key: cord-0984339-4rt0w5ij
authors: Medhat, Mohamed A.; El-Kassas, Mohamed; Karam-Allah, Haidi; Al Shafie, Ahmed; Abd-Elsalam, Sherief; Moustafa, Ehab; Hassany, Sahar M.; Salama, Marwa; Abd Elghafar, Mohamed S.; Sayed, Hamdy; Badr, Mohamed; Kamal, Dalia T.; Shamseldeen, Ahmed; Ossimi, Ashima'a; Moaz, Inas; El-deen Esmael, Hossam; Ezz Eldin, Azza M.; Ezzat, Sameera; Abdelghaffar, Hossam; Abdelghaffar, Khaled
title: Sofosbuvir/Ledipasvir in Combination or Nitazoxanide Alone are Safe and Efficient Treatments for COVID-19 Infection: A Randomized Controlled Trial for Repurposing antivirals
date: 2022-05-06
journal: Arab J Gastroenterol
DOI: 10.1016/j.ajg.2022.04.005
sha: bda3561e67207dbbd1f06ea7b32233c123db3d32
doc_id: 984339
cord_uid: 4rt0w5ij

BACKGROUND AND STUDY AIMS: Currently, there is no therapy approved for COVID-19. We evaluated the efficacy and safety of sofosbuvir/ledipasvir and nitazoxanide for the treatment of patients with COVID-19 infection. PATIENTS AND METHODS: A multicenter, open-label randomized controlled trial included one hundred and ninety patients with non-severe COVID-19 infection. Patients were randomized into three groups. All groups received standard care supportive treatment (SCT). In addition, group 1 received sofosbuvir/ledipasvir, and group 2 received nitazoxanide. Follow-up by reverse-transcriptase polymerase chain reaction (RT-PCR) was done at intervals of 5, 8, 11, and 14 days. The primary endpoint was viral clearance. RESULTS: Viral clearance was significantly higher in the sofosbuvir/ledipasvir and nitazoxanide groups compared to the SCT group in all follow-up intervals (p < 0.001). In the sofosbuvir/ledipasvir arm, 36.9% showed early viral clearance by day 5. By day 14, 83.1% of the sofosbuvir/ledipasvir group, 39.7% of the nitazoxanide group, and 19.4% of the SCT group tested negative for SARS-CoV-2. Sofosbuvir/ledipasvir and nitazoxanide treatment were the only significant factors in Cox regression of negative RT-PCR with the highest OR (17.88, 95% CI: 6.66–47.98 and 2.59, 95% CI: 1.11–6.07, respectively). No mortality or serious adverse events were recorded. CONCLUSION: The addition of sofosbuvir/ledipasvir or nitazoxanide to the SCT results in an early and high viral clearance rate in mild and moderate patients with COVID-19. These drugs represent a safe and affordable treatment for COVID-19.

January 2020, it was identified as the cause of an epidemic of pneumonia in the Chinese city of Wuhan, which led to a subsequent global pandemic. [2] To date, there is no specific effective antiviral treatment for COVID- 19 . In most cases, the disease has a mild or moderate course, although up to 5%-10% of the patients develop a severe, potentially life-threatening disease. A global effort is being made to find an effective treatment against COVID-19. These efforts include trying lopinavir (LPV)/ritonavir (RTV), interferon (IFN) β-1a, hydroxychloroquine/chloroquine [3] , and ivermectin [4] , which have not proven to be successful.

Coronaviruses are large, positive-sense RNA viruses, including alpha, beta, delta, and gamma genera [5] . The coronavirus replication machinery is a large multi-subunit complex [6] . The most highly conserved protein in all known RNA viruses is the viral monomeric RNA-dependent RNA-polymerase (RdRp). The [6] re are now several drugs, such as favipiravir (FVP) [7] and remdesivir [8] , which bind to the RdRp active site and have been approved to treat other RNA viral diseases.

The available data about SARS-CoV-2 shows a viral genome replication similar to that of the hepatitis C virus (HCV) [9] . The virus enters the cell by endocytosis, is uncoated, and open reading frames 1a and 1b (ORF1a and ORF1b) of the positive-strand RNA is translated to produce nonstructural proteins, including cysteine and serine proteases, helicase, and RdRp.

Recent data suggest that HCV direct-acting antiviral drugs (DAAs), which are used in HCV management, might be effective against the SARS-CoV-2 virus through RdRp targeting [10, 11] .

Sofosbuvir is a HCV-DAA with NS5B RdRp inhibitory activity. It has been used in combination with several NS5A protein inhibitors, such as daclatasvir or ledipasvir, with high efficacy for HCV treatment [12] . The introduction of DAAs was considered a milestone in HCV eradication in Egypt, with a sustained virological response rate of almost 100%, using certain DAA combinations [13] .

Molecular docking was performed to test several HCV-DAAs, including sofosbuvir, against SARS-CoV-2 RdRp, and the results were promising. Therefore, DAAs might have inhibitory activity against the newly emerged coronavirus [14] .

Nitazoxanide, a drug developed initially as an antiprotozoal agent, has a broad-spectrum antiviral activity and can inhibit the replication of a wide range of RNA and DNA viruses in cell-culture assays. It exhibits in vitro activity against MERS-CoV and several coronaviruses [15] . In humans, a Phase IIb/III clinical trial found that oral administration of nitazoxanide reduced the duration of clinical symptoms and viral shedding compared to placebo in individuals with influenza [16] .

Currently, sufficient data are not available on the efficacy of DAAs or nitazoxanide in treating patients with COVID-19 infection. Therefore, we aimed to evaluate the efficacy and safety of sofosbuvir/ledipasvir and nitazoxanide to treat COVID-19 infection. Both drugs are inexpensive and available throughout the world.

All patients in this multicenter open-label randomized controlled trial were COVID-19 positive.

COVID-19 diagnosis was based on positive reverse-transcriptase polymerase chain reaction (RT-PCR) testing for SARS-CoV-2 on nasopharyngeal swabs. Patients either attended, 1-2 days after diagnosis, to the outpatient clinic or were admitted to the quarantine hospitals of Helwan University Hospital, Al-Rajhy Liver University Hospital, and Tanta University Hospital, Egypt.

Patients were included between July 2020 and October 2021 and were assigned randomly according to a randomization number generated by an independent statistician and provided in 

The participants were assigned randomly according to a randomization number generated by an independent statistician and provided in pre-sealed envelopes. Individuals involved in randomization and masking had no involvement in the rest of the trial.

The trial was conducted in accordance with the principles of the Declaration of Helsinki. Ethical approvals were obtained from the relevant ethics committees in the participating hospitals.

Before conduction, this study was registered on the clinicaltrials.gov website, with registration number NCT04498936.

Data were collected and analyzed using Statistical Package for Social Science program for statistical analysis (version 13; Inc., Chicago. IL). The means, standard deviations, frequencies, and percentages were used as appropriate. Chi-square was used to measure the association between qualitative variables. One-way analysis of variance test was used to compare the three groups having normally distributed quantitative data, whereas the Kruskal-Wallis test was used when these data were not normally distributed. Kaplan-Meier survival analysis was utilized to explore the duration needed to get an RT-PCR negative event. If the patient had two consecutive negative RT-PCR, the time to event was considered the date of the first one. The Fisher exact test was then done to compare the three groups at each point of time. The Cox regression model gave an adjusted hazard ratio (odds ratio (OR)) and 95% confidence interval of the effect of the different risk factors for survival. A p-value of <0.05 was considered statistically significant.

The sponsor of this study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. All authors had full access to all the data in the study, and the corresponding author had final responsibility for the decision to submit for publication.

The study included one hundred and ninety patients in the three groups. Group 1 included 70 patients who received sofosbuvir/ledipasvir with SCT; Group 2 included 77 patients who received nitazoxanide with SCT; Group 3 included 73 patients who received SCT alone. As shown in Table 1 , there was no significant difference in the baseline demographic characteristics of the three groups except for gender (p =0.035). More than half were females in Group 1 (54.3 %) and Group II (64.9%), whereas males predominated in Group 3. The common comorbidities were hypertension and diabetes mellitus, with the highest frequency in Group III, but without significant difference (p = 0.471 and 0.445, respectively). Other comorbidities, such as obstructive lung disease, chronic liver or kidney diseases, and rheumatic heart disease, were of lower frequency (n = 9).

The comparison between the studied groups regarding symptoms and signs is shown in Table 2 .

No significant differences were found among groups regarding fever, heart rate, respiratory rate, blood pressure, and oxygen saturation, as shown in Table 2 . Oxygen therapy, steroids, and anticoagulants were used more frequently in Group 3, followed by Groups 2 then group 1, without statistical difference. The baseline laboratory data are summarized in Table 4 .

Lymphocyte percentages were significantly lower in Group 2 (p = 0.049), while direct bilirubin was significantly lower in Group 1 ( p = 0.013). Laboratory markers of infection, such as CRP, LDH, ferritin, and D-dimer, were not statistically different among all groups.

The cumulative incidence of negative RT-PCR following treatment, analyzed by the intention to treat method, reveals an overall significant difference among all groups at each follow-up interval (p < 0.001) (Figure 1 ). The highest percentage of negative RT-PCR at each follow-up was observed with group 1 (sofosbuvir/ledipasvir therapy), then group 2 (nitazoxanide), followed by group 3 (SCT). In group 1, 42.9% of the patients tested negative on day 5. By the end of the follow-up, at day 14, 85.7% of the patients who received sofosbuvir/ledipasvir, 35.1% of group 2, and 16.4% of group 3 tested negative by RT-PCR (Figure 1) . Similarly, the log-rank curve of the cumulative time to viral clearance ( Figure 2) shows that sofosbuvir/ledipasvir had a significantly higher treatment value compared with nitazoxanide and SCT (p < 0.001). The pairwise log-rank curve for the cumulative incidence of negative RT-PCR showed that sofosbuvir/ledipasvir is significantly higher than nitazoxanide (p < 0.001) and SCT (p < 0.001) and that nitazoxanide is significantly higher than SCT (p = 0.01). Nitazoxanide also had a higher curative rate than SCT (p = 0.01) ( Figure 2 ). The Breslow test compared the survival rates among the three groups, which was statistically significant (p < 0.001). Accordingly, sofosbuvir/ledipasvir therapy resulted in significantly earlier and higher rates of viral clearance than both nitazoxanide and SCT.

The Cox regression analysis ( however, sofosbuvir is better absorbed orally [22] . The efficacy of remdesivir was tested in randomized trials, but the results are contradictory [23, 24] . SARS-CoV-2 has an exonucleasebased proofreader to maintain viral genome integrity. Any antiviral drug targeting SARS-CoV-2

RdRp must display a certain resistance level to this proofreading activity. Sofosbuvir terminated RNA resists removal by the exonuclease to a substantially higher level than RNA terminated by remdesivir [25] .

Our study indicates a positive effect of sofosbuvir/ledipasvir as a treatment for COVID-19.

Approximately 85.7% of patients who received sofosbuvir/ledipasvir tested negative by the end of the study (day 14). The cumulative time to negative RT-PCR was significantly shorter in the sofosbuvir/ledipasvir group than in SCT (p < 0.001). No mortality or need for ICU admission was recorded in our cohort. Our study corroborates a smaller study that reported that the addition of sofosbuvir/ledipasvir to SCT accelerated clinical response [26] .

Daclatasvir is an NS5A protein inhibitor, similar to ledipasvir, and is used in combination with sofosbuvir with high efficacy in HCV management. Recently, the Roozheh et al. study on sofosbuvir/daclatasvir in mild cases of COVID-19 reported no significant alleviation of symptoms after seven days of therapy compared with the control. Fewer hospitalizations were observed in the sofosbuvir/daclatasvir group but without statistical significance [27] .

Sofosbuvir/daclatasvir treatment of patients with severe COVID-19 was reported to significantly lower hospitalization time, time in ICU, and mortality rate compared with ribavirin treatment [28] . Likewise, Sadegh et al. concluded that the addition of sofosbuvir and daclatasvir to SCT for 14 days in moderate or severe cases of COVID-19 significantly reduced the duration of hospital stay, resulting in a lower mortality rate compared with SCT alone [29] .

In the current study, we also evaluated the efficacy of nitazoxanide in the treatment of COVID-19. Negative RT-PCR was observed in 36.4% of the nitazoxanide group compared with 16.4% of the SCT group (p = 0.01). The broad-spectrum antiviral activity of tizoxanide, the active metabolite of nitazoxanide, is attributed to interference with host-regulated pathways involved in viral replication rather than a virus-targeted mechanism. These pathways might include IFN or mammalian target of rapamycin complex 1 (mTORC1) signaling [30, 31] . Nitazoxanide also upregulates host defense mechanisms that viruses target to bypass host cellular defenses [15] . In addition, nitazoxanide inhibits the production of cytokines, such as tumor necrosis factor (TNF)α and interleukin (IL)-2, IL-4, I-5, IL-6, IL-8, and IL-10 in peripheral blood mononuclear cells [15] .

Similar to the current study, Rocco et al. evaluated nitazoxanide in patients with mild COVID-19, finding that symptom resolution did not differ between nitazoxanide administered as 500 mg three times a day and the placebo group after five days of therapy. However, early nitazoxanide therapy was safe and reduced the viral load significantly. Swabs collected were negative in 29.9% of the patients in the nitazoxanide arm versus 18.2% in the placebo arm (p = 0.009) [32] .

Another study showed that early usage of nitazoxanide in combination with Ribavirin, Ivermectin, and Zinc supplements was associated with a significantly shorter duration for SARS-COV2 clearance from the nasopharynx than symptomatic therapy [33] .

Antiviral therapy for COVID-19 infection shows variable viral clearance results among different drugs. A double-blinded randomized controlled trial (RCT) revealed no superiority of Remedsivir over placebo in time to clinical recovery, 28-day mortality, or viral clearance [20] . In the efficacy of the LPV/RTV and arbidol against novel coronavirus infection (ELACOI) trial with a single-blinded RCT, including 44 patients with mild-to-moderate COVID-19, no differences in the time to negative RT-PCR test among the LPV/RTV, umifenovir, and control groups (8.5, 7, and 4 days, respectively) were reported [34] .

The rate of RT-PCR negative conversion in hospitalized patients with COVID-19 who received LPV/RTV was similar to those who received the SCT at different time points [35] . Although the use of FVP in SARS-CoV-2 infected patients was associated with significant clinical and radiological improvement, it was not superior to the SCT regarding viral clearance [36] .

However, in a multicenter randomized Phase II/III clinical trial, FVP enabled SARS-CoV-2 viral clearance was observed in 62.5% of patients within four days versus 30% in the SCT group [37] .

However, the combined use of FVP with inhaled IFN-α showed viral clearance at a significantly shorter duration when compared with combined LPV/RTV and inhaled IFN-α (4 days vs. 11 days) [38] . In a recently published study, adding sofosbuvir/daclatasvir to the SCT for COVID-19 was associated with faster PCR negativity and shorter hospital stay [39] . Molnupiravir is an antiviral drug which was initially used as a possible treatment of influenza viruses and viral encephalitis caused by alphaviruses like Venezuelan, Eastern and Western equine encephalitic viruses [40, 41] . Some in vitro and in vivo studies showed ability of molnupiravir to inhibit replication, reduce the viral load of SARS-CoV-2 virus, in addition to improvement the pulmonary function ( [42, 43] . A recently published systematic review about the use molnupiravir in COVID-19 showed that it is benifecial in reducing hospitalization or death in mild COVID-19, but its role in moderate to severe COVID-19 is debatable [44] . A novel SARS-COV-2 protease inhibitor (PAXLOVID™) produced by Pfizer Inc. showed that it can lead to a 89% reduction in COVID-19-related hospitalization or death compared to placebo [45] .

In the current study, sofosbuvir/ledipasvir resulted in earlier viral clearance than nitazoxanide and SCT. Importantly, this could shorten the duration of infectivity and the risk of viral transmission from infected patients, reducing the rapid viral spread observed in this pandemic.

Although there is a significant difference among the groups regarding gender, this factor had no significant impact on the viral clearance in the Cox regression analysis. Notably, both drugs in the regression analysis were the only significant factors affecting the viral clearance, and sofosbuvir/ledipasvir had much higher odds of obtaining viral clearance than nitazoxanide. This could result from the targeting of two viral proteins by the combination of sofosbuvir/ledipasvir, thus enhancing its antiviral activity and reducing the ability of the virus to develop resistance.

Reporting the clinical improvement as a marker for treatment efficacy might not be accurate because non-severe cases of COVID-19 have good clinical outcomes and recovery, even when using SCT regimens alone. Our primary endpoint was viral clearance by serial RT-PCR testing on nasopharyngeal swabs. Our primary endpoint enabled us to conclude that sofosbuvir/ledipasvir resulted in faster COVID-19 virus elimination. However, the small sample size of this study is a limitation that should be stated. Further multicenter studies are needed to evaluate the efficiency of sofosbuvir/ledipasvir and nitazoxanide to treat severe COVID-19

cases. Also, comparing the efficacy of different types of sofosbuvir-based DAAs therapy, such as sofosbuvir/daclatasvir, in the management of COVID-19 is recommended.

In conclusion, the addition of sofosbuvir/ledipasvir or nitazoxanide (to a lesser extent) to SCT results in an earlier and higher viral clearance rate in mild and moderate COVID-19 compared with SCT alone. Sofosbuvir/ledipasvir shows the highest efficacy in promoting viral clearance.

These drugs are well tolerated for the duration of therapy. Thus, they represent a promising, safe, and affordable treatment in the management of COVID-19. 

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Alkaline phosphatase; ALT: Alanine aminotransferase; APTT: Activated partial thromboplastin time; AST: Aspartate aminotransferase; CRP: C-reactive protein; ESR: Erythrocytic sedimentation rate

Hb: Hemoglobin; HCT: Hematocrit; INR: International normalized ratio; LDH; Lactate dehydrogenase; PLT: Platelets; WBCs: White blood cells