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Synthetic oligosaccharides can replace animal-sourced low–molecular weight heparins

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Science Translational Medicine  06 Sep 2017:
Vol. 9, Issue 406, eaan5954
DOI: 10.1126/scitranslmed.aan5954
  • Fig. 1. Chemical structures and synthetic scheme of synthetic LMWHs, 12-mer-1 and 12-mer-2.

    (A) Chemical structures of 12-mer-1 and 12-mer-2. The name of each residue (A to L) is indicated at the top of the panel. The differences between two 12-mers are the number of 3-O-sulfo groups present: one in 12-mer-1 in residue C and two in 12-mer-2 in residues C and G (highlighted). pNP represents p-nitrophenyl group. (B) Synthetic schemes for 12-mer-1 and 12-mer-2 using shorthand symbols. Each reaction step uses different enzymes and chemicals: a, PmHS2 (heparosan synthase 2 from Pasteurella multocida) and UDP-GlcNTFA; b, PmHS2 and UDP-GlcA; c, LiOH; d, N-sulfotransferase (NST) and 3′-phosphoadenosine 5′-phosphosulfate (PAPS); e, C5-epimerase (C5-epi), 2-O-sulfotransferase (2-OST), and PAPS; f, 6-OST-1, 6-OST-3, and PAPS; g, 3-O-sulfotransferase isoform 1 (3-OST-1) and PAPS; h, 3-OST-5 and PAPS. Full synthetic schemes using chemical structures are shown in fig. S1.

  • Fig. 2. In vitro, ex vivo, and in vivo analysis of the anticoagulant activity of 12-mers.

    (A) FXa inhibition curves of 12-mers and UFH in vitro. Data are means ± SD (n = 4). (B) Neutralization of FXa activity of 12-mers by protamine in vitro. In addition to 12-mers, UFH was included as a positive control, and fondaparinux and enoxaparin were included as negative controls. Data are means ± SD (n = 4). (C) Reversibility of anti-FXa activity by protamine in an ex vivo experiment. The statistical significance for the comparison of each sample with and without protamine is indicated (P < 0.001). (D) Reduction of clot weight in a venous thrombosis mouse model treated with PBS, enoxaparin, and 12-mer-1. (E) Plasma concentration of thrombin anti-thrombin (TAT) complexes and (F) FXa activity in control (AA) and sickle BERK (SS) mice injected with PBS or 12-mer-1 (2 mg/kg) subcutaneously every 8 hours for 7 days (n = 8 to 13). Blood was collected 2 hours after the last injection. Data are means ± SD. *P < 0.05, ***P < 0.001, ****P < 0.0001. n.s., not significant.

  • Fig. 3. Clearance of 12-mer-1 in kidney I/R injury model in C57BL/6J mice.

    (A) Kidney I/R experimental scheme. (B) Plasma activity of FXa in mice subjected to kidney I/R injury (30 min of ischemia time followed by 24 hours of reperfusion) and injected subcutaneously with UFH (n = 6; 3 mg/kg), 12-mer-1 (n = 6; 1.5 mg/kg), or 12-mer-2 (n = 6; 0.3 mg/kg). (C) Plasma activity of FXa in sham-operated mice and mice subjected to different ischemia periods (20, 25, and 30 min). Mice received subcutaneous injection of PBS or 12-mer-1 (0.3 mg/kg) after 24 hours of reperfusion, and plasma was collected 2 hours later (n = 6 for each group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 4. Clearance of 12-mer-1 from a nonhuman primate model.

    M. mulatta monkeys were treated with 12-mer-1 at a dose of 250 μg/kg either intravenously (IV) (A) or subcutaneously (SC) (B). Blood samples were collected before dosing (at t = 0 hour) and at 30, 60, and 120 min after dosing (for IV group) or at 1, 2, 4, and 6 hours after dosing (for SC group). The plasma concentration of 12-mer-1 was determined by measuring the activity of FXa, which was converted into the concentration of 12-mer-1 by a standard curve, as shown in fig. S21. Data are average ± range (n = 2).

  • Table 1. Toxicological results and organ weights.
    ParametersControl (n = 5)3600 μg/kg (n = 5)Historical control values*
    White blood cells (×103/μl)9.9 ± 1.17.8 ± 1.09.6 ± 3.0
    Red blood cells (×106/μl)8.3 ± 0.28.1 ± 0.38.5 ± 0.6
    Hemoglobin (g/dl)15.1 ± 0.414.8 ± 0.315.9 ± 0.9
    Blood urea nitrogen (mg/dl)18.0 ± 2.217.8 ± 2.418 ± 3.2
    Creatinine (mg/dl)0.40 ± 0.010.42 ± 0.030.46 ± 0.12
    Albumin (g/dl)3.54 ± 0.063.62 ± 0.083.4 ± 0.17
    Total bilirubin (mg/dl)0.11 ± 0.010.10 ± 0.010.12 ± 0.024
    Alanine aminotransferase (U/liter)43.6 ± 8.138.6 ± 2.446 ± 15.3
    Total protein (g/dl)6.18 ± 0.166.28 ± 0.116.2 ± 0.41
    Body weight (g)268.4 ± 4.2271.4 ± 5.9
    Organ–to–body weight ratio
      Brain0.69 ± 0.040.71 ± 0.02
      Adrenal glands0.026 ± 0.0140.020 ± 0.004
      Heart0.41 ± 0.040.41 ± 0.02
      Kidneys0.73 ± 0.050.70 ± 0.04
      Liver3.04 ± 0.093.04 ± 0.11
      Spleen0.25 ± 0.010.25 ± 0.02
      Testes1.36 ± 0.041.29 ± 0.06
      Thyroid/parathyroid0.0062 ± 0.00110.0058 ± 0.0011

    *The historical control value was obtained from the analysis of ~1750 individual normal rats.

    Body weight and organ weight were measured on day 8.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/9/406/eaan5954/DC1

      Materials and Methods

      Fig. S1. Chemoenzymatic synthetic scheme for 12-mer-1 and 12-mer-2.

      Fig. S2. Anion exchange HPLC chromatograms of 12-mer-1 and 12-mer-2.

      Fig. S3. High-resolution MS analysis of 12-mer-1 and 12-mer-2 by HILIC-FT MS.

      Fig. S4. 1H and 13C NMR spectra of 12-mer-1.

      Fig. S5. 1H-1H COSY and 1H-13C HSQC NMR spectra of 12-mer-1.

      Fig. S6. 1H-1H NOESY and 1H-1H TOCSY NMR spectra of 12-mer-1.

      Fig. S7. 1H-13C HMBC spectrum of 12-mer-1.

      Fig. S8. 1H NMR/13C NMR chemical shift assignments (in ppm) of 12-mer-1.

      Fig. S9. 1H and 13C NMR spectra of 12-mer-2.

      Fig. S10. 1H-1H COSY and 1H-13C HSQC spectra of 12-mer-2.

      Fig. S11. 1H-1H NOESY and 1H-1H TOCSY spectra of 12-mer-2.

      Fig. S12. 1H-13C HMBC spectrum of 12-mer-2.

      Fig. S13. 1H NMR/13C NMR chemical shift assignments (in ppm) of 12-mer-2.

      Fig. S14. Determining the structure of 12-mer-2 by heparin lyase II digestion.

      Fig. S15. Determination of the 3-O-sulfo group in 12-mer-2 by partial heparin lyase II digestion.

      Fig. S16. Confirmation of the location of the 3-O-sulfo group in 12-mer-2 by partial heparin lyase I digestion.

      Fig. S17. Comparison of 2D 1H-1H COSY with 1H NMR.

      Fig. S18. Comparison of 2D 1H-13C HSQC of 12-mer-1 and 12-mer-2.

      Fig. S19. Comparison of 2D 1H-1H TOCSY and NOESY spectra of 12-mer-2.

      Fig. S20. The effect of 12-mer-1 on the activity of FXa in mice.

      Fig. S21. In vitro assay method and standard curve of 12-mer-1 for pharmacodynamic studies in nonhuman primates.

      Fig. S22. Elimination profiles of 12-mer-1 in primate through intravenous or subcutaneous administration at 500 μg/kg.

      Table S1. Additional toxicological data and organ and body weight ratios.

      References (5257)

    • Supplementary Material for:

      Synthetic oligosaccharides can replace animal-sourced low–molecular weight heparins

      Yongmei Xu, Kasemsiri Chandarajoti, Xing Zhang, Vijayakanth Pagadala, Wenfang Dou, Debra Moorman Hoppensteadt, Erica M. Sparkenbaugh, Brian Cooley, Sharon Daily, Nigel S. Key, Diana Severynse-Stevens, Jawed Fareed, Robert J. Linhardt,* Rafal Pawlinski,* Jian Liu*

      *Corresponding author. Email: jian_liu{at}unc.edu (J.L.); rafal_pawlinski{at}med.unc.edu (R.P.); linhar{at}rpi.edu (R.J.L.)

      Published 6 September 2017, Sci. Transl. Med. 9, eaan5954 (2017)
      DOI: 10.1126/scitranslmed.aan5954

      This PDF file includes:

      • Materials and Methods
        Fig. S1. Chemoenzymatic synthetic scheme for 12-mer-1 and 12-mer-2.
        Fig. S2. Anion exchange HPLC chromatograms of 12-mer-1 and 12-mer-2.
        Fig. S3. High-resolution MS analysis of 12-mer-1 and 12-mer-2 by HILIC-FT
        MS.
        Fig. S4. 1H and 13C NMR spectra of 12-mer-1.
        Fig. S5. 1H-1H COSY and 1H-13C HSQC NMR spectra of 12-mer-1.
        Fig. S6. 1H-1H NOESY and 1H-1H TOCSY NMR spectra of 12-mer-1.
        Fig. S7. 1H-13C HMBC spectrum of 12-mer-1.
        Fig. S8. 1H NMR/13C NMR chemical shift assignments (in ppm) of 12-mer-1.
        Fig. S9. 1H and 13C NMR spectra of 12-mer-2.
        Fig. S10. 1H-1H COSY and 1H-13C HSQC spectra of 12-mer-2.
        Fig. S11. 1H-1H NOESY and 1H-1H TOCSY spectra of 12-mer-2.
        Fig. S12. 1H-13C HMBC spectrum of 12-mer-2.
        Fig. S13. 1H NMR/13C NMR chemical shift assignments (in ppm) of 12-mer-2.
        Fig. S14. Determining the structure of 12-mer-2 by heparin lyase II digestion.
        Fig. S15. Determination of the 3-O-sulfo group in 12-mer-2 by partial heparin
        lyase II digestion.
        Fig. S16. Confirmation of the location of the 3-O-sulfo group in 12-mer-2 by
        partial heparin lyase I digestion.
        Fig. S17. Comparison of 2D 1H-1H COSY with 1H NMR.
        Fig. S18. Comparison of 2D 1H-13C HSQC of 12-mer-1 and 12-mer-2.
        Fig. S19. Comparison of 2D 1H-1H TOCSY and NOESY spectra of 12-mer-2.
        Fig. S20. The effect of 12-mer-1 on the activity of FXa in mice.
        Fig. S21. In vitro assay method and standard curve of 12-mer-1 for pharmacodynamic studies in nonhuman primates.
        Fig. S22. Elimination profiles of 12-mer-1 in primate through intravenous or subcutaneous administration at 500 μg/kg.
        Table S1. Additional toxicological data and organ and body weight ratios.
        References (5257)

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