Tocilizumab in Covid-19 Interstitial Pneumonia: A Phase II Pilot Study

Background: Multiple studies have been conducted to investigate Tocilizumab in patients with cOVID-19 pneumonitis. However, published reports show conicting results, largely due to weak retrospective designs and heterogeneity in critical methodological issues. Methods: This open-label trial was structured according to the Simon’s optimal two-stage design in order to clarify which patients could really benet from anti-IL6 strategies and how a future randomized trial should be designed to provide reliable and unequivocal results. 46 patients received a single infusion of Tocilizumab. Inclusion criteria were: SARS-CoV2 infection diagnosed by rt-PCR, multifocal interstitial pneumonia, need of oxygen therapy (FiO2 50%) to maintain SO2 >93%, recent (within the last 24 hours) worsening of lung function. Clinical outcomes were established a priori to assess whether a patient responded to treatment. A low number of carefully chosen clinical and biological markers was measured in order to test their predictive values. Primary end point was early and sustained clinical response. Results: Twenty-one (46%) patients fullled pre-dened response criteria. Lower levels of IL-6 at 24 hours after tocilizumab infusion (p=0.049) and higher baseline values of PaO2/FiO2 (p=0.008) predicted a favorable clinical response. Patients not improving at 72 hours were also non-responder at day 7. 11/25 of non-responder patients were intubated and 7 died. High levels of vWF were detected in all sera, with a tendency towards higher concentrations in the non-responder group. Conclusions: Objective clinical response rate overcame the pre-dened threshold of 30%. Ecacy of tocilizumab to improve respiratory function in selected patients with severe COVID-19 pneumonitis warrants investigations in randomized trials. Trial registration:


Introduction
Multifocal interstitial pneumonia represents the most common cause of admission in intensive care units (ICU) and death during SARS-CoV2 infection. Available data, mostly collected on other coronavirus infections with similar clinical behaviour, highlight an intense "cytokine storm" with a consequent in ammatory in ltrate of pulmonary interstitium, macrophage activation, giant cells formation and subsequent extended alveolar damage[i] , [ii], [iii] .
Tocilizumab, an anti-IL-6 receptor monoclonal antibody, has proved effective in rheumatoid arthritis, as well as in diseases characterized by an intense systemic in ammatory activation [iv] , [v] . Moreover, small case series from China and Italy suggest that IL-6 blockade induces rapid clinical improvement in patients affected by COVID-19 interstitial pneumonia [vi] , [vii], [viii], [ix] . However, con icting results have been recently reported, in particular in patients affected by more severe disease. Indeed, favorable effect of the drug on mortality and risk of intubation[x] has not been con rmed in other studies [xi] , [xii] .
Weak retrospective designs, together with heterogeneity in enrolled population characteristics and outcome de nition and measurement hamper a reliable clinical data interpretation.
To investigate which patients with severe and worsening pneumonia could bene t of a single intravenous infusion of Tocilizumab, we designed a multicentre, prospective, open-label trial, structured according to the 'Simon's optimal two-stage design' with the goals: 1. to con rm that a signi cant proportion of patients can obtain a rapid and relevant improvement of respiratory function; 2. to explore clinical and serological parameters useful to predict clinical response; 3. to acquire elements useful to design future unequivocal randomized trials

Study Design
On March 12 th , 2020 the Internal Ethical Board of Ospedali Riuniti-Ancona (Italy) authorized off-label treatment of patients affected by SARS-CoV2 related severe interstitial pneumonia with tocilizumab.
The study was structured according to Simon's optimal two-stage design [i] . Fourteen patients (variable: n1) entered the rst stage of the trial. Enrollment would have been stopped and the drug rejected if <10% of the patients (variable: P0) had met the primary end point 72 hours after the administration of tocilizumab. Conversely, the study would have been continued until at least 46 patients were enrolled in total (variable: n). The drug would have been rejected in the case of a positive response rate <30% (variable: P1) of the patients completing the study.
The values of variables n1, n, P0, and P1 were set to obtain a probability ≤0.01 of accepting a drug worse than P0 after the rst stage and a probability ≤0.2 of rejecting a drug better than P1 at the end of the study.

Study Population
Between March 12 th and March 26 th , 46 consecutive patients were treated.
Inclusion criteria were: patients 18 to 90 years of age; SARS-CoV2 Infection diagnosed by rt-PCR, multifocal interstitial pneumonia con rmed by Rx or chest CT-scan, need of oxygen therapy (FiO2 50%) to maintain peripheral oxygen saturation (SO2) >93%, recent (within the last 24 hours) worsening of lung function, de ned as a decrease of SO2>3 % points and/or decrease of the ratio of PaO2 to FiO2 (P/F) >50% and/or P/F below 150 mm/Hg. Exclusion criteria were: severe heart failure, active bacterial infection, hematological malignancy, neutrophil count <x10 9 /L, platelet count <50x10 9 /L, Alanine Aminotransferase (ALT) 5-fold above the upper normal level (UNL), inability to give a valid informed consent.

Drug administration
Tocilizumab was administered as a single infusion of 8mg/Kg (max 800mg), after a premedication with 40 mg of 6-Methylprednisolone. Written informed consent was obtained before the infusion.
Concomitant therapies were allowed according to local protocols. No patient received systemic corticosteroids. All patients were on oxygen therapy, administered either by Ventimask (V) or C-PAP.

Assessment
Complete clinical evaluation, assessment of SO2, arterial blood gas analysis (when possible), recording of both FiO2 and type of ventilation were performed at baseline, and after tocilizumab infusion at 24 and 72 hours and day 7. Complete blood count, ALT, D-dimer, and creatinine. were also performed at the same time points.

CT-Scan
High Resolution chest CT-scan (HRCT) was performed at baseline in 33 cases and at day +7 in 21 patients.
Two independent Pulmonologists, with 10 and 12 years of experience, evaluated the images blinded for sequence and nal decisions was reached by consensus.
Assessment of radiological pattern, and de nition of the extent of parenchymal involvement were performed and scored as described [ii] , [iii].
The extent of parenchymal involvement at HRCT was rst scored by visually evaluating the percentage of lesions' involvement at lobar basis according to a 5-point categorical scale. A Total Severity Score (TSS; range from 0 to 20) was obtained by summing the scores of the ve lobes.

Response criteria
The primary outcome was the rate of responder patients. A patient was a-priori de ned as responder if ful lling either criteria 1 or 2 AND criteria 3 of the ones listed below: 1. Improvement of oxygen saturation by more than 3% points and/or increase in P/F by 50% and/or increase P/F above 150 mmHg 72 hours after tocilizumab AND persistence of this improvement at day 7; 2. No worsening of respiratory function as de ned in the inclusion criteria at 72 hours AND improvement of oxygen saturation by more than 3% points and/or increase in P/F>50% and/or increase P/F above 150 mmHg at day 7; 3. No need of endotracheal ventilation for all or CP for those not requiring it at baseline Secondary outcomes were: 1. rate of admission to intensive care unit for endotracheal intubation or evidence of multiple organ dysfunction; 2. death; 3. rate of severe drug-related adverse events.

Adverse Events
Patients were examined every day until discharge and all possible adverse events were recorded. Adverse events were classi ed according to U.S. Department of Health And Human Services criteria [iv] .

Statistical analysis
Data were expressed as median (and range or Interquartile range) unless otherwise stated. Comparisons were made using Mann-Whitney U-test or chi-square test as appropriate. Two multivariate logistic regression analyses were performed using the primary outcome as dependent binary variable and the possible prognostic factors as independent variables. As possible prognostic variables we considered age, sex, number of comorbidities, PaO2/FiO2 at baseline, heparin and HYQ as co-treatments, and either IL-6 at baseline or IL-6 at 24h. A signi cance level alpha=0.05 will be used for all the statistical analyses.

Characteristics of Patients
Since ve out of the rst 14 patients met the primary outcome, enrollment was completed up to a total of 46 cases. Demographic and clinical characteristics are summarized in Table 1. Median age was 67.5 (range 34-89) years and median disease duration before the drug infusion was 9.5 (range 2-21) days. Thirty-three patients (72%) were male and 13 (28%) were female; all Caucasian.
All patients were non-smokers, frequently affected by hypertension (63%). Five (11%) patients were affected by type II diabetes, 3 (7%) by renal failure, 2 (4%) by chronic heart failure. About half of observed patients had no co-morbidities at the time of enrolling.
All the subjects were affected by pneumonitis requiring high-ow oxygen therapy. Twenty-ve (63%) patients had severe respiratory failure, characterized by a P/F ratio <150 mmHg and 30 (65%) were on C-PAP.
HRCT, available in 33 (72%) patients, revealed a diffuse pulmonary involvement and 23 (69%) had a TSS of 8 or more, typical of the most severe patterns 12 .
Laboratory tests performed at baseline showed a moderate increase of D-dimer, lymphocytopenia and a slight elevation of ALT.

Treatment Response
According to the a priori criteria, 72 hours after tocilizumab 20 (43.5%) patients had an objective improvement and maintained the improvement in lung function at day 7. In 14 patients improvement was already apparent 24 hours after drug administration. One further patient was stable after 72 hours and improved at day 7. Overall, 21 (45.6%) of the enrolled patients could be classi ed as responder. None underwent endotracheal intubation, admission to ICU or died.
At day 7, twenty-ve patients were non-responder, although three of them had shown a transient improvement after 72 hours. Eleven (44%) of 25 were intubated a median of 2 (range 0-9) days after treatment and 4 died. Of the remaining 14 patients, 3 died and 11 were still classi ed as non-responder.
All responder patients were discharged after a median of 21 days (IQ range 17-27) after tocilizumab infusion, compared to 25.5 days (IQ range 20-34.25) of the sixteen non-responder patients. Two patients are still intubated 60 days after treatment.
Patients with P/F >150 mmHg at baseline (P=0.003) and lower CT-scan TSS (p=0.008) were most likely to bene t from tocilizumab ( Table 2).
Higher levels of IL-6 at baseline correlated with poor response (p=0.02). At 24 and 72 hours, an evident increase in IL-6 serum levels were observed both in responder and in non-responder subjects. However, the increase of IL-6 serum levels was clearly greater in non-responders (Fig 1 and Table 4).
No signi cant differences emerged between responder and non-responder groups regarding age distribution, sex, number of comorbidities, concomitant therapy with HYQ or LMWH and baseline levels of lymphocytes or D-dimer (Table 2).
No differences regarding to different CT-scan patterns emerged from the comparison between responders and non-responders.

Markers of endothelial and alveolar damage
Increased levels of vWF were detected in sera at baseline and 72 hours after drug infusion, particularly in non-responder group. Thrombomodulin levels remained stable within the normal limits at baseline and after tocilizumab, in both groups.
The serum levels of SP-D, a glycoprotein secreted by type II pneumocytes and a potential surrogate marker of alveolar damage, were elevated at baseline in 35% of patients and the concentrations rose overtime, particularly in non-responder patients (Fig 1 and Table 4).

Multivariate analysis
In a multivariable model, lower IL-6 levels measured at 24 hours (p=0.049), and higher baseline values of P/F (p=0.008) were associated with better response to the drug (Table 3). In the alternative model, IL-6 levels at baseline did not emerge as independent variable.

Safety
Adverse events were reported in 29 (63%) of patients (Table S1). The most common was an increase in aminotransferases. Four patients reported a 3-fold ALT elevation from baseline and 10 patients a 5-fold increase at day 7. All these patients showed a normalization of liver enzymes within 14 days. No case of hepatic failure was observed.
In three patients (one responder and two non-responders) signi cant neutropenia (<1x10 9 /L) appeared more than 7 days after drug infusion. Two patients (one still neutropenic) were discharged without overt bacterial infections. The third patient was a non-responder and had a severe infection caused by Corynebacterium Jeikeium and Clostridium di cile one month after tocilizumab infusion and despite normal neutrophil count.
Six bacterial infections were reported in 6 patients, all after admission to the ICU. The relationship with the experimental drug was considered uncertain by the investigators.
Three patients, on hydroxychloroquine and lopinavir-ritonavir combination, suffered clinically relevant arrhythmias. One patient, had a new episode of atrial brillation (AF) soon after tocilizumab infusion, with a spontaneous return to normal sinus rhythm within 24 hours. A second patient developed AF 24 hours after tocilizumab, and since this occurred with the worsening of respiratory function he was transferred to the ICU and intubated. The third patient developed AF within 24 hours after infusion and underwent successful pharmacological cardioversion.
After the detection of pulmonary embolism nine days after tocilizumab, one non-responder patient was anticoagulated and was discharged home once clinically improved.
Finally, in one hypertensive 77-year-old man a transient worsening of moderate-severe renal failure was observed.
No reaction has been reported during or after parenteral administration of tocilizumab.

Discussion
The present study shows that 7 days after a single dose of tocilizumab, 21 patients with severe pneumonia ful lled the responder criteria and none required admission to ICU or died. These patients were part of the 23 patients subgroup who had improved at 72 hours. The responder rate (46%) overcame the prede ned threshold of 30%, and thus the results justify future phase III randomized trials.
All 23 patients who did not improve at 72 hours were also non-responder at day 7. All the patients who died were in the non-responder subgroup.
These data indicate that the response obtained 24-72 hours after the administration of tocilizumab to patients with severe pneumonia foreshadows the likely prognosis.
At variance with what reported by others[i], high baseline values of IL-6 did not predict the clinical outcome of our patients, rather more informative were the serum levels at 24 hours which better re ected the actual generation of IL-6 and thus the severity of the in ammation, as previously described in chronic in ammatory diseases [ii]. The greater increase of the cytokine serum levels in patients who did not respond to tocilizumab support the hypothesis that IL-6 plays a major role in the pathogenesis of organ damage in this infection, as also reported by others[iii] ,[iv],[v], [vi] .
While the increasing levels of SP-D accurately illustrate the on-going lung damage, vWF and thrombomodulin performed differently, the former higher in more severely affected patients at 72 hours, the latter stable and within the normal limits at all time points. Since vWF is produced constitutively in endothelium, while thrombomodulin is an integral membrane protein expressed on the surface of endothelial cells, our ndings suggest that at least an abnormal breakdown of vWF may be involved in the microvascular damage in Covid-19 patients.
Thus, the conclusions that can be drawn from these set of data are: i. a signi cant portion of severely ill and deteriorating patients responded quickly to tocilizumab infusion; ii. Lower levels of IL-6, particularly 24h after drug infusion, higher P/F ratio and less extensive CT manifestations correlated with a better response; iii. patients not improving after 24-72 hours, as well as those with unfavorable prognostic factors, should either receive a second administration of tocilizumab, as it has been proposed for patients with the cytokine release syndrome following the chimeric antigen receptor (CAR) T cell therapy [vii] or switched to other treatment regimen Several adverse events occurred in our patients, not unexpectedly given the severity of the clinical conditions at baseline. Bacterial infections could be ascribed to the risk factors associated to the prolonged hospital stay or the viral infection itself, which rendered them susceptible to infections. The three episodes of AF, which occurred within 24 hours from tocilizumab, seem to point to a direct relationship with the medication. However, it is di cult to consider tocilizumab the sole culprit since: i. arrhythmias are frequent in COVID-19 patients[viii]; ii. Tocilizumab seems to reduce the incidence of disorders of cardiac rhythm and cardiac events in in ammatory diseases[ix] , [x] ; iii all three patients' cotreatments could have, at least, contributed to the rhythm abnormalities.
The most relevant elements of our prospective study are the following: i. treatment of a homogeneous population of patients; ii. enrollment of patients with severe disease manifestations and poor prognosis; iii. pre-speci ed clinically relevant outcomes; iv. sample size determined to provide the power required by the Simon's optimal two-stage design.
Our study has, however, some weaknesses. First, it was a small, open-label trial without a control group. It cannot be ruled out that the improvement observed in some patients was due to a milder disease.
However, in our opinion, the enrollment of patients with severe pneumonia and recent worsening of lung function in the previous 24 hours, and appraising as responder only those who rapidly ameliorated after treatment, mitigated this problem. Second, due to the frantic days of the pandemic, there was quite a number of missing data, albeit equally distributed between the responder and non-responder subgroups. Complete reports were, however, available from two-thirds of the enrolled patients. Third, the small study group makes the multivariate analysis exposed to the risk of "random effects", thus, the data should be compared with those of on-going prospective studies.

Conclusion
A single infusion of 8mg/Kg of tocilizumab appears a promising strategy to rapidly improve respiratory function in patients with severe and rapidly worsening COVID-19 lung disease, particularly with a P/F ratio >150mm/hg and lower generation of IL-6.