WO2017175132A1

WO2017175132A1 - The use of cationic derivative of dextran for inhibition of herpes simplex viruses

Info

Publication number: WO2017175132A1
Application number: PCT/IB2017/051918
Authority: WO
Inventors: Maria Nowakowska; Krzysztof Pyrc; Krzysztof Szczubialka; Justyna CIEJKA; Aleksandra MILEWSKA; Magdalena PACHOTA
Original assignee: Uniwersytet Jagiellonski
Priority date: 2016-04-04
Filing date: 2017-04-04
Publication date: 2017-10-12
Also published as: PL416749A1; PL235490B1

Abstract

The use of dextran cationically modified by quaternary ammonium groups of structure I for the treatment and prophylaxis of diseases caused by herpes simplex viruses, wherein dextran is modified using glycidyltrimethylammonium chloride (GTMAC).

Description

The use of cationic derivative of dextran for inhibition of herpes simplex viruses

The subject of the invention is the use of dextran polymer for the prophylaxis and treatment of infections caused by human herpesvirus.

Herpes simplex viruses type 1 and 2 (HSV-1 and HSV-2, respectively) belong to the Herpesviridae family encompassing more than 100 viruses, eight of which affect humans ¹. They are also widespread in human population, as it is estimated that 50-90% of the population worldwide may be infected ². HSV-1 is mainly associated with the watery blisters in the mouth area (lips, tongue, oral mucosa), while HSV-2 mostly affects genital region ^{3 4}. Both of them are, however, also associated with potentially fatal viral encephalitis and stromal keratitis, an ocular disease, which is a leading cause of cornea-derived blindness in developed countries ^5"7.

The HSV virion is a spherical, complex structure, about 200 nm in diameter. The inner core, containing the viral DNA stabilized by polyamines, is encapsulated by an icosahedral capsid, which is further coated with an unstructured protein layer, called the tegument. The whole structure is surrounded by a lipid bilayer decorated with viral surface glycoproteins. HSV is a linear dsDNA virus, with genome of 152 and 155 kbp for HSV-1 for HSV-2, respectively. Two major genomic regions - L (long) and S (short) - both flanked with inverted repeats may be distinguished. The HSV genome encodes for at least 90 transcriptional units, which are sorted into 3 classes: a - immediate early, β - early, and γ - late genes. The a genes are located in the inverted repeats of both L and S components and encode for proteins regulating expression of early and late genes. The β and γ genes are distributed within the L and S region

8

Primary HSV infection begins with epithelial cells, usually oral or ocular for HSV-1 and genital for HSV-2. The virus is easily spread through direct contact, i.e., kissing or sexual intercourse. After a productive infection in the mucosal tissue, HSV proceeds to enter nerve endings and, employing retrograde transport, makes its way to the dorsal root neurons nuclei, where it establishes latency - a state of lifelong infection. This possibility, a hallmark of the Herpesviridae family, is facilitated by a failure of immediate early genes transcription caused by ineffective axonal transport of viral regulation factors, such as VP 16 and IFN-a activity in sensory neurons ^{9 10}. Once the virus reaches the latent state, the infection seems to be incurable and may be reactivated, either spontaneously or by stress stimuli, such as fever, UV exposure, or hormonal changes ^u.

No effective HSV vaccine is available, and therefore current research is oriented towards the development of antiviral compounds limiting the primary infection and supporting further treatment. Currently, the approved anti-HSV therapies involve mainly nucleoside analogues, such as acyclovir (ACV), which interfere with viral DNA synthesis reducing virus replication and shedding. Low bioavailability of this drug led to the development of derivatives with better pharmacokinetic parameters, e.g., selenoaciclovir, valacyclovir, famciclovir, and ganciclovir ^12"14. Most of these drugs undergo bioconversion within the cell to ACV. An alternative for treatment of some ACV-resistant HSV infections is foscarnet, which hampers viral polymerase activity by binding to the pyrophosphate binding site ¹⁵. All mentioned compounds are oriented on the same molecular target, and therefore some cross resistance is observed ^{16 1V}. For these reasons, there is a need for the development of novel anti -herpes drugs with different mechanism of action. The site of entry of herpesviruses seems to be a promising target, as inhibition of early steps of the virus infection may limit infection, but also virus transmission. In the current study we made an effort to develop the non-toxic cationic polymer that could prevent virions from entering the cell via the formation of multivalent interactions either with the virion (direct inactivation) or with the cell.

Application of polymers for inhibition of HSV infection was proposed before and involved synthetic polycations and polyanions, as well as peptides/proteins. The examples of the polycations which showed anti-HSV activity are poly-L-lysine and poly-L-arginine derivatives were shown to inhibit early stages of HSV replication¹⁸, poly(amidoamines) substituted with agmatine¹⁹, viologen dendrimers²⁰, and a copolymer of methyl methacrylate, N,N-dimethylaminoethyl methacrylate, and butyl methacrylate (Eudragit®, El 00) which destabilized virus membrane²¹. The examples of polyanions which inhibit HSV were dendrimers containing sulfonic groups ²², poly(sodium styrene sulfonate) (PSSS)²³ and its copolymer with maleic acid²⁴, and telomerized co-acryloyl anionic surfactants²⁵. Among natural polymers having anti-HSV activity were sulfated lignins²⁶, sulfated cellulose nanocrystals (CNC)²⁷, peptides and aptamers, which interfered with virus - cell interactions ^28"30, and cationic polysaccharides conjugated with oligoamines via reductive amination ³¹.

Amine and ammonium derivatives of natural polysaccharides (e.g., dextran, pullulan) also have been studied. It has been shown that dextran substituted with amine groups, in particular dextran-propyl- 1,3 -diamine, effectively inhibits HSV-1 virus replication, while this compound modified by the conversion of a primary amine group to a trimethylammonium group (monoquaternary ammonium propyl-1,3- diamine, MQ-propane- 1,3 -diamine) did not show this activity³¹, in complete contrast to structurally similar cationic derivatives of dextran being the object of the present application.

According to some studies, entry inhibitors include also sulfonated galactans and fucoidans, dextran sulfate, heparin, and sulfonated lignins.

EP0066379 describes a composition for combating HSV type I and II, comprising a dextran sulfate sodium salt with molecular weight in the range of 4100 to 25000 and containing at least 0.2 equivalent of S0₃Na per glucopyranosyl unit.

WO2000024784 application discloses dextran derivatives showing biological activity towards HSV, namely dextranpolyaldehyde-resorcine.

The aim of the invention described in the present application is to provide a new effective drug for the treatment and prophylaxis of infections caused by herpes simplex viruses.

Subject Matter of the Invention

The subject of the invention is the use of dextran cationically modified by quaternary ammonium groups of structure I for the treatment and prophylaxis of diseases caused by HSV-1 virus. Preferably, dextran modified by glycidyltrimethylammonium chloride (GTMAC) is used.

Structure I

Preferably, dextran with molecular weight from 6 to 100 kDa is used. Cationically modified dextran has the degree of substitution in the range of 20-100%, preferably 20-60%, more preferably 25-60%.

The use includes polymers based on dextran in the form of ointment or drops applied topically to the skin or to the eye, orally, intraperitoneally, or intravenously in the systemic treatment.

The invention has been illustrated with figures which present preferred embodiments:

Fig. 1 shows IR-ATR spectrum of cationically modified dextrans with the weight of 6 kDa, Fig. 2 is IR-ATR spectrum of cationically modified dextrans with the weight of 40 kDa, Fig. 3 is IR-ATR spectrum of cationically modified dextrans with the weight of 100 kDa, Fig. 4 illustrates the cytotoxicity of polymers. Cell viability is shown as % of the value for the control sample, i.e. not treated with polymers.

Fig. 5 shows the antiviral activity of GTMAC-modified dextrans.

Fig. 6 illustrates the relation between the anti-HSV-1 activity and the degree of substitution. Fig. 7 presents cytotoxicity of the tested polymers.

Fig. 8 shows influence of DS and MW of DEXxDSy on their anti-HSV-1 activity.

Fig. 9 shows concentration-dependent antiviral activity of DEX100DS42 against HSV-1

(A,C) and HSV-2 (B,D).

Fig. 10 shows inhibition of HSV-1 replication cycle at different stages.

Fig. 11 shows UV-Vis absorption spectra of Azure A in the presence of 0.2 mg/ml of HS and different concentrations of DEX100DS42 | g/ml].

Fig. 12 illustrates dependence of the relative concentration of free (unbound) HS on the weight ratio of DEX100DS42 and HS.

Fig. 13 presents HSV-1 attachment in the presence of DEX100DS42, as determined with flow cytometry.

Fig. 14 shows modified dextrans block HSV-1 attachment to susceptible cells.

Fig. 15 shows subcellular localization of dextran derivatives.

Embodiments

The subject of the invention has been presented in more detail in the embodiments.

Example 1

Synthesis of cationic dextrans

0.5 g of dextran with the molecular weight of 6 kDa (DEX6, Sigma-Aldrich), 40 kDa (DEX40, Sigma-Aldrich), or 100 kDa (DEXIOO, Sigma-Aldrich) was dissolved in 25 ml of distilled water, followed by the addition of suitable amount (Table 1) of glycidyltrimethylammonium chloride (GTMAC, Sigma-Aldrich). The solution was alkalized by adding 1 ml 5 M solution of NaOH. The mixture was intensively stirred with magnetic stirrer and heated under reflux for 4 hours at the temperature of 80°C. Upon synthesis, the reaction mixture was cooled to the room temperature, and neutralized with HCl solution. The solution was transferred to a dialysis tube with the molecular weight cutoff value of 3.5 kDa and the dialysis was carried out against water until the conductivity of the said solution decreased below 10 μ8. Obtained polymers were isolated with freeze-drying. These were denoted by acronyms in which the first number is the molecular weight in kilodaltons, while the second is the degree of substitution (DS) in percentage.

Example 2

Elemental analysis-based determination of the degree of substitution in cationic dextrans

Dextran and its cationic derivatives were subjected to the elemental analysis in order to determine the degree of substitution (DS) of glucose units with GTMAC groups, defined as a number of GTMAC groups per 1 glucose unit in dextran. The results are reported in Table 2.

Table 2. Results of elemental analysis of obtained cationic derivatives of dextran

Polymer C f%l H f%l N f%l N/C

DEX6DS0 40.75 6.544 0 0

DEX6DS23 42.49 6.98 1.550 0.0365

DEX6DS37 42.33 6.632 2.24 0.0529

DEX6DS41 41.23 7.214 2.32 0.0563

DEX6DS50 43.55 7.387 2.83 0.0650

DEX6DS52 43.38 7.491 2.87 0.0662

DEX6DS64 41.96 7.379 3.17 0.0754

DEX6DS78 42.62 7.681 3.64 0.0853

DEX40DS0 40.97 6.526 0 0 DEX40DS24 39.60 7.033 1.48 0.0373

DEX40DS26 40.89 7.103 1.64 0.0401

DEX40DS28 39.61 6.866 1.71 0.0432

DEX40DS29 39.56 7.058 1.72 0.0434

DEX40DS37 41.00 7.333 2.16 0.0527

DEX40DS51 41.30 7.587 2.71 0.0656

DEX40DS64 39.58 7.430 2.99 0.0757

DEX100DS0 40.97 6.526 0 0

DEX100DS21 40.30 7.039 1.35 0.0334

DEX100DS33 38.67 7.283 1.86 0.0480

DEX100DS35 41.53 7.372 2.12 0.0510

DEX100DS41 38.37 7.261 2.19 0.0571

DEX100DS42 40.13 7.323 2.32 0.0577

DEX100DS55 38.41 7.298 2.65 0.0690

DEX100DS59 41.91 7.613 3.04 0.0724

DEX100DS71 41.69 7.846 3.37 0.0808

DEX100DS74 40.61 7.726 3.36 0.0827

DEX100DS81 41.27 8.09 3.59 0.0869

DEX100DS82 40.94 7.943 3.60 0.0878

DEX100DS98 40.59 7.932 3.93 0.0967

Example 3

IR spectra for cationic derivatives of dextran

In order to confirm dextran substitutions with GTMAC, IR spectra were measured. The presence of GTMAC groups in modified dextran is demonstrated by a band at about 1475cm^"1. The selected results are reported in Table 3 and Fig. 1-3).

Table 3.

Example 4

Zeta potential of cationic derivatives of dextran Zeta potential was measured with NanoZetasizer instrument from Malvern. The measurements were performed for samples at the concentration of 1 mg/ml in 0.015 M NaCl. The results are reported in Table 4.

Table 4. Zeta potential of 1 mg/ml solutions of cationic derivatives of dextran in 0.015 M

NaCl.

Polymer DS Zeta potential [11]

DEX6DS0 0 1.80 ± 1.78

DEX6DS23 23 +9.48 ± 1.80

DEX6DS37 37 +11.5 ±2.29

DEX6DS41 41 +9.69+1.73

DEX6DS50 50 +19.5 + 0.59

DEX6DS59 59 +16.4+1.31

DEX6DS64 64 +18.6+1.99

DEX6DS78 78 +8.77 + 0.90

DEX40DS0 0 -1.87 + 0.64

DEX40DS24 24 +7.06+1.23

DEX40DS26 26 +14.0+1.61

DEX40DS28 28 +17.1 ± 1.48

DEX40DS29 29 +9.06 + 2.93

DEX40DS37 37 +15.4 + 0.95

DEX40DS51 51 +17.9+1.51

DEX40DS64 64 +33.2+1.65

DEX40DS66 66 +22.4 + 2.15

DEX100DS0 0 -2.85 ± 0.94

DEX100DS21 21 +9.78 + 2.58

DEX100DS33 33 +22.8 + 3.27

DEX100DS35 35 +25.2+1.42

DEX100DS41 41 +21.5 + 2.21

DEX100DS42 42 +15.7+1.17 DEX100DS55 55 +22.9 ± 1.31

DEX100DS59 59 +22.1 ±0.56

DEX100DS71 71 +29.5 + 0.55

DEX100DS74 74 +25.5 + 0.93

DEX100DS81 81 +23.4 + 0.20

DEX100DS82 82 +35.3 + 2.00

DEX100DS98 98 +24.2 + 4.31

Example 5

Effect of cationically modified dextrans on the cell viability

The cytotoxicity of cationic derivatives of dextran was determined using XTT assay. The assay was performed on Vero E6 cell line which is permissive for HSV-1 virus. This assay is based on the determination of mitochondrial enzymes' activity by testing the ability thereof to reduce a substrate to a colored product. The cell viability was determined with measuring the absorbance at 450 nm in relation to the control which was represented by cells cultured in medium without additives. Cells were cultured for 2 days in a mixture of MEM medium, with the addition of Hank's salt, and M199 medium, with the addition of Eagle's medium in 2: 1 ratio, supplemented with 3% heat-inactivated fetal bovine serum (FBS), as well as penicillin and streptomycin, with the addition of the tested polymer.

Results presented in Fig. 4 show the cytotoxicity of the synthesized polymers at various concentrations.

Example 6

Effect of cationically modified dextrans on the replication of human herpesvirus type I (HS V- 1)

Upon the determination of compound concentrations without toxic effect on cells, their effect on HSV-1 virus replication was tested. Cells were pre-incubated with dextrans at suitable concentrations for 30 min and, subsequently, still in the presence of dextrans, were infected with the virus at TCID5o=400. After 2 hours, unbound virus particles were removed by washing 3 times with PBS, and the incubation was performed, also in the presence of dextrans, for the next 2 days. Subsequently, DNA was isolated from cell medium and used as a matrix in real-time PCR. Dextrans' ability to inhibit HSV-1 replication was determined as a decrease in virus copy number per millilitre of medium.

Fig. 5 shows the antiviral activity of GTMAC-modified dextrans. For the analysis, GTMAC- modified dextrans with various molar weight (6 , 40, and 100 kDa) were used. Suitable unmodified dextrans were used as controls (ctrl). For all compounds the concentration used was 500 μg/ml.

Based on the diagram, it can be clearly seen that GTMAC-modified dextran inhibits HSV-1 multiplication when compared with the suitable control sample. Next, the effect of the degree of GTMAC substitution on the antiviral activity at the constant molar weight of dextran (6 kDa) was determined.

Fig. 6 shows the relation between the anti-HSV-1 activity and the degree of substitution. Three different degrees of polymer substitution were used: 23%, 37%, and 52%. The presented diagram clearly shows that the antiviral activity of the polymer correlates with the degree of substitution.

Example 7

Cationic derivatives of dextran (DEXxDSy) as effective inhibitors of HSV-1 and HSV-2 infection.

Dextran (DEX, MW of 6, 40 and 100 kDa, from Leuconostoc spp., Sigma), glycidyltrimethylammonium chloride (GTMAC, >90% Sigma), heparan sulfate (HS), Azure A chloride (Fluka, Fluka standard), PBS (tablet, Sigma), fluorescein 5(6)-isotiocyanate (FITC, >90% (HPLC), Sigma), sodium chloride (analytical grade, POCh), NaOH (analytical grade, POCh), HC1 (pure, POCh) were used as received. Water was distilled twice and deionized using the Millipore Simplicity system. FTIR spectra were obtained on a Bruker IFS 48 spectrometer. Elemental analysis (EA) was performed on a EuroEA 3000 Elemental Analyzer. The UV/VIS absorption spectra were obtained at room temperature in 1-cm quartz cuvettes using the single beam diode array Hewlett-Packard 8452A spectrophotometer with a resolution of 2 nm in the range of 190-820 nm. Zeta potential measurements were performed using Zetasizer Nano ZS instrument (Malvern Instruments). The sample was illuminated with a 633-nm laser and the intensity of light scattered at an angle of 173° was measured with an avalanche photodiode.

Synthesis of cationically modified dextran (DEX)

The synthesis of cationic derivatives of dextran is presented in Scheme 1. Cationic derivatives of DEX were obtained by the substitution of hydroxyl groups with GTMAC as described previously ³² with some modifications. We have applied two synthetic protocols. In the first synthetic procedure, 0.5 g of DEX was dissolved in 25 ml of distilled water, then NaOH (2.5 mmol) was added. The solution was stirred with a magnetic stirrer and heated to 60°C or 80°C. In the next step different volumes of GTMAC were added and the mixtures were kept at 60°C or 80°C for 4 h while being stirred. The MW values of DEX, volume of GTMAC and the reaction temperature used to obtain each DEX derivative are given in Table SI (see Supporting Information). The products are denoted as DEXxDSy where x is the MW of DEX in kDa and y is the degree of substitution defined as the number of GTMAC groups attached to DEX per 100 glucose repeating units. In the second synthetic procedure both the reaction time and GTMAC volume were varied (Table S2). The first polymer sample in each series was obtained by reacting 2 g of DEX dissolved in 100 ml of distilled water with initial volume of GTMAC (20 or 40 ml) for 1 or 2 h. Then, a defined volume of the reaction mixture was withdrawn to isolate a polymer sample while to the rest of the reaction mixture the defined volume of GTMAC was added and the resulting mixture was heated while mixing for the defined time period. The withdrawal/GTMAC addition/heating cycles were repeated twice or thrice. To isolate the polymer the reaction mixtures were neutralized to pH~7 by the addition of 1 M HC1, cooled and transferred to dialysis tubes (MW cutoff values of 3.5, 12.8 and 14 kDa, depending of the MW of DEX). The dialysis was carried out against distilled water until the conductivity of the liquid surrounding the tube decreased below 10 μ8. The polymers obtained were isolated from the solution using the freeze-drying technique. The substitution of DEX with GTMAC was confirmed using EA, IR spectroscopy and zeta potential measurements. The DS values of the polymers obtained covered almost the whole possible range, i.e. from 2 to 98.

Synthesis of fluorescently labeled DEXxDSy

DEX6DS41, DEX40DS37 and DEX100DS40 were fluorescently labeled as described previously³³. Briefly, 50 mg of the respective polymer was dissolved in 2 ml of warm DMSO and 1 drop of pyridine and 3 mg of FITC in 2 ml of DMSO were added. The mixture was heated to 95°C for 2 hours under continuous stirring. Then, DMSO was removed by dialysis against water and the product was lyophilized.

Determination of the degree of substitution (DS) of DEX derivatives with GTMAC

The DS of the DEXxDSy was determined with elemental analysis (EA). The ratio of N/C in each polymer was determined and the DS was calculated using the following equation:

DS [%] = 100 (457.34 (N/C)³ + 3.007 (N/C)² + 5.5951 (N/C)) where N/C is the ratio of the mass fraction of nitrogen to carbon

Studies on the interactions between the DEXxDSy and HS using a colorimetric method The mixtures containing a constant concentration of HS (0.2 mg/ml) and 1 x PBS solutions of various DEXxDSy at different concentrations (0.06-0.27 mg/ml) were prepared. The mixtures were shaken for 10 min and centrifuged at 3000 rpm for 10 min to separate the insoluble complex of HS with DEXxDSy. Then, 0.1 ml of the supernatant was added to the mixture of 0.9 ml of 1 x PBS and 1 ml of 8 10^"5 M Azure A solution and the UV-VIS spectra were measured.

Cell culture

Vero E6 cells (Cercopithecus aethiops kidney epithelial, ATCC: CRL-1586) were routinely maintained under Dulbecco-modified Eagle's medium (DMEM, high glucose, Life Technologies) supplemented with 3% heat-inactivated fetal bovine serum (FBS, Life Technologies), penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C in atmosphere containing 5% C0₂.

Virus preparation and titration

HSV-1 strain 17+ and HSV-2 strain HG52 were obtained from Public Health England (0104151v and 0104152v, respectively). Virus stocks were generated by infecting Vero E6 cells monolayers for 48 h, then lysing the cells with two freeze-thaw cycles. Collected lysates were aliquoted and stored at -80°C. Mock samples were prepared in the same manner, using mock-infected cells. Virus stocks were quantified by titration on Vero E6 cells (48 h infection, 37°C), according to Reed and Muench method ³⁴. Following a 48 h incubation the number of cytopathic effect (CPE)-positive wells was counted and the TCID50 was calculated.

Cell viability assay

Cell viability was assessed on Vero E6 cell line using XTT Cell Viability Assay Kit (Biological Industries) according to vendor's protocol. Briefly, cells were seeded in 96-well plates and cultured for 48 h. Subsequently medium was discarded and cells were overlaid with fresh medium supplemented with DEXxDSy polymers or control samples. Culture was carried out for 48 h at 37°C and after that incubation medium was refreshed again and 25 μΐ of activated XTT reagent was added to each well. Following a 2 h incubation at 37°C, media were transferred to a new 96-well plate and absorbance was measured at λ=480 nm using a spectrophotometer (Flexstation 3, Molecular Devices). The results were normalized against a control of untreated cells (100% viability).

Virus replication assay (Assay 0)

Vero E6 cells were seeded in 6-well plates and cultured for 2 days at 37°C in atmosphere supplemented with 5% of CO2. Subsequently, media was discarded and cells were overlaid with fresh media supplemented with DEXxDSy polymers or control samples. Following a 30 min incubation, cells were infected with HSV-1 or HSV-2 virus at TCID50 = 400/ml, or mock-infected. Infection was carried out for 2 h at 37°C. Subsequently, unbound virus particles were removed by triple washing with sterile 1 ^χ PBS, and culture media supplemented with polymers were applied. Samples were analyzed 2 days post-inoculation

(P i ).

Plaque assay

24 h prior to the infection cells were seeded in 6-well plates. On day 0 sub-confluent cells were infected with 10-fold serial dilutions of the virus and incubated for 1 h at 37°C. Subsequently, cells were washed once with 1 ^χ PBS and overlaid with 2 ml of media supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 0.33% methylcellulose. After 72 h, media were discarded and 1 ml of 0.1% crystal violet solution in 50:50 water : ethanol mixture was added to each well. Following a 10 min incubation at room temperature, cells were washed twice with water and plaques were counted.

Mechanism of anti-HSV action of DEXxDSy

A series of assays was employed to determine the mechanism of antiviral activity of DEXxDSy. The tests differ mainly in the sequence in which the cells, the virus and the polymer may interact.

Assay I: Does the compound inactivate the virus?

Virus stock samples were incubated with DEXxDSy polymers (500 μg/ml) for 1 h at 22°C under constant mixing. Subsequently, the samples were diluted to decrease the polymer concentration below its active range (25 μg/ml). In negative control samples cell culture medium was added instead of the polymer solution. The samples were then titrated on fully confluent Vero E6 cells to assess viral yield. This test indicates whether the antiviral effect results from a direct interaction between the polymer and the virus.

Assay II: Does the compound protect the cell?

Fully confluent Vero E6 cells were overlaid with polymer solution and incubated for 1 h at 37°C. Subsequently, the polymer was removed by triple wash with 1 x PBS and virus at TCID50 = 400/ml or mock at equal volume was added. After 2 h infection at 37°C, the virus was washed away, fresh culture medium was applied, and the cells were cultured for 48 h. Samples of cell culture supernatant were collected and viral yield was assessed. As no unbound polymer is present during the exposure to virus, inhibition of infection in this test indicates the influence of the polymer on the cell (e.g., cell surface coating, induction of innate immune responses).

Assay III: Does the compound block virus - receptor interaction?

Fully confluent Vero E6 cells were chilled on ice, overlaid with an ice-cold solution of the DEXxDSy polymer (500 μg/ml) containing the virus at TCID50 = 400/ml or mock at equal volume in cell culture medium. Under these conditions, virions can attach to their receptor on the cell surface, but cannot enter the cell, as the intracellular transport is inhibited at lower temperatures. Following 1 h incubation at 4°C, the polymer solution was removed, cells were washed thrice with ice-cold 1 x PBS to remove any unbound polymers and virions, overlaid with fresh medium and cultured for 48 h at 37°C. Then, samples of cell culture supernatant were collected and viral yield was assessed. If the polymer inhibits the virus-receptor interaction, a decline in the viral yield is observed compared to the control.

Assay IV: Does the compound hamper infection at later stages?

Fully confluent Vero E6 cells were infected with virus at TCID50 = 400/ml or mock for 2 h at 4°C. Subsequently, cells were rinsed thrice with 1 x PBS to remove unbound virus particles and fresh medium supplemented with the polymer was added. Cells were cultured for 48 h at 37°C, then medium samples were collected and viral yield was assessed. Since the polymer is absent during the early stages of infection (virus adsorption/attachment and entry), a decline in the viral yield compared to the control in this test indicates that the antiviral activity of the polymer is due to inhibition of later stages of virus replication cycle (e.g., virus replication, assembly or egress). qPCR

Viral DNA was isolated from supernatants using Viral DNA/RNA Isolation Kit (A&A Biotechnology, Poland). Virus yield was then determined by quantitative real-time PCR (qPCR). The reaction was carried out in a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad), in 10 μΐ reaction mixture consisting of 1 x Kapa Probe Fast qPCR Master Mix, specific probe labeled with 6-carboxyfluorescein (FAM) and Black Hole Quencher 1 (BHQ1) (sequence: 5' FAM CCG CCG AAC TGA GCA GAC ACC CGC GC BHQ1 -3 ', 100 nM), and primers (450 nM each, sequences: sense primer 5 '-CAT CAC CGA CCC GGA GAG GGA C-3', anti-sense primer 5' GGG CCA GGC GCT TGT TGG TGT A-3') and 2.5 μΐ of viral DNA in 10 μΐ. The temperature profile included 3 min at 95°C, followed by 37 cycles of 2 s at 95°C and 20 s at 60°C. For quantification DNA standards were prepared. Briefly, fragment of DNA polymerase gene, conserved among HSV-1 and HSV-2 strains, was amplified using the primers mentioned above and cloned into pTZ57R/T (Thermo Scientific, Poland) plasmid using InsTAclone PCR cloning kit (Thermo Scientific, Poland). The plasmid was propagated in E. coli TOP 10 (Life Technologies, Poland), purified with GeneJET Plasmid Miniprep Kit (Thermo Scientific, Poland) and linearized by digestion with Kpnl restriction enzyme. Concentration of the linearized DNA was determined spectrophotometrically and the number of copies per milliliter was calculated. Six subsequent serial dilutions were then used as qPCR reaction template.

Flow cytometry

Vero E6 cells were seeded in 6-well plates and cultured for 2 days at 37°C in atmosphere containing 5% CO2. For analysis, cells were trypsinized and fixed with 4% PFA in 1 x PBS for 20 min. Subsequently, the cells were permeabilized by 20 min incubation in 0.1% Triton X100 in 1 x PBS and non-specific binding was blocked by overnight incubation with 5% BSA in 1 x PBS at 4°C. Protein labelling was carried out by 2 h incubation with primary rabbit anti-HSV VP5 antibody (20-HR50, Fitzgerald Industries, USA) diluted 1 :500, followed by lh incubation with secondary goat anti-rabbit antibody, conjugated with AlexaFluor 488 (A11001, Invitrogen, Poland). After washing with 0.1% Tween-20 in 1 x PBS, the cells were re-suspended in 1 x PBS and analyzed with FACS Calibur (Becton Dickinson) using Cell Quest software. Confocal microscopy

Vero E6 cells were seeded on coverslips in 6-well plates and cultured for 2 days at 37°C with 5% C0₂. For analysis of virus adhesion and entry, Assays II and III were carried out, respectively. Immediately after washing away unbound virions, cells were fixed with 4% PFA in 1 x PBS for 20 min. Subsequently, the cells were permeabilized by 20 min incubation in 0.1% Triton X100 in 1 x PBS and non-specific binding sites were blocked by overnight incubation in 5% BSA in 1 x PBS at 4°C. To visualize virus particles, cells were incubated for 2 h with primary rabbit anti-HSV VP5 antibody (20-HR50, Fitzgerald Industries, USA) diluted 1 :500 in 1 x PBS with 0.5% Tween 20, followed by 1 h incubation with secondary goat anti -rabbit antibody, conjugated with AlexaFluor 488 (A11001, Invitrogen, Poland). F-actin was co-stained with AlexaFluor 633 -conjugated phalloidin (Invitrogen, Poland). Nuclear DNA was labelled with 0.1 μg/ml 4',6'-diamidino-2-phenylindole (DAPI) (Sigma- Aldrich, Poland). Immunostained cultures were mounted on glass slides with ProLong Diamond Antifade Mountant (Life technologies, Poland). Fluorescent images were acquired under a ZEISS LSM 710 (release version 8.1) confocal microscope and acquired with ZEN 2012 SP1 (black edition, version 8.1.0.484) software. Stacks acquisition parameters were as follows: frame size 1024 x 1024, step size 0.35 μπι, pixel size 0.10 μπι. For image processing ImageJ FIJI version was used.

Statistical analysis

All the experiments were performed in triplicates and the results are expressed as mean ±SD. To determine the significance of the obtained results a comparison between groups was made using the Student's t test. P values <0.05 were considered significant.

Synthesis of DEXxDSy derivatives

In order to systematically study the subject, an extended library of polymers was synthesized, varying in the chain's length and the content/density of positively charged GTMAC residues. Cationic DEX derivatives (DEXxDSy) were synthesized by the GTMAC epoxide ring opening in basic conditions followed by the substitution of the hydrogen atom of the hydroxyl groups with GTMAC. The synthesis of cationic derivatives of DEX is presented in Scheme 1. The structure of the DEXxDSy polymers was confirmed using EAand FTIR spectroscopy. EA revealed the presence of nitrogen in the obtained polymers, which unambiguously confirms the successful substitution of DEX with GTMAC. DS values were calculated based on the elemental composition of the polymers.

OH CH₃

I

N⁺-CH₃

CH₃

Scheme 1. Synthesis of the cationic derivatives of DEX.

In the FTIR spectra of all GTMAC-substituted polymers a weak band at 1477 cm^"1 appeared that may be attributed to the presence of asymmetric angular binding of the methyl groups in GTMAC, thereby confirming substitution of DEX with GTMAC. The zeta potential was determined for the synthesized polymers (1 mg/ml solution in 0.015 M NaCl at 25°C). All measurements were carried out in triplicate. As expected, the DEX derivatives have a positive zeta potential resulting from the presence of the cationic quaternary amino groups of GTMAC attached to the main chain. The values of zeta potential ranged from +7 to 35 mV and they generally increased with increasing DS of the polymers studied.

Cytotoxicity

In order to evaluate the usability of developed compounds, cytotoxicity of DEXxDSy was determined using XTT assay, which relies on the ability of mitochondrial enzymes to convert the substrate (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) into colored formazan salts. The assay was performed on Vero E6 cell line which is an accepted model for HSV-1 and HSV-2 infection. Briefly, cells were incubated for 48 hours in the presence of different DEXxDSy polymers. Results presented in Fig. 7 clearly show that the polymers of lower MW are characterized by lower toxicity. Increase in the polymer toxicity with increasing MW may be explained by the fact that longer polymer chain adopts the globular conformation with high surface density resulting from large number of charged residues present. It is also very clear that the increase in charge density increases polymer antiviral activity. Thus, there is a need to optimize the system to ensure the highest antiviral activity while maintaining low toxicity. It was found that DEX100DS42 had optimal activity/toxicity properties and therefore it was chosen for mechanistic studies.

In Fig. 7 cell viability was assessed using XTT assay 2 days p.i. Background fluorescence was subtracted for all the samples. Cell viability was calculated in reference to untreated cells (control). Derivatives of dextran with MW of 6 kDa (A), 40 kDa (B) and 100 kDa (C) with various DS were tested in the range of concentrations 50 - 2000 μg/ml. Results are presented as average ± SD. Similar results were obtained in at least three independent experiment.

Antiviral activity of DEXxDSy

The entry of virus into host cells is the first and critical step in the viral pathogenesis ³⁵'³⁶. It is mediated by an interplay of viral and cellular proteins, which differ in the number and characteristics between viral species. HSV-1 encodes several glycoproteins responsible for cell surface binding and virus entry. Glycoproteins gB and gC are responsible for initial binding of the virus at cell surface, involving HS proteoglycans (HSPGs). Virions devoid of these glycoproteins demonstrate significantly impaired infectivity ³⁵. gB was also found to bind PILR-a, a particle considered to be a HSV-1 coreceptor ³¹. Glycoprotein D (gD) is considered to be the main entry mediator and several receptors for gD have been identified. The most important are nectin-1 and nectin-2, herpesvirus entry mediator (HVEM) and 3-0 sulfated HSPG ^38-41. The in vivo experiments carried out using single and double knockout mice have demonstrated the dependence of HSV-2 on nectin-1 for entry and spread ⁴² and HSV-1 on HSPG receptor for their entry to the cells and spread. Interestingly, the in vitro experiments carried out using human corneal fibroblasts have shown that, unlike HSV-1, HSV- 2 does not use the HSPG receptor for cell penetration ⁴³. Another two glycoproteins taking part in HSV-1 entry, gH and gL form a heterodimer, which dissociates upon binding to its receptor, ανβό- or avP8-integrin. This interaction induces a change in gH conformation which induces virus entry into the cell and directs the virus to acidic endosomes ⁴⁴.

In order to assess inhibitory activity of the tested polymers, virus replication assay was performed. Fig. 8 demonstrates the decreased virus replication in the presence of both cationically modified 40 kDa DEX (DEX40DSy) and in the presence of cationically modified 100 kDa DEX (DEXlOODSy), for polymers with DS > -20-40%. The anti-HSV-1 activity of both polymers was similar in this assay. On the other hand, cationically modified 6 kDa DEX showed no significant antiviral activity in the range of DS values studied. The decrease in the virus yield is presented as log removal value (LRV), showing the relative decrease in the number of virus DNA copies in the sample treated with the compound, compared to the untreated control sample. In Fig. 8 cationic dextrans with MW of 6, 40 and 100 kDa with different DS were present throughout the infection. Two days p.i. the amount of viral DNA in cell culture supernatant was assessed by qPCR. Difference in viral yield is presented on y-axis as log removal value (LRV), showing the relative decrease in the amount of virus in cell culture media, compared to the control. Results are presented as average ± SD. Similar results were obtained in at least three independent experiment.

Virus replication assay (Assay 0) was conducted as described above with varying concentrations of the polymer. Then, viral yield in samples was quantified by titration or qPCR. The results are presented in Figure 9. Calculated IC50 values were 37.07 μg/ml for HSV-1 and 48.56 μg/ml for HSV-2 as determined by virus titration and 31.71 μg/ml for HSV- 1 and 17.99 μg/ml for HSV-2 as determined by qPCR.

In Fig. 9 viral yield in samples was quantified by titration (A, B) or qPCR (C, D). For virus titration the virus quantity is presented as TCID50, and for qPCR as number of viral DNA copies per milliliter. Results are presented as average ± SD. Similar results were obtained in at least three independent experiment.

Mechanism of action

An effort was made to elucidate the antiviral mechanism of action of modified dextrans. For this, described above set of functional assays was used. For mechanistic studies DEX100DS42 polymer was used.

First, the influence was investigated of DEX100DS42 on HSV-1 virions (Assay I). No significant reduction of viral titer was detected. Next, the possibility that the polymer renders the cell resistant to the infection was tested (Assay II). Decrease in virus yield (-50%) was noted using both techniques. Subsequently, the ability of DEX100DS42 to block viral adhesion to the cell was examined (Assay III). A decrease in viral titer reaching -90% for virus titration and 100%> for plaque assay was observed. Finally, it was tested whether DEX100DS42 may interfere with virus replication and release (Assay IV). Decrease in virus titer (-50%)) was observed. The results of the tests carried out using the functional assays are presented in Fig. 10, where following functional assays described in the methods section HSV-1 was quantified by titration (A) and by plaque assay (B). Obtained results were normalized to the control sample of polymer-untreated cells. Results are presented as average ± SD. Similar results were obtained in at least three independent experiment.

Interaction between the cationic DEX and HSV attachment receptor

The interaction between DEXxDSy polymers and HS was studied. HS is an anionic sulfated polysaccharide (glycosaminoglycan, GAG) which serves as an attachment factor for HSV viruses. Further, a variant of HS in which 3-O-sulfo groups are present constitute one the three receptors used by HSV-1 to enter the cell ³⁸'⁴⁰. The ability of the studied polymers to bind HS was assessed with a colorimetric method using Azure A, a cationic dye. Azure A in the solution of HS forms aggregates absorbing at 513 nm, while the monomeric form of the dye (prevalent in the absence of HS) absorbs at 630 nm. Addition of the DEXxDSy polymer caused the disruption of dye aggregates resulting in the increase in intensity of the absorption at 630 nm (monomelic band) and in the decrease of absorption at 513 nm (aggregate absorption band). The plot showing changes in the absorption spectra of Azure A upon addition of HS is shown in Fig. 11.

Based on the results of these experiments one can conclude that the cationic DEX derivatives form complexes with HS in 1 x PBS buffer (pH=7.4) what is consistent with our previous observation that cationic dextran derivatives reverse the anticoagulant activity of heparin in vitro and in vivo due to its complexation¹³. It was also possible to quantify the complexation process. Fig. 12 shows the dependence of the concentration of free (unbound) HS on the mass ratio of DEX100DS42 to HS present in solution. The amount of DEX100DS42 required to bind 1 mg of HS was calculated to be equal to 1.30 mg.

Obtained data demonstrated clearly that HS is complexed with all DEXxDSy polymers obtained. The general trend was that cationic DEXxDSy of a given MW bound HS stronger when their DS was higher. Among DEXxDSy of comparable DS the derivatives of DEX with MW of 40 kDa showed the strongest HS binding properties.

To confirm whether the interactions between cationic dextrans and HS present on the cell surface play a role in the inhibition of virus entry, flow cytometry analysis was carried out, as described in Methods section. Data was normalized against a control of cells infected without pre-incubation with dextran. The signal recorded from the infected sample treated with DEX100DS42 was vastly limited, reaching the intensity observed for mock-treated sample. The results are presented in Fig. 13 and suggest that interaction between modified dextrans and HS present at the cell surface hinders the viral adhesion to the host's cell. In Fig. 13 cells were pre-incubated with the polymer for 1 h, then infected with HSV-1 or mock sample for 2 h in the absence of DEX100DS42. vir ctr: cells not treated with the polymer and infected with HSV-1, mock: cells not infected with HSV-1, vir+DEX100DS42: cells treated with the polymer and infected with HSV-1. Value on y axis represents the number of cells that internalized the virus, relative to the control sample (not treated with the polymer). Results are presented as average ± SD. Similar results were obtained in at least three independent experiment.

To additionally verify the ability of cationic dextrans to inhibit HSV attachment to cell surface, confocal images were obtained for cells prepared as described for Assay II. After washing off the unbound virus particles, the cells were fixed and stained for visualization of HSV and F-actin. Fig. 14 demonstrates maximal projections of XY stacks. Cells pre-incubated with mock (A) or 500 μg/ml DEX100DS42 (B) were incubated with HSV-lvirions. Blue - DNA, red - f-actin, green - HSV-1. Maximal projections of collected stacks are presented. Scale bar 10 μπι. Similar results were obtained in at least three independent experiment.

Even though our results clearly show that developed polymers should interfere with early steps of virus replication, inhibition of virus replication was also observed. One may assume that observed inhibition of virus replication at the later stages of infection may result from multi-cycle replication and blockade of subsequent transmission events, however, an additional mechanism involving interruption of virus assembly, packing or releasing cannot be ruled out. Mechanistic studies indicated that the GTMAC-substituted dextrans hamper virus infection by interference with virus binding to the cell surface. Such decrease in virus density on susceptible cells will most likely limit virus entry and virus infectivity, as observed in vitro. One may also assume polymers may hamper entry of HSV-1 via HS receptor during ocular infections. We believe that the developed polymers are able to interfere with HSV-1 (and possibly also HSV-2) transmission and limit virus spread between cells and as such may be used for prophylaxis or in combination with replication inhibitors.

Subcellular localization of dextran derivatives

To verify, whether polymers may enter the cell fluorescent derivatives of DEXxDSy were used Briefly, cells were incubated with 250 μg/ml of fluorescently labeled DEXxDSy for 1 h at 37°C, then cells were rinsed with 1 ^χ PBS, fixed and stained with phalloidin and DAPI as described above. Even though all tested polymers (DEX6DS41, DEX40DS37, and DEX100DS40) were present inside the cell, the intracellular presence of DEX100DS40 seemed to be limited, compared to the polymers of lower MW. However, detailed study should be carried out to quantify the polymer internalization. The results are presented in Fig.15. Fluorescent derivatives of DEX₆DS₄i (A), DEX₄₀DS₃v (B) and DEX100DS40 (C). Cells were incubated with the compounds for 1 h at 37°C. Blue - DNA, red - f-actin, green - dextran. Images present single slices of XY stacks. Scale bar 10 μιη. Similar results were obtained in at least three independent experiment.

Cationic derivatives of DEX with different molecular mass (MW) and degree of substitution (DS) were obtained and studied as potential anti-herpes agents. They were found to interact with HS and the efficiency of binding is related to the degree of cationic modification. The analysis of obtained results showed that evaluated polymers can efficiently inhibit HSV-1 entry to the cell.

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Claims

1. The use of dextran cationically modified by quaternary ammonium groups of structure I for the treatment and prophylaxis of diseases caused by herpes simplex viruses.

Structure I

2. The use according to claim 1, wherein dextran is modified using glycidyltrimethylammonium chloride (GTMAC).

3. The use according to claim 1 or 2, wherein dextran with molecular weight from 6 to 100 kDa is used.

4. The use according to claims 1-3, wherein cationically modified dextran has the degree of substitution in the range of 20-100%, preferably 20-60%, more preferably 25-60%).

5. The use according to claims 1-4, wherein cationically modified dextran is used in the form of ointment or drops applied topically to the skin or to the eye, orally, intraperitoneally, or intravenously in the systemic treatment.