Clinical Effect of Dialyzable Leukocyte Extract in Suspected or Confirmed Cases of COVID-19 (FUTURE-T)

Dialyzable Leukocyte Extract (DLE) (Transferon oral®) is a dialyzed extract obtained from
human leukocytes. Its active ingredient is a complex mixture of low molecular weight peptides
obtained from dialysis (12 kDa), and subsequent ultrafiltration (10 kDa) of lysed leukocyte
and platelet concentrates. This product is manufactured by Laboratory of Scientific Research
(Pharma-FT) at the Nacional School of Biological Sciences (ENCB), National Polytechnic
Institute (IPN) using a patented method in Mexico (MX/a/2011/013852), USA (US9328152B2), EU
(EPA-126128), Canada (2860260), Peru (000969-2014/DIN) and Colombia (14138326).

DLE is a complex drug, the active principle is multiple peptide units. Thus, DLE cannot be
fully characterized, requiring the application of analytical techniques that can describe the
overall behavior of all of its components (peptide polydispersity).

Two orthogonal techniques have been used to determine the molecular weight of the peptide
components of DLE based on their migration through a polymer matrix. The molecular weight of
10 kDa was determined by polyacrylamide gels electrophoresis under denaturing conditions
(SDS-PAGE), while a molecular weight lower than 15 kDa was determined by molecular exclusion
chromatography.

The peptide nature of DLE has been demonstrated using an aminogram method. This method
includes the acidic hydrolysis of the peptide components until free amino acids are obtained,
which are derivatized and identified based on the retention time of an amino acid standard
using reverse phase chromatography (RP-UPLC). Additionally, the peptides of this drug were
determined to be highly soluble by RP-UPLC coupled to UV, while mass spectometry, coupled to
RP-UPLC, exhibited consistent ionization patterns.

Non-clinical and clinical studies relevant to this research. Experimentally, in a murine
model of virus infection, administration of DLE showed a decrease in serum concentrations for
TNF and IL-6, accompanied by an increase of IFN. In humans, viral etiology is responsible for
90-95% of cases of respiratory tract infections. The common viruses involved in respiratory
diseases are rhinoviruses (∼50%), coronaviruses (∼10%), adenoviruses, respiratory syncytial
virus, influenza, and parainfluenza viruses (which generally add up between 10% to 15%),
human metapneumovirus (<5 %). The first evidence that DLE might have a therapeutic effect in
respiratory infections arose from the analysis of a series of cases (36 adults and 63
children) with allergic rhinitis who received DLE in addition to the standard treatment. 11%
of adults and 25.4% of children reported a decrease in the frequency of respiratory
infections. Similarly, in a series of 70 adults and 54 children with persistent moderate
asthma who received treatment with DLE as adjuvant therapy, 44% of adults and 31% of children
reported a decrease in the frequency of respiratory infections, in addition to the
improvement of allergic symptoms. In a study of 52 individuals, the effectiveness of DLE was
evaluated by reducing the severity and frequency of infectious symptoms. That study showed a
decrease in the frequency and in the severity of the infectious respiratory symptoms (77.1%)
in pediatric patients. In comparison, 82.4% of adults decrease the frequency and/or diminish
the severity of infectious respiratory symptoms. In a clinical follow-up of pediatric
patients with sepsis to whom DLE was added to the standard treatment decrease in C-reactive
protein levels, reduction in the neutrophil count at 72 hours after hospitalization, and an
increase in the survival rate of 30% was reported.

SARS-CoV-2 induces significant lung damage due to uncontrolled and dysregulated inflammatory
activity, then immunomodulatory drugs have been suggested as treatment. The findings related
to DLE, support their immunomodulatory capacity as a potential therapeutic tool in infectious
respiratory diseases with a prominent inflammatory component.

Primary goal. To generate information on the efficacy of DLE as an aid in symptomatic
treatment, by reducing the signs and symptoms of acute respiratory infection
(suspected/confirmed cases of COVID-19).

Secondary goals.

1. To evaluate clinical deterioration and respiratory alarm data.

2. To assess the duration of the clinical manifestations.

3. To explore cytokine changes

4. To obtain data on the safety of DLE as an aid in the symptomatic treatment of acute
respiratory infection (suspected/confirmed cases of COVID-19).

5. To generate information to validate the contingency scale (CS) to assess the severity of
acute respiratory disease (suspected/confirmed cases of COVID-19).

Hypothesis. The addition of DLE to the symptomatic treatment will reduce the signs and
symptoms of acute respiratory infection.

The recruitment method will be achieved electronically.

The acute respiratory infection will be classified according to its clinical symptoms,
following the Mexican Clinical Guideline in which COVID-19 disease is divided into
non-serious and severe cases. The clinical evaluation of the patient will be assessed by a CS
designed for this study, and includes three major items subdivided into minor items (range 0
to 4 in each one):

General symptoms: Fever, Headache Superior airway respiratory symptoms: Sneezing, nasal
congestion, runny nose Lower airway respiratory symptoms: Cough, Dyspnea, and Pain or Sense
of constriction or oppression in the chest.

Statistics. Four populations will be considered for the analysis: randomized individuals in
the study or full analysis set (FAS), safety population (SP), the population of the
intention-to-treat (ITT), and population per-protocol (PP).

The FAS population will consist of all individuals who were randomized into the study (they
are assigned a random number). The ITT population will consist of all patients who were
randomized, who received at least one dose of the study drug, and who had at least one
assessment of the primary or secondary endpoints after the baseline assessment. Following the
principle of the intention of treatment, patients will be analyzed according to the treatment
assigned during randomization. The SP will consist of all patients who received at least one
dose of the study drug and who have at least one safety assessment after the baseline
assessment. The patients will be analyzed according to the treatment they received. As a
clarifying note, the statement that the patient has no adverse events constitutes a safety
assessment. The PP will consist of all patients in the ITT population who complete all
visits, who have an accurate evaluation of the primary efficacy variable, and who do not
contain significant protocol violations.

Statistical methods. Demographic data, clinical history, and key baseline efficacy variables
for all randomized patients will be summarized by treatment group in tables (number and
percentage) for qualitative variables; and in tables containing the average, standard
deviation, median, minimum and maximum, by treatment group, for the quantitative variables. A
comparison of groups at the baseline visit will be analyzed using Chi-square tests for
qualitative variables and two-sample student t-tests for quantitative variables (p-values
will be provided for descriptive purposes only). Additionally, the medical history,
concomitant medications, comorbidities will be summarized using frequency tables.

Primary outcome. The primary result of efficacy will be the measurement of the score of the
CS at the end of treatment or early termination, in the ITT population. The algorithm of the
imputation of the last observation carried forward (LOCF) will be applied in the case of
patients who withdraw, discontinue therapy, or withdraw from the study. The primary analysis
will be performed using the ITT population. This analysis will also be performed in the PP
population to assess the robustness of the results. The primary outcome will be analyzed with
a covariance model that includes the treatment group, baseline value, and center.

The exploratory hypothesis of the primary objective is to establish whether there could be a
difference in the decrease in the score of the CS at the end of treatment, under a
superiority hypothesis approach described below:

Ho: µPlacebo - µDLE>0 Ha: µPlacebo - µDLE<0

Possible superiority of the DLE addition as an aid to symptomatic treatment relative to
symptomatic/placebo treatment will be established if the upper limit of the 2-sided 95%
confidence interval for the difference µPlacebo-µDLE is less than 0.

Secondary outcomes. The appearance of respiratory alarm data will be identified in the study
groups, presenting the results in a frequency chart with the proportion of patients who
presented alarm data by group. It will be analyzed using the Chi-Square test. The duration of
clinical manifestations corresponds to the number of days with any of the signs and symptoms
described in the CS. Descriptive statistics will be performed in each treatment group; the
comparison will be made using the student's t-test. Immunological changes are defined as
changes in the total leukocyte count, total lymphocytes, ESR, and serum concentration of CRP,
procalcitonin, D-dimer, ferritin, CK, myoglobin, IL-6, TNF, interferons, and specific
antibodies for the virus. Descriptive statistics will be presented by treatment group vs.
visit, as well as the proportion of patients who show normalization of values during
treatment.

Sample size. Due to the lack of information on the efficacy of the study drug in cases of
acute respiratory infection with COVID-19, the statistical calculation of the sample size
does not apply. However, as a descriptive exercise, given the current contingency by COVID-19
pandemic, an estimation of the sample size is proposed in this study based on the CS that has
been specifically designed for this study. Contingency score ranges from 0 to 32, considering
that the study population is non-severe patients, the scoring average will correspond to the
order of 16 points with a standard deviation of 4. It has also been assumed that the scoring
behavior is that of a continuous variable with a normal distribution.

The sample size calculation procedure is by means of the Julious 2004/FARTSSIE22 equation for
superiority designs in parallel with two groups (DLE vs. Placebo). According to the following
values:

a. Type I error: 0.05 (5%); b. Type II error: 0.2 (20%). Power: 1-b: 0.8 (80%) Proposed
standard deviation: 4 Difference to detect between groups: 1 The sample size relationship
between groups: 1:1 (balanced) The sample size calculated is 253 per group. An additional 10%
(q) will be included to offset withdrawals from the study or treatment; therefore, 562
patients (nt) 281 will be randomized by treatment in order to have the estimated number of
patients for the hypothesis test. nt=n/1-q =506/0.9=562

Procedures to explain missing data. Missing data for the variables that are measured on a
single occasion cannot be substituted, and that individual cannot be included in the
analysis. For data that is measured on more than one time, in case of missing data, the last
value measured after the baseline will be taken into account. (LOCF)

Procedures for reporting any deviation from the statistical plan. If there are deviations
from the original statistical plan, these will be described and justified in the final
report.

Selection of individuals to be included in the analysis. All randomized individuals who have
received at least one dose of the investigational product under the maxim of ITT will be
included in the analysis.

Access to information. The study will be monitored by specific personnel; internal and
external inspections and audits will be allowed and facilitated, allowing access to
information while maintaining the confidentiality of the study individuals.