阅读说明：本技术 安全的牛肝素、制备方法和应用 (Safe bovine heparin, preparation method and application ) 是由 R·S·D·阿奎诺 P·A·D·S·穆拉渺 L·N·D·梅德利诺斯 E·P·维拉诺瓦 A·M 于 2019-02-11 设计创作，主要内容包括：本发明涉及用于放大规模生产安全的牛肝素的制备方法,及其生产方法和应用,所述安全的牛肝素由不同选择的未分级的牛肝素聚合物组成,其具有低6-O-脱硫酸化的葡糖胺含量和猪样抗凝血活性以及鱼精蛋白中和。这种安全的牛肝素(SB肝素)具有与猪肝素(临床使用参考)可比的结构和功能,作为安全的药学产品可预防临床使用障碍,允许其作为可互换的药物使用。(The present invention relates to a process for the preparation of safe bovine heparin consisting of differently selected unfractionated polymers of bovine heparin with a low 6-O-desulphated glucosamine content and porcine-like anticoagulant activity and protamine neutralization for large scale production, as well as to a process for its production and use. The safe bovine heparin (SB heparin) has a structure and a function comparable to those of porcine heparin (clinical use reference), and can prevent clinical use disorder as a safe pharmaceutical product, allowing it to be used as an interchangeable drug.)

1. Safe bovine heparin (sb heparin) sodium comprising a preparation of bovine intestinal mucosal heparin.

2. Unfractionated heparin with low 6-O-desulphated glucosamine and porcine-like clinical therapeutic properties comprising a preparation of bovine intestinal mucosal heparin.

3. Heparin having anticoagulant activity and protamine neutralization comparable to porcine heparin, contained in a preparation of bovine intestinal mucosal heparin.

4. Bovine heparin according to claims 1-3 wherein said structural composition contains a high trisulfated disaccharide unit and a low 6-O-desulphated glucosamine polymer.

5. The bovine heparin of claims 1-3 wherein the activity of about 190UI/mL is statistically similar to porcine heparin.

6. The preparation of bovine heparin according to claims 1-5, comprising the steps of:

-solubilization of bovine intestinal mucosa in running buffer;

-purification by ion chromatography on different resins;

-washing and drying after filtration to obtain the safe bovine heparin (SB heparin) sodium; porcine-like heparin; and highly anticoagulant bovine heparin.

7. The process for the preparation of bovine heparin according to claim 6, which comprises the step of mass production of a productive chromatography system; a salt exclusion method was used.

8. Bovine heparin according to claims 1-5 comprising anticoagulant, antithrombotic and halal agents.

9. Bovine heparin according to claims 1-5 comprising injections of anticoagulant, antithrombotic and halal agents.

10. Bovine heparin according to claims 1-5 comprising anti-inflammatory and/or anti-metastatic agents, inflammatory and cancer treatments.

11. Bovine heparin according to claims 1-5, comprising a source of raw materials for the production of different types of low molecular weight heparin sodium.

Technical Field

The invention relates to the preparation of new and safe bovine heparin, which is a safe anticoagulant drug (media) from the intestinal mucosa of a cow, and relates to the field of biotechnology medicine of drug discovery and manufacture.

Background

Glycosaminoglycans (GAGs) are linear, complex heteropolysaccharides found as components of Extracellular Matrix (EMC), cell surface and intracellular space (intracellular space). The history of GAGs dates back to the 19 th century when Chondroitin Sulfate (CS) was first identified as a component of cartilage and its structure was further elucidated (DAVIDSON and MEYER, 1954). CS is a polymer consisting of repeating disaccharide units of glucuronic acid and N-acetylgalactosamine, which can be sulfated at the 4-or 6-position of N-acetylgalactosamine [ → 4- β -GlcA- (1 → 3) - β -GalNAc- (4S) (6S) -1 → ]. Subsequent studies have shown that CS is widely distributed in nature, similar to other GAGs described further.

Surprisingly, in the early 20 th century, studies on procoagulant phospholipids in various tissue extracts have found anti-coagulant GAGs. The alcohol extract of dog liver produced a "fat soluble" fraction with unexpected anticoagulant effect. The anticoagulant effect is associated with the presence of carbohydrates known as heparin (hepar is the liver in greek) (Howell and Holt,1918) (Howell, 1925). Later, the use of NMR analysis techniques indicated that heparin consisted of repeating disaccharide units of iduronic acid and N-acetylglucosamine, which were predominantly sulfated [ → 4- β -IdoA- (2S) -1- (1 → 4) - α -GlcNAc- (NS) (6S) -1 → ] (Cifonelli and Dorfman,1962) at the N-, 6-O-, 2-O-positions.

Despite limited information about structure and biological function, the demand for anticoagulant drugs has driven crystalline forms of heparin to be tested in clinical trials and into the pharmaceutical market (Wardrop and kelling, 2008), demonstrating its clinical relevance and demand as an anticoagulant drug.

Later developments in chromatographic and NMR analysis techniques could elucidate the motifs (motifs) required for specific GAG-protein binding interactions. HPLC and NMR analysis of growth factor-bound heparin showed that 2-O sulfated iduronic acid (Habuchi et al, 1992) was essential for specific growth factor interactions (bFGF binding rather than for FGF-2, FGF-2 instead requiring 6-O sulfated N-acetylglucosamine (Maccarana et al, 1993)). On the other hand, the polysaccharide binding site that mediates heparin-antithrombin III binding and inhibition requires rare 3-O-sulfation on N-acetylglucosamine, among other structural modifications (Lindahl et al, 1980). These early examples show the significant significance of heparin structure in regulating its interactions and determining its biological function.

Although dog liver was the original source of heparin, from a more practical point of view, analysis directed to production showed that heparin preparations extracted from different tissues showed high concentrations of this GAG in the bovine intestine and lung, which was selected as an early source of heparin for clinical use. Bovine unfractionated heparin (UFH) was first marketed in 1939 on the american health systems market and was sold for over 50 years thereafter.

UFH heparin from bovine sources was spontaneously withdrawn from the major market in the early/mid 80 s due to the risk of Bovine Spongiform Encephalopathy (BSE) contamination, making porcine mucosal heparin almost a proprietary supply of the global heparin market.

However, the worldwide high use of such life-saving drugs poses a shortage risk for heparin production, as its source is limited to porcine sources. Furthermore, since heparin is mainly produced from one animal source (swine) and geographical area (china), the risk of shortage due to raw material supply changes is higher.

As the knowledge of BSE analysis and purification techniques advances, the risk of BSE contamination is better understood. Recent studies have shown that different steps of heparin purification are able to completely remove bovine spongiform encephalopathy causing agents (agents) from crude heparin (Bett et al, 2017), demonstrating that this is currently a controlled risk. Similar progress was shown in the BSE virulence factor assay, facilitating quality control of bovine-derived purified heparin.

Recent market demand coupled with scientific advances in this field has led to the market re-introduction of bovine heparin as a common sense. The regulatory body published the need for the market to reintroduce bovine heparin (Szajek et al, 2016).

However, despite reducing the risk of BSE, bovine heparin preparations still show lower quality and activity, which is a continuing problem for their reintroduction. Heparin extracted and purified from bovine mucosa has structural differences compared to porcine heparin (reference drug). Bovine mucosal heparin showed a lower 6-O-sulfation and a higher N-acetylation rate (glucosamine residues) than porcine heparin (Aquino et al, 2010). Associated with these structural differences are the observed lower anticoagulant activity of whole plasma or purified coagulation factors Xa and IIa in vitro, lower anticoagulant activity of the plasma of mice treated ex vivo, and lower anticoagulant activity in vivo in animal models of venous thrombosis. These results have been confirmed by other studies (Santos et al, 2014, Tovar et al, 2016). The lower anticoagulant activity of bovine heparin was also demonstrated in plasma of dialyzed patients compared to patients treated with porcine heparin (Tovar et al, 2013).

Despite having lower anticoagulant activity, bovine heparin-like product also had a higher bleeding tendency as shown by higher blood loss rates in animal models. Furthermore, it was observed that a higher concentration of protamine (heparin antidote) was required to neutralize bovine heparin anticoagulant activity (Aquino et al, 2010). Combining the two observations suggests a determinant of the mechanism of the increase in clinically detected bleeding events observed in the brazilian hygiene system following replacement of porcine heparin with bovine heparin (Melo et al, 2008; Junqueira et al, 2011). Therefore, this structural and functional difference poses a critical risk for clinical use compared to porcine heparin.

In summary, despite the need for a sanitary system and the development of BSE prevention, the reintroduction of bovine heparin in the market is constrained by its lower anticoagulant activity and higher bleeding characteristics. Therefore, it is essential to determine the structure/function of a polydisperse heterogeneous population of unfractionated bovine heparin, with the goal of being able to scale-up a new batch (pol) of high quality bovine heparin with similar clinical activity as the reference product on the market (porcine heparin), avoiding variations during clinical administration which have been shown to have a detrimental effect on the survival of patients. Furthermore, patent searches on SPACENET and USPTO do suggest that any existing patent (innovation with the ability to produce bovine heparin with structural and functional activity similar to porcine heparin) exposes a lack of knowledge in this field (table 1).

Table 1 shows the most relevant patents in this field after search on SPACENET and USPTO. Different approaches to improve heparin purification have been published; however, there is no way to convert a bovine heparin preparation to porcine-like clinical quality.

Disclosure of the invention

The present invention provides a process for obtaining unfractionated heparin preparations from bovine intestinal mucosa with low 6-O-desulphated glucosamine content, giving heparin preparations with structure and anticoagulant activity comparable to porcine mucosal heparin (market reference product), herein referred to as safe bovine heparin (SB heparin).

Detailed analysis of the intestinal mucosal heparin fraction showed a high 6-O-desulphated glucosamine content, which is related to: lower anticoagulant activity and higher protamine (antidote) neutralizing concentrations are required.

The process of the invention comprises fractionating intestinal mucosa-derived bovine heparin with a refining ion exchange step. The ion exchange step is carried out with sufficient salt elution concentration to provide bovine heparin with a structure and anticoagulant activity comparable to porcine heparin.

As described below, these bovine heparin preparations have a low 6-O-desulphated glucosamine content as compared to conventional intestinal mucosal bovine heparins.

SUMMARY

Safe bovine heparin, which is unfractionated heparin from bovine intestinal mucosa with low 6-O-desulphated glucosamine (6-O-desulphated glucosamine) content and porcine-like (porcine-like) anticoagulant activity and protamine neutralization, and methods of making and using the same.

Drawings

Figure 1 shows the structure of unfractionated bovine heparin compared to porcine heparin. FIG. 1A shows a one-dimensional NMR spectrum of swine and cattle; FIG. 1B shows the percentage calculation of each one-dimensional peak from different commercial preparations of porcine and bovine heparin; figure 1C shows the predicted structure of porcine and bovine heparin.

Figure 2 shows the function of unfractionated bovine heparin (activity and protamine neutralization) compared to porcine heparin. FIG. 2A shows the in vitro anticoagulant activity of porcine and bovine heparin; FIG. 2B shows the anticoagulant activity of porcine and bovine heparin in vivo; figure 2C shows protamine neutralization of porcine and bovine heparin.

Figure 3 shows a comparison between porcine and bovine derived heparins based on their activity and bleeding tendency. Figure 3A shows venous antithrombotic activity in rat vena cava using stasis (statis) and hypercoagulable thrombosis models. Different doses of bovine or porcine heparin were administered and allowed to circulate for 5 minutes. Then, thromboplastin (5mg kg-1 body weight) was slowly injected intravenously, and 0.7cm of a divided luminal venous segment was ligated. After 20 minutes of stasis, the thrombus formed was dried and weighed. Results are expressed as% of the thrombus weight, 100% representing the absence of any inhibition of thrombus formation (weight of thrombus without heparin administration). Fig. 3B shows citrated blood samples (citrated blood samples) collected from the carotid artery before and 5 minutes after intravascular administration of heparin. Plasma was then assayed for aPTT ex vivo. Results are expressed as the ratio of clotting times after administration of different heparin doses (T1) and administration of control (saline treated animals) (T0). No values above 10 were detected in the assay. Figure 3C shows different doses of heparin injected into rats. After 5 minutes, the rat tail was cut 3mm from the tip and immersed in 40ml of distilled water at room temperature. Blood loss was determined after 60 minutes by measuring hemoglobin in the water. Results are expressed as μ L of blood loss. The insert in panels (Panel) A and C shows a curve of dose versus response based on anticoagulant activity (IU kg-1). For clarity, only one Standard Error (SE) bar is shown in the panel. For bovine heparin versus porcine heparin, all results were using the Mann-Whitney rank sum test, expressed as mean SE, n ═ 5, × p <0.01 and × p < 0.05. Arrows in panels A and C inset indicate the heparin dose (100IU kg-1 body weight) required to achieve complete inhibition of thrombosis.

Figure 4 shows the neutralization of heparin by protamine. Heparin (0.1IU ml-1) was incubated with increasing concentrations of protamine and then mixed with 10nM antithrombin and 2nM factor Xa in 40. mu.l TS/PEG buffer. After incubation at 37 ℃ for 60 seconds, the remaining factor Xa activity is determined with a chromogenic substrate (A)₄₀₅nm min-1)。

Figure 5 shows the optimization of SB heparin production on an ion exchange chromatography 1mL column coupled to an HPLC system.

Figure 6 shows optimization of scale-up SB heparin production on an ion exchange chromatography 10mL column coupled to an HPLC system.

Figure 7 shows the structure (one-dimensional hydrogen nuclear magnetic resonance map) and function (whole plasma (aPTT) and purified coagulation factor (thrombin and Xa) assays.

Modes for carrying out the invention

The present invention relates to bovine mucosal derived unfractionated heparin preparations (which exhibit comparable structure and anti-coagulant activity to porcine mucosal heparin), methods of their preparation and use, prepared from commercially available heparin. "safe bovine heparin" or "SB heparin" refers to preparations with low 6-O-desulphated glucosamine content, having porcine mucosal heparin-like structure and anticoagulant activity.

In the context of the present invention, the anticoagulant activity of heparin is related to the inhibition of the coagulation factors Xa and IIa by antithrombin III (AT).

In one aspect, the invention relates to a method for preparing bovine heparin having a clinical therapeutic effect (including anticoagulant activity and protamine neutralization) similar to that of porcine heparin.

By Nuclear Magnetic Resonance (NMR)¹H and¹³c one-dimensional (1D) and two-dimensional (2D) profiling of commercially available preparations of porcine and bovine heparin revealed that porcine heparin consists mainly of trisulfated disaccharide units and bovine heparin consists of highly 2-sulfated iduronic acid residues, but bovine heparin lacks 6-O-sulfation on glucosamine units.

Detailed analysis of heparins with different structural compositions indicates that the presence of disaccharides lacking 6-O-sulphation on the glucosamine of bovine heparin is associated with lower anticoagulant activity, higher bleeding and lower protamine neutralisation rate.

More specifically, it was determined that bovine heparin had lower anticoagulant activity in the whole human plasma experiment (aPTT) when compared to porcine heparin (fig. 2A). aPTT is aPTT reagent (bovine phospholipid reagent) and CaCl for inducing blood coagulation by human plasma and various heparin concentrations₂Performed, this was recorded on a coagulometer. Results are expressed as the ratio of clotting times in the presence (Ti) or absence (To) of heparin. Use of bases 5^thA parallel calibration curve of the International heparin Standard (229IU mg-1) obtained from the national institute for bioscience and control (Portsba, UK) estimates anticoagulant activity in IU mg-1. Solutions of bovine and porcine heparin were prepared on a weight basis and showed similar hexuronic acid content when examined by the carbazole reaction.

In fact, the lower anticoagulant activity of bovine heparin compared to porcine heparin was demonstrated using an assay with purified coagulation factors (fig. 2B and 2C). Factor Xa and thrombin (anti-Xa and anti-IIa assays) were used in the presence of a chromogenic substrate (S2238 for thrombin, S-2222 for factor Xa) and absorbance was recorded at 405nm for 300 seconds using a microplate reader. The rate of change in absorbance is proportional to the activity of the remaining thrombin or factor Xa in solution. Using a parallel standard curve based on the international heparin standard (229 units mg-1), the anti-IIa and anti-Xa activities are reported in units of mg-1.

On the other hand, using rats having rabbit brain thromboplastin as a stimulator of thrombus formation, it was confirmed that bovine heparin had lower anticoagulant/antithrombotic activity in vivo compared to porcine heparin (fig. 3A). Following institutional guidelines for animal care, and experimental rats (both sexes, body weight 200g, 5 animals per dose) were anesthetized and different doses of heparin were injected into the right carotid artery and allowed to circulate for 5 minutes. Isolating the inferior vena cava and slowly injecting a brain thromboplastin (5mg kg-1 body weight) intravenously; after 1 minute, 0.7cm of isolated vena cava was clamped using distal and proximal sutures. After 20 minutes of stasis, the thrombus formed in the enclosed section was carefully removed, rinsed with phosphate buffered saline, dried at 60 ℃ for 1 hour, and weighed. The mean thrombus weight was obtained from the mean weight of each group and was then expressed as a percentage of the weight, 100% representing the absence of any inhibition of thrombus formation (weight of thrombus without heparin administration).

Rat plasma injected with bovine heparin showed lower anticoagulant activity compared to porcine heparin, as evidenced by citrated blood samples collected from the carotid artery before and 5 minutes after intravascular administration of heparin (fig. 3B). Plasma was assayed for aPTT ex vivo. Results are expressed as the ratio of clotting times after administration of different heparin doses (T1) and controls (saline treated animals) (T0).

Bleeding tendency is an important aspect during anticoagulant therapy, presenting a significant side effect with a risk of death during its clinical use. Analysis of bleeding tendency after intravascular administration of bovine heparin in rats showed a higher blood loss rate compared to administration of bovine heparin (fig. 3C). It is important to note that the analysis showed that both heparins had the same dose-dependent bleeding induction on a weight basis. However, the curve based on anticoagulant activity (inset) clearly shows that bovine heparin is twice as effective in inducing bleeding as porcine heparin. Since heparin is used clinically based on its anticoagulant activity, bovine heparin has twice the bleeding tendency than porcine heparin, making it a significant health risk.

During the clinical use of heparin, neutralization of heparin requires the appropriate dose of protamine at the end of extracorporeal circulation or when drug overdose is detected. Heparin, such as bovine heparin and porcine heparin, with different chemical and biological properties may exhibit different protamine neutralization profiles. Protamine was added to bovine heparin or porcine heparin at similar doses (on an IU basis), and heparin neutralization was assessed based on anti-Xa activity, demonstrating that bovine heparin requires significantly higher doses of protamine than porcine heparin to achieve neutralization (fig. 4). This change in protamine neutralization is a key aspect of commercial bovine heparin products because it is difficult to interpret this change during clinical procedures, thereby presenting a potential risk to the health of the patient.

These findings establish new correlations between heparin origin, structure and function. More specifically, it was determined that heparin fragments containing 6-O-desulphated glucosamine were associated with lower anticoagulant activity and protamine neutralization and higher hemorrhagic effects.

Based on these findings, an innovative process was developed to remove the heparin chain containing 6-O-desulphated glucosamine from bovine heparin purified from the intestinal mucosa, allowing purified bovine heparin to have a structure and function comparable to porcine heparin. Surprisingly, examination of different purification strategies shows that removal of heparin chains containing 6-O-desulphated glucosamine can be performed using an optimized chromatography protocol with ion exchange chromatography. More specifically, the production of these new trisulphated rich heparins from bovine mucosa is accomplished using a scalable purification protocol using a synthetic methacrylate (methacrylate) -based polymer matrix with long linear polymer chains carrying functional ligands of the trimethylammonium ethyl group (trimethylamonium ethyl).

The present invention shows that the use of an ion exchange chromatography purification step enables the production of safe bovine heparin (SB heparin), which is a purified bovine heparin, having a low heparin polymer containing 6-O-desulphated glucosamine and a high trisulphated disaccharide unit, with a structure and function comparable to porcine heparin. Due to its similarity to the market reference (porcine heparin), this is considered to be a safe bovine heparin for clinical use, allowing for interchangeable use in clinical medicine.

To remove the heparin polymer containing 6-O-desulphated glucosamine, an ion exchange column TMAE HICAP from MERCK was used coupled to the HPLC system (shown optimized in FIGS. 5 and 6). Commercially available intestinal mucosal bovine heparin, with a specific activity (specific activity) of about 100IU/mL, was used as a starting material. The heparin powder was diluted in running buffer (20mM Tris, with 0.02-0.1M NaCl, pH 7.2) and applied to a TMAE column at a flow rate of 3.5 ml/min. Elution of the bound heparin was performed by an initial washing step followed by two stepwise conditions with salt concentrations ranging from 0.5M to 2.0M NaCl. The first elution step produced heparin containing low anticoagulant and high 6-O-desulphated glucosamine, which was then discarded. Collecting the second peak; the salts were removed by dialysis against distilled water and lyophilized. The structure and function of the purified bovine heparin was then analyzed in comparison to porcine heparin and commercially available bovine heparin.

Analysis of purified bovine heparin showed that this purification method provided bovine heparin with a structure and anticoagulant activity similar to porcine heparin. Structural analysis by nuclear magnetic resonance demonstrated that heparin polymers containing 6-O-desulphated glucosamine were removed from the SB heparin preparation process, as evidenced by the lack of the disulphated 6-O-desulphated glucosamine disaccharide residue represented by peak C (FIG. 7A). Thus, this innovative manufacturing process is effective in removing 6-O-desulphated glucosamine polymers from enteroheparins, producing heparin preparations with a structure similar to porcine heparins.

More importantly, purified bovine heparin showed comparable anticoagulant activity to porcine heparin (fig. 7B). In vitro anticoagulation assays using whole human plasma showed that the purified bovine heparin had slightly lower anticoagulation activity than porcine heparin. However, the anticoagulant assay with purified coagulation factors Xa and IIa showed that the anticoagulant activity of purified bovine heparin was slightly higher than porcine heparin, but could be considered statistically similar. Assays performed using purified coagulation factors are those accepted by the united states pharmacopeia as the official determinant of anticoagulant activity.

Thus, the present disclosure is directed to the production of a preparation of bovine heparin in the intestine with a structure and function similar to porcine heparin by using a single purification pre-amplification step, which is considered safe bovine heparin (SB heparin) once it can be used interchangeably in clinical medicine.

In aspects of the invention, a lab scale purification protocol using a single bead carrier (monobeas support) linked to a quaternary aminoethyl functional ligand also produces trisulfated-rich bovine heparin with porcine-like structure and function. These results indicate that other ion exchange resins can be used to produce heparin with low 6-O-desulphated glucosamine after the process is optimized. Furthermore, in aspects of the invention, the purification step may be used in any step of the purification process.

Examples

The following examples are intended to illustrate, but not limit, the present invention.

The starting material was bovine heparin (Extrasul S.A.) from the intestinal mucosa with a specific activity of approximately 100IU/mL and a structure rich in 6-O-desulphated glucosamine. An amount of 30mg of bovine heparin was diluted in 3mL of running buffer (20mM Tris pH 7.2 with 100mM NaCl).

TMAE HICAP 1mL column was equilibrated in an HPLC system with 10 column volumes running buffer and 3mL bovine heparin sample was applied at a flow rate of 3.5 mL/min. After washing with 5 column volumes of running buffer, the polymer containing 6-O-desulphated glucosamine was removed by a 5 column volume NaCl step (0.93M NaCl) followed by a 5 column volume wash. Elution was performed with a second 5 column volumes of 2M NaCl and the trisulfated enriched heparin polymer was collected. The collected samples were dialyzed against distilled water and lyophilized.

The purified sample was diluted in distilled water and the concentration was determined by quantifying uronic acid by carbazole reaction. The samples were then submitted to nuclear magnetic resonance structural analysis, which indicated a porcine heparin-like structure that lacked the 6-O-desulphated glucosamine peak previously observed in commercially available bovine heparin. The activity was shown to be about 190mL IU/mL using purified coagulation factor assays (Xa and thrombin), statistically similar to the activity of 180IU/mL porcine heparin. The recovery on an activity (IU units) basis was 85%, which is a determinant of heparin preparation production.

It is shown here that the process of improving bovine heparin production yields safe bovine heparin (SB heparin) with a structure and function comparable to porcine heparin (market reference) at a yield of 85%. This innovation allows for the inexpensive production of high quality bovine heparin, which can be used interchangeably with porcine heparin in clinical medicine. This has a crucial health system value for the whole world heparin production (prevention of shortage risk) and the halal market.

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