Academic Editor: Sang Heui Seo
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of Coronavirus disease 2019 (COVID-19), which was announced as a pandemic leading to devastating economic and medical burden worldwide. The virus attacks the organ system across the body by binding to its receptor (for example, angiotensin converting enzyme 2) on the surface of the host cell of various organs. The patients present with a variety of pathological symptoms ranging from fever, cough and cytokine storm to acute respiratory distress syndrome (ARDS). Many combination therapies have been developed to combat the disease, via blocking one or more processes of the viral life cycle and/or relieving host complications simultaneously. In this review, the progress of those combination therapies containing at least one small molecule is updated. We believe it’ll provide significant inspiration for further development of treatment strategy against SARS-CoV-2, especially its mutant variants.
COVID-19 is an infectious disease caused by SARS-CoV-2 virus. It had killed more than 6.12 million of persons worldwide as of 29 March 2022. While the majority of patients with COVID-19 will recover without special treatment, a minority will manifest severe symptoms requiring hospitalization and even suffer serious complications, including ARDS, which may induce multi-organ dysfunction. Considering SARS-CoV-2 is a unprecedented virus and radical treatment against the resulting disease remain to be developed, a clear interpretation of the viral life cycle is essential for designing prophylactic and/or therapeutic strategies that target one or more processes of its life cycle. SARS-CoV-2 affects human starting from entering the nasopharynx by attaching to angiotensin converting enzyme 2 (ACE2) receptor-enriched epithelial cells of the nasal and oral mucosa. Then it goes down and attacks the lung, where the infection mainly takes place. The following processes include endocytosis, replication, transcription, assembly and release of virus .
SARS-CoV-2 invades human cells through interaction of the receptor binding domain (RBD) of its spike (S) protein with the host ACE2 on the cell surface . The SARS-CoV-2 S protein belongs to class 1 viral fusion protein and it means that the S protein should be cleaved into S1 and S2 subunits by a fusion protein before functioning normally. S1 contains the receptor binding domain (RBD), which directly binds to the peptidase domain (PD) of ACE2, whereas S2 is responsible for the fusion between the viral envelope and the host cell membrane and internalization by endocytosis with ACE2 [3, 4]. Various proteases including cathepsins, trypsin, human airway trypsin-like proteases, furin and transmembrane protease serine protease (2/4) (TMPRSS) have been reported to cleave S protein in a proteolytic way in those harmful coronaviruses since 2003 [5, 6, 7, 8]. That a specific furin-like cleavage site was discovered in the S-protein genome sequence of SARS-CoV-2 indicates furin and furin-like proteases may be involved in the S protein cleavage . We have known that SARS-CoV-2 uses TMPRSS2 as well as furin or furin-like proteases, for its interaction with the ACE2 receptor and entry into the host cell. The study by Hoffmann et al.  proposed a rational role of TMPRSS2 in proteolysis of S protein to S1 and S2 subunits for S protein priming and camostat mesylate, an inhibitor of TMPRSS2, blocked SARS-CoV-2 infection of lung cells. Furthermore, the data showed that both furin and TMPRSS2 could not replace each other functionally and suppression of either of them might interfere virus to bind to host cells. Indeed, many experimental pieces of evidence recently have made clear that these two proteins act synergistically in viral entry and infectivity and shed light on the combination of furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2 . Further processing that the S1 subunit is removed in host cell endosomes is promoted by cathepsins, which eventually assists the fusion of viral envelope with the host membranes, viral RNA release, and replication . Overall, virus S protein, ACE2 and the host proteases play the essential roles in SARS-CoV-2 entry in host cells. Other than ACE2, several other receptors were also discovered including DC-SIGN (also known as CD209), L-SIGN (also known as CD209L or CLEC4M), SIGLEC1 (also known as CD169, sialoadhesin or Siglec-1) [12, 13] and neuropilin-1 (NRP1, known to bind furin-cleaved substrates) .
In case of the release of the viral genome into the host cell, the SARS-CoV-2 RNA acts as messenger RNA (mRNA) and undergoes translation by using host cell machinery . The viral genome comprises a positive-sense, single-stranded RNA molecule with about 29,900 nucleotides which encode about 31 proteins: nonstructural proteins (NSPs; 16 proteins), structural proteins (4 proteins), and accessory proteins (11 proteins) . The structural proteins consist of S protein, envelope (E) and membrane (M) proteins which form the viral envelope, and nucleocapsid (N) protein which connects with the virus genome. After entering host cell, the NSPs domain is first translated into two polypeptides, both of which are further processed to produce papain like protease (PLpro) (NSP3), main protease (Mpro) (also known as 3-chymotrypsin-like protease (3CLpro); NSP5) , and RNA-dependent RNA polymerase (RdRp; NSP12) . Interestingly, the process is promoted by host proteases at the first stage, and then, is strengthened by PLpro and Mpro. The viral RdRp is key for the replication of genetic material. The viral genome and the N structural protein are produced in the host cell cytoplasm, while other viral structural proteins including S, M, and E are finally synthesized in the endoplasmic reticulum and transferred to the Golgi apparatus. The viral RNA–N complex and S, M, and E proteins are then packaged together to form a virion by a budding process. Following packaging, the complete virus particles are transported to the cell surface in vesicles and released by exocytosis [19, 20]. These newly released virions continue their life cycle in other healthy cells.
The shortest and the longest average incubation period of SARS-CoV-2 in China
are 1.8 days and 12.8 days, respectively, mostly ranging from 3 to 7 days [21, 22]. As those with SARS and MERS, most patients with COVID-19 have a specific
ground glass appearance on chest computed tomographic (CT) scans . Besides,
patients with COVID-19 exhibit similar biopsy features to that seen in SARS-CoV
and MERS-CoV patients . Presentations of SARS-CoV-2 frequently emerged
include fever (
As aforementioned above, SARS-CoV-2 enters into host cells via binding to the receptor ACE2. ACE2 exists generally in many organs and tissues, including nasal epithelium, oral mucosa, kidney, brain, lungs, etc., which indicates SARS-CoV-2 can attack multiple organs aside from the lung . Indeed, SARS-CoV-2 causes a wide variety of symptoms across organ systems in patients with COVID-19. The involvement of the lungs by SARS-CoV-2 might result in ARDS, requiring intubation and admission to the intensive care unit. Individuals with life-threatening SARS-CoV-2 disease demonstrate related cytokine release syndrome (CRS) . CRS appears to be a hazardous factor involved in different inflammatory pathways hastening lung parenchymal impairment and thromboembolism . Severe lung involvement and mortality may be predicted early through lymphocytopenia and elevated signs of inflammatory factors . Particularly, neurogenic pulmonary edema can be seen in invalids with extreme COVID-19 pneumonia. It is also defined as a non-cardiogenic interstitial pulmonary edema with a distribution of the peripheral lung zone that can be found in viral pneumonia .
SARS-CoV-2 is a potent inducer of inflammatory cytokines. The virus can activate immune cells and induce the secretion of inflammatory cytokines and chemokines directing to pulmonary vascular endothelial cells. Surge of cytokines can cause a cytokine storm and extensive inflammation all over the body of patients. Organ damage following SARS-CoV-2 invasion may be attributed to the cytokine storm or cytokine cascade . Although respiratory failure from ARDS is the utmost reason for death, several death cases were reported to die from circulatory failure as a result of myocardial damage, which indicated the infection of SARS-CoV-2 might cause fulminant myocarditis . Neurological syndromes caused by COVID-19 include anosmia and dysgeusia, ataxia, headache, dizziness, and unconsciousness . The neuron-invading potency and possible role of SARS-CoV-2 in the patients with acute respiratory failure was first proposed by Li et al. . Although COVID-19 is characterized by respiratory presentations, the relative virus can also attack the alimentary system. Most patients suffer diarrhea, nausea, or vomiting . SARS-CoV-2 is also considered a causative agent for thyroiditis or thyrotoxicosis. And SARS-CoV-2 may be a potential trigger for autoimmune thyroid disease . Once infected by SARS-CoV-2, various key organs across the body can be attacked, and possibly result in multiple organ involvement. More complications about this viral infection can be read in another review .
The whole viral lifetime involves attachment and entry to the host cell, translation, replication and release, during which time the patients manifest various symptoms ranging from cough to cytokine storm, even multi-organ dysfunction, etc. In order to combat the disease COVID-19 efficiently, not only the stages of the viral life cycle, but the complications should be considered for potential therapeutic intervention. As long as immunologic complications like macrophage activation syndrome (MAS) occur, anti-viral monotherapy is not enough and additional anti-inflammatory treatment should be added. Early detection and proper treatment of MAS and cytokine storm will reduce the incidence and mortality in COVID-19 patients. Therefore, a combination therapy with dual or multiple drugs encompassing one or more targets could be given more favor. Ideally, the combined use of drugs should have at least dual functions: inhibiting or killing the virus and relieving the complicated symptoms of infected patients. The former function is performed by anti-viral drugs which block RNA synthesis and virus invasion, and bind to receptor proteins on the surface of cells, and cell cycle protein, etc. The latter function is served mainly by anti-inflammatory drugs which control cytokine production, break down the basement membrane, regulate outer mitochondrial membrane permeability, stimulate activated B-cell and T-cell proliferation, etc. Other drugs serving the latter function include anti-oxidant, immunomodulator and relating symptom-relieving drugs.
Combination therapies have the advantage to improve treatment efficiency while
decreasing concentration of individual drug. For example, remdesivir at 5.05
Drug combination therapy has been proved useful in treating virus infection disease . Recently it also showes some application potential on COVID-19 patients. Some studies demonstrate that combination therapy for COVID-19 outpatients might decrease hospitalization and death by 89%. Particularly, in some cases, combination therapy displays excellent outcome. COVID-19 patients sometimes may suffer several devastating conditions, such as cytokine cascade, organ damage, and thrombosis. McCullough and co-authors recommended that combination therapy should be a vital standard for management of those with these devastating conditions . It is exciting that combination therapy is effective against some new SARS-CoV-2 variants, which makes this treatment strategy invaluable especially when the SARS-CoV-2 virus presents fast-mutating characteristics.
In this review, we aim to summarize the outcome of the combination therapies against COVID-19. We focus on the combination therapies that contain at least one small molecule, such as remdesivir, umifenovir, or hydroxychloroquine (HCQ). While those combination therapies with both or more biomacromolecules, like antibody, nanobody, convalescent plasma or some other therapeutic proteins like interferon, are not included. You may read another relative review for this kind of combination therapies . We deem that this review would provide an option for the scientific and rational therapeutic alliance against COVID-19.
In this section, we will summarize those combination therapies with two or more small molecules. Each combination therapy may comprise two virus-directed antivirals, one virus-directed antiviral and another host-directed, one antiviral and another complication-relieving drug (anti-inflammatory, antioxidant, etc.), one antiviral and its pharmacokinetic enhancer, or other antimicrobials, etc. And according to the composition, they are categorized to 6 groups, all of which will be presented in the next 6 chapters, respectively. Many studies of combination therapy involve remdesivir, so we give a brief introduction of it first. Remdesivir is a nucleotide analog and the high resemblance between its triphosphate form and adenosine triphosphate (ATP) enables it to function as a competing inhibitor of RNA synthesis (RdRp inhibitor). Remdesivir shows great potential in inhibting all the coronaviruses including SARS-CoV-2 . Beigel et al.  completed a double-blind trial and they found that about half patients (total 1062 participants) treated with remdesivir had a shorter recovery time. Their data also indicate that remdesivir may prevent deterioration of the disease. Given the positive results of the trial, remdesivir became the first antiviral to be authorized by the Food and Drug Association for emergency use for hospitalized adult patients at the risk of serious illness . Following the approval, the clinical performance of the drug is also closely supervised and updated with the new evidence . It was found that remdesivir could lead to renal failure or liver dysfunction during therapeutic process of COVID-19 [48, 49]. Furthermore, a solidarity trial guided by the World Health Organization (WHO) demonstrated remdesivir had little or no benefit on hospitalized patients with COVID-19 . Thus, on 20 November 2020, WHO recommended against its use in spite of state of illness of hospitalized patients. Nonetheless, recently, on 22 April 2022 WHO updated the conditional recommendation for the use of remdesivir in patients with non-severe COVID-19 at the highest risk of hospitalization. There have been many clinical trials on remdesivir in a completed, terminated or recruiting stage, which were designed in combination with other agents such as Interferon beta-1b, Interferon beta-1a, Tocilizumab, Lopinavir/Ritonavir, Merimepodib, DWJ1248, baricitinib, or dexamethasone (https://www.clinicaltrials.gov/ct2/results?cond=COVID19&term=remdesivir&cntry=&state=&city=&dist=).
SARS-CoV-2 proteases Mpro and PLpro are promising targets for antiviral drug
development for their vital roles in coronavirus replication. MG-101, a Mpro
inhibitor, significantly improved the antiviral activity against SARS-CoV-2 Delta
variant, when combined with PLpro inhibitor Sitagliptin. Similarly, enhanced
anti-SARS-CoV-2 activity was also observed in combination therapy of two Mpro
inhibitors, such as MG-101 plus Lycorine HCl or Nelfinavir. MG-101, combined with
Lycorine HCl or Nelfinavir mesylate, led to a 3–4 log reduction in virus titer
|Drugs for Combination||Targets or action mechanisms||Results||Reference|
|MG-101 + Sitagliptin||Mpro + PLpro||Improved the antiviral effect against SARS-CoV-2 Delta variant|||
|MG-101 + Lycorine or Nelfinavir||Mpro||Enhanced anti-SARS-CoV-2 activity|||
|GC376 + remdesivir||Mpro + RdRp||Additive antiviral activity|||
|Molnupiravir + nirmatrelvir||RdRp + Mpro||Synergisitic antiviral activity|||
|An indole derivative+ remdesivir||Mpro||Synergistic activity|||
|Corilagin + remdesivir||RdRp (with different mechanisms)||Additive inhibitory effect|||
|SOF + DCV||RdRp||Clinical recovery rate was increased and hospitalization length reduced|||
|SOF + DCV; SOF + DCV + ribavirin||RdRp||Favoring this combination therapy||[57, 58]|
|VEL + SOF + the national standard of care*||Mpro + RdRp||(1) Safe; (2) did not improve the clinical status or reduce mortality||[59, 60]|
|Linoleic acid (LA) + remdesivir||SARS-CoV-2 S glycoprotein||Synergistic in inhibiting SARS-CoV-2 replication||[61, 62]|
|Glycyrrhizin (GR) + boswellic acid (BA)||SARS-CoV-2 S glycoprotein||(1) Reduction of systemic inflammation; (2) reduced risks of hospitalization and mortality, being safe, well tolerated, and widely available||[63, 64, 65]|
|Cepharanthine (CEP) + nelfinavir (NFV)||SARS-CoV-2 S protein + Mpro||Synergistic to limit SARS-CoV-2 proliferation|||
|NAC + CBS or BSS||Cysteine enzymes (proteases) including PLpro, Mpro, helicase (Hel) and ACE2||Significantly diminished the viral load of lung and the pathologic condition|||
|* The national standard of care comprises hydroxychloroquine and lopinavir/ritonavir as well as supportive care.|
As aforementioned, some host proteases facilitate SARS-CoV-2 entry in host cell
and the virus rely on host translation system for replication, thus it’s also
vital to block host relative protease and translation associated enzymes for
combating SARS-CoV-2. IMU-838, a developmental dihydroorotate dehydrogenase
(DHODH) inhibitor in phase II for autoimmune disease, showed enhanced in
vitro anti-SARS-CoV-2 activity when combined with remdesivir  (Table 2, Ref. [2, 40, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88]). Biering et al.  identified
compound B02, a human RAD51 inhibitor, exhibiting antiviral synergy with
remdesivir after screening a library of FDA-approved and well-studied preclinical
and clinical chemicals (Table 2). Besides, a synergy between remdesivir and
emetine (an anti-protozoan drug against ameobiasis) was noticed that remdesivir
|Drugs for Combination||Targets or action mechanisms||Results||Reference|
|IMU-838 + remdesivir||DHODH + RdRp||Enhanced in vitro anti-SARS-CoV-2 activity|||
|B02 + remdesivir||Human RAD51 + RdRp||Antiviral synergy|||
|Emetine + remdesivir||A protein synthesis inhibitor + RdRp||64.9% inhibition in viral yield|||
|Baricitinib + remdesivir||Janus kinase + RdRp||Reducing recovery time and accelerating improvement|||
|Baricitinib + corticosteroids||Janus kinaser + anti-inflammation||Strongly recommended by WHO||https://www.who.int/teams/health-care-readiness/covid-19/therapeutics|
|Tipifarnib + Omipalisib (GSK2126458)||Farnesyltransferase + phosphoinositide 3-kinases||Strong synergistic effects||[40, 74, 75]|
|Omipalisib + remdesivir; tipifarnib + remdesivir||Phosphoinositide 3-kinases + RdRp; Farnesyltransferase + RdRp||Strong synergistic effects|||
|Calpeptin + remdesivir||Cysteine proteinase + RdRp||Enhanced the anti-SARS-CoV-2 activity|||
|Lenvatinib + remdesivir||Host RTK + RdRp||Exhibited striking synergistic effect|||
|Camostat + enzalutamide or ARD-69||TMPRSS2 serine protease + anti-androgen or androgen receptor degrader||More efficacious in blocking the entry|||
|Raloxifene + tilorone||A heparin/HS-binding drug + a pan-antiviral agent||Synergistic against SARS-CoV-2-induced cytopathic effect|||
|Fluoxetine + GS-441524||Acid sphingomyelinase + RdRp||Synergistic antiviral effect|||
|HCQ + azithromycin||An alkalinizing lysosomatropic drug + antibiotic||(1) Synergic; (2) a better clinical status and a quicker virus eradication; (3) no clinical benefit for the treatment of the hospitalized patients with severe COVID-19; (4) a greater QT interval||[81, 82, 83, 84]|
|HCQ + lopinavir + ritonavir||An alkalinizing lysosomatropic drug + anti-HIV drug||Minimal in vitro antiviral activity|||
|HCQ + zinc supplements||An alkalinizing lysosomatropic drug + an essential micronutrient||No additive effect|||
|MEDS433 + dipyridamole (DPY)||DHODH + the pyrimidine salvage pathway||Restored the anti-SARS-CoV-2 activity of MEDS433|||
|Camostat mesylate + E-64d||TMPRSS2 + endosomal cysteine proteases cathepsin B and L||Complete inhibition of the SARS-CoV-2 cell entry||[2, 88]|
|Camostat mesylate + HCQ||TMPRSS2 + an alkalinizing lysosomatropic drug||Relative clinical trials have been withdrawn or in an unknown status||NCT04355052, NCT04338906|
MEDS433 is a new inhibitor of the human dihydroorotate dehydrogenase (hDHODH), a key enzyme of the de novo pyrimidine biosynthesis pathway. The pyrimidine salvage pathway may attenuate the antiviral efficacy of an hDHODH inhibitor through transporting nucleosides from extracellular medium . Thus, a combination strategy was adopted with MEDS433 and dipyridamole (DPY), the latter inhibiting the pyrimidine salvage pathway. And this combination strategy restored the lost anti-SARS-CoV-2 activity of MEDS433 in the presence of exogenous uridine . Camostat mesylate is a clinically efficient serine protease inhibitor and active against TMPRSS2 . Complete inhibition of the SARS-CoV-2 cell entry was realized when camostat mesylate and E-64d, an inhibitor of endosomal cysteine proteases cathepsin B and L (CatB/L), were added . However, two clinical trials (NCT04355052, NCT04338906) exploring the combination remedy of TMPRSS2 inhibitor camostat mesylate and HCQ have been withdrawn or in an unknown status (Table 2).
PF-07321332 (Nirmatrelvir) is an oral antiviral drug developed by Pfizer. Ritonavir is able to slow the metabolism of PF-07321332 by cytochrome enzymes, therefore maintaining higher concentrations of the primary drug. Thus PF-07321332 and ritonavir was used as a combination medication in Phase III studies and was marketed as Paxlovid for the treatment of COVID-19. Now Paxlovid has been utilized in market across the world since it was able to reduce the risk of hospitalization or death by 89% compared with placebo  (Table 3, Ref. [98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112]). WHO has strongly recommended the use of nirmatrelvir/ritonavir in patients with non-severe illness at the highest risk of hospitalization, however provided conditional recommendation against the use of them in patients with non-severe illness at a low risk of hospitalization (published 22 April 2022) (https://www.who.int/teams/health-care-readiness/covid-19/therapeutics). Cobicistat is an FDA-approved drug that can boost the activity of major drug via blocking the activity of cytochrome P450-3As (CYP3As) and P-glycoprotein (P-gp). Recently, it was reported to inhibit SARS-CoV-2 replication through suppressing the fusion of the viral S-glycoprotein to the cell membrane. In combination with remdesivir, cobicistat exhibited a synergistic antiviral effect in vitro and decreased viral titers and disease progression in Syrian hamsters . Darunavir, a protease inhibitor and its pharmacokinetic enhancer, cobicistat, work as a whole to treat HIV infection. A pilot study was conducted at Shanghai Public Health Clinical Center (SPHCC) to preliminarily evaluate the efficacy and safety of darunavir/cobicistat in treating COVID-19 pneumonia. There was no any tendency of improvement observed in the darunavir/cobicistat group in comparison with the control group, although it was well tolerated (clinicaltrials.gov: NCT04252274) (Table 3) .
|Drugs for Combination||Targets or action mechanisms||Results||Reference|
|PF-07321332 (Nirmatrelvir) + ritonavir||Mpro + CYP3A||Decrease the risk of hospitalization or death by 89%|||
|Cobicistat + remdesivir||CYP3As + P-gp + S-glycoprotein + RdRp||Decreased viral titers and disease progression|||
|Darunavir + cobicistat||A protease inhibitor + CYP3As+ P-gp||No any trend of improvement|||
|Quercetin + Vitamin C (VC)||Anti-inflammatory + a broad spectrum antiviral agent||Synergistic antiviral, antioxidant and immunomodulatory effects|||
|Plitidepsin + dexamethasone||host cell’s eEF1A + anti-inflammation||A phase III trial is underway|||
|Standard of care (SOC) + remdesivir + dexamethasone||RdRp + anti-inflammation||A reduction in 30-day mortality|||
|Methylprednisolone + remdesivir||RdRp||(1) Prevented body weight loss and inflammation; (2) dampened viral protein expression and viral load|||
|olfactory rehabilitation + palmitoylethanolamide + luteolin||NA*||Effective in improving recovery of olfactory function|||
|Cannabidiol (CBD) + terpenes||An anti-inflammatory molecule + anti-microbials||Demonstrated mild to moderate antivirus effect||[106, 107]|
|Pentoxifylline + oxypurinol||TNF-
||Just a suggestion, no experimental data|||
|VD + DPP-4i||Anti-inflammatory + immunomodulatory||A perspective, no experimental data||[109, 110, 111]|
|Dapsone + doxycycline||Suppress production of various cytokines + anti-microbial||Just a suggestion, no experimental data|||
|* NA, not available.|
In plants, quercetin is a flavonoid compound, produced from the phenylpropanoid pathway and ultimately derived from phenylalanine. There is a tremendous amount of literature supporting its anti-inflammatory and antiviral properties, especially against several respiratory viruses in both in vitro and in vivo experiments (Table 3) . Vitamin C (VC) is a broad spectrum antiviral agent and an inhibitor of aerobic glycolysis. Treatment with quercetin in combination with VC provided synergistic antiviral, antioxidant and immunomodulatory effects due to overlapping antiviral and immunomodulatory properties and the capacity of VC to regenerate quercetin (Table 3) . Plitidepsin is a cyclic depsipeptide known for its anti-tumor and antiviral activity, mainly acting on isoforms of the host cell’s eukaryotic-translation-elongation-factor-1-alpha (eEF1A). Through blocking eEF1A and therefore translation of essential viral proteins, it exhibits anti-SARS-CoV-2 potential. A phase III trial is underway to compare the plitidepsin/dexamethasone remedy with the standard of care in moderate hospitalized patients (ClinicalTrials.gov Identifier: NCT04784559) . A comparative study of the effectiveness of remdesivir/dexamethasone plus standard of care (SOC) versus SOC alone was proceeded in a clinical trial, and the result showed a reduction in 30-day mortality with the combination treatment . In the hamster model of SARS-CoV-2 infection, treatment with methylprednisolone suppressed viral induction of proinflammatory cytokines but enhanced RNA replication of SARS-CoV-2. Although weight reduction, along with nasal and pulmonary inflammation, was relieved with methylprednisolone monotherapy, both viral loads enhancement and antibody response weakening also accompanied. On the contrary, a combination therapy methylprednisolone/remdesivir not only restrained weight reduction and inflammation, but also dampened viral protein expression and viral loads. Furthermore, the suppression of methylprednisolone on antibody response was also attenuated in this combination therapy . Approximately 30% of COVID-19 patients were reported to have obstinate smell or taste dysfunction as prolonged sequalae of infection. Treatment combining olfactory rehabilitation with oral supplementation with palmitoylethanolamide and luteolin was effective in improving recovery of olfactory function, especially in those patients with longstanding olfactory dysfunction . Cannabidiol (CBD) is widely available as medicinal compounds with various applications, most involved in modulating the inflammation processes . Santos et al.  have evaluated the anti-infection effect of the combination of CBD with terpenes, as an anti-inflammatory chemical and anti-microbial, respectively. The virucide effectiveness of CBD and terpene-based six formulations were tested in different cell lines and the result demonstrated mild to moderate antivirus effect .
Pentoxifylline is an inhibitor of TNF-
The pathophysiology of SARS-Cov-2 relates to inflammation, immune dysregulation, coagulopathy, and endothelial dysfunction. No single therapeutic agent can manage all these pathophysiologic conditions. Hence, a randomized open-label trial was initiated to investigate a triple combination remedy (aspirin, atorvastatin, and nicorandil) with anti-inflammatory, antithrombotic, immunomodulatory, and vasodilator properties against COVID-19 in India  (Table 4, Ref. [115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132]). Procter et al. [116, 117] have evaluated the effects of multidrug combination therapy on high-risk patients. At least two anti-SARS-CoV-2 agents (zinc, HCQ, ivermectin) and one antibiotic (azithromycin, doxycycline, ceftriaxone) were used as well as inhaled budesonide and/or intramuscular dexamethasone. And it was concluded that early ambulatory multidrug therapy was associated with low rates of hospitalization and death (Table 4) [116, 117]. The outcome of a triple therapy (zinc, azithromycin, and HCQ) was evaluated in COVID-19 patients. No adverse cardiac events and notably fewer hospitalizations were observed .
|Drugs for Combination||Targets or action mechanisms||Results||Reference|
|Aspirin + atorvastatin + nicorandil||Anti-inflammatory, antithrombotic, immunomodulatory and vasodilator properties||NA*|||
|Two antivirals of (zinc, HCQ, ivermectin) + one antibiotic of (azithromycin, doxycycline, ceftriaxone) + budesonide + dexamethasone||Multiple targets||Low rates of hospitalization and death||[116, 117]|
|Zinc + azithromycin + HCQ||Multiple targets||No adverse cardiac events and notably fewer hospitalizations|||
|Umifenovir (arbidol) + LPV/r||Inhibition of membrane fusion + anti-HIV||Viral load vanished in 94% patients of the combination group, compared to 52.9% of the monotherapy group (LPV/r)||[119, 120]|
|Shufeng Jiedu Capsules + umifenovir||Multiple targets||The more rapid subsidence of a fever and better resolution of pneumonia symptoms|||
|HCQ + VC + VD + Zinc||An alkalinizing lysosomatropic drug + broad-spectrum antiviral||Ongoing||NCT04335084|
|Clofazimine + remdesivir||An anti-tuberculosis drug + RdRp||Synergistic antiviral activity|||
|Mefloquine + artesunate, artesunate + amodiaquine, artemether + lumefantrine, artesunate + pyronaridine, or dihydroartemisinin + piperaquine||Antimalarial drug||Mefloquine/artesunate demonstrated the strongest antiviral activity with % inhibition of 72.1
|Mefloquine + Nelfinavir||Blocking viral entry + Mpro inhibitor||Synergistic antiviral activity|||
|Nitazoxanide + favipiravir||A commercial antiprotozoal agent + RdRp||Underway||NCT04918927|
|ATV+ RTV||An HIV-1 protease inhibitor + Cytochrome P450 3A||More potent|||
|LPV/r+azithromycin||Anti-HIV + antibiotic||The most effective combination group among 8 groups of combination drugs|||
|LPV/r||Anti-HIV drug||No significant benefit and more gastrointestinal adverse events|||
|DOXY + HCQ||Antibiotic + an alkalinizing lysosomatropic drug||Reduction in recovery time and mortality and lower rate of transfer to hospital|||
|DOXY+ ivermectin||Antibiotic||Recovered earlier|||
|DOXY+ VC||Antibiotic + broad-spectrum antiviral||Suggestion|||
|Minocycline + HCQ||Antibiotic + an alkalinizing lysosomatropic drug||Just an appeal for further clinical studies|||
|Itraconazole + remdesivir||Antifungals + RdRp||Synergistically prohibited the production of SARS‐CoV‐2 particles in vitro|||
|* NA, not available.|
Umifenovir (also termed as arbidol), an antiviral agent with broad spectrum, functions primarily through inhibition of membrane fusion between the viral envelope and host cell membrane, therefore, suppressing viral entry and infection . Deng et al.  conducted a retrospective cohort study to compare arbidol and lopinavir/ritonavir (LPV/r) combination treatment for COVID-19 patients with LPV/r alone. They found that viral load vanished after 14 days in 94% patients of the combination group, compared to 52.9% of the monotherapy group (LPV/r) . Recently, another retrospective cohort study was carried out to understand the clinical effectiveness and safety of Shufeng Jiedu Capsules (a Chinese herbal compound composed of eight medicinal plants ) in combination with umifenovir (Arbidol) for common-type COVID-19. The subsidence of a fever was observed more rapidly and the chest CT scan also showed better resolution of pneumonia symptoms in the combination treatment group than that in the control group (treated with arbidol hydrochloride capsules alone) . A Phase II interventional study testing whether treatment with HCQ, Vitamin C, Vitamin D, and Zinc can prevent symptoms of COVID-19 is ongoing (NCT04335084).
Clofazimine was discovered as an anti-tuberculosis drug and later used for the
treatment of leprosy . Clofazimine, in combination with remdesivir,
exhibited synergistic antiviral activity in vitro and in vivo,
and restricted viral shedding from the upper respiratory tract (Table 4)
. The antiviral activity of several antimalarial artemisinin-based
combination therapies (ACT), including mefloquine/artesunate,
artesunate/amodiaquine, artemether/lumefantrine, artesunate/pyronaridine, or
dihydroartemisinin/piperaquine, was tested in vitro against a SARS-CoV-2
strain (IHUMI-3) in Vero E6 cells. Mefloquine/artesunate demonstrated the
strongest antiviral activity with % inhibition of 72.1
Doxycycline (DOXY) is a semisynthetic, second-generation class of tetracycline with a wide spectrum of antimicrobial activity. The activity of DOXY/HCQ combination therapy was studied in a series of fifty-four high-risk COVID-19 patients. And the clinical experience of this case series indicated a reduction in recovery time and death rate, and lower rate of transfer to hospital after treatment with DOXY/HCQ . A double-blind and randomized interventional trial of a combination of DOXY and ivermectin was carried out with 400 participants. Patients with mild-to-moderate COVID-19 infection treated with ivermectin plus DOXY recovered earlier than those with placebo, were less possible to deteriorate, and were more inclined to be SARS-CoV-2 negative by RT-PCR at the end of the treatment (NCT04523831) . It is suggested that co-administration of doxycycline and vitamin C shows more benefit against COVID-19 . Minocycline is another semisynthetic, second-generation derivative of tetracycline with an activity against lots of microorganisms . It also inhibits many proinflammatory cytokines, which are common in severe and complicated COVID-19 cases . Gautam et al.  analyzed the pros and cons of the cocktail HCQ/minocycline in treating moderate to severe COVID-19 patients and called upon public and private healthcare bodies to implement large well-designed clinical studies for generating more convincing suggestions.
Interferons (IFNs) are glycoproteins with potential immunomodulatory and
hormone-like functions . Both IFN
|Drugs for Combination||Targets or action mechanisms||Results||Reference|
||Immunomodulatory + antiviral||Viral eradication rates, fever subsidence, recovery time and safety characteristics were improved|||
||Immunomodulatory + antiviral||Oxygenation increase, survival advantage and discharging of sever COVID-19|||
||Immunomodulatory + antiviral||Neither clinical improvement nor reduction of SARS-CoV-2 load|||
||Immunomodulatory + antiviral||Exhibited superior performance|||
||Immunomodulatory + anti-HIV drug||A decline in the hospital time and acceleration of viral eradication|||
||Immunomodulatory + antivirals||Safe and superior to LPV/r alone|||
||Immunomodulatory + anticoagulant||Suppressed SARS-CoV-2 infection in an additive fasion|||
||Immunomodulatory + SARS-CoV-2 inhibitors||A strong synergy in inhibiting SARS-CoV-2 infection|||
|Remdesivir + tocilizumab + dexamethasone||Antiviral (small molecular and antibody) + anti-inflammation||Timely resolution of the cytokine storm and subsequent improvement in ARDS symptoms and eventual recovery|||
|Remdesivir + convalescent plasma or mAbs||Antiviral (small molecular and antibody)||High viral clearance||[156, 157, 158, 159, 160]|
|Ruxolitinib + eculizumab||A Janus kinase (JAK) 1/2 inhibitor + an anti-C5a complement monoclonal antibody||Significant improvements in respiratory symptoms and radiographic pulmonary lesions and reduction of circulating D-dimer|||
|Favilavir + tocilizumab||Antiviral + an anti–interleukin-6 monoclonal antibody||Its status has not been updated for 2 years||NCT04310228|
|Immunoglobulin + steroid pulses||Immunoglobulin + anti-inflammation||Useful in single-kidney transplanted patient with COVID-19|||
|Sarilumab + standard of care (including corticosteroids)||IL6 alfa-receptor antibody + anti-inflammation||Not more effective|||
|Dexamethasone + anti- SARS-CoV-2 mAbs||Anti-inflammatory + antiviral antibody||Synergistic|||
|GRFT + EK1||An antiviral + spike S2 subunit||Strong synergistic effect|||
|Bromelain + acetylcysteine||Virus glycoproteins||Synergistically weakened the infectivity||[166, 167]|
|Bromelain + curcumin||Prevent entry of SARS-CoV-2 into cells and interfere with viral replication||A proposal|||
COVID-19 may developed as a chronic disease in patients with some types of immunodeficiency. In this condition, remdesivir monotherapy is frequently ineffective, but the combination of remdesivir with antibody-based therapeutics holds promise. Remdesivir, combined with tocilizumab (an anti–interleukin-6 monoclonal antibody) and dexamethasone, was administered at the early stage of the disease, resulting in timely resolution of the cytokine storm and subsequent improvement in ARDS symptoms and eventual recovery . Combination of remdesivir with convalescent plasma or anti-SARS-CoV-2 monoclonal antibodies (mAbs) achieved high viral clearance [156, 157]. Successful outcomes with this combination therapy have also been demonstrated in other similar patients, such as with B-cell depleted , chronic lymphocytic leukemia  and X-linked agammaglobulinemia . Considering that ruxolitinib, a Janus kinase (JAK) 1/2 inhibitor and eculizumab, an anti-C5a complement monoclonal antibody, function by acting on different but correlative pathological pathways, a combination therapy containing both of them was conceived to test its effect on SARS-CoV-2-related ARDS. And the results showed that patients treated with the combination obtained significant improvement in respiratory symptoms and radiographic pulmonary lesions and reduction in circulating D-dimer concentrations compared to the best available therapy group. Since the number of participants was small, only 7 in this study, the authors suggested further clinical studies with larger populations . NCT04310228 is a clinical trial assessing the curative effect and security of favilavir in combination with tocilizumab. But its status has not been updated for 2 years. It was proved useful that high-dose intravenous immunoglobulin plus steroid pulses in treating a case of COVID-19 pneumonia patient with a single-kidney transplanted requiring mechanical ventilation and hemodialysis . Sarilumab is a recombinant human immunoglobulin monoclonal antibody of IL6 alfa-receptor. In hospitalized patients with COVID-19 pneumonia, an early treatment with sarilumab combined with standard of care (including corticosteroids) was not more efficatious than current standard of care alone . We have known that from previous studies glucocorticoid treatment brought beneficial anti-inflammatory effects, with virus replication being rather strengthened. This outcome inspired us to apply glucocorticoid together with virus-neutralizing mAbs. Based on histopathology and bulk and single-cell transcriptomic analysis, Wyler et al.  demonstrated the useful therapeutic effects of them and their potential as synergistic combination therapy in hamster models of moderate and severe COVID-19.
griffithsin (GRFT), a lectin isolated from the red alga Griffithsia sp, can recognize mannose with high specifity and has a broad-spectrum antiviral activity . Combining GRFT and EK1, a pan-CoV fusion inhibitor targeting SARS-CoV-2 spike S2 subunit, exhibited strong synergistic effect against pseudotyped and live SARS-CoV-2 infection .
Bromelain exists mainly in the stem of the pineapple plant (Ananas comosus) and contains a number of enzymes that enable it to hydrolyze glycosidic bonds. Experimental studies have shown that bromelain exhibits unique immunomodulatory activity through various pathways . Acetylcysteine is a powerful antioxidant and able to reduce disulfide bonds. Bromelain and acetylcysteine combination regimen (BromAc) showed synergistic action against glycoproteins by breakage of glycosidic linkages and disulfide bonds. Thus the combination therapy, BromAc, synergistically weakened the infectivity of SARS-CoV-2 cultured on Vero cells . Further study indicated strong mucolytic and anti-inflammatory effect of BromAc ex vivo in tracheal aspirates from severe patients with COVID-19 . Curcumin (diferuloylmethane) is a natural phenol found in turmeric (Curcuma longa), a member of the ginger family of plants . Interestingly, curcumin has been shown in silico studies to prevent entry of SARS-CoV-2 into cells and interfere with viral replication, while bromelain may also prohibit viral entry demonstrated by a recent experimental study . Notably, bromelain can markedly increase the absorption of curcumin, thereby compensating for its poor absorption drawback. Based on the analysis, Kritis et al.  reported to highlight the potential value of the synergistic effect of bromelain and curcumin in the prevention of severe COVID-19.
The outbreak of COVID-19 has caused devastating economic and medical burden worldwide and seriously affects the living styles of most people. It has been more than two years since the first emergence of SARS-CoV-2 infected cases in Wuhan, China, during which time many scientists are trying to develop effective vaccines and therapies including antibodies and oral small molecules, at an unprecedented scale and pace. And fortunately, there have been several vaccines, antibodies and oral small molecules prove effective as prophylaxis or remedy in combating COVID-19. However, since its outbreak, SARS-CoV-2 has developed a variety of mutations, and different mutants have been identified in all four structural proteins and other viral proteins . Mutations in the SARS-CoV-2 RBD or N-terminal domain (NTD) may endow these strains with enhanced replication and/or transmission ability, which lead to escape from antibody recognition and attenuated neutralizing activity of mAbs . Besides, some new SARS-CoV-2 variants can also infect those people who have been vaccinated once or more. In theory, combination therapy may have additive or synergistic activity in preventing infection by escape mutants compared to monotherapy, thus is highly emphasized. However, the present studies on combination therapy against SARS-CoV-2 mutant strains focus on mAbs, the small molecule drugs scarcely being involved.
In a combination therapy, one drug may endow another more power to counter the disease. Ideally, the pharmacokinetic profiles as well as the pharmacodynamics characteristics of individual drug are improved when combined used. As aforementioned above, effective concentration of each drug would be reduced below the maximal plasma concentration, which therefore attenuates toxicity resulted by high concentration. However, in many cases, there were limited data regarding the pharmacokinetic profiles of individual drug in the combination therapies. We call for more attention on the drug interaction and the resulting pharmacokinetic profiles in the future research.
The virus invades the organ systems across the body, which triggers a variety of complications (concomitant symptoms), ranging from fever to multiple organ dysfunction syndrome, etc. Sometimes, the severe complications are fatal, needing to be tackled as a matter of urgency. Thus it is not enough for a combination therapy to counter just the disease-causing agent. An effective treatment strategy for COVID-19 should take comprehensive consideration of both the host symptom and the pathogenic microorganism. So we are a bit more optimistic about the therapeutic effect of those combination therapies targeting both the virus life cycle and host complications.
This review updates the progress of dual and multiple drug combination therapies (containing at least one small molecule drug) against COVID-19. The antiviral mechanism of each combination, especially the target of each component, and the outcome is highlighted. We can see that these combinations target the same viral enzyme with different action mechanisms, different viral enzymes playing key roles in virus life cycle, different enzymes from virus and host cell facilitating virus survival, or host complication-relating pathways. Also we find that some combination therapies were proposed as medical hypotheses or perspective based on previous knowledge about the pharmacology of individual drug, most of which deserve further exploration by experiment. Others were validated by clinical trials, animal experiments, cell or enzyme assays. Many combination therapies exhibited positive (additive or synergistic) outcome awaiting further efficiency clarification in SARS-CoV-2-relating animal model or clinical trial. Though some combinations turned out to be no effect, even toxic, they are still of guiding value in clinical practice, especially for those combinations of repurposing drugs. In summary, this review is hopeful for facilitating us to select a proper anti-SARS-CoV-2 combination therapy for further research or clinical treatment.
QL conceived the topic and wrote the manuscript. YZ assisted in writing and organizing the draft. JZ supervised the work and approved the final draft. All authors read and approved the final draft.
JZ was supported by the Thousand Young Talents Program of China, the National Natural Science Foundation of China (Grant No. 31770795; Grant No. 81974514), and the Jiangxi Province Natural Science Foundation (Grant No. 20181ACB20014).
The authors declare no conflict of interest.
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