Novel method for the isolation, purification, and characterization of a heparin-like substance derived from the mucus of Achatina fulica and its use

Abstract

Heparin-like substances (HLS) have a structure similar to that of heparin, a highly sulfated glycosaminoglycan (GAG). The polysaccharide, a product of mast cells, has been isolated from animal tissues (porcine intestinal mucosa, bovine lung) and also has anticoagulant activity and activates antithrombin and protease inhibitors. Herein, methods for the isolation, purification and characterization of HLS from snail mucus (Achatina fulica) and its use are disclosed. This HLS can be used to develop simple, sensitive and specific tests for the biological activity of anti-SARS-CoV-2 (COVID-19) and anti-PD-L1 against breast cancer cells.

Description

Technical Field

[0001] Medicine Biology

Background Art

[0002] Heparin-like substances (HLS) are closely related to glycosaminoglycans (GAGs) consisting of disaccharide repeating units in which uronic acid (IdoA or β-D-glucuronic acid (GlcA)) is alternately (1→4) glycosidically linked to glucosamine (α-D-N-sulphoglucosamine (GlcNS) or α-D-N-acetylglucosamine (GlcNAc)) (Linhardt, 2003). HLS have been described in various clinical situations, which are generally attributed to circulating glycosaminoglycans, mainly heparin (HP) and dermatan sulphate (DS). This highly sulphated glycosaminoglycan (i.e., heparin) has a 102-year history as an antithrombotic drug and is still one of the most widely prescribed pharmaceuticals today. As a pharmaceutical, it is used as an anticoagulant (antithrombotic drug). Specifically, it is also used in the treatment of heart attacks and unstable angina and is administered by intravenous or subcutaneous injection. Other uses include use in test tubes and kidney dialysis devices.

[0003] Heparin (also known as unfractionated heparin (UFH)) is produced by basophils and mast cells in all mammals. Commercially available preparations currently most commonly originate from the mucosal lining of porcine intestines and have a long production cycle. However, in some countries including Argentina, Brazil and India, heparin of bovine origin is still recognized for religious reasons. In the past, no research has been done on novel methods for the isolation, purification and characterization of heparin-like substances from Achatina fulica mucus, and their use (which is the same as the present invention), because the records lack descriptions of those techniques.

[0004] The present invention provides a new method for the isolation, purification, and characterization of HLS from Achatina fulica slime as a new alternative source that is simple to operate, has a short production cycle, has no religious or health problems, and avoids the risk of future heparin and HLS shortages. This alternative resource of this raw material can be bred in an organic environment at home. This resource can be managed as an industrial and agricultural harvest so that it can be used for further use and it is easy to isolate and purify the active fraction from the starting material. The present invention provides a relative test report on the use of HLS for anti-SARS-CoV-2 and anti-PD-Ll activity against breast cancer cells, including anti-migratory activity of breast cancer cells.

[0005] JY van der Meer (2017). From Farm to Pharma: An Overview of Industrial Heparin Manufacturing Methods, this literature shows that many chemicals are added in the porcine / bovine / sheep heparin manufacturing process and it is closely related to health problems.

[0006] Haiying Liu et al. (2009). Lessons learned from the contamination of heparin, this literature emphasizes the heparin contamination crisis of heparin analogs as potential heparin contaminants.

[0007] Szajek A.Y. et al. (2016) The US regulatory and pharmacopeia response to the global heparin contamination crisis, this literature reports that the porcine heparin manufacturing process has not changed substantially since it was introduced.

[0008] Patent Document CN105,001,353A discloses an optimized purification technology for sodium heparin crude, without using any crude heparin isolation method that is a longer and more complex process.

[0009] Patent Document EP0113040A2 discloses an ultrafiltration process using two membranes for the purification and fractionation of heparin, which is superior to the prior art.

[0010] Patent Document KR0170064B1 discloses an earthworm extract containing heparan sulfate. The present invention uses an earthworm tissue source and different extraction methods. Precipitation of GAG with trichloroacetic acid (TCA), potassium acetate, and cetylpyridinium chloride can be found as dangerous residues in this process.

[0011] Patent Document IN2502DEL1996A discloses a process for the isolation of a new highly specific 9-O-acetylated sialoglycoconjugate binding lectin (Achatinin-H) from Achatina fulica earthworms useful for the diagnosis of visceral leishmaniasis, using different isolation methods and a blood lymph source.

[0012] U Lindahl (2020). Heparin - An old drug with multiple protential targets in Covid - 19 therapy. This report shows that treatment with low molecular weight heparin (LMWH) reduces mortality in severe patients.

[0013] Andrade et al. (2013). A heparin - like compound isolated from a marine crab rich in glycuronic acid 2 - O - sulfate present low anticoagulant activity.

[0014] Li Fu et al. (2016). Bioengineered heparins and heparan sulfates disclose the use of recombinant techniques in chemoenzymatic synthesis and metabolic engineering to address intrinsic impurities, source tissue constraints, and inadequate control of the manufacturing process.

[0015] Griffin et al. (1995). Isolation and characterization of Heparan sulfate from crude porcine intestinal mucosal peptidoglycan heparin disclose a longer and more complex process.

[0016] Linhardt et al. (1995). Dermatan sulfate as a potential therapeutic agent of anticoagulant and antithrombotic agents.

Disclosure of the Invention

[0017] The present invention is an innovative strategic research for an alternative source of heparin-like substance (HLS) derived from Achatina fulica mucus, as well as for its new biological activities as anti-PD-L1 and anti-SARS-CoV2 (COVID-19) infection. The present invention provides a method for simply and easily isolating, purifying, and characterizing active fractions from starting materials, with a short production cycle, high production yield, reliable quality, no religious or health problems, and avoiding the risk of future shortages of heparin and HLS. The most important advantage of the present invention is that the source of raw materials can be used to produce an unlimited amount of medicine.

[0018] To achieve the above object, the technical scheme of the present invention is as follows. (1) As shown in FIGS. 1 and 2 below, the present inventors have developed a novel method for isolating, purifying, and characterizing a heparin-like substance (HLS) from Achatina fulica mucus.

[0019] 1.1 Preparation of Sulfated Glycosaminoglycan (GAG) from Achatina fulica Mucus 1 L of mucus was collected from the feet and mantle of live Achatina fulica and then lyophilized by a lyophilization process. This dried mucus powder was defatted with 3 volumes of acetone with shaking overnight. After lyophilization, the mucus powder (15 - 20 g) was suspended in 5 volumes of sodium acetate buffer (pH 5.5) containing 5 mM EDTA and 5 mM cysteine. Papain enzyme (50 mg, 3 times each, 3.2 units / mg solid) was added. Then, the reaction mixture was incubated at 55 °C with shaking for 48 hours. This digestion mixture was stopped by heating at 100 °C for 5 minutes, and then this suspension was centrifuged at 5000 g for 30 minutes at room temperature, and the supernatant (which contains GAG) was collected.

[0020] 1.2 Isolation and Purification of Sulfated Glycosaminoglycan (GAG) from Digested Achatina fulica Mucus The papain-digested sample was isolated and purified by anion-exchange chromatography on a DEAE-crosslinked agarose beads column (HiTrap DEAE FF, GE healthcare). Elution was performed stepwise in the concentration range of 0.1 M - 1.0 M NaCl in 50 mM sodium acetate buffer (pH 5.5). Elution was monitored at 210 nm, and the flow rate was set at 0.5 mL / min. Three elution fractions, (1) non-interacting fraction (F1, yield 1.2% (w / w)), (2) low-sulfated GAG-containing fraction (F2, yield 6.9% (w / w)), and (3) high-sulfated GAG-containing fraction (F3, yield 14% (w / w)) were collected. The isolated fractions were thoroughly desalted by chromatography on a HiTrap desalting column (GE healthcare) and lyophilized to obtain a pale yellow powder.

[0021] 1.3 The characteristics of the disaccharide repeat were discovered. The specific disaccharide composition of the GAG isolated from snail mucus can be studied using enzymatic digestion and HPLC. The active sulfated GAG fraction (F3) was prepared as follows, as shown in Figure 3. The F3 oligosaccharides with a high sulfated GAG content were depolymerized at 37 °C with shaking for 24 h using 1 mIU of heparitinase II and heparitinase III in 0.3 mL of 50 mM Tris-HCl buffer (pH 7.2) and 10 mM CaCl2. The reaction mixture was then heated in boiling water for 5 min, centrifuged at 5000 g for 10 min, and lyophilized. This heparitinase digestion product was injected into an analytical SAX-HPLC column (Phenomenex, 0.46×25 cm, Torrells CA) to monitor the reaction. A linear gradient of 0.1–1.0 M NaCl was run over 40 min at a flow rate of 1.0 mL / min, and detection was set at 232 nm to monitor the unsaturated disaccharides of uronic acid. In Figure 4, the major peak of the active sulfated GAG fraction (F3) disaccharide observed at peak 4 (17.89 min), which exceeds 73% of the composition, corresponds to the disaccharide repeating unit of chondroitin sulfate, ΔHexA(2S)-GlcNAc. Furthermore, ΔHexA(2S)-GlcNAc(6S) (peak 7) and ΔHexA(2S)-GlcNSO3(6S) (peak 8) were also observed as components of 15% and 12%, respectively. All are hereinafter referred to as the "snail heparin-like substance (snail HLS)".

[0022] (2) In vitro studies against SARS-CoV-2 were reported. The life cycle of SARS-CoV-2 has been reported, and each step of virus infection and replication has been targeted for drug discovery. In particular, the first step as a virus particle utilizes the binding of the spike protein to the receptor, which is ACEII (angiotensin-converting enzyme II), as shown in Figure 5, and antiviral activity was evaluated based on the inhibition of spike ACE2 binding by a competitive immunoassay. The inhibitory activity of the mean 50% inhibitory concentration (IC 50 ) was calculated (Figure 6). This data indicates that the slug HLS showed strong inhibitory activity against the SARS-CoV-2 spike RBD region.

[0023] (3) Anti-PD-L1 activity against breast cancer cell line (MDA-MB231) has been reported. Inhibition of the programmed cell death ligand 1 (PD-L1) and programmed cell death 1 (PD-1) immune checkpoints by monoclonal antibodies has been shown to be successful in cancer. Binding of PD-L1 to PD-L1 inhibits T cell effector function, resulting in an immunosuppressive state. The expression of PD-L1 in cancer cells plays an important role in cancer immune evasion and cancer progression. The development of compounds that downregulate PD-L1 expression has been studied. The slug HLS was found to be able to show downregulation of PD-L1 both at the gene expression and protein levels in breast cancer cells MDA-MB231, as shown in Figure 7.

[0024] (4) Anti-migration activity of breast cancer cells has been reported. Metastasis is the most characteristic stage of cancer and causes dysfunction of tissues and other organs throughout the body. In order to study the properties of the slug HLS, the inventors used a scrap-assay to study the inhibition of cancer cell migration, as shown in Figure 8, and found that the slug HLS derived from Achatina fulica can inhibit the migration of breast cancer cells using a migration assay.

[0025] (5) It has been reported that cat snail HLS increases the ability of the cytotoxic effect of T cells against MDA-MB231 cells. Assay of the cytotoxicity of T cells against cancer cells. Peripheral blood mononuclear cells (PBMCs) derived from healthy donor screening will be collected from the Blood Bank Section Maharaj Nakorn Chiang Mai Hospital. And the PBMCs were isolated by density gradient centrifugation (Ficoll). To stimulate the PBMCs, 6-well plates were coated with anti-CD3 by pre-incubating the plates with 1 μg / ml antibody in PBS for 4 hours. Furthermore, at the start of the culture experiment, soluble anti-CD28 antibody (1 μg / ml) and IL-2 (10 ng / ml) were added. After pretreatment with MDA-MB231 for 48 hours with or without cat snail HLS (0 - 200 μg / ml), the medium was changed, and the cancer cells were co-cultured with T cells to be activated (the ratio of tumor cells to lymphocytes was 1:10). The wells of the activated T cells were washed twice with PBS to remove the T cells, then the living cancer cells were fixed, stained with crystal violet, eluted with 20% acetic acid, and the absorbance was measured at 590 nm. To determine whether the downregulation of PD-L1 on MDA-MB231 cells by cat snail HLS changes immune-mediated cytotoxicity, T cells isolated from peripheral blood mononuclear cells of healthy volunteers were incubated with MDA-MB231 cells pretreated with cat snail HLS for 48 hours. Then, they were co-cultured for 24 hours, and the surviving cancer cells were marked with crystal violet solution. The T cells slightly reduced the survival rate of cancer cells in the absence of cat snail HLS compared with the control (without T cells). However, the treatment with cat snail HLS (200 μg / ml) reduced the survival rate of cancer cells in MDA-MB231 cells co-cultured with T cells by approximately 33.78% compared with the control group (with T cells), as shown in Figure 9. This finding obtained suggested that the downregulation of PD-L1 induced by cat snail HLS activated T cells by cancer cells and increased the effect of killing cancer cells.

Brief Description of the Drawings

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Claims

**Claim 1** A novel method for isolating, purifying, and characterizing a heparin-like substance (HLS) from the mucus of the African giant snail (Achatina fulica). (1) Preparation of sulfated glycosaminoglycan (GAG) from snail mucus: 1 L of mucus was collected from the foot and mantle of live snails and then lyophilized by a lyophilization process. This dry mucus powder was defatted with three times the amount of acetone while shaking overnight. After lyophilization, the mucus powder (15 - 20 g) was suspended in five times the amount of sodium acetate buffer (pH 5.5) containing 5 mM EDTA and 5 mM cysteine. Papain enzyme (3.2 unit / mg solid) at 50 mg (three times each) was added. Then, the reaction mixture was incubated at 55°C with shaking for 48 hours. This digestion mixture was stopped by heating at 100°C for 5 minutes, and then this suspension was centrifuged at 5000 rpm for 30 minutes at room temperature, and the supernatant (which contains GAG) was collected. (2) Isolation and purification of sulfated glycosaminoglycan (GAG) from digested snail mucus: The papain-digested sample was isolated and purified by anion exchange chromatography on a DEAE-crosslinked agarose bead column (HiTrap DEAE FF, GE healthcare). Elution was performed stepwise in the concentration range of 0.1 M - 1.0 M NaCl in 50 mM sodium acetate buffer (pH 5.5). Elution was monitored at 210 nm, and the flow rate was set at 0.5 mL / min. Three elution fractions were collected: (1) non-interacting fraction (F1, yield 1.2% (w / w)), (2) fraction with low sulfated GAG content (F2, yield 6.9% (w / w)), and (3) fraction with high sulfated GAG content (F3, yield 14% (w / w)). The isolated fractions were thoroughly desalted by chromatography on a HiTrap desalting column (GE healthcare) and lyophilized to obtain a pale yellow powder. (3) Characteristics of disaccharide repeats were discovered: The specific disaccharide composition of GAGs isolated from snail mucus can be studied using enzymatic digestion and HPLC. An active sulfated GAG fraction (F3) was performed. F3 oligosaccharides with a high sulfated GAG content were incubated in 0.3 mL of 50 mM Tris-HCl buffer (pH 7.2) and 10 mM CaCl ２ 2 in the presence of 1 mIU of heparitinase II and heparitinase III, and depolymerized at 37 °C for 24 hours with shaking. The reaction mixture was then heated in boiling water for 5 minutes, centrifuged at 5000 rpm for 10 minutes, and lyophilized. This heparitinase digestion product was injected onto an analytical SAX-HPLC column (Phenomenex, 0.46×25 cm, Torrence CA) to monitor the reaction. A linear gradient of 0.1–1.0 M NaCl was run over 40 minutes at a flow rate of 1.0 mL / min, and detection was set at 232 nm to monitor unsaturated disaccharides of uronic acid. The major peak of the active sulfated GAG fraction (F3) disaccharide, observed at peak 4 (17.89 minutes) and exceeding 73% of the composition, corresponds to the disaccharide repeating unit of akeran sulfate, ΔHexA(2S)-GlcNAc. Additionally, ΔHexA(2S)-GlcNAc(6S) (peak 7) and ΔHexA(2S)-GlcNSO3(6S) (peak 8) were also observed as components at 15% and 12%, respectively. All are hereinafter referred to as "snail heparin-like substances (snail HLS)".

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