Targeting de Novo Pyrimidine Biosynthesis by Leflunomide for the Treatment of COVID-19 Virus Disease

Leflunomide is a potent inhibitor of the enzyme DHODH and is already licenced to treat
rheumatoid arthritis. Importantly, this drug has dual anti-viral and anti-inflammatory
properties which is advantageous in COVID-19 infected patients, as more progressive stages of
the disease involve augmented responses from the host inflammatory mechanisms leading to
serious complications and death.

Based on the current understanding of the pathogenesis of COVID-19 infection and its spectrum
of clinical manifestation it is evident that a significant proportion of those infected
develop pneumonia, systemic inflammatory response and cardiovascular complications with high
morbidity and mortality. The progression from initial mild symptoms (usually fever, fatigue
and cough) to severe pneumonia requiring oxygen support or mechanical ventilation often takes
one to two weeks after the onset of symptoms. The progression leading to a such deterioration
is thought to be related to the kinetics of viral replication culminating in an exaggerated
surge in inflammatory mediator release, called "cytokine storm".

Host-targeting antiviral (HTA) strategies have many advantages to fight against a broad
spectrum of viruses compared to direct-acting antiviral drugs. These include blocking the
viral replication and overcoming the potential of viral mutagenesis simultaneously.

Concept The quest for optimal HTA strategies enabled the investigators to identify a crucial
enzyme called human dihydroorotate dehydrogenase (DHODH) that limits pyrimidine synthesis.
This is a vital metabolic process utilised by viruses for replication.

In preclinical models of cell and animal infection by SARS-CoV-2 the DHODH inhibitors
exhibited multiple actions including attenuation of viral genome replication, suppression of
the inflammatory reaction and the release of pro-inflammatory cytokines and chemokine. These
drugs also offered protection against mortality in mice when administered at later stages of
infection when direct acting antivirals offered no survival benefit. The hypothesis is that
DHODH inhibitors may have the advantage in treatment of COVID-19 disease even at its advanced
stage compared to the drugs with either standalone anti-viral or anti-inflammatory
properties.

To this end, collaborators in China have conducted a pilot study in COVID-19 patients in
Wuhan, the epicentre of China COVID-19 outbreak, with moderate clinical symptoms. This study
has demonstrated feasibility with a favourable clinical outcome and good safety profile
within the leflunomide treatment arm. Patients receiving leflunomide had a shorter clinical
recovery time and a reduction in clinical inflammatory biomarkers with no side effects
observed.

Leflunomide This is an immunosuppressive agent whose efficacy and safety profiles are well
established in treatment of rheumatoid arthritis. In 76 000 patients treated with leflunomide
there were 16 potential cases of pancytopenia and 9 cases of serious skin reactions. It has
few GI side effects including derangement in liver function, associated with long-term use.

The recommended dosage of leflunomide in COVID-19 is a loading dose of 100 mg/day for 3 days,
followed by 10-20 mg once daily depending on liver functions (patients with ALT/AST levels
above upper limits of normal (ULN) reference range will have 10mg instead of 20mg). This
regimen is routinely used in the treatment of rheumatoid arthritis providing effective blood
concentration and few adverse reactions.

Leflunomide not only inhibits SARS-CoV-2 viral genome replication but also suppresses the
release of pro-inflammatory cytokines and chemokines, suggesting its potential advantage in
treating the SARS-CoV-2-induced COVID-19 disease compared to the drugs with standalone
anti-viral or anti-inflammatory properties.

Rapidly replicating viruses rely on the de novo pyrimidines biosynthesis to acquire
sufficient pyrimidines for genome replication. By inhibiting the host target DHODH,
leflunomide can block the de novo pyrimidine biosynthesis by inhibiting the rate limiting
step for the viral nucleic acid (DNA and RNA) replication process. The in-vitro data have
shown it to be effective against a broad-spectrum of viruses.

Objectives and Outcome Measures/ Endpoints The main trial objective is to evaluate the
clinical efficacy and safety of 10 days oral leflunomide treatment starting within 2 weeks of
moderate to critical illness symptoms' onset in patients with confirmed COVID-19 infection.

Additionally, the study will evaluate the impact of leflunomide on:

1. the rate of viral clearance

2. the extent of lung injury, based on CT imaging and on the need of invasive/non-invasive
ventilation and respiratory support.

3. myocardial, kidney, and liver function based on the need for inotropic/vasoactive and
mechanical support and renal replacement therapy.

4. assessment of respiratory, myocardial and kidney injury assessed by standardised
physiological and biomarker criteria including emerging molecular and inflammatory
footprints of COVID-19 disease Based on the measured variables models to predict the
severity of the disease as well as clinical outcomes will be built.

Trial Design The World Health Organisation (WHO) "master protocol" i.e. the R&D blueprint
Therapeutic Trial Synopsis for COVID-19 was taken into account when designing the trial. This
is illustrated in how the trial is conducted i.e. i) using a 2 stage approach of including a
pilot phase before a main trial, ii) target population, ii) participant eligibility, iv)
primary and secondary outcomes / endpoints, and v) the trial assessments needed to meet the
objectives of ensuring clinical benefit and patient safety of the new therapy being
investigated.

The number of patient visits and assessments conducted are kept to a minimum but sufficient
to address both the immediate and long term risk to public health. This takes into account
conventional standard operating procedures, Good Clinical Practice (GCP), national and
international clinical trial regulations.

DEFEAT-COVID is a multi-site, international, interventional, pragmatic, parallel group
design, open label, randomised CTIMP with a pilot phase that will allow us to adapt
procedures and assessments if required.

Trial Setting The study is primarily conducted at secondary and tertiary healthcare settings
in England and India. At each local clinical centre (LCC), the principal investigator (P)I
will be responsible for trial activities but much of the work will be carried out by medical
staff providing care to patients with COVID-19 within the hospital and by hospital research
nurses, medical students and other staff when delegated and with appropriate education,
training, and experience.

The landscape of the COVID infection in London and other regions in the UK is being
monitored. Additional sites will be invited to contribute to the trial upon recommendation by
the Steering and Trial Management Committees taking into consideration of developing national
hotspots, second wave of the pandemic etc.