[1]{Talles Bispo da Silva}%
\author[1]{Miguel Neto}%
\author[1]{Lucas da Cruz}%
\author[1]{Matheus Rocha}%
\author[1]{Mônica de Melo}%
\author[1]{Diogo Batista}%
\author[1]{Amanda Rodrigues}%
\affil[1]{Universidade Federal de Alagoas - Campus Arapiraca}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
The new coronavirus (Sars-Cov-2) appeared in China in late 2019 and with
it an intense search for effective pharmacological tools in the
treatment of COVID-19. This virus can cause from mild symptoms, such as
dry cough, fever and tiredness, to more severe symptoms, such as
respiratory failure and systemic shock. Part of this problem is due to
the increase in the neutrophil/lymphocyte ratio and serum levels of
several inflammatory cytokines and chemokines. Other blood markers also
deflagrate the inflammatory condition, among them C-reactive protein,
ferritin and D dimers. Additionally, the history of chronic and/or acute
hyperglycemia is an independent predictor for morbidity and mortality in
patients with severe acute respiratory syndrome. In this pandemic
scenario, numerous new and other drugs already available on the market
have become therapeutic tools. Biguanide class drugs, classically used
in the treatment of type 2 diabetes mellitus, received great notoriety
for potentially useful systemic effects in the clinic, in cardiovascular
level and against cancer. Some of their properties may be useful in the
fight against Sars-Cov-2, such as the reduction of mRNA expression
levels of inflammatory cytokines, chemokines and by the attenuation of
NF-\selectlanguage{greek}κ\selectlanguage{english}B activation. These actions reduce the inflammatory response in the
individual and the damage from a severe inflammatory response. Given the
morbidity and mortality, partly attributed to the cardiovascular damage
of the COVID-19, the objective of this work was to review the systemic
effects of biguanides, as well as their therapeutic potential as an
adjuvant.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

COVID-19 is an infectious disease caused by the new coronavirus
(SARS-CoV-2), was first reported in Wuhan, China, later reaching
pandemic proportions. Patients may present symptoms (fever, dry cough
and tiredness), severe (dyspnea, hypoxia and/or great pulmonary
involvement) and critical (respiratory failure, systemic shock and/or
multiple organ failure) {[}1{]}. Respiratory discomfort syndrome, an
incident complication, affected 15.6\% of patients in a study that
collected data from 1,099 patients with COVID-19 in China {[}2{]}.
Despite the incessant search for effective drugs in their clinical
treatment, no drug has yet been validated {[}3,4{]}. Several classes of
drugs and strategies are being evaluated or developed aiming at the
clinical management of COVID-19, such as antibiotics, antiviral,
anti-inflammatory, antibodies, anticoagulants, anti-fibrotic, oxygen
therapy, and immunomodulation, among others. Possibly, different
treatment modalities have different efficiencies according to the stage
of the disease and its manifestation {[}4{]}. Thus, it is necessary to
understand the pathophysiology of the disease, as well as the affected
organs and systems, in order to improve the identification of
therapeutic targets and, thus, rigorously define the ideal treatment.

In the context of the search for adequate pharmacotherapy, biguanides,
buformin (BUF), metformin (MET) and phenformin (PHEN) appear as possible
adjuvant therapy for the treatment of COVID-19, due to their
comprehensive pharmacological properties {[}5{]}. Only MET is used in
large scale {[}6{]}, appearing as the first therapeutic line for type 2
diabetes mellitus (DM2) and one of the most prescribed drugs worldwide
{[}7{]}.

Antihelmintic, antimalarial and antiviral effects of biguanides have
been described, the latter being dependent on the administration of high
doses {[}8,9{]}. Considering recent publications of this pharmacological
class, its potential off label use, pharmacological advances that allow
the administration of drugs by alternative routes, as well as the demand
for new therapeutic tools for COVID-19, this study aimed to review the
pharmacological effects of biguanides, as well as its therapeutic
potential in viral infections.

\textbf{METHODOLOGY}

This is a narrative review of the literature performed in the PUBMED and
Virtual Health Library databases through the combination of descriptors
in health sciences (Table 1). After applying the search and selection
criteria, original studies were included, without language and
publication time restrictions, dealing with biguanides and their
pharmacological effects. Those who did not have a control group were
excluded, when the intervention was only in association, when it did not
specify the treatment of data, as well as duplicity. The data were
extracted: author, year of publication, objectives, method, experimental
design, experimental model, follow-up time, intervention, posological
scheme, main results and bibliographic references (submitted to the
cited criteria). No evaluation by the Research and Ethics Committee was
necessary.

\textbf{IMPACT OF HYPERGLYCEMIA ON COVID-19}

Evidence points to clinical and pathophysiological complications related
to SARS (Severe Acute Respiratory Syndrome) in hyperglycemic patients,
either by SARS-CoV or SARS-CoV-2. Analysis of clinical and biochemical
characteristics of 135 patients who died of SARS-CoV, 385 survivors and
19 with non-SARS pneumonia, indicated a history of chronic and/or acute
hyperglycemia as an independent predictor for morbidity and mortality in
patients with SARS, and revealed metabolic control as the best
prognostic factor {[}10{]}.

A cohort of more than 7000 Sars-CoV- 2 infected patients (with or
without DM2) showed correlated hyperglycemia with worse prognosis and
higher risk of mortality {[}11{]}. This pathological condition favors
inflammation and abnormal immune response, contributing to the
development and progression of radiological findings {[}12{]}. When
comparing groups of infected patients: without a history of diabetes
(1), with secondary hyperglycemia (2) and diabetics (3), it was noticed
that the proportion of critical patients and mortality in 2 and 3 was
higher than in 1, besides needing a longer hospitalization time
{[}13{]}.

A retrospective analysis reinforced DM as the greatest risk for negative
outcomes in COVID-19 infected {[}12{]}. However, acute or stress
hyperglycemia may lead to additional complications in these patients
(Figure 1). The glycosylation of the ACE2 receptor (Angiotensin 2
converting enzyme) facilitates the intrusion of the virus into the cell
{[}14{]}, since the spike protein of the virus binds to this receptor
and promotes the fusion of the viral membrane with the host membrane
{[}15{]}. In addition, monocytes and macrophages are the immune cells
most present in the lungs. This virus effectively infects monocytes from
peripheral blood and increases the expression of ACE2. Infected
monocytes increase IFN expression \selectlanguage{greek}α, β \selectlanguage{english}and \selectlanguage{greek}λ \selectlanguage{english}and pro-inflammatory
cytokines associated with the ''cytokine storm'' triggered by
SARS-CoV-2. It was also demonstrated that these monocytes increased the
function of HIF-1, a strong inducer of glycolysis and transcription of
IL-1\selectlanguage{greek}β, \selectlanguage{english}contributing to the pro-inflammatory state. In addition, changes
in oxidative metabolism were observed in critically ill patients with
COVID-19, by reducing oxygen consumption in infected monocytes and
increasing production of mitochondrial ROS (mtROS). Thus, it is
suggested that infected monocytes can promote epithelial cell death in
an mtROS/HIF-1\selectlanguage{greek}α \selectlanguage{english}dependent manner {[}16{]}. Under conditions of
hyperglycemia during infection, inhibition of T-cell proliferation may
occur, resulting in dysfunction and lymphopenia {[}16-18{]}. There was
also an increase in viral load, expression of ACE2 and IL-1\selectlanguage{greek}β \selectlanguage{english}in a
glucose dependent dose. In this sense, hyperglycemia promotes increased
viral replication and expression of cytokines {[}16{]}.

Based on the data found so far, hyperglycemia should not be neglected at
the time of admission, but should be adequately treated, aiming at
better outcomes in diabetic patients or not, contaminated by
COVID-19{[}11{]}. Biguanides can represent auxiliary tools in treatment,
thanks to their anti-hyperglicemiant, cardio-protective and modulating
properties of the immune system, in addition to their potential
antiviral effect.

\textbf{PHARMACOLOGICAL ASPECTS OF METFORMIN AND ITS ANALOGUES}

Biguanides are anti-hyperglicemiant agents {[}5{]}. MET promotes
reduction in hepatic glucose production, increased muscle uptake,
decreased gastrointestinal absorption, in addition to being an insulin
sensitizer {[}19{]}. The complex I of the electron transport chain was
the first identified target of this drug, which acts as partial
inhibitor of the mitochondrial complex {[}20-23{]}. The main effect of
this inhibition is the activation of the AMPK and its pathways, which
occurs through the stimulation of the regulatory subunit \selectlanguage{greek}γ \selectlanguage{english}for the high
levels of AMP/ADP {[}24,25{]}.

The pharmacological aspects of MET were summarized in Chart 1. In a
pharmacokinetic context, it differs from other biguanides by not
suffering hepatic metabolism. The PMAT (plasma monoamine membrane
transporter), located in the luminal side of the enterocytes, is one of
the responsible for capturing the MET from the intestine. Moreover, the
OCT transporters (organic cation carrier) have great importance in
tissue distribution, particularly for MET. The OCT3 transporters deserve
to be highlighted, because they are located in the brush border of the
enterocytes and participate in the capture of the drug. The transport of
MET and PHEN from the blood to the hepatocytes is mediated mainly by
OCT1. On the other hand, the renal uptake of MET is mediated by OCT2
{[}27{]}. Urine excretion occurs via MATE1 and MATE2 (multidrug and
toxin extrusion proteins transporters) {[}28{]}. The distribution volume
of MET is 63 to 276L after an intravenous administration, while for a
daily oral administration of 2g it is approximately 600L {[}27{]}. Its
half-life with physiological renal function is approximately five hours
{[}27,28{]}, and the maximum recommended daily dose 2.55g {[}19{]}.

PHEN and BUF present bioavailability of 40 to 50\% after oral
administration, accumulating mainly in the liver, pancreas, kidneys and
muscles. Approximately 50\% of PHEN is metabolized in the liver, which
generates inactive metabolites. This way, the ''slow metabolizers''
present a higher risk of developing lactic acidosis, the most important
adverse effect of biguanides. PHEN is eliminated through urine and bile
{[}5{]}.

MET is a safe, well tolerated and accepted drug {[}19{]}, since the
adverse effects usually occur during the beginning of therapy and are
resolved spontaneously {[}27{]}. Lactic acidosis occurs in five out of
100,000 individuals, generally being reported in patients with low
tissue perfusion (e.g., sepsis, myocardial infarction and congestive
heart failure) {[}27,28{]}. Renal insufficiency is the most common
comorbidity present in patients with lactic acidosis associated with
MET, being reported especially in those with high plasma creatinine
levels (\textgreater{}3mg/dL) {[}28,30{]}. MET should not be used in
moderate or severe renal insufficiency, or in those with mild but not
stable renal failure {[}30{]}. Moreover, in severe cases of Sars-CoV-2
infection, there may be a need for drug interruption due to the presence
of hypoxemia and hemodynamic instability, which increases the risk of
lactic acidosis {[}31{]}. Adverse effects and contraindications have
been listed in Chart 1.

The need for unconventional routes for the administration of biguanides
is of great clinical importance, as it can direct the drug to the target
organ/tissue and decrease possible systemic adverse effects. This is
because the MET, after oral ingestion, is unable to be effectively
distributed in the lungs {[}32{]}. Existing limitations trigger the need
for different formulations and administration routes for biguanides.
Drug administration systems (micro and nanoparticles, liposomes,
niosomes, among others) are very useful to overcome the difficulties
associated with conventional pharmaceutical forms. Among the potential
advantages are the protection of the active against enzymatic
degradation, reduction of side effects, reduction of the need for
repetition of doses and alleviation of discomfort generated by the
administration of the drug. Additionally, modulation of the release of
the active principle(s) in the organism, control of the place where they
are released, as well as an increase in the relative bioavailability of
the drugs can be obtained {[}33{]}.

Several studies point out the use of MET in unconventional ways, as in
the topical use for the treatment of skin lesions or in the form of
suppositories in patients with colorectal adenomas{[}34{]}. Experimental
studies, in humans and animals, point MET as a promising agent in the
treatment and prevention of lung diseases, but which end up having as a
barrier the low concentration of the drug in the lungs{[}32{]}.
Menendez, Quirantes-Pin\selectlanguage{ngerman}é and Rodríguez-Gallego et al. (2014), address
the need for alternative formulations of biguanides related to the site
where action is desired, such as inhalation use in lung cancer{[}35{]}.
Berstein (2018) ratifies the administration of MET not only by oral
route, highlighting the need to take into account its pharmacokinetics,
in addition to its different serum and tissue concentrations when used
by different routes{[}34{]}. In addition, inhibition of mitochondrial
complex I by the drug may be useful to reduce oxidative stress and lung
injury {[}36{]}. Thus, the use of inhaled biguanides to direct its
actions may present great pharmacological potential, including for
COVID-19.

\textbf{BIGUANIDES: NEW ANTIVIRAL TOOL?}

The search for the antiviral activity of biguanides is not recent. Still
in the 60's, the broad spectrum of action of this pharmacological class
was already being noticed {[}8,9{]}. From several experiments,
anti-helmintic, antiviral and antimalarial effects were identified.
However, it was found that the high doses necessary for the antiviral
activity of the modified biguanides made the clinical use of the
compounds known at that time impracticable {[}8{]}. In the last decade,
several studies have been carried out in order to verify the effect of
biguanides in the aid of antiviral therapies, with MET as the main
representative. Some of these studies approach the benefits of the drug
in the antiviral treatment indirectly, due to its ability to reduce the
body's resistance to insulin, which ends up improving body defenses. It
was verified that in patients co-infected with HIV and HCV, whose
metabolic and/or inflammatory variables were significantly altered, the
treatment with MET was well tolerated and significantly increased the
sensitivity of peripheral tissues to insulin {[}37{]}. Very beneficial
effect since viral infections affect glucose metabolism, leading to
insulin resistance and development of DM2 in predisposed individuals
{[}38{]}.

By adding MET to alphapeginterferon-2a and ribavirin treatment in
individuals with chronic Hepatitis C genotype 1 and insulin resistance,
there was an improvement in insulin sensitivity. Additionally, the
treatment increased the sustained viral response rate of patients with a
good safety profile {[}39{]}.

A quasi-experimental study selected 138 individuals with chronic
hepatitis C who presented insulin resistance and divided three groups:
A, treated with interferon and ribavirin; B, plus MET; and C interferon,
ribavirin and pioglitazone. After 48 weeks, the sustained viral response
was higher in B (p = 0.003) {[}40{]}.

When used in combination with lamivudine or interferon alpha-2b, MET
increased inhibition of surface antigen expression and replication of
hepatitis B virus, ratifying the possibility of synergism with other
antivirals {[}41{]}.

An in vitro study found that, compared with non-infected control cells,
ZIKV infection resulted in decreased AMPK in endothelial cells (dengue
and hepatitis C viruses are also known to inhibit AMPK activation).
Consequently, ZIKV-infected cells were exposed to AMPK pharmacological
activators (such as AICAR) and MET. These compounds did not exert direct
antiviral activity nor impaired viral adsorption/entry, but reduced (p
\textless{} 0.001) ZIKV infection. According to the authors, AMPK
activation exerts a powerful antiviral response that restricts
replication in infected endothelial cells by inhibiting glycolysis
induced by ZIKV, an essential source of energy and basic elements for
replication. Additionally, this activation enhances the innate antiviral
response in ZIKV-infected cells. It is worth noting, however, that the
compound AICAR inhibited the replication of ZIKV at a higher rate
compared to MET (p \textless{} 0.001) {[}42{]}.

The activation of AMPK inhibits the protein kinase mTOR. This effect
provoked by biguanides can also bring benefits on antiviral therapies.
It was verified that the mitogenic stimulation caused by mTOR
accelerates mortality induced by influenza in animals, increasing the
susceptibility of alveolar cells type II to infection. Due to the
inhibitory property of mTOR, it was suggested the use of biguanides for
the treatment of influenza {[}43{]}.

In order to avoid lactic acidosis, the use of inhaled pharmaceutical
forms for treatment has been proposed. However, an important aspect
emphasized by the authors regarding this form of administration was that
the inhaled MET could require a large amount of powder delivery to the
lungs, resulting in reduced complacency, bronchospasm and cough. BUF was
pointed out as an adequate substitute, because it has eight times more
potency than MET, making possible to reduce the amount of particulate
material delivered to the lungs and reducing the occurrence of lactic
acidosis as an adverse effect {[}43{]}.

A study whose objective was to verify drugs with potential to be used in
the treatment of pregnant women with COVID-19 cites the use of MET in
the therapy directed to the host. This type of therapy aims at
activating the body's protective immune response and suppressing the
overactive inflammatory response. This method is considered safe and
effective, capable of reducing the immunopathology and improving the
immune response, in addition to being suitable for pregnant women with
the disease {[}44{]}.

\textbf{EFFECTS OF BIGUANIDES ON THE IMMUNE SYSTEM}

\textbf{I} \textbf{IMMUNOSTIMULATION}

It was suggested that MET may present antagonistic actions according to
the pathological state of the submitted organism. In individuals with
cancer, it has been shown to activate both the innate and the adaptive
immune system. Such immuno-stimulatory effects were able to inhibit the
progression of the tumor or even to eradicate it, besides producing an
anti-metastatic effect in several animal models {[}45{]}. This
immuno-stimulatory action was demonstrated in a series of studies chosen
for this review and a detailed description of its effects was
schematized in Chart 2.

MET has received considerable attention in the field of oncology since
2005, after epidemiological publication highlighting the reduction in
the risk of neoplasias. From this, several observational studies
corroborate the antineoplastic effects in diabetics, including liver,
colorectal, pancreas, and stomach and esophagus cancer {[}24{]}.
Anisimov (2015), in his meta-analysis, stated that after a single dose
of MET, its peak plasma concentration in patients with DMT2 ranged from
4-15\selectlanguage{greek}µ\selectlanguage{english}M, and that most in vitro studies used doses of MET between 0.5-50
mM to suppress tumor growth, apparently much more than in humans.
However, in vivo studies in several types of rats demonstrated an
antitumor effect for several cancers with doses lower than 500 mg/kg, a
value that produces plasma concentration equivalent to that produced by
the maximum dose in humans (2500 mg/kg), comparing the body surface and
weight of mice with 20g and human with 60kg {[}68{]}.

The mechanisms associated with the benefits of MET against cancer
continue to be discussed {[}20{]}. The decrease in the production of ATP
induced by the drug may be intolerable for cells sensitive to energy
stress, and may lead to cell apoptosis {[}46, 47{]}. The consequent
lower production of ROSs is one of the mechanisms that lead to a lower
incidence of cancer, activating AMPK and its related pathways {[}49{]}.
Pryor and Cabreiro (2015) stated that the protective effects of MET are
attenuated by the pharmacological inhibition of AMPK or its absence
{[}24{]}. MET can also inhibit mTORC1 via Rag GTPase, regardless of the
activation of AMPK {[}47{]}. MTOR inhibition decreases protein synthesis
and growth, inducing cell cycle arrest and apoptosis {[}25{]}.

There is a hypothesis that the activation of AMPK can also confer
resistance against cancer, as it was observed in cells without LKB1 of
lung adenocarcinoma, after restoration of AMPK {[}24, 47{]}.
Pecinova\emph{et al.} (2017) approached that the viability of cancer
cells (rho0) without mitochondrial DNA is also affected by MET
treatment, which makes the actions related to the respiratory chain
questionable {[}50{]}.

\textbf{IMMUNOSUPPRESSION}

In pathological contexts other than cancer, paradoxically MET can have
immunosuppressive effects, both on innate and adaptive immunity
{[}45{]}. These effects are comprehensive and are schematically
illustrated in Chart 2. The study by Diaz et al. (2017) involving obese
patients with DMT2 deserves an observation. In it, it was observed that
B-cell functions in response to vaccines were recovered and antibody
response increased after MET treatment, although the pathological
context of DMT2 has exactly the opposite effect to that observed. These
effects, although they can be categorized as immuno-stimulators, are due
to an anti-inflammatory action of the drug, capable of improving the
functions of the B cells {[}53{]}.

The role of AMPK in inflammation and polarization of macrophages was
explored in a study using MET, compound C (an AMPK inhibitor) and AICAR
for in vitro intervention. They found that in obese rats, AICAR
apparently decreased the percentage of M1 macrophages and increased
markedly M2 macrophages, suggesting that activation of AMPK plays a
vital role in macrophage polarization {[}69{]}. \emph{Chung, Nicol,
Cheng et al} demonstrated the blocking of many effects caused by MET in
the presence of compound C, suggesting that activation of AMPK is
necessary to decrease pro-inflammatory cytokines, protective effect,
decrease levels of nitric oxide synthase (iNOS) and cyclooxygenase-2
(COX-2) {[}54{]}.

Several studies report the action of MET on inflammatory mediators by
reducing mRNA expression levels of inflammatory cytokines TNF\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}α,
\selectlanguage{english}IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, chemokines or by attenuating NF-\selectlanguage{greek}κ\selectlanguage{english}B activation {[}52, 55,
56{]} (Chart 2). This effect arouses great interest in severe cases of
COVID-19, by the elevation of levels of inflammatory markers in the
blood (C-reactive protein, ferritin and D-dimers), increase in the
neutrophil/lymphocyte ratio and of several inflammatory cytokines and
chemokines {[}70{]}. Mice with acute respiratory distress syndrome,
accumulation of neutrophils and increased levels of inflammatory
cytokines showed reduction of these indicators of lung damage after MET
treatment (v.o. 50 mg/Kg for 7 days) {[}71{]}. Thus, the use of MET as a
potential therapeutic tool for COVID-19 is highlighted. However, it is
essential to understand, initially, the systemic inflammatory responses
induced by SARS-CoV-2 and how these pathways are modulated by the
different therapies used today.

\textbf{MITOCHONDRIAL EFFECTS}

Mitochondria are the organelle and key place for understanding the main
effects of biguanides, especially of MET {[}20{]}. Understanding their
capture becomes fundamental to the understanding of their action
mechanisms and applications. The MET molecule is positively charged and
its capture by the mitochondria depends on the energetic status of this
organelle {[}20, 21, 24, 50{]}. Lower respiratory activity associated
with an increase in membrane potential induced by inhibition of complex
I, may help in the capture and accumulation of MET in the mitochondria
{[}21{]}. Therefore, the concentration may be 100 to 300 times higher
within this organelle at a potential between 120 and 150mV, compared to
cytosol {[}20, 50{]}. Moreover, compared to the extracellular medium,
this concentration can be 1000 times higher in the mitochondria {[}20,
23{]}. Therefore, milli-molar scales concentrations of MET are
achievable, despite micro-molar concentrations of cytoplasm {[}23{]}. It
is worth noting that its uptake is favored by the action of complex I
due to the increase in potential, however, the drug inhibits this
complex, decreasing its subsequent uptake {[}20, 23{]}.

The I complex of the electron transport chain was the first identified
target of MET. It is known to have two forms: A (activated) and D
(deactivated) {[}21{]}. The exact location of the biguanide connection
still arouses debate. According to Pecinova et al. (2017), biguanides do
not bind to any specific site of dehydrogenesis in the respiratory chain
{[}50{]}. However, Bridges et al. (2014) concluded that MET is a
reversible non-competitive ubiquinone binding site inhibitor. In
addition, this compound exhibits greater inhibition in the non-catalytic
state of the complex (D) {[}20{]}. The possible site of MET in this
complex is an amphipathic region at the interface of the hydrophilic and
membrane regions, where redox processes initiate proton translocation.
MET would keep the complex in an open loop conformation, state D {[}20,
24{]}. Matsuzaki and Humphries (2015) observed that 25 mM of MET was
able to inhibit 12.1\% of the activity of complex I in state A, and
about 40\% in state D induced by heating, while the same concentration
inhibited 44.6\% of the activity of complex III-IV, and 59.0\% of the
activity of NADH oxidase (combination of activities of I-III-IV).
Bridges et al. (2014) approached that MET presents a CI50 of 19.4 mM
(\selectlanguage{ngerman}±1.4) over the bovine heart mitochondrial complex I (Bos taurus)
{[}20{]}. PHEN at 200 \selectlanguage{greek}µ\selectlanguage{english}M is able to inhibit 10\% and 35-40\% of complex
I activity in states A and D, respectively {[}21{]}. The main effect of
the inhibition of complex I related to MET is the activation of AMPK and
its pathways {[}24, 25{]}. AMPK favors a cellular catabolic state,
regulating several metabolic factors that lead to the restoration of the
energy balance {[}23{]}.

Some effects of MET, including the redox state of the cell, may vary
depending on the time or concentration used. While short-term
administration has caused little inhibition of complex I activity,
long-term is associated with benefits such as reduced oxidative stress
and increased antioxidant defenses, resulting in lower incidence of
organism damage {[}58{]}. Regarding the concentration, a change in the
redox state of the mitochondria was observed comparing concentrations of
100 \selectlanguage{greek}µ\selectlanguage{english}M and 500 \selectlanguage{greek}µ\selectlanguage{english}M. In 500 \selectlanguage{greek}µ\selectlanguage{english}M, as observed for retenone, the mitochondria
was in a lower NADH/NAD+ state; while in 100 \selectlanguage{greek}µ\selectlanguage{english}M, in a more oxidized
state {[}72{]}. Other effects are described in Chart 2.

\textbf{CARDIOVASCULAR PROTECTION ASSOCIATED WITH METFORMIN}

Sars-Cov-2 can cause or aggravate cardiovascular events in patients with
or without comorbidities. A meta-analysis of six Chinese studies, with
1527 patients with COVID-19, reported prevalence of diabetes (9.7\%),
cardio-cerebrovascular disease (16.4\%) and hypertension (17.1\%) among
the participants. Recent data showed that systemic inflammation and
increase of catecholamine caused by COVID-19 can lead to acute
atherosclerotic plaque rupture, resulting in acute coronary syndrome. In
addition, coagulation cascade deregulation may occur with micro-thrombus
formation in terminal organs {[}73{]}. In view of this, the important
role that MET can play in the treatment and functional recovery of
cardio-cerebrovascular diseases, such as stroke and myocardial
infarction (MI), conditions present in the severe form of COVID-19 is
verified.

Most studies that evaluate the mechanisms of action of MET, when used in
stroke, use as a resource the middle cerebral artery occlusion (MCAO) in
rats, transitory or permanently. When transient, they usually cause
ischemia between 60 and 90 minutes, followed by reperfusion. The daily
doses of MET used vary between 10 and 300 mg/kg. It was reported that
the moment of administration (pre- or post-stroke), the duration of
treatment (acute or chronic) and the degree of activation of AMPK are
important factors in physiological responses to the ischemia generated
in the brain {[}74{]}. MET-induced AMPK activation provides protection
against brain ischemia (Chart 2) {[}62{]}. There is a lot of discussion
about pre-stroke treatment with MET. While the acute treatment three
days before the experimental stroke caused more damage from the
infarction after 24h of the MCAO because it caused deleterious
activation of the AMPK, the treatment for three weeks provided reduction
of the acute infarction 24h after the occlusion by reducing this
activation {[}62{]}. It was observed that these post-ischemic
alterations can be mediated by neuronal nitric oxidase (nNOS), since its
exclusion abolished the deleterious and beneficial effects presented in
each case {[}62, 74{]}. It has also been demonstrated that pre-treatment
for two weeks with MET provides neuroprotection by activating AMPK. On
the other hand, it was observed that the acute pre-treatment 24h before
pre-conditioning induced by permanent occlusion of the middle cerebral
artery conferred the same effect 24h after ischemia {[}62{]}. The
divergence in the results may be related to the different MET doses and
the ischemia models used. The effects of the treatment in post-stroke
are better established, suggesting that there is not only reduction of
acute infarction, but also long-term functional recovery. The mechanisms
that explain the effect of the drug in post-stroke are described in
Chart 2 {[}62, 63{]}

The vast majority of studies that evaluated the cardiovascular effects
of MET induced MI in rats by occlusion of the left anterior descending
artery, causing ischemia (30 to 35 minutes) followed by reperfusion, or
through administration of isoproterenol (doses between 25 and 250
mg/kg). One study reported that the drug uses several biochemical
pathways to realize its effects. Attenuation of mitochondrial
respiratory decoupling in mice treated with MET compared to those
treated with saline solution was found. In addition, AMPK has been
proven to be an important regulator of myocardial energy balance,
activated also in I/R lesions. Activation during reperfusion maintains
cardiac viability by limiting apoptosis. Other mechanisms act to
minimize cardiovascular damage through nitric oxide (NO) production
{[}64{]}, of adenosine receptor stimulus {[}64, 65{]}, the suppression
of TLR4 signaling {[}66{]} and the reduction of cardiac remodeling and
neutrophilic activity {[}67{]} (Chart 2).

The clinical use of MET to reduce cardiovascular diseases, such as MI
and stroke, needs further investigation. A case-control study with
patients of MI (n=413), stroke (n=247) and case-control (n=443), in
which all used insulin, followed the effect of MET and oral hypoglycemic
agents. It was observed that the use of MET compared to non-use provided
a lower risk of stroke (OR 0.54; 95\%IC, 0.31-0.95), however, the same
result was not obtained for MI (OR 0.85; 95\%IC, 0.55-1.30) {[}75{]}.
The clinical benefit of this biguanide was also evaluated at MI, where
380 non-diabetic patients (n=191, MET 500mg twice a day and n=189,
placebo), who presented MI with ST segment superstructure submitted to
primary percutaneous coronary intervention (PCI), did not obtain
improvement in the left ventricle ejection fraction after 4 months of
treatment {[}76{]}. This same group was analyzed after two years, where
it was also found absence of clinical benefits {[}77{]}. More promising
results are seen in patients who have some pre-established metabolic
disorder and who needed an elective PCI without having done previous
treatment with the drug. In these, the levels of CK-MB and cardiac
troponin I were reduced and, after one year of follow-up, the cardiac
events were less frequent {[}78{]}.

\textbf{CONCLUSION}

Biguanides, especially metformin, have a cardiovascular pharmacological,
immunomodulatory, antiviral and antioxidant effect, thus, they are a
potential therapeutic tool supporting the treatment of COVID-19. Thus,
experimental studies are necessary to confirm its effects in relation to
SARS-CoV-2 and the clinical evolution of the disease.

\textbf{ACKNOWLEDGEMENTS}

We thank Dra Amanda Rodrigues and Dr Diogo Batista for their support and
guidance.

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\textbf{TABLE AND CHARTS}

\textbf{Table 1.} Descriptor strategies used in the search for studies
through databases.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Subject} & \textbf{Main descriptors} & \textbf{Combination
descriptors (AND)} & \textbf{Exclusion descriptors (NOT)}\tabularnewline
\midrule
\endhead
\textbf{Hyperglycemia} & hyperglycemia, hyperglycemic & coronavirus &
-\tabularnewline
\textbf{Farmacology} & biguanides, metformin & absorption,
administration, biotransformation, clearance, distribution, dosage,
elimination & -\tabularnewline
\textbf{Mitochondrial effect} & mitocondria & biguanides, metformin &
-\tabularnewline
\textbf{Antiviral effect} & antiviral & biguanides, buformin, metformin
& -\tabularnewline
\textbf{Imune system} & inflamation & biguanides, metformin & cancer,
tumor\tabularnewline
& immune system & biguanides, metformin, pharmacology &
neoplasm\tabularnewline
& cancer, neoplasm & biguanides, metformin & -\tabularnewline
\textbf{Cardiovascular system} & myocardial infarction & biguanides,
metformin & diabetes mellitus\tabularnewline
& stroke & biguanides, metformin & diabetes mellitus\tabularnewline
\bottomrule
\end{longtable}

\textbf{Chart 1.} Summary of the pharmacological aspects of metformin.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\textbf{PHARMACOLOGICAL PARAMETERS} & \textbf{INFORMATIONS} &
\textbf{REF}\tabularnewline
\midrule
\endhead
\textbf{Absorption and bioavailability} & After oral administration is
absorbed predominantly by the small intestine and presents
bioavailability of about 50\%. & 26-29\tabularnewline
\textbf{Distribution} & Distributed and concentrated mainly in the
kidneys, adrenal glands, pancreas and liver. Lower concentrations of MET
are found in the lungs and muscles. &\tabularnewline
\textbf{Biotransformation} & MET does not suffer hepatic metabolism.
&\tabularnewline
\textbf{Excretion} & Approximately 30-50\% of an oral dose of MET is
excreted unchanged in the urine over a period of 24 hours and about 30\%
of the dose is excreted unchanged in the faeces. &\tabularnewline
\textbf{Adverse effects} & Nausea, vomiting, flatulence, diarrhea and
abdominal pain, asthenia, headache, skin rash, ovulation induction and
lactic acidosis. &\tabularnewline
\textbf{Contraindications} & Drug allergy, creatinine clearance above
1.4 mL/min in women and 1.5 mL/min in men, metabolic acidosis, lactic
acidosis, iodinated contrast, hypoxemia, dehydration, sepsis, surgery
and liver disease. &\tabularnewline
\bottomrule
\end{longtable}

\textbf{Source:} 26-29. \textbf{Subtitle:} REF = reference.

\textbf{Chart 2.} Summary of the pharmacodynamic effects found for
biguanides.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
& \textbf{EFFECTS} & \textbf{REF}\tabularnewline
\midrule
\endhead
\textbf{Immunostimulation} & MET: & -\tabularnewline
& hyperinsulinemia and insulin-related pathways & 24, 25\tabularnewline
& regulation of inflammatory processes &\tabularnewline
& | energy stress, which leads to apoptosis of certain cancer cells &
46, 47\tabularnewline
& | of the Warburg effect & 24, 47, 48\tabularnewline
& inhibition of the mTORC1 pathway & 47\tabularnewline
& | caspases 3 and 7 & 25\tabularnewline
& | Bad and Bax expression &\tabularnewline
& | of Bcl-2 and Bcl-xl &\tabularnewline
& | of VEGF production &\tabularnewline
& | of cyclin D1 and E2F1 expression &\tabularnewline
& | of fatty acid synthesis in prostate cancer cells & 24,
47\tabularnewline
& anti-folate effect &\tabularnewline
& | P53 phosphorylation and its regulator, MDMX (indicating cell cycle
arrest) &\tabularnewline
& | cell resistance of lung adenocarcinoma without LKB1 after AMPK
restoration &\tabularnewline
& | of ROS production & 49\tabularnewline
& inhibition of tumor growth in obese and pre-diabetic rats
&\tabularnewline
& | of TGF-\selectlanguage{greek}β &\tabularnewline
& | epithelial-mesenchymal transition &\tabularnewline
& regulation of the expression of Notch receptors &\tabularnewline
& | viability of rho0 cancer cells (without mitochondrial DNA) &
50\tabularnewline
& | DNA methylation using S-adenosyl-methionine in breast cancer cells &
51\tabularnewline
& | sensitivity to chemotherapy in cancer stem cells & 25\tabularnewline
& | sensitivity to radiation therapy & 24\tabularnewline
& higher sensitivity of MET in cells with high Myc expression in cancer
suppression & 46\tabularnewline
& inhibition of M2 macrophage polarization and induction of change from
M2 to M1 polarization & 45\tabularnewline
& | the amount of myeloid suppressor cells and their migration to tumors
&\tabularnewline
& | number of Treg lymphocytes &\tabularnewline
& | the amount of memory T lymphocytes &\tabularnewline
\textbf{Immunosuppression} & MET: & -\tabularnewline
& inhibition of monocyte differentiation into macrophages &
45\tabularnewline
& | infiltration of monocytes or macrophages in diseased tissues
&\tabularnewline
& | anti-inflammatory T cells (like Th2, for example) or
immunosuppressive (Treg, for example) &\tabularnewline
& | inflammatory T cells (such as Th1 or Th17, for example)
&\tabularnewline
& | of inflammatory cytokine expression and | of anti-inflammatory
cytokines &\tabularnewline
& inhibition of function and | number of neutrophils & , 45,
52\tabularnewline
& improvement in B lymphocyte function and | antibody production &
53\tabularnewline
& | of M1 macrophages and | of M2 macrophages & 45, 54\tabularnewline
& suppression of phosphatase and tensin homolog (PTEN), a tumor
suppressor gene, in aortic smooth muscle cells & 55\tabularnewline
& | expression of messenger RNA from inflammatory cytokines (TNF\selectlanguage{greek}α,
\selectlanguage{english}IL-1\selectlanguage{greek}α, \selectlanguage{english}IL-1\selectlanguage{greek}β \selectlanguage{english}and IL-6), chemokines and NF-\selectlanguage{greek}κ\selectlanguage{english}B & 52, 55, 56\tabularnewline
& | in the amount of Th2 or Treg lymphocytes and | in the amount and
infiltration of Th1 and Th17 lymphocytes in diseased tissues &
56\tabularnewline
& | of autoantibodies in relevant models of autoimmune diseases
&\tabularnewline
& | in the concentration of components associated with neutrophil
extracellular traps (NETs) & 57\tabularnewline
\textbf{Autophagy and Longevity} & MET: & -\tabularnewline
& | SIRT3 levels (associated with | biogenesis, mitochondrial function,
| senescence of cardiomyocytes, etc.) & 58\tabularnewline
& | reverse transcriptase (hTERT) telomerase activity (associated with |
senescence in endothelial cells) &\tabularnewline
& | senescence markers: p16, p21, p27 and p53 &\tabularnewline
& | the \selectlanguage{greek}β-\selectlanguage{english}oxidation regulatory protein, ACAD10, associated with |
nematode survival & 22\tabularnewline
& | in the expression of FGF21, a mitokine capable of improving the
lipid profile, glucose uptake, etc. & 59\tabularnewline
& PHEN: & -\tabularnewline
& | autophagy & 60\tabularnewline
\textbf{Mitochondrial and Associated Effects} & MET, PHEN: &
-\tabularnewline
& | of the activity of mitochondria complex I and, therefore, of
oxidative phosphorylation & 21, 49\tabularnewline
& | in the production of ROS by complex I & 20, 21, 23, 49,
50\tabularnewline
& decrease in ROS production by SDH and mGPDH & 50\tabularnewline
& MET: & -\tabularnewline
& inhibition of mitochondrial glycerophosphate dehydrogenase (mGPDH) &
20, 50, 61\tabularnewline
& inhibition of ATP synthase and succinate dehydrogenase (SDH) &
50\tabularnewline
& activation of AMPK due to energy stress & 24, 25\tabularnewline
& inhibition of acetyl-CoA-carboxylase and | expression of lipogenic
genes & 24\tabularnewline
& | gluconeogenesis in hepatocytes & 23-25\tabularnewline
& | oxidative stress and | antioxidant defenses with long-term therapy &
58\tabularnewline
& improvement of superoxide desmutase levels in patients with DM2 &
52\tabularnewline
\textbf{Cardiovascular Effects} & MET: & -\tabularnewline
& protection against cerebral ischemia via nuclear factor related to
erythroid 2 (Nrf2) or inhibition of the NF-\selectlanguage{greek}κ\selectlanguage{english}B cascade &
62\tabularnewline
& recovery of post-stroke function and ischemia damage &\tabularnewline
& improvement of angiogenesis with chronic post-stroke treatment &
63\tabularnewline
& macrophage polarization to M2 with chronic post-stroke treatment
&\tabularnewline
& | production of nitric oxide (NO) by means of nitric oxide synthase
endothelial (eNOS) in myocardial infarction (MI) & 64\tabularnewline
& stimulation of the adenosine receptor, increasing its synthesis,
decreasing reperfusion damage & 64, 65\tabularnewline
& inhibition of TLR4 signaling in MI (indicating | of the production of
inflammatory mediators and left ventricular dysfunction) &
66\tabularnewline
& | cardiac remodeling, neutrophilic activity and myocardial injury in
MI with chronic pretreatment & 67\tabularnewline
\bottomrule
\end{longtable}

\textbf{Source:} 20-25, 45-67. \textbf{Subtitle:} REF = reference; MET =
metformin; PHE = phenformin; | = decrease; | = increase.

\textbf{Hosted file}

\verb`Figure - 1.docx`  available at \url{https://authorea.com/users/470463/articles/562595-metformin-and-its-analogues-as-a-therapeutic-tool-for-covid-19-a-narrative-review}

\selectlanguage{english}
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\end{document}

