Thrombin

In the internal and exterior coagulation pathways, histidine suppresses thrombin activity, deprives thrombin of its capacity to cleave fibrinogen, and stops fibrin from forming as well as the polymerization process of fibrin monomers by cross-linking [1]. By opposing thrombin, Hirudin decreases apoptosis in human microvascular endothelial cells and suppresses p-JAK2 expression [1]. At high concentrations, Hirudin suppresses the growth of human microvascular endothelial cells and the VEGF-Notch pathway [1]. TGF-β1-induced aberrant proliferation and fibrosis in HK-2 cells can be counteracted by hirudin (3–10 mg/mL) [1]. Hirudin suppresses the ERK1/2 pathway, decreases oxidative stress, inhibits angiotensin II-induced cardiac fibroblasts, and regulates variables associated to fiber in a dose-dependent manner [1].

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Li et al. reported that Hirudin exhibited antitumor effect when targeting cells of the LN229 and U251 cell lines in vitro. It significantly suppressed the growth of LN229 and U251 cell lines, with the IC 50 of 30 and 15 mM, respectively. In addition, hirudin also increased the apoptosis in these glioma cells by down-regulating the expression of Bcl-2 and increased cell cycle arrest at the G1 phase. Notably, the content of pERK1/2 was decreased after hirudin administration, as well as the level of the protein Cyclin D1, which suggested that down-regulation of ERK/MAPK signaling pathway might be a crucial mechanism of hirudin in treating gliomas (Zhao, 2015).

Recently, Hirudin was found to possess inhibitory property on hepatocellular carcinoma, and the underlying mechanism might be implicated in suppressing angiogenesis. Li et al. reported that Hirudin could inhibit the proliferation, apoptosis, migration and invasion of HepG2 cells in a dose-dependent manner. In addition, the expressions of VEGF mRNA and protein were significantly down-regulated after the treatment of hirudin (Li et al., 2016), which is consistent with previous studies that the reduction of hepatocellular carcinoma angiogenesis owes to the inactivation of VEGF pathway (Morse et al., 2019). Furthermore, hirudin is a specific inhibitor of thrombin, which can inhibit angiogenesis stimulated by thrombin and reduce the expression of VEGF. Hemangioma is a lesion caused by the abnormal proliferation of vascular endothelial cells and pericyte, which could be intervened and treated by antiangiogenic drugs (Ji et al., 2014). A study in 2015 revealed that 4 U/ml Hirudin could effectively inhibit the proliferation and promote the apoptosis of mouse EOMA hemangioma cells in vitro, which showed an obvious dose-effect relationship (Yang et al., 2015).
Besides natural Hirudin, rH was proved to have antitumor effects in Hep-2 human laryngeal cancer (LC) cells via suppressing angiogenesis. [1]

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Hirudin reduces the expression of inflammatory factors in TGF-β-induced renal tubular epithelial cells [2]
After the induction of TGF-β in human HK-2 cells, mouse IMCD3 cells and rat NRK-52E cells, the levels of IL-1β, IL-6 and TNF-α in those cells were increased, which were then gradually decreased when those cells were treated with Hirudin from 0.5 mg/ml to 1 mg/ml (Fig. 5A–C).

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Hirudin reduces the occurrence of EMT in TGF-β-induced renal tubular epithelial cells [2]
E-cad, N-cad and slug are critical proteins responsible for the occurrence of EMT. As shown in Fig. 6A–C, the expression of N-cad and slug was increased and E-cad expression was decreased when HK-2 cells, IMCD3 cells and NRK-52E cells were treated with TGF-β. What’s more, Hirudin treatment from 0.5 mg/ml to 1 mg/ml could improve the expression of N-cad and slug and depress the E-cad expression in those cells.

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Hirudin reduces the incidence of fibrosis in TGF-β induced renal tubular epithelial cells [2]
As shown in Fig. 7A–C, the expression of collagen-I, FN and α-SMA was increased in HK-2 cells, IMCD3 cells and NRK-52E cells induced by TGF-β. When those cells were treated with Hirudin from 0.5 mg/ml to 1 mg/ml, the expression of collagen-I, FN and α-SMA in cells were gradually decreased.

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Hirudin reduces the apoptosis of TGF-β-induced renal tubular epithelial cells [2]
The apoptosis of HK-2 cells, IMCD3 cells and NRK-52E cells was detected by Hoechst staining. The apoptosis of HK-2 cells, IMCD3 cells and NRK-52E cells induced by TGF-β was enhanced while alleviated when those cells were treated with Hirudin from 0.5 mg/ml to 1 mg/ml (Fig. 8A–C).

In rats, hindrin decreases the inflammatory response and boosts the survivability of random skin flaps [1]. Following laser surgery, hinddin helps SD rats' wounds recover [1]. In a mouse model of unilateral ureteral obstruction (UUO), hinddin (10 and 15 mg/kg; gavage, thrice daily for 21 days) reduces renal interstitial fibrosis to lessen tubular damage and inflammation [2].

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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. [1]

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Renal interstitial fibrosis (RIF) often occurs in many chronic kidney diseases (CKD). Hirudin now is applied to treat fibrosis in some organs. In this study, we verified the treatment effects of hirudin on RIF in vivo and in vitro with the underlying mechanism. The RIF in vivo was the unilateral ureteral obstruction (UUO) model and RIF in vitro was the renal tubular epithelial cells induced by TGF-β. The renal pathological changes and renal fibrosis were observed by hematoxylin and eosin (H&E) staining and Masson staining. The α-SMA in renal tissues was detected by immunohistochemistry. The inflammatory factors were analyzed by the ELISA assay. The cell apoptosis was observed by TUNEL assay. The related proteins of fibrosis, epithelial-mesenchymal transition (EMT) and apoptosis were assessed by western blot analysis. The experimental data demonstrated that hirudin decreased fibrosis, EMT, inflammation and cell apoptosis in renal tissues of UUO rats and TGF-β-induced renal tubular epithelial cells. Furthermore, Hirudin also reduced the expression of collgen-I, FN, α-SMA, N-cad, slug, E-cad, IL-1β, IL-6 and TNF-α in mice serum and TGF-β-induced renal tubular epithelial cells. The apoptosis related proteins (pro-caspase3, pro-caspase9, bcl2 and bax) expression was also down-regulated in renal tissues of UUO rats. In conclusion, hirudin depressed the fibrosis in renal tissues and renal tubular epithelial cells by inhibiting the inflammation, regulating the related proteins of fibrosis and ETM and decreasing the apoptosis of renal tubular epithelial cells. These findings may offer an effective treatment method for RIF [2].

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Hirudin reduces renal fibrosis in renal tissues induced by UUO model [2]
The pathologic changes of renal tissues were observed by H&E staining. UUO induced remarkable renal damage with many dilated and atrophic tubules. The hirudin intervention partially alleviated the above renal damages (Fig. 1A). There was obvious interstitial inflammation and collagen deposition in renal tissues induced by UUO model, as shown by Masson staining. The α-SMA expression assessed by immunohistochemistry was upregulated in renal tissues of UUO mice. The hirudin intervention suppressed the upregulation of α-SMA and collagen deposition in UUO mice (Fig. 1B and C).

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Hirudin regulates the expression of fibrosis-related proteins and ECM in renal tissues induced by UUO model [2]
The expression of fibrosis-related proteins and ECM were assessed by western blot analysis, and the former ones (collagen-I, FN and α-SMA) were obviously increased in renal tissues induced by UUO model while dwindled when UUO mice were treated with hirudin from 10 mg/kg to 15 mg/kg (Fig. 2A). The expression of ETM proteins was shown in Fig. 2B. The expression of N-cad and slug was increased and E-cad was decreased significantly in renal tissues induced by UUO model and hirudin gradually reversed the expression of N-cad, slug and E-cad from 10 mg/kg to 15 mg/kg.

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Hirudin decreases the expression of inflammatory factors in serum induced by UUO model [2]
The ELISA assay was used to detect the levels of IL-1β, IL-6 and TNF-α in the mouse serum. And, the levels of IL-1β, IL-6 and TNF-α in the mouse serum were upregulated in the serum of UUO mice. Hirudin treatment from 10 mg/kg to 15 mg/kg for the UUO mice could decline the levels of IL-1β, IL-6 and TNF-α in the serum (Fig. 3).

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Hirudin decreases the apoptosis of renal tubular cells in UUO mice [2]
The apoptosis of renal tubular cells in renal tissues induced by UUO model was showed by TUNEL assay. The apoptosis of renal tubular cells was increased in renal tissues of UUO mice and reduced when UUO mice were treated with hirudin from 10 mg/kg to 15 mg/kg (Fig. 4A). Accordingly, the expression of cleaved caspase3, cleaved caspase9 and bax was upregulated and the expression of bcl2 was downregulated in renal tubular cells of UUO mice. In addition, the expression of cleaved caspase3, cleaved caspase9, bax and bcl2 in renal tubular cells of UUO mice was reversed after the hirudin treatment. The expression of pro-caspase3 and pro-caspase9 in four groups was not changed (Fig. 4B).

Cell culture and cell induction [2]
The human HK-2 cells, mouse IMCD3 cells and rat NRK-52E cells were bought from ATCC. All three cell lines were incubated in the 90% high glucose DMEM and 10% FBS at 37 °C with 5% CO2. Then, the three kinds of cells were treated with 5 ng/ml TGF-β for 48 h to establish the vitro model.

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Hematoxylin and eosin (H&E) staining [2]
The kidney of mice was fixed in 4% paraformaldehyde for 48 h, then embedded in paraffin and cut into slices. The sections were put into xylene for 20 min, ethanol for 5 min and 75% ethanol for 5 min. Subsequently, the sections were put into hematoxylin for 3 min, washed with tap water and dehydrated by ethanol for 5 min. Finally, the sections were stained with eosin that was sealed with neutral gum.

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Masson staining [2]
The steps of dewaxing were the same as HE staining. The sections were soaked in potassium dichromate overnight and washed with tap water. Then, the sections were dyed with hematoxylin for 3 min, washed with tap water, dyed with ponceaux for 10 min and rinsed with tap water. Subsequently, the sections were stained with phosphomolybdic acid solution and aniline blue solution for 3 min respectively, which sealed with neutral gum.

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Immunohistochemistry [2]
The steps of dewaxing were the same as HE staining. The sections were heated for antigen retrieval at 95 °C for 30–45 min. Then, sections were cooled down at room temperature and soaked in 3% H2O2 methanol solution for 10 min. The goat serum was added to the sections to block at 25 °C for 10 min. Subsequently, the primary antibody, rabbit anti-α-SMA antibody, secondary antibody, biotin-labeled goat anti-rabbit antibody and horseradish peroxidase (HRP)-labeled streptavidin working solution were successively added to the sections. Then, the sections were colored with DAB Color Development Kit and counterstained with hematoxylin, followed by the rinse of tap water. Finally, the sections were blocked with neutral gum and observed under a microscope.

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Western blot analysis [2]
The renal tissues and renal tubular epithelial cells were obtained and lysed with the appropriate RIPA lysate. Then the supernatant of mixture was obtained after high speed centrifugation and BCA kits were used to detect the protein concentration in different groups. The required protein volume per lane was calculated according to the measured concentration and protein solution was boiled for 10 min. The proteins were separated with 10% SDS-PAGE and transferred by PVDF membrane. The PVDF membrane was sealed in the skim milk at 25 °C for 1 h. The primary antibodies for the protein detection of renal tissues were collgen-I, FN, α-SMA, E-cad, N-cad, slug, E-cad, cleaved caspase3, cleaved caspase9, pro-caspase3, pro-caspase9, bcl2 and bax. The primary antibodies for the protein detection of renal tubular epithelial cells were collgen-I, FN, α-SMA, E-cad, N-cad and slug. After PVDF membrane was incubated with primary antibodies overnight at 4 °C, PVDF membrane was washed by TBST and treated with rabbit horseradish peroxidase-linked IgG second antibody at 25 °C for 1 h. After the addition of ECL solution, PVDF membrane was exposed in dark and scanned by Bio-Rad gel documentation system. Finally, Quantity One software was used to analyze the area and gray value of each strip.

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Enzyme-linked immunosorbent assay (ELISA) [2]
Blood was taken from eyeball of mice and centrifuged at 5,000 rpm for 5 min to separate the serum. The levels of IL-1β, IL-6 and tumor necrosis factor α (TNF-α) in mice serum were determined with ELISA Kits. A Model 680 Microplate Reader was applied to obtain the optical density (OD) values at 450 nm.

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TUNEL assay The steps of dewaxing were the same as HE staining. Next, sections were incubated with Proteinase K at 25 °C for 15–30 min. After being washed by PBS twice, each section was added with 100 μL TdT reaction solution, which was incubated in a wet box at 37 °C for 1 h. Each section was washed by PBS for three times and incubated with 50 μL streptavidin fluorescein-dUTP reaction solution at 37 °C for 1 h. Finally, each section was sealed with 50 μL anti-fluorescence quenching agent for 30 min, which were then observed by a fluorescence microscopy.

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Hoechst staining [2]
The steps of dewaxing were the same as HE staining. The sections were placed in oscillating table to be washed by PBS twice. When the sections were sucked dry, they were treated with 0.5 ml Hoechst 33,258 for 5 min in oscillating table. The above cleaning procedure was repeated. Then, the sections were placed on the slide, added a drop of anti-quench sealing solution and covered with a clean slide. Blue nucleis of apoptotic cells could be observed by a fluorescence microscope.

Animal/Disease Models: Male balb/c (Bagg ALBino) mouse: with underwent unilateral ureteral ligation (UUO)[2]
Doses: 10 and 15 mg/kg
Route of Administration: po (oral gavage); 10 and 15 mg/kg, one time/day for 21 days
Experimental Results: diminished renal damages and suppressed the upregulation of α-SMA, collagen deposition in UUO mice. Increased the level of fibrosis (collagen-I, FN, α-SMA), N- cad, slug and E-cad in UUO mice. diminished the level of IL-1β, IL-6 and TNF-α, apoptosis of renal tubular cells in UUO mice. diminished the expression of inflammatory factors, the occurrence of EMT, the incidence of fibrosis and the apoptosis of TGF-β-induced renal tubular epithelial cell.

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In this experiment, mice underwent unilateral ureteral ligation (UUO) was adopted as the animal model. Forty male balb/c mice (25 g ± 3 g weight) were kept in SPF animal room with alternating light and dark for 12 h/time with humidity 60% and temperature 23 ± 3 °C and free access of water. The mice were randomly divided into four groups, including control group, UUO group, UUO + Hirudin (10 mg/kg) group and UUO + Hirudin (15 mg/kg) group, with ten mice in each group. All mice were weighed on the electronic balance to calculate the required anesthetic dose and then intraperitoneally injected with 2% pentobarbital (50 mg/kg). In the UUO group, mice were fixed on the rat board in the supine position and the abdominal skin of mice was disinfected with medical alcohol. The right abdominal incision (1.75 ± 0.25 cm) was taken and the peritoneum was opened. The right ureter was exposed and separated before its ligation with silk thread near the renal hilum and bladder. When no obvious bleeding appeared in the surgical field, the intestinal tract was rectified and the wound was sutured. In the UUO + Hirudin (10 mg/kg) group and UUO + Hirudin (15 mg/kg) group, mice were disposed with same surgery in UUO group and given the 10 mg/kg and 15 mg/kg Hirudin respectively by gavage once daily for duration of the study. The mice in the control group were not given any treatment. During the experiment, all the mice were free to eat and drink. On the 21th day after the operation, 1 ml blood was taken from eyeball of mice which then were sacrificed by cervical dislocation method and their right kidneys were obtained. [2]

Absorption, Distribution and Excretion
In nine healthy volunteers (both male and female), a single intravenous bolus injection of 0.4 mg/kg lepirudin resulted in a peak plasma concentration (Cmax) of 2924 ng/mL, a time to peak concentration (tmax) of 0.17 h, and an area under the curve (AUC0-∞) of 2500 ng•h/mL. In healthy male volunteers, single intravenous infusions of 0.1, 0.15, and 0.2 mg/kg lepirudin over 6 hours resulted in peak plasma concentrations (Cmax) of 111, 203, and 2446 ng/mL, respectively, with AUCs of 612, 1184, and 1446 ng•h/mL, respectively. The bioavailability after injection was 100%. Furthermore, it has been reported that the bioavailability of lepirudin is almost 100% after subcutaneous injection. Lepirudin is primarily excreted in the urine (48.3%). Approximately 35% of lepirudin is excreted unchanged, with the remaining metabolites accounting for smaller proportions (M1 2.5%, M2 5.4%, M3 3.9%, M4 1.6%). The steady-state volume of distribution of lepirudin was 12.2 L in healthy young adults (n=18, 18–60 years old), 18.7 L in healthy older adults (n=10, 65–80 years old), 18.0 L in subjects with renal impairment (n=16, creatinine clearance <80 mL/min), and 32.1 L in patients with heparin-induced thrombocytopenia (n=73). Lepirudin is primarily distributed in the extracellular fluid. The clearance of lepirudin is directly proportional to the glomerular filtration rate. On average, the clearance of lepirudin is 164 mL/min. The clearance in healthy young adults (n=18, 18–60 years old) was 139 mL/min, with women having a clearance 25% lower than men. The clearance rate in healthy elderly subjects (n=10, 65–80 years old) was 139 mL/min, approximately 20% lower than in younger patients. This is likely due to the lower creatinine clearance in older patients. The clearance rate in subjects with impaired renal function (n=16, creatinine clearance <80 mL/min) was 61 mL/min, and in patients with heparin-induced thrombocytopenic purpura (n=73) was 114 mL/min.

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Metabolism/Metabolites
As a polypeptide, lepilurodine is expected to be metabolized by the sequential cleavage of amino acids by extrarenal proteases with carboxypeptidase and dipeptidase-like activities. C-terminal cleavage of lepilurodine amino acids (amino acids 1 to 65) yields four metabolites with antithrombotic activity: M1 (amino acids 1 to 64), M2 (amino acids 1 to 63), M3 (amino acids 1 to 62), and M4 (amino acids 1 to 61).

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Biological Half-Life
The initial half-life of lepirudin is approximately 10 minutes, and its terminal half-life is 1.3 hours in young, healthy volunteers. The elimination kinetics of lepirudin are first-order; plasma concentrations increase proportionally with the intravenous dose of lepirudin. In patients with severely impaired renal function (creatinine clearance < 15 mL/min), elimination half-lives of up to 2 days have been detected.

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Pharmacokinetics[1]
Several pharmacokinetic studies have been conducted on hirudin and recombinant hirudin. Pharmacokinetic studies of hirudin have been conducted in a variety of animals, including rats, rabbits, dogs, and humans; detailed pharmacokinetic parameters for these studies are shown in Table 2. Due to the polypeptide structure of hirudin, oral administration is difficult to achieve effective concentrations. Therefore, hirudin is usually administered parenterally to improve bioavailability. The pharmacokinetic behavior of hirudin in different animals tends to be consistent. Following intravenous administration, the pharmacokinetics of hirudin followed a two-compartment open model (Markwardt et al., 1988b; Richter et al., 1988; Kaiser et al., 1990). The absorption half-life (t1/2α) indicates that hirudin rapidly distributes from the central compartment to the peripheral compartment. Furthermore, the elimination half-life (t1/2β) is approximately 1 hour, indicating rapid excretion and metabolism of hirudin. Additionally, hirudin exhibits high bioavailability. Following subcutaneous injection, hirudin clearance reached almost 100%, and the concentration-time curves showed that its plasma pharmacokinetics conformed to a one-compartment model (Nowak et al., 1988). Compared to intravenous administration, the elimination half-life (t1/2) was significantly prolonged in rats (2.1 h) and dogs (3.03 h). Similar to natural hirudin, recombinant hirudin (rH) rapidly distributes into the extravascular space, with a t1/2α of 0.08 to 0.25 h. Furthermore, the elimination half-life after intravenous injection is relatively long. Notably, in dogs, most of the administered hirudin and rH are cleared by the kidneys in their active form (Nowak et al., 1988). In addition, in healthy volunteers, renal clearance and degradation account for 90% of systemic clearance (Greinacher and Lubenow, 2000).
Other routes of administration of rH besides subcutaneous and intravenous injection have also been investigated. In rats, after intranasal spray administration, the in vivo process of rHV2 conformed to a single-compartment model, with a relative bioavailability of 28.53% (Zhang et al., 2006). In addition, Zhang et al. reported that the relative bioavailability (FR) of rHV2 liposomes and rHV2 saline after intranasal administration was 12.36% and 1.83%, respectively. These results indicate that the pharmacokinetics and bioavailability of rH are greatly affected by the dosage form (Zhang et al., 2007). In addition, Liu et al. compared the pharmacokinetics of rH in rats under four different routes of administration (tracheal, buccal, nasal and rectal) and found that... compared with the other three routes, the pulmonary administration route is more suitable for systemic administration of rHV2 (Liu et al., 2005).

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Preparation of hirudin[1]
In order to reduce adverse reactions and improve the bioavailability of hirudin, a variety of formulations have been reported in recent years, such as micelles, nanoparticles, TiO2 nanotube systems, polyamide dendritic polymers, etc. It is worth noting that the characteristics of these new formulations are mainly focused on prolonging circulation time, targeting thrombi, continuous administration and targeting specific diseases.

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Prolonged circulation time[1]
Hirudin can be rapidly distributed into the intercellular space after intravenous injection, but its half-life is short; repeated injections are required to maintain its therapeutic effect. However, repeated injections have some disadvantages (such as high cost and bleeding risk), which greatly limits the clinical application of hirudin. Hirudin. Bovine serum albumin (BSA) nanoparticles are considered a promising carrier that can solve these problems (Green et al., 2006). In 2015, researchers successfully synthesized and characterized hirudin-BSA nanoparticles using desolvation technology. These nanoparticles have good encapsulation efficiency and certain sustained-release capacity, which can control the release of hirudin, thereby prolonging its antithrombotic effect, indicating that hirudin-BSA nanoparticles may be applied to the clinical treatment of thrombosis. However, the pharmacokinetics and safety of these novel formulations still need further study (Jing et al., 2016). In addition to nanoparticles, polydopamine-modified titanium dioxide nanotube systems have also been used to prolong the release of hirudin. In 2018, Yang et al. reported that a polydopamine-modified titanium dioxide nanotube system could prolong the release of bivalirudin and improve its efficacy. It exhibited good blood compatibility both in vitro and in vivo. Furthermore, this system effectively reduced thrombus formation by inhibiting the denaturation and adhesion of platelets, fibrinogen, and other blood components (Yang et al., 2018). Although hirudin, after modification with platelet-targeting peptides such as RGD, can inhibit platelet aggregation, its short half-life remains a problem to be solved. In recent years, polyionic complex (PIC) micelles have attracted much attention due to their complementary and unique properties (Yang et al., 2009), which can prolong the circulation time of drugs in vivo. In this study, PIC micelles loaded with recombinant hirudin variant 2 (rHV2) were prepared. These micelles consisted of methoxy polyethylene glycol-grafted chitosan (mPEG-g-chitosan) and arginine-glycine-aspartic acid conjugated polyethylene glycol-grafted chitosan. (RGD-PEG-g-chitosan) was prepared by Wang et al. in 2010. These PIC micelles loaded with rHV2 had an average particle size of 41.9 ± 1.8 nm and an encapsulation efficiency of 81.08 ± 0.85%, which prolonged the average retention time of rHV2 and specifically bound to platelets. In addition, these micelles also exhibited effective anticoagulant and platelet aggregation inhibition effects, indicating that RGD-PIC micelles can achieve platelet-targeted delivery and long circulation of rHV2 (Wang et al., 2010).

Effects During Pregnancy and Lactation
◉ Overview of use during lactation Lepirudine has been discontinued in the United States. Limited information suggests that the concentration of lepirudine in breast milk is extremely low at daily doses up to 100 mg. Due to its large molecular weight, it is not expected that the infant will absorb it from breast milk. Lepirudine is not expected to have any adverse effects on breastfed infants, especially those older than 2 months. ◉ Effects on breastfed infants One infant was breastfed for 3 months while receiving therapeutic lepirudine treatment starting at 7 weeks of age. No bleeding events occurred. ◉ Effects on lactation and breast milk As of the revision date, no relevant published information was found. Protein binding In human plasma, lepirudine is bound to approximately 3% of the protein. Toxicity [1] Toxicity studies of hirudin and its derivatives have been conducted in several animal studies. Previous studies have shown that subcutaneous injection of 100 mg/kg hirudin for 8 days in rats resulted in pleural, leptomeningeal, and peritoneal hemorrhage. Furthermore, in subchronic toxicity studies in rats and dogs, daily administration of hirudin at doses ranging from 1 mg/kg to 5 mg/kg resulted in a dose-dependent increase in bleeding tendency lasting up to 3 months. Notably, antibody production was observed after administration of extremely high doses of hirudin in dogs, but these antibodies did not neutralize the effects of hirudin (Nowak, 2002). Interestingly, similar antibodies have also been detected in humans who have taken hirudin long-term (Eichler et al., 2000). However, antibody-bound hirudin still exhibits antithrombin activity as before, with a longer half-life, suggesting that these antibodies may be a storage form of hirudin. Furthermore, the median lethal dose (LD50) of orally administered lyophilized hirudin powder exceeded 10.0 g/kg, and there were no significant differences in micronucleus rate and sperm abnormality rate between the hirudin groups (2.5, 5.0, and 10.0 g/kg) and the control group (Huang et al., 2010). In addition, lyophilized hirudin powder did not exhibit maternal toxicity, embryotoxicity, or teratogenicity in rats at doses of 312.5, 1250, and 5000 mg/kg (Huang et al., 2011). Additionally, some studies have investigated the toxicity of recombinant hirudin (rH). Lu et al. found that rH (1.0, 3.0, and 6.0 mg/kg) significantly prolonged clotting time, thrombin time, and activated partial thromboplastin time in rhesus monkeys, but these effects were reversible within 24 hours after administration (Lu et al., 2004). These studies indicate that although hirudin has mild bleeding side effects, its toxicity is extremely low, thus it has broad application prospects and can be used for the prevention and treatment of various diseases.

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72941487 Mammalian (unspecified species) LD50 Intravenous injection >50 mg/kg Chinese Pharmacuetical Journal., 26(396), 1991

[1]. Pharmacological Activities and Mechanisms of Hirudin and Its Derivatives - A Review. Front Pharmacol. 2021 Apr 16;12:660757.

[2]. Hirudin improves renal interstitial fibrosis by reducing renal tubule injury and inflammation in unilateral ureteral obstruction (UUO) mice. Int Immunopharmacol. 2020 Apr;81:106249.

Lepirudin is a heterocyclic peptide consisting of 65 amino acids linked in sequence and cyclized by three disulfide bonds between cysteine residues 6-14, 16-28, and 22-39. It is a highly specific thrombin inhibitor used to treat heparin-induced thrombocytopenia. It is both an EC 3.4.21.5 (thrombin) inhibitor and an anticoagulant. It is a polypeptide, heterocyclic peptide, and organic disulfide. Lepirudin is a recombinant hirudin composed of 65 amino acids and acts as a highly specific direct thrombin inhibitor. Natural hirudin is an endogenous anticoagulant found in medicinal leeches (Hirudo medicinalis). Lepirudin is produced by yeast cells and has a structure essentially the same as natural hirudin, differing in that it lacks a sulfate group at the 63rd tyrosine residue and that the isoleucine at position 1 (N-terminus) is replaced by leucine. Lepirudin was used to treat heparin-induced thrombocytopenia (HIT), an immune response associated with a high risk of thromboembolic complications. HIT is caused by the expression of immunoglobulin G (IgG) antibodies that bind to a complex formed by heparin and platelet factor 4. This activates endothelial cells and platelets, promoting thrombus formation. Bayer ceased production of Lepirudin (Refludan) on May 31, 2012. Lepirudin is a yeast-derived recombinant polypeptide associated with hirudin, a naturally occurring anticoagulant derived from leeches. Lepirudin binds directly to and inactivates thrombin, resulting in a dose-dependent prolongation of activated partial thromboplastin time (aPTT) and prothrombin time (PT). The drug's mechanism of action is independent of antithrombin III and is not inhibited by platelet factor 4. Natural hirudin is a class of highly homologous heteropeptides produced in trace amounts by medicinal leeches (Hirudo medicinalis). Drug Indications Lepirudin is indicated for anticoagulation therapy in adult patients with acute coronary syndromes (ACS), such as unstable angina and non-ST-segment elevation myocardial infarction. In ACS patients, Lepirudin should be used in combination with aspirin. Lepirudin is also indicated for anticoagulation therapy in patients with heparin-induced thrombocytopenia (HIT) and related thromboembolic diseases to prevent further thromboembolic complications. For anticoagulation therapy in adult patients with type II heparin-induced thrombocytopenia and thromboembolic diseases requiring parenteral antithrombotic therapy. Diagnosis should be confirmed by a heparin-induced platelet activation test or equivalent test. Mechanism of Action Lepirudin is a direct thrombin inhibitor used for anticoagulation therapy in patients contraindicated for heparin. Thrombin is a serine protease involved in the blood coagulation cascade, formed by the cleavage of prothrombin. Active thrombin cleaves fibrinogen to generate fibrin monomers, which polymerize to form fibrin clots. Lepirudin binds to the catalytic and substrate-binding sites of thrombin, forming a stable, irreversible, non-covalent complex. This blocks the protease activity of thrombin, thereby inhibiting the coagulation process. Each Lepirudin molecule binds to only one thrombin molecule, unlike heparin, and it inhibits the activity of thrombin in both thrombus-bound and free states.
Pharmacodynamics
Lepirudin is a recombinant hirudin, a highly specific thrombin inhibitor. Its activity is measured in antithrombin units (ATU), which refers to the amount of Lepirudin required to neutralize one World Health Organization α-thrombin (89/588) standard unit. The activity of Lepirudin is 16,000 ATU/mg. A single lepirudine molecule binds to a thrombin molecule, blocking its prothrombotic activity. This drug prolongs activated partial thromboplastin time (aPTT) and prothrombin time (PT/INR) in a dose-dependent manner, and its mechanism of action is independent of antithrombin III. Platelet factor 4 does not inhibit lepirudine. The pharmacodynamic effects of lepirudine are assessed by measuring the prolongation of aPTT. No saturation effect was observed at the highest tested dose (0.5 mg/kg, intravenous bolus). Because high thrombin time values (200 seconds) can be detected even at low doses, thrombin time is not considered suitable as a routine indicator for lepirudine monitoring. Due to the high risk of bleeding, which may be life-threatening, concomitant use of thrombolytic therapy and lepirudine is not recommended. For patients at risk of bleeding, physicians should weigh the risks and benefits of lepirudine use. Patients weighing less than 50 kg have a particularly high risk of bleeding and therefore require dose reduction. Patients with renal insufficiency have an even higher risk of bleeding adverse events. Hirudin is a single-chain polypeptide produced by leeches, containing approximately 65 amino acids (7 kDa), with a neutral hydrophobic N-terminus, an acidic hydrophilic C-terminus, and a compact hydrophobic core region. Recombinant hirudin lacks Tyr-63 sulfation modification and is called "desulfurized hirudin." It forms a stable non-covalent complex with α-thrombin, thereby eliminating its ability to cleave fibrinogen. Significant research achievements have been made in hirudin over the past few decades, including its clinical applications, the development of derivatives, the exploration of novel pharmacological activities, and the research of new formulations. Successful clinical applications of hirudin derivatives: With the development of genetic engineering technology, the production challenges of natural hirudin have been successfully solved, which has greatly promoted the successful clinical application of lepirudin, disiludin, and bivalirudin. Lepirudin has been approved for the treatment of heparin-induced thrombocytopenia (HIT), usually administered as a 0.4 mg/kg intravenous bolus followed by an intravenous infusion at a rate of 0.15 mg/kg/h (Greinacher and Lubenow, 2001). It reduces HIT-related adverse events by 72%, providing effective anticoagulation therapy and promoting rapid platelet recovery in HIT patients. It is important to note that the bolus and infusion rates should be adjusted according to the patient's serum creatinine levels (Greinacher et al., 1999). Diasiludine is used to prevent deep vein thrombosis (DVT) in patients undergoing total hip replacement (THR) and total knee replacement (TKR), administered subcutaneously. Previous studies have reported that in patients undergoing elective hip replacement surgery, 15 mg (subcutaneously every 12 hours) of disiludin is superior to low-dose unfractionated heparin and low molecular weight heparin in preventing venous thromboembolism (VTE) (Eriksson et al., 1997; Bergese et al., 2013). Another direct thrombin inhibitor, bivalirudin, is also effective in preventing thromboembolic complications. In a reported trial, bivalirudin was initially administered as a bolus injection of 1.0 mg/kg intravenously, followed by a continuous intravenous infusion at a rate of 2.5 mg/kg/h for 4 hours; subsequently, the bolus dose was reduced to 0.75 mg/kg, followed by a continuous infusion at a rate of 1.75 mg/kg/h for 4 hours. The results showed that 98% of patients had successful surgery and 96% had good clinical outcomes (Sun et al., 2017). In addition, bivalirudin is also undergoing phase II and III multicenter trials as an alternative anticoagulant for cardiopulmonary bypass and off-pump cardiac surgery in patients with or without HIT (Joseph et al., 2014). [1]

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Future Prospects:
Although research on hirudin has covered pharmacology, pharmacology, formulation and clinical applications, we have found some gaps, particularly in the study of dose-response relationships, toxicity and safety of its derivatives and formulations. This section focuses on potential directions for future research on hirudin.
Given the diverse pharmacological activities of hirudin, elucidating its dose-response relationships will help determine the clinical treatment doses for different diseases in the future. It is noteworthy that hirudin exhibits different activities and even opposite biological effects at different concentrations. For example, VEGF expression increased when hirudin was used to treat flap necrosis; however, VEGF expression was inhibited when hirudin was used to treat diabetic complications and tumors. This difference may be due to the different doses of hirudin used in the two studies. Low concentrations (1 and 4 ATU/ml) of hirudin promote cell proliferation and microangiogenesis by upregulating VEGF, which is crucial for wound repair. However, at higher concentrations (7 ATU/ml), the promoting effect of hirudin on the VEGF/Notch signaling pathway gradually weakens and transforms into an inhibitory effect. Correspondingly, VEGF expression decreases, indicating that high concentrations of hirudin can inhibit cell growth and exert anti-inflammatory effects, which is beneficial for the treatment of diabetic complications and tumors. Similarly, the effect of hirudin on the ERK1/2 pathway is bidirectional. At a dose of 2 ATU, hirudin significantly increases the expression level of ERK1/2 phosphorylation; activated ERK promotes the transcription of VEGF signaling molecules by regulating the expression of AP-1 and c-Jun, thereby promoting angiogenesis in ischemic flap rats. However, at other doses (20, 40, and 80 μg/ml), hirudin downregulates the expression of ERK1/2 phosphorylation, thereby participating in the treatment of fibrosis. Table 4 provides detailed information on the pharmacological activities, routes of administration, effective doses, and molecular mechanisms of hirudin. It is worth noting that, in addition to concentration, different routes of administration and different animal models or cells used in different studies may also lead to differences in biological activity. Therefore, more in vivo studies are needed to elucidate the dose-response relationship of hirudin. [1]

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Although hirudin has been successfully used in clinical thrombosis treatment, its short half-life and bleeding risk remain limiting factors, so future research could focus on structural modification. Perhaps, fusing the recognition peptides identified from FXIa and FXa with these drugs is an effective way to reduce the bleeding risk. In addition, research on hirudin derivatives and formulations is still in its early stages and further research is needed to reveal their effects, toxicity, and potential mechanisms.
In conclusion, hirudin has a wide range of pharmacological activities and great potential for clinical application, and deserves more in-depth and comprehensive research. [1]

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Hirudin has a strong inhibitory effect on thrombin and has anticoagulant, antithrombotic, antitumor, and antifibrotic functions. Hirudin can reduce the deposition of fibrin-associated antigens in the glomeruli, inhibit the proliferation of glomerular mesangial cells and glomerulosclerosis, and reduce proteinuria and hypoalbuminemia, thereby improving renal function. Hirudin may downregulate the expression of TGF-β and α-SMA, reduce the proliferation and activation of fibroblast growth factors and fibroblast phenotypic transformation, thereby preventing and treating renal interstitial fibrosis. This study found that hirudin can reduce the inflammatory response and EMT of renal tubular epithelial cells, thereby inhibiting the fibrosis and apoptosis of renal tubular epithelial cells. Hirudin inhibits the fibrosis of renal tissue and renal tubular epithelial cells by inhibiting inflammation, regulating fibrosis-associated proteins and extracellular matrix (ECM) and reducing renal tubular epithelial cell apoptosis. However, natural products usually have the potential risk of liver damage. Since the study found that natural products have a protective effect against kidney damage, we will also investigate the effects of natural products on the liver in future studies. [2]

C287H440N80O111S6

6979.42396068573

6977.967

138068-37-8

Hirudin;8001-27-2

118856773

H-Leu-Thr-Tyr-Thr-Asp-Cys(1)-Thr-Glu-Ser-Gly-Gln-Asn-Leu-Cys(1)-Leu-Cys(2)-Glu-Gly-Ser-Asn-Val-Cys(3)-Gly-Gln-Gly-Asn-Lys-Cys(2)-Ile-Leu-Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys(3)-Val-Thr-Gly-Glu-Gly-Thr-Pro-Lys-Pro-Gln-Ser-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln-OH
L-leucyl-L-threonyl-L-tyrosyl-L-threonyl-L-alpha-aspartyl-L-cysteinyl-L-threonyl-L-alpha-glutamyl-L-seryl-glycyl-L-glutaminyl-L-asparagyl-L-leucyl-L-cysteinyl-L-leucyl-L-cysteinyl-L-alpha-glutamyl-glycyl-L-seryl-L-asparagyl-L-valyl-L-cysteinyl-glycyl-L-glutaminyl-glycyl-L-asparagyl-L-lysyl-L-cysteinyl-L-isoleucyl-L-leucyl-glycyl-L-seryl-L-alpha-aspartyl-glycyl-L-alpha-glutamyl-L-lysyl-L-asparagyl-L-glutaminyl-L-cysteinyl-L-valyl-L-threonyl-glycyl-L-alpha-glutamyl-glycyl-L-threonyl-L-prolyl-L-lysyl-L-prolyl-L-glutaminyl-L-seryl-L-histidyl-L-asparagyl-L-alpha-aspartyl-glycyl-L-alpha-aspartyl-L-phenylalanyl-L-alpha-glutamyl-L-alpha-glutamyl-L-isoleucyl-L-prolyl-L-alpha-glutamyl-L-alpha-glutamyl-L-tyrosyl-L-leucyl-L-glutamine (6->14),(16->28),(22->39)-tris(disulfide)
PEPTIDE1{L.T.Y.T.D.C.T.E.S.G.Q.N.L.C.L.C.E.G.S.N.V.C.G.Q.G.N.K.C.I.L.G.S.D.G.E.K.N.Q.C.V.T.G.E.G.T.P.K.P.Q.S.H.N.D.G.D.F.E.E.I.P.E.E.Y.L.Q}$PEPTIDE1,PEPTIDE1,6:R3-14:R3|PEPTIDE1,PEPTIDE1,16:R3-28:R3|PEPTIDE1,PEPTIDE1,22:R3-39:R3$$$

LTYTDCTESGQNLCLCEGSNVCGQGNKCILGSDGEKNQCVTGEGTPKPQSHNDGDFEEIPEEYLQ
H-LTYTDC(1)TESGQNLC(1)LC(2)EGSNVC(3)GQGNKC(2)ILGSDGEKNQC(3)VTGEGTPKPQSHNDGDFEEIPEEYLQ-OH

Typically exists as solid at room temperature

65 °C

-41.3

100

122

166

484

18600

63

S1C[C@@H](C(N[C@@H](C(C)C)C(N[C@@H]([C@@H](C)O)C(NCC(N[C@@H](CCC(=O)O)C(NCC(N[C@@H]([C@@H](C)O)C(N2CCC[C@H]2C(N[C@@H](CCCCN)C(N2CCC[C@H]2C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(NCC(N[C@@H](CC(=O)O)C(N[C@@H](CC2C=CC=CC=2)C(N[C@@H](CCC(=O)O)C(N[C@@H](CCC(=O)O)C(N[C@@H]([C@@H](C)CC)C(N2CCC[C@H]2C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(=O)O)CCC(N)=O)=O)CC(C)C)=O)CC2C=CC(=CC=2)O)=O)CCC(=O)O)=O)CCC(=O)O)=O)=O)=O)=O)=O)=O)=O)=O)CC(=O)O)=O)CC(N)=O)=O)CC2=CNC=N2)=O)CO)=O)CCC(N)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)NC([C@H](CCC(N)=O)NC([C@H](CC(N)=O)NC([C@H](CCCCN)NC([C@H](CCC(=O)O)NC(CNC([C@H](CC(=O)O)NC([C@H](CO)NC(CNC([C@H](CC(C)C)NC([C@H]([C@@H](C)CC)NC([C@@H]2CSSC[C@@H](C(N[C@@H](CCC(=O)O)C(NCC(N[C@@H](CO)C(N[C@@H](CC(N)=O)C(N[C@@H](C(C)C)C(N[C@H](C(NCC(N[C@H](C(NCC(N[C@@H](CC(N)=O)C(N[C@H](C(N2)=O)CCCCN)=O)=O)=O)CCC(N)=O)=O)=O)CS1)=O)=O)=O)=O)=O)=O)NC([C@H](CC(C)C)NC([C@@H]1CSSC[C@@H](C(N[C@H](C(N[C@H](C(N[C@@H](CO)C(NCC(N[C@@H](CCC(N)=O)C(N[C@@H](CC(N)=O)C(N[C@H](C(N1)=O)CC(C)C)=O)=O)=O)=O)=O)CCC(=O)O)=O)[C@@H](C)O)=O)NC([C@H](CC(=O)O)NC([C@H]([C@@H](C)O)NC([C@H](CC1C=CC(=CC=1)O)NC([C@H]([C@@H](C)O)NC([C@H](CC(C)C)N)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O

FIBJDTSHOUXTKV-BRHMIFOHSA-N

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(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S,3S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-1-[(2S)-6-amino-2-[[(2S)-1-[(2S,3R)-2-[[2-[[(2S)-2-[[2-[[(2S,3R)-2-[[(2S)-2-[[(1R,6R,9S,12S,15S,18S,24S,27S,33S,36S,39R,44R,47S,53S,56S,59S,67S,73S,76S)-15,76-bis(4-aminobutyl)-44-[[(2S)-2-[[(4R,7S,10S,13S,19S,22S,25S,28R)-28-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-amino-4-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-3-hydroxybutanoyl]amino]-3-carboxypropanoyl]amino]-10-(2-amino-2-oxoethyl)-13-(3-amino-3-oxopropyl)-22-(2-carboxyethyl)-25-[(1R)-1-hydroxyethyl]-19-(hydroxymethyl)-7-(2-methylpropyl)-6,9,12,15,18,21,24,27-octaoxo-1,2-dithia-5,8,11,14,17,20,23,26-octazacyclononacosane-4-carbonyl]amino]-4-methylpentanoyl]amino]-12,56,73-tris(2-amino-2-oxoethyl)-9,67-bis(3-amino-3-oxopropyl)-36-[(2S)-butan-2-yl]-18,47-bis(2-carboxyethyl)-24-(carboxymethyl)-27,53-bis(hydroxymethyl)-33-(2-methylpropyl)-8,11,14,17,20,23,26,29,32,35,38,45,48,51,54,57,60,62,65,68,71,74,77-tricosaoxo-59-propan-2-yl-3,4,41,42-tetrathia-7,10,13,16,19,22,25,28,31,34,37,46,49,52,55,58,61,63,66,69,72,75,78-tricosazabicyclo[37.22.17]octaheptacontane-6-carbonyl]amino]-3-methylbutanoyl]amino]-3-hydroxybutanoyl]amino]acetyl]amino]-4-carboxybutanoyl]amino]acetyl]amino]-3-hydroxybutanoyl]pyrrolidine-2-carbonyl]amino]hexanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-4-yl)propanoyl]amino]-4-oxobutanoyl]amino]-3-carboxypropanoyl]amino]acetyl]amino]-3-carboxypropanoyl]amino]-3-phenylpropanoyl]amino]-4-carboxybutanoyl]amino]-4-carboxybutanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbonyl]amino]-4-carboxybutanoyl]amino]-4-carboxybutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-methylpentanoyl]amino]-5-oxopentanoic acid

Lepirudin; Refludan; Lepirudin recombinant; 1-Leu-2-thr-63-desulfohirudin; Hbw 023; Lepirudin [INN:BAN]; UNII-Y43GF64R34; 138068-37-8;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)