A randomized phase 2 study on demeclocycline in patients with mild-to-moderate COVID-19

Tetracyclines exhibit anti-viral, anti-inflammatory, and immunomodulatory activities via various mechanisms. The present study investigated the efficacy and safety of demeclocycline in patients hospitalized with mild-to-moderate COVID-19 via an open-label, multicenter, parallel-group, randomized controlled phase 2 trial. Primary and secondary outcomes included changes from baseline (day 1, before the study treatment) in lymphocytes, cytokines, and SARS-CoV-2 RNA on day 8. Seven, seven, and six patients in the control, demeclocycline 150 mg daily, and demeclocycline 300 mg daily groups, respectively, were included in the modified intention-to-treat population that was followed until day 29. A significant change of 191.3/μL in the number of CD4+ T cells from day 1 to day 8 was observed in the demeclocycline 150 mg group (95% CI 5.1/μL–377.6/μL) (p = 0.023), whereas that in the control group was 47.8/μL (95% CI − 151.2/μL to 246.8/μL), which was not significant (p = 0.271). The change rates of CD4+ T cells negatively correlated with those of IL-6 in the demeclocycline-treated groups (R = − 0.807, p = 0.009). All treatment-emergent adverse events were of mild-to-moderate severity. The present results indicate that the treatment of mild-to-moderate COVID-19 patients with demeclocycline elicits immune responses conducive to recovery from COVID-19 with good tolerability. Trial registration: This study was registered with the Japan Registry of Clinical Trials (Trial registration number: jRCTs051200049; Date of the first registration: 26/08/2020).

conducted between September 1, 2020 and March 31, 2022 at four institutes in Osaka, Japan.The last entry was on September 22, 2021, when the delta variant of SARS-CoV-2 was prevalent in Japan.Twenty-three hospitalized patients who provided informed consent and were screened were randomized into three groups: a control group (n = 7), demeclocycline 150 mg daily group (n = 8), and demeclocycline 300 mg daily group (n = 8) (Fig. 1).Among the 23 randomized patients, one in the demeclocycline 150 mg group and one in the demeclocycline 300 mg group did not receive the study drug and, thus, were excluded from efficacy and safety analyses.Furthermore, one patient in the demeclocycline 300 mg group was excluded from the safety analysis due to the withdrawal of consent.Therefore, 20 patients (control group (n = 7), demeclocycline 150 mg group (n = 7), and demeclocycline 300 mg group (n = 6)) were included in the modified intention-to-treat (mITT) population.
Baseline characteristics of patients.The baseline characteristics of the 20 patients in the mITT population were similar across groups (Table 1).The lowest oxygen saturation (SpO 2 ) concentration was 95% at room air in each group.All patients in each group were diagnosed with pneumonia by chest imaging (chest X-ray or computed tomography).The most frequent coexisting conditions were hypertension (20.0%) and diabetes mellitus (20.0%).The median duration from symptom onset to the initiation of the study treatment was 7.5 days (range, 4 to 19 days).www.nature.com/scientificreports/Preventing the exacerbation of COVID-19.Kaplan-Meier estimates of the cumulative incidence of dexamethasone treatment due to COVID-19 exacerbations by day 8 were 42.9% in the control group (95% CI 7.6% to 75.7%), 14.3% in the demeclocycline 150 mg group (95% CI 0.5% to 49.1%), and 40.0% in the demeclocycline 300 mg group (95% CI 3.1% to 78.6%) (Fig. 2A).Subsequently, on or after day 8, there were no changes in the cumulative incidence of dexamethasone treatment within each group.Furthermore, no significant differences were observed in the time to dexamethasone treatment between the control group and demeclocycline 150 and 300 mg groups (p = 0.139 and 0.388, respectively).To minimize the effects of T cell responses, the concurrent administration of dexamethasone was prohibited during the course of the study.Consequently, the study treatment was discontinued upon the administration of dexamethasone.Concurrent treatments in each group are shown in Supplementary Table 1.Kaplan-Meier estimates of the cumulative incidence of continuous oxygen therapy due to respiratory failure in COVID-19 by day 8 were 16.7% in the control group (95% CI 0.5% to 54.9%) and 0.0% in the demeclocycline treatment groups (Fig. 2B).Thereafter, on or after day 8, there were no changes in the cumulative incidence of continuous oxygen therapy within each group.Moreover, no significant differences were observed in the time to continuous oxygen therapy between the control group and demeclocycline 150 and 300 mg groups (p = 0.159 and 0.181, respectively).
Average changes in oxygen saturation measured by pulse oximetry (SpO 2 ) were 0.7% in the demeclocycline 150 mg group, 0.2% in the demeclocycline 300 mg group, and 0.2% in the control group (Supplementary Fig. 1).Differences in the average change in SpO 2 from the control group to the demeclocycline 150 and 300 mg groups were 0.5% (95% CI − 0.8% to 1.8%) and 0.0% (95% CI − 2.2% to 2.3%), respectively, which were not significant (p = 0.205 and p = 0.487, respectively).The National Early Warning Score (NEWS) and seven-category ordinal scale are shown in Supplementary Tables 2, 3, and Supplementary Fig. 2. The highest rate (85.7%) of patients discharged without treatment by day 29 was observed in the demeclocycline 150 mg group.  2 and Supplementary Fig. 3).There were no significant differences in the rate of the negative conversion of SARS-CoV-2 RNA between the control group and demeclocycline 150 and 300 mg groups (p = 0.343 and 0.333, respectively).

SARS-CoV
T cell and cytokine responses after treatment.T cell and cytokine responses were analyzed from blood samples collected at baseline (prior to the study treatment on day 1) and on day 8. Blood samples collected on day 8 were obtained prior to the administration of dexamethasone, if applicable.Due to the administration of dexamethasone prior to blood sample collection on day 8, samples were not obtained from two patients in the control group, one in the demeclocycline 150 mg group, and two in the demeclocycline 300 mg group.Furthermore, a blood sample from one patient in the demeclocycline 300 mg group was not obtained due to patient withdrawal.The study treatment was discontinued before day 8 due to dexamethasone or oxygen therapy 2 1 2 The average change rates of CD8 + T cells from day 1 to day 8 were 44.1% in the control group, 40.7% in the demeclocycline 150 mg group, and 57.4% in the demeclocycline 300 mg group (as shown in Table 3).The difference in the average change rate of CD8 + T cells between the control group and demeclocycline 150 mg group was -3.48% (95% CI − 81.95% to 74.99%), which was not significant (p = 0.539).Since no significant differences were observed between the control group and demeclocycline 150 mg group, a comparison of the average change rate of CD8 + T cells between the control group and demeclocycline 300 mg group was not performed according to the closed testing procedure.Regarding the average change in the CD8 + T cell count from day 1 to day 8, the differences between the control group and groups treated with demeclocycline at doses of 150 and 300 mg were 10.1/μL (95% CI − 149.6/μL to 169.8/μL) and 36.3/μL(95% CI − 225.0/μL to 297.6/μL), respectively (as shown in Table 3).These differences were not significant (p = 0.445 and p = 0.373, respectively).However, average changes in CD8 + T cells from day 1 to day 8 showed slight variations, particularly in the demeclocycline 150 mg group, which was recorded as 109.5/μL(95% CI − 1.6/μL to 220.6/μL; p = 0.026), whereas average changes were 135.7/ μL in the demeclocycline 300 mg group (95% CI − 301.6/μL to 572.9/μL; p = 0.157) and 99.4/μL in the control group (95% CI − 60.5/μL to 259.3/μL; p = 0.080) (as shown in Table 3).Changes in HLA-DR + CD8 + T cell counts from day 1 to day 8 in the demeclocycline-treated groups were 71/μL, 54/μL, and 31/μL, respectively (Table 4).These results indicate that CD8 + T cells exhibited a weak response following the treatment with demeclocycline.
In contrast to CD8 + T cells, a greater number of disparities were observed in CD4 + T cells between the control group and demeclocycline-treated groups.The average change rates of CD4 + T cells from day 1 to day 8 were Table 3. Changes in and change rates of T cells from day 1 to day 8 of the study treatment.3).Differences in the average change rate of CD4 + T cells between the control group and demeclocycline 150 and 300 mg groups were 24.55% (95% CI − 70.53% to 119.63%) and 36.05%(95% CI − 121.62% to 193.72%), respectively, which were not significant (p = 0.287 and p = 0.298, respectively).
These results indicate that CD4 + T cells showed a response following the administration of demeclocycline.Furthermore, the change rates of CD4 + T cells in the demeclocycline-treated groups correlated with those of CD8 + T cells and CD19 + B cells (R = 0.831, p = 0.006, and R = 0.765, p = 0.016, respectively) (Supplementary Fig. 4).An analysis of the relationship between the change rates of T cells (CD4 + and CD8 + ) and cytokines (IL-6, TNFα, and IFN-γ) in the demeclocycline-treated groups revealed no correlations, except for a negative correlation between the change rates of CD4 + T cells and IL-6 (R = -0.807,p = 0.009) (Fig. 3A and B).In conjunction with these results, patients treated with demeclocycline exhibited an increase in CD4 + T cell counts, which correlated with a decrease in IL-6 levels following treatment, suggesting the attenuation of COVID-19.
At the initiation of the study treatment, SARS-CoV-2 antibodies in three patients were positive.The durations from symptom onset to the initiation of the study treatment for these patients were seven, eleven, and thirteen days, respectively.Among patients whose antibodies were negative at treatment initiation, there was a 75.0%(3/4) positivity rate for SARS-CoV-2 antibodies in the control group, 83.3% (5/6) in the demeclocycline 150 mg group, and 100% (2/2) in the demeclocycline 300 mg group on day 8 of the study treatment.These results suggest that B cell counts and SARS-CoV-2 antibodies slightly increased in each group.

COVID-19 symptoms.
On day 1, the most prevalent symptom among each group was cough, with a prevalence of 57.1% (4/7) in the control group, 71.4% (5/7) in the demeclocycline 150 mg group, and 83.3% (5/6) in the demeclocycline 300 mg group (as shown in Supplementary Fig. 6).There was a reduction in the percentage of patients with cough from day 1 to day 15 across all groups.Additionally, there were patients reporting symptoms of chest pain, dyspnea on exertion, and dysgeusia on day 1 within each group.The percentage of patients with these symptoms also decreased from day 1 to day 15.

Safety.
Treatment-emergent adverse events (TEAEs) were reported by one (14.3%),one (14.3%),and three (50.0%)patients in the control, demeclocycline 150 mg, and demeclocycline 300 mg groups, respectively (as shown in Table 5).The severity of all TEAEs was mild to moderate.Notably, hyperuricemia was observed in four patients, all of whom were concurrently taking favipiravir.Treatment-related adverse events (TRAEs) were reported by zero (0.0%) and two (33.3%)patients in the demeclocycline 150 and 300 mg groups, respectively.The two TRAEs reported in the demeclocycline 300 mg group were rash and diarrhea.

Discussion
In the present study, the administration of low-dose demeclocycline for patients with mild-to-moderate COVID-19 was well tolerated and demonstrated potential in mitigating the exacerbation of COVID-19.The number of CD4 + T cells significantly increased in individuals with mild-to-moderate COVID-19 who were administered 150 mg of demeclocycline on a daily basis.The change rate of CD4 + T cells in the demeclocycline treatment group positively correlated with those of CD8 + T cells and CD19 + B cells, and negatively correlated with IL-6 levels.These results suggest that patients with COVID-19 treated with demeclocycline exhibit immune responses indicative of recovery from the virus.
Patients with mild-to-moderate COVID-19 were recruited in the present study.Immunological responses varied based on the severity of the disease.In the RECOVERY trial, no clear benefit of the dexamethasone treatment was observed among patients with COVID-19 who did not require oxygen support.However, the mortality rate was lower among patients receiving oxygen support who were administered dexamethasone than in the group receiving standard care 14 .These findings may be partially attributed to the immunosuppressive effects of dexamethasone on immune responses in patients with mild-to-moderate COVID-19.Antiviral T cell responses play a vital role in the recovery of patients with mild-to-moderate COVID-19 16 .Therefore, based on previous findings, it was hypothesized that tetracyclines may be effective therapeutic agents for mild-to-moderate COVID-19, particularly regarding enhancements in T cell responses 24 .
In the present study, a significant increase was observed in the population of CD4 + T cells, including activated HLA-DR + CD4 + T cells, among patients with mild-to-moderate COVID-19 who were treated with demeclocycline.Previous studies indicated that CD4 + T cells, rather than CD8 + T cells, were the first to respond in patients with mild COVID-19 during the first two weeks of symptom onset 25 .Although CD4 + and CD8 + T cells both respond in patients with mild COVID-19 26,27 , it is possible that CD4 + T cells respond first, followed by CD8 + T cells.Furthermore, in the present study, the change rate in CD4 + T cells positively correlated with that in CD8 + T cells, but negatively correlated with that in IL-6, which is consistent with previous findings 28,29 .Additionally, IL-6 levels were shown to be elevated in patients with severe COVID-19 and decreased during the recovery phase of COVID-19 30 .These findings support the hypothesis that demeclocycline may be an effective treatment for COVID-19 by increasing the population of CD4 + T cells, which correlated with a decrease in IL-6.
The present study had a number of limitations that need to be addressed.We did not conduct an analysis of SARS-CoV-2-specific T cells, which is crucial for understanding the immunological response to SARS-CoV-2 in patients with COVID-19.Furthermore, due to the small cohort size, it was challenging to evaluate clinical responses to the demeclocycline treatment.During the course of this clinical study, we encountered significant difficulties in both patient enrollment and study treatment completion.To minimize the effects of T cell responses, patients who had been vaccinated against COVID-19 were excluded from the study, and the concurrent use of corticosteroids, including dexamethasone, was prohibited during study treatment.Since the majority of Japanese citizens had rapidly received COVID-19 vaccines, there were a limited number of eligible COVID-19 patients who had not been vaccinated.Furthermore, all of the COVID-19 patients enrolled in this study were diagnosed with pneumonia via chest imaging and were generally indicated for dexamethasone treatments when demeclocycline treatments were terminated.We estimated that 8 patients were needed in each three groups, and actually, a total of 23 patients were randomized for the present study.However, the discontinuation of the study treatment, attributed to the administration of dexamethasone, resulted in a reduction in the cohort of patients assessed for T cell responses.Contrary to our hypothesis of CD8 + T cell responses, CD4 + T cells increased in the demeclocycline treated group.Nevertheless, a significant difference was not observed for the change rate of CD4 + T cells, as influenced by relatively small cohort size.Therefore, further large cohort studies are needed to confirm our results.
In summary, the present study suggests that patients with mild-to-moderate COVID-19 treated with low-dose demeclocycline exhibited T cell responses that were favorable for recovery from COVID-19.However, further studies with larger sample sizes are needed to confirm and expand upon these results.

Methods
Study design.The present study was an open-label, multicenter, parallel-group, randomized controlled clinical trial involving patients diagnosed with mild-to-moderate COVID-19.Participants with mild-to-moderate COVID-19 were planned to be randomly assigned in a 1:1:1 ratio to one of three groups: a control group (normal treatment), a group receiving 150 mg of demeclocycline once daily, and a group receiving 300 mg of demeclocycline (150 mg twice daily).Individuals allocated to the demeclocycline groups received a daily dose of 150 mg once daily or 150 mg twice daily for a period of 14 days.
This clinical trial was conducted in accordance with the guidelines set forth in the Declaration of Helsinki.The study protocol received approval from the Certified Review Board of Osaka University (CRB5180007) and each individual participating institution.Written informed consent was obtained from patients prior to their inclusion in the study.This study was registered with the Japan Registry of Clinical Trials as jRCTs051200049 (https:// jrct.niph.go.jp/ en-latest-detail/ jRCTs 05120 0049).
Patients.We prospectively identified patients aged between 20 and 75 years with SARS-CoV-2 infection confirmed by a positive reverse transcription-polymerase chain reaction (RT-PCR) or SARS-CoV-2 antigen tests that were approved by the Ministry of Health, Labour and Welfare in Japan.Inclusion criteria were mild-tomoderate COVID-19 without the need for oxygen support.Exclusion criteria were an SpO 2 concentration < 93% at room air and any COVID-19 vaccination prior to randomization.
Treatment.Patients in the demeclocycline 150 mg daily group and demeclocycline 300 mg daily group (administered as 150 mg twice daily) received the study drug orally in the form of demeclocycline 150 mg capsules for a period of 14 days.The concurrent use of corticosteroids, including dexamethasone, immunosuppressants, and immunostimulants, or biologics, such as vaccines and antibody drugs, was prohibited during the study treatment, except for sotrovimab or casirivimab plus imdevimab.If a patient required the administration of dexamethasone due to the exacerbation of COVID-19, the study treatment was discontinued.
Randomization.The patients were randomly assigned to one of three groups; control, demeclocycline 150 mg daily, demeclocycline 300 mg daily groups.The allocation ratio was set at 1:1:1.We used stratified randomization by institute with permuted blocks (block size = 3).In this study, an open-label design was applied.The reason for this design was that the primary endpoint was the enhancement of the T cell response, which is an objective measure devoid of evaluator bias.The statistician, who was responsible for this study, generated the allocation list confidentially and implementation was managed via the electronic data capture system (Datatrak Enterprise Cloud ® ).
Outcomes and assessments.The primary and secondary outcomes of the present study included changes from baseline (day 1, prior to the study treatment) in T cells, cytokines, and SARS-CoV-2 RNA on day 8.The primary outcome was the change rate of peripheral CD8 + T cells.Secondary outcomes included the change in CD8 + T cells, change rates of CD4 + T cells, CD19 + B cells, cytokines (IL-6, TNF-α, and IFN-γ), and the rate of the negative conversion of SARS-CoV-2 RNA.The measurement of CD4 (anti-CD4-FITC, clone SK3, Becton Dickinson), CD8 (anti-CD8-FITC, clone SK1, Becton Dickinson), and CD19 (anti-CD19-FITC, clone B4, Beckman Coulter) was performed using FACS Calibur (Becton Dickinson) at LSI Medience (Tokyo, Japan).In the middle of the study, to analyze activated T cells, the measurement of HLA-DR (anti-HLA-DR-PE, clone

Figure 2 .
Figure 2. (A) Kaplan-Meier curves for the cumulative incidence of the dexamethasone treatment due to COVID-19 exacerbations.(B) Kaplan-Meier curves for the cumulative incidence of continuous oxygen therapy due to respiratory failure in COVID-19.

Figure 3 .
Figure 3. Relationships between change rates of T cells and cytokines in demeclocycline treatment groups (n = 9).(A) Correlations between the change rates of CD8 + T cells and cytokines (IL-6, TNF-α, and IFN-γ) in the demeclocycline treatment groups.(B) Correlations between the change rates of CD4 + T cells and cytokines (IL-6, TNF-α, and IFN-γ) in the demeclocycline treatment groups.

Table 2 .
Negative conversion of saliva SARS-CoV-2 RNA levels on day 8.Control group

Table 5 .
Adverse events (safety analysis population) over 29 days.