Hesperidin and Diosmin for Treatment of COVID-19

Research Background and Rationale At the end of December 2019, pneumonia of unknown origin
was detected in the hospitals of Wuhan city, China, and reported to the WHO country office
for the first time [1-3]. After a few days, the Chinese government has confirmed the
human-to-human transmission of the new infectious respiratory disease [4]. At the end of
January 2020, the WHO declared the outbreak of severe acute respiratory syndrome (SARS),
caused by a novel coronavirus (SARS-CoV-2), as an international public health emergency. The
disease termed coronavirus 19 (COVID-19) rapidly transmitted from China to all over the world
and subsequently the WHO declared it a global pandemic disease. The virulent virus structure
is closely related to (SARS-CoV) strain with a single-stranded positive-sense RNA
composition[5].

This pandemic disease is particularly of major importance to the whole world and especially
to countries with a heavy population like Egypt. There is a critical need for emergent,
continuous, and cost-effective health care delivery to infected people. Early detection and
strategies for prevention of progression of COVID-19 would make a major difference for
infected patients and would also be economically beneficial for a resource-constrained
country.

People infected with COVID-19 may have no symptoms but still, act as a source of infection to
other surrounding persons. The most common clinical manifestations following infection range
from mild symptoms of (generalized fatigue, dry cough, low-grade fever, and sore throat) to
severe symptoms of (typical severe acute respiratory distress syndrome (ARDS) and
pneumonia)[6]. Although the tremendous scientific research effort is focusing mainly on the
use of antiviral drugs, certain drug repurposing, and vaccine production for the treatment of
COVID-19 patients, there is no specific cure or vaccine for treatment up till now. New drug
development is a time-consuming process so that drug repositioning may be the optimum
solution to control this pandemic infection.

Selected Drugs Hesperidin is a common flavone glycoside found in citrus fruit such as lemons
and sweet oranges[7, 8]. Hesperidin has several pharmacological activities such as
anti-atherogenic, antihyperlipidemic, antidiabetic, venotonic, cardioprotective,
anti-inflammatory, and antihypertensive actions. The anti-inflammatory activity of hesperidin
was mainly attributed to its antioxidant defense mechanism and suppression of
pro-inflammatory cytokine production[7]. Hesperidin exhibited anti-viral activity against the
influenza virus through a significant reduction of virus replication. Treatment of infected
cells with hesperidin enhanced cell-autonomous immunity via activation and upregulation of
p38 and JNK expression which is essential for cell defense mechanisms against influenza
virus[9].

Hesperidin has been used as an herbal medicine for a long time. The safety of hesperidin was
confirmed by FASEB (Federation of American Societies of Experimental Biology) upon request of
the FDA. Toxicity studies have confirmed the high safety profile of hesperidin after oral
intake. Results from oral toxicity studies showed the absence of adverse side effects after
oral hesperidin ingestion of more than 2g /kg [10].

Daflon 500 mg is a marketed tablet dosage form containing a micronized flavonoid mixture of
50 mg of hesperidin and 450 mg of diosmin which used as vasoprotective venotonic agent[10].
This hesperidin mixture is characterized by its high safety profile. Continues oral
administration for hesperidin mixture to rats for 13 and 26 weeks, using a very high dose of
35-fold of the daily dosage showed no toxicity with a high LD50 value of more than 3 g/kg
body weight. Clinical trials used more than 2850 patients treated with the hesperidin mixture
for a period of 6 weeks to 1 year showed normal hematological parameters, hepatic and renal
functions with no signs of toxicity[11].

Hesperidin role in prevention and treatment of COVID 19 was recently published by our
research team in Medical Hypotheses journal [12].

Unraveling host cellular receptors used for cellular entry of COVID-19 will provide possible
lines for attack. Cell entry of COVID-19 depends on two consecutive steps, firstly binding of
the viral spike (S-protein) to host cellular receptors followed by priming of S-protein by
cell proteases. Recently, researchers showed that COVID-19 uses the ACE-2 receptor for entry
[13] and the serine protease TMPRSS2 for priming of S-protein. Camostat mesylate, a serine
protease inhibitor drug blocked virus entry and was used as a COVID-19 treatment in
Japan[14].

COVID-19 binds to the ACE-2 receptor through its specific Spike-receptor binding domain (RBD)
sequence to form the SARS-CoV-2-RBD-ACE-2 complex. The proposed computational activity of 78
anti-viral drugs against the human ACE2 receptor was screened using homology modeling. This
study showed that hesperidin is the only compound that could target the binding interface
between SARS-CoV-2 Spike and ACE2 human receptors. based on virtual screening, hesperidin may
disrupt the interaction of ACE2 with RBD of SARS-CoV-2 thus block its entry into the lung
cells [15]. Therefore, hesperidin can be used as a promising prophylactic agent against
COVID-19 infection.

Host antiviral responses against COVID-19 infection depend on the activation of both the
immune systems and cellular self-defense mechanisms. Immunity plays a major role in the
protection of the host against viral infection. The occurrence of immune over-response or
immune deficiency is responsible for the condition of infected patients becoming critical or
severe[16]. The anti-viral activity of hesperidin against the influenza virus involves its
role in the activation of the mitogen-activated protein kinase (MAPK) pathway. The MAPK host
defense cascade contributes to efficiently restraining viral replication, spread, and
minimizing tissue damage[9]. A recent study showed that the interferon-MAPK pathway played an
important role in the COVID-19 immune response[16]. Therefore, hesperidin by its activation
to host immunity my help against COVID-19 viral replication and hence its progression which
will improve the patient outcome.

Patients infected with COVID-19 exhibited what is called "cytokine storm" which initiated
primarily as an inflammatory response and resulted in uncontrolled over-production of soluble
markers of inflammation. Available evidence showing that cytokine storm, is a major cause for
the development of ARDS. Cytokine storm involves the release of various immune-active
molecules such as Interferons (e.g. IFNγ), interleukins (e.g. IL-1β, IL-2, IL-6), chemokines,
and tumor necrosis factor-alpha (TNF-α ) [17].

Hesperidin with its high anti-inflammatory activity inhibited the secretion of
pro-inflammatory cytokines such as IFN-γ and IL-2[18]. Besides, hesperidin inhibited IL 1β
stimulated inflammatory responses by inhibiting the activation of the NF κB signaling
cascade[19]. It also played a major rule in suppressing the release of inflammatory markers
such as (TNFα and IL-6) in type 2 diabetic patients[20]. Therefore, it can be used as
adjuvant therapy to control the severe inflammatory reaction against COVID-19.

Activation of coagulation pathways following the immune response to COVID-19 infection
promotes clot formation. The proposed mechanism of formation of micro thrombosis involves the
occurrence of procoagulant-anticoagulant imbalance, platelet activation, and converting
fibrinogen to fibrin. Disseminated intravascular coagulation predisposes to the development
of multiorgan failure especially in severe infected cases[21].

A prophylactic dose of heparin (with low molecular weight, LMWH) is recommended for
protection against venous thromboembolism in COVID-19 hospitalized patients[21]. In this
context, it is essential to highlight the role of concomitant administration of hesperidin
and diosmin mixture with heparin for protection against thromboembolism. Results from
previous clinical trials that used Daflon 500 mg with LMWH confirmed the significant effect
of this combination compared to LMWH alone in preventing the incidence of pulmonary embolism
and deep vein thrombosis. Therefore, co-administration of LMWH and Daflon 500 mg can
significantly inhibit clot formation and prevent disease progression [22].

Diagnostic criteria The viral research institution in China has conducted preliminary
identification of the SARS-CoV-2 through the classical Koch's postulates and observing its
morphology through electron microscopy[1]. So far, the golden clinical diagnosis method of
COVID-19 is nucleic acid detection in the nasal and throat swab sampling or other respiratory
tract samplings by real-time PCR and further confirmed by next-generation sequencing Clinical
classification of patients Patients can be grouped into three categories: asymptomatic, upper
respiratory tract infection (URTI) when presenting with rhinitis, pharyngitis, or isolated
low-grade fever and myalgia, and lower respiratory tract infections (LRTI) when presenting
with symptoms of pneumonia or bronchitis[3].

Side effects of (Hesperidin and Diosmin mixture, Daflon®) Like all medicines, Daflon 500 mg
can cause side effects, although not everybody gets them. Some cases of commonplace
gastrointestinal disorders and neurovegetative disorders (feeling of discomfort) have been
described, which do not require stopping the treatment.

Safety concerns of (Hesperidin and Diosmin mixture, Daflon®) Daflon 500 mg tablets is
POSSIBLY SAFE for most people when taken by mouth for up to 6 months. The safety of using it
for a longer period of time is unknown. Side effects include stomach pain and upset,
diarrhea, and headache.

Contraindications of (Hesperidin and Diosmin mixture, Daflon®) No reported contraindications.

Interactions of (Hesperidin and Diosmin mixture, Daflon®) No sever drug-drug interactions,
only moderate and minor interactions

- Celiprolol: (Moderate drug-drug interaction) Hesperidin may reduce the absorption of
celiprolol.

- Diltiazem: (Moderate drug-drug interaction) Hesperidin may reduce the absorption of
diltiazem.

- Verapamil: (Moderate drug-drug interaction) Hesperidin may increase the absorption of
verapamil.

- Antihypertensive drugs: (Moderate drug-drug interaction) Hesperidin may lower high blood
pressure might cause your blood pressure to go too low.

- Anticoagulant / Antiplatelet drugs: (Moderate drug-drug interaction) Hesperidin may slow
blood clotting which might increase the chances of bruising and bleeding.

- Sedative medications (Benzodiazepines) (Moderate drug-drug interaction) Hesperidin may
cause too much sleepiness.

Special Precautions & Warnings:

- Pregnancy and breast-feeding: Hesperidin is POSSIBLY SAFE for pregnant or breast-feeding
women when taken by mouth with diosmin.

- Bleeding disorder: Hesperidin might slow blood clotting and increase the risk of
bleeding. In theory, hesperidin might make bleeding disorders worse.

- Low blood pressure: Hesperidin might lower blood pressure. In theory, taking hesperidin
might make blood pressure become too low in people who already have low blood pressure.

- Surgery: Hesperidin might prolong bleeding. There is concern that hesperidin might
increase the risk of bleeding during and after surgical procedures. Stop taking
hesperidin at least 2 weeks before a scheduled surgery.

Research Objectives

This research proposal was employed as a practical strategy for providing a suitable drug or
drug combination for possible treatment of COVID-19 infected patients. This drug may help to
prevent the progression of respiratory complications. This can be achieved through different
goals as following:

1. Screening of different drugs related to different pharmacological classes depending on
its possible activity against COVID-19 virus.

2. Providing cost-effective and easy-to-implement treatment strategy for infected patients
and/or patients with high risk of developing respiratory failure.

3. Finally, this clinical strategy remains an important goal in improving Egyptian health
state which can save their life meanwhile save a lot of money.

Scope of Work The scope of work will be conducted through

1. Use of newly repurposed drug (Daflon 500 mg) for treatment of COVID-19 infected people.

2. Evaluation of the effect of the drug on the symptomatic treatment of mild COVID-19
patients through a clinical trial designed according to WHO (Clinical management of
severe acute respiratory infection (SARI) when COVID-19 disease is suspected) interim
guidance published at 13 March 2020.

3. Investigating the impact of the drug on the prevention of sever compilations such as
Acute Respiratory Distress Syndrome (ARDS).

Randomized double-blind controlled Parallel study of (Hesperidin and Diosmin mixture,
Daflon®) for treatment of Covid-19 newly diagnosed Patients in Egypt.

Study design This study will be a double blind randomized controlled parallel study Study
population and Methods Setting This study will be on Patients currently being granted at
designated hospitals affiliated to Ministry of Higher Education. Patients will be randomized
to either receive Daflon versus control group who will receive standard care Patients
Hospitalized patients with confirmed COVID-19 will included in this study according to the
following criteria; Adult (18-65 Years old), both sexes, PCR positive in nasopharyngeal
sample at admission. In addition, inclusion criteria will involve newly diagnosed
asymptomatic or with upper respiratory tract infection (URTI) patients who will present with
rhinitis, pharyngitis, or isolated low-grade fever and myalgia.

Informed consent Before being included in the study, patients meeting inclusion criteria will
give their written informed consent to participate to the study. An information document that
clearly indicates the nature of the study and the risks or side effects and the benefits
associated with the participation to the study will be given to each participant. The study
will be conducted in accordance with the Ethical standards of Helsinki Declaration. The
protocol will be submitted to the National Ethic Committee for reviewing and approval. This
trial will be registered as Clinical Trial.

Procedure Patients will be seen at baseline for enrolment, initial data collection and
treatment at day-0. The follow up duration will be 28 days or till PCR negative. Starting
from day 1, each day, patients will receive standardized care or Daflon 1000 mg, three times
daily for 10 days. Symptomatic treatment and antibiotics as a measure to prevent bacterial
superinfection will be provided based on clinical judgment for all participants. All
participants will be submitted to blood samples collection at base line and after the follow
up period for RT- PCR assay and to evaluate the change in serum IL1β, TNF-α, hsCRP. CBC will
be done for Lymphocyte count. Changes in respiratory rate and PaO2 will be assessed.
Furthermore, mortality rate will be calculated.

Outcomes Primary Outcome Measures: is RT-PCR negative Secondary Outcome Measures include:
changes in respiratory rate and PaO2, change in serum IL1β, TNF-α, hsCRP, Lymphocyte count,
mortality rate and occurrence of side-effects.

Randomized double-blind controlled Parallel study of (Hesperidin and Diosmin mixture,
Daflon®) for treatment of Covid-19 newly diagnosed Patients in Egypt.

Study design: Randomized double-blind controlled Parallel study Study Type: Interventional
Clinical Trial Enrolment: 50 participants in each arm Allocation: Randomized Assignment:
Parallel Intervention Model Description: Two parallel groups randomly asserted Masking:
Double (Participant, Investigator) Primary Purpose: Treatment or efficacy plus safety
Estimated Study Start Date: 01/07/2020 Estimated Primary Completion Date: 15/07/2020
Estimated Study Completion Date: 01/10/2020 Arms

Experimental:

1000mg of Daflon three times daily for 10 days

Active Comparator:

Standard care delivered in the isolation hospitals. Outcomes

Primary Outcome Measures:

PCR negative

Secondary Outcome Measures:

Changes in respiratory rate Change in patients PaO2 Change in serum IL1β Change in serum
TNF-α Change in serum hsCRP CBC for Lymphocyte count Reported side effects Mortality rate

Inclusion Criteria:

Confirmed cases of Covid-19 (all by RT-PCR) Newly diagnosed asymptomatic or with upper
respiratory tract infection (URTI) patients who will present with rhinitis, pharyngitis, or
isolated low-grade fever and myalgia, Adult (18-65 Years old) Both sexes

Exclusion Criteria:

Patients with bleeding disorders Patients with low to very low blood pressure Patients after
surgery Immunocompromised patients taking medication upon screening