key: cord-0986322-aibo2wsx
authors: Cruz, L. R.; Baladron, I.; Rittoles, A.; Diaz, P. A.; Valenzuela, C.; Santana, R.; Vazquez, M. M.; Garcia, A.; Chacon, D.; Thompson, D.; Perera, G.; Gonzalez, A.; Reyes, R.; Torres, L.; Perez, J.; Vazquez, D. M.; Lemus, G.; Rosales, M.; Ramon, A. C.; Perez, G. V.; Guillen, G.; Muzio, V.; Perera, Y.; Perea, S.; group, ATENEA-Co-300
title: Treatment with an Anti-CK2 Synthetic Peptide Improves Clinical Response in Covid-19 Patients with Pneumonia. A Randomized and Controlled Clinical Trial
date: 2020-09-15
journal: nan
DOI: 10.1101/2020.09.03.20187112
sha: 9761018c62f5d80401031e5af5690f44f3185d0d
doc_id: 986322
cord_uid: aibo2wsx

Background: The recently discovered instrumental role of CK2 in the SARS-Cov2 infection has pointed out this protein kinase as a promising therapeutic target in Covid-19 disease. Accordingly, anti-SARS-Cov2 activity has been reported by CK2 inhibitors in vitro, however any anti-CK2 clinical approach has been assessed in Covid-19 patients so far. Here, we investigated the putative clinical benefit of CIGB-325, an anti-CK2 peptide previously used in cancer patients, which was added to the standard-of-care to treat Covid-19. Methods: A monocentric, randomized standard-of-care controlled trial of intravenous CIGB-325 in adults hospitalized with Covid-19. Twenty patients were randomly assigned to receive CIGB-300 (2.5 mg/kg/day during 5-consecutive days) plus standard-of-care (10 patients) or standard-of-care (10 patients). Primary outcomes were time to viral clearance defined as the time (in days) to have nasopharyngeal SARS-Cov2 negative PCR and clinical response. This trial is registered with https://rpcec.sld.cu/trials/RPCEC00000317-En (Code: IG/CIGB300I/CV/2001). Results: Most of the patients had initial positive by chest-computed tomography (CT). CIGB-325 treatment reduced both number of pulmonary lesions and lesion's extent compared to control group in seven days. Taking into account the Covid-19 chest-CT abnormalities, CIGB-325 was also superior to control as well as in terms of proportion of patients with such clinical benefit. Improvement of clinical status was experienced in 50% of patients in the CIGB-325 group and 25% in control. Accordingly, systemic levels of CPK, LDH and CRP were lowered at day 7 by treating with this anti-CK2 peptide. Both therapeutic regimens were similar respect to SARS-Cov2 clearance in nasopharynx swabs over the time. Conclusion: Our study revealed that consecutive-5 day regimen of intravenous CIGB-325 at 2.5 mg/kg quickly improved the chest-CT outcomes over standard-of-care. This is the first report describing signs of clinical benefit of an anti-CK2 approach in Covid-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide 58

Papilloma Virus (HPV) [24] , and Herpes simplex-1 (HSV-1) [25] . Furthermore, CK2 88 also regulates a number of signaling cascades such as NF-κB [26] , JAK / STAT [27] , and 89 PTEN / PI3K / Akt-PKB [28] , that viruses hijacked to guaranteed their propagation. 90 Therefore, it is expected that pharmacological intervention of CK2 may impact on viral 91

replication. Recently, CK2 has been found to be directly targeted by the SARS-Cov2 92 nucleocapsid protein and they both co-localize along the filopodia protrusions that 93 promote virus egress and rapid cell-to-cell spread across epithelial mono-layers of 94 infected cells [29] . Additionally, inhibition of CK2 in those experiments demonstrated 95 the instrumental role of this protein kinase for SARS-Cov2 infection in vitro [29] . 96

Considering the scientific rationality of inhibiting CK2 in SARS-Cov2 infection and 97 taking advance of CIGB-325 (formerly CIGB-300) as an anti-CK2 synthetic peptide 98 previously already assessed in different Phase I-II in cancer patients, we investigated the 99 putative clinical benefit of this peptide in Covid-19 patients. Particularly, safety and 100 tolerability of intravenous delivery of CIGB-325 has been previously assessed from 0.2 101 to 12.8 mg/kg [30, 31] as well as the consecutive-5 day regimen of administration. 102

In this clinical study we administered intravenous CIGB-325 at 2.5 mg/kg along with 103 standard-of-care for treating SARS-Cov2 positive patients in Cuba which is based on 104 alpha 2b-IFN plus kaletra/hydroxyquinoline. Preliminary data indicated that combination 105 of CIGB-325 with standard-of-care improved chest-CT outcomes in Covid-19 patients 106 with pneumonia at day 7. Other signs of clinical benefit for this therapeutic regimen were 107 also registered in our study. This is the first clinical study where an anti-CK2 approach is 108 investigated in Covid-19 disease. 109 . 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) preprint

The copyright holder for this this version posted September 15, 2020 . . https://doi.org/10.1101 size, the difference between groups (for clinical and chest-CT evaluations, proportion of 154 patients with reduction of score and number of lesions) was estimated from the Bayesian 155 point of view, performing 10,000 simulations and specifying non-informative prior 156 distributions. The analysis was performed using SPSS 25.0 software and EPIDAT 3.1. 157 . 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) preprint

The copyright holder for this this version posted September 15, 2020. .

Between June 1, 2020, and June 16, 2020, twenty SARS-Cov2 positive patients 160 underwent randomization in the hospital "Luis Diaz Soto" in Havana, Cuba. Ten were 161 assigned to receive intravenous CIGB-325 + standard-of-care (Group I) and 10 to receive 162 standard-of-care as control (Group II). To diminish the chance of disease progression, the 163 Cuban Program for managing Covid-19 patients is based on the application of standard-164 of-care immediately upon confirmation of nasopharyngeal SARS-Cov2 diagnosis 165 irrespectively of having symptoms or not. The median number of days between SARS-166 Cov2 diagnosis, hospitalization and treatment initiation was 2 days. All the patients 167 completed the treatment as planned except one patient from Group I who experienced 168 some concomitant moderate histaminergic adverse events during the first CIGB-325 169 intravenous delivery and dose was decreased to 1.6 mg/kg for remaining days as 170 indicated in the clinical protocol. 171

The mean age of patients was 45.35 years and 70% were male (Table 1) . Fourteen 172 patients (70%) were asymptomatic at the enrollment and 9 (45%) at the starting day of 173 treatment. Fifty percent of patients (5/10) had moderate disease and 20% (2/10) were 174 severe in the CIGB-325 group while 40% of patients (4/10) had moderate disease and 175 none severe case at the starting day of treatment (Table 1) . 176

Overall, 25% of patients had hypertension, 25% had obesity and none had type 2 177 diabetes. Other comorbidities were anemia (1/20), glaucoma (1/20), hypothyroidism 178

(1/20), cancer (1/20). The most substantial imbalance in baseline characteristics was 179 observed between the CIGB-325 group and control respect to age and coexisting high 180 risk conditions which were more unfavorable in the CIGB-325 group (Table 1) . 181

Data from baseline chest-CT analysis showed that 80% of patients in the CIGB-325 182 group had positive chest-CT according to presence of ground-glass opacity, 183 consolidation, mixed pattern and affectation of more than three pulmonary lobules. In the 184 control group, 50% of patients had positive chest-CT with presence of consolidation, 185 mixed pattern and affectation of less than two pulmonary lobules. 186

Concerning hematological and biochemical baseline parameters in all patients, abnormal 187 levels were observed in hemoglobin (25%), platelets (15%), neutrophils (50%), 188 . 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) preprint

The copyright holder for this this version posted September 15, 2020. . lymphocytes (40%), aspartate aminotransferase (ASAT) (20%), alanine aminotransferase 189 (ALAT)(40%), ferritin (35%), creatinine (20%), and glycemia (30%). Other parameters 190 were in the normal range. 191

The dynamic conversion from positive to negative of SARS-Cov2 RT-PCR results was 193 analyzed in both groups of treatments at 0, 3, 7, and 14 days. No significant differences 194 were observed in the median time, 11 days  8.0 for CIGB-325 plus standard care and 12 195 days  6 for standard care alone (p = 0.614). Thus, time to SARS-Cov2 viral clearance in 196 the nasopharynx swabs behaved similarly for both treatments over the time. 197

The clinical status was classified in categories of asymptomatic, mild, moderate or severe 198 disease and it was followed up until ending treatment and thereafter for both groups. In 199 the intent-to-treat population of CIGB-325 group at day 6 there were 2/10 patients that 200 remained asymptomatic since initiation of treatment, 2/10 changed from severe to 201 moderate, 1/10 from moderate to asymptomatic, 1/10 from mild to asymptomatic and 202 4/10 did not change their clinical status during the treatment. Otherwise, in the control 203 6/10 patients remained asymptomatic since initiation of treatment, 1/10 changed from 204 mild to asymptomatic and 3/10 did not change their clinical status during the treatment. 205

Additionally, chest-CT analysis was performed to investigate the effect of CIGB-325 206 over the Covid-19 pulmonary lesions. For that purpose, both number and lesion's extent 207 at day 0 and after treatment (day 7) were compared just in those patients analyzed per 208 protocol (Table 2) . Importantly, CIGB-325 treatment significantly reduced the median 209 number of pulmonary lesions from 9.5  10 (day 0) to 5.5  10 (day 7) (p = 0.042). 210

Conversely, no substantial change was observed in the control group. Proportion of 211 patients with reduction of pulmonary lesions was higher in the CIGB-325 group 212 compared with control according to the Bayesian analysis (pDif > 0; 0.951) ( Table 2) . 213

The effect over lesion's extent showed in Table 3 including those patients analyzed per 214 protocol indicates that CIGB-325 treatment also reduced the median of lesion's extent 215 after consecutive-5 day regimen although not significantly. Otherwise, any kind of 216 reduction was observed in the control group. Importantly, proportion of patients with 217 reduction of lesion's extent was higher in the CIGB-325 group (4/7) compared with 218 control (1/9) (pDif > 0; 0.982) (Bayesian analysis). 219 . 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) preprint

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The overall chest-CT response was also analyzed considering the number of lesions, 220 lesion's extent and the pattern of Covid-19 typical chest-CT abnormalities ( Table 4 ). The 221 analysis per protocol showed that 50% (3/6) of patients in the CIGB-325 group had a 222 favorable overall chest-CT response compared with 28.6% (2/7) in the control (pDif > 0; 223 0.796) (Bayesian analysis). Importantly, any patient had progression in the CIGB-325 224 group and 28.6% (2/7) in the control group did it. Representative images of chest-CT 225 evolution from one CIGB-325 treated patient are shown in Figure 1 . Data from safety analysis indicated that intravenous CIGB-325 added to standard-of-care 235 did increase both frequency of adverse events and patients with adverse events (Table 5) . 236

Particularly, pruritus, flushing and rash were increased by CIGB-325 treatment in the 237 100, 80 and 60 % of patients respectively. As previously observed during CIGB-325 238 intravenous administration, intensity of this kind of adverse events was mild and/or 239 moderate in all of the patients. 240

. 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. protective effect from tissue damage. High serum CPK levels have been associated to 284 rhabdomyolysis, weakness and heart injury in Covid-19 patients; therefore, these findings 285 also support the clinical benefit of using CIGB-325 in this viral disease. Accordingly, 286 LDH and CRP serum levels which are considered inflammatory markers were also 287 reduced after CIGB-325 treatment although no significantly. 288 Improvement of the clinical status at day 7 also trended to be superior by adding CIGB-289 325 to the standard care where 50% (4/8) patients experienced change towards a more 290 favorable clinical category. Otherwise, standard care alone improved clinical status in 291 25% (1/4). However, as this analysis takes into account only symptomatic patients in 292 both groups, confirmation of the CIGB-325 effect in larger size population must be of 293 major priority in future studies of this anti-CK2 peptide in The dynamic conversion from positive to negative of SARS-Cov2 RT-PCR at the 295 nasopharyngeal swabs was very similar for both groups of treatments over the time. 296

Addition of CIGB-325 to the standard care did not shorten the time to viral clearance 297 respect to the standard care at least in the regimen tested in our clinical trial. Other 298 protocols with higher frequency of CIGB-325 administration merit to be explored for that 299 purpose in future trials. Nonetheless, the usefulness and feasibility of the viral end point 300 . 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 this version posted September 15, 2020 . . https://doi.org/10.1101 in nasopharyngeal swabs to assess anti-SARS-Cov2 drugs has not been established yet. 301

Instead, clinical endpoints seem to be more informative in the Covid-19 disease [11, 15] . 302

The safety profile of CIGB-325 plus standard care was very similar to that observed for 303 CIGB-325 alone in which a predominant pattern of transient histaminergic-like side 304 effects has been observed [30] . However, intensity of the side effects was mild and/or 305 moderate in all the patients and total resolution of them was achieved in no more than one 306 hour after CIGB-325 administration. Thus, our study confirms that combination of 307 intravenous CIGB-325 at 2.5 mg/kg with standard care for Covid-19 is a manageable and 308 safe strategy to treat patients with this infectious disease. 309

All in all, our study gave important clues suggesting a potential quick clinical benefit at 310 day 7 by using our anti-CK2 approach which has not been reported so far in Recently, a clinical trial with RDV has reported clinical benefit in terms of the number of 312 days to recovery of the hospitalized Covid-19 patients with lower respiratory tract 313 involvement in 11 days (95% confidence interval [CI], 9 to 12), [15] , however evidences 314 of chest-CT improvement at day 7 were not provided. 315

Finally, integration of our data leads to the speculation that CIGB-325 might prevent 316 COVID-19 getting worse by inducing an antiviral effect in the lungs and preventing 317 damage caused by the virus. This notion merits further verification in future larger 318 clinical trials of CIGB-325 in Covid-19 patients. 319

Limitations 320

In this exploratory study with a small sample size, randomization allowed a balance 321 between the groups regarding the allocation of treatments but not regarding the 322 prognostic variables. 323 . 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) preprint

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Combination of CIGB-325 anti-CK2 peptide with standard-of-care based on alpha 2b-325 IFN/kaletra/hydroxychloroquine was adequately tolerated and superior in improving a 326 quick chest-CT response in Covid-19 patients. On the contrary, SARS-Cov2 dynamics in 327 nasopharyngeal swabs was similar for both groups over the time. Our clinical findings 328 support future studies to continue to improve patient outcomes in Covid-19 by optimizing 329 CIGB-325 regimens and combining with other antiviral agents. This is the first clinical 330 study using an anti-CK2 approach in Covid-19 and our findings suggest that CK2 331 inhibition can be an antiviral strategy with favorable risk-benefit to fight SARS-Cov2 332 infection. 333

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The copyright holder for this this version posted September 15, 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. (which was not certified by peer review) preprint

The copyright holder for this this version posted September 15, 2020. . * Per protocol population defined as patients who completed the originally allocated and patients that have TAC at the beginning and at the end of treatment.

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The copyright holder for this this version posted September 15, 2020. . * Per protocol population defined as patients who completed the originally allocated and patients that have TAC at the beginning and at the end of treatment . 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) preprint

The copyright holder for this this version posted September 15, 2020. . 0.796 * Per protocol population defined as patients who completed the originally allocated and patients that have TAC at the beginning and at the end of treatment.

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