End-Site-Specific Conjugation of Enoxaparin and Tetradeoxycholic Acid Using Nonenzymatic Glycosylation for Oral Delivery

Article abstract of DOI:10.1021/acs.jmedchem.6b00936

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.

Full text of DOI:10.1021/acs.jmedchem.6b00936

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Article
End-site specific conjugation of enoxaparin and tetra-deoxycholic
acid using non-enzymatic 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
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00936 • Publication Date (Web): 09 Nov 2016
Downloaded from http://pubs.acs.org on November 10, 2016
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End-site specific conjugation of enoxaparin and
tetra-deoxycholic acid using non-enzymatic
glycosylation for oral delivery
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Jooho Park1,2, Ok Cheol Jeon3, Jisuk Yun4, Hwajung Nam4, Jinha Hwang4, Taslim A. Al-Hilal2,
Kwangmeyung Kim2, Kyungjin Kim4, Youngro Byun1,5*
Author affiliations:
1Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University,
Seoul 151-742, South Korea
2Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and
Technology, Seoul 136-791, South Korea
3B&L DELIPHARM, Seoul 151-742, South Korea
4ST Pharm Research & Development Center, HyeopRyeok Road, Siheung-Si, Gyeonggi-do,
South Korea
5Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of
Convergence Science and Technology, Seoul National University, Seoul 151-742, South Korea
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ABSTRACT
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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 tetra-oligomer of deoxycholic acid (TetraDOCA; TD) at the end-site of enoxaparin via non-
enzymatic 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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Keywords: Enoxaparin; End-site specific; Glycosylation; Oral delivery; Bile acid conjugate;
Deep vein thrombosis
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Introduction
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Anticoagulant therapy with heparin is widely used in healthcare therapeutics 1, 2
.
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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
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.
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 (12000-15000 Da). LMWHs are generally prepared by controlled
chemical or enzymatic 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
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related to glycosylation reaction. About 80 % of the end components of enoxaparin contain
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reducing ends 16, 17
.
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Glycosylation is an important reaction in the field of glycochemistry for synthesizing
natural glycoproteins and glycans. Chemical or non-enzymatic 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 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 non-
enzymatic glycosylation has shown great possibilities for polysaccharide end-specific
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conjugation 23, 24
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The clinical use of enoxaparin can be extended to non-enzymatic 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
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completely eliminated after commercial processes and, the tail end of polysaccharide of
nadroparin is not chemically active either 1, 33-35. Moreover, in designing a drug with a
polysaccharide conjugate, the side-conjugation 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
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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. Firstly, the end
site reactivity of heparin was confirmed using different kinds of LMWHs and Benedict’s reagent.
Secondly, we optimized the synthesis processes to synthesize new tetraDOCA (TD). Then TD
was conjugated to the tail end of enoxaparin by non-enzymatic 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.
Results
Confirmation of end site-specific activity
The end site-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
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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 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 end-specific reactivity.
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Figure 1. (A) Molecular structure of enoxaparin, (B) Non-enzymatic 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.
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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.
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Figure 2. Chemical synthesis of EnoxaTD by non-enzymatic glycosylation. The total synthesis
process was optimized to 7 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.
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The final conjugate with enoxaparin and TD was characterized in different ways.
EnoxaTD is the enoxaparin conjugate with TD by using non-enzymatic 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.
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).
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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).
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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: 1TB6), (E) Anti FXa activity, (F) The 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)
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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 of 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.
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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
Analysis of end-specific conjugation by 2D NMR
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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 non-reducing end of its molecular structure (Figure 5B). Two-dimensional (2D)
1H-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 non-reducing end in enoxaparin because of the relatively small amount (about
80%) and existence of a reactive contributing structure. Firstly, as shown in Fig. 1, the reducible
end in heparin has a resonance or contributing structure, so it has reactivity and can disrupt the
strength of proton peak. Secondly, 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 was
disappeared in anomeric region after end-specific conjugation (Figure 5C). On the other hand,
the proton in non-reducing end of enoxaparin was still remained.
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Figure 5 (A) 1H NMR of enoxaparin with anomeric region, (B) The molecular structure of
anomeric carbons in non-reducing and reducing end of enoxaparin (NE: The anomeric carbon in
non-reducing end, RE: The anomeric carbons in reducing end), (C) The HSQC NMR results of
enoxaparin and EnoxaTD. The point from the reactive anomeric carbon in reducing end of
enoxaparin (RE1) was disappeared after conjugation.
PK study
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The main motive for the chemical glycosylation of enoxaparin was to increase the
bioavailability of non-absorbable 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.
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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.
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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
(P.O.) group
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Table 1. The results of PK study with enoxaparin and EnoxaTD in SD rats
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a
c
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Dose
Cmax
AUC0-8 h
t1/2
MRT e
F f
(%)
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(mg/kg)
(IU•h/mL)
(IU/mL)
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(h)
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Enoxaparin (I.V.)
EnoxaTD (I.V.)
Enoxaparin (P.O.)
EnoxaTD (P.O.)
EnoxaTD (P.O.)
1
1
1.53 0.19
1.46 0.34
0.64 0.18
2.01 0.13
3.04 0.75
2.59 1.15
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2.18 0.12
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-
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0.17 0.05
0.42 0.02
0.60 0.08
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3.73 0.84
3.13 0.11
3.07 0.16
4.2
26.3
19.9
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a Maximum plasma concentration
b Area under the curve
d Half-life of materials
e Mean residence time
f Bioavailability
DVT and bleeding experiments
The therapeutic effect of EnoxaTD was evaluated by using a venous thrombosis animal
model with SD rats. 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
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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.
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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 seconds was
observed after cutting. The rat’s tail was cut using a scalpel blade after administration of
enoxaparin and EnoxaTD. The primary clotting (bleeding) time for the control group was 225.2
57.0 seconds. Contrastively, the primary clotting time for groups treated with EnoxaTD (oral, 1
h) and enoxaparin (subcutaneous, 1 h) were 332.0 74.7 seconds and 380.0 51.6 seconds,
respectively (Figure 8A). The detailed profile of bleeding time of enoxaparin and EnoxaTD is
shown in Figure 8B and 8C.
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Figure 7 (A) Inhibition effects in DVT model by using enoxaparin and EnoxaTD in rats. **p <
5
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0.001 vs control group.
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Figure 8 (A) The primary bleeding time was calculated after cutting the tail, (B) Clotting time
in enoxaparin treated group (S.C.) (C) Clotting time in EnoxaTD treated group (P.O.).
Discussion
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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 animal-derived
agent, it is not absorbed in the gastrointestinal (GI) tract.
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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 polysaccharides 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
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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.
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The focus of this study was on the end-specific conjugation of enoxaparin and its
availability of an oral delivery. The end-specific conjugation was made by non-enzymatic
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 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 high-affinity substrate, induced
the receptor-like functional transformation of ASBT 33. This phenomenon allowed the apical-to-
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basal 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.
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Based on 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 anti-thrombogenic 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 be considered in tandem with
bleeding time; uncontrolled bleeding could be indicative of apparent toxicity. Both Enoxaparin
and EnoxaTD showed controlled bleeding effect with a similar pattern in the animal study.
Conclusion
The present study showed that enoxaparin has an end-specific activity related to
glycosylation. Using the non-enzymatic glycosylation, enoxaparin was conjugated to the
optimized TD for oral delivery. Orally active EnoxaTD shows a promising therapeutic potential
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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, N-Hydroxysuccinimide, N,N'-Dicyclohexylcarbodiimide, sodium
cyanoborohydride, sodium nitrite and 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
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mmol) was dissolved in methanol, and then 2,2-dimethoxypropan (70 mL) and HCl (18 mL) was
added to the solution. After 3 h 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. Compound 1 and 2 were reacted by N,N'-
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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℃ 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). The compound 6 (130 mg) in DMF (7 mL) was mixed with it, and they
were reacted at 60 ℃ 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
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Benedict’s reagent was used to confirm the non-enzymatic glycosylation and aldehyde
group in enoxaparin. It was prepared using anhydrous sodium carbonate (10 g), sodium citrate
(17.3 g) and copper (II) sulphate 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℃.
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The synthesized conjugates in each step were characterized using NMR and mass
spectrometer. ¹H NMR spectra was measured at 500 MHz using D2O and DMSO-d6 as co-
solvent (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 an 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).
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After 4 h reaction without light, the solution and precipitate (in EnoxaTD) were separated by
centrifuge. Then they were lysophilized using a freeze-dryer, and followed by analyses by NMR
and Matrix-Assisted Laser Desorption Ionization Mass Spectrometer (MALDI-TOF). Their dried
weights were used for calculation of the conjugation ratio (fragment analysis) (n=3).
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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 were 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 (non-essential amino acids), sodium bicarbonate
and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic 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 96 well, and
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then the plate was additionally incubated at 37 °C for 1 h. The cell viability was calculated from
the absorbance value (OD 450 - 600 nm) and compared with that of the untreated group (n=3).
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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 in at each time point. Blood samples were immediately mixed with
sodium citrate solution (50 µL) and kept in ice bath. The plasma samples were centrifuged for 20
min (2500 g). 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 (by
subcutaneous) or EnoxaTD (by oral), the rats were anesthetized with ketamine and xylazine.
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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).
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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 pre-warmed (37
ºC) 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 seconds.
Statistical analysis
Statistical analysis was performed by using one-way analyses of variance followed by
Bonferroni’s tests.
Conflicts of Interest
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No conflicts of interest were disclosed.
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AUTHOR INFORMATION
Corresponding Author
*To whom correspondence should be addressed:
Youngro Byun, Ph.D.
Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of
Biopharmaceutical Sciences and Technology and College of Pharmacy, Seoul National
University, Seoul 151-742, South Korea. Tel.: +82 2 880 7866; fax: +82 2 872 7864
E-mail address: yrbyun@snu.ac.kr
ACKNOWLEDGMENT
This work was supported by grants of Korea Drug Development Fund (KDDF-201408-
03). This study was also supported by a grant from Basic Science Research Program (grant no.
2010-0027955) of the National Research Foundation of Korea (NRF) and Cancer Control (grant
no. 1420390), Ministry of Health and Welfare.
ABBREVIATIONS
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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, non-reducing end; RE, reducing end; TD,
tetraDOCA; UFH, unfractionated heparin
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