Glycyrrhizin, an active component of liquorice roots, = and replication of SARS-associated coronavirus

Glycyrrhizin,=20 an active component of liquorice = roots, and=20 replication of SARS-associated coronavirus

J Cinatl, B Morgenstern, G Bauer, P Chandra, H Rabenau, H W Doerr =

The outbreak = of SARS=20 warrants the search for antiviral compounds to treat the disease. At = present, no=20 specific treatment has been identified for SARS-associated coronavirus infection. We assessed the antiviral = potential=20 of ribavirin, 6-azauridine, pyrazofurin, mycophenolic acid,=20 and glycyrrhizin against two clinical isolates of coronavirus (FFM-1 and FFM-2) from patients with = SARS=20 admitted to the clinical centre of Frankfurt University, Germany. Of all = the=20 compounds, glycyrrhizin was the most active in inhibiting replication of = the=20 SARS-associated virus. Our findings suggest that glycyrrhizin should be = assessed=20 for treatment of SARS.

Lancet 2003; = 361:=20 2045-46

A new coronavirus has been identified in patients with = severe=20 acute respiratory syndrome (SARS).¹ SARS is an infectious = disease=20 with a high potential for transmission to close contacts. The outbreak = of SARS=20 in several countries has led to the search for active antiviral = compounds to=20 treat this disease.

Here, we = assessed the=20 antiviral activities of ribavirin, = 6-azauridine, pyrazofurin, mycophenolic acid,=20 and glycyrrhizin against two clinical isolates of coronavirus (FFM-1 and FFM-2) from patients with = SARS=20 admitted to the clinical centre of Frankfurt University, Germany. All = the=20 compounds are available commercially and have been used in patients for = their=20 antiviral, antitumour, and immunosuppressive = activity.=20 We visually scored cytopathogenicity induced = by the=20 virus 72-96 h after infection in 96-well microplates=20 on confluent layers of Vero cells. The selectivity index was determined = as the=20 ratio of the concentration of the compound that reduced cell viability = to 50%=20 (CC₅₀) to the concentration of the compound needed to inhibit = the=20 cytopathic effect to 50% of the control = value=20 (EC₅₀). We determined the cytotoxicity of=20 the drugs with an MMT cell-proliferative Kit = I (Roche,=20 Mannheim, Germany).

Ribavirin and mycophenolic acid, inhibitors of inosine monophosphate dehydrogenase, did not = affect=20 replication of the SARS-associated coronaviruses=20 (SARS-CV) (table). The inhibitors of orotidine=20 monophosphate decarboxylase, 6-azauridine = and pyrazofurin, inhibited replication of SARS-CV at = non-toxic=20 doses with selectivity indices of 5 and 12, respectively. The most = potent=20 inhibitor of SARS-CV replication in Vero cells was glycyrrhizin, which = had a=20 selectivity index of 67.

EC₅₀*=20 (mg/L)

CC₅₀*=20 (mg/L)

Selectivity=20 index

Compound

6-azauridine

16=B78 = (2=B79)

104=20 (18)

Pyrazofurin

4=B72=20 (0=B757)

52=20 (9=B76)

Mycophenolic=20 acid

>50

Ribavirin

>1000

Glycyrrhizin

After = virus=20 adsorption

600=20 (72)

>20000=86

>33

During = and after=20 virus

300=20 (51)

>20000

>67

adsorption

During = virus=20

2400=20 (410)

>20000

>8=B73

adsorption

EC₅₀=3Deffective=20 concentration of compound needed to inhibit the =

cytopathic = effect to 50%=20 of control value. CC₅₀=3Dcytotoxic=20 concentration

of the = compound=20 that reduced cell viability to 50%. NC=3Dnot calculable. =

*Mean = (SD) of=20 eight assays. =86At the maximum concentration used =

(20 = 000 mg/L) a=20 20-30% reduction of cell viability was = recorded.

Activity of=20 compounds against SARS-associated coronavirus in=20 Vero cell cultures

In addition = to=20 inhibition of virus replication, glycyrrhizin inhibits adsorption and=20 penetration of the virus--early steps of the replicative cycle. Glycyrrhizin was less effective = when=20 added during the adsorption period than when added after virus = adsorption=20 (EC₅₀ 600 mg/L vs 2400 = mg/L,=20 respectively). Glycyrrhizin was most effective when given both during = and after=20 the adsorption period (EC₅₀ 300 mg/L).

The figure = shows the=20 effect of glycyrrhizin on replication of SARS-CV in Vero cells. We = detected=20 replication of SARS-CV with serum samples from patients with SARS. = Expression of=20 viral antigens was much lower in cultures treated with 1000 mg/L of = glycyrrhizin=20 than in any other culture; high concentrations of glycyrrhizin (4000 = mg/L)=20 completely blocked replication of the virus (figure).

Effect = of=20 glycyrrhizin on replication of SARS-associated coronavirus in Vero cells

Cells = were fixed=20 with 60 parts methanol to 40 parts acetone72 h after infection. = Virus was=20 detected in serum from the patient with SARS by peroxidase staining. (A) mock infected = cells. (B)=20 infected cells without treatment. (C) infected cells treated with = 4000=20 mg/L glycyrrhizin. (D) infected cells treated with 1000 mg/L = glycyrrhizin.=20

In a = comparison of the=20 antiviral potential of 6-azauridine, ribavirin, and=20 glycyrrhizin against several pathogenic flaviviruses,=20 Crance and collegues² showed that = ribavirin and 6-azauridine were active but not = selective=20 inhibitors when assessed with regard to inhibition of cell growth. = Glycyrrhizin=20 had a low selectivity index, but was a significantly potent inhibitor of = replication of all the viruses tested. The EC₅₀ reported by = these=20 authors for glycyrrhizin was 316-625 mg/L (added twice during the = incubation=20 period of 7 days). Taking into consideration that the compounds were = added twice=20 during the total period of incubation, the EC₅₀ for = glycyrrhizin we=20 recorded (table) indicates a higher sensitivity of SARS-CV to this drug = than=20 that recorded by Crance and colleagues. =

The = mechanism of=20 glycyrrhizin's activity against SARS-CV is unclear. Glycyrrhizin affects = cellular signalling pathways such as protein = kinase C;=20 casein kinase II; and transcription factors such as activator protein 1 = and=20 nuclear factor 3Dkappa=20 B. Furthermore, glycyrrhizin and its aglycone=20 metabolite 18=DF glycyrrhetinic acid upregulate expression of inducible nitrous oxide = synthase and production of nitrous oxide in=20 macrophages.³ Nitrous oxide inhibits replication of several=20 viruses--eg, Japanese encephalitis = virus⁴=20 (a member of the Flaviviridae family), which = can also=20 be inhibited by glycyrrhizin.² Our preliminary results show = that=20 glycyrrhizin induces nitrous oxide synthase = in Vero=20 cells and that virus replication is inhibited when the nitrous oxide = donor (DETA=20 NONOate) is added to the culture medium. =

Glycyrrhizin = has=20 previously been used to treat patients with HIV-1 and chronic hepatitis = C virus.=20 The resulting low concentrations of P24 antigen in patients with HIV-1 = who were=20 given this compound has been attributed to upregulation of chemokines.=20 Infrequent side-effects such as raised blood pressure and hypokalaemia were reported in some patients after = several=20 months of glycyrrhizin treatment. Treatment of SARS should only be = needed for a=20 short time. Since the side-effects of this compound are known and can be = controlled for, proper monitoring could lead to effective use of = glycyrrhizin as=20 a treatment for SARS. Booth and colleagues⁵ reported that = ribavirin had many toxic effects when given to = patients with=20 SARS, including haemolysis (76% of patients) = and a=20 drastic reduction of haemoglobin (49% of = patients).=20 However, although high doses of glycyrrhizin have been used in clinical = trials,=20 this compound had few toxic effects compared with the other regimens, = and the=20 drug was reported to be clinically effective.

Future = search for=20 compounds of therapeutic interest against SARS will be greatly = facilitated by=20 establishing growth of SARS-CV in human cells.

1 Drosten C, Gunther S, = Preiser W, et al. Identification of a novel coronavirus in patients with severe acute = respiratory=20 syndrome. N Engl J Med .=20 2003; 348: 1967-76. [PubMed]

2 Crance JM, Scaramozzino N, Jouan A, Garin D. = Interferon,=20 ribavirin, 6-azauridine, and glycyrrhizin: = antiviral=20 compounds active against pathogenic flaviviruses.=20 Antiviral Res 2003; 58: 73-79. = ;[PubMed]

3 Jeong HG, Kim JY. Induction of inducible nitric = oxide synthase expression by 18=DF-glycyrrhetinic acid = in=20 macrophages. FEBS Lett 2002; 513: 208-12.&n= bsp;[PubMed]

4 Lin YL, = Huang YL, Ma=20 SH, et al. Inhibition of Japanese encephalitis virus infection by nitric = oxide:=20 antiviral effect of nitric oxide on RNA virus replication. J = Virol 1997; 71: 5227-35.&= nbsp;[PubMed]

5 Booth CM, = Matukas LM, Tomlinson GA, et al. Clinical features = and=20 short-term outcomes of 144 patients with SARS in the greater Toronto = area.=20 JAMA 2003; 289: 1-9. [PubMed]

Institute of = Medical=20 Virology, Frankfurt University Medical School, Paul-Ehrlich Str 40, D-60596 Frankfurt, Germany = (Prof J = Cinatl PhD, B Morgenstern, G Bauer, Prof P Chandra = PhD, Prof=20 H Rabenau PhD, Prof H W Doerr MD)

Correspondence to:=20 Prof J Cinatl (e-mail:mailto:cinatl@em.uni-frankfurt= .dee)