University of Groningen Deciphering the antiviral potential of tomatidine towards mosquito-borne viral infections Troost-Kind, Berit DOI: 10.33612/diss.161786279 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2021 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Troost-Kind, B. (2021). Deciphering the antiviral potential of tomatidine towards mosquito-borne viral infections. University of Groningen. https://doi.org/10.33612/diss.161786279 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 15-10-2021
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University of Groningen
Deciphering the antiviral potential of tomatidine towards mosquito-borne viral infectionsTroost-Kind, Berit
DOI:10.33612/diss.161786279
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.
Document VersionPublisher's PDF, also known as Version of record
Publication date:2021
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):Troost-Kind, B. (2021). Deciphering the antiviral potential of tomatidine towards mosquito-borne viralinfections. University of Groningen. https://doi.org/10.33612/diss.161786279
CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.
Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.
The antiviral effi cacy of tomatidine towards dengue
virus in vivo
Berit Troost-Kinda, Suzanne J. Kapteinb, Johan Neytsb, Izabela A. Rodenhuis-Zyberta, Jolanda M. Smita
aDepartment of Medical Microbiology and Infection Prevention, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands
bDepartment of Microbiology, Immunology and Transplantation, Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, Leuven,
AbstractDengue virus (DENV) causes the most common arboviral disease worldwide with an estimated yearly global incidence of 390 million infections of which approximately 96 million are clinically apparent. There are four DENV serotypes (DENV-1 to 4), which all cause disease ranging from mild to life-threatening manifestations. Despite the high disease burden, there is no antiviral treatment available. Multiple antiviral compounds towards DENV have been identified within the last decades, but only very few have shown protective effects in clinical trials. We previously demonstrated that tomatidine exhibits potent antiviral activity towards DENV, Zika virus and chikungunya virus in vitro. Here, we evaluate the antiviral potential of tomatidine towards DENV in a non-lethal AG129 mouse model. Tomatidine (50 mg/kg) was given to mice once a day or twice a day with two doses of 25 mg/kg for 6 days via oral gavage. Mice were challenged with DENV one day after initiating tomatidine treatment and the viral load was measured at day 5 post-infection in the plasma, spleen, kidney and liver. In this experimental set-up no differences in viral load between tomatidine-treated and control mice were seen. Subsequent analysis of the tomatidine concentration in the plasma revealed an average concentration of 59 ng/mL, which is approximately 6 times lower than the EC50 value of tomatidine in vitro. Given the low plasma concentration of tomatidine in mice we conclude that further optimization of the in vivo model and treatment regime is required to elucidate the antiviral potential of tomatidine in vivo.
IntroductionDengue virus (DENV) is an arthropod-borne virus (arbovirus), which is transmitted to humans by the mosquito vector Aedes egypti1. DENV belongs to the Flaviviridae family and exists as four serotypes (DENV-1 to 4)2. The virus has drastically re-emerged within the last decades and although it is mainly endemic in the subtropical and tropical regions of the world, DENV also circulates in the south of Europe for the last 10 years3,4. Due to its high number of infections, which is estimated to be 390 million per year, it has been declared the most important human arbovirus infection worldwide1,5. Annually, approximately 96 million individuals develop clinical symptoms, which usually include high fever, severe headache, eye pain, muscle pain, bone pain and rashes5. In 500,000 to 1 million cases, the infection leads to severe disease, which is characterized by capillary leakage, pleural effusion, severe bleeding and organ impairment and is potentially fatal6.
Despite the high burden of DENV, there is no antiviral treatment available to date. The overall aim of antiviral therapy is to reduce the virus titer early in infection thereby alleviating disease symptoms later in infection7. In our previous work, we have identified the natural steroidal alkaloid tomatidine as a potent inhibitor of DENV, Zika virus and chikungunya virus infectivity in vitro8,9. Tomatidine was found to exhibit potent antiviral activity towards all four DENV serotypes in Huh7 cells with EC50 values in the low micromolar range12. Moreover, time-of-addition studies demonstrated that tomatidine predominantly acts at a post-entry step of the virus replication cycle.
Tomatidine is derived from the stems and leaves of unripe green tomatoes and is the aglycone metabolite of tomatine, which serves as a defense molecule towards different pathogens in the tomato plant10. Within the last decades, tomatidine was reported to have many health beneficial properties such as an anti-metastatic, anti-inflammatory, anti-atherosclerotic, anti-
The antiviral efficacy of tomatidine towards DENV in vivo
osteoporosis and anti-microbial activity as well as protection from age-dependent muscle atrophy and ischemic neuronal damage8,9,11–22. The anti-inflammatory, anti-fungal and anti-atherosclerotic effect and the protection from muscle atrophy have already been demonstrated in mice. In these studies, tomatidine was mostly administered orally, via supplementation of standard chow16,20,23. No toxicity was reported up to a dose of 50 mg/kg. The pharmacokinetic properties of tomatidine are ill understood to date. Only one report on the protective effect of tomatidine on muscle atrophy quantified the plasma concentration of tomatidine as 287 ng/ml after two month of continuous tomatidine treatment (0.05% w/w) supplemented to the standard chow20.
Here, we evaluated the antiviral activity of tomatidine in a non-lethal AG129 mouse model of DENV infection. AG129 mice are deficient in the interferon-a/b and g receptors and therefore allow for efficient DENV replication24. Mice were treated with tomatidine (up to 50 mg/kg) via oral gavage once or twice a day for 6 days and treatment was initiated one day prior to DENV infection. The RNA load in plasma, spleen, kidney and liver, as well as the tomatidine concentration in plasma was determined at 5 days post-infection (dpi). In this experimental set-up, no effect of tomatidine was seen on the viral load in plasma and tissues. Furthermore, the achieved plasma levels of tomatidine were found to be very low and approximately 10 times lower than the in vitro EC50 value. Taken together, the lack of efficacy on viremia in the mice appears to be the result of the low exposure of tomatidine. Therefore, it is too soon to disregard tomatidine as an antiviral compound and future studies are required to optimize the dosage of tomatidine and the treatment regime in mice.
Results
Tomatidine has no effect on the viral titer in plasma, spleen, kidney and liver of dengue virus-infected AG129 mice
In order to evaluate the antiviral activity of tomatidine in vivo, a non-lethal AG129 mouse model of DENV infection was used. The mice were treated with 50 mg/kg of tomatidine dissolved in corn oil or corn oil as vehicle control via oral gavage twice per day with a half dose of 25 mg/kg or once per day with a full dose of 50 mg/kg respectively. Oral gavage was chosen as most in vivo reports on tomatidine are based on oral administration16,20,23. Corn oil was selected as solvent due to the hydrophobic nature of tomatidine. The highest reported dosage of tomatidine in vivo is 50 mg/kg16,18. Hence, we used this concentration as our dosage in vivo. Mice were pre-treated with tomatidine for one day and challenged with 102 PFU of DENV-2 intraperitoneally (Rega strain). Intraperitoneal injection is a commonly used route of administration for DENV infection in vivo24. A non-lethal infectious dose of 102 PFU was chosen as the replication dynamics closely reflect that of a natural human infection. A schematic overview of the experimental setup is given in Figure 1A. The weight of the mice was measured daily to monitor the well-being of the mice and to ensure that tomatidine does not induce toxicity. Throughout the treatment and infection period, the weight of the mice remained constant in all treatment groups (Figure 1A) indicating that tomatidine-induced weight loss did not occur. The highest virus titer was observed in the plasma with 4.19±0.5 Log10 GEC/mL in the vehicle control group. For spleen, kidney and liver, the Log10 RNA copy numbers of the control group were 2.39±1.3, 3.47±0.5 and 2.21±0.4 Log10 GEC/mL, respectively (Figure 1C). Unfortunately, comparable virus titers were observed in the tissues upon treatment with two daily doses of 25 mg/kg or one daily dose of 50 mg/kg tomatidine.
Figure 1. Tomatidine does not reduce DENV infectivity in vivo. (A) Outline of experimental setup.11-week-old female AG129 mice were treated with 50 mg/kg tomatidine daily in one single dose or two half doses of 25 mg/kg via oral gavage starting one day prior to DENV infection. Mice were infected with 102 PFU/mL of DENV-2 intraperitoneally. (B) Th e weight of the mice was taken daily from the point of tomatidine treatment until the end-point of the experiment at 5 days post-infection. (C) At day 5 post-infection, mice were sacrifi ced and samples of plasma, spleen, kidney and liver were analyzed for their viral GEC/mL via RT-qPCR. Each treatment group consisted of fi ve mice. Data is presented as mean±SEM.
The antiviral efficacy of tomatidine towards DENV in vivo
Plasma levels of tomatidine are very low at 5 days post-infection
To better understand the reason for the lack of antiviral activity of tomatidine in this in vivo model, we next determined the tomatidine concentration in the plasma samples of mice collected at the endpoint (i.e. 6 days after start of treatment) treated with the 50 mg/kg tomatidine once daily via LC-MS. The plasma concentration of tomatidine per mouse varied from 26 to 108 ng/mL. Two of the 4 samples were below 50 ng/mL, the lowest point of the standard curve (Supplementary Figure S1). Based on the 4 samples, the average tomatidine concentration was 59 ng/mL (Figure 2), which corresponds to a concentration of 0.13 µM (Figure 2). Therefore, the tomatidine concentration as determined in the mouse plasma was on average 6 times lower than the EC50 values determined in vitro in Huh7 cells8.
1 2 3 40
50
100
150
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atid
ine
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mL]
Mouse
Mouse ng/mL µM
1 63 0.14
2 39 0.09
3 26 0.06
4 108 0.24
Figure 2. Plasma level of tomatidine in mice is low. The tomatidine concentration in plasma samples of 4 DENV-infected mice treated once daily with 50 mg/kg tomatidine was determined via liquid chromatography/mass spectrometry. Insert: Tomatidine concentration for each mouse presented as ng/mL and µM. Data is presented individually for each mouse.
DiscussionHere, we describe a first pilot experiment to evaluate the in vivo antiviral efficacy of tomatidine towards DENV. Antiviral efficacy was evaluated in a non-lethal AG129 mouse model of DENV infection and mice were treated daily with 50 mg/kg dissolved in corn oil via oral gavage. At these conditions, no reduction in viral load was seen. The tomatidine plasma concentration in the mice treated once per day with 50 mg/kg tomatidine was 59 ng/mL after six days of consecutive tomatidine treatment. This concentration is 6 times lower than the in vitro EC50 value. Although it is difficult to translate in vitro EC50 values to effective in vivo doses, literature suggests that the in vivo effective dose should be approximately 4 times the determined EC50 value26. Therefore, it seems plausible that the low bioavailability of tomatidine may have been the limiting factor in this study.
In most of the reported in vivo studies, tomatidine was administered via supplementation to the standard chow16,20,23. One study reported a plasma concentration of 287 ng/mL after 2 months of unlimited access to standard chow supplemented with 0.05% (w/w) tomatidine20. In our study, we administered tomatidine via oral gavage to control the intake of tomatidine and with the aim to increase the plasma concentration. The results, however, show that lower
plasma concentrations were achieved compared to intake of tomatidine via standard chow. This might be due to various reasons including differences in the treatment duration. Yet, it becomes evident that oral administration of tomatidine-containing standard chow and tomatidine dissolved in corn oil may not lead to sufficiently high plasma concentrations.
Solvents are known to play a crucial role in the treatment outcome of in vivo studies as they are able to influence the absorption, distribution, metabolism and elimination of the test compounds27,28. We used corn oil due to the hydrophobic nature of tomatidine and since this solvent was used before to study the role of tomatidine in skeletal muscle atrophy12. Earlier in vivo antifungal studies on tomatidine used DMSO, ethanol or cyclodextrin complexation yet no efficacious results were obtained18. Importantly, the authors did observe potent antifungal activity when tomatidine was incorporated into nanoparticles18. Although the obtained plasma concentrations were not reported, it is clear that incorporation of tomatidine into solid lipid nanoparticles prepared with fluconazole has beneficial effects. Therefore, in order to enhance the bioavailability of tomatidine in in vivo studies, we propose to use tomatidine-containing nanoparticles in future studies.
The treatment frequency is another important factor that can influence the effectiveness of antiviral drugs. For celgosivir for example, in vivo anti-DENV activity in a non-lethal AG129 model was only seen after increasing the treatment frequency from two to four times per day29. Given the unknown stability and pharmacokinetic profile of tomatidine in vivo, it might be possible that a treatment frequency of 12 to 24 h is too low to achieve a sufficient accumulation and bioavailability of the compound. Hence, a more detailed investigation of the pharmacokinetic profile of tomatidine is warranted to better understand the bioavailability of tomatidine and to predict an efficacious treatment regime.
Another aspect that should be considered for future studies is the virus strain. Our previous in vitro studies on tomatidine were performed using the DENV-2 strain 16681, whereas in this in vivo model another DENV-2 strain (Rega strain) was used8. The Rega strain is not registered in GenBank but shares 99% sequence homology with a South Korean isolate DENV-2/KBPV-VR-29. When aligning the sequence of DENV-2/KBPV-VR-29 and DENV-2 16681 using nucleotide and protein blast, a 92.2% and 97.2% sequence homology was found, respectively. Hence, it may be possible that the differences in genome sequence between the two DENV-2 strains are responsible for the lack of antiviral activity of tomatidine. In this regard it is important to note that we found antiviral activity towards all four DENV serotypes, which have a sequence homology between 60 to 75%8,30,31. Nevertheless, since we cannot rule out that specific amino acid sequences within the Rega strain are responsible for the lack of an antiviral effect we recommend in vitro testing of the Rega strain prior to the start of new in vivo experiments.
Altogether, the potential of tomatidine as an antiviral treatment option for DENV infection should be further evaluated. Future studies should aim to increase the plasma concentration of tomatidine without inducing toxicity by investigating different solvents and treatment regimes. This, together with pharmacokinetic studies on the compound will help to design a more suitable in vivo set-up to further assess the antiviral potential of tomatidine towards DENV infections.
The antiviral efficacy of tomatidine towards DENV in vivo
Materials and methods
Chemicals
Tomatidine hydrochloride was purchased from Sigma Aldrich (St. Louis, Missouri, USA) and dissolved in corn oil to a stock solution of 5 mg/mL. The tomatidine solution was prepared every day.
Virus
The dengue virus serotype 2 (Rega strain) was obtained from the Institute Pasteur (Paris, France). This strain is not yet available in the GenBank, but shows 99% sequence homology with the DENV-2/KBPV-VR-29 isolate from South Korea (Accession Number GenBank KP406804).
Mouse model of DENV infection and tomatidine treatment
All mouse experiments were performed at KU Leuven with approval of the Ethical Committee of the University of Leuven [P047/2017]. Female AG129 mice (11-weeks old) were treated twice daily with 25 mg/kg or once daily with 50 mg/kg tomatidine dissolved in corn oil or corn oil only (vehicle control) via oral gavage daily for 6 days. Intraperitoneal injection with 102 PFU/mL DENV-2 was performed one day after tomatidine treatment was initiated. The weight of the mice was monitored daily. At 5 dpi, mice were euthanized by pentobarbital (Nembutal) injection. Blood serum and tissue samples of spleen, kidney and liver were collected and stored at -80°C until further use.
Quantification of viral RNA via Q-RT-qPCR
For viral RNA isolation from serum samples, the NucleoSpin RNA virus kit (Macherey-Nagel, Dueren, Germany) was used according to the manufacturers protocol. For tissue samples, RLT lysis buffer (Qiagen, Germantown, Maryland, USA) was added and tissues were homogenized using an automated homogenizer (Precellys24, Bertin Technologies, Montigny Le Bretonneux, France). Subsequently, homogenates were cleared via centrifugation and RNA was extracted via the RNeasy minikit (Qiagen) according to the manufacturer’s protocol and eluted in RNase-free water. Genome-equivalent CHIKV copies (GEC) were determined via Q-RT-qPCR as previously described25.
Mass spectrometry analysis for tomatidine quantification in mouse plasma
Plasma levels of tomatidine were measured via liquid chromatography/mass spectrometry (LC-MS) on an Acquity UPLC (Waters, Milford, Massachusetts, USA) coupled to a Maxis QTOF (Bruker, Billerica, Massachusetts, USA). For quantification, tomatidine was dissolved in 100% methanol (MeOH) as a stock solution of 9.4 mg/mL and serial dilutions were generated in methanol to 500, 200, 100 and 50 ng/mL. The standard curve was generated by mixing each solution with 100 µL blank serum. Then tomatidine was extracted by adding 400 µL methanol to precipitate the proteins. After centrifugation for 2 min at 2,000 RCF the supernatant was dried using a nitrogen flow. Tomatidine extraction from the plasma samples was performed in the same way. Finally, all standards and samples were reconstituted in 100 µL of 40% MeOH in water with 0.1% formic acid (FA). LC-MSMS was performed using an electrospray interface in positive ion mode with an end plate offset of 500 V and a drying
temperature of 200°C. Data was collected in the multiple reaction monitoring mode of m/z 416.3 to m/z 161.1 with a collision-induced dissociation energy of 45. For LC separation a Cortecs C18 100x2.1 mm, 2.7 µm column (Waters) was used at a temperature of 55°C. The sample injection volume was 10 µL and gradient separation was performed at a flow rate of 0.3 ml/min. Solvent A was water with 0.1% FA and solvent B was acetonitrile with 0.1% FA. Solvent B was increased from 5% to 95% B in 5 min, held for 3 min at 95% and then decreased back to 5% B at 8.1 min. The total run time was 10 min and the retention time for tomatidine was 3.8 minutes.
Statistical analysis
All data was analyzed with GraphPad Prism (La Jolla, CA, USA) and data are presented as mean ±SEM. Statistical differences were evaluated via Student’s t-test.
Supplementary material
y = 11638xR² = 0.9961
0
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Supplementary Figure S1. Standard curve of tomatidine. For quantification, tomatidine was dissolved in 100% methanol as a stock solution of 9.4 mg/mL. Serial dilutions were performed in methanol to 500, 200, 100 and 50 ng/mL To generate the standard curve each dilution was mixed with 100 µL blank mouse serum.
The antiviral efficacy of tomatidine towards DENV in vivo
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