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Abstract Background Diffuse large B-cell lymphoma (DLBCL) is a malignant tumour. Although some standard therapies have been established to improve the cure rate, they remain ineffective for specific individuals. Therefore, it is meaningful to find more novel therapeutic approaches. Macrophage polarisation is extensively involved in the process of tumour development. Recombinant hirudin (rH) affects macrophages and has been researched frequently in clinical trials lately. Our article validated the regulatory role of rH in macrophage polarisation and the mechanism of PAR-1 by collecting clinical samples and subsequently establishing a cellular model to provide a scientifically supported perspective for discovering new therapeutic approaches. Method We assessed the expression of macrophage polarisation markers, cytokines and PAR-1 in clinical samples. We established a cell model by co-culture with THP-1 and OCI-Ly10 cell. We determined the degree of cell polarisation and expression of validation cytokines by flow cytometry, ELISA, and RT-qPCR to confirm the success of the cell model. Subsequently, different doses of rH were added to discover the function of rH on cell polarisation. We confirmed the mechanism of PAR-1 in macrophage polarisation by transfecting si-PAR-1 and pcDNA3.1-PAR-1. Results We found higher expression of M2 macrophage markers (CD163 + CMAF+) and PAR-1 in 32 DLBCL samples. After inducing monocyte differentiation into M0 macrophages and co-culturing with OCI-Ly10 lymphoma cells, we found a trend of these expressions in the cell model consistent with the clinical samples. Subsequently, we discovered that rH promotes the polarisation of M1 macrophages but inhibits the polarisation of M2 macrophages. We also found that PAR-1 regulates macrophage polarisation, inhibiting cell proliferation, migration, invasion and angiogenic capacity. Conclusion rH inhibits macrophage polarisation towards the M2 type and PAR-1 regulates polarisation, proliferation, migration, invasion, and angiogenesis of DLBCL-associated macrophages.

Background: Diffuse large B-cell lymphoma (DLBCL) is a malignant tumour. Although some standard therapies have been established to improve the cure rate, they remain ineffective for specific individuals. Therefore, it is meaningful to find more novel therapeutic approaches. Macrophage polarisation is extensively involved in the process of tumour development. Recombinant hirudin (rH) affects macrophages and has been researched frequently in clinical trials lately. Our article validated the regulatory role of rH in macrophage polarisation and the mechanism of PAR-1 by collecting clinical samples and subsequently establishing a cellular model to provide a scientifically supported perspective for discovering new therapeutic approaches. Method: We assessed the expression of macrophage polarisation markers, cytokines and PAR-1 in clinical samples. We established a cell model by co-culture with THP-1 and OCI-Ly10 cell. We determined the degree of cell polarisation and expression of validation cytokines by flow cytometry, ELISA, and RT-qPCR to confirm the success of the cell model. Subsequently, different doses of rH were added to discover the function of rH on cell polarisation. We confirmed the mechanism of PAR-1 in macrophage polarisation by transfecting si-PAR-1 and pcDNA3.1-PAR-1. Results: We found higher expression of M2 macrophage markers (CD163 + CMAF+) and PAR-1 in 32 DLBCL samples. After inducing monocyte differentiation into M0 macrophages and co-culturing with OCI-Ly10 lymphoma cells, we found a trend of these expressions in the cell model consistent with the clinical samples. Subsequently, we discovered that rH promotes the polarisation of M1 macrophages but inhibits the polarisation of M2 macrophages. We also found that PAR-1 regulates macrophage polarisation, inhibiting cell proliferation, migration, invasion and angiogenic capacity. Conclusion: rH inhibits macrophage polarisation towards the M2 type and PAR-1 regulates polarisation, proliferation, migration, invasion, and angiogenesis of DLBCL-associated macrophages. [ABSTRACT FROM AUTHOR]

BSTRACT Background. Acute pulmonary embolism (APE) is classified as a subset of diseases that are characterized by lung obstruction due to various types of emboli. Current clinical APE treatment using anticoagulants is frequently accompanied by high risk of bleeding complications. Recombinant hirudin (R-hirudin) has been found to have antithrombotic properties. However, the specific impact of R-hirudin on APE remains unknown. Methods. Sprague-Dawley (SD) rats were randomly assigned to five groups, with thrombi injections to establish APE models. Control and APE group rats were subcutaneously injected with equal amounts of dimethyl sulfoxide (DMSO). The APE+R-hirudin low-dose, middle-dose, and high-dose groups received subcutaneous injections of hirudin at doses of 0.25 mg/kg, 0.5 mg/kg, and 1.0 mg/kg, respectively. Each group was subdivided into time points of 2 h, 6 h, 1 d, and 4 d, with five animals per point. Subsequently, all rats were euthanized, and serum and lung tissues were collected. Following the assessment of right ventricular pressure (RVP) and mean pulmonary artery pressure (mPAP), blood gas analysis, enzyme-linked immunosorbnent assay (ELISA), pulmonary artery vascular testing, hematoxylin-eosin (HE) staining, Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, immunohistochemistry, and Western blot experiments were conducted. Results. R-hirudin treatment caused a significant reduction of mPAP, RVP, and Mal- ondialdehyde (MDA) content, as well as H2O2 and myeloperoxidase (MPO) activity, while increasing pressure of oxygen (PaO2) and Superoxide Dismutase (SOD) activity. R-hirudin also decreased wall area ratio and wall thickness to diameter ratio in APE rat pulmonary arteries. Serum levels of endothelin-1 (ET-1) and thromboxaneB2 (TXB2) decreased, while prostaglandin (6-K-PGF1_) and NO levels increased. Moreover, R-hirudin ameliorated histopathological injuries and reduced apoptotic cells and Matrix metalloproteinase-9 (MMP9), vascular cell adhesion molecule-1 (VCAM-1), p-Extracellular signal-regulated kinase (ERK)1/2/ERK1/2, and p-P65/P65 expression in lung tissues.Conclusion. R-hirudin attenuated pulmonary hypertension and thrombosis in APE rats, suggesting its potential as a novel treatment strategy for APE. [ABSTRACT FROM AUTHOR]

After orthopedic surgeries, such as hip replacement, many patients are prone to developing deep vein thrombosis (DVT), which in severe cases can lead to fatal pulmonary embolism or major bleeding. Clinical intervention with high-dose anticoagulant therapy inevitably carries the risk of bleeding. Therefore, a targeted drug delivery system that adjusts local DVT lesions and potentially reduces drug dosage and toxic side effects important. In this study, we developed a targeted drug delivery platelet-derived nanoplatform (AMSNP@PM-rH/A) for DVT treatment that can simultaneously deliver a direct thrombin inhibitor (DTI) Recombinant Hirudin (rH), and the Factor Xa inhibitor Apixaban (A) by utilizing Aminated mesoporous silica nanoparticles (AMSNP). This formulation exhibits improved biocompatibility and blood half-life and can effectively eliminate deep vein thrombosis lesions and achieve therapeutic effects at half the dosage. Furthermore, we employed various visualization techniques to capture the targeted accumulation and release of a platelet membrane (PM) coating in deep vein thrombosis and explored its potential targeting mechanism. Development of a targeted delivery platelet-derived nanoplatform for treating deep vein thrombosis by delivering direct thrombin inhibitors and factor Xa inhibitors, with the ability to be tracked using digitalization and visualization technologies. [Display omitted] [ABSTRACT FROM AUTHOR]

Hirudin, an acidic polypeptide secreted by the salivary glands of Hirudo medicinalis (also known as “Shuizhi” in traditional Chinese medicine), is the strongest natural specific inhibitor of thrombin found so far. Hirudin has been demonstrated to possess potent anti-thrombotic effect in previous studies. Recently, increasing researches have focused on the anti-thrombotic activity of the derivatives of hirudin, mainly because these derivatives have stronger antithrombotic activity and lower bleeding risk. Additionally, various bioactivities of hirudin have been reported as well, including wound repair effect, anti-fibrosis effect, effect on diabetic complications, anti-tumor effect, anti-hyperuricemia effect, effect on cerebral hemorrhage, and others. Therefore, by collecting and summarizing publications from the recent two decades, the pharmacological activities, pharmacokinetics, novel preparations and derivatives, as well as toxicity of hirudin were systematically reviewed in this paper. In addition, the clinical application, the underlying mechanisms of pharmacological effects, the dose-effect relationship, and the development potential in new drug research of hirudin were discussed on the purpose of providing new ideas for application of hirudin in treating related diseases.

• In situ sulfated recombinant hirudin-I was obtained through periplasmic co-expression system. • In situ sulfation significantly increased the anti-thrombotic activity of recombinant hirudin-I. • Column free purification method for recombinant hirudin-I was developed. Hirudin, a blood anticoagulant, is the most potent natural thrombin inhibitor of leech origin. Its application is limited because it is difficult to obtain abundant natural hirudin directly from the leech. Although some bioengineering methods can significantly increase the production of hirudin, the reduced efficacy of recombinant hirudin (rH) remains a critical shortcoming. The lack of sulfation of tyrosine 63 in rH is an important cause of its inadequate performance. This article is the first report of periplasmic co-expression of an rH-I analogue with arylsulfotransferase (ASST) in E. coli BL21(DE3). Co-expressed rH-I analogue with sulfate donor substrate (p -nitrophenyl sulfate potassium) showed anticoagulant (rabbit and goat serum) activity twice more than rH-I analogue expressed without ASST, indicating its potential periplasmic sulfation. Moreover, purified rH-I analogue showed above 4.5 times higher anticoagulant activity compared to therapeutic anti-thrombotic heparin (HE). At the same time, pH-dependent differential solubility was employed to purify rH analogues from fermentation broth, which is a simple, fast and inexpensive purification technology, and can potentially be used for larger scale purification. This will also greatly improve the application of rH in clinical treatment. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Recombinant hirudin variant-2 (HV2) was conjugated with palmitic acid (PAL). • The selectivity of this reaction was improved in mixed aqueous-DMSO solution. • Three mono-PAL-HV2 isomers was efficiently separated by prep-HPLC. • Mono-PAL-HV2-3 showed the highest selectivity and the highest in vitro bioactivity. • The modification site of mono-PAL-HV2-3 was identified as Lys 27 of HV2. Abstract In this study, chemical modification of recombinant hirudin variant-2 (HV2) with palmitic acid (PAL) was proposed as an alternative approach to circumvent the limitations of PEGylation. To facilitate a sufficient contact of the hydrophilic HV2 to the hydrophobic PAL, thereby improving the reaction specificity to achieve the desired mono-PAL-HV2 with high retained bioactivity, the reaction was developed in mixed aqueous-organic solutions. Compared with HV2 conjugation with PAL-benzotriazole (PAL-BTA) in mixed aqueous-NMP solution, the conjugation of HV2 with PAL- N -hydroxysuccinimide (PAL-NHS) in mixed aqueous-DMSO solution could improve the site-specific conjugation of one PAL molecule to a particular lysine residue. Furthermore, the reaction mixture of the latter was further purified by preparative liquid chromatography. Three mono-PAL-HV2 isomers were obtained and retained 36%, 4% and 89% of the in vitro anticoagulant activity of unmodified HV2, respectively. One of the mono-PAL-HV2 isomers, namely, mono-PAL-HV2-3, was isolated with the highest selectivity and exhibited the highest in vitro anticoagulant bioactivity. Modification site analysis of mono-PAL-HV2-3 revealed that a single PAL molecule was conjugated at Lys27 of HV2. This study presented a successful PAL modification in which site-specific reaction was improved to achieve the desired mono-PAL-HV2 with highly retained bioactivity in mixed aqueous-organic solutions. [ABSTRACT FROM AUTHOR]

Abstract: In this study, a combined optimization method was developed to optimize the N‐terminal site‐specific PEGylation of recombinant hirudin variant‐2 (HV2) with different molecular weight mPEG‐propionaldehyde (mPEG‐ALD), which is a multifactor‐influencing process. The HV2‐PEGylation with 5 kDa mPEG‐ALD was first chosen to screen significant factors and determine the locally optimized conditions for maximizing the yield of mono‐PEGylated product using combined statistical methods, including the Plackett–Burman design, steepest ascent path analysis, and central composition design for the response surface methodology (RSM). Under the locally optimized conditions, PEGylation kinetics of HV2 with 5, 10, and 20 kDa mPEG‐ALD were further investigated. The molar ratio of polyethylene glycol to HV2 and reaction time (the two most significant factors influencing the PEGylation efficiency) were globally optimized in a wide range using kinetic analysis. The data predicted by the combined optimization method using RSM and kinetic analysis were in good agreement with the corresponding experiment data. PEGylation site analysis revealed that almost 100% of the obtained mono‐PEGylated‐HV2 was modified at the N‐terminus of HV2. This study demonstrated that the developed method is a useful tool for the optimization of the N‐terminal site‐specific PEGylation process to obtain a homogeneous mono‐PEGylated protein with desirable yield. [ABSTRACT FROM AUTHOR]

To improve the therapy efficacy of recombinant hirudin variant-2 (HV2), its PEGylation was investigated using linear mPEG-succinimidyl carbonate (mPEG-SC) and branched mPEG 2 - N -hydroxysuccinimide (mPEG 2 -NHS). The reaction mixtures of PEGylation were analyzed by RP-HPLC and the mono-PEG-HV2 products were purified by anion exchange chromatography (IEC). Effects of linear and branched PEG on the hydrolysis kinetics of the PEG reagent, the PEGylation kinetics of HV2 and the in vitro and in vivo bioactivity of mono-PEG-HV2 were investigated. The RP-HPLC and IEC analyses showed that linear and branched PEG-HV2 with identical molecular weight had different chromatographic behaviors. The reaction kinetics showed that branched mPEG 2 -NHS displayed higher hydrolysis rate but lower PEGylation rates than linear mPEG-SC. Consequently, HV2 conjugated with mPEG 2 -NHS required a greater molar ratio of PEG to HV2 than that of mPEG-SC to achieve the identically desired yield of mono-PEG-HV2. The in vitro and in vivo bioactivities of mono-PEG-HV2 showed that branched PEG-HV2 had higher therapeutic efficacy than linear PEG-HV2 with identical molecular weight. The in vivo bioactivity of mono-B-PEG40k-HV2 (mono-PEG-HV2 derived from 40 kDa branched mPEG 2 -NHS) had a markedly longer duration in rabbits than did unmodified HV2, which showed its potential to be developed as a candidate antithrombotic drug. [ABSTRACT FROM AUTHOR]

In this study, an integrated process was developed for successive solid-phase PEGylation of recombinant hirudin variant-2 (HV2) and separation of PEGylated HV2 species on an anion exchange chromatography column (so-called in situ PEGylation). The effects of different PEG sizes, ion exchange resins and reaction conditions on in situ PEGylation were investigated. The results showed that in situ PEGylation efficiently integrates the reaction, separation and purification into a single-unit operation using the same column. In situ PEGylation could improve the selectivity of PEGylation reactions by significantly reducing the formation of multi-PEG-HV2. The pore sizes and internal surface structures of different resins had a significant impact on the yield of mono-PEG-HV2. In contrast to liquid-phase PEGylation, the yield of mono-PEG-HV2 decreased as PEG size increased during the in situ PEGylation process, indicating that in situ PEGylation is a pore diffusion-controlled process. The in vitro and in vivo anticoagulant activities of mono-PEG-HV2 derived from in situ PEGylation were higher than those from liquid-phase PEGylation, indicating that in situ PEGylation could enhance the bioactivity retention of mono-PEG-HV2. The results of this study demonstrated that in situ PEGylation can be used as an effective approach for the development of PEGylated protein drugs. [ABSTRACT FROM AUTHOR]

Blood-contacting materials with good mechanical property, excellent anticoagulant function and promoting effect on endothelialization are in great demand for clinical application such as vascular grafts in treating cardiovascular diseases. In this study, electrospinning nanofiber scaffolds of polycaprolactone (PCL) were functionalized by oxidative self-polymerization of dopamine (PDA) on the surface followed by the modification of anticoagulant recombinant hirudin (rH) molecules. The morphology, structure, mechanical property, degradation behavior, cellular compatibility and blood compatibility of the multifunctional PCL/PDA/rH nanofiber scaffolds were evaluated. The diameter of the nanofibers was between 270-1030 nm. The ultimate tensile strength of the scaffolds was around 4 MPa and the elastic modulus increased with the amount of rH. The degradation tests in vitro indicated that the nanofiber scaffolds began to crack on the 7th day, but still maintained the nanoscale architecture within a month. The cumulative release of rH from the nanofiber scaffold was up to 95.9 % at 30th day. The functionalized scaffolds promoted the adhesion and proliferation of endothelial cells, while resisting platelet adhesion and enhancing anticoagulation effects. The hemolysis ratios of all scaffolds were <2 %. The nanofiber scaffolds are promising candidates for vascular tissue engineering., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

To improve the bioactivity of recombinant (r) Hirudin, the orthogonal pair MjBTyrRS/tRNATyr cua (made up of the boronophenylalanine, tRNA and tRNA synthetase), was selected to incorporate boronophenylalanine site-specifically into rHirudin at the 63 sites in an Escherichia coli system in response to the TAG codon. Following fusion with the gIII signal peptide and a hexahistidine tag, the modified protein was secreted into Luria-Bertani culture medium and purified by nickel-nitrilotriacetic acid affinity chromatography following a gel filtration column. In a 200 ml flask, the yield of boronophenylalanine-modified hirudin was 10 mg l-1 and that of rHirudin was 19 mg l-1. The authenticity of the purified proteins was verified using matrix-assisted laser desorption ionization time of flight mass spectroscopy and antithrombin activity assays. The results revealed that the antithrombin activity of the boronophenylalanine-modified hirudin to human thrombin was more enhanced than that of rHirudin. The modified hirudin demonstrated stronger proliferation inhibiting ability on fibroblast L929 cells compared with that of rHirudin. [ABSTRACT FROM AUTHOR]

Serious loss of polyethylene glycol (PEG) reagent is confronted during PEGylation of proteins in mildly acidic aqueous solution. In this study, a novel approach of PEGylation of recombinant hirudin variant-2 (HV2) with monomethoxy-PEG-succinimidyl carbonate (mPEG-SC) was developed in six mixed aqueous–organic solutions. The PEGylation efficiency in each mixed solutions was greater than that in pure aqueous solution. Structure analysis of HV2, hydrolysis kinetics of mPEG-SC, and PEGylation kinetics of HV2 in different solutions revealed that solvent effects occurred in mixed aqueous–organic solutions. According to the dominant solvent effect of PEGylation acceleration and mPEG-SC hydrolysis inhibition, the selected mixed aqueous–organic solutions could be divided into different types (PEGylation-driven, mPEG-SC hydrolysis-driven, both PEGylation and mPEG-SC hydrolysis-driven). In aqueous-DMSO solutions, the desired yield (approximately 50%) of mono-PEG-HV2 was achieved under the optimized reaction conditions. The optimal usage of PEG reagent could decrease 3–5 fold compared with that of PEGylation in pure aqueous solution. The mono-PEG-HV2 could restore its structure and bioactivity after being purified into aqueous solution. This study demonstrated that PEGylation of proteins in mixed aqueous–organic solutions could be used as an alternative method to PEGylation in pure aqueous solution. [ABSTRACT FROM AUTHOR]

In this article, the surface of intraocular len material PMMA was first aminated for activation on which some polar groups generated such as CN, COO−, OH, NH3+, etc. Then the anticoagulant drugs recombinant hirudin (rH) was grafted with amido bonds to look forward to resist the adsorption of nonspecific protein or cells in tear, even the cataract. The detailed analysis and discussion about the grafting quantity, molography, wettability, electric charges, chemical structure, and the dynamic adsorption of protein Fn on the material surface were carried on by the technology of ultraviolet photometric, contact angle, solid Zeta potential, X-ray photoelectron spectroscopy, and quartz crystal microbalance. The surface with a certain amount of rH modification existed more hydrophilic due to the amphiphilic structure than before, on which the protein adsorption was the most unstable. The results indicated that the rH modification improved the resistance of PMMA to nonspecific adsorption of protein Fn to achieve the expectative effect. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 878-886, 2015. [ABSTRACT FROM AUTHOR]

Highlights: [•] We constructed a reaction kinetic model for the PEGylation of HV2 with mPEG-SC. [•] We have successfully used the established model to optimize the PEGylation reaction. [•] We found several important process parameters and their mathematical equations. [•] The proposed reaction kinetic model is applicable to other PEGylation and similar biochemical reactions. [ABSTRACT FROM AUTHOR]

We have assessed the anti-inflammatory, anti-oxidative and anti-coagulant effects of locally applied natural and recombinant hirudin in a random skin flap rat model. Thirty Wistar rats with venous congested skin flaps were randomly divided into two treatment groups and a control group to receive subcutaneous injections of natural hirudin (6 U), recombinant hirudin (6 U) or physiological saline, respectively. Superoxide dismutase, malondialdehyde and endothelin levels as well as flap survival rates of the skin flaps were measured after surgery. Compared to the control group, the treatment groups had significant higher superoxide dismutase levels and lower malondialdehyde and endothelin levels in the skin flaps. The surviving areas of the flaps were larger in the treatment groups than the control group. Our results demonstrated that hirudin could improve skin flap survival through its anti-inflammatory, anti-oxidative and anti-coagulant activities. [ABSTRACT FROM PUBLISHER]

Abstract: Two biodegradable cationic lipids, stearylamine and DC-Chol, were chosen to investigate the effect of cationic lipids on the in vitro and in vivo characteristics of hydrophilic proteins or peptides of low isoelectric point. Thrombin inhibitor recombinant hirudin variant-2 (rHV2) was selected as the model drug. The cationic lipids were found to achieve higher entrapment efficiency of rHV2 in liposomes than zwitterionic lipids. The positively charged liposomes became less positive and relatively stable in serum after loading rHV2. The cationic liposomes induced sustained release of rHV2 in the presence of plasma, significantly prolonged the antithrombotic efficacy and plasma level of rHV2 after intravenous injection in rats in comparison with neutral lipid liposomes, especially for stearylamine group. Both clotting times correlated well with plasma rHV2 levels. No serious adverse events were observed and physical state of rats was satisfactory for all the formulations. Electrostatic interaction between negative charge of rHV2 and cationic liposomes was confirmed and it might affect all the characteristics of rHV2 loaded cationic vehicles. The findings suggest that cationic liposomes may be a potential sustained-release delivery system for parenteral administration of hydrophilic proteins or peptides with low isoelectric point to prolong efficacy and improve bioavailability. [Copyright &y& Elsevier]

Abstract: Here we investigated thermo-sensitive Pluronic® F127 (PF127) hydrogel for the controlled release of peptide and protein drugs after subcutaneous injection, using an antithrombotic polypeptide, recombinant hirudin variant-2 (rHV2), as the model drug. The in vitro release experiment performed with a membrane-less model at 37 °C showed that the release of antithrombotic activity of rHV2 from PF127 gel followed zero-order kinetics and correlated well with the weight percentage of PF127 dissolved, indicating a dissolution-controlled release mechanism. The in vivo result obtained after subcutaneous injection of rHV2-loaded PF127 gel in normal rats demonstrated that PF127 gel improved the bioavailability, prolonged the antithrombotic effect of rHV2, and induced detectable plasma rHV2 concentration for a longer time in comparison with rHV2 aqueous solution. Differential scanning calorimetry, dynamic light scattering and Fourier transform infrared spectroscopy provided evidence of the interaction between PF127 and rHV2, but such interaction was unlikely to interfere the feasibility of this drug delivery system. Our current in vitro and in vivo study suggested that PF127 gel may be useful as an injectable delivery vehicle for peptides and proteins with short half-lives to prolong their therapeutic effect, increase their bioavailability and improve the clinic outcome. [Copyright &y& Elsevier]

Abstract: One of recombinant hirudin variants, rHV2, a polypeptide used as an anticoagulant agent in clinic, was administered to anesthetized rats via intratracheal, buccal, nasal and rectal routes. Prolongation in clotting time and thrombin time was measured to calculate pharmacological bioavailability. Plasma concentration of rHV2 was determined using a chromogenic thrombin substrate assay and pharmacokinetic parameters were obtained on the basis of a non-compartmental model. Intravenous administration was also performed as the gold standard by which the other routes were compared. Difference in pharmacological bioavailability (P.A.), bioavailability (F) and absorption rate of rHV2 was found for the four non-parenteral routes. The rank order for both P.A. and F was intratracheal>nasal>buccal>rectal. Absorption was more rapid after both intratracheal and rectal administration (t max ∼20–40min), compared with that after nasal and rectal administration. It is evident that the pulmonary route is preferable to other three routes for successful systemic delivery of rHV2. [Copyright &y& Elsevier]

Several of the newly developed anti-Xa and anti-IIa agents have been shown to influence the International Normalized Ratio (INR) values. During phase I trials with normal healthy volunters and phase II study patients who were given warfarin and con-comitant anti-IIa or anti-Xa agents, it has been reported that INR values were falsely elevated. It is of critical importance to know of the effects of these agents on INR to avoid dosage errors. To study the influence of these agents on INR, we used several anti-IIa agents (argatroban, recombinant hirudin, efegatran, and PEG-hirudin) and anti-Xa drugs (pentasaccharides such as fondaparinux and idra-parinux, DX-9065a and JTV-803). The anti-IIa drugs were supplemented in citrated plasma at a concentration of 0 to 1 µg/mL level and anti-Xa drugs in the range of 0 to 25 µg/ml. The IC50 values for each of these agents were calculated. Four different commercially available prothrombin time (PT) reagents were used to perform the PT assays and to calculate the relative INR values. Direct synthetic factor IIa and Xa inhibitors exhibited a concentration-dependent increase in the INR values. Hirudin, efegatran, and PEG-hirudin showed a weaker effect, whereas argatroban showed a much higher elevation of the INR values. Synthetic indirect anti-Xa agents such as the pentasaccharide did not show any effect on the INR values. Furthermore, prothrombin time reagents with high ISI values exhibited disproportionally higher INR values for both the direct anti-Xa and anti-IIa agents. Elevation of INR values has therapeutic implications when non-oral anticoagulant drugs are used in combination with drugs such as warfarin. Because of the false elevation of INR values with some of the non-oral anticoagulant drugs, patients who are on concomitant warfarin therapy should be carefully evaluated for their corresponding INR values for proper dosing. To avoid dosing errors it is best not to use the INR values in the... [ABSTRACT FROM AUTHOR]

The purification and identification of recombinant hirudin (r-hirudin) (rHV2-Lys47) and its several C-terminal proteolytic degradation derivatives, produced by Pichia pastoris, were described. The high-purity rHV2-Lys47 of above 99% and its three degradation products were obtained by a straightforward two-step chromatography procedure, a combination of cation exchange and reverse phase chromatography, with a recovery yield of 74% for hirudin. The purified rHV2 had the predicted N-terminal amino acid sequence and the derivatives were the degradation products of hirudin, short of one to three amino acid residues at C-terminal. [ABSTRACT FROM AUTHOR]

: ObjectivesThe effects of ximelagatran, an oral direct thrombin inhibitor (DTI), recombinant hirudin (r-hirudin) and enoxaparin on thrombin generation and platelet activation were studied in humans.: BackgroundRecombinant hirudin (parenteral DTI) and enoxaparin (low molecular weight heparin) have been demonstrated to be clinically effective in acute coronary syndromes. Ximelagatran is currently under investigation for the prevention and treatment of thromboembolism. The shed blood model allows for the study of thrombin generation and platelet activation in humans in vivo.: MethodsThis was an open-label, parallel-group study involving 120 healthy male volunteers randomized to receive one of three oral doses of ximelagatran (15, 30 or 60 mg), r-hirudin (intravenous) or enoxaparin (subcutaneous) at doses demonstrated to be clinically effective in acute coronary syndromes, or to serve as a control. Thrombin generation (prothrombin fragment 1+2 [F1+2] and thrombin-antithrombin complex [TAT]) and platelet activation (β-thromboglobulin [β-TG]) biomarkers were studied using a shed blood model involving blood collection from skin incisions made using standardized bleeding time devices.: ResultsOral ximelagatran, intravenous r-hirudin and subcutaneous enoxaparin rapidly and significantly (p < 0.05) decreased F1+2, TAT and β-TG levels in shed blood, indicating inhibition of thrombin generation and platelet activation. Statistically significant concentration (melagatran, the active form of ximelagatran)-response relationships for F1+2 (p = 0.005), TAT (p = 0.005) and β-TG (p < 0.001) levels, with IC50s of 0.376 (F1+2), 0.163 (TAT) and 0.115 (β-TG) μmol/l, were detected. Melagatran showed dose-proportional pharmacokinetics with low variability. All drugs were well tolerated.: ConclusionsOral administration of the DTI ximelagatran resulted in a rapid inhibition of both thrombin generation and platelet activation in a concentration-dependent manner using a human shed blood model. The inhibition of thrombin generation by 60 mg ximelagatran was comparable to that observed with doses of r-hirudin and enoxaparin demonstrated to be effective for the treatment of acute coronary syndromes. [Copyright &y& Elsevier]

Background: The number of indications for recombinant human hirudin lepirudin therapy has increased in recent years, and now includes acute coronary syndromes and heparin-induced thrombocytopenia. Hence, point of care monitoring appears desirable for therapy with lepirudin. As CoaguChek Plus (CCP) provides a rapid bedside test to monitor therapy with other anticoagulants, we aimed to determine its suitability for lepirudin therapy. Methods: Forty-four healthy volunteers received a 2 ng/kg endotoxin infusion (to induce coagulation) together with clinically relevant doses of lepirudin or heparin in a prospective, placebo-controlled, randomised fashion. Measurements of CCP-partial thromboplastin time (aPTT) were compared to laboratory STA-aPTT. Results: As expected, baseline values of CCP-aPTT were shorter than STA-aPTT. Lepirudin increased CCP-aPTT 3-fold, and STA-aPTT 2-fold 1 h after bolus infusion. During lepirudin infusion, the correlation between CCP-aPTT and STA-aPTT was excellent (r=0.86–0.92). Both methods were equally sensitive to over-anticoagulation with heparin. Acute systemic inflammation had little effects on CCP-aPTT. Conclusion: CCP-aPTT is suitable for longitudinal point of care monitoring of lepirudin therapy. As baseline values of CCP-aPTT are shorter than STA-aPTT, it is recommended not to indiscriminately change between methods in the follow-up of individual patients. [Copyright &y& Elsevier]