Phase 1 clinical trial for intravenous administration of mesenchymal stem cells derived from umbilical cord and placenta in patients with moderate COVID-19 virus pneumonia: Results of Stage 1 of the study

Background: Mesenchymal stem cells can serve as a therapeutic option for COVID-19. Their immunomodulatory and anti-inammatory properties can regulate the exaggerated inammatory response and promote recovery of lung damage. Methods: Phase-1, single-centre open-label, prospective clinical trial was conducted to evaluate the safety and ecacy of intravenous administration of mesenchymal stem cells derived from umbilical cord and placenta in moderate COVID-19. The study was done in 2 stages with total 20 patients. Herein, the results of stage 1 including rst 10 patients receiving 100 million cells on day 1 and 4 with a follow up of 6 months have been discussed. Results: No adverse events were recorded immediately after the administration of MSCs or on follow up. There was no deterioration observed in clinical, laboratory and radiological parameters. All symptoms of the study group resolved within 10 days. Levels of inammatory biomarkers such as NLR, CRP, IL 6, ferritin and D-dimer improved in all patients after intervention along with improved oxygenation demonstrated by improvement in the SpO2 / FiO2 ratio and PaO2 / FiO2 ratio. None of the patients progressed to severe stage. 9 out of 10 patients were discharged within 9 days of their admission. Improvements were noted in chest x-ray and chest CT scan scores at day 7 in most patients. No post-covid brosis was observed on chest CT 28 days after intervention and Chest X ray after 6 months of the intervention. Conclusion: Administration of 100 million mesenchymal stem cells in combination with standard treatment was found to be safe and resulted in prevention of the cytokine storm, halting of the disease progression and acceleration of recovery in moderate COVID-19.


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was rst identi ed in 2019 in Wuhan, Hubei Province, China. Due to its highly contagious nature, the World Health Organization (WHO) declared COVID-19 a global pandemic in January 2020. (1) COVID-19 spread quickly around the world and has affected millions of people so far primarily affecting the respiratory tract and lungs. The clinical spectrum of COVID-19 ranges from asymptomatic infection to critical illness. Pathophysiological studies have reported cytokine dysregulation and hyperin ammation associated with viral infection in severe and critical illness. (2) Elevated levels of pro-in ammatory cytokines result in a cytokine storm causing lung tissue edema, air exchange dysfunction, acute respiratory distress (ARDS), lung brosis, secondary infection, multiorgan failure often leading to death. Pre-existing comorbidities such as diabetes, high blood pressure, obesity, etc result in escalated complications. Mortality rate is higher in these patients who demonstrate cytokine storm. (4) Many treatment strategies have been recommended since the diagnosis of COVID-19 and lot of research is ongoing to establish a cure for the disease. Pharmacological management for treatment of COVID-19 includes hydroxychloroquine, azithromycin, remdesivir, favipiravir, to immunomodulators e.g. steroid and IL-6 antagonists (tocilizumab, itolizumab) (5). Although, these agents showed promising results in invitro studies, the mortality rate remains high in moderate and severe COVID-19. Hence, other options are being evaluated either as drug therapy or cell therapy e.g. SARS-CoV-2 entry inhibitors, Fusion inhibitors, RdRp inhibitors, NSP-15 inhibitors, umbilical cord MSCs, etc. (6) Due to its immunomodulatory properties, mesenchymal stem cells (MSC) have the potential to combat COVID-19 infection. In the past, MSC treatment was found to be safe and effective in various conditions such as pulmonary, orthopedic, neurologic, cardiac conditions, etc. It has also shown promise in other viral infections like Human immunode ciency virus (HIV), hepatitis B virus, virus associated ARDS, in uenza amongst others. (7)(8)(9)(10). Patients with mild COVID-19 usually recover at home, with supportive care and isolation. Infection-hospitalization ratio is estimated to be 0.4% for individuals younger than 40 years and 9.2% for those older than 60 years. 10-20% of infected individuals progress to severe or critical stage due to cytokine storm and about 3.5% is the mortality rate. (11)(12) As cytokine storm is known to be one of the major reasons associated with mortality in COVID-19, immunoregulatory agents may result in a positive outcome. (13) Therefore, we need to evaluate whether MSCs can help preventing the progression in moderate stage patients, help in faster recovery or reduce mortality.
This study was conducted to evaluate the safety and e cacy of mesenchymal stem cells derived from umbilical cord and placenta in moderate COVID-19 pneumonia. The primary endpoint of the study was to evaluate safety of the MSCs administered while the secondary endpoint was to evaluate the e cacy of these MSCs in moderate COVID-19 patients.

Study design and participants
This study was a Phase 1, single-centre open label, prospective clinical trial conducted at Lokmanya Tilak Municipal General (LTMG) Hospital and Medical College, Mumbai, India in collaboration with NeuroGen Brain and Spine Institute and ReeLabs Pvt. Ltd. The study was conducted in 2 stages with total 20 patients. Stage 1 of the study including rst 10 patients receiving 100 million cells has been completed with 6 months follow up. The dose was escalated to 200 million cells on day 1 and day 4 for stage 2 including next set of 10 patients which is still ongoing. Herein, the results of stage 1 with rst 10 patients have been discussed. The protocol of the study was approved by the Central Drugs Standard Control Organisation (CDSCO), Ministry of Health & Family Welfare, Government of India and the Institutional Ethics Committee of LTMG Hospital and Medical College. This clinical trial has been registered with the Clinical Trial Registry-India (CTRI) as CTRI/2020/08/027043. A written informed consent was obtained from all the patients and relatives before the intervention. An independent data safety and monitoring board (DSMB) was constituted to monitor any adverse events.
Inclusion Criteria: • Patients admitted with RT-PCR con rmed COVID-19 illness. • Patients with other severe co-morbidities like cancer, chronic renal, chronic liver failure and chronic cardiac failure. This will not include diabetes, hypertension, etc • Participating in any other clinical trial Pre-Intervention Assessment: In stage 1 (100 million cells), 10 patients who met the inclusion and exclusion criteria were recruited in the study. All these patients underwent a detailed assessment prior to the intervention. Their medical history was recorded alongwith clinical assessment. Serological, biochemical and hematological tests which included Complete blood count, Renal function test (BUN, creatinine and GFR), Liver function test, Serum Creatine Kinase (CK), Creatine Kinase Myocardial Band (CK-MB), Troponin and myoglobin test, Serum electrolytes test, Serum HbA1Ctest, Serum Fibrinogen test, D-dimer test, Serum C-reactive protein test, NT-pro BNP, HIV, HCV and HBV test was conducted. Arterial blood gas analysis (ABG), Electrocardiogram (ECG) was performed along with Chest X-ray and Chest CT scan.

Intervention:
Cell transplantation Cells used for this study were a mixture of MSCs derived from human umbilical cord blood and placenta. They were obtained from ReeLabs, Mumbai, a cGMP facility with a cord blood banking license and Form 29. The procurement and banking of these cells was done as per government guidelines. Cell culture medium used was StemProTM MSC SFM XenoFree for cell culture. All 10 patients received two doses of 100 million cells each on day 1 and day 4 along with standard treatment. Cells were suspended in 100 ml of normal saline and were administered intravenously over forty minutes with a speed of ~40 drops per minute.
Standard Treatment: All patients received standard medication which included antibiotics (cephalosporin, ivermectin, doxycycline), one antiviral (Lopinavir/ritonavir or Favipiravir or Remdisivir), low molecular weight heparin, methylprednisolone, vitamin supplements (vitamin E, vitamin C, zinc, multivitamins) and antacid (pantoprazole). Other medicines were administered for symptomatic relief such as paracetamol for fever, antitussive for cough, etc. Supplemental oxygen for patients who had oxygen saturation (SpO2) below 95% on room air. None of the patients received Tocilizumab.

Data collection:
Clinical parameters were measured daily by the clinical staff from Day 1 to discharge. Laboratory parameters, ABG and ECG were performed before the intervention (Day 1) followed by Day 2, 4, 6, 8 and 14. Chest X-Ray and Chest CT scan was performed on Day 1, 7, 14 and 28. Data was recorded in the case record form and electronically.

Data Analysis:
A detailed analysis was performed to study the outcome of the intervention. Neutrophil-Lymphocyte Ratio, CRP, IL-6, Ferritin, D-Dimer, SpO2/FiO2 ratio and PaO2/FiO2 ratio were analysed. Median of their values was calculated and a line graph with standard error was plotted to study the overall trend in the study population. Resolution of clinical symptoms was evaluated by calculating frequency of symptoms present on Day 1 and average number of days taken for resolution of these symptoms. Chest X rays were scored subjectively based on visual assessment by an expert radiologist. X -ray ndings before the treatment were noted and the days at which improvement was seen and complete resolution was seen was computed and used for analysis. Comparison of Chest CT scores on Day 1, 7, 14,21 and 28 days was performed.

Results
Demographics (Table 1) Table 1 Demographic data of the study group Follow up duration 6 months The frequency of symptoms of COVID-19 were observed in the recruited participants of this study. (Table  2) Table 2 Frequency of symptoms of COVID-19 observed in the patients recruited for this study. At 6 months, follow up X-rays were performed for 7 patients which did not reveal any signi cant abnormality. One patient had expired due to cardiac arrest after 3.5 months post intervention (unrelated to cell therapy).
Secondary Endpoint: E cacy Evaluation E cacy of intervention was assessed by evaluating the time taken for COVID-19 symptoms to resolve, changes in levels of biomarkers, oxygenation and radiological ndings.
1. Time to symptom resolution ( Table 3) Before completion of Day 14, all patients were clinically stable and discharged.

Changes in levels of biomarkers (A) Neutrophil-Lymphocyte Ratio
Neutrophil-Lymphocyte ratio is a cost-effective in ammatory biomarker used to study the progression of COVID-19. Increased NLR may be indicative of excessive in ammation and immune suppression caused due to SARS-CoV-2. Median of NLR was calculated for each patient and a line graph was plotted. In the study group, NLR increased on Day 2, decreased on Day 4 and showed an increase again on Day 6. However, Day 6 onwards median NLR reduced consistently. Overall, a decreasing trend was observed.

(D) Ferritin
The median ferritin levels were slightly higher than the normal range on Day 1 which further increased on Day 2. However, after administration of MSCs the levels of ferritin started to reduce and were found to be within normal range on Day 6. (Figure 7,8). In 4 out of 10 patients the levels showed a decreasing trend but did not normalize till Day 14. Ferritin is a key mediator of immune dysregulation, via direct immunesuppressive and pro-in ammatory effects, contributing to the cytokine storm which further results in a fatal outcome. However, as recorded in this study the median ferritin levels which were demonstrating an increasing trend started to reduce after intervention. This can be attributed to the immunomodulatory effects of MSCs.

(E) D-Dimer
The levels of D-dimer in the blood increased from Day 1 to Day 6, after which it sharply decreased and did not differ from normal values. This suggests that MSCs promote the rapid elimination of intravascular hemocoagulation, which effectively prevents microthrombosis of the lung vessels (Figure 9, 10).
3. Improvement in markers of Oxygenation: The   There are no approved treatments for Covid-19 but some medications including antiviral, antiin ammatory have shown to be bene cial. There is an urgent need to explore treatment strategies that can prevent patients from worsening clinically and progressing to severe or critical stage. Due to its immunomodulatory properties, cell therapy has a potential to halt the progression of the disease and accelerate the recovery process.
To evaluate the safety and e cacy of cell therapy in COVID-19, we administered 10 patients with moderate illness with a mixture of 100 million MSCs derived from umbilical cord and placenta.
Rationale for use of MSCs derived from umbilical cord and placenta Cellular therapy has been studied widely for treating various conditions, including pulmonary, immunological, haematological, cardiac, neurological, hepatic, endocrine, musculoskeletal, skin, and ophthalmological diseases. Multiple pro-survival pathways converge and death-inducing pathways are attenuated on a cellular level to bring about improvements in the lungs. Inhibition of pulmonary brosis and reduced collagen deposition by these cells (56-58) may lead to decreased edema and opacity in chest X-rays. Further, MSCs trapped in pulmonary circulation undergo differentiation over the long term to yield a multitude of alveolar cell types that integrate into the pulmonary tissue and improve lung function; (41,59) we may thus also expect improved tissue microarchitechture in the chest CT scans along with improved air exchange and lung function.

Antimicrobial properties
Along with immunomodulation and regeneration, MSCs also possess antimicrobial properties. Besides, these cells have shown to be ACE-2 negative and therefore cannot be infected by SARS-CoV-2. (60) Angiogenesis VEGF secreted by stem cells is a pro-angiogenic factor. Angiogenesis is critical for tissue regeneration.
(61) In the lung, angiogenesis is crucial since the blood-air interface is the source of oxygenation and oxygen delivery to the body. Through secretion of VEGF, stem cells can help recovery from lung injury.
Animal studies have shown that administration of VEGF improved aeration and prevented the development of respiratory distress syndrome and mortality in premature animals. Clinical outcome of this study Use of MSCs signi cantly decreased the time required for COVID-19 symptoms to resolve. (Table 2).
It has been reported that the median duration for symptom resolution of Covid 19 patients is16 days, (77) however, we noticed that all symptoms of our study group resolved within 10 days. Cough, sore throat, sputum, chest pain, loss of appetite, taste and smell, giddiness, resolved within 5 days. However, generalized fatigue, shortness of breath and requirement for supplemental oxygen was resolved within 10 days. 9 out of 10 patients were discharged within 9 days of their admission. Along with improved clinical symptoms, levels of in ammatory biomarkers such as C-reactive protein, interleukin 6, ferritin and Ddimer also improved in all patients after intervention. There was no deterioration observed in clinical and laboratory parameters. None of the patients progressed to severe stage of Covid. Improved oxygenation was recorded in all the patients which was demonstrated by improvement in the SpO2 / FiO2 ratio and PaO2 / FiO2 ratio. None of the patients showed any major or minor adverse events immediately after intervention or on follow up after 6 months. One patient with history of diabetes mellitus expired due to cardiac arrest after 3.5 months post intervention which was unrelated to cell therapy. Cardiac

Radiological ndings
Radiological investigations like Chest X-ray and Chest CT scan showed no adverse effects of the MSCs transplantation on lung tissue. In addition, no post-covid brosis was observed on Chest CT post 28 days of the treatment and Chest X ray post 6 months of the treatment.
On chest X-ray, 8 out of 10 patients showed signi cant bilateral lung in ltrates. 7 (87.5%) out of these 8 patients resolved completely in average 19 days. Improvement was noted in the radiographs at Day 7 in most patients. Warissara Kiththiworaphongkich, 2021 showed that the improvement was seen in X-rays post 13 days of the illness. (80) However, in this study improvement was seen post 7 days of treatment suggesting early resolution of lung pathology.
Temporal changes in the lung tissue involvement studied previously on a CT scan shows that CT scores peak during illness days 6-11. (81,82) In this study, 90% of the patients receiving MSCs transplantation showed reduction in the CT score at day 7 and the scores continued to reduce thereafter, suggesting better resolution of lung pathology post treatment.

Conclusion
Stage 1 of this phase 1 clinical trial wherein 10 patients with moderate COVID-19 infection were administered two doses of MSCs (100 million cells each on Day 1 and Day 4) was found to be safe and effective. No adverse events were noted. Early resolution of symptoms, improved in ammatory markers, improved oxygenation and halting of disease progression observed in the study population after intervention, can be attributed to the ability of MSCs to immunomodulate, reduce in ammation, prevent cytokine storm, promote angiogenesis, improve oxidative stress and oxygenation in moderate COVID-19.
The combination of MSCs derived from umbilical cord and placenta with standard treatment may be further explored as an effective therapeutic strategy for COVID-19. The regenerative and repair potential of these cells may also help in better post Covid recovery. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Competing Interests: 2. Wang C, Huang P, Wang L, Shen Z, Lin B, Wang Q, Zhao T, Zheng H, Ji W, Gao Y, Xia J. Temporal changes of COVID-19 pneumonia by mass evaluation using CT: a retrospective multi-center study.