Anti-SARS-CoV-2 Activity of Rhamnan Sulfate from Monostroma nitidum.

The COVID-19 pandemic is a major human health concern. The pathogen responsible for COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), invades its host through the interaction of its spike (S) protein with a host cell receptor, angiotensin-converting enzyme 2 (ACE2). In addition to ACE2, heparan sulfate (HS) on the surface of host cells also plays a significant role as a co-receptor. Our previous studies demonstrated that sulfated glycans, such as heparin and fucoidans, show anti-COVID-19 activities. In the current study, rhamnan sulfate (RS), a polysaccharide with a rhamnose backbone from a green seaweed, Monostroma nitidum, was evaluated for binding to the S-protein from SARS-CoV-2 and inhibition of viral infectivity in vitro. The structural characteristics of RS were investigated by determining its monosaccharide composition and performing two-dimensional nuclear magnetic resonance. RS inhibition of the interaction of heparin, a highly sulfated HS, with the SARS-CoV-2 spike protein (from wild type and different mutant variants) was studied using surface plasmon resonance (SPR). In competitive binding studies, the IC50 of RS against the S-protein receptor binding domain (RBD) binding to immobilized heparin was 1.6 ng/mL, which is much lower than the IC50 for heparin (~750 ng/mL). RS showed stronger inhibition than heparin on the S-protein RBD or pseudoviral particles binding to immobilized heparin. Finally, in an in vitro cell-based assay, RS showed strong antiviral activities against wild type SARS-CoV-2 and the delta variant.

Anti-Influenza A Virus Activity of Rhamnan Sulfate from Green Algae Monostroma nitidum in Mice with Normal and Compromised Immunity.

Influenza viruses cause a significant public health burden each year despite the availability of anti-influenza drugs and vaccines. Therefore, new anti-influenza virus agents are needed. Rhamnan sulfate (RS) is a sulfated polysaccharide derived from the green alga Monostroma nitidum. Here, we aimed to demonstrate the antiviral activity of RS, especially against influenza A virus (IFV) infection, in vitro and in vivo. RS showed inhibitory effects on viral proliferation of enveloped viruses in vitro. Evaluation of the anti-IFV activity of RS in vitro showed that it inhibited both virus adsorption and entry steps. The oral administration of RS in IFV-infected immunocompetent and immunocompromised mice suppressed viral proliferation in both mouse types. The oral administration of RS also had stimulatory effects on neutralizing antibody production. Fluorescent analysis showed that RS colocalized with M cells in Peyer's patches, suggesting that RS bound to the M cells and may be incorporated into the Peyer's patches, which are essential to intestinal immunity. In summary, RS inhibits influenza virus infection and promotes antibody production, suggesting that RS is a potential candidate for the treatment of influenza virus infections.

Structural characterization and anti-lung cancer activity of a sulfated glucurono-xylo-rhamnan from Enteromorpha prolifera.

A sulfated glucurono-xylo-rhamnan (EP-3-H) was purified from a green alga, Enteromorpha prolifera. EP-3-H and its oligomers were characterized by high performance liquid chromatography, mass spectrometry and one and two-dimensional nuclear magnetic resource spectroscopy. The structural analysis showed EP-3-H has a backbone of glucurono-xylo-rhamnan, branches with glucuronic acid and sulfated at C3 of rhamnose and/or C2 of xylose. The inhibition of EP-3-H on human lung cancer A549 cell proliferation in vitro and its therapeutic effects in BALB/c-nu mice in vivo were determined to evaluate the anti-lung cancer activity of EP-3-H. The tumor inhibition level was 59 %, suggesting that EP-3-H might be a good candidate for the treatment of lung cancer. Surface plasmon resonance (SPR) studies revealed the IC50 on the binding of fibroblast growth factors, (FGF1 and FGF2), to heparin were 0.85 and 1.47 mg/mL, respectively. These results suggest that EP-3-H inhibits cancer proliferation by interacting with these growth factors.

Rhamnan sulfate extracted from Monostroma nitidum attenuates blood coagulation and inflammation of vascular endothelial cells.

Rhamnan sulfate (RS) is a polysaccharide with a rhamnose backbone isolated from Monostroma nitidum. Like heparin, it exerts anticoagulant activity in the presence of antithrombin. Endothelial cells facilitate the crosstalk between blood coagulation and vascular inflammation. In this study, we compared the effect of RS with that of heparin on blood coagulation and vascular endothelial cells in the presence or absence of inflammatory factors, using human umbilical vein endothelial cells. We found that RS significantly enhances inhibition of thrombin and factor Xa in the presence of antithrombin as well as heparin, and that RS inhibits tissue factor expression and von Willebrand factor release from the endothelial cells treated with or without lipopolysaccharide, tumor necrosis factor-α, or thrombin. Heparin did not show any effects on endothelial cell inflammation. Our findings suggest that RS, like heparin, is an antithrombin-dependent anticoagulant and, unlike heparin, is a potent anti-inflammatory agent acting on vascular endothelial cells.

Potential Utilization of a Polysaccharide from the Marine Algae Gayralia oxysperma, as an Antivenom for Viperidae Snakebites.

Worldwide, snakebites have serious implications for human health. The administration of antivenom is the official treatment used to reverse the toxic activities of envenomation. However, this therapy is not efficient to treat the local effects, leading to the amputation or deformity of affected limbs. As such, alternative treatments are needed. Here, we analyze the ability of a polysaccharide from the green marine alga Gayralia oxysperma (Go3) to inhibit the effects of venom from Bothrops jararaca and Lachesis muta. B. jararaca or L. muta venoms were incubated together with sulfated heterorhamnans from Go3, and the in vitro (coagulation, proteolytic, and hemolytic) and in vivo (hemorrhagic, myotoxic, edematogenic, and lethal) activities of venoms were assessed. Additionally, Go3 was injected before and after the injection of venoms, and the toxic activities were further tested. When incubated with the venoms, Go3 inhibited all activities, though results varied with different potencies. Moreover, Go3 neutralized hemorrhagic, myotoxic, and edematogenic activities when injected before or after injection with B. jararaca and L. muta venom. Go3 also blocked the coagulation of plasma in mice caused by the venoms in an ex vivo test. Therefore, Go3 has the potential to be used as antivenom for B. jararaca and L. muta bites, notably exhibiting higher efficacy on L. muta venom.

In vitro radiation responses of human intracranial meningiomas & their modifications by 2-deoxy-D-glucose.

Radiation responses of 16 human intracranial meningiomas [hemangiopericyte, 4; fibrous, 4; transitional, 4; vascular, 3; and meningiothelial, 1] and their modifications by 2-deoxy-D-glucose (2-DG) have been studied in vitro using organ cultures derived from post-operative tumour specimens. Treatment induced cytogenetic damage viz., micronuclei formation and DNA content dispersion, were analysed as indices of the radiation damage. Results showed that the 60Co gamma ray induced micronuclei frequencies in meningiomas varied over a large range (0.7-6.2%). Presence of 2-DG (at equimolar concentration with glucose) for 4 h after irradiation, increased the radiation damage by a factor of up to 2.8 in nearly 70 per cent of the cases, although a decrease was observed in 20 per cent cases. These observations can be explained on the basis of the energy linked modulations of the competitive processes of repair and fixation of radiation induced DNA lesions. The results of the study suggest that the therapy of meningiomas could be improved by combining radiotherapy with administration of 2-DG.

Repair inhibition in tumours irradiated with fast protons and negative pions.

Solid Ehrlich mouse tumours were irradiated in the 80 MeV proton beam of the Paul-Scherrer-Institute. The tumour volume was measured as a function of time after irradiation and two experimental endpoints were determined: local tumour control and minimal tumour volume after irradiation. The application of 2-deoxy-D-glucose (2-DG; 2 mg/kg) increased the radiation effect of protons by a factor of 1.4. The same tumour system was used with negative pions. Human tumours are usually irradiated with a mixed radiation produced by the "spot-scan-technique". This radiation quality was simulated in the mouse experiment by two successive irradiations with a spot of densely ionizing peak pions and a spot of sparsely ionizing plateau pions. Application of 2-DG raised the radiation effect due to the sparsely ionizing component again by a factor of 1.4. This indicates that clinical results in radiotherapy might be improved by application of 2-DG during the treatment.

Inhibition of established rat fibrosarcoma growth by the glucose antagonist 2-deoxy-D-glucose.

Sarcoma cells exhibit higher rates of glycolysis than normal tissues and may be dependent on glucose utilization for growth. Accordingly, we tested the ability of the glucose antimetabolite 2-deoxy-D-glucose (2-DG) to inhibit the growth of an established methylcholanthrene-induced rat fibrosarcoma in three groups of F344 rats with increasing subcutaneous inoculations of tumor (2 X 10(6) cells, 1 X 10(7) cells, and 1 mm tumor fragments). Rats were randomized to receive 2-DG or saline solution at doses of 0.75 gm/kg, 1.5 gm/kg, or 1.75 gm/kg, beginning 3 days after tumor implantation and continuing for 10 days. Tumors were removed and weighed on day 14. We measured tissue [14C]-2-DG levels in tumor, brain, liver, and muscle after intraperitoneal injection of radiolabeled 2-DG. In these same tissues we determined the activity of glucose-6-phosphatase (G-6-Pase), an enzyme which dephosphorylates the intracellular glycolytic inhibitor 2-DG-6-phosphate, thus reversing the antitumor effect of 2-DG. All groups treated with 2-DG had a significant reduction in tumor weight of 50% to 70% when compared with saline solution-treated controls. Toxicity was substantial at the highest dose of 2-DG, but minimal toxicity was noted at intermediate and low doses. Tumor had the greatest uptake of [14C]-2-DG, with low levels of G-6-Pase leading to prolonged retention and highest tissue levels of radiolabeled 2-DG. Use of 2-DG inhibits established sarcoma growth because it is rapidly transported into tumors, cannot be metabolized after phosphorylation, and is dephosphorylated and released slowly from tumor cells. Rat sarcoma growth is dependent on glucose utilization and can be effectively inhibited by glucose antagonism.

Efficacy of glycoprotein inhibitors alone and in combination with trifluridine in the treatment of murine herpetic keratitis.

The present study examined the anti-herpetic effect of the glycoprotein inhibitors, hydroxynorvaline and 2-deoxyglucose, alone and in combination with trifluridine on murine ocular herpes. Following ocular inoculation with a large dose of HSV-1 RE strain (10(6) pfu), ICR mice were treated during the acute infection with different therapeutic regimens, and their efficacy was evaluated by ocular virus titers, clinical grading of blepharo-conjunctivitis and histological evaluation of stromal keratitis and iridocyclitis. The results following a large dose HSV-1 inoculum demonstrated that trifluridine was the best single therapeutic agent. Hydroxynorvaline and 2-deoxyglucose had no effect at all. Combination therapy of the glycoprotein inhibitors with trifluridine was no better than trifluridine alone. The mouse HSV-1 keratitis model proved to be an effective, economical alternative to the rabbit model for the evaluation of new antiviral agents.

Hyperthermia and radiation in vivo: effect of 2-deoxy-D-glucose.

Since hypoxic cells rely heavily on glucose metabolism for energy, 2-deoxy-D-glucose (2-DG), an inhibitor of anaerobic glycolysis, would be expected to increase tumor cell killing by heat and thus enhance the effect of concurrent radiation. In order to test this hypothesis two types of BALB/c mouse tumors, one induced by subcutaneous injection of 10(6) herpes virus Type 2-transformed (H238) cells and the other by injection of 1.6 X 10(5) 1,2-dimethylhydrazine-transformed (#51) cells in the right thigh, were subjected to radiation, 2-DG, and heat used singly and in various combinations. Control mice were injected with saline. Three to four weeks after inoculation the mice were assigned to one of eight treatment groups (28 mice/group) so that average tumor volume/group before treatment would be equivalent. A single 2000 rad dose of radiation 3 hr prior to heat and 2-DG injected intraperitoneally at 1 g/kg 30 min before heating were given to some of the groups. Localized heat at 43.5 +/- 0.1 degrees C for 30 min, when used, was administered by means of a water bath. Rectal temperatures were kept below 39 degrees C, whereas intratumor temperatures reached a maximum of 42 degrees C. After treatment, tumor volume, mouse weight, and mortality were noted twice a week for four weeks. In both tumor models, mice receiving radiation plus heat, and radiation plus heat plus 2-DG, had significantly smaller tumors over the entire 4 to 28 day range after treatment than saline-injected control mice. In addition, in the H238 tumor model, addition of 2-DG to treatment with radiation and heat resulted in significantly smaller tumors at 25 days. 2-DG alone or in combination with heat (without radiation) resulted in significantly smaller H238 cell-induced tumors at day 28 post-treatment when compared to the saline controls. The H238 tumor-bearing mice experienced a significant (4.7%) loss in total body weight after heating. It could be that heating trauma produced dehydration and possibly also decreased caloric intake to an extent which could be measured in weight loss. This observation, however, was not made in the heated mice in the #51 tumor model.

Mouse neoplasia and immunity: effects of radiation, hyperthermia, 2-deoxy-D-glucose, and Corynebacterium parvum.

Radiation (XRT), hyperthermia, 2-deoxy-D-glucose (2DG), and Corynebacterium parvum were given in various combinations to BALB/c mice injected with herpes virus type 2-transformed (H238) cells. Addition of heat significantly increased the antitumor effects of XRT, and the combination of XRT + 2DG + heat resulted in the highest incidence of complete tumor regression. Enhanced activity of phytohemagglutinin-responsive T lymphocytes and natural killer cells capable of killing YAC-1 tumor cells was noted in some of the treatment groups while tumor volume was similar for all of the groups. This enhancement was most likely to be achieved when heat was included as part of the treatment protocol.

The lack of effect of tunicamycin and 2-deoxy-D-glucose on corneal stromal herpes in rabbits.

A rabbit model for herpes simplex virus (HSV) stromal keratitis, produced by intrastromal injection of live virus, was used to evaluate the effects of tunicamycin and 2-deoxy-D-glucose therapy. In vivo and in vitro evidence suggests that HSV strains that produce stromal disease secrete relatively large amounts of highly antigenic glycoproteins. Also, various studies have shown that tunicamycin and 2-deoxy-D-glucose inhibit the production of complete HSV-specific glycoproteins. Thus, these drugs might be capable of mitigating the clinical manifestations of HSV stromal keratitis by reducing the antigenic load. However, when topical therapy with tunicamycin and/or 2-deoxy-D-glucose was begun in rabbit eyes, the day after intrastromal inoculation of live RE strain HSV and several days before the appearance of stromal disease, no difference in the clinical course of herpetic ocular disease was seen between the experimental (treated) and control (untreated) groups.

Failure of 2-deoxy-D-glucose and 5-thio-D-glucose to kill hypoxic cells of two murine tumors.

We have administered the glycolysis inhibitors 2-deoxy-D-glucose and 5-thio-D-glucose to C3H/HeJ mice bearing KHT or 16/C transplantable tumors to seek evidence for hypoxic cell toxicity in vivo. The drugs were given (a) with or without insulin, (b) as large single doses or as multiple hourly injections, and (c) alone or immediately after the tumors had received radiation to kill most of the aerobic cell population. Tumor response was assessed by growth delay or by lung colony assay. Limiting toxicity of 2-deoxy-D-glucose and 5-thio-D-glucose was neurological, leading to seizures and/or death, and this toxicity was increased by insulin. The drugs had at most minimal effects on the growth of either untreated or irradiated tumors at maximal tolerated doses. Despite the known selective toxicity of these glucose analogues for hypoxic cells in tissue culture, we have found them to be ineffective in killing hypoxic cells of two murine tumors.

Gastric acid secretion and the changes of blood gastrin, glucose and insulin levels following 2-deoxy-D-glucose administration in hypersecretory and normosecretory duodenal ulcer patients.

Changes in gastric acid output, as well as the concentration of serum gastrin, blood glucose and serum insulin were measured in hypersecretory and normosecretory duodenal ulcer patients following 2-deoxy-D-glucose (2-DG) administration. The stimulation of gastric acid secretion caused by 2-DG was accompanied by a rise in serum gastrin and blood glucose concentrations. The values of serum gastrin measured during the test were significantly higher in hypersecretors. Despite high glucose level, there was no significant increase in serum insulin concentration both in hypersecretors and in normosecretors.

Failure of 2-deoxy-D-glucose in the treatment of experimental cutaneous and genital infections due to herpes simplex virus.

The effectiveness of 2-deoxy-D-glucose (2-DG) was evaluated in the treatment of cutaneous infections with herpes simplex virus type 1 (HSV-1) in mice and genital infections with HSV type 2 (HSV-2) in mice and guinea pigs. Groups of mice were inoculated in the lumbosacral or orofacial area with HSV-1 and treated topically three times a day with 0.2% or 0.5% 2-DG solution beginning 3 hr after inoculation. No effect on skin lesions, mortality, or latency was observed. Mice were inoculated intravaginally with HSV-2 and treated intravaginally three times a day with 0.2%-5% 2-DG in solution or suspended in miconazole nitrate cream beginning 6 hr, 24 hr, or 48 hr after inoculation. Replication of HSV-2 in the vagina and final mortality were not affected. Guinea pigs were infected intravaginally with HSV-2 and treated both intravaginally and topically on the external genitalia four times a day with 1% or 5% 2-DG in miconazole nitrate cream. Treatment initiated just prior to development of lesions (on day 3 after inoculation) did not alter vaginal virus replication, lesion development and severity, or virus titers in lesions.

Lack of efficacy of 2-deoxy-D-glucose in the treatment of experimental herpes genitalis in guinea pigs.

Topical treatment of herpes genitalis in female guinea pigs with 2-deoxy-D-glucose in either agarose gels or miconazole nitrate ointments failed to prevent the development of genital lesions or to reduce the mean titers of recoverable virus in vaginal swabs from infected animals. In contrast, phosphonoacetic acid was therapeutically effective.

Chemotherapeutic value of 2-deoxy-D-glucose in infectious bovine rhinotracheitis viral infection in calves.

Daily injection of 2-deoxy-D-glucose (2-dG) had no protective effect against respiratory tract infection in calves caused by infectious bovine rhinotracheitis virus. It also did not reduce the severity of this infection. Ocular instillation of the drug, however, markedly reduced the severity of viral-induced conjunctivitis and keratoconjunctivitis. The drug was effective when given at the time of ocular infection or after clinical conjunctivitis developed.