Methods for producing bovine-derived heparin and derivatives thereof, pharmaceutical compositions, and applications

Abstract

The present invention relates to the pharmaceutical industry and introduces innovative methods for producing highly active pharmaceutical heparin products using conventional crude bovine heparin (HBI) as the starting material. The methods include the production of (1) crude bovine heparin (crude SBH), (2) Active Pharmaceutical Ingredients (API) of unfractionated bovine heparin (SBH- UFH API), and (3) API of bovine low-molecular-weight heparin (B-LMWH), for preparing pharmaceutical compositions comprising them. By utilizing cetylpyridinium chloride (CPC) for selective precipitation, these methods achieve enhanced control over purity and anticoagulant potency, yielding products comparable to porcine-derived heparins. Additionally, the use of benzethonium chloride (CBZ) for producing B-LMWH demonstrates the expanded applicability of quaternary ammonium compounds and the flexibility of this invention. This practical, cost-effective process is suitable for large-scale manufacturing and addresses the need for diversified heparin sources, reducing dependency on porcine products and mitigating supply chain risks. It is particularly significant for markets requiring non-porcine medical products, such as those adhering to Halal laws, thus meeting a critical demand in the global pharmaceutical industry.

Description

[0001] METHODS FOR PRODUCING BOVINE-DERIVED HEPARIN AND DERIVATIVES THEREOF, PHARMACEUTICAL COMPOSITIONS, AND APPLICATIONS FIELD OF THE INVENTION

[0002] The present invention relates to the pharmaceutical industry, specifically to the production of highly active and safe crude bovine heparin (Crude SBH) and Active Pharmaceutical Ingredients (API) of safe bovine unfractionated heparin (SBH-UFH API) and bovine low-molecular-weight heparin (B-LMWH API) used for preparing anticoagulants medicines pharmacologically equivalent to conventional porcine counterparts’ medicines. The methods developed introduce innovative techniques for the refinement of heparin production processes from bovine sources, aiming to enhance the anticoagulant properties and safety profiles of the resulting products, enhancing the efficiency and sustainability of producing bovine heparin-based medications. The methods potentially reduce reliance on traditional porcine sources and address the demand for safer, more reliable anticoagulant therapies in healthcare industry, meeting to the needs of Muslim markets.

[0003] BACKGROUND

[0004] Heparin is a critical anticoagulant medication extensively used in various medical procedures, including in surgery, for prevention and treatment of thrombotic disorders, including myocardial infarction, pulmonary embolism, stroke and venous thrombosis. Figure 1 demonstrates the crucial interaction between heparin and antithrombin with its specific pentasaccharide sequence. This interaction is key to heparin’s mode of action to prevent the coagulation process, where it enhances the inhibition of critical enzymes like activated thrombin (Factor Ila) and activated Factor Xa by binding in antithrombin, leading to significant anticoagulant effects.

[0005] The current production of heparin relies primarily on porcine intestinal mucosa. This dependence on a single species introduces risks, such as supply chain vulnerabilities and cultural or dietary restrictions that prohibit the use of porcine products, such as Muslims. Additionally, the geographical concentration of production in regions like China and the reliance on a single animal source significantly increase the risks to the global supply chain. Such dependencies make the supply susceptible to disruptions caused by regional diseases like African Swine Fever and global health crises like COVID-19, which can lead to sudden shortages and intensify the need for diversified sources of heparin.

[0006] The scenario is worsened by the increase in global demand for heparin and its derivatives driven by an aging population and a rise in cardiovascular diseases. These factors underscore the necessity for alternative sources, such as bovine heparin.

[0007] The proposed production method of the present invention offers a strategic advantage by incorporating bovine sources, which are less affected by diseases prevalent in porcine populations and have a broader geographical distribution for sourcing. It reduces potential supply chain disruptions, ensuring a more stable and reliable supply of heparin in the face of global health emergencies. This approach not only addresses immediate supply concerns but also contributes to long-term pharmaceutical security and sustainability.

[0008] Further, bovine derived heparin offers a culturally acceptable alternative under various religious dietary laws, such as Halal, which is observed by approximately 25% of the global population. This source is particularly advantageous as cattle are more abundantly available in many regions, including Brazil, potentially decreasing the dependency on imported porcine heparin and enhancing pharmaceutical security. However, despite its benefits, the production of bovine heparin has encountered challenges in achieving comparable efficacy and safety as the porcine one.

[0009] Historically, differentiating between sources of heparin is essential, since they have distinct structural characteristics which directly influence their biological and pharmacological properties. The 1 D1H NMR spectra of conventional bovine and porcine unfractionated heparins highlight its structural nuances through signals corresponding to specific anomeric protons. These differences are pivotal for pharmacopeial identification (Figure 2). Specifically, a distinct difference is observed by the C1 signal observed in the bovine heparin in comparison to the porcine one, which is related to 6-O-desulfaed disaccharides. Further the disaccharide composition analysis using 2D1H / 13C HSQC NMR spectra provides quantitative insights into the specific sugar units predominant in each type, which play a critical role in their respective anticoagulant function (Figure 3). Together, these analyses confirm structural differences between the two heparins, mainly expressed by the lower presence of 6-O-sulfated disaccharide units in the bovine heparin when compared to its porcine counterpart.

[0010] The comparative evaluation of the anticoagulant activities of bovine and porcine heparins by quantification of the inhibition potency against Factor Ila using chromogenic substrate assays, displays the lower activity of the bovine heparin and reflects the practical implications of structural differences on clinical effectiveness.

[0011] The clinical relevance of these heparins was further studied by testing their interaction with protamine, a commonly used antidote for heparin during medical procedures. The assessment of protamine efficacy in neutralizing the anticoagulant effects showed that bovine heparin requires higher amounts of protamine to be inactivated. This difference in protamine neutralization have been associated with real-life clinical problems, such as hemorrhagic events during post-anticoagulation therapy.

[0012] The differences in bovine-derived compared to porcine-derived heparin also restrict its use as a raw material to produce low-molecular-weight heparin (LMWH), such as enoxaparin. This poses a crucial challenge for the health system due to the limited supply and risk of shortages of enoxaparin-like products, which accounts for approximately 90% of the overall heparin market revenues.

[0013] Enoxaparin is the main low-molecular-weight-heparin (LMWH) commonly used as an anticoagulant to prevent and treat thrombosis. The average molecular weight of enoxaparin is approximately 4,500 Daltons, ranging between 3,800 and 5,000 Daltons due to its heterogeneous nature. This lower molecular weight, compared to unfractionated heparin, allows for more predictable pharmacokinetics and pharmacodynamics, resulting in improved bioavailability, a longer half-life, and reduced risk of adverse effects such as heparin-induced thrombocytopenia (HIT). These properties make enoxaparin a preferred choice for many clinical applications, with a broader use as an outpatient medication.

[0014] A 1 D1H NMR analysis is essential for the detailed characterization of enoxaparin structure (Figure 7), including identification of anhydro sugars, which are critical components formed during the depolymerization process of heparin to create enoxaparin. These anhydro sugars possess unique anomeric signals, distinguishable in the NMR spectra, which help to confirm their presence and quantifying their proportion in the mixture. Other anomeric signals provide detailed information on sulfation patterns and the distribution of glucosamine and glucuronic acid residues, which in turn directly influence the anticoagulant activity of enoxaparin.

[0015] Figure 8 illustrates the anticoagulant capacity of enoxaparin, measured through anti-FI la and anti-FXa activities, which are crucial for its pharmacological validation and therapeutic application. Enoxaparin has reduced anti-Flla activity compared to unfractionated heparin (UFH) due to its lower molecular weight and reduced capacity to bridge antithrombin and thrombin, while retaining a strong anti-FXa activity. Thus, enoxaparin has a higher anti- FXa to anti-Flla ratio, favoring the inhibition of Factor Xa over thrombin, contributing to a more predictable anticoagulant effect with a reduced risk of bleeding.

[0016] Enoxaparin combines characteristics related to an easier administration of an anticoagulant therapy when compared to UFH: can be administered via subcutaneous injection once or twice daily, without the need for continuous intravenous infusion or frequent dose adjustments required for UFH; lower or no-monitoring requirements unlike UFH, which requires regular monitoring of the activated partial thromboplastin time (aPTT) to adjust dosing. Due to high safety and efficiency, enoxaparin typically does not require routine laboratory monitoring in most patients. This favorable safety profile, ease of use, and effective anticoagulation make enoxaparin suitable for outpatient management of conditions like deep vein thrombosis (DVT), pulmonary embolism (PE), and prevention of thrombosis in high-risk patients, among others uses. Overall, this enoxaparin characteristics highlights the relevance and importance of new sources for this vital medication.

[0017] Together, these figures and analyses underscore the significant advancements and ongoing innovations in the field of heparin research. They highlight the necessity for continual development in the production and application of heparin derivatives to meet the diverse and evolving needs of medical practice, particularly in enhancing safety and efficacy in anticoagulation therapy with bovine-derived products.

[0018] Therefore, to overcome these challenges, the production process of the present invention employs the use of the quaternary ammonium compound cetylpyridinium chloride (CPC)-mediated purification technique. CPC is a quaternary ammonium compound capable of binding to and precipitate the heparin from aqueous solutions. Extensive research and development associated with the study of heparin's structural peculiarities from different animal origins have led to this innovative use of CPC as a purification process. Although previous works (Demore, B. et al. 1998 - “Determination of heparin in aqueous solutions.” Journal of clinical pharmacy and therapeutics vol. 23,5: 381 -4. doi:10.1046 / j.1365-2710.1998.00177.x) have shown that CPC can bind to heparin, indicating its potential as a precipitation reagent, this is the first time that CPC has been used as a purification method to isolate heparin pools with specific high-anticoagulant structures.

[0019] The innovative approach of the present invention enables precise control over the purification process, allowing to produce heparin and its derivatives with the desired purity, structural and anticoagulant characteristics. This method represents a significant advancement over traditional extraction techniques and can be applied to conventional crude bovine heparin (HBI) to produce SBH and its derivatives.

[0020] Long research developed by our group discovered a process to produce highly active and safe crude bovine heparin (Crude SBH). Adaptations applied to the abovementioned process also allowed the production the following Active Pharmaceutical Ingredients (API): safe unfractionated bovine heparin API (SBH-UFH API) and a novel bovine-derived low-molecular-weight heparin API (B-LMWH API) (Figure 9). The term "Safe Bovine Heparin (SBH)" refers to bovine heparin produced to be similar to porcine heparin, thereby reducing clinical complications that arise from differences in pharmacokinetics and pharmacodynamics between the different drugs. By optimizing these parameters, the process ensures a high-yield heparin production process that meets rigorous pharmaceutical standards, making it a more effective and reliable method for large-scale manufacturing.

[0021] Several studies and patents have unsuccessfully explored various methods for producing bovine heparin and its derivatives with high anticoagulant activities. The patent US704573A (1968) describes a method involving the autolysis of heparin-containing animal tissue followed by the extraction of acidic polysaccharides and their precipitation using CPC. The method further includes fractionation through selective elutions using sodium or potassium chloride solutions and subsequent purification. Additionally, Lindahl (Lindahl, U. (1970) - Biochemical Journal, 116(1 ), 27-34. DOI:

[0022] 10.1042 / bj1160027) outlines a procedure for heparin production from bovine liver using potassium chloride and CPC as precipitation agents. However, none of these attempts address the selective precipitation of heparin chains, nor the production of a fraction with enhanced anticoagulant activity. These data show that CPC have been used before during heparin production merely as a nonspecific precipitation agent. Similarly, the patent US4654327A (1985) focuses on forming complexes between heparin and quaternary ammonium salt ions, including CPC, to enable oral administration of a heparin product. However, it also lacks the method for producing high-anticoagulant activity heparin from bovine sources. The patent W002 / 08295 (2001 ) study involves the use of CPC for complexation with heparin, followed by depolymerization and purification of low-anticoagulant heparin preparations. However, it does not render a high-anticoagulant activity heparin. The patent US20210113607A1 (2021 ) study outlines an enzymatic modification method to increase the 3-O-sulfation on conventional bovine heparin, enhancing its anticoagulant properties. Despite its innovative approach, this method is not economically viable for large-scale production due to excessive costs and not sufficient to produce a bovine heparin similar to the porcine one. Similarly, Linhardt (Linhardt, R.J. (2024) - "Enzymatic Methods for Producing High-Anticoagulant Bovine Heparin," Journal of Thrombosis and Haemostasis) worked on enzymatic methods for producing high-anticoagulant bovine heparin which highlights the potential of bovine heparin sources, but is process limited by excessive costs and economic impracticality for large-scale production.

[0023] Nogueira’s dissertation (Nogueira, A.V. (2013) - "Comparative Study of the Anticoagulant and Antithrombotic Activities of Bovine and Porcine Heparins," PhD Dissertation, University of Sao Paulo) compares the anticoagulant and antithrombotic activities of bovine and porcine heparins of different molecular weights. It involves the use of ultrafiltration for fractionation but does not utilize CPC or address the selective precipitation of a heparin with high anticoagulant activity. While relevant, this study teaches away from the current invention due to the absence of CPC in its methodology and the different focus on ultrafiltration.

[0024] Similar efforts have been made unsuccessfully to produce bovine LMWH comparable to porcine LMWH (enoxaparin). The patents WO2017032276A1 (2015) and CN105237657A (2015) both address the preparation of low- molecular-weight heparin (LMWH) from bovine sources. WO2017032276A1 describes a method for preparing enoxaparin sodium from bovine intestinal mucosa; however, it did not yield a high-quality final product. CN105237657A outlines a method for producing LMWH from new bovine species, emphasizing on the extraction and purification processes to enhance the anticoagulant properties of the heparin produced, but it relies on a complex process that lacks economic viability. Also, CN102585038A describes a method for producing LMWH from bovine lung tissue. However, similar to WO2017032276A1 , this method also faces challenges in achieving economic viability and producing a final product that consistently meets pharmaceutical standards. Thus, none of the previous studies were able to produce a bovine LMWH similar to the porcine one.

[0025] In contrast, the present invention introduces a novel approach by employing cetylpyridinium chloride (CPC) as a key reagent to be used as a scalable purification process and enhancing the overall safety and quality of this anticoagulant. Its ability to selectively remove impurities and low active heparin chains is more effective than traditional methods. Consequently, the final product meets stringent regulatory standards for pharmaceutical products, ensuring superior quality and safety. This innovation process extends to the development of bovine low-molecular-weight heparin (B-LMWH), which is the first to use a cost-effective method while adhering to different pharmacopeial standards. By adapting, the CPC method can be used as a controlled depolymerization technique for the bovine heparin extraction. It is possible to produce a bovine-derived LMWH that mirrors the anticoagulant properties of widely used porcine LMWH, such as enoxaparin. This adaptation further demonstrates the versatility and effectiveness of the CPC precipitation method.

[0026] Regulatory considerations are integral to this development, given the increasing emphasis on the traceability and safety of pharmaceutical ingredients. The clear, controlled, and reproducible nature of the CPC production method aligns well with these regulatory frameworks, facilitating a smoother pathway to market approval.

[0027] In conclusion, the innovative method for producing Crude SBH, SBH- UFH API and B-LMWH API not only meets current market and health needs but also paves the way for future advancements in the field of anticoagulants with an economically viable innovative large scale production process. It opens new opportunities for the development of similar drugs and establishes the technology as a fundamental component for further research and development in biopharmaceutical manufacturing, positioning it as a viable alternative to traditional heparin sources. This development could significantly impact the production diversity of heparin-derived products, reducing reliance on specific raw materials and promoting supply security in the pharmaceutical industry.

[0028] SUMMARY OF THE INVENTION

[0029] This invention pertains to novel methods to produce highly active and safe (1 ) crude bovine heparin (Crude SBH) and active pharmaceutical ingredients (APIs) of (2) unfractionated bovine heparin (SBH-UFH API) and (3) bovine low-molecular-weight-heparin (B-LMWH API) from a conventional bovine crude heparin (HBI), designed to achieve a product similar in quality and function to existing porcine-derived heparins. The method utilizes a new purification process comprising a selective precipitation with quaternary ammonium compounds, such as cetylpyridinium chloride and benzethonium chloride.

[0030] The innovation fundamentally transforms the traditional approaches to produce heparins by incorporating an innovative selective precipitations method which can use cetylpyridinium chloride (CPC), a quaternary ammonium salt compound with effective heparin precipitation capabilities from aqueous solutions. This process enables the selective precipitation of heparin with specific characteristics, allowing for enhanced control over the purity, composition and anticoagulant characteristics of the product. The method is designed to be practical, cost-effective, and economically viable for large-scale manufacturing.

[0031] By addressing the challenges associated with the production of bovine heparin, such as achieving comparable efficacy and safety profiles to porcine heparin, this method stands to significantly diversify the sources of heparin available on the global market. It reduces dependency on porcine sources and mitigates risks associated with supply chain vulnerabilities, such as those highlighted by regional disease outbreaks or geopolitical tensions. The process is particularly significant for markets requiring non-porcine medical products, such as those adhering to Halal laws. This invention not only has the potential to stabilize heparin supply lines globally but also opens new venues for the development of heparin products that are more accessible and acceptable across distinct cultural and dietary spectra.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 - Heparin-antithrombin interaction. Demonstrates how heparin's potent anticoagulant activity results from its ability to inactivate key coagulation enzymes, particularly thrombin (Fl la) and Factor Xa (FXa), by enhancing antithrombin (AT) activity through its interaction with a specific heparin pentasaccharide sequence.

[0034] Figure 2 - One-dimension (1 D)1H nuclear magnetic resonance (NMR) spectra of bovine (HBI-UFH) and porcine (HPI-UFH) unfractionated heparins. Presents the 1 D1H NMR spectra comparing bovine (HBI-UFH) and porcine (HPI-UFH) unfractionated heparins. The spectra identify anomeric protons indicative of structural differences critical for pharmacopeial identification of heparin sources. Signals annotated on the panel correspond to anomeric protons (H1 ) from units of a-GIcNS linked to [3-GlcA (D1 ), a-GlcN,6diS (A1 ) and a-GIcNS (C1 ) linked to a-ldoA2S (11 ) and from the CH3 of the N-acetyl group of a-GIcN (CH3 heparin). Integrals of H1 signals A1 and C1 on the1H NMR spectra of HBI-UFH and HPI-UFH allowed to calculate the ratio between a- GlcN,6diS linked to a-ldoA2S and a-GIcNS linked to a-ldoA2S units, which is key signals that help distinguish between bovine and porcine heparins as required by the Brazilian Pharmacopeia.

[0035] Figure 3 - Disaccharide composition of unfractioned heparin from bovine intestine (HBI-UFH) and heparin from porcine intestine (HPI-UFH). Quantifies the proportions of specific disaccharides within bovine and porcine heparins using 2D1H / 13C HSQC NMR spectra (not shown). This analysis highlights the relative content of disaccharides composing HBI-UFH and HPI-UFH: [a- ldoA2S^a-GlcN,6diS], [a-ldoA2S^a-GlcNS], and the critical disaccharide from the antithrombin-binding pentasaccharide sequence [[3-GlcA^a-GlcN,3,6triS], which is key to heparin’s anticoagulant function.

[0036] Figure 4 - Evaluation of anticoagulant activity of HBI-UFH and HPI-UFH. Assesses the anticoagulant efficacy of both heparins through in vitro enzymatic assays, measuring anti-FI la potency by hydrolyzing chromogenic substrates. The activities are quantified in International Units (IU) per milligram, standardized against the 6th International Heparin Standard from the NIBSC.

[0037] Figure 5 - Protamine neutralization of HBI-UFH and HPI-UFH. Evaluates the effectiveness of protamine in neutralizing the anticoagulant action of HBI- UFH and HPI-UFH. This in vitro assessment involves measuring the residual anti-Flla activity post-exposure to increasing concentrations of protamine, highlighting its clinical relevance in reversing heparin’s effects during medical procedures.

[0038] Figure 6 - Molecular weight distribution of enoxaparin. Presents size exclusion chromatograms comparing enoxaparin (solid line) and the Heparin Sodium Molecular Weight Calibrant from the NIBISC (pointed lines). The chromatograms, analyzed using HPLC with refractive index detection, facilitate the calculation of average molecular weight (MW) and the proportion of components with mass < 2000 Da (M<2000Da), mass 2000-8000 Da (M2000- 8000Da) and mass > 8000 Da (M>8000Da). These parameters are crucial for complying with pharmacopeial standards for enoxaparin in Brazil, Europe, and the United States. Figure 7 - 1 D1H NMR spectrum of the LMWH enoxaparin. Displays the 1 D1H NMR spectrum highlighting structural features of enoxaparin. Annotated signals include H1 (AU1) and H4 (AU4) from unsaturated A4,5UA2S units at nonreducing terminals and H1 from a-GlcN,6diS (A1 ) linked to a-ldoA2S (11 ), along with signals from the CH3 of the N-acetyl group of a-GIcN (CH3 enoxaparin), illustrating the molecular composition and confirmation of structure.

[0039] Figure 8 - In Vitro Anticoagulant Activity of Enoxaparin. Evaluates the anti-FI la and anti-FXa potencies of enoxaparin through in vitro enzymatic assays using specific chromogenic substrates. The anticoagulant activities, expressed in International Units (IU) / mg, are derived using calibration against the 6th International Heparin Standard from the NIBISC, ensuring accurate and reliable potency assessment.

[0040] Figure 9 - Innovative bovine heparins production processes. This figure illustrates the 3 innovative purification processes through CPC purification for producing Crude SBH (1 ), SBH-UFH API (2) and B-LMWH API (3).

[0041] Figure 10 - 1 D1H NMR spectra of Crude SBH. Signals annotated correspond to anomeric protons (H1 ) from units of a-GIcNS linked to [3-GlcA (D1 ), a-GIcN, 6diS (A1 ) and a-GIcNS (C1 ) linked to a-ldoA2S (11 ) and from the CH3 of the N-acetyl group of a-GIcN of both heparin and dermatan sulfate impurity (CH3 heparin+DS). The signals annotated as (*) correspond to contaminations with ethanol from production processes. The panel shows spectra and A1 / C1 ratios of the crude SBHs produced in different conditions (Crude SBH-1 and Crude SBH-2), the conventional crude bovine heparin used as starting material for producing the crude SBHs (Crude HBI), and a typical preparation of crude porcine heparin (Crude HPI) for comparison purposes.

[0042] Figure 11 - Dermatan sulfate impurities of Crude SBH. Presence of dermatan sulfate (DS) in crude SBHs was assessed by galactosamine limit analysis in total hexosamine with high pressure ion chromatography (HPIC) method described in the Monograph of sodium heparin of the United States Pharmacopeia.

[0043] Figure 12 - 1 D1H NMR spectra of SBH-UFH API. Signals annotated on the panel correspond to anomeric protons (H1 ) from units of a-GIcNS linked to p-GIcA (D1 ), a-GlcN,6diS (A1 ), and a-GIcNS (C1) linked to a-ldoA2S (11 ), and from the CH3 of the N-acetyl group of a-GIcN of both heparin and dermatan sulfate impurity (CH3 heparin + DS). The signals annotated as (*) correspond to contaminations with ethanol from production processes. The panel shows spectra and C1 / A1 ratios of the SBH-UFH API, the conventional crude bovine heparin used as starting material for producing SBH-UFH (Crude HPI), porcine UFH (HPI-UFH), and conventional bovine UFH (HBI-UFH) for comparison purposes.

[0044] Figure 13 - Dermatan sulfate impurity of SBH-UFH API. Presence of glycosaminoglycan impurities, especially dermatan sulfate (DS), in SBH-UFH API was assessed by following the ion-exchange chromatography method before and after treatment with nitrous acid required for identification of sodium heparin by the Brazilian Pharmacopeia.

[0045] Figure 14 - Interaction of a pharmaceutical formulation prepared with SBH-UFH API with antithrombin (AT). Dissociation constants (Kd) of the bindings of AT with the SBH-UFH API formulation (SBH-UFH), porcine UFH (HPI-UFH), and conventional bovine UFH (HBI-UFH) were calculated based on the intrinsic enhancement of fluorescence promoted by the titration of AT binding with the heparins.

[0046] Figure 15 - Antithrombotic activity of a pharmaceutical formulation prepared with SBH-UFH API. In vivo assays based on inducing the formation of thrombus in the veins of mice (venous thrombosis) and monitoring their evolution using an intra-vital fluorescence microscope. Veins of animals treated with 1 mg / kg of the SBH-UFH API formulation (SBH-UFH), porcine UFH (HPI- UFH), and conventional bovine UFH (HBI-UFH) did not occlude up to 60 minutes of monitoring. Figure 16 - Neutralization of a pharmaceutical formulation prepared with SBH-UFH with protamine. In vitro neutralization was assessed by measuring the residual anti-FI la activities of the SBH-UFH API formulation (SBH-UFH) and fixed doses of porcine UFH (HPI-UFH) challenged against increasing concentrations of protamine.

[0047] Figure 17 - 1 D1H NMR spectrum of B-LMWH API. Signals annotated on the panel for B-LMWH API, Enoxaparin, and HBI-LMWH spectra correspond to H1 (AU1) and H4 (AU4) from unsaturated A4,5UA2S units at nonreducing terminals, H1 from a-GlcN,6diS (A1 ) linked to a-ldoA2S (11 ), and from the CH3 of the N-acetyl group of a-GIcN (CH3 enoxaparin). For signals annotated on the panel of Crude HBI spectrum see the description for Figure 10.

[0048] Figure 18 - Molecular weight of B-LMWH API. Size exclusion chromatograms of the Heparin Sodium Molecular Weight Calibrant from the NIBSC (dotted lines) and B-LMWH API, Enoxaparin, and HBI-LMWH (solid lines), obtained with a set of analytical columns linked to an HPLC system by monitoring refractive index, used to calculate the parameters of average molecular weights (MW) and proportions of components with mass < 2000 Da (M<2000Da), mass 2000-8000 Da (M2000-8000Da), and mass > 8000 Da (M>8000Da) required for identification of enoxaparin sodium by the Brazilian Pharmacopeia (BP).

[0049] Figure 19 - Interaction of a pharmaceutical formulation prepared with B- LMWH API with antithrombin (AT). Dissociation constants (Kd) of the bindings of AT with B-LMWH API formulation (B-LMWH), Enoxaparin, and HBI-LMWH were calculated based on the intrinsic enhancement of fluorescence promoted by the titration of AT binding with the LMWHs.

[0050] Figure 20 - Antithrombotic activity of a pharmaceutical formulation prepared with B-LMWH API. In vivo assays based on inducing the formation of thrombus in the veins of mice (venous thrombosis) and monitoring their evolution using an intra-vital fluorescence microscope. The panel shows the occlusion time of veins in animals treated with 10 mg / kg of B-LMWH API formulation (B-LMWH), Enoxaparin, and HBI-LMWH over 60 minutes of monitoring.

[0051] Figure 21 - 1 D1H NMR spectra of Crude SBH produced using benzethonium chloride (CBZ). Signals annotated correspond to anomeric protons (H1 ) from units of a-GIcNS linked to [3-GlcA (D1 ), a-GlcN,6diS (A1 ) and a-GIcNS (C1 ) linked to a-ldoA2S (11 ) and from the CH3 of the N-acetyl group of a-GIcN of both heparin and dermatan sulfate impurity (CH3 heparin+DS). The signals annotated as (*) correspond to contaminations with ethanol or CBZ from production processes. The panel shows spectra and A1 / C1 ratios of the crude bovine heparin used as starting material (Crude HBI) and Crude SBHs produced in different conditions, using a fixed amount of CBZ and different concentrations of NaCI: 0.5 M (CBZ + 0.5 M NaCI), 1 .0 M (CBZ + 1 .0 M NaCI) and 1.5 M (CBZ + 1.5 M NaCI).

[0052] DETAILED DESCRIPTION

[0053] This invention describes an innovative process for using conventional crude heparin (Crude HBI) as starting material to produce highly active (1 ) Crude SBH, (2) SBH-UFH API and (3) B-LMWH API, using a selective precipitation method with quaternary ammonium compounds, such as cetylpyridinium chloride [CPC] and benzethonium chloride, to produce high quality products with function similar to porcine-derived heparins (see Figure 9).

[0054] The processes to obtain high-activity bovine heparins involve using conventional crude bovine heparin (Crude HBI) solution, 0.5% to 5.0% CPC solutions, and NaCI concentrations ranging from 0.5 M to 2.0 M. This method enables the selective precipitation of highly active crude heparins, providing enhanced control over the purity, overall structure and anticoagulant characteristics for use as pharmaceutical raw material for producing Active Pharmaceutical Ingredients (API) of both Unfractionated Heparin (UFH) and Low-molecular-weight Heparin (LMWH).

[0055] In an embodiment the present invention discloses (1 ) the process for obtaining crude safe bovine heparin (Crude SBH) from conventional crude bovine heparin (Crude HBI), which has ratio between a-D-glucosamine N- sulfate and a-D-glucosamine N, 6-disulfate units less than 35, comprising the following steps: a) Forming heparin-CPC complex: Add conventional crude bovine heparin (crude HBI) into a solution with 0.5% to 5.0% cetylpyridinium chloride (CPC) and 0.5 M to 2.0 M NaCI, resulting in a turbid supernatant due to the reaction between heparin and CPC. Sodium chloride (NaCI) disrupts less-charged heparin-CPC complexes, selectively precipitating heparin chains with high anticoagulant activity. b) Precipitating heparin-CPC complex: Add 10 to 20% of the reaction volume in ethanol. c) Separating heparin-CPC complex: Supernatant containing non-complexed low-activity heparin chains and non-complexed CPC is separated by centrifugation, membrane filtration and / or siphoning. d) Solubilizing Crude SBH: Resuspend the high-activity heparin-CPC precipitate in 0.5 M to 3.0 M NaCI. e) Precipitating Crude SBH: Add one to 3 volumes of absolute ethanol and separate the precipitate by centrifugation, membrane filtration and / or siphoning. f) Washing Crude SBH: Wash the precipitate twice with 80% ethanol. g) Preserving Crude SBH: Drying the washed precipitate at 50° C to 100 ° C in drying oven and mechanically triturating the Crude SBH.

[0056] In another embodiment the present invention discloses (2) a method for producing Active Pharmaceutical Ingredient of highly active safe bovine unfractionated heparin (SBH-UFH API) from conventional crude bovine heparin (Crude HBI), which has ratio between a-D-glucosamine N-sulfate and a-D- glucosamine N, 6-disulfate units equal or less than 35 and achieves anticoagulant activity equal to or greater than 160 lU / mg in anti-Flla inactivation assays, for preparing parenteral (intravenous or subcutaneous) pharmaceutical compositions containing 1 ,000 to 20,000 heparin lUs API and water for injectables or other pharmaceutically acceptable vehicles, excipients, or carriers, comprising the following steps: a) Viral inactivating: Subjecting conventional crude bovine heparin (Crude HBI) to the method as described above to viral inactivation by varying pH from 12 to 3 with NaOH and HCI, respectively, at 40° to 60° C temperature, followed by membrane filtration with vacuum filtration pump; b) Bleaching: Bleaching by adding 10% to 20% KMnC (w / w), at 20° to 80° C temperature, followed by celite filtration with vacuum filtration pump; c) Purifying: Precipitating SBH-UFH API by adding 5% to 10% NaCI (w / v), adjusting pH from 6 to 7 with and then adding 40% to 80% (v / v) ethanol, followed by membrane filtration with vacuum filtration pump; d) Preserving: Drying at 50° C to 100 ° C in drying oven, and mechanically triturating SBH-UFH API.

[0057] In a further embodiment the present invention discloses (3) a method for producing Active Pharmaceutical Ingredient of Bovine Low-molecular-weight Heparin (B-LMWH API) from conventional crude bovine heparin (crude HBI), which achieves anticoagulant activity greater than 15 and less than 40 lU / mg in anti-Flla and greater than 80 and less than 130 lU / mg in anti-FXa inactivation assays, for preparing parenteral (subcutaneous) pharmaceutical compositions containing 10 to 100 mg API and water for injectables or other pharmaceutically acceptable vehicles, excipients, or carriers, comprising the following steps: a) Precipitating: Subjecting conventional crude bovine heparin (crude HBI) to the method as described above, and precipitating by adding benzethonium chloride 1 :3 to 4 (w / w) at pH from 6 to 7 and 40°C to 80°C temperature; b) Solubilizing: Solubilizing the benzetonium heparinate in dimethylformamide (DMF) at 35°C to 50°C temperature and then adding benzyl chloride 1 .0:0.5 to 2.0 (g / mL); c) Forming: Precipitating the benzyl ester heparinate at 1 :1 to 2 ratios (v / v) by adding sodium acetate in methanol 10% (v / v); d) Depolymerizing: Solubilizing the benzyl ester heparinate in water at 40°C to 60°C temperature and then adding NaOH to reach a final concentration of 0.05 to 0.15 M and incubating for 60 to 120 minutes; e) Purifying: Neutralizing the solution to pH from 7.0, adding from 10% to 20% NaCI (w / v) and precipitating the B-LMWH API by adding 2 to 3 volumes of ethanol (v / v) and then separating precipitated B-LMWH API by centrifugation, membrane filtration and / or siphoning; f) Preserving: Drying at 60° C to 80 ° C in drying oven, and mechanically triturating B-LMWH API.

[0058] Next, the present invention will be described in detail through examples. It is necessary to emphasize that the invention is not limited to this example, but that it also includes changes and modifications within the limits in which it operates.

[0059] EXAMPLES

[0060] The following examples illustrate the innovative processes developed for the production of heparin and its derivatives from an intestinal mucosa bovine source, focusing on achieving high purity and potent anticoagulant activity. These processes utilize a novel cetylpyridinium chloride (CPC) purification step, which is a critical innovation step, enabling enhanced control over the product's structural and functional characteristics. This step can be applied at various stages of production to produce different forms of heparin derivatives, including Crude SBH, SBH-UFH API and B-LMWH API.

[0061] In Figure 9 is demonstrated the overall process flow, highlighting the processes (1 , 2 and 3) including the innovative CPC-mediated purification step that it was adapted to function at different stages of the process of heparin production to render distinct products. The versatility and effectiveness of this low-cost innovative purification technique can be applied independently at the non-GLP (Good Laboratory Practices) and non-GMP (Good Manufacturing Practices) production of crude SBH (Process 1 ) and at the GLP and GMP production of SBH-UFH API (Process 2) or B-LMWH API (Process 3), which consists in three distinct production lines from independent production plants. By integrating this step, the process not only enhances the purification but also optimizes the yield and efficacy of the heparin derivatives. Furthermore, this invention introduces a direct method for producing B-LMWH API using benzethonium chloride (CBZ), a reagent already employed in enoxaparin production. This innovation is highly relevant as it streamlines the production process, minimizes changes to the existing production line, and significantly enhances manufacturing feasibility.

[0062] In the subsequent examples, detailed descriptions of each step in the production and analysis of these novel bovine heparin intermediates are provided. These examples emphasize the practical application and benefits of the quaternary ammonium compounds purification method, demonstrating its significant impact on producing high-quality heparin products suitable for various pharmaceutical applications.

[0063] Example 1 : Production of Crude SBH

[0064] To produce crude bovine heparin with enhanced activity (Crude SBH), 1 gram of conventional crude bovine heparin (Crude HBI) produced from bovine intestinal mucosa was precisely weighed and dissolved in a 10 mL volumetric flask with water, achieving a 100 mg / mL solution. This solution was then homogenized with 20 mL of a 0.5 to 2.0 M NaCI solution. Following this, a 10 mL of a 5% to 15% CPC solution was carefully added and homogenized. Ethanol was introduced into the solution until the concentration reached 10% to 20% of the initial volume, followed by centrifugation at 3200 rpm for 12 minutes to isolate the precipitate of interest.

[0065] The precipitate was then treated with 20 mL of a 0.5 to 3.0 M NaCI, agitated for 30 minutes until fully dissolved, followed by the addition of 20 to 60 mL of ethanol and submitted to another round of centrifugation at 3200 rpm for 10 minutes. The precipitate underwent two washes with 20 mL of 80% ethanol, with centrifugation at 3200 rpm after each wash. After drying, approximately 400-500 mg of two Crude SBHs were obtained. To characterize the structural properties of Crude SBH, Figure 10 presents the 1 D1H NMR spectra, revealing signals corresponding to anomeric protons from a-GIcNS linked to [3-GlcA, a-GIcN, 6diS, and a-GIcNS linked to a- ldoA2S, as well as signals from the CH3 of the N-acetyl group of a-GIcN and dermatan sulfate impurity. The Crude SBH samples displayed a C1 / A1 *100 signal ratio in 1 D1H NMR of 25 and 19, in contrast to the approximately 50 C1 / A1 ratio before the purification process. These spectra and the A1 / C1 ratios of crude SBHs produced under different conditions were comparable with that of crude porcine heparin.

[0066] The physical characteristics and purity of Crude SBH were further examined and displayed in table 1 , which highlights non-compendial requirements such as the absence of protein and nucleotide impurities, commonly seen in certificates of analysis (COA) of crude heparins (in italic) of the crude SBHs produced in different conditions (named Crude SBH-1 and Crude SBH-2) and the conventional crude bovine heparin used as starting material for crude SBHs (Crude HBI) production. This comparison of Crude SBHs produced under different conditions with conventional crude bovine heparin demonstrated the improved purity of our product. Table 1. Physical characteristics and purity of crude SBH.

[0067]

[0068] Dermatan sulfate impurities, which can affect the efficacy and safety of heparin, were analyzed using high-pressure ion chromatography (HPIC) as described in the monograph of sodium heparin of the United States Pharmacopeia. Figure 11 shows the results of this analysis, confirming that Crude SBHs of the present invention meets stringent impurity standards.

[0069] Anticoagulant activity is a critical parameter for heparin efficacy, anticipating the activity of future purified API material. Table 2 shows the anticoagulant activity of Crude SBHs using the activated partial thromboplastin time assay (APTT) with ovine plasma, which is universally employed to determine potency of crude heparins, and which is known to overestimate the potency of bovine heparins. Both conventional APTT and Anti-Fl la assays were employed to measure the anticoagulant activity of Crude SBHs produced under different conditions (Crude SBH-1 and Crude SBH-2), comparing them with conventional crude bovine heparin used as starting material for producing the crude SBHs (Crude HBI).

[0070] Table 2. Anticoagulant activity of crude SBH.

[0071] To assess the efficiency of the crude SBHs production process, Table 3 illustrates the mass, hexuronic acid content, and anticoagulant activity yields of the production processes of crude SBH-1 and crude SBH-2 relative to conventional crude bovine heparin used as starting material (crude HBI). This data demonstrates the yield and effectiveness of the present innovative production method.

[0072] Table 3. Yield of Crude SBH producing process.

[0073] The production and analysis of crude bovine heparin using the present invention’s innovative CPC-based purification process demonstrated significant improvements in purity, structural characteristics, and anticoagulant activity of crude SBH. The method effectively addresses the challenges associated with producing high-quality bovine heparin, providing a viable alternative to traditional porcine-derived heparins. This process not only enhances the yield and efficacy of crude heparin but also ensures compliance with stringent pharmaceutical standards, making it a promising solution for the heparin market.

[0074] Example 2: Production of SBH-UFH API

[0075] Here, conventional bovine crude heparin (crude HBI) was used to produce an intermediate Active Pharmaceutical Ingredient (API) safe bovine heparin (SBH-UFH API). As an initial step, the crude HBI was complexed with CPC, precipitated in the presence of NaCI, precipitated and washed with ethanol, and then dried and preserved as Crude SBH.

[0076] A 10 mL solution of 100 mg / mL Crude SBH was prepared at a temperature of 50°C with constant stirring. Viral inactivation was performed by adjusting the pH to 12 with NaOH for 4 hours at 50°C under stirring, then adjusting the pH to 3 with 37% HCI for 4 hours at 50°C under stirring, followed by membrane filtration. Bleaching was carried out by adding 4 mL of a 40 mg / mL KMnO4 solution at 70°C for 2 hours and then for 15 hours at 25°C with constant stirring. Filtration with celite was performed at 50-55°C followed by membrane filtration using a Buchner funnel. After filtration, NaCI was added in a proportion of 5% w / v, and then the pH was adjusted to 6-7 with 37% HCI for the selective precipitation step. Next, 60% v / v ethanol was added, and the solution was kept under stirring for 30 minutes and then left to stand for 8 hours at 4°C. The precipitate containing SBH-UFH API was then filtered with a membrane and dried for 24 hours at 55°C. This critical step effectively removed impurities and achieved the desired molecular weight distribution, ensuring the product met the necessary pharmaceutical standards.

[0077] The physical-chemical characteristics and purity of the resulting SBH- UFH API were meticulously analyzed to confirm its suitability for pharmaceutical use. Structural analyses with 1 D1H NMR spectra (Figure 12) revealed that SBH-UFH API is highly pure and has a composition similar to porcine UFH (HPI-UFH), remarkably higher a-GIcN, 6diS linked to a-ldoA2S and reduced a- GIcNS linked to a-ldoA2S unities, when compared to conventional bovine heparin (HBI). Thus, the SBH-UFH API showed a C1 / A1 ratio within the compendial specifications for porcine UFH.

[0078] As shown in Table 4, the SBH-UFH API adhered to the compendial requirements for physical characteristics and purity, including the absence of protein and nucleotide impurities and bacterial endotoxins, as specified by the Brazilian Pharmacopeia.

[0079] Table 4. Physical characteristics and purity of SBH-UFH API A vital aspect of ensuring product purity involved assessing glycosaminoglycan impurities, particularly dermatan sulfate (DS). Figure 13 demonstrates the minimal presence of DS impurities in SBH-UFH API, as assessed by ion-exchange chromatography before and after nitrous acid treatment. This analysis, required for the identification of sodium heparin by the Brazilian Pharmacopeia, confirmed the high purity of SBH-UFH.

[0080] Next, the anticoagulant activity of SBH-UFH API was rigorously evaluated. Table 5 highlights the anti-FI la and anti-FXa potencies of SBH-UFH in lU / mg, determined through parallel line assays using the 6th International Heparin Standard from the NIBSC. These values were used to calculate the anti-FI la / anti-FXa ratio, essential for identifying sodium heparin by the Brazilian Pharmacopeia.

[0081] Average anti-Flla and anti-FXa potencies and anti-Flla / anti-FXa ratios of porcine UFH (HPI-UFH) and conventional bovine UFH (HBI-UFH) were included for comparison purposes. The results indicated that SBH-UFH API exhibited comparable anticoagulant activity to porcine UFH (HPI-UFH) API, different from the conventional bovine UFH (HBI-UFH), validating its therapeutic potential.

[0082] Table 5. Anticoagulant activity of SBH-UFH API. Understanding the interaction between heparin and antithrombin (AT) is crucial for assess anticoagulant function. For this purpose, a pharmaceutical formulation was prepared using SBH-UFH API by dissolving it in water for injections at a dose of 5,000 International Units of heparin (lUs) in sterile conditions. Figure 14 details the dissociation constants (Kd) of the bindings of AT with the SBH-UFH formulation (SBH-UFH), porcine UFH (HPI-UFH), and conventional bovine UFH (HBI-UFH). These constants, calculated based on the intrinsic enhancement of fluorescence promoted by the titration of AT binding with the heparins, underscored a strong interaction of SBH-UFH with AT, essential for its efficacy.

[0083] The antithrombotic activity of the SBH-UFH formulation was then assessed through in vivo assays, where thrombus formation was induced in the veins of mice and monitored using an intra-vital fluorescence microscope. Figure 15 illustrates that veins of animals treated with 1 mg / kg of SBH-UFH formulation (SBH-UFH), porcine UFH (HPI-UFH), and conventional bovine UFH (HBI-UFH) did not occlude within 60 minutes, demonstrating the effective antithrombotic properties of SBH-UFH.

[0084] Additionally, the ability of protamine to neutralize the anticoagulant activity of the SBH-UFH formulation was evaluated. Figure 16 shows the in vitro neutralization results, measuring residual anti-FI la activities after exposing the SBH-UFH formulation to increasing concentrations of protamine. This data indicated that protamine could effectively neutralize the SBH-UFH formulation similarly to porcine UFH (HPI-UFH), ensuring its safety in clinical settings.

[0085] Finally, the yield of the SBH-UFH API production process was analyzed, focusing on mass, hexuronic acid content, and anticoagulant activity (anti-FI la). Table 6 presents these yields relative to the conventional crude bovine heparin used as the starting material (Crude HBI). The data demonstrated the efficiency and high yield of the SBH-UFH production process. Table 6. Yield of SBH-UFH API production process

[0086]

[0087] In conclusion, the production of the API of SBH-UFH through this innovative process yielded a high-purity, high-potency formulated heparin that meets rigorous pharmaceutical standards. The comprehensive analyses confirmed that SBH-UFH API is a viable intermediate product for producing enhanced bovine derived anticoagulant medicines for treatment of thromboembolic diseases and use in surgical and non-surgical procedures requiring extracorporeal circulation, showcasing its potential to meet the stringent requirements of the medical field.

[0088] Example 3: Production of B-LMWH API

[0089] Conventional bovine crude heparin (crude HBI) was used to produce an intermediate Active Pharmaceutical Ingredient (API) of a bovine Low Molecular Weight Heparin (B-LMWH API), who can be used for treatment and prophylaxis of thromboembolic diseases, including myocardial infarction, pulmonary embolism, stroke and venous thrombosis, in a hospital or outpatient setting. As an initial step, the crude HBI was complexed with CPC, precipitated in the presence of NaCI, washed with ethanol and then dried and preserved as Crude SBH. Following, the next steps of B-LMWH API production included the viral inactivation, bleaching, purification and preservation, as described in Example 2.

[0090] Further, the following steps involves using the fragmentation conditions described above to break down the heparin molecules into lower molecular weight fragments while maintaining their anticoagulant properties. First, A 5 mL solution of 200 mg / mL purified crude SBH was prepared and a precipitation procedure was used by adding 8.75 mL benzethonium chloride solution 1 :3 to 4 (w / w), at pH 7 and 60°C temperature, and then centrifuging the tubes at 3200 rpm for 10 minutes. After, 1.2 g of benzethonium heparinate was solubilized in dimethylformamide (DMF) and then added to 2.4 mL of benzyl chloride and incubated for 24 hours at 46° C. Benzyl ester heparinate was formed by adding one volume of 10% sodium acetate in methanol and incubating for 2 hours at room temperature and then separated by centrifuging the tubes at 3200 rpm for 10 minutes. Following, 800 mg of benzyl ester heparinate were dissolved in 16 mL water and 1 mL of 1 .4 M NaOH solution added, incubated for 90 minutes at 50° C temperature and then neutralized to a pH of 7.0. Afterwards, 1 .7 g NaCI and 34 mL ethanol were added, incubated for 12 hours, and then centrifuged at 3200 rpm for 10 minutes. Precipitated B- LMWH API was washed twice with 20 mL of 80% ethanol, with centrifugation at 3200 rpm after each wash, dried at 60°C temperature and triturated.

[0091] The physical-chemical characteristics and purity of B-LMWH API are critical for its pharmaceutical application. 1 D1H NMR spectra (Figure 17) of B- LMWH API and enoxaparin presents coincident signals for fingerprinting units from unsaturated A4,5UA2S at nonreducing terminals, a-GIcN, 6diS linked to a- ldoA2S and the CH3 of the N-acetyl group, confirming its purity and structural equivalence.

[0092] Table 7 presents compendial requirements for physical-chemical characteristics and purity, including the absence of protein and nucleotide impurities and bacterial endotoxins, as specified by the Brazilian Pharmacopeia for sodium enoxaparin. The data demonstrates that B-LMWH API meets these stringent standards, ensuring its suitability for pharmaceutical use. Table 7. Physical characteristics and purity of B-LMWH API

[0093]

[0094] The molecular weight distribution of B-LMWH API is another important parameter, as it influences the drug's pharmacokinetics and pharmacodynamics. Figure 18 shows the size exclusion chromatograms of B- LMWH API, enoxaparin, and HBI-LMWH compared to the Heparin Sodium Molecular Weight Calibrant from the NIBSC. These chromatograms, obtained using HPLC and monitoring the refractive index, allow for the calculation of average molecular weights (MW) and the proportions of components with mass < 2000 Da (M<2000Da), mass 2000-8000 Da (M2000-8000Da), and mass > 8000 Da (M>8000Da), as required for the identification of sodium enoxaparin by the Brazilian Pharmacopeia. This analysis confirms that, different than HBI- LMWH, B-LMWH API has a molecular weight profile similar to that of enoxaparin, indicating its potential efficacy and safety.

[0095] Table 8 evaluates the anticoagulant activity of B-LMWH API by measuring its anti-FI la and anti-FXa potencies in lU / mg, calculated based on parallel line assays performed with the 6th International Heparin Standard from the NIBSC. These values are used to calculate the anti-FXa / anti-Flla ratio, which is also required for the identification of sodium enoxaparin by the Brazilian Pharmacopeia (BP). The data includes average anti-FI la and anti-FXa potencies and anti-FI la / anti-FXa ratios for comparison with enoxaparin and HBI- LMWH, demonstrating that B-LMWH API possesses comparable anticoagulant properties to enoxaparin. On the contrary, HBI-LMWH shows lower anti-FI la and anti-FXa, demonstrating the importance of the present innovation for the production of a Bovine LMWH similar to the porcine one.

[0096] Table 8. Anticoagulant activity of B-LMWH API The interaction of LMWH with antithrombin (AT) is crucial for assess its anticoagulant function. For this purpose, a pharmaceutical formulation was prepared using B-LMWH API by dissolving it in water for injections at a dose of 40 mg in sterile conditions. Figure 19 details the dissociation constants (Kd) of the bindings of AT with B-LMWH formulation (B-LMWH), enoxaparin, and HBI- LMWH, calculated based on the intrinsic enhancement of fluorescence promoted by the titration of AT binding with the LMWHs. This analysis indicates a strong interaction of the B-LMWH formulation with AT, essential for its anticoagulant activity.

[0097] The antithrombotic activity of B-LMWH formulation is assessed through in vivo assays, where thrombus formation is induced in the veins of mice (venous thrombosis) and monitored using an intra-vital fluorescence microscope. Figure 20 shows the occlusion time of veins in animals treated with 10 mg / kg of B-LMWH formulation (B-LMWH), enoxaparin, and HBI-LMWH over 60 minutes of monitoring, demonstrating the antithrombotic efficacy of B- LMWH.

[0098] Finally, the yield of the B-LMWH API production process is analyzed in terms of mass, hexuronic acid content, and anticoagulant activity (anti-FI la). Table 9 presents these yields relative to the conventional crude bovine heparin (Crude HBI) used as the starting material. This data demonstrates the efficiency and high yield of the B-LMWH production process. Table 9. Yield of B-LMWH API production process.

[0099] The production of B-LMWH API through this innovative process yields a high-purity, high-potency heparin derivative that meets rigorous pharmaceutical standards. The comprehensive physical, chemical, and biological analyses confirm that B-LMWH API is suitable for preparing a bovine LMWH medicine comparable to enoxaparin in terms of efficacy and safety, making it a viable alternative for pharmaceutical use.

[0100] Example 4: Production of B-LMWH API with a different quaternary ammonium compound

[0101]

[0001] Conventional bovine crude heparin (crude HBI) was used to produce Crude SBH using a quaternary ammonium compound other than cetylpyridinium chloride (CPC), as this is identified as a key production step. Thus, the production of Crude SBH with benzethonium chloride (CBZ) as a quaternary ammonium compound relies on selecting highly active unfractionated bovine heparin. This step is crucial in ensuring the production of high-quality B-LMWH, as it significantly influences the consistency and effectiveness of the final product. Therefore, different conditions for using CBZ were tested to identify the heparin chains required for the production process.

[0102] This process involves using a specific ratio of heparin to CBZ, typically at 1 :1 to 4 (v / v) in solutions of 100 mg / mL and 200 mg / mL, respectively. The solution is prepared by combining conventional crude bovine heparin (Crude HBI) and different NaCI concentrations (Figure 21 ), followed by the addition of CBZ. This sequence ensures proper interaction and precipitation of the heparin- CBZ complex. Upon precipitation, the mixture is centrifuged to separate the highly active heparin. Once separated, this highly active heparin chains were dissolved with a 2M NaCI solution and reprecipitated using two volumes of absolute ethanol. The resultant precipitate was then washed twice with 80% ethanol to ensure purity. The different fraction with various NaCI concentrations were dried at 60° C and triturated.

[0103] To characterize the structural properties of Crude SBH produced with CBZ, Figure 21 presents the 1 D1H NMR spectra. The Crude SBH samples produced with fixed amount of CBZ and 0.5 M, 1 .0 M and 1 .5 M NaCI concentrations displayed a C1 / A1 ratio 39.5, 32.1 and 19.4, respectively, and the Crude HBI used as start material 51 .4, confirming that CBZ can be used to produce Crude SBH.

[0104] The prolongation of the process from Example 3 after using this CBZ as the precipitating method yield Crude SBH with quality to ensure production of B- LMWH, similar to the porcine enoxaparin. The efficacy of various quaternary ammonium compounds, including CBZ, which is commonly used in the production of enoxaparin-like LMWH, is demonstrated.

[0105] This approach greatly expands the manufacturing possibilities for producing high-quality B-LMWH, while minimizing risks and reducing the need for equipment and technical adaptations. This significant innovation alters specific and unique conditions to produce B-LMWH similar to porcine enoxaparin, offering substantial technical advantages for the industrial production of this new medication.

Claims

CLAIMS1. Method for producing crude safe bovine heparin (Crude SBH) from conventional crude bovine heparin (crude HBI) CHARACTERIZED by using quaternary ammonium compounds, such as cetylpyridinium chloride (CPC) or benzethonium chloride (CBZ).

2. Method, in accordance with the claim 1 , CHARACTERIZED by the quaternary ammonium compound is preferably cetylpyridinium chloride (CPC), and comprising the following steps: a. precipitating conventional crude bovine heparin (HBI) with 0.5% to 5.0% cetylpyridinium chloride (CPC), 0.5 M to 2.0 M NaCI and 10 to 20% (v / v) ethanol solutions to form a heparin-CPC complex; b. separating the insoluble heparin-CPC complex from the aqueous solution by centrifugation, membrane filtration and / or siphoning; c. dissociating CPC from the heparin-CPC complex with 0.5 to 3.0 M NaCI solutions to recover soluble Crude SBH; d. precipitating soluble Crude SBH by adding one to three volumes of absolute ethanol; e. washing Crude SBH with 80% ethanol; and f. drying, at 60° C in drying oven, and mechanically triturating Crude SBH.

3. Method for producing Active Pharmaceutical Ingredient of safe bovine unfractionated heparin (SBH-UFH API) from conventional crude bovine heparin (crude HBI), CHARACTERIZED by comprising the following steps: a. subjecting conventional crude bovine heparin (crude HBI) to the method as described in claim 1 , to viral inactivation by varying pH from 12 to 3 with NaOH and HCI, respectively, at 40° to 60° C temperature, followed by membrane filtration with vacuum filtration pump; b. Bleaching the crude SBH by adding 10% to 20% KMnCU (w / w), at 20° to 80° C temperature, followed by celite filtration with vacuum filtration pump;c. precipitating SBH-UFH API, by adding 5% to 10% NaCI (w / v), adjusting pH from 6 to 7 with and then adding 40% to 80% (v / v) ethanol, followed by membrane filtration with vacuum filtration pump; and d. drying, at 50° to 100° C in drying oven, and mechanically triturating SBH-UFH API.

4. Method for producing Active Pharmaceutical Ingredient of Bovine Low Molecular Weight Heparin (B-LMWH API) from conventional crude bovine heparin (crude HBI), CHARACTERIZED by comprising the following steps: a. Subjecting conventional crude bovine heparin (crude HBI) to the method as described in claim 2, and precipitating by adding benzethonium chloride 1 :3 to 4 (w / w) at pH from 6 to 7 and 40°C to 80°C temperature; b. Solubilizing the benzetonium heparinate in dimethylformamide (DMF) at 35°C to 45°C temperature and then adding benzyl chloride 1 .0:0.5 to 2.0 (g / mL); c. Precipitating the benzyl ester heparinate at 1 : 1 to 2 ratio (v / v) by adding sodium acetate in methanol 10% (v / v); d. Solubilizing the benzyl ester heparinate in water at 40°C to 60°C temperature and then adding NaOH to reach 0.05 to 0.15 M final concentration and incubating for 60 to 120 minutes; e. Neutralizing the solution to pH 7.0 by adding HCI, adding NaCI to a final concentration of 10 to 20% (w / v) and precipitating the B-LMWH API by adding 2 to 3 volumes of ethanol (v / v); f. Separating precipitated B-LMWH API by centrifugation, membrane filtration and / or siphoning; and g. drying, at 60 to 80sC in drying oven, and mechanically triturating B- LMWH API.

5. Safe crude bovine heparin (crude SBH), produced by the method defined in claim 1 , CHARACTERIZED by the fact that has ratio between a-D- glucosamine N-sulfate and a-D-glucosamine N, 6-disulfate units less than 35.

6. Active Pharmaceutical Ingredient of safe bovine unfractionated heparin (SBH-UFH API), produced by the method defined in claim 2, CHARACTERIZED by the fact that has ratio between a-D-glucosamine N- sulfate and a-D-glucosamine N, 6-disulfate units less than 35 and anticoagulant activity equal to or greater than 160 International Units of heparin (lUs) per mg of heparin in antithrombin-mediated activated thrombin inactivation assays.

7. Active Pharmaceutical Ingredient of Bovine Low Molecular Weight Heparin (B-LMWH API) produced by the method defined in claim 3, CHARACTERIZED by the fact that it achieves anticoagulant activity greater than 15 and less than 40 lUs per mg in antithrombin-mediated activated thrombin inactivation assays and greater than 80 and less than 130 lUs per mg in antithrombin-mediated activated factor Xa inactivation assays.

8. Use of safe crude bovine heparin (crude SBH), according to claim 4, CHARACTERIZED by the fact that it is pharmaceutical raw material for producing Active Pharmaceutical Ingredients (API) of both Unfractionated Heparin (UFH) and Low Molecular Weight Heparin (LMWH).

9. Use of Active Pharmaceutical Ingredient of safe bovine unfractionated heparin (SBH-UFH API), as defined in claim 5, CHARACTERIZED by the fact that it is for preparing anticoagulant medicine for treatment of thromboembolic diseases.

10. Use of Active Pharmaceutical Ingredient of Bovine Low Molecular Heparin (B-LMWH API), as defined in claim 6 CHARACTERIZED by the fact that it is for preparing anticoagulant medicine for treatment and prophylaxis of thromboembolic diseases, including myocardial infarction, pulmonary embolism, stroke and venous thrombosis, in a hospital or outpatient setting.

11. Pharmaceutical composition comprising SBH-UFH API, as defined in claim 5, and water for injectables or other pharmaceutically acceptable vehicle, excipient, or carrier, with an amount of 1 ,000 to 20,000 International Units of heparin (lUs), wherein the parenteral via is intravenous or subcutaneous.

12. Pharmaceutical composition comprising B-LMWH API, as defined in claim 6, water for injectables or other pharmaceutically acceptable vehicle, excipient, or carrier, with an amount of 10 to 100 mg, wherein the parenteral via is subcutaneous.

Images