Lithocholic Acid Oleate Preparative Synthesis and Its Formulation with Lithocholic Acid as a Preventive Antiviral: In Vitro and In Vivo Assays Against HSV-1 as a Viral Infection Model

Abstract

The discovery and design of antiviral agents have gained unprecedented significance due to the emergence of global health threats. The use of synthetic chemistry has enabled the modification of existing molecules and the creation of entirely novel compounds. In our laboratory, we have enzymatically synthesized a novel bioconjugate, lithocholic acid oleate (LO), derived from lithocholic acid (LCA), a bile acid that has been proven by researchers to exhibit antiviral activity in vitro. The study presented herein describes the preparative synthesis, formulation, and evaluation of LO both in vitro and in vivo for its antiviral activity against human herpes simplex virus 1 (HSV-1) as a model of viral infection. Evaluation of cytotoxicity using A549 cells indicated that a combination of LO (400 μM) and LCA (30 μM) exhibited a favorable safety profile while effectively inhibiting HSV-1 infection comparable to acyclovir treatment. Furthermore, in the in vivo assay, animals treated with an oily formulation containing 7% LO; 0.50% LCA; and 3% oleic acid (OA), 48 h prior to virus exposure, showed results even superior to a 5% acyclovir commercial formulation in terms of scar formation and wound recovery. These promising results enable the development of new preventive products against HSV-1 and probably other viruses.

Keywords: HSV-1 antivirals; lithocholic acid; lithocholic acid oleate; preparative synthesis.

Figure 1

LO reaction solvent screening. 1. LCA, 2. OA, 3. Reaction in 2-methyl-2-propanol, 4. Reaction in tetrahydrofuran, 5. Reaction in dimethyl sulfoxide, 6. Reaction in 2-methyl-2-butanol, 7. Control reaction of OA in 2-methyl-2-propanol, 8. Control reaction of LCA in 2-methyl-2-propanol. The reaction was performed using an OA molar excess: 100 mM of LCA and 150 mM of OA.

Figure 2

Reaction kinetics of LO preparative synthesis and purification. (A). Reaction kinetics time 1. 0 h, 2. 6 h, 3. 8 h, 4. 12 h, 5. 24 h, and 6. 48 h, 7. LCA and OA standards. The reaction was performed using a molar excess of LCA: 150 mM of LCA and 100 mM of OA. TLC was revealed using 0.75% (w/v) KMnO₄, 0.056% (w/v) NaOH, and 10% (w/v) K₂CO₃. (B). LO purification. 1. Remaining LCA after petroleum ether extraction, 2. Purified LO extracted with petroleum ether. TLC was revealed using 10% (w/v) phosphomolybdic acid in absolute ethanol.

Figure 3

LO, NMR spectra. (A). LO structure. (B). Carbon 13 (¹³C) spectrum. 1′ = 173.1 ppm; 9′ and 10′ = 130.6 ppm; 3 = 73.9 ppm. (C). Proton (¹H) NMR spectrum. 9′ and 10′ = 5.34 ppm; 3 = 4.61 ppm; 2′ = 2.35, 2″ = 2.33 ppm. ppm values corresponding to characteristic ¹³C and ¹H NMR peaks were estimated using Chemdraw.

Figure 4

Cytotoxicity of LO and LCA in A549 cells. The cytotoxicity (CC50) was expressed as the concentration at which 50% of the cells died in DMEM containing varying concentrations of the tested molecules. Graphs show the mean values of two experiments (n = 4). The error bars represent the standard deviation.

Figure 5

Inhibition of HSV-1 in A549 cells. (A): Representative photographs from the cytopathic effect (CPE) inhibition assay at 72 h post-infection. A549 cells were pretreated for 24 h with different treatments and then infected with HSV-1. C−: Uninfected A549 cells; C+: A549 cells infected with HSV-1, showing CPE characterized by cell detachment (yellow arrow) and syncytia formation (red arrow). LO: A549 cells treated with 400 µM LO; LCA: A549 cells treated with 30 µM LCA; LO + LCA: A549 cells treated with 400 µM LO in combination with 30 µM LCA; acyclovir: A549 cells treated with 20 µM acyclovir; OA: A549 cells treated with 250 µM oleic acid (original magnification 10×). (B): Inhibition of HSV1 by viral plaque reduction assay. The images show plaque formation due to HSV-1 infection (C+) and the absence or reduction in plaques following treatment. A significant reduction in plaque formation was observed with LO or LCA treatment, while complete inhibition was achieved with the LO + LCA combination, as well as with acyclovir treatment.

Figure 6

Infectious viral yield reduction assay (A) and genomic copies (B) obtained from qPCR detection. A: Results are plotted as the log₁₀ of the viral yield, calculated using the TCID₅₀ method. HSV-1 represents the untreated, HSV-1-infected cultures. **** p < 0.0001, *** p < 0.0005 one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test when compared with the untreated control. B: Data are presented as the mean of genomic copies quantified using q-PCR from three replicates ± SD.

Figure 7

Time evolution of the mean lesion score. Lesion scoring was performed using a nominal scale based on clinical presentation as follows: 0, no lesion; 1, discrete vesicles; 2, two or more open lesions; 3, separate ulcerations; and 4, Zoster band formations. The first lesions appeared on day 4 post-inoculation, with all groups exhibiting some form of lesion by day 6. Group T2 showed the worst clinical outcome, whereas groups T4 and T5 had the least severe clinical presentations. Zoster band formation was observed on day 7 post-inoculation in group T2 and on day 12 in group T5. Overall, groups T4 and T5 demonstrated the best clinical recovery, characterized by improved outcomes and faster healing. The area under the curve (AUC) was analyzed using Graphad Prism 8. Groups were compared using ANOVA analysis with a post hoc test, Sydak’s multiple comparisons test. **** p < 0.0001, *** p < 0.0002, * p< 0.01 and ns: no statistical differences. There was no statistically significant difference in AUC values between the T5 group (Acyclovir) (25.40 ± 4.21) and T4 group (group pre-treated with LO + LCA 48 h before virus inoculation) (20.00 ± 3.44).

Figure 8

Representative images of groups T4 and T5 showing infection recovery during the experiment. We found that wound healing was more effective in group T4 compared to T5, with a noticeable reduction in scar formation and improved skin repair. (A): HSV-1 infected mice without treatment at 11 dpi; (B): HSV-1 infected mice and treated with LO + LCA at 11 dpi; (C): HSV-1 infected mice treated with acyclovir at 11 dpi; (D): HSV-1 infected mice without treatment at 18 dpi; (E): HSV-1 infected mice and treated with LO + LCA at 18 dpi; (F): HSV-1 infected mice treated with acyclovir at 18 dpi.