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Nov 9, 2016 - Seoul National University, Seoul 151-742, South Korea. •S Supporting ... characteristic of enoxaparin is that its end component consis...
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End-Site-Specific Conjugation of Enoxaparin and Tetradeoxycholic Acid Using Nonenzymatic Glycosylation for Oral Delivery Jooho Park,†,‡ Ok Cheol Jeon,§ Jisuk Yun,∥ Hwajung Nam,∥ Jinha Hwang,∥ Taslim A. Al-Hilal,‡ Kwangmeyung Kim,‡ Kyungjin Kim,∥ and Youngro Byun*,†,⊥ †

Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, South Korea Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 136-791, South Korea § B&L DELIPHARM, Seoul 151-742, South Korea ∥ ST Pharm Research & Development Center, HyeopRyeok Road, Siheung-Si, Gyeonggi-do, South Korea ⊥ Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul 151-742, South Korea ‡

S Supporting Information *

ABSTRACT: Heparin and low molecular weight heparins (LMWHs) have been the drug of choice for the treatment or the prevention of thromboembolic disease. Different methods are employed to prepare the LMWHs that are clinically approved for the market currently. In particular, enoxaparin, which has a reducing sugar moiety at the end-site of polysaccharide, is prepared by alkaline depolymerization. Focusing on this end-site-specific activity of LMWHs, we conjugated the tetraoligomer of deoxycholic acid (TetraDOCA; TD) at the end-site of enoxaparin via nonenzymatic glycosylation reaction. The end-site-specific conjugation is important for polysaccharide drug development because of the heterogeneity of polysaccharides. This study also showed that orally active enoxaparin and tetraDOCA conjugate (EnoxaTD) had therapeutic effect on deep vein thrombosis (DVT) without bleeding in animal models. Considering the importance of end-specific conjugation, these results suggest that EnoxaTD could be a drug candidate for oral heparin development.

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depolymerization from UFH.11,12 Nadroparin, dalteparin, and reviparin can be prepared by deaminative cleavage with nitrous acid, while tinzaparin can be obtained using heparinase.13 Currently, enoxaparin sodium is the best-selling heparin in the market.14 Among the different LMWHs, enoxaparin has been prepared by alkaline depolymerization with benzethonium salt. This method of using controlled depolymerization of natural heparin from porcine intestinal mucosa is unique; it contributed to gain a maximum market of LMWHs.15 One characteristic of enoxaparin is that its end component consists of a “reducing end”, referring to a monosaccharide residue end of a polysaccharide taking the acetal form, a configuration also related to glycosylation reaction. About 80% of the end components of enoxaparin contain reducing ends.16,17

INTRODUCTION Anticoagulant therapy with heparin is widely used in healthcare therapeutics.1,2 Thrombotic disorders such as deep vein thrombosis, pulmonary embolism, and stroke are potential lethal diseases, requiring patients at high risk to be treated by anticoagulants such as heparin.3 Heparin has been the drug of choice for the treatment or the prevention of thromboembolic disease and is also a safe carbohydrate drug extracted from animal sources.4−6 Among different forms of heparins, low molecular weight heparin (LMWH) is more effective and has more predictable effects than unfractionated heparin (UFH) on preventing blood coagulation because of its lower bleeding risk, higher bioavailability, and predictable effects.7−9 There are several kinds of clinically approved LMWHs, which were introduced in the past decades.10 The average molecular weight of LMWHs is usually 4000−5000 Da, which is lower than that of UFH (12 000−15 000 Da). LMWHs are generally prepared by controlled chemical or enzymatic © 2016 American Chemical Society

Received: June 23, 2016 Published: November 9, 2016 10520

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Figure 1. (A) Molecular structure of enoxaparin. (B) Nonenzymatic glycosylation of polysaccharide and glucose. Enoxaparin, which has a reducing sugar moiety at the end of structure, reacted with Benedict’s reagent, and then the color of solution was changed.

Glycosylation is an important reaction in the field of glycochemistry for synthesizing natural glycoproteins and glycans. Chemical or nonenzymatic glycosylation can also serve as a general strategy for modulating the end of monosaccharides.18,19 Glycosylation with oligosaccharides is one of the most challenging chemical techniques in organic synthesis; however, in the case of polysaccharides, the glycosyl donors of polysaccharides usually used in glycosylation are too stable and rare to be modified by chemical reaction due to their enormous size and dynamics.20−22 Nevertheless, the end conjugation of oligosaccharides using nonenzymatic glycosylation has shown great possibilities for polysaccharide endspecific conjugation.23,24 The clinical use of enoxaparin can be extended to nonenzymatic glycosylation reaction. Chemical end-site-specific conjugation has provided several advantages such as monitoring in vivo tissue distribution by dye conjugation maintaining its biological action.25 Furthermore, biological functional groups might be introduced into the end-site of enoxaparin. The oral delivery of enoxaparin by conjugation would increase its therapeutic use because the application of heparin has always been limited by its requirement for injections.26 In our previous studies, we introduced several kinds of nadroparin and bile acid conjugates.27−30 It was shown that DOCA conjugation promotes the intestinal absorption of nadroparin, which is a well-known LMWH.31,32 We had also designed a nadroparin and tetraDOCA conjugate to facilitate the oral administration of nadroparin; however, this particular conjugation is not end-site-specific because the end-site aldehyde group of nadroparin is completely eliminated after commercial processes and the tail end of polysaccharide of nadroparin is not chemically active either.1,33−35 Moreover, in

the design of a drug with a polysaccharide conjugate, the sideconjugation of nadroparin is not preferred due to heparin’s heterogeneity and complexity. On the other hand, since the recognition of heparin by antithrombin III depends on a specific heparin sequence, the precise end-site-specific conjugation would be highly preferable for its therapeutic effect and drug development.6,36 In the present study, we designed enoxaparin and tetraDOCA conjugate (EnoxaTD) using chemical glycosylation for end-site-specific reactivity for oral heparin delivery. First, the end-site reactivity of heparin was confirmed using different kinds of LMWHs and Benedict’s reagent. Second, we optimized the synthesis processes to synthesize new tetraDOCA (TD). Then TD was conjugated to the tail end of enoxaparin by nonenzymatic glycosylation. The conjugate was confirmed by several methods of characterization. Finally, we evaluated the oral absorption of EnoxaTD and therapeutic anticoagulant effects of EnoxaTD in animal models.

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RESULTS Confirmation of End-Site-Specific Activity. The endsite-specific activities of glucose, enoxaparin, and tinzaparin were evaluated using Benedict’s reagent (blue alkaline solution). LMWHs such as enoxaparin, nadroparin, and tinzaparin have the same major sequences in the middle of molecular structure, although the end-site of each polysaccharide is distinctly different due to different degrading patterns of unfractionated heparin. Among them, the molecular structure of enoxaparin is shown in Figure 1A. Since enoxaparin has a reducing sugar moiety at the end-site of polysaccharide, it reduced Cu2+ to Cu+ in Benedict’s reagent when forming the precipitate of Cu2O (Figure 1B). Glucose, which was used as 10521

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Figure 2. Chemical synthesis of EnoxaTD by nonenzymatic glycosylation. The total synthesis process was optimized to seven steps. In the last step, enoxaparin was conjugated with compound 6, and then NaBH3CN was added to reduce the Schiff base into second amine in the product.

The molecular binding between the antithrombin III (ATIII) and EnoxaTD was also virtually simulated, which showed that the end-site-specific conjugation would not affect the biological activity of enoxaparin (Figure 3D). After the conjugation, the anticoagulant activity (94.7 ± 4.6 IU/mL) of EnoxaTD was not significantly reduced compared to that (103.3 ± 10.4 IU/mL) of enoxaparin (Figure 3E). The end-site-specific conjugation of EnoxaTD, confirmed by using Benedict’s reagent, was compared with that of enoxaparin. The color of EnoxaTD solution (40 mg/mL) was slightly changed due to the presence of unreacted enoxaparin, whereas the color of enoxaparin (40 mg/mL) had completely changed (Figure 3F). In addition, we evaluated cell viability using CCK-8 assay to determine the cytotoxicity of EnoxaTD. Madin−Darby canine kidney (MDCK) cells were used for viability test because they are suitable to form stable monolayers just like epithelial cells and usually used in various membrane permeability studies. The results showed that EnoxaTD was not cytotoxic, and the viability of MDCK cell was not decreased even when EnoxaTD was applied at different concentrations (100−2000 μg/mL) for 24 h (Figure 3G). Characterization with Depolymerization. EnoxaTD was degraded and analyzed to find out the molecular ratios of the product and its degraded constituents. After depolymerization with nitrous acid, water-insoluble materials were precipitated in water (Figure 4A). The property of water-insoluble materials to be dissolved in methanol and ethanol is also observed for TD. EnoxaTD was degraded in water and analyzed by using MALDI-TOF. The degraded constituents of enoxaparin and EnoxaTD reacted similarly in water (Figure 4B). Also, the NMR peaks of water-insoluble material from EnoxaTD were matched with the NMR peaks of TD, as shown in Figure 4C.

control, was rapidly changed by the reaction but tinzaparin was not. Although it is generally known that polysaccharides are not active to Benedict’s reagent compared with monosaccharides, we found that enoxaparin could react with Benedict’s reagent in a prolonged reaction time, so we were able to prove its endspecific reactivity. Synthesis and Characterization. All synthesized compounds were initially evaluated by using TLC, NMR, and mass chromatography. The optimized synthesis process is shown in Figure 2: the seven steps summarized here were required to synthesize EnoxaTD from L-lysine and enoxaparin. At first step, two different lysine conjugates were prepared. Then, the flexible peptide with four primary amines was synthesized. All the synthetic processes were optimized for commercial production. The final conjugate with enoxaparin and TD was characterized in different ways. EnoxaTD is the enoxaparin conjugate with TD by using nonenzymatic glycosylation, and its molecular structure is provided in Figure 3A. The proton NMR result of it was not clear in water (D2O). However, it can show the apparent peaks related to enoxaparin and TD in water/organic (DMSO-d6, 75%) cosolvent mixture (Figure 3B). On the other hand, the conjugation ratio was calculated by using the sulfuric acid assay, NMR, and the acid depolymerization assay (Figure 3C). The coupling ratio of enoxaparin and TD in EnoxaTD was 0.72 ± 0.13, 0.84 ± 0.17, and 0.83 ± 0.04 by sulfuric acid assay, NMR, and acid depolymerization assay (fragment analysis), respectively. The mixtures of various molecular ratios of enoxaparin and TD were used in NMR and the sulfuric assay to calculate the conjugation ratio. On the other hand, the acid depolymerization assay depended on the mass of degraded products after nitrous acid depolymerization. 10522

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Figure 3. (A) Molecular structure of EnoxaTD. (B) 1H NMR data of EnoxaTD. (C) Conjugation ratio was determined in different methods. (D) Computer simulation of antithrombin and EnoxaTD (dp5) to visualize the end-specific conjugation effect from antithrombin−heparin complex (PDB code 1TB6). (E) Anti-FXa activity. (F) Reaction of enoxaparin and EnoxaTD with Benedict’s reagent (40 mg/mL). (G) Cell viability with MDCK cell in high dose (100−2000 μg/mL).

Analysis of End-Specific Conjugation by 2D NMR. The end-specific conjugation of enoxaparin was determined by 2D NMR after the confirmation with Benedict’s reagent. In particular, the anomeric carbons in enoxaparin were analyzed to confirm the conjugation (Figure 5A). Enoxaparin has anomeric carbons in the reducing and nonreducing end of its molecular structure (Figure 5B). Two-dimensional (2D) 1 H−13C HSQC (heteronuclear single-quantum coherence) NMR was used to analyze the protons at the end of enoxaparin. The strength of proton peak in the reducing end is significantly lower than that of the nonreducing end in enoxaparin because of the relatively small amount (about 80%) and existence of a reactive contributing structure. First, as shown in Figure 1, the reducible end in heparin has a resonance or contributing structure, so it has reactivity and can disrupt the strength of proton peak. Second, the value from the HPLC analysis does not exactly match up with the peaks in NMR because of the relativity and complexity of 2D-COSY NMR. The 2D HSQC NMR results showed that the proton (RE) that existed at the end of enoxaparin disappeared in the anomeric region after end-specific conjugation (Figure 5C). On the other hand, the proton in nonreducing end of enoxaparin still remained. PK Study. The main motive for the chemical glycosylation of enoxaparin was to increase the bioavailability of nonabsorbable enoxaparin in oral delivery. The oral absorption of bile acid conjugates and their absorption mechanism were confirmed in the previous studies.33,34 In those studies,

tetraDOCA (compound 6) showed the highest binding affinity to ASBT when compared to monoDOCA, bisDOCA, triDOCA, and tetraDOCA, and the conjugate of enoxaparin and tetraDOCA showed the highest oral bioavailability. Because enoxaparin cannot be measured directly in the plasma, the anti-FXa profile was used to study pharmacokinetic profiles. Intravenously injected EnoxaTD in rats showed similar effects to that of enoxapain in vivo (Figure 6A). However, the orally administrated enoxaparin shows lower absorption than EnoxaTD when orally delivered (Figure 6B), as indicated by their respective maximum concentrations of 0.60 ± 0.08 IU/ mL and 0.17 ± 0.05 IU/mL. The detailed pharmacokinetic profile of enoxaparin and EnoxaTD is given in Table 1. DVT and Bleeding Experiments. The therapeutic effect of EnoxaTD was evaluated by using a venous thrombosis animal model with SD rats (Figure 7). Normal saline was administered orally as control. The wet weight of the thrombus in the vena cava was 49.3 ± 10.8 mg in the control group. The weights of mice treated with 10 mg/kg of enoxaparin (subcutaneous), 5 mg/kg of EnoxaTD (oral), and 10 mg/kg of EnoxaTD (oral) were 11.4 ± 2.7, 12.5 ± 2.0, and 6.05 ± 2.1, respectively. The group administered orally with EnoxaTD showed reduced thrombus formation due to the oral absorption of EnoxaTD. The side effects of anticoagulants were taken into consideration when the primary bleeding time in rats was calculated. Primary bleeding that did not exceed 600 s was observed after cutting. The rat’s tail was cut using a scalpel 10523

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Figure 4. (A) Scheme of molecular reaction by nitrous acid depolymerization and the picture of results. (B) Similar MALDI-TOF results from water-soluble degradation products. (C) 1H NMR data of water-insoluble degradation products from EnoxaTD compared to that of TD.

saccharides such as starches and dextrins do not react with Benedict’s reagent considering that the reaction rarely happens only at the ends of long carbohydrate chains. EnoxaTD exhibits similar properties as enoxaparin except for its oral uptake property in the GI tract owing to end-specific conjugation. End-specific conjugation is an important property to consider in polysaccharide-based drug development because conjugation itself cannot change the therapeutic properties of polysaccharides. In the case of EnoxaTD, its anti-FXa activity is maintained even after the conjugation by end-specific conjugation. In addition, the TD moiety in EnoxaTD does not affect the molecular binding between the heparin saccharide (dp5) and the heparin binding site in antithrombin because the responsible binding force in this case is van der Waals force, whereas heparin usually binds heparin-binding proteins by electrostatic interaction. Since hydrophobic effect is revealed only with solubilizers or organic solvents such as DMF or DMSO, the water fearing property of TD can be used for the analysis of EnoxaTD. After depolymerization of EnoxaTD with nitrous acid, the hydrophobic part of EnoxaTD is precipitated out from the water solution. The mass and NMR spectra of water-insoluble materials were similar to those of TD, demonstrating that TD in EnoxaTD is expelled after depolymerization.

blade after administration of enoxaparin and EnoxaTD. The primary clotting (bleeding) time for the control group was 225.2 ± 57.0 s. In contrast, the primary clotting times for groups treated with EnoxaTD (oral, 1 h) and enoxaparin (subcutaneous, 1 h) were 332.0 ± 74.7 s and 380.0 ± 51.6 s, respectively (Figure 8A). The detailed profile of bleeding time of enoxaparin and EnoxaTD is shown in Figure 8B and Figure 8C.

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DISCUSSION Several anticoagulants such as UFH, LMWH, VLWMH, and vitamin K antagonist are presently available in the field of clinical thromboembolism, along with new FXa inhibitors. These newly developed direct factor Xa inhibitors, including rivaroxaban, apixaban, and edoxaban, have been used to treat patients with venous thromboembolic (VTE) disorders, since they are orally available. The oral delivery of anticoagulants is also clinically important because it can be commonly used for prevention. While heparin is a safe, effective, and animalderived agent, it is not absorbed in the gastrointestinal (GI) tract. Benedict’s solution, developed by an American chemist Stanley Rossiter and clinically used as the reagent of choice for measuring sugar content, can detect reducing sugars with free aldehyde or ketone groups.37 It is well-known that poly10524

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Figure 6. (A) Anti-FXa activity of intravenously injected enoxaparin and EnoxaTD in rats. (B) Anti-FXa activity of orally administered enoxaparin and EnoxaTD: (∗) p < 0.05 vs enoxaparin (po) group.

DOCA and ASBT in the GI tract. Previous studies showed that heparin is not orally available without DOCA conjugation because heparin is a very hydrophilic macromolecule with poor bioavailability.30,31 In this study, compound 6 (tetraDOCA) was conjugated to enoxaparin so that enoxaparin could be successfully absorbed in animal studies. In the previous study, we found that EnoxaTD, being a highaffinity substrate, induced the receptor-like functional transformation of ASBT.33 This phenomenon allowed the apical-tobasal transport of ASBT/EnoxaTD complexes by forming vesicles. In the cytoplasm, ASBT/EnoxaTD complexes were dissociated when EnoxaTD interacted with ileal bile acid binding protein (IBABP). This process eventually led to the exocytosis of EnoxaTD and prevented its entry into the lysosomal compartment. Therefore, the functional transformation of ASBT that induced vesicular transport enabled the transport of macromolecular EnoxaTD. On the basis of the successful oral delivery of EnoxaTD, we further investigated its therapeutic effects in a disease model of DVT and bleeding. DVT, the formation of blood clot in a deep vein, predominantly found in the leg, leads to pulmonary embolism, which is a potentially life-threatening complication. For decades, heparin has been the drug of choice for the effective prevention of DVT. Considering the fact that heparin is only available in paranteral injection, the orally active form of heparin would be clinically important not only for its therapeutic effect but also for prophylactic treatment purposes. In this study, we evaluated the antithrombogenic effect of EnoxaTD in DVT disease model. The efficacy of enoxaparin and EnoxaTD was enough to prevent DVT in rats. However, as antithrombotic agents are associated with the increased occurrence of bleeding, the therapeutic effect of heparin should

Figure 5. (A) 1H NMR of enoxaparin with anomeric region. (B) Molecular structure of anomeric carbons in nonreducing and reducing ends of enoxaparin (NE, the anomeric carbon in nonreducing end; RE, the anomeric carbons in reducing end). (C) HSQC NMR results of enoxaparin and EnoxaTD. The point from the reactive anomeric carbon at the reducing end of enoxaparin (RE1) disappeared after conjugation.

The focus of this study was on the end-specific conjugation of enoxaparin and its availability of an oral delivery. The endspecific conjugation was made by nonenzymatic glycosylation with enoxaparin; it was also confirmed by using Benedict’s reagent and 2D HSQC NMR result. The end-specific conjugation is important for heparin and enoxaparin because the molecular structure of heparin is heterogeneous. Although heparins have a complicated structure, the molecular structures of their end-site are clear. Therefore, the end-specific reaction could increase the chance of new drug development with heparins. Another reason for end-specific conjugation is that this conjugation could maintain the activity of enoxaparin and make an oral delivery possible. The oral delivery of EnoxaTD was evaluated by PK study. The oral delivery of EnoxaTD was confirmed by FXa assay, as enoxaparin cannot be measured directly in the plasma. The results of PK experiment revealed higher bioavailability, Cmax, and AUC values for EnoxaTD than for enoxaparin. The oral uptake of enoxaparin is based on the interaction between 10525

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Table 1. Results of PK Study with Enoxaparin and EnoxaTD in SD Rats dose (mg/kg) enoxaparin (iv) EnoxaTD (iv) enoxaparin (po) EnoxaTD (po) EnoxaTD (po) a

1 1 10 5 10

Cmax a (IU/mL)

AUC0−8h b (IU·h/mL)

t1/2 c (h)

± ± ± ± ±

2.59 ± 1.15 2.18 ± 0.12

0.17 ± 0.05 0.42 ± 0.02 0.60 ± 0.08

1.53 1.46 0.64 2.01 3.04

0.19 0.34 0.18 0.13 0.75

MRT d (h)

F e (%)

3.73 ± 0.84 3.13 ± 0.11 3.07 ± 0.16

4.2 26.3 19.9

Maximum plasma concentration. bArea under the curve. cHalf-life of materials. dMean residence time. eBioavailability.

enoxaparin and EnoxaTD showed controlled bleeding effect with a similar pattern in the animal study.

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CONCLUSION The present study showed that enoxaparin has an end-specific activity related to glycosylation. By use of the nonenzymatic glycosylation, enoxaparin was conjugated to the optimized TD for oral delivery. Orally active EnoxaTD shows a promising therapeutic potential for treating DVT without toxicity. Therefore, given the importance of oral delivery, EnoxaTD could be used in long-term maintenance therapy for preventing blood coagulation.

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EXPERIMENTAL SECTION

Materials. Enoxaparin sodium was obtained from Hebei Changshan Biochemical Pharmaceutical Co. (Shijiazhuang, China). Tinzaparin was purchased from the Nanjing Biochemical Pharmaceutical Company (Nanjing, China). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, 2,2-dimethoxypropan, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), deoxycholic acid, ethylenediamine, L-lysine, Nhydroxysuccinimide, N,N′-dicyclohexylcarbodiimide, sodium cyano-

Figure 7. Inhibition effects in DVT model by using enoxaparin and EnoxaTD in rats.: (∗∗) p < 0.001 vs control group.

be considered in tandem with bleeding time; uncontrolled bleeding could be indicative of apparent toxicity. Both

Figure 8. (A) The primary bleeding time was calculated after cutting the tail. (B) Clotting time in enoxaparin treated group (sc). (C) Clotting time in EnoxaTD treated group (po). 10526

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borohydride, and sodium nitrite were purchased from Sigma Chemical Co. (St. Louis, MO). Methanol, ethanol, tetrahydrofuran, and DMF were purchased from Merck (Darmstadt, Germany). DMEM high glucose medium, fetal bovine serum, penicillin−streptomycin, and trypsin−EDTA were obtained from GIBCO (Grand Island, NY). Coatest anti factor Xa assay kit was purchased from Chromogenix (Milano, Italy). Synthesis. EnoxaTD was prepared by enoxaparin and tetraDOCA (TD) conjugation. TD was synthesized by optimizing the methods used previously.34,38 Briefly, L-lysine (10 g, 54.75 mmol) was dissolved in methanol, and then 2,2-dimethoxypropane (70 mL) and HCl (18 mL) were added to the solution. After 3 h of reaction, compound 1 was concentrated under reduced pressure. In the other batch, L-lysine (20 g, 109.5 mmol) was dissolved in water (160 mL) and THF (160 mL). It was stirred with sodium carbonate and di-tert-butyl dicarbonate, and di-boc-lysine was extracted from it using ethyl acetate. Compounds 1 and 2 were reacted by N,N′-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) in ethyl acetate. Because compound 1 has two primary amine group, compound 3 was generated in a single step. After deprotection with acetyl chloride, compound 4 and NHS-activated deoxycholic acid were mixed overnight at 4 °C to synthesize compound 5. It was purified by column chromatography, and then the conjugate was stirred with ethylenediamine (EDA) at room temperature for 3 days to get compound 6. To synthesize EnoxaTD using glycosylation, enoxaparin (50 mg) was dissolved in distilled water (1 mL). Compound 6 (130 mg) in DMF (7 mL) was mixed with it, and they were reacted at 60 °C for 48 h. Sodium cyanoborohydride was added to the solution, followed by 4 h of further reaction. The product was purified three times by reprecipitation in cold ethanol. Finally, EnoxaTD was dissolved in distilled water and then lyophillzed for 3 days to get white powder. The purity of products in all synthetic processes was confirmed by TLC and HPLC. The purity of all synthesized compounds was above 95%. Characterization. Benedict’s reagent was used to confirm the nonenzymatic glycosylation and aldehyde group in enoxaparin. It was prepared using anhydrous sodium carbonate (10 g), sodium citrate (17.3 g), and copper(II) sulfate pentahydrate (1.73 g) in distilled water (100 mL).37 Glucose, enoxaparin, and tinzaparin (100 mg) were dissolved in Benedict’s solution (1 mL) and then heated at 75 °C. The synthesized conjugates in each step were characterized using NMR and mass spectrometry. 1H NMR spectra were measured at 500 MHz using D2O and DMSO-d6 as cosolvent (DMSO-d6, 75%). The calculation process using NMR analysis and sulfuric acid assay was introduced in the previous studies.39,40 In both cases, the mixtures with various molecular ratios of enoxaparin and TD were prepared. In the NMR analysis, the conjugation ratio was calculated by analyzing the NMR peaks at 0.8−1.5 and 5.0−6.5 ppm. In the sulfuric acid assay, the product and mixtures (enoxaparin and TD) were reacted with sulfuric acid in water at 70 °C. After heating, the absorbance (420 nm) of solution was measured by using a UV/vis spectrometer. The molecular structures of the materials were visualized by ChemBioDraw Ultra 12.0 (Cambridge Soft Corporation) and PyMOL 1.7.0.1 (DeLano Scientific). The factor Xa complex was simulated by AutoDock Vina 1.0.3 and MGLTools-1.4.6 (The Scripps Research Institute) using the crystal structure of the antithrombin−thrombin− heparin ternary complex (Protein Data Bank [PDB] code, 1TB6).41,42 The conjugation ratio of TD to enoxaparin was evaluated using a nitric acid degradation method. First, EnoxaTD was dissolved in distilled water (100 mg/mL). Sodium nitrite solution (20 mg/0.2 mL) and HCl solution (1 N, 0.8 mL) were added to each heparin solution (1 mL). After a 4 h reaction without light, the solution and precipitate (in EnoxaTD) were separated by centrifuge. Then they were lysophilized using a freeze-dryer, followed by analyses by NMR and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS). Their dried weights were used for calculation of the conjugation ratio (fragment analysis) (n = 3). Anti-FXa Chromogenic Assay. The therapeutic effect of enoxaparin is correlated with the inhibitory effect of factor Xa. Anti

factor Xa activity was measured by following the manufacter’s protocol. Briefly, the solution (100 μL) with enoxaparin or EnoxaTD was mixed with 100 μL of antithrombin III solution, and then they were incubated at 37 °C for 3 min. After the reaction with 100 μL of factor Xa (FXa), they were incubated for 3 min with the substrate (200 μL). Anti-FXa activity of EnoxaTD was calculated using the values at 405 nm using a UV/vis spectrometer (n = 3). Cell Viability Test. The cytotoxicity of EnoxaTD was evaluated using MDCK cells in varying concentrations. MDCK cells were cultured in a DMEM high glucose medium supplemented with 10% (v/v) FBS, penicillin−streptomycin, NEAA (nonessential amino acids), sodium bicarbonate, and HEPES (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid). When a 96-well plate was confluent with MDCK cells, the medium was replaced with DMEM containing EnoxaTD with different concentrations. After incubation at 37 °C for 24 h, the medium was removed, DMEM (100 μL) with 10 μL of cell counting kit-8 (CCK-8) solution was added to each of the 96 wells, and then the plate was additionally incubated at 37 °C for 1 h. The cell viability was calculated from the absorbance value (OD of 450−600 nm) and compared with that of the untreated group (n = 3). Pharmacokinetic Study. All procedures for the animal experiments were approved by the Committee on the Use and Care on Animals according to the regulations of the Institutional Animal Ethics Committee of the Seoul National University. The pharmacokinetics of enoxaparin and EnoxaTD were studied on the basis of anti factor Xa activity levels in the plasma of rats. Sprague−Dawley (SD) rats (Orientbio Inc., Seongnam, Korea) were fasted for 12 h before oral administration. EnoxaTD was dissolved in drinking water with solubilizers including labrasol and poloxamer 188. After oral administration of the materials, the blood for plasma preparation was collected from the vein of rats by using a capillary at each time point. Blood samples were immediately mixed with sodium citrate solution (50 μL) and kept in an ice bath. The plasma samples were centrifuged for 20 min (2500g). The resulting plasma samples were frozen and stored below −20 °C until analyzed by anti-FXa chromogenic assay. Deep Vein Thrombosis (DVT) Study. The animal experiment for DVT was designed and prepared in the previous study.29,43 Briefly, SD rats (250−280 g) were purchased and kept in air controlled room. They were fed with normal mouse diet for 1 week and then fasted for 12 h. After administration of enoxaparin (subcutaneous) or EnoxaTD (oral), the rats were anesthetized with ketamine and xylazine. After a surgical procedure to gain access into the abdominal cavity, the vena cava of rat was loosely tied. On the other hand, the other blood vessels around the vena cava were completely tied. The warmed human plasma (1 mL/kg) was injected via the tail vein 1 h after administration, followed by the ligation of the vena cava. The weight of the isolated thrombus from the vena cava was measured 3 h after administration (n = 4). Ex Vivo Animal Study To Monitor Bleeding Effects. C57BL/6 mice (7 weeks old male, Orientbio Inc.) were anesthetized with ketamine and xylazine by intraperitoneal injection. The tail of each mouse was immersed in a prewarmed (37 °C) 50 mL tube containing normal saline for 5 min; 3−4 mm of the distal tip of the tail was cut off using a scalpel blade and immediately inserted back into the tube. At this point, venous blood was detected flowing in the tube, and the primary bleeding time (clotting time) was measured using electrical stop-clocks (n = 5). The maximum duration of primary bleeding was 600 s. Statistical Analysis. Statistical analysis was performed by using one-way analyses of variance followed by Bonferroni’s tests.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00936. Molecular formula strings (CSV) 10527

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of low molecular weight heparins: structure and activity differences. J. Med. Chem. 1990, 33, 1639−1645. (13) Cosmi, B.; Palareti, G. Old and new heparins. Thromb. Res. 2012, 129, 388−391. (14) Melnikova, I. The anticoagulants market. Nat. Rev. Drug Discovery 2009, 8, 353−354. (15) Ingle, R. G.; Agarwal, A. S. A world of low molecular weight heparins (LMWHs) enoxaparin as a promising moiety–a review. Carbohydr. Polym. 2014, 106, 148−153. (16) Fu, L.; Zhang, F. M.; Li, G. Y.; Onishi, A.; Bhaskar, U.; Sun, P. L.; Linhardt, R. J. Structure and activity of a new low-molecular-weight heparin produced by enzymatic ultrafiltration. J. Pharm. Sci. 2014, 103, 1375−1383. (17) Bianchini, P.; Liverani, L.; Spelta, F.; Mascellani, G.; Parma, B. Variability of heparins and heterogeneity of low molecular weight heparins. Semin. Thromb. Hemostasis 2007, 33, 496−502. (18) Sola, R. J.; Griebenow, K. Chemical glycosylation: new insights on the interrelation between protein structural mobility, thermodynamic stability, and catalysis. FEBS Lett. 2006, 580, 1685−1690. (19) Stallforth, P.; Lepenies, B.; Adibekian, A.; Seeberger, P. H. 2009 Claude S. Hudson Award in Carbohydrate Chemistry. Carbohydrates: a frontier in medicinal chemistry. J. Med. Chem. 2009, 52, 5561−5577. (20) Galonic, D. P.; Gin, D. Y. Chemical glycosylation in the synthesis of glycoconjugate antitumour vaccines. Nature 2007, 446, 1000−1007. (21) Bohe, L.; Crich, D. A propos of glycosyl cations and the mechanism of chemical glycosylation; the current state of the art. Carbohydr. Res. 2015, 403, 48−59. (22) Crich, D. Mechanism of a chemical glycosylation reaction. Acc. Chem. Res. 2010, 43, 1144−1153. (23) Durand, G.; Seta, N. Protein glycosylation and diseases: Blood and urinary oligosaccharides as markers for diagnosis and therapeutic monitoring. Clin. Chem. 2000, 46, 795−805. (24) Nigudkar, S. S.; Demchenko, A. V. Stereocontrolled 1,2-cis glycosylation as the driving force of progress in synthetic carbohydrate chemistry. Chem. Sci. 2015, 6, 2687−2704. (25) Hansen, S. U.; Miller, G. J.; Cole, C.; Rushton, G.; Avizienyte, E.; Jayson, G. C.; Gardiner, J. M. Tetrasaccharide iteration synthesis of a heparin-like dodecasaccharide and radiolabelling for in vivo tissue distribution studies. Nat. Commun. 2013, 4, 2016. (26) Goldberg, M.; Gomez-Orellana, I. Challenges for the oral delivery of macromolecules. Nat. Rev. Drug Discovery 2003, 2, 289− 295. (27) Park, K.; Kim, Y. S.; Lee, G. Y.; Nam, J. O.; Lee, S. K.; Park, R. W.; Kim, S. Y.; Kim, I. S.; Byun, Y. Antiangiogenic effect of bile acid acylated heparin derivative. Pharm. Res. 2007, 24, 176−185. (28) Kim, S. K.; Lee, D. Y.; Lee, E.; Lee, Y. K.; Kim, C. Y.; Moon, H. T.; Byun, Y. Absorption study of deoxycholic acid-heparin conjugate as a new form of oral anti-coagulant. J. Controlled Release 2007, 120, 4− 10. (29) Kim, S. K.; Lee, D. Y.; Kim, C. Y.; Nam, J. H.; Moon, H. T.; Byun, Y. A newly developed oral heparin derivative for deep vein thrombosis: non-human primate study. J. Controlled Release 2007, 123, 155−163. (30) Hwang, S. R.; Seo, D. H.; Al-Hilal, T. A.; Jeon, O. C.; Kang, J. H.; Kim, S. H.; Kim, H. S.; Chang, Y. T.; Kang, Y. M.; Yang, V. C.; Byun, Y. Orally active desulfated low molecular weight heparin and deoxycholic acid conjugate, 6ODS-LHbD, suppresses neovascularization and bone destruction in arthritis. J. Controlled Release 2012, 163, 374−384. (31) Kim, S. K.; Vaishali, B.; Lee, E.; Lee, S.; Lee, Y. K.; Kumar, T. S.; Moon, H. T.; Byun, Y. Oral delivery of chemical conjugates of heparin and deoxycholic acid in aqueous formulation. Thromb. Res. 2006, 117, 419−427. (32) Park, J. W.; Jeon, O. C.; Kim, S. K.; Al-Hilal, T. A.; Lim, K. M.; Moon, H. T.; Kim, C. Y.; Byun, Y. Pharmacokinetic evaluation of an oral tablet form of low-molecular-weight heparin and deoxycholic acid conjugate as a novel oral anticoagulant. Thromb. Haemostasis 2011, 105, 1060−1071.

AUTHOR INFORMATION

Corresponding Author

*Phone: +82 2 880 7866. Fax: +82 2 872 7864. E-mail: [email protected]. ORCID

Youngro Byun: 0000-0002-3863-2236 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Korea Drug Development Fund (Grant KDDF-201408-03). This study was also supported by Basic Science Research Program (Grant 2010-0027955) of the National Research Foundation of Korea (NRF) and Cancer Control (Grant 1420390), Ministry of Health and Welfare.

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ABBREVIATIONS USED CCK, cell counting kit; DOCA, deoxycholic acid; DVT, deep vein thrombosis; EnoxaTD, enoxaparin tetraDOCA conjugate; FXa, factor Xa; LMWH, low molecular weight heparin; MDCK, Madin−Darby canine kidney; NR, nonreducing end; RE, reducing end; TD, tetraDOCA; UFH, unfractionated heparin

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REFERENCES

(1) Linhardt, R. J.; Gunay, N. S. Production and chemical processing of low molecular weight heparins. Semin. Thromb. Hemostasis 1999, 25 (Suppl. 3), 5−16. (2) Tyrrell, D. J.; Kilfeather, S.; Page, C. P. Therapeutic uses of heparin beyond its traditional role as an anticoagulant. Trends Pharmacol. Sci. 1995, 16, 198−204. (3) White, R. H. The epidemiology of venous thromboembolism. Circulation 2003, 107, I4−I8. (4) Horlocker, T. T.; Heit, J. A. Low molecular weight heparin: biochemistry, pharmacology, perioperative prophylaxis regimens, and guidelines for regional anesthetic management. Anesth. Analg. 1997, 85, 874−885. (5) Birkmeyer, N. J. O.; Finks, J. F.; Carlin, A. M.; Chengelis, D. L.; Krause, K. R.; Hawasli, A. A.; Genaw, J. A.; English, W. J.; Schram, J. L.; Birkmeyer, J. D.; Michigan Bariatric Surgery Collaborative.. Comparative effectiveness of unfractionated and low-molecular-weight heparin for prevention of venous thromboembolism following bariatric surgery. Arch. Surg. 2012, 147, 994−998. (6) Imberty, A.; Lortat-Jacob, H.; Perez, S. Structural view of glycosaminoglycan-protein interactions. Carbohydr. Res. 2007, 342, 430−439. (7) Geerts, W. H.; Jay, R. M.; Code, K. I.; Chen, E.; Szalai, J. P.; Saibil, E. A.; Hamilton, P. A. A comparison of low-dose heparin with low-molecular-weight heparin as prophylaxis against venous thromboembolism after major trauma. N. Engl. J. Med. 1996, 335, 701−707. (8) Planes, A. Review of bemiparin sodium–a new second-generation low molecular weight heparin and its applications in venous thromboembolism. Expert Opin. Pharmacother. 2003, 4, 1551−1561. (9) Chapman, T. M.; Goa, K. L. Bemiparin: a review of its use in the prevention of venous thromboembolism and treatment of deep vein thrombosis. Drugs 2003, 63, 2357−2377. (10) Agnelli, G.; George, D. J.; Kakkar, A. K.; Fisher, W.; Lassen, M. R.; Mismetti, P.; Mouret, P.; Chaudhari, U.; Lawson, F.; Turpie, A. G. Investigators, S.-O. Semuloparin for thromboprophylaxis in patients receiving chemotherapy for cancer. N. Engl. J. Med. 2012, 366, 601− 609. (11) Coyne, E. From heparin to heparin fractions and derivatives. Semin. Thromb. Hemostasis 1985, 11, 10−12. (12) Linhardt, R. J.; Loganathan, D.; al-Hakim, A.; Wang, H. M.; Walenga, J. M.; Hoppensteadt, D.; Fareed, J. Oligosaccharide mapping 10528

DOI: 10.1021/acs.jmedchem.6b00936 J. Med. Chem. 2016, 59, 10520−10529

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Article

(33) Al-Hilal, T. A.; Chung, S. W.; Alam, F.; Park, J.; Lee, K. E.; Jeon, H.; Kim, K.; Kwon, I. C.; Kim, I. S.; Kim, S. Y.; Byun, Y. Functional transformations of bile acid transporters induced by high-affinity macromolecules. Sci. Rep. 2014, 4, 4163. (34) Al-Hilal, T. A.; Park, J.; Alam, F.; Chung, S. W.; Park, J. W.; Kim, K.; Kwon, I. C.; Kim, I. S.; Kim, S. Y.; Byun, Y. Oligomeric bile acidmediated oral delivery of low molecular weight heparin. J. Controlled Release 2014, 175, 17−24. (35) Rabenstein, D. L. Heparin and heparan sulfate: structure and function. Nat. Prod. Rep. 2002, 19, 312−331. (36) Roy, S.; El Hadri, A.; Richard, S.; Denis, F.; Holte, K.; Duffner, J.; Yu, F.; Galcheva-Gargova, Z.; Capila, I.; Schultes, B.; Petitou, M.; Kaundinya, G. V. Synthesis and biological evaluation of a unique heparin mimetic hexasaccharide for structure-activity relationship studies. J. Med. Chem. 2014, 57, 4511−4520. (37) Benedict, S. R. A reagent for the detection of reducing sugars. 1908. J. Biol. Chem. 2002, 277, e5. (38) Park, J. W.; Jeon, O. C.; Kim, S. K.; Al-Hilal, T. A.; Jin, S. J.; Moon, H. T.; Yang, V. C.; Kim, S. Y.; Byun, Y. High antiangiogenic and low anticoagulant efficacy of orally active low molecular weight heparin derivatives. J. Controlled Release 2010, 148, 317−326. (39) Nichifor, M.; Carpov, A. Bile acids covalently bound to polysaccharides 1. Esters of bile acids with dextran. Eur. Polym. J. 1999, 35, 2125−2129. (40) Park, J.; Jeong, J. H.; Al-Hilal, T. A.; Kim, J. Y.; Byun, Y. Size controlled heparin fragment-deoxycholic acid conjugate showed anticancer property by inhibiting VEGF165. Bioconjugate Chem. 2015, 26, 932−940. (41) Li, W.; Johnson, D. J.; Esmon, C. T.; Huntington, J. A. Structure of the antithrombin-thrombin-heparin ternary complex reveals the antithrombotic mechanism of heparin. Nat. Struct. Mol. Biol. 2004, 11, 857−862. (42) Trott, O.; Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2010, 31, 455− 461. (43) Kim, S. K.; Lee, D. Y.; Kim, C. Y.; Moon, H. T.; Byun, Y. Prevention effect of orally active heparin derivative on deep vein thrombosis. Thromb. Haemostasis 2006, 96, 149−153.

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