key: cord-1036615-qsrmr1dm
authors: Blumberg, E. A.; Tebas, P.; Frank, I.; Marshall, A.; Chew, A.; Veloso, E. A.; Carulli, A.; Rogal, W.; Gaymon, A. L.; Schmidt, A. H.; Barnette, T.; Jurek, R.; Noorchashm, H.; Hwang, W.-T.; Noll, J. H.; Fraietta, J. A.; June, C. H.; Hexner, E. O.
title: Phase 1 Trial of Cyclosporine for Hospitalized Patients with COVID-19
date: 2021-06-15
journal: nan
DOI: 10.1101/2021.06.10.21258714
sha: 863a290ca1ce62404c245f8ea6dee6c66091b506
doc_id: 1036615
cord_uid: qsrmr1dm

Coronavirus Disease 2019 (COVID-19) remains a global health emergency with limited treatment options, lagging vaccine rates and inadequate healthcare resources in the face of an ongoing calamity. The disease is characterized by immune dysregulation and cytokine storm. Cyclosporine A (CSA) is a calcineurin inhibitor that modulates cytokine production and may have direct antiviral properties against coronaviruses. To test whether a short course of treatment was safe in COVID-19 patients, we treated 10 hospitalized, oxygen requiring, non-critically ill patients with CSA at a starting dose of 9mg/kg/day. Five patients experienced adverse events, none were serious, and transaminitis was most common. No subject enrolled in this trial required intensive care unit (ICU)-level care and all patients were discharged alive from the hospital. Further, CSA treatment was associated with significant reductions in serum cytokines and chemokines important in COVID-19 hyper-inflammation, including CXCL10. In conclusion, short courses of CSA appear safe and feasible in COVID-19 patients requiring oxygen and therefore, may be a useful adjunct in resource-poor or resource-limited health care settings.

SARS-CoV-2, the causative agent of COVID-19, is a novel coronavirus that induces an acute respiratory disease with systemic complications that range from minimally symptomatic, self-limited disease to critical illness and death. The COVID-19 pandemic is an ongoing global health crisis. Although vaccine development has been rapid, safe and effective, treatment of disease has largely suffered from a paucity of effective antiviral drugs and variable impact of antiinflammatory agents, some of which are both costly and associated with long-term immunosuppression. Global infection rates continue to rise, vaccines are lagging, and case fatality rates vary globally but remain in the 1-10% range. As of June 2021, global infections exceeded 170 million, with over 3.7 million dead [1] . Severe pneumonia occurs in approximately 15% of cases and drives mortality. Access to critical care resources such as ventilators or even supplemental oxygen remains an unmet need in some areas more than a year into the pandemic.

COVID-19 is characterized by immune dysregulation or cytokine storm, an orchestrated response involving infected cells, effector T cells, macrophages and other innate immune cells, and the cytokines/chemokines produced that collectively result in widespread lung inflammation [2] . Hospitalized patients with severe COVID-19 exhibit high serum levels of interleukin (IL)-2, IL-7, IL-10, granulocyte colony-stimulating factor (G-CSF), tumor necrosis factor (TNF), C-X-C motif chemokine ligand 10 (CXCL10), monocyte chemoattractant protein 1 (MCP1), and macrophage inflammatory protein (MIP)1 α [3] [4] . Systemic elevations of these cytokines, Creactive protein (CRP), and ferritin accompanied by lymphopenia are frequently observed in patients with COVID-19, and are also hallmarks of patients with hemophagocytic lymphohistiocytosis), also referred to as macrophage activation syndrome (HLH) [5] .

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The copyright holder for this preprint this version posted June 15, 2021 Calcineurin inhibitors are a class of non-cytotoxic immunosuppressive drugs that selectively impair T cell function by blocking Nuclear factor of activated T cells (NFAT) signaling and downstream cytokine production. A drug screen conducted at the University of Pennsylvania identified cyclosporine as an active antiviral agent in human lung cells, blocking entry of SARS-CoV-2 [6] . Currently cyclosporine A (CSA), tacrolimus and sirolimus are widely used to prevent rejection in solid organ transplant and for the treatment of arthritis and psoriasis;

CSA is also the backbone of most protocols treating HLH. Given the shared inflammatory pathways seen in both HLH and the immune dysregulation seen in severe COVID-19 infection, we hypothesized that CSA could be an effective anti-inflammatory agent for the treatment of COVID-19-associated immunopathology.

Furthermore, CSA is not myelosuppressive and has also been shown to have direct antiviral effects by inhibiting coronavirus replication. [6, 7] . Thus, we asked whether, if properly timed in patients with COVID-19, CSA would be sufficiently safe so that ultimately it could serve as a broad-spectrum inhibitor to help control SARS-CoV-2 infection, decrease severity of cytokine storms and improve outcomes. This early intervention with a well characterized and approved medication might be of particular importance in resource-poor or resource-limited areas. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

The study treatment, cyclosporine (modified, Gengraf ® ) capsules (25 and 100mg) was given at an initial starting dose of 9mg/ kg/day orally divided into dosing every 12 hours, with a maximum dose of 400mg/dose for all subjects. An oral solution (100mg/mL) or intravenous (IV) formulation (Sandimmune ® ; 3mg/kg/day given by continuous infusion) was also available for patients unable to swallow capsules. Therapeutic drug monitoring was performed on day 2 and every Monday, Wednesday and Friday during active dosing. Subsequent cyclosporine dosing was adjusted to target a trough level of 200 to 300ng/mL without a maximum dose level. The intended duration of administration was up to 14 days with planned treatment discontinuation if mechanical ventilation was required. Treatment was held for significant elevations in creatinine or transaminases and discontinued for all patients at the time of hospital discharge.

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This was a study to assess the safety of CSA in subjects with COVID-19, as measured by treatment-related adverse events (AEs), on study ICU transfer, secondary infections, and need for mechanical ventilation or increase in supplemental oxygen requirements. Samples for exploratory measurements (e.g., serum cytokine levels) were also collected.

This safety study also defined the following events as ones that would trigger a pause of the study: moderate to severe superinfection, severe microangiopathy or Posterior Reversible leuko-Encephalopathy syndrome (PRES), also known as Reversible Posterior Leukoencephalopathy syndrome (RPLS), and subject death.

Clinical laboratory tests at screening/enrollment and post-CSA administration included complete blood counts and assessment of C-reactive protein (CRP) levels. Routine blood analysis was performed using a fully automated cell counter in the University of Pennsylvania Hematology Laboratory. CSA concentration was measured in whole blood samples using a validated Liquid Chromatography-Tandem Mass Spectrometry method .

For cytokine measurements, serum samples stored at -80ᵒC were thawed and centrifuged at 2,000 × g for 5 minutes. The LEGENDplex™ COVID-19 Cytokine Storm Panels 1 and 2 (BioLegend) were used to determine cytokine concentrations, with sample analysis performed using an LSRFortessa flow cytometer (BD Biosciences). Serum was diluted 1:2 and the procedure was carried out according to the manufacturer's instructions with the addition that capture beads were inactivated in 4% paraformaldehyde for 15 minutes at room temperature and washed once in . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 15, 2021. ; https://doi.org/10.1101/2021.06.10.21258714 doi: medRxiv preprint wash buffer after completion of the staining protocol in a Class II Biosafety Cabinet under BSL-2+ conditions.

The initial trial was designed to enroll approximately 25 patients for a target of maximum 20 patients treated with CSA. We estimated that 20% of enrolled subjects would not receive CSA, primarily due to unanticipated rapid clinical deterioration or improvement. The sample size would enable us to provide a reasonable precision to rates of adverse events: a sample size of 20, the halfwidth of 90% exact confidence interval for an AE rate would be no more than 20%. After study activation, accrual rate tracked with infection rates, and when infection rates were declining, lengths of hospital stays were decreasing and vaccines became widely available a decision was made to close the study. With a reduced sample size of 10 patients, we have probability of 0.9 that the true AE rate would be <26%, <40%, if 0 or 1 AEs are observed. Descriptive statistics were computed for all endpoints.

Serum cytokine levels were analyzed between time points using two-sided paired Student's t-tests. Statistical analyses were performed using Prism 8 GraphPad (GraphPad Software). P values < 0.05 were considered to be statistically significant.

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The copyright holder for this preprint this version posted June 15, 2021. ; https://doi.org/10.1101/2021.06.10.21258714 doi: medRxiv preprint

Eleven patients consented to the study and 10 patients were treated (Figure 1) . The median age was 57.5 years ( Table 1) . All were oxygen requiring with a median National Early Warning Score of 3 (range 2-8).

While the optimal dose is unknown for this indication, for this initial safety study a standard transplant target dose was selected to achieve a trough level of 200 to 300ng/ml. All patients received only the oral capsule formulation of CSA. The median number of days of treatment was 4 (range 2 -6), median doses received was 8 (range 3 -11). Trough levels (Figure 2 ) ranged from 83 to >500 ng/ml. The target trough level was achieved in 80% of patients, and 80% of patients had dose modifications based on therapeutic drug monitoring.

The trial opened on June 22, 2020, when remdesivir was still being investigated and continued through its approval as well as the introduction of dexamethasone as part of our standard institutional treatment protocol. All patients received both remdesivir and dexamethasone as part of standard of care treatment.

Five patients (50%, CI, 22% to 78%) experienced AEs, ( Table 2) . Transaminitis was the most common AE, there was one event each of headache and creatinine increase. Two patients discontinued treatment due to AEs. No patients required ICU-level care and all patients were . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 15, 2021. ; https://doi.org/10.1101/2021.06.10.21258714 doi: medRxiv preprint discharged alive from the hospital. There were no events of PRES/RPLS or microangiopathy, and no serious AEs.

Serum specimens were analyzed to quantify circulating pro-inflammatory cytokine/chemokine s levels using multiplex bead-based immunoassays. In most of the 10 patients accrued (n = 6), samples were collected twice, typically from day -7 to 0 (baseline) and at day 3 post-CSA administration. A subset of patients (n = 5) had cytokine measurements performed more than once after enrollment. At baseline, we detected high levels of multiple pro-inflammatory cytokines and chemokines known to characterize COVID-19-associated hyper-inflammation [3] ( Figure 3A) . Significant or near significant reductions in CXCL10, IL-10, IL-7 and IL-8 were observed on day 3 following CSA administration, relative to pre-treatment time points ( Figure   3B ). The overall inflammatory cytokine signature continued to decrease from day 3 to day 7 post-CSA administration (Figure 3A) , in association with reductions in body temperature ( Figure 3C) and non-cardiac CRP levels ( Figure 3D) . Reductions in pro-inflammatory cytokine production coincided with increased white blood cell (WBC; Figure 3E ) and absolute lymphocyte counts (ALC; Figure 3F ).

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The copyright holder for this preprint this version posted June 15, 2021. ; https://doi.org/10.1101/2021.06.10.21258714 doi: medRxiv preprint Discussion CSA is approved by the US FDA for prophylaxis of organ rejection in solid organ transplant recipients, treatment of rheumatoid arthritis and severe psoriasis. CSA, given for a short course to hospitalized patients with COVID-19 and requiring oxygen, is feasible and appears safe.

AEs were consistent with the known safety profile of CSA in other populations [8] [9, 10] . The majority of AEs were mild and no stopping rules were met. Notably, all patients improved and none required mechanical ventilation. This initial study was not designed to determine efficacy as to whether CSA impacted outcomes. An ideal point of intervention for CSA may be after a sufficient amount of time has passed following acquisition that the humoral immune response to SARS-CoV-2 has been primed, but potentially aberrant immune activation has not yet occurred. This is in contrast to immunologically naïve patients, because administration of CSA prior to infection can be deleterious [15] [10].

One advantage to the use of CSA is the cost effectiveness of this intervention for resource limited settings. Drug acquisition costs are low and this medication is widely available in both pill and liquid formulations, making it easy to administer orally to diverse populations. It is likely that lower doses can be given without a need for therapeutic drug monitoring. Because remdesivir is a substrate for cytochrome P450 3A4 (CYO3A4), organic anion transporting polypeptide 1B1 (OATP1B1), and P-glycoprotein 1 (P-gp) in vitro (reviewed in [11, 12] ), there is a potential for a significant drug interaction with CSA. However, based on our experience, this interaction appears to be manageable. Additionally, given the short recommended duration of therapy with remdesivir and the likelihood that short courses of cyclosporine will also be beneficial, we do not anticipate . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 15, 2021. significant AEs related to the co-administration of these medications. More experience with diverse populations will help further clarify this issue.

Several reports have revealed that the hyper-inflammatory response associated with COVID-19 is a major cause of disease severity and death. In this study, we detected decreased levels of several pro-inflammatory cytokines/chemokines, including CXCL10, following CSA administration compared to baseline time points. Continuously high levels of CXCL10 have been previously associated with increased viral load, loss of respiratory function, lung injury and a fatal outcome in SARS-CoV-2 infection [13] . Furthermore, it has been proposed that CXCL10 may mediate the aberrant immune response that drives the duration of mechanical ventilation in COVID-19 patients with acute respiratory distress syndrome (ARDS) [14] . CXCL10 has also been associated with disease severity of H5N1, H1N1, SARS-CoV, and MERS-CoV [15] [16] [17] [18] . Notably, CSA is a robust inhibitor of CXCL10-induced NFATc1 activation and this is a mechanism by which CXCL10 regulates the recruitment of inflammatory cells in rheumatoid arthritis [19] . Thus, modulation of CXCL10 through short course CSA treatment may be a promising therapeutic approach to prevent progression to COVID-19 related ARDS.

There are several limitations to this study. Given the variable epidemiology of COVID-19 and the changing therapeutics during our study period, our enrollment was impacted and the number studied is small. The small numbers also affected our ability to estimate whether there was a significant clinical effect for our patients, independently of those related to corticosteroids and remdesivir.

In summary, we show that CSA is a safe and potentially effective therapeutic intervention for patients with SARS-CoV-2 infection. Its anti-inflammatory properties, wide availability, safety . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 15, 2021. ; https://doi.org/10.1101/2021.06.10.21258714 doi: medRxiv preprint and low cost make it a particularly attractive modality for use in resource-limited settings. A prospective randomized controlled trial testing the efficacy of cyclosporine for the treatment of COVID-19 pneumonia is registered in Spain [20] . Based on our results further large-scale trials are warranted to explore the safety and benefits of this intervention globally.

acknowledged for day-to-day running of the CPU, sample processing, and management of the extensive patient sample bank. We thank Lester Lledo and Joan Gilmore in the Clinical Trials Unit of the Center for Cellular Immunotherapies, and Dr. Laurel Glaser for assistance with data compilation, as well as Drs. Sara Cherry and Ronald Collman for helpful advice.

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EudraCT number 2020-002123-11. EFFICACY OF CYCLOSPORINE IN THE  TREATMENT OF COVID-19 PNEUMONIA: A RANDOMIZED CONTROLLED TRIAL. https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-002123-11/ES, 2020.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) (6), Diabetes (5), hypertension (4), asthma/obstructive lung disease (2), cancer history (1), cardiomyopathy (1) . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) Grade   1  2  3  Total  Alanine aminotransferase increased  1  0  1  2  Aspartate aminotransferase increased  0  0  1  1  Creatinine increased  0  1  0  1  Hypertriglyceridemia  1  0  0  1  Hypoalbuminemia  1  0  0  1  Hypomagnesemia  1  0  0  1  Headache  0  1  1  2  Total  4  2  3  9   Table 2 . Adverse Events . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 15, 2021. ; https://doi.org/10.1101/2021.06.10.21258714 doi: medRxiv preprint

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