Preparation and evaluation a new generation of low molecular weight heparin.

Enoxaparin is widely used in clinic, but it has some disadvantages. For example, its anticoagulant activity is weaker compared with heparin and it can not be effectively neutralizad by protamine sulfate (PS) in case of bleeding. Therefore, in this work, a new generation of low molecular weight heparin (NG-LMWH) was prepared.The NG-LMWH was prepared with the method of alkaline ß-elimination followed by gel chromatography. Estimating the molecular weight of the NG-LMWH by GPC-HPLC, it has a remarkably low polydispersity index and narrow molecular weight distribution. The polydispersity index of NG-LMWH was 1.052, which was lower than heparin (1.5) and enoxaparin (1.279). Anti-FXa and anti-FIIa potency of NG-LMWH was much higher than that of Enoxaparin, and close to that of heparin, which was determined by chromogenic substrate method. To test the degree of anti-FXa or anti-FIIa potency neutralized by PS, equivalent anti-FXa or anti-FIIa activity doses of different anticoagulant in plasma were titrated with increasing amounts of PS in plasma. The results indicate that NG-LMWH was more efficiently neutralized by PS than enoxaparin.The efficacy of anti-thrombus of NG-LMWH was superior to enoxaparin and the effect was dose dependent, which was evaluated with rat carotid artery thrombosis and inferior vena cava thrombosis model. The results of pharmacokinetics in New Zealand rabbits showed that the pharmacokinetic characteristics of NG-LMWH were similar to enoxaparin. The NG-LMWH prepared in this work has both advantages of heparin and enoxaparin with more effective and safer anticoagulation than enoxaparin.

The design and synthesis of new synthetic low-molecular-weight heparins.

Low-molecular-weight heparin (LMWH) has remained the most favorable form of heparin in clinics since the 1990s owing to its predictable pharmacokinetic properties. However, LMWH is mainly eliminated through the kidney, which limits its use in renal-impaired patients. In addition, the anticoagulant activity of LMWH is only partially neutralized by protamine. LMWH is obtained from a full-length, highly sulfated polysaccharide harvested from porcine mucosal tissue. The depolymerization involved in LMWH production generates a broad distribution of LMWH fragments (6-22 sugar residues). This, combined with the various methods used to produce commercial LMWHs, results in variable pharmacological and pharmacokinetic properties. An alternative chemoenzymatic approach offers a method for the synthesis of LMWH that has the potential to overcome the limitations of current LMWHs. This review summarizes the application of a chemoenzymatic approach to generate LMWH and the rationale for development of a synthetic LMWH.

Preparation and optimization of N-trimethyl-O-carboxymethyl chitosan nanoparticles for delivery of low-molecular-weight heparin.

The aim of this study was preparation, optimization and in vitro characterization of nanoparticles composed of 6-[O-carboxymethyl]-[N,N,N-trimethyl] (TMCMC) for oral delivery of low-molecular-weight heparin. The chitosan derivative was synthesized. Nanoparticles were prepared using the polyelectrolyte complexation method. Box-Behnken response surface experimental design methodology was used for optimization of nanoparticles. The morphology of nanoparticles was studied using transmission electron microscopy. In vitro release of enoxaparin from nanoparticles was determined under simulated intestinal fluid. The cytotoxicity of nanoparticles on a Caco-2 cell line was determined, and finally the transport of prepared nanoparticles across Caco-2 cell monolayer was defined. Optimized nanoparticles with proper physico-chemical properties were obtained. The size, zeta potential, poly-dispersity index, entrapment efficiency and loading efficiency of nanoparticles were reported as 235 ± 24.3 nm, +18.6 ± 2.57 mV, 0.230 ± 0.03, 76.4 ± 5.43% and 12.6 ± 1.37%, respectively. Morphological studies revealed spherical nanoparticles with no sign of aggregation. In vitro release studies demonstrated that 93.6 ± 1.17% of enoxaparin released from nanoparticles after 600 min of incubation. MTT cell cytotoxicity studies showed no cytotoxicity at 3 h post-incubation, while the study demonstrated concentration-dependent cytotoxicity after 24 h of exposure. The obtained data had shown that the nanoparticles prepared from trimethylcarboxymethyl chitosan may be considered as a good candidate for oral delivery of enoxaparin.

A latent reactive handle for functionalising heparin-like and LMWH deca- and dodecasaccharides.

d-Glucosamine derivatives bearing latent O4 functionality provide modified H/HS-type disaccharide donors for a final stage capping approach enabling introduction of conjugation-suitable, non-reducing terminal functionality to biologically important glycosaminoglycan oligosaccharides. Application to the synthesis of the first O4-terminus modified synthetic LMWH decasaccharide and an HS-like dodecasaccharide is reported.

Preparation and Characterization of Low Molecular Weight Heparin by Liquid Phase Plasma Method.

An liquid phase plasma process system was applied to the production of low molecular weight heparin. The molecular weight of produed heparin decreased with increasing liquid phase plasma treatment time. The abscission of the chemical bonds between the constituents of heparin by liquid phase plasma reaction did not alter the characteristics of heparin. Formation of any by-products due to side reaction was not observed. It is suggested that heparin was depolymerized by active oxygen radicals produced during the liquid phase plasma reaction.

Isolation of low-molecular-weight heparin/heparan sulfate from marine sources.

The glycosaminoglycan (heparin and heparan sulfate) are polyanionic sulfated polysaccharides mostly recognized for its anticoagulant activity. In many countries, low-molecular-weight heparins have replaced the unfractionated heparin, owing to its high bioavailability, half-life, and less adverse effect. The low-molecular-weight heparins differ in mode of preparation (chemical or enzymatic synthesis and chromatography fractionations) and as a consequence in molecular weight distribution, chemical structure, and pharmacological activities. Bovine and porcine body parts are at present used for manufacturing of commercial heparins, and the appearance of mad cow disease and Creutzfeldt-Jakob disease in humans has limited the use of bovine heparin. Consequently, marine organisms come across the new resource for the production of low-molecular-weight heparin and heparan sulfate. The importance of this chapter suggests that the low-molecular-weight heparin and heparan sulfate from marine species could be alternative sources for commercial heparin.

Proteoglycans and glycosaminoglycans in misfolded proteins formation in Alzheimer's disease.

Misfolded protein amyloid-beta protein (Aß) and tau protein are two high hallmarks of Alzheimer's disease (AD), representing significant targets in treating AD. Researches on mechanisms of the two proteins inducing neuron dysfunctions provide therapeutic strategies of AD, including inhibition of Aß production and aggregation, acceleration of Aß clearance as well as reduction of tau hyperphosphorylation. Proteoglycans (PGs) consist of a core protein and glycosaminoglycans (GAGs) chains, with enormous structural diversity due to variation in the core protein, the number of GAGs chains as well as extent and position of sulfation. Considerable evidences have indicated that PGs and GAGs play important roles in Aß and tau processing. Numbers of GAGs and analogues have potential therapeutic function in AD. In this Review, we focus on the relationship of PGs and GAGs with misfolded proteins in AD and their potential therapeutic implications.

Oral delivery of a potent anti-angiogenic heparin conjugate by chemical conjugation and physical complexation using deoxycholic acid.

Angiogenesis, the formation of new blood vessels, plays a pivotal role in tumor progression and for this reason angiogenesis inhibitors are an important class of therapeutics for cancer treatment. Heparin-based angiogenesis inhibitors have been newly developed as one of such classes of therapeutics and possess a great promise in the clinical context. Taurocholate conjugated low molecular weight heparin derivative (LHT7) has been proven to be a potent, multi-targeting angiogenesis inhibitor against broad-spectrum angiogenic tumors. However, major limitations of LHT7 are its poor oral bioavailability, short half-life, and frequent parenteral dosing schedule. Addressing these issues, we have developed an oral formulation of LHT7 by chemically conjugating LHT7 with a tetrameric deoxycholic acid named LHTD4, and then physically complexing it with deoxycholylethylamine (DCK). The resulting LHTD4/DCK complex showed significantly enhanced oral bioavailability (34.3 ± 2.89%) and prolonged the mean residence time (7.5 ± 0.5 h). The LHTD4/DCK complex was mostly absorbed in the intestine by transcellular pathway via its interaction with apical sodium bile acid transporter. In vitro, the VEGF-induced sprouting of endothelial spheroids was significantly blocked by LHTD4. LHTD4/DCK complex significantly regressed the total vessel fractions of tumor (77.2 ± 3.9%), as analyzed by X-ray microCT angiography, thereby inhibiting tumor growth in vivo. Using the oral route of administration, we showed that LHTD4/DCK complex could be effective and chronically administered as angiogenesis inhibitor.

Pharmacological effects and clinical applications of ultra low molecular weight heparins.

Heparin, one of the common anticoagulants, is clinically used to prevent and treat venous thromboembolism (VTE). Though it has been the drug of choice for many advanced medical and surgical procedures with a long history, the adverse events, such as bleeding, heparin-induced thrombocytopenia (HIT), allergic reactions, follow. Therefore, low molecular weight heparins (LMWHs) and ultra low molecular weight heparins (ULMWHs), with lower molecular weights, higher anti-FXa activity, longer half-life times and lower incidence of adverse events than unfractionated heparin (UFH), were researched and developed. Fondaparinux, a chemically synthesized ULMWH of pentasaccharide, has the same antithrombin III (AT-III)-binding sequence as found in UFH and LMWH. In addition, AVE5026 and RO-14, another two ULMWHs, are obtained by selective chemical depolymerization. In this paper, we review the preparation process, pharmacological effects and clinical applications of fondaparinux, AVE5026 and RO-14.

Homogeneous low-molecular-weight heparins with reversible anticoagulant activity.

Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to treat thrombotic disorders, but impurities, structural heterogeneity or functional irreversibility can limit treatment options. We report a series of synthetic LMWHs prepared by cost-effective chemoenzymatic methods. The high activity of one defined synthetic LMWH against human factor Xa (FXa) was reversible in vitro and in vivo using protamine, demonstrating that synthetically accessible constructs can have a critical role in the next generation of LMWHs.

Ultrasonic-assisted preparation of a low molecular weight heparin (LMWH) with anticoagulant activity.

Low molecular weight heparin (LMWH) is currently used as an anticoagulant agent and constitutes an alternative to unfractionated heparin, which is the cause of serious adverse drug reaction such as heparin-induced thrombocytopenia (HIT). Commercially available LMWH is produced by enzymatic depolymerization that is costly or by chemical methods that are generally carried out under conditions that could imply side reactions that reduce final product efficiency and yields. In this work, we present the use of a physicochemical method for the production of LMWH. This method consists in the use of hydrogen peroxide-catalyzed radical hydrolysis assisted by ultrasonic waves. LMWH that are produced using this physicochemical method have an average molecular weight and anticoagulant properties (Anti-Xa and Anti-IIa) that are comparable to some of commercial LMWH that are currently used. Ultrasonic-assisted radical depolymerization of heparin leads to products with a remarkably low polydispersity index. Moreover, in comparison to other LMWH such as those produced by enzymatic ß-elimination, this physicochemical depolymerization of heparin induces fewer oligosaccharides with less than five monosaccharide units. This contributes to the better preservation of the ATIII pentasaccharide binding sequence, which results in a high Anti-Xa/Anti-IIa ratio (1.86). However, LMWH obtained using this physicochemical method have a lower degree of sulfation than other LMWH, which seems to be the cause of a lower Anti-Xa and Anti-IIa activity (143.62±5.42 and 77.07±4.4, respectively).

Preparation and application of a 'clickable' acceptor for enzymatic synthesis of heparin oligosaccharides.

A 'clickable' disaccharide was prepared by treating the aldehyde precursor with hydroxylamine, followed by the catalytic hydrogenation and diazotransfer reaction. This disaccharide was successfully applied to the elongation of the backbone construction of ultralow molecular weight (ULMW) heparins using two bacterial glycosyl transferases, N-acetyl glucosaminyl transferase from Escherichia coli K5 (KfiA) and heparosan synthase-2 (pmHS2) from Pasteurella multocida.

Cyclic RGDyk-conjugated LMWH-taurocholate derivative as a targeting angiogenesis inhibitor.

LMWH-taurocholate derivative (LHT7) has been reported as a novel angiogenesis inhibitor, due to its ability to bind to several kinds of growth factors, which play critical roles in tumor angiogenesis. In this study, we have highlighted the enhanced antiangiogenic activity of LHT7, by using cyclic RGDyk (cRGD), a targeting moiety that was chemically conjugated to LHT7 via amide bond. The SPR study revealed that cRGD-LHT7 bound to α(v)ß(3) integrin as strongly as cRGD, and it bound to VEGF as strongly as LHT7. Importantly, in vitro anti-angiogenesis studies revealed that cRGD-LHT7 had a significant inhibition effect on HUVEC tubular formation. Finally, cRGD-LHT7 showed a greater inhibitory efficiency on the tumor growth in the U87MG xenograft model than the original LHT7, which was owed to its ability to target the tumor cells. All of these findings demonstrated that cRGD-LHT7 targeted α(v)ß(3) integrin-positive cancer cells and endothelial cells, resulting in a greater anti-angiogenesis effect on the solid tumors.

Pharmacokinetics of a paclitaxel-loaded low molecular weight heparin-all-trans-retinoid acid conjugate ternary nanoparticulate drug delivery system.

Amphiphilic low molecular weight heparin-all-trans-retinoid acid (LHR) conjugate, as a drug carrier for cancer therapy, was found to have markedly low toxicity and to form self-assembled nanoparticles for simultaneous delivery of paclitaxel (PTX) and all-trans-retinoid acid (ATRA) in our previous study. In the present study, PTX-loaded LHR nanoparticles were prepared and demonstrated a spherical shape with particle size of 108.9 nm. Cellular uptake analysis suggested rapid internalization and nuclear transport of LHR nanoparticles. In order to investigate the dynamic behaviors and targeting ability of LHR nanoparticles on tumor-bearing mice, near-infrared fluorescent (NIFR) dye DiR was encapsulated into the nanoparticles for ex vivo optical imaging. The results indicated that LHR nanoparticles could enhance the targeting and residence time in tumor site. Furthermore, in vivo biodistribution study also showed that the area under the plasma concentration time curve (AUC (0→inf)) values of PTX and ATRA for PTX-loaded LHR nanoparticles in tumor were 1.56 and 1.62-fold higher than those for PTX plus ATRA solution. Finally, PTX-loaded LHR nanoparticles demonstrated greater tumor growth inhibition effect in vivo without unexpected side effects, compared to PTX solution and PTX plus ATRA solution. These results suggest that PTX-loaded LHR nanoparticles can be considered as promising targeted delivery system for combination cancer chemotherapy to improve therapeutic efficacy and minimize adverse effects.

Chemoenzymatic synthesis of the next generation of ultralow MW heparin therapeutics.

Heparin, a sulfated glycosaminoglycan, is a widely used injectable anticoagulant. This polysaccharide is a natural product extracted from porcine intestinal tissue. A specific pentasaccharide sequence is responsible for heparin's high affinity towards anti-thrombin III, which undergoes a conformational change and, as a result, inhibits the blood coagulation Factor Xa, a critical serine protease at the convergence on the intrinsic and extrinsic activation pathway of the coagulation cascade. Due to its structural complexity and heterogeneity, the synthesis of the anti-thrombin III-binding sequence of heparin has been limited to a few approaches. The heparin contamination crisis in 2007 has motivated the development of alternative methods for the efficient preparation of safe heparin products. In this article, we discuss the current methods and recent advances in heparin and low MW heparin syntheses and the recent successful chemoenzymatic preparation of ultralow MW heparins.

Chemoenzymatic synthesis of homogeneous ultralow molecular weight heparins.

Ultralow molecular weight (ULMW) heparins are sulfated glycans that are clinically used to treat thrombotic disorders. ULMW heparins range from 1500 to 3000 daltons, corresponding from 5 to 10 saccharide units. The commercial drug Arixtra (fondaparinux sodium) is a structurally homogeneous ULMW heparin pentasaccharide that is synthesized through a lengthy chemical process. Here, we report 10- and 12-step chemoenzymatic syntheses of two structurally homogeneous ULMW heparins (MW = 1778.5 and 1816.5) in 45 and 37% overall yield, respectively, starting from a simple disaccharide. These ULMW heparins display excellent in vitro anticoagulant activity and comparable pharmacokinetic properties to Arixtra, as demonstrated in a rabbit model. The chemoenzymatic approach is scalable and shows promise for a more efficient route to synthesize this important class of medicinal agent.

Estudos de medicamentos biosimilares: [revisão] / Studies on biosimilar medications: [review]

No Brasil, o registro de novos medicamentos é feito apenas quando a agência reguladora - Agência Nacional de Vigilância Sanitária (Anvisa) - se satisfaz plenamente com as evidências de sua qualidade, eficácia e segurança, apresentadas por uma indústria farmacêutica que pleiteie esse registro. Com o vencimento de patentes, empresas farmacêuticas se sentem atraídas pela produção medicamentos biológicos chamados de biosimilares ou biogenéricos ou simplesmente genéricos, cuja aprovação pode resultar em redução de custos de tratamento. Mas é preciso que o biosimilar seja, pelo menos, igualmente eficaz e seguro e sem contaminantes em relação ao original. Consensos recentes apontam diretrizes para estabelecer critérios de eficácia e segurança desses medicamentos. Estudos pré-clínicos in vitro e in vivo, procedência da matéria-prima e estudos clínicos fase I, II e III são preconizados para registro do produto biosimilar no mercado internacional. As heparinas de baixo peso molecular encontram-se nessa situação. Nesta revisão, abordamos especificamente esse tipo de medicamento, o que pode servir de parâmetro para outros biosimilares.

In Brazil, the registration of new drugs is carried out only when the regulatory agency (Anvisa, acronym in Portuguese) is fully satisfied with the evidence of their quality, efficacy and safety, presented by a pharmaceutical industry that strive for this registration. With the patent expiration, pharmaceutical companies are attracted to produce biological medicines called biosimilar or biogenerics or simply generics, whose approval may result in reduced treatment costs. But it is necessary that the biosimilar be, at least, equally effective and safe and without contaminants in relation to the original. Recent consensus guidelines aim to establish criteria for efficacy and safety of these medicines. Preclinical studies in vitro and in vivo, the origin of raw materials and clinical studies phase I, II and III are recommended for biosimilar medicine registration in the international market. Low molecular weight heparins are found in this situation. In this review we specifically addressed this type of medicine, which could serve as a benchmark for other biosimilar medicines.

Low-molecular-weight heparin from Cu2+ and Fe2+ Fenton type depolymerisation processes.

Hydrogen peroxide (H2O2) and Cu(OAc)2 or FeSO4 (Fenton type reagents) perform heparin (Hep) depolymerisation to low-molecular-weight heparin (LMWH) following a radical chain mechanism. Hydroxyl (OH) radicals which are initially generated from H2O2 reduction by transition metal ions abstract hydrogen atoms on the heparin chain providing carbon centred radicals whose decay leads to the depolymerisation process. The main depolymerisation mechanism involves Hep radical intermediates that cleave the glycosidic linkage at unsulphated uronic acids followed by a 6-O-nonsulphated glucosamine, thus largely preserving the pentasaccharide sequence responsible for the binding to antithrombin III (AT). Both the transition metal ions influence the overall efficiency of the radical chain processes: Fe2+ acting as a catalyst, while Cu2+ acts as a reagent. LMWHs, especially those afforded by Cu2+, are somewhat unstable to the usual basic workup. However, this lack of stability can be eliminated by a previous NaBH4 reduction. Furthermore, with Cu2+, the process is much more reproducible than with Fe2+. Therefore, for the process of Fenton type depolymerisation of heparin, the use of Cu(OAc)2 is clearly preferable to the more "classical" FeSO4. The resulting activities and characteristics of these LMWHs are peculiar to these oxidative radical processes. In addition, LMWH provided by H2O2/Cu(OAc)2 in optimised conditions was found to posses anti-Xa and anti-IIa activities comparable to those of LMWHs currently in clinical use.

Inhibitors of slit protein interactions with the heparan sulphate proteoglycan glypican-1: potential agents for the treatment of spinal cord injury.

1. The heparan sulphate proteoglycan glypican-1 is a major high-affinity ligand of the Slit proteins. 2. Messenger RNA for both Slit-2 and glypican-1 is strongly upregulated and coexpressed in the reactive astrocytes of injured adult brain, suggesting a possible function of Slit proteins and glypican-1 in the adult central nervous system as significant components of the inhibitory environment that prevents axonal regeneration after injury. 3. Based on the hypothesis that adverse effects on axonal regeneration may be due to a glypican-Slit complex or the retention of glypican-binding C-terminal proteolytic processing fragments of Slit at the injury site, we used ELISA to examine a number of small molecules and low molecular weight heparin analogues for their ability to inhibit glypican-Slit interactions. 4. Our studies have led to the identification of several potent inhibitors with a favourable therapeutic profile that can now be tested in a spinal cord injury model. Among the most promising of these are a low molecular weight heparin produced by periodate oxidation and having no significant anticoagulant activity, the chemically sulphonated yeast-derived phosphomannan PI-88 and a number of randomly derivatized water-soluble sulphated dextrans.