The synergistic effects of betulin with acyclovir against herpes simplex viruses

doi:10.1016/j.antiviral.2004.05.006

Antiviral Research

Volume 64, Issue 2, November 2004, Pages 127-130

https://doi.org/10.1016/j.antiviral.2004.05.006 Get rights and content

Abstract

Betulin, a pentacyclic triterpenoid, was isolated from the bark of Betula papyrifera. The antiviral efficacies of betulin on herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) were evaluated using viral plaque reduction assays on Vero cells. The results indicate that betulin is active against both HSV-1 and HSV-2 infections with the 50% effective concentrations (EC₅₀) of 0.40 and 4.15 μg/ml, respectively. The cytotoxicity of betulin was examined on Vero cells using a neutral red uptake assay. The 50% cytotoxic concentration (CC₅₀) of betulin was 73.1 μg/ml. A synergistic antiviral effect between betulin and acyclovir (ACV) was determined by drug combination studies. Strong and moderate synergistic antiviral effects were observed for betulin and ACV against HSV-1 when the concentrations of ACV and betulin were higher than 0.068 and 0.4 μg/ml, respectively. At the concentrations lower than these, additive effect was found. Synergistic antiviral effects were also found against HSV-2 at higher concentrations than for HSV-1, i.e. 0.45 μg/ml of ACV combined with 8.4 μg/ml of betulin.

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Citation Excerpt :
For example, the first effective antiviral drug, acyclovir, which is used in the treatment of HHV infections, is a DNA polymerase inhibitor [24]. Research shows that betulin combined with acyclovir exhibits synergistic effects against HHV-1 and HHV-2 viruses [25]. Considering the results of research on the antiviral activity of betulin 1, it is appropriate to obtain betulin 1 derivatives containing the common structural elements of widely available drugs.
The search for new methods of antiviral therapy is primarily focused on the use of substances of natural origin. In this context, a triterpene compound, betulin 1, proved to be a good starting point for derivatization. Thirty-eight betulin acid ester derivatives were synthetized, characterized, and tested against DNA and RNA viruses. Several compounds exhibited 4- to 11-fold better activity against Enterovirus E (compound 5 EC₅₀: 10.3 μM) and 3- to 6-fold better activity against Human alphaherpesvirus 1 (HHV-1; compound 3c EC₅₀: 17.2 μM). Time-of-addition experiments showed that most of the active compounds acted in the later steps of the virus replication cycle (e.g., nucleic acid/protein synthesis). Further in-silico analysis confirmed in-vitro data and demonstrated that interactions between HHV-1 DNA polymerase and the most active compound, 3c, were more stable than interactions with the parent non-active betulin 1.
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Betulinic acid (1) has been modified to ionic derivatives (2–5) to improve its water solubility and biological activities. The binding properties of these derivatives with respect to human serum albumin (HSA) was examined and found to be similar to current anti-HIV drugs. These compounds did not inhibit HIV reverse transcriptase, however, 1, 2 and 5 inhibited herpes simplex type 2 (HSV-2) replication at concentrations similar to those reported for acyclovir (IC₅₀ ∼0.1–10 μM) and with minimal cellular cytotoxicity. IC₅₀ values for antiviral activity against HSV-2 186 were 1.6, 0.6, 0.9, 7.2, and 0.9 μM for compounds 1–5, respectively.
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Experiments to assess the cytotoxicity of TE and pentacyclic triterpenes indicated a low toxic behaviour in cell cultures (RC 37) according to Halle and Göres (1987). This is in accordance with Gong et al. (2004), who demonstrated also a low cytotoxicity for betulin in cell culture on Vero cells. We have analyzed the in vitro inhibitory effect of TE and related pentacyclic triterpenes on HSV-1 strains using plaque reduction assays.
Antiviral agents frequently applied for treatment of herpesvirus infections include acyclovir and its derivatives. The antiviral effect of a triterpene extract of birch bark and its major pentacyclic triterpenes, i.e. betulin, lupeol and betulinic acid against acyclovir-sensitive and acyclovir-resistant HSV type 1 strains was examined. The cytotoxic effect of a phytochemically defined birch bark triterpene extract (TE) as well as different pentacyclic triterpenes was analyzed in cell culture, and revealed a moderate cytotoxicity on RC-37 cells. TE, betulin, lupeol and betulinic acid exhibited high levels of antiviral activity against HSV-1 in viral suspension tests with IC₅₀ values ranging between 0.2 and 0.5 μg/ml. Infectivity of acyclovir-sensitive and clinical isolates of acyclovir-resistant HSV-1 strains was significantly reduced by all tested compounds and a direct concentration- and time-dependent antiherpetic activity could be demonstrated. In order to determine the mode of antiviral action, TE and the compounds were added at different times during the viral infection cycle. Addition of these drugs to uninfected cells prior to infection or to herpesvirus-infected cells during intracellular replication had low effect on virus multiplication. Minor virucidal activity of triterpenes was observed, however both TE and tested compounds exhibited high anti-herpetic activity when viruses were pretreated with these drugs prior to infection. Pentacyclic triterpenes inhibit acyclovir-sensitive and acyclovir-resistant clinical isolates of HSV-1 in the early phase of infection.
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Short communicationThe synergistic effects of betulin with acyclovir against herpes simplex viruses

Abstract

Bioorg. Med. Chem. Lett.

Adv. Enzyme Regul.

J. Clin. Virol.

Antiviral Res.

Antiviral Res.

Bioorg. Med. Chem. Lett.

Antiviral Res.

Fitoterapia

Bioorg. Med. Chem. Lett.

Inhibitory effects of triterpenoids and sterols on human immunodeficiency virus-1 reverse transcriptase

Lipids

Short communication
The synergistic effects of betulin with acyclovir against herpes simplex viruses