Journal of Entrepreneurial Health Sciences and Innovation (JEHSI)™
Combined Use of Oral Lysosomotropic Agents to Hinder the Onset,
Transmission and Progression of COVID-19: An Open Randomized Clinical Trial
Dr Gabriel Pulido-Cejudo1,2*, Peter Humphries2, Dr Bratati Kar1,2 and Dr Khadija El Abdaimi1,2
1International Centre for Advancement of Health Regional Innovation and Science, ICAHRIS
*corresponding author, gabriel.pulido-cejudo@icahris.org
2Canadian Federation of Breast Diseases, CFBD
Abstract
To date, approximately 1.3 million people have been infected with SARS-CoV-2, the causative agent of COVID-19, of which almost 70,000 have perished. Regardless of its relatively lower fatality rate (5.36%) by comparison to SARS-CoV (9.6%) and MERS-CoV (34.4%), SARS-CoV-2 has led to more deaths than MERS-CoV and SARS-CoV combined. In a concerted effort to abrogate the onset, transmission and progression of COVID-19, the clinical use of a combination of known lysosomotropic agents such as Chlorpromazine and Hydroxychloroquine Sulphate in combination with Azithromycin or Doxycycline is a potential and immediate viable approach [1] that has been moved forward within Canada and the United States. This is based on the capacity of the aforementioned lysosomotropic agents to effectively increase the acidic intravesicular pH of endosomal structures believed to be involved in the late viral entry of SARS-CoV-2 which require an acidic pH for processing and effective viral replication. This intervention clinical trial involves the use of oral Chlorpromazine and Hydroxychloroquine Sulphate in combination with Azithromycin to confirm the most suitable therapeutic combinations for the effective treatment of confirmed cases of COVID-19 patients as determined by RT-PCR using standard biological fluids.
Aim of the Open Randomized Clinical Trial
Determine the potential clinical benefits of a combination of Chlorpromazine and Azithromycin or of Hydroxychloroquine Sulphate in combination with Azithromycin to hinder the progression and infectivity of SARS-CoV-2 at clinically proven safe concentrations.
1. Introduction
Details provided in our recent publication indicate that the first onset of pneumonia of unknown origin, currently known as COVID-19, might have taken place on December 8 2019 in Wuhan, the capital city of the province of Hubei in central China.[1] This first episode was followed by a cluster of seven patients accepted at the intensive care unit (ICU) of the Wuhan Jin Yin-Tan Hospital at the beginning of the pneumonia outbreak between December 20 and December 29.[1]
Based on oral, anal swabs, blood samples and bronchoalveolar lavage fluids (BALF) from one of these critically ill patients, the first viral cultures and genomic sequences of a novel coronavirus were performed and reported.[1,2] Peng Zhou and colleagues tentatively named this virus novel coronavirus 2019 (2019-nCoV).
After isolating more strains which were 99.9% identical to each other from 4 additional patients, they identified 2019-nCov as the causative agent of the pneumonia outbreak of unknown origin affecting inhabitants of the city of Wuhan, China.[1,2]
Independent genomic analysis confirmed 2019-nCov as a member of Severe Acute Respiratory Syndrome-Related Coronavirus (SARS-CoVs) responsible for the acute respiratory distress syndrome and pneumonia epidemic outbreak in China.[2-5]
Taking the collective genomic information and corresponding detailed analysis, the Coronaviridae Study Group (CSG) of the International Committee on Taxonomy of Viruses (ICTV) confirmed 2019-nCoV as a "sister group" or clade to human and bat Severe Acute Respiratory Syndrome Coronaviruses (SARS-CoVs) and rename it SARS-CoV-2.[1,6]
Concomitantly, WHO proceeded to rename the disease caused by SARS-CoV-2 as Coronavirus Disease 2019 with the acronym of COVID-19, the current common name associated with the ongoing pandemic outbreak.[1,6]
Furthermore, genomic sequence analysis equally revealed a high level of homology (96.2%) shared between human SARS-CoV-2 and the bat SARSr-CoV RaTG13 strain suggesting a potential direct zoonotic transmission of SARS-CoV-2 from bats to humans without the need of an intermediate animal reservoir.[1,2]
Although pangolins or scaly anteaters were initially suspected to be an intermediate animal reservoir of SARS-CoV-2[7], virome data sets from pangolins and SARS related coronaviruses from bats disproved this possibility.[8]
Out of the group of Severe Acute Respiratory Syndrome-Related Coronavirus (SARS-CoVs), two other coronavirus, namely SARS-CoV the causative agent of SARS and MERS-CoV the viral pathogen responsible for MERS, have been related to two other major acute respiratory distress syndrome (ARDS) outbreaks.[1,9,10]
The current spillover of SARS-CoV-2 from bats to humans is equally supported by the remarkably greater homology between SARS-CoV-2 and the bat SARSr-CoV RaTG13 strain (96.2%)[2] by comparison to those found between SARS-CoV-2 and SARS-CoV (79.6%)[2,6] and between SARS-CoV-2 and MERS-CoV (50%)[6], respectively.
Interestingly, however, SARS-CoV the causative agent of SARS and SARS-CoV-2 the pathogen causing COVID-19 share very similar receptor binding domains (RBDs) both binding to their putative receptor Angiotensin Converting Enzyme 2 (ACE2).[1,11-13] These similarities are not shared with MERS-CoV the infectious agent causing MERS which binds to CD26 (dipeptidyl peptidase 4 [DPP4]).[1,14]
From a clinical perspective, this high degree of homology is crucial for the design of novel selective bio-therapeutics using gene-editing technologies as well as immediate therapeutic strategies in combination with other compounds currently available.[1]
Based on the strongest affinity of SARS-CoV-2 RBD to its putative ACE2 receptor when compared to that of SARS-CoV[1,11-13] as well as the proposed non-Clathrin non-Caveolae receptor-mediated endocytosis and pH dependent intracellular processing of SARS-CoV-2RBD/ACE2 complexes, on March 27, 2020 we moved forward with the potential clinical use of known lysosomotropic agents to hinder SARS-CoV-2 infectivity and transmission.[1]
The present clinical trial protocol provides the optimal combinations and set up of an open randomized clinical trial using lysosomotropic agents (PO) for the prophylactic prevention and attenuation of SARS-CoV-2 infectivity and progression of COVID-19.
Briefly, a combination of Chlorpromazine and Azithromycin or of Hydroxychloroquine Sulphate in combination with Azithromycin will be tested in patients who have either tested positive for SARS-CoV-2 as determined by Real Time Polymerase Chain Reaction (RT-PCR) or individuals who have been exposed to those being infected and have been clinically confirmed as suffering from COVID-19.
2. Rationale
The three-dimensional structure of SARS-CoV-2 Receptor Binding Domain (RBD) displays a quasi-identical molecular spatial conformational dynamics to that of SARS-CoV.[15] This high degree of similarity is crucial because both SARS-CoV and SARS-CoV-2 are known to bind to Angiotensin Converting Enzyme 2 (ACE2) as their putative receptor.[1,11-13]
Briefly, as previously described[1], SARS-CoV-2/ACE2 receptor complexes are likely to gain entry to lower respiratory host cells through non-Clathrin non Caveolae endocytic vesicles. The apparent higher affinity and avidity of SARS-CoV-2 RBD towards ACE2 at pH 7[16], could explain in part the high level of transmission due to an effective early entry processing of endocytic vesicles.
However, the strong binding of SARS-CoV-2 RBD to ACE2 at pH 7, can equally be used to hinder subsequent pH-dependent intracellular viral processing. It is possible that by chemically increasing the acidic pH of endocytic vesicles containing SARS-CoV-2/ACE2 complexes with the use of lysosomotropic agents, late endocytic processing and effective viral replication could be abrogated.[1]
Based upon our experience with reversal of intracellular iron release from Transferrin/Transferrin Receptor complexes through an intracellular increase of pH within late endosomes, we proposed the use of two common and clinically approved lysosomotropic agents such as Chlorpromazine and Hydroxychloroquine Sulphate to attenuate viral replication and infectivity of SARS-CoV-2.
In addition, due to the synergistic effect found between Hydroxychloroquine Sulphate and Doxycycline[1] in the treatment of bacterial endocarditis, the use of doxycycline or azithromycin in patients infected with SARS-CoV-2 proves to be essential in the clinical management of COVID-19 patients.
Despite of the relatively lower fatality rate of SARS-CoV-2, which as of to date stands at 5.36%, by comparison to those of SARS-CoV (9.6%) and MERS-CoV (34.4%), SARS-CoV-2 has led to significantly more deaths, currently sitting at 69, 428, than SARS-CoV and MERS-CoV combined.[1]
Apart from enacting social distancing measures in most countries, including Canada, Mexico and the United States, the potential use of lysosomotropic agents in the general population for primary prophylactic purposes could translate into the mitigation and potential abrogation of the infectivity and transmission of SARS-CoV-2/COVID-19. Patients enrolled in this clinical trial will enable us to determine the efficacy, safety and efficiency of the proposed clinical combinations of approved lysosomotropic agents such as Chlorpromazine or Hydroxychloroquine Sulphate together with Azithromycin or Doxycycline.
3. Study Design
3.1 Coordination of the Study
Primary and Principal Investigator in Canada:
Dr Gabriel Pulido-Cejudo
Chief Scientist
ICAHRIS Research
gabriel.pulido-cejudo@icahris.org
Co-Investigators in Canada and United States:
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Peter Humphries CIO Canadian Federation of Breast Diseases peter.humphries@icahris.org |
Dr Khadija El Abdaimi Clinical Scientist ICAHRIS Research k.elabdaimi@icahris.org |
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Dr Bratati Kar Research Scientist ICAHRIS Research b.kar@icahris.org |
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3.2 Chief Study Characteristics
Main characteristics of the study are as follows:
3.2.1 Study type: Randomized controlled two arms interventional clinical trial.
3.2.2 Expected Number of Patients per Centre: Ongoing open, aiming at an optimal enrolment of 600 individuals with an expeditious participation of 60 patients.
3.2.3 Allocation of participants to experimental treatment: Participants to the ongoing two-arm interventional clinical trial will be assigned to corresponding experimental protocols using the Simple randomization method.
4. Inclusion and Exclusion Criteria
4.1 Inclusion Criteria
Individuals that comply with the following screening criteria will be included in the study:
4.1.1 Being 18 years of age and older of all genders and self-described sexual categorization.
4.1.2 Reactive to SARS-CoV-2/COVID-19 testing by Real Time PCR (RT-PCR)[2] within the first 48 hours after being confirmed as a COVID-19 positive patient using upper or other respiratory sample. SARS-CoV-2/COVID-19 RT-PCR positivity using other standardized body samples will also be considered acceptable.
4.1.3 Presenting a combination of at least three of the following symptoms:
4.1.3.1 Fever > 37.9°C
4.1.3.2 Cough
4.1.3.3 Chest tightness / pain
4.1.3.4 Dyspnoea
4.1.3.5 Expectoration
4.1.3.6 Malaise
4.1.3.7 Dizziness
4.1.3.8 Palpitations
4.1.4 Asymptomatic patients suffering from or already hospitalized for:
4.1.4.1 Diabetes
4.1.4.2 Emphysema / pulmonary disorders (this group will be excluded from taking Chlorpromazine)
4.1.4.3 Liver disorders
4.1.4.4 Kidney disorders
4.1.5 Otherwise healthy individuals or front-line health professionals exposed to SARS-CoV-2/COVID-19 positive asymptomatic and or to patients currently being treated for COVID-19.
4.1.6 Signed Informed Consent Form (Appendix 1) by patient or legal representative.
4.2 Exclusion Criteria
Individuals with the following health profile will be excluded:
4.2.1 Patients with advanced stages of COVID-19 not capable of taking oral medications and/or taking medications with potential adverse interactions with Chlorpromazine, Hydroxychloroquine Sulphate, Azithromycin or Doxycycline.
4.2.2 Pregnant or breast feeding women.
4.2.3 Individuals requiring haemodialysis.
4.2.4 Patients with autoimmune diseases, malaria, porphyria cutanea tarda, rheumatoid arthritis or any other disease for which they are currently taking Hydroxychloroquine Sulphate.
4.2.5 Patients currently taking Azithromycin or Doxycycline.
4.2.6 Patients suffering from any mental disorder for which they are currently taking Chlorpromazine or for which Chlorpromazine is contraindicated.
4.2.7 Patients with known allergies against or suffering from a current medical condition for which Chlorpromazine, Hydroxychloroquine Sulphate, Azithromycin or Doxycycline has been contraindicated.
4.2.8 Individuals showing a Prolonged QT as revealed by electrocardiogram (QTc ≥ 470 milliseconds for women and QTc ≥ 450 milliseconds for men.
4.2.9 Patients with reduced left ventricular function at less than 30% ejection fraction.
4.2.10 Individuals suffering from retinopathy or glaucoma.
4.2.11 Individuals already enrolled in any other clinical trial.
5. Arms and Interventions
5.1 Enrolment and immediate desirable sample size patient population
Enrolment will proceed immediately with a desirable population of 600 individuals with a minimum of 10% (60) of this sample size to commence Simple randomization and treatment. A two arm intervention and control group will proceed as follows:
5.1.1 Control: This group will receive standard hospital care and practice with no experimental treatment.
5.1.2 Chlorpromazine and Azithromycin: This experimental group will receive both oral Chlorpromazine and Azithromycin or if required, Chlorpromazine and Doxycycline as described in Table 1.
| Experimental Treatment | Chlorpromazine | Azithromycin* |
| Day 1 | 200 mg twice a day | 500 mg once a day |
| Day 2-5 | 200 mg twice a day | 250 mg once a day |
| Day 6-7 | 200 mg once a day | |
| Day 8-10 | 100 mg once a day |
Table 1 Patients in this intervention group will commence within 48 hours of being diagnosed with COVID-19 (please refer to Section 4.1.2 of Inclusion Criteria). These patients should not be offered acetaminophen or any other medication contraindicated while taking Chlorpromazine.
*If during the course of the experimental treatment adverse reactions against Azithromycin were to be developed, the following doses of Doxycycline will be used: Day 1: 200 mg twice a day. Day 2-5: 200 mg once daily.
5.1.3 Hydroxychloroquine Sulphate and Azithromycin: This experimental group will receive both oral Hydroxychloroquine Sulphate and Azithromycin or if required, Hydroxychloroquine Sulphate and Doxycycline as described in Table 2.
| Experimental Treatment | Hydroxychloroquine Sulphate | Azithromycin* |
| Day 1 | 200 mg twice a day | 500 mg once a day |
| Day 2-5 | 200 mg twice a day | 250 mg once a day |
| Day 6-7 | 200 mg twice a day | |
| Day 8-10 | 100 mg once a day |
Table 2 Patients in this intervention group will commence within 48 hours of being diagnosed with COVID-19 (please refer to Section 4.1.2 of Inclusion Criteria).
*If during the course of the experimental treatment adverse reactions against Azithromycin were to be developed, the following doses of Doxycycline will be used: Day 1: 200 mg twice a day. Day 2-5: 200 mg once daily.
6. Viral infectivity and RT-PCR Assays from Study Participants
6.1 Determination of SARS-CoV-2 in vitro Infectivity from Nasopharyngeal Swab Samples
Aliquots of 0.5ml of eluents from nasopharyngeal swab samples obtained from all participants will be used for in vitro infectivity assays preferably using Vero E6 or Huh7 cells lines as per originally described by Peng Zhou et al.[2] Original nasopharyngeal swab sample eluents as well as supernatants of in vitro infected cells (Vero E6 or Huh7) will be assessed for SARS-CoV-2 by RT-PCR.[2]
6.2 Routine Assessment of SARS-CoV-2 in Nasopharyngeal Swab Samples from Study Participants
As part of the daily clinical assessment of enrolled patients during the 14 days of experimental treatment, determination of SARS-CoV-2 in nasopharyngeal swab samples will be performed every second day using RT-PCR[2] for a total of 7 determinations per patient. Additional clinical information considered valuable and pertinent by the Principal Investigator and Co-Investigators from the Participating Centre during routine evaluation of enrolled individuals will also be used to assess the overall impact of the experimental treatment by comparison to the control group.
7. Data Gathering and Analysis
7.1 Centric Digitalization of Data Entry and Analysis
A powerful centric digital platform, the Health Profile Navigator™ (HPN™), will be used throughout the study to:
7.1.1 Collect baseline patient information;
7.1.2 Perform data capturing and analysis;
7.1.3 Define inclusion and exclusion criteria;
7.1.4 Perform study data entry and control;
7.1.5 Perform post-clinical trial surveillance and follow up.
These parameters will be securely gathered within a special SARS-CoV-2/COVID-19 Data Operational Tab (DOT).
7.2 Data Characterization and Statistical Analysis
Sample characterization will be realized by performing distribution graphics of important variables and calculating summary statistics. We will compare the demographic characteristics of those participating in each arm of the study and within the control sample population.
A Chi-square test will be used to assess the effect of experimental group assignment on the proportion of patients achieving significant improvement over the control group (those achieving an undetectable viral load faster than the control group). We will also conduct multivariate analyses of factors, including the intervention arms, associated with the primary interventions.
If results indicate that certain factors in the original multivariate model have no effect, only key factors will be kept in the final model. We will assess changes in mediating factors from baseline to exit by Chi-squared test or Mantel-Haenszel and Chi-squared test for categorical measurements and paired t test or Wilcoxon rank sum test for continuous measures.
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