Gastrointestinal Absorption of Recombinant Hirudin-2 in Rats

Yan, Xueying; Wang, Xiangtao; Xue-nong, Zhang; Zhang, Qiang

doi:10.1124/jpet.103.056655

Cited by 9 publications

(4 citation statements)

References 38 publications

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“…Hirudin possesses valuable therapeutical potential as anticoagulant and antithrombotic agent because it is the most potent thrombin-specific inhibitor so far known and does not inhibit other enzymes in the blood coagulation or fibrinolytic pathways (Sohn et al 2001). Interestingly, hirudin can be taken orally (Yan et al 2004;Cen et al 2006). To test whether hirudin can be expressed in the food-grade bacterium L. lactis for oral administration, HV3 gene was fused to SP310mut2 signal peptide sequence (Ravn et al 2003), and extracellularly secreted in the food-grade lactic acid bacterium L. lactis by using a nisin-controlled gene expression system.…”

Section: Introductionmentioning

confidence: 99%

Extracellular secretion of anticoagulant peptide hirudin in Lactococcus lactis using SP310mut2 signal peptide

Huang

Zhang

et al. 2011

Biotechnol Lett

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Hirudin can be used as an oral anticoagulant and antithrombotic agent. The hirudin variant III gene, derived from the medicinal leech, Hirudo medicinalis, was fused to SP310mut2 signal sequence and expressed by a nisin-controlled gene expression system in Lactococcus lactis which was then grown in a 7 l fermenter. After induction with 8 ng nisin ml(-1), the product was secreted into the culture medium and accumulated up to ~2.7 mg l(-1). MALDI-TOF/MS and anticoagulant activity analyses on the purified product confirmed its authenticity. This is the first demonstration that hirudin can be extracellularly secreted and correctly processed in L. lactis.

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Section: Introductionmentioning

confidence: 99%

Extracellular secretion of anticoagulant peptide hirudin in Lactococcus lactis using SP310mut2 signal peptide

Huang

Zhang

et al. 2011

Biotechnol Lett

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“…Therefore, considerable effort has been directed towards developing alternative administration routes other than injection. Although recombinant hirudin-1 (rHV1) and rHV2 can be absorbed in the gastrointestinal tracts of rats after duodenal and oral administration, the absorption was limited or it varied depending on the analytical methods used, which indicated that the results were unreliable [6,7] . As a convenient method of administration, nasal delivery has many benefits relative to oral administration, including the avoidance of the liver first-pass effect and a higher bioavailability.…”

Section: Introductionmentioning

confidence: 99%

Permeation-enhancing effects of chitosan formulations on recombinant hirudin-2 by nasal delivery in vitro and in vivo

Zhang

Ma²,

Lü³

et al. 2005

Acta Pharmacologica Sinica

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Aim: To investigate the enhancing effects of chitosan with or without enhancers on nasal recombinant hirudin‐2 (rHV2) delivery in vitro and in vivo, and to evaluate the ciliotoxicity of these formulations. Methods: The permeation‐enhancing effect of various chitosan formulations was estimated by using the permeation coefficient of fluorescein isothiocyanate recombinant hirudin‐2 (FITC‐rHV2) across the excited rabbit nasal epithelium in vitro. The effect was further evaluated by measuring the blood concentration level after nasal absorption of FITC‐rHV2 in rats. The mucosal ciliotoxicity of different formulations was evaluated with an in situ toad palate model. Results: Chitosan at a concentration of 0.5% with or without various enhancers significantly increased the permeability coefficient (P) and relative bioavailability (Fr) of FITC‐rHV2 compared with the blank control. The addition of 1% sodium dodecylsulfate, 5%Brij35, 5%Tween80, 1.5% menthol, 1% glycyrrhizic acid monoammonium salt (GAM) or 4% Azone into the 0.5% chitosan solution resulted in a further increase in absorption (P<0.05) compared with 0.5% chitosan alone. But co‐administration of chitosan with 5% hydroxyl‐propyl‐beta‐cyclodextrin(HP‐β‐CD), 5%lecithin or 0.1% ethylenediamine tetraacetic acid (EDTA) was not more effective than using the 0.5% chitosan solution alone. Chitosan alone and with 5% HP‐β‐CD, 0.1% EDTA, 1% GAM or 5%Tween80 was relatively less ciliotoxic. Conclusion: Chitosan with or without some enhancers was able to effectively promote the nasal absorption of recombinant hirudin, while not resulting in severe mucosal ciliotoxicity. A chitosan formulation system would be a useful approach for the nasal delivery of recombinant hirudin.

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“…That is comparable to the representative highyield of rHV2-Lys 47 in S. cerevisiae (500 mg l -1 ), which was reached by fed-batch cultivation for 57 h [14], and 1.5 g l -1 of secreted rHIR in methylotrophic yeast P. pastoris, which was achieved by fed-batch cultivation for 65 h after methanol induction [18,23]. However, it can be seen that the long cultivation period is absolutely needed for attaining high level of expression with the yeast system, and this will inevitably increase the preparation cost and complexity of manufacturing.…”

Section: Discussionmentioning

confidence: 91%

Enhanced secretion of adhesive recognition sequence containing hirudin III mutein in E. coli

Tan

Wu-tong

et al. 2007

Mol Biotechnol

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It has been previously shown that Escherichia coli L-asparaginase II (L-ASP) signal peptide is capable of being utilized to direct extracellular secretion of hirudin III (HV3) in shake flask. In this study HV3 muteins R33G34D35(S36)-HV3 were generated by introduction of adhesive recognition sequence RGD(S) into the non-functional region of HV3. The resultant recombinants were cultivated on 30 l bioreactor scale using L-ASP signal peptide expression system and the optimized fed-batch cultivation was well established. After cultivation for approximately 11 h the secreted product accumulated up to approximately 1 g l(-1), which means 17-fold increase in productivity compared to initial expression in shake flask. N-terminal analysis, pI measurement, and MALDI mass spectral analysis on mutein R33G34D35S36-HV3 confirmed the authenticity of the product. Compared to wild-type HV3 and R33G34D35HV3, the mutein R33G34D35S36-HV3 exhibits the improved pharmacological activity. Collectively, a novel secretion strategy using L-ASP signal peptide for the rapid, efficient and cost-effective production of HV3 mutein possessing improved pharmacological activity on bioreactor scale has been well established. Using this expression system downstream processing becomes very simple because secreted product is mature, soluble, active, and without N-terminal extension of Met, which is quite critical for most therapeutic protein to reduce the side effect in clinic use. Thus, it provides a promising alternative for extracellular production of other difficult-to-express protein for biopharmaceutical use.

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Gastrointestinal Absorption of Recombinant Hirudin-2 in Rats

Abstract: To investigate the absorption of recombinant hirudin-2 (rHV2) after oral administration to rats and its possible absorption mechanism, a series of experiments were carried out. The degradation of 125

Cited by 9 publications

References 38 publications

Extracellular secretion of anticoagulant peptide hirudin in Lactococcus lactis using SP310mut2 signal peptide

Extracellular secretion of anticoagulant peptide hirudin in Lactococcus lactis using SP310mut2 signal peptide

Permeation-enhancing effects of chitosan formulations on recombinant hirudin-2 by nasal delivery in vitro and in vivo

Enhanced secretion of adhesive recognition sequence containing hirudin III mutein in E. coli

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