TKTL1 inhibitors for antiviral therapy

Abstract

At least one inhibitor of transketolase TKTL1 is used for the treatment of a viral infection, in particular an RNA viral infection, in particular also a SARS-CoV-2 infection, in a patient.

Description

[Technical Field]

[0001] The present invention relates to inhibitors of the transketolase TKTL1 (hereinafter abbreviated as "TKTL1 inhibitors") for use in antiviral therapy, in particular in the treatment of RNA viruses, in particular also in the treatment of SARS-CoV-2 infections and Covid-19 disease.

[0002] In 2019, a novel coronavirus named SARS-CoV-2 was reported in China, infecting people and spreading rapidly. Some infected individuals develop a disease called COVID-19, causing lung damage, which can lead to oxygen deficiency and difficulty breathing. In March 2020, the WHO declared the SARS-CoV-2 outbreak a pandemic. Unlike influenza, SARS-CoV-2 infection can progress asymptomatically, meaning that seemingly healthy individuals can transmit the virus to others. This makes containment of SARS-CoV-2 extremely difficult. In addition to the asymptomatic course of infection, many individuals infected with SARS-CoV-2 exhibit flu-like symptoms such as cough, sore throat, diarrhea, or fever. Unlike influenza, symptoms such as loss of taste and other symptoms or deficits related to neuronal function are also observed. Such neurological symptoms, such as cognitive impairment, fatigue, and chronic fatigue syndrome, persist long after acute SARS-CoV-2 infection has resolved and pose a major health problem for infected individuals. A subgroup of SARS-CoV-2-infected individuals also develop pneumonia, sometimes bilateral pneumonia. SARS-CoV-2 infection can lead to multiple organ failure and fatal outcomes. In severe cases of SARS-CoV-2 infection, an overreaction—that is, an overly aggressive immune system response—often results, leading to immune cells migrating to the lungs and inhibiting their function. Medications that inhibit immune system activity, such as dexamethasone, have a positive effect on the course of the disease. However, a high proportion of patients with severe SARS-CoV-2 infection die. Currently, no fully effective treatments are available to prevent or treat severe cases of Covid-19 disease.

[0003] In the case of many other viral diseases, there are still no active ingredients that are effective enough to combat the viral infection in patients, such as the lack of a suitable treatment for dengue fever caused by a virus or severe herpes infections of the eyes that can lead to blindness.

[0004] Most therapeutic control strategies for viral diseases known in the art aim to neutralize the virus itself, such as preventive treatment with vaccines or palliative and curative treatment with the administration of antiviral active ingredients. What these strategies have in common is their attack on virus-specific structures (epitopes / enzymes). However, viruses have the ability to change their genomes in a very short time, allowing them to adapt very quickly to new environmental conditions. This is currently evident with new variants of the SARS-CoV-2 virus, which can spread better, provide better protection from the body's own immune system, provide better protection from vaccination, or even result in increased infection mortality. Therefore, therapeutic strategies based on virus-specific structures may quickly fail because many naturally occurring viral mutants have emerged that are resistant to such active ingredients or vaccines. Furthermore, it is essentially impossible to predict which viral mutations or viruses will emerge next and pose a threat to humans in the future. Therefore, it is very difficult, even nearly impossible, to develop and plan virus-specific therapeutic strategies in a timely manner.

[0005] Viral genomes are composed of either RNA or DNA, and the SARS-CoV-2 virus belongs to the RNA virus family.

[0006] After infecting cells and releasing viral RNA, RNA viruses replicate (amplify) this viral RNA to produce as many copies as possible. This replication absolutely requires ribose-5-phosphate (R5P) from the host cell.

[0007] DNA viruses also require R5P for replication in host cells because deoxyribose-5-phosphate, an essential component of DNA, is synthesized from ribose-5-phosphate. Thus, both RNA and DNA viruses depend on host cell R5P synthesis.

[0008] In mammalian cells, R5P is provided via the pentose phosphate pathway (PPP), and according to prevailing theory, this can occur via both the oxidative and non-oxidative portions of the PPP.

[0009] Transketolase (TKT) is a key enzyme in the PPP. It forms a dimer and is activated by the binding of a thiamin diphosphate molecule and a divalent cation. This transketolase enzyme activity can be altered by the formation of a heterodimer from TKT and the transketolase-like-1 (TKTL1) protein (Li et al., 2019).

[0010] Li et al. (2019) described how the formation of TKTL1-TKT heterodimers allows cells to reprogram their R5P metabolism. When more R5P is needed, which is primarily the case for nucleotide and DNA synthesis during the cell's S phase, increased TKTL1 levels increase the formation of TKTL1-TKT heterodimers, which promote R5P production. In other words, Li et al. demonstrated that the basal R5P supply of cells is provided via TKT-TKT homodimers. When cells require even greater amounts of R5P, TKTL1 expression and the resulting formation of TKT-TKTL1 heterodimers initiate a "turbo program" that promotes increased R5P formation to enable cell division and, ultimately, cell proliferation.

[0011] A study by Bojkova et al. (2020) using SARS-CoV-2 infected cells of the human cell line Caco-2 showed that the cellular PPP is activated in SARS-CoV-2 infected cells.

[0012] Bojkova et al. (2020) also investigated whether SARS-CoV-2 replication could be affected by TKT inhibitors. The use of the TKT inhibitor benfoxythiamin (B-OT), a prodrug of the TKT inhibitor oxythiamin (B-OT), an inhibitory analog of thiamin, inhibited SARS-CoV-2 viral replication in host cells. However, a B-OT concentration of at least 5 mM was required to inhibit SARS-CoV-2 viral growth. Translated to humans, this corresponds to a B-OT dose of at least 1 g of B-OT per kg of body weight, i.e., 70 g of B-OT for a 70 kg human. This value is outside the range of active ingredient typically or exceptionally used and far exceeds the tolerable dose of oxythiamin known in the prior art for humans, which can be converted to B-OT. Therefore, those skilled in the art assume that B-OT and TKT inhibitors per se are not viable options in the context of active ingredients for virus inhibition.

[0013] The present invention is based on the problem of providing a drug for combating viral infections, in particular RNA virus infections, which is also suitable in particular for combating the SARS-CoV-2 virus.

[0014] The solution to this problem consists in providing at least one inhibitor of transketolase TKTL1 (hereinafter abbreviated as "TKTL1 inhibitor") for use in the medical therapeutic treatment of viral infections (antiviral treatment), in particular in the treatment (treatment) of RNA virus infections, in particular also in the treatment (treatment) of SARS-CoV-2 infection and related diseases such as, for example, COVID-19.

[0015] In other words, the solution to this problem lies in the technical teaching of the use of at least one inhibitor of transketolase TKTL1 (abbreviated as "TKTL1 inhibitor") as an active ingredient of a drug having an antiviral effect, in particular a virostatic (virus-inhibiting) effect.

[0016] Definitions of terms: - the term "inhibitor" is used herein in the context of an agent (i.e. in principle any agent), in particular any material substance, i.e. a substance or mixture of substances, including a pharmaceutical composition, that is suitable for directly or indirectly bringing about (causing) a decrease in the activity of a TKTL1 polypeptide in an individual or in the cells of an individual. These agents or materials include in particular: (a) Inhibitors of transcription of the TKTL1 gene, for example, substances that bind to the TKTL1 promoter and thereby limit or prevent its activity, such as resveratrol, which inhibits the TKTL1 promoter in a dose-dependent manner. (b) Inhibitors of TKTL1-mRNA translation, for example inhibitory RNAs such as antisense constructs, siRNAs, sh-RNAs, ribozymes, etc. (c) inhibitors of the enzymatic reaction of TKTL1 and / or the TKTL1-TKT heterodimer complex, in particular active ingredients which limit or block this enzymatic reaction. These include in particular antagonists of the cofactors of the TKTL1 polypeptide, such as, for example, antithiamine compounds. (d) Inhibitors of the formation of the TKTL1-TKT heterodimer complex, in particular active ingredients that limit or prevent the formation of the heterodimer. - The term "RNA" is used herein as synonymous with ribonucleic acid. - The term "DNA" is used herein to refer to deoxyribonucleic acid.

[0017] The present invention is based on the surprising realization that inhibition of the enzyme TKTL1 in human cells, and thus inhibition of the enzymatic activity of the TKTL1 / TKT heterodimer, significantly inhibits the growth (replication) of viral RNA or viral DNA, while the host cell itself remains viable and the human body does not suffer permanent and irreversible damage.

[0018] Because cell division and therefore proliferation are essential for the human body, inhibiting TKTL1 and / or the TKTL1 / TKT heterodimer to the extent necessary to slow or prevent viral replication, according to Bojkova et al. (2020), would be expected to result in severe side effects, including inhibition of cell division and even death. Furthermore, precisely because transketolases, particularly TKTL1, are considered fundamentally involved in cell survival as key enzymes in the control of the cell cycle and cell division, the severe side effects predicted by conventional wisdom made them unsuitable targets for antiviral therapeutic strategies.

[0019] However, it was surprisingly found that inhibition of the enzymatic activity of the TKTL1 / TKT heterodimer in virus-infected human host cells prevents or advantageously inhibits the massive (potent) increase in ribose-5-phosphate (R5P) production required for the biosynthesis of viral nucleic acids and thus the propagation of viral nucleic acids (RNA or DNA) via host cell metabolism, while maintaining the amount of R5P required for host cell survival.

[0020] In other words, the use of TKTL1 inhibitors in the treatment of viral infections, particularly SARS-CoV-2 infections, is based on the surprising realization that TKTL1 inhibitors can significantly inhibit or reduce the biosynthesis of viral nucleic acids and therefore viral growth (viral replication) in human host cells without endangering the survival of the host cells and therefore without risking damage to the human body.

[0021] The use of TKTL1 inhibitors according to the present invention disrupts the metabolism of host cells and thus the entire human body, so that there are no longer enough ribose-5-phosphate building blocks (R5P) available for viral replication, and thus viral growth is slowed down or completely prevented without irreversibly damaging the cells and the entire human body. This has the advantage that the virus itself is not the (direct) target of the active ingredient, but rather the metabolism of the host cell, and therefore any viral mutations do not impair the success of the application according to the present invention.

[0022] The use of TKTL1 inhibitors according to the present invention is a therapeutic strategy that targets factors derived from infected host cells and required for virus proliferation, rather than virus-specific structures. Therefore, firstly, it can be used with promise against a wide range of viruses, and secondly, an antiviral therapeutic strategy is available that is effective against viral mutations (viral mutants). This also enables protection against viruses that may emerge in the future. The present invention enables protection against viruses without knowing the viral RNA or DNA sequence. Therefore, compared to vaccination, there is no lead time required for drug development, and treatments will become available soon. This will enable protection of humans and mammals in general from existing and future viruses and diseases that are or may be derived from viruses. For the first time, existing protection against viral diseases will be realized.

[0023] In a preferred embodiment, the inhibitor is a substance or a mixture of substances that can specifically restrict or inhibit the transcription of TKTL1 gene and is suitable for this purpose.In this regard, particularly, substances that can bind to the TKTL1 promoter of cells and thereby restrict or inhibit its activity and are suitable for this purpose are considered.In practice, resveratrol has been shown to be a suitable inhibitor of TKTL1 promoter (Kumar B, 2018).

[0024] In a similarly preferred embodiment, the inhibitor is a substance or mixture of substances suitable for specifically inhibiting the translation of TKTL1-mRNA. In this regard, at least one substance selected from the group consisting of TKTL1-mRNA-specific antisense constructs, TKTL1-mRNA-specific siRNAs, TKTL1-mRNA-specific sh-RNAs, TKTL1-mRNA-specific ribozymes and other TKTL1-mRNA-specific inhibitory RNAs is particularly considered.

[0025] In a further preferred embodiment, the inhibitor is a substance or mixture of substances suitable for limiting or blocking the enzymatic reaction of TKTL1 and / or the TKTL1-TKT heterodimer complex. In this regard, in particular at least one substance selected from the group of antithiamine compounds and other antagonists, in particular antagonists from the group of cofactors of the enzyme TKTL1, is considered.

[0026] In one embodiment that has been tested, the TKTL1 inhibitor is an inhibitory thiamine analog. All known transketolase enzymes, including TKTL1, are functionally dependent on thiamine (vitamin B1) as a coenzyme. For example, thiamine analogs, such as oxythiamine, act as thiamine antagonists and can be used to inhibit transketolase (see EP 1354961).

[0027] A preferred inhibitory thiamine analog is the substance benphoxythiamine (B-OT).

[0028] B-OT is a precursor ("propharmacon," "prodrug") of oxythiamine, which can be administered orally and releases oxythiamine immediately after absorption by mammalian organisms. B-OT can reach every cell in every part of the body via the bloodstream. In vivo pharmacokinetic data show that oxythiamine can cross the blood-brain barrier and, as a result, can also be used successfully in the treatment of viral infections of the brain.

[0029] The chemical structure (structural formula) of benphoxythiamine is known, for example, from EP 1 354 961 A1.

[0030] Surprisingly, it was found that a B-OT concentration more than 1000-fold lower than the lowest concentration described by Bojkova et al. (2020) at the cell culture level for the human Caco-2 cell line is sufficient to inhibit the enzymatic activity of TKTL1 in the TKTL1 / TKT heterodimer in the human body. In their study, Bojkova et al. (2020) showed that a concentration of at least 5 mM B-OT was required to inhibit SARS-CoV-2 viral growth in SARS-CoV-2-infected Caco-2 cell lines. However, unexpectedly and surprisingly for those skilled in the art, during the course of the research underlying the present invention, it was shown that administration of less than 35 mg of B-OT per 70 kg of patient body weight per day, i.e., less than 0.5 mg per kg of body weight per day, correspondingly, less than 1 nanomolar (1 nM) of B-OT per kg of body weight per day, is sufficient to inhibit SARS-CoV-2 viral growth in the human body.

[0031] Thus, according to the present invention, in the case of benphoxethiamine as a TKTL1 inhibitor, B-OT is applied / administered at a dose / administration of 7 μg to 430 μg (μg = micrograms), advantageously 14 μg to 215 μg, and particularly preferably 14 μg to 130 μg of B-OT per kg of patient body weight per day. In other words, the dose / administration of the TKTL1 inhibitor benphoxethiamine per kg of patient body weight per day is 7 μg≦μg B-OT≦430 μg, advantageously 14 μg≦μg B-OT≦215 μg, and particularly preferably 14 μg≦μg B-OT≦130 μg.

[0032] Generally, according to the present invention, in the case of benfoxythiamine as a TKTL1 inhibitor, benfoxythiamine is used in a dose of less than 0.5 mg, advantageously less than 0.3 mg per kg of patient body weight per day.

[0033] The production of benphooxythiamine (B-OT) according to EU GMP guidelines for human and veterinary medicines is well established in the art, allowing the use of benphooxythiamine in mammals (e.g., dogs, cats), especially humans.

[0034] In a further preferred embodiment, the TKTL1 inhibitor is a substance or mixture of substances suitable for limiting or preventing the formation (production) of the TKTL1-TKT heterodimer complex.

[0035] The present invention will now be described in more detail with reference to exemplary embodiments using tables and figures. [Brief explanation of the drawings]

[0036] [Figure 1] FIG. 1 shows the reduction of SARS Cov-2 viral load and viability of Caco-2 cells after inhibition of TKTL1 translation using TKTL1-specific siRNA. [Figure 2] FIG. 1 shows the reduction of SARS Cov-2 viral load and viability of Caco-2 cells after inhibition of TKT translation using TKT-specific siRNA. [Figure 3] FIG. 1 shows the reduction of human cytomegalovirus (HCMV) viral load and viability of Caco-2 cells after inhibition of TKTL1 translation using TKTL1-specific siRNA. [Figure 4] FIG. 1 shows the reduction of human cytomegalovirus (HCMV) viral load and viability of Caco-2 cells after inhibition of TKT translation using TKT-specific siRNA. [Figure 5] FIG. 1 shows the dose-dependent reduction of SARS-Cov-2 viral load and viability of Caco-2 cells after inhibition of the TKTL1 promoter with different doses of resveratrol.

[0037] The reduction in viral load is shown in each case as the indicated percentage reduction.

[0038] Example 1: Comparison of (a) siRNA inhibition of TKTL1 with (b) siRNA inhibition of TKT in SARS-CoV-2-infected mammalian cells SARS-CoV-2-infected Caco-2 cells were transfected with (a) human TKTL1-specific siRNA and (b) human TKT-specific siRNA, and (c) an siRNA negative control (here, AllStars negative control siRNA from Qiagen, which, according to the manufacturer's information, has no homology to any known mammalian gene) using a transfection reagent consisting of polycationic and neutral lipids (here, METAFECTENE® from Biontex Laboratories GmbH, Munich). (siRNAs, or small interfering RNAs, are short RNA molecules that do not code for proteins and that bind to complementary single-stranded RNA molecules, thereby preventing their normal function.)

[0039] The following siRNA sequences were used: (a) TKTL1 specific: 1. 5'-GGAGUUGCAUGUGGAAUGG-3' (Diaz-Morelli et al. 2016) 2. 5'-UUAUUCACGAAGGAAACACUU-3' (Heller et al. 2018) 3. 5'-UAAAUAACCAUAGUUUCUGGU-3' (Heller et al. 2018) (b) TKT-specific: 5'-AAUGAUGGCUGUUGGCUGGTT-3' (Lu et al. 2017).

[0040] Effective silencing (i.e., gene silencing, gene expression knockdown by RNA interference) of (a) TKTL1 and (b) TKT in SARS-CoV-2-infected and siRNA-transfected Caco-2 cells was demonstrated using reverse transcriptase quantitative PCR (RTqPCR) and Western blot analysis. To measure and quantify TKTL1 / TKT mRNA and TKTL1 / TKT protein, RTqPCR and Western blot analysis were performed in SARS-CoV-2-infected and siRNA-transfected Caco-2 cells 72 and 96 hours after transfection.

[0041] The replication capacity of SARS-CoV-2 virus after transfection of Caco-2 cells with different siRNAs was determined by immunostaining of SARS-CoV-2 spike (S) protein. Spike protein staining was used to calculate the percentage of viral inhibition in cells transfected with (a) TKTL1 or (b) TKT siRNA compared to cells transfected with (c) siRNA negative control.

[0042] The cell viability of SARS-CoV-2-infected Caco-2 cells transfected with different siRNAs was counted by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay.

[0043] The results of these studies are shown in Table 1 and Figures 1 and 2. [Table 1]

[0044] Depending on the different siRNA sequences, the following effects were observed on the reduction of viral load and viability of SARS-CoV-2 infected Caco-2 cells compared to Caco-2 cells transfected with AllStars negative control siRNA:

[0045] In cells transfected with TKTL1-specific siRNA (compared to cells transfected with a non-targeting control duplex), TKTL1 translation was inhibited, and inhibiting TKTL1 translation at a level that still allowed cells to remain viable significantly reduced the ability of SARS-CoV-2 virus to replicate.

[0046] Thus, these experiments demonstrate that SARS-CoV-2 viral replication can be significantly inhibited by inhibiting TKTL1 without affecting cell viability.

[0047] A parallel approach using TKT-specific siRNA as an inhibitor of TKT-mRNA translation demonstrated that inhibition of TKT translation also led to a significant reduction in viral load in SARS-CoV-2-infected Caco-2 cells. However, cell viability was also significantly reduced. This suggests that the human therapeutic window for TKT inhibition to inhibit SARS-CoV-2 viral replication largely overlaps with the damage caused to human cells and is virtually nonexistent. In other words, the degree of TKT inhibition required to inhibit SARS-CoV-2 viral replication is so great that human cells are severely damaged to the point of death. When transketolase TKT inhibitors are administered at doses that result in the inhibition of SARS-CoV-2 viral replication, the damage to the human body is so severe that the therapeutic use of TKT inhibition to inhibit SARS-CoV-2 viral replication in humans must be ruled out.

[0048] Conclusion: The results show that applying TKTL1-specific siRNA as an inhibitor of TKTL1 to SARS-CoV-2-infected Caco-2 cells prevents viral replication within the host cells, and the host cells survive in the process.

[0049] Parallel in vitro studies with human cytomegalovirus (HMCV) (see Example 2 herein) confirm that siRNA inhibition of TKTL1 can block viral replication within host cells without compromising host cell viability.

[0050] Example 2: Comparison of (a) siRNA inhibition of TKTL1 with (b) siRNA inhibition of TKT in human cytomegalovirus (HCMV)-infected mammalian cells We investigated the effects of siRNA inhibition of TKTL1 and TKT in Caco-2 cells infected with human cytomegalovirus (HCMV). As described by Dal Pozzo et al. (2008), we used a recombinant HCMV-EYFP virus encoding enhanced yellow fluorescent protein (EYFP) to infect Caco-2 cells. This allows us to determine the replication capacity of HCM viruses using a fluorescence-based antiviral assay (e.g., Dal Pozzo et al. (2008)).

[0051] Caco-2 cells were transfected with the siRNA sequences of TKTL1 and TKT shown in Example 1. Effective silencing was demonstrated using RTqPCR (reverse transcriptase quantitative PCR) and Western blot analysis. These analyses were performed 72 and 96 hours after transfection. The viability of HMCV-infected cells was determined by MMT assay.

[0052] The replication capacity of HCM virus after transfection of Caco-2 cells with different siRNAs was determined using a fluorescence-based antiviral assay (Dal Pozzo et al. (2008)). The EYFP signal of the recombinant virus was used to calculate the percentage of viral inhibition in cells transfected with (a) TKTL1 or (b) TKT siRNA compared to cells transfected with (c) the siRNA negative control. The results are shown in Figures 3 and 4.

[0053] These results also confirm the results obtained in Example 1 using SARS-CoV-2 in HMCV-infected cells: inhibition of TKTL1 using TKTL1-specific siRNA as an inhibitor prevents HMCV virus replication in host cells, resulting in host cell survival. In contrast, TKT inhibition using TKT-specific siRNA also results in a significant reduction in viral load in HMCV-infected Caco-2 cells, but at the expense of significantly reduced cell viability.

[0054] Example 3: Use of B-OT as a TKTL1 inhibitor in human cytomegalovirus (HCMV)-infected mammalian cells We further investigated the susceptibility of HCMV replication to B-OT in human mammalian cells. To this end, we performed experiments using B-OT as described in Dal Pozzo et al. (2008): human embryonic lung fibroblasts (HEL) were infected with a recombinant HCMV-EYFP virus that additionally encodes enhanced yellow fluorescent protein (EYFP) (Dal Pozzo et al. (2008)). As an indicator of viral proliferation after HCMV-EYFP infection, EYFP protein luminescence was measured (excitation wavelength: 485 nm / emission: 530 nm). Here, a constant, low, cell-associated background luminescence, measured automatically, was subtracted from the fluorescence emission values. To examine the antiviral effect of B-OT, HCMV-EYFP-infected cells were incubated with different B-OT concentrations (0–20 mM B-OT) for 48 h. A dose-dependent inhibition of fluorescence (as an indicator of viral proliferation) was observed. The concentration of substance required to halve EYFP emission compared to the untreated control (0 mM B-OT) was defined as the 50% inhibitory concentration (IC50) in the fluorescence-based antiviral assay. The IC50 obtained here was 0.5 mM B-OT.

[0055] Example 4: Use of B-OT as a TKTL1 inhibitor to inhibit SARS-CoV-2 viral replication in the human body It is known from Zhu et al. (2020) that in patients with SARS-CoV-2 infection and Covid-19 disease, the severity of the disease can be assessed by the clinical values ​​of immunoinflammatory markers. High levels of the proinflammatory cytokines IL-6 and / or C-reactive protein (CRP) indicate severe disease and a very high risk of disease progression. Due to its pleiotropic properties, the proinflammatory cytokine IL-6 plays a key role in the "cytokine storm" described in patients infected with SARS-CoV-2. Its constitutive expression causes organ damage and severe pain (Zhu et al. (2020); Gupta et al. (2020)).

[0056] As part of individual cure trials in hospitalized patients with SARS-CoV-2 infection and COVID-19 disease, B-OT administered once daily at less than 20 mg per 70 kg of patient body weight, i.e., a concentration of less than 0.3 mg per kg of body weight, or correspondingly, less than 1 nanomolar (1 nM) per kg of body weight, resulted in a significant reduction in viral load as detected by PCR over a 7-day period, as well as in the inflammatory parameters C-reactive protein (CRP) and interleukin-6 (IL-6): after 7 days of B-OT treatment, CRP levels decreased from an average of 77 pg / ml to 5 pg / ml, and IL-6 levels decreased from an average of 63 pg / ml to 5 pg / ml.

[0057] Example 5: Use of resveratrol as a TKTL1 inhibitor to inhibit viral growth in SARS-CoV-2 infected Caco-2 cells Resveratrol (molecular formula C) is an organic compound in the polyphenol group. 14 H 12 O3) is known to those skilled in the art as an inhibitor of the TKTL1 promoter. Kumar B. (2018) reported that treatment of HeLa cells with 50 μM resveratrol for 48 hours reduced TKTL1 promoter activity by 75% compared to untreated cells. However, this also resulted in a significant decrease in HeLa cell viability.

[0058] In the study described in Example 5, resveratrol was used to inhibit viral replication in SARS-CoV-2-infected Caco-2 cells. To this end, SARS-CoV-2-infected Caco-2 cells were treated with different resveratrol concentrations for 24 hours. RTqPCR was performed to confirm the effect of reduced promoter activity on TKTL1 mRNA production. The RTqPCR results showed a decrease in TKTL1 mRNA depending on the resveratrol concentration used: the higher the concentration, the greater the decrease. Furthermore, as described in Example 1 herein, the viability of Caco-2 cells was determined by MTT assay, and the replication capacity of SARS-CoV-2 virus was determined by immunostaining of the SARS-CoV-2 spike (S) protein. SARS-CoV-2-infected Caco-2 cells not treated with resveratrol served as a control.

[0059] The replication capacity of SARS-CoV-2 virus under resveratrol treatment decreased with increasing doses of resveratrol compared to the control, but the viability of SARS-CoV-2-infected Caco-2 cells remained at a sufficient level. Thus, treatment with 25 μM resveratrol for 48 hours reduced the replication capacity of SARS-CoV-2 virus by 68% compared to the control, while Caco-2 cell viability remained at 93%. The results are shown graphically in Figure 5.

[0060] Non-patent literature cited: Bojkova D, Costa R, Bechtel M, Ciesek S, Michaelis M, Cinatl jr. J (2020): Targeting pentose phosphate pathway for SARS-CoV-2 therapy. bioRxiv preprint https: / / doi.org / 10.1101 / 2020.08.19.257022 Dal Pozzo F, Andrei G, Daelemans D, Winkler M, Piette J, De Clercq E, Snoeck R, (2008): Fluorescence-based antiviral assay for the evaluation of compounds against vaccinia virus, varicella zoster virus and human cytomegalovirus. Journal of Virological Methods 151 66-73. doi: 10.1016 / j.jviromet.2008.03.025 Diaz-Moralli S, Aguilar E, Marin S, Coy JF, Dewerchin M, Antoniewicz MR, Meca-Cortes O, Notebaert L, Ghesquiere B, Eelen G, Thomson TM, Carmeliet P, Cascante M. (2016): A key role for transketolase-like 1 in tumor metabolic reprogramming. Oncotarget 7(32):51875-51897; doi: 10.18632 / oncotarget.10429; PMID: 27391434. Gupta K. K., Khan M. A, Singh S. K (2020): Constitutive Inflammatory Cytokine Storm: A Major Threat to Human Health. Journal of Interferon & Cytokine Research. Jan 2020.19-23. http: / / doi.org / 10.1089 / jir.2019.0085 Heller S, Maurer GD, Wanka C, Hofmann U, Luger AL, Bruns I, Steinbach JP, Rieger J. (2018): Gene Suppression of Transketolase-Like Protein 1 (TKTL1) Sensitizes Glioma Cells to hypoxia and Ionizing Radiation. Int J Mol Sci. 2018 Jul 25;19(8):2168. Kumar B (2018): Resveratrol inhibits expression of cancer-specific pentose phosphate pathway enzyme TKTL1. Asian Journal of Pharmaceutical and Clinical Research 11:332. DOI: 10.22159 / ajpcr.2018.v11i6.25021 Li Y, Yao CF, Xu FJ, Qu YY., Li JT, Lin Y, Cao ZL, Lin PC, Xu W, Zhao SM, & Zhao JY (2019): APC / C CDH1 synchronizes ribose-5-phosphate levels and DNA synthesis to cell cycle progression. Nature Communications (2019) 10:2502; https: / / doi.org / 10.1038 / s41467-019-10375-x Lu H, Zhu H. (2017): Effect of siRNA-mediated gene silencing of transketolase on A549 lung cancer cells. Oncol Lett. 2017; 14(5): 5906-5912. DOI: 10.3892 / ol.2017.6916. Zhu Z, Cai T, Fan L, Lou K, Hua X, Huang Z, Gao G. (2010): Clinical value of immune-inflammatory parameters to assess the severity of coronavirus disease 2019. Int J Infect Dis. 2020 Jun; 95: 332-339. http: / / doi: 10.1016 / j.ijid.2020.04.041

Claims

1. 1. A composition comprising benphoxythiamine for use as an inhibitor of the enzyme transketolase-like 1 (TKTL1) and / or the TKTL1-TKT heterodimer complex in the treatment of viral infections in humans, wherein the benphoxythiamine is used in a dose of 7 μg to 430 μg per kg of patient body weight per day.

2. 2. The composition of claim 1, wherein benphoxythiamine is used in a dose of 14 μg to 215 μg per kg of patient body weight per day.

3. 2. The composition of claim 1, wherein benphoxythiamine is used in a dose of 14 μg to 130 μg per kg of patient body weight per day.

4. The composition of claim 1 further comprising oxythiamine.

5. The composition of claim 1, wherein the human viral infection is an RNA virus infection or SARS-CoV-2 infection.

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