European Journal of Medicinal Chemistry 86 (2014) 310e317
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Invited review
Current research on hyaluronic acid-drug bioconjugates Haiqun Zhang a, Siling Huang b, Xiaoye Yang a, Guangxi Zhai a, * a b
Department of Pharmaceutics, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua Xilu, Jinan 250012, China Bloomage Freda Biopharm Co., Ltd., Jinan 250101, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 December 2013 Received in revised form 4 August 2014 Accepted 25 August 2014 Available online 27 August 2014
Hyaluronic acid (HA) is a mucopolysaccharide acid composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine. Based on numerous characteristics such as viscoelastic properties, water-binding ability, biocompatibility and non-immunogenicity, HA has been approved by FDA for biological and medical applications. In addition, multifarious receptors of HA like CD44, RHAMM and TSG6 are over-expressed on the surface of malignant cells, which play important roles in targeting ability. Bioconjugates linking drugs to HA could improve solubility, prolong half-life, provide active targeting capability and then increase the bioavailability of these coupled drugs by pro-drug strategy. Therefore, a large number of HA-drug bioconjugates have been studied. The purpose of this review was to summarize these HA-drug bioconjugates and further discuss synthetic methods and the relevant application in pharmaceuticals. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Hyaluronic acid Pro-drug Bioconjugates Synthesis Evaluation
1. Introduction Based on biocompatibility, biodegradability, nonimmunogenicity, water-bonding property and receptors such as CD44, RHAMM, TSG6, HA has drawn lots of attention in pharmacy [1e6]. In addition, HA held many significant biological functions such as stabilizing and organizing extracellular matrix, regulating cell adhesion and motility [7e9]. Therefore, HA and its derivatives have been studied widely [10]. In the biopharmaceutics classification system, there is a kind of drugs suffering from low bioavailability because of the low solubility and low permeability. A prodrug strategy of HA-drug conjugates could make full use of the superiorities of HA to make up for many deficiencies of these drugs. In the past few decades, investigators have devoted numerous efforts to develop HA-drug bioconjugates to enhance targeting ability, improve bioavailability and weaken adverse effects [11,12]. For example, HA-Exendin 4 and HA-Insulin bioconjugates were studied for the treatment of diabetics [13e19], which were prepared by peptide covalently combined with HA to prolong the short half-life, enhance stability and therapeutical effect [14,20e24]. Through vast trials and attempts, great achievements have been made. The aim of this paper was to present an overview of synthesis and evaluation of HA-drug bioconjugates. The structural formulas
* Corresponding author. E-mail addresses: [email protected] (H. Zhang), [email protected] (G. Zhai). http://dx.doi.org/10.1016/j.ejmech.2014.08.067 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.
and corresponding thumbnails of HA and all drugs were showed in Table 1 and structural formulas of linkers in HA-drugs bioconjugates were showed in Table 2. Detailed examples were illustrated and discussed as reference for future research and application. 2. HA-anticancer drug bioconjugates 2.1. HA-anticancer drug bioconjugates with linker Owing to steric hindrance, HA and anticancer drugs could not be directly combined covalently. Therefore, the bioconjugates need linkers the process needs of synthesis, which are discussed as follows in details. 2.1.1. HA-paclitaxel bioconjugate Paclitaxel (PTX), initially extracted from the bark of pacific yew, is a powerful anticancer drug [25,26]. However, many disadvantages such as poor solubility, toxic side effects and drug-resistance limited the application of PTX. A lot of investigations have been conducted to overcome these disadvantages [27]. Luo et al. prepared HA-PTX conjugate by the dihydrazide method [28] (Fig. 1A). PTX was combined to HA with anhydride and hydrazide as spacers. HA was activated by 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and reacted with adipic dihydrazide (ADH) to obtain HA-ADH. PTX was reacted with succinic anhydride (Suc) in the presence of pyridine for 3 days at room temperature to obtain the 20 -hemisuccinate PTX derivative (PTX-Suc) [29]. Then the PTX-Suc
H. Zhang et al. / European Journal of Medicinal Chemistry 86 (2014) 310e317 Table 1 Structural formulas and thumbnail of HA and drugs.
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H. Zhang et al. / European Journal of Medicinal Chemistry 86 (2014) 310e317
was activated by carbodiimide coupling reagent composed of diphenylphosphoryl chloride, NHS and triethylamine, and reacted with N-hydroxysuccinimide (NHS) to obtain PTX-NHS ester [30]. HA-ADH and PTX-NHS eater were mixed at pH 6.5 for 24 h to obtain HA-PTX conjugate. In the study of cell toxicity, the HA-PTX conjugate showed the strong toxicity on ovarian cancer, breast cancer, and colon tumor cells, which could over-express receptors of HA. While the conjugate had no effect on mouse fibroblast cells without these receptors [31]. In the cell uptake assays, the result suggested that the HA-PTX conjugate entered into the cell within several minutes and then gradually appeared in the nucleus of ovarian cancer, breast cancer, and colon tumor cells. Conversely, a slow uptake process was showed in fibroblast cells group. The conjugate had lower IC50, e.g. the half inhibitory concentration, than free Taxol and HA-ADH. In addition, the conjugate exhibited high drug loading (up to 15%) and solubility, strong cytotoxicity and targeting capability. Moreover, HA-PTX conjugates were prepared and investigated by other synthesis methods. Rosato and colleagues prepared HAPTX conjugate with halogenated acid as the linker for fighting superficial bladder cancer [32] (Fig. 1B). PTX was activated by EDC and reacted with 4-bromobutyric acid (4-BBa). The intermediate product mixed with HA-thiobarbituric acid under anhydrous conditions for 7 days to obtain HA-PTX conjugate. Besides, this conjugate was prepared by Suc and polyamine as the spacer [33] (Fig. 1C). A 20 -hemisuccinate derivative of PTX was prepared and activated by NHS and dicyclohexylcarbodiimide (DCC) to get the activated ester. HA was activated by EDC and linked to ethyleneimine (EI) at pH 5.0 to obtain an amine-modified HA. The two
intermediate products were reacted to form HA-PTX conjugate. Lee et al. further developed HA-PTX conjugate [34] (Fig. 1D). HA was desalted by dialysis primarily. HA and poly(ethylene glycol) dimethyl ether (dmPEG) were mixed in deionized water to prepare HA/dmPEG. The HA/dmPEG was dialyzed and freeze-dried to get complex powder. This powder was used to improve the solubility of HA in organic solvents such as DMSO/DMF. Then, this HA/dmPEG complex powder was activated by DCC and 4dimethylaminopyridine (DMAP) and reacted with PTX in anhydrous DMSO to obtain the final HA-PTX conjugate. Based on a series of evaluations on these conjugates, HA-PTX conjugates showed many improvements such as improved solubility, biocompatibility, active targeting ability, high antitumor activity and cell uptake ability. 2.1.2. HA-Doxorubicin bioconjugate Doxorubicin (DOX), an anthracycline antibiotic, is an important anticancer agent with extensive biological activities [35,36]. However, the occurrence of renal toxicity, hepatic toxicity, cardiactoxicity and foaming phenomenon limited the application of DOX [37]. Bioconjugates of DOX and biocompatible materials like HA could decrease or compromise the toxicity and had active targeting ability [38,39]. Hence, these superiorities might provide a platform for HA-DOX conjugate to deliver DOX selectively to breast cancer cells and lymphatic cells. HA-DOX conjugate was made for treating breast cancers [40]. HA and ADH was dissolved in deionized water by activation of EDC to prepare HA-ADH. Then HA-ADH was reacted with DOX at pH 6.5 in sodium phosphate buffer for 2 h. The HADOX conjugate solution was dialyzed until no change of color.
H. Zhang et al. / European Journal of Medicinal Chemistry 86 (2014) 310e317 Table 2 Structural formulas of linker in HA-drugs bioconjugates.
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H. Zhang et al. / European Journal of Medicinal Chemistry 86 (2014) 310e317
HA
COOH
+ Suc +EI
4-BBa+ADH
Suc +ADH
+
PTX
PTX
OCO
Suc+ADH PTX
NH
OCO
CO
No linker
OH
HA
4-BBa +ADH
PTX
PTX NH
CO
OCO
OCO
HA
HA Suc +EI
NH
CO
HA
Fig. 1. Schematic view of HA linking with PTX by Suc and ADH (A), 4-bromobutyric acid and ADH (B), Suc and EI(C), direct link (D).
Properties of this conjugate were further evaluated. The half-life (t1/ of DOX releasing from the conjugate lasted for 172 h at pH 7.4, 110 h at pH 6.0 and 45 h at pH 5.0, respectively, which indicated the pH-sensitivity of linker of the HA-DOX conjugate. The Cmax value, peak concentration of drug, of HA-DOX (0.130 ± 0.020 mg/mL within 30 min) was lower than that of free DOX (2.580 ± 0.670 mg/mL within 5 min), which revealed lower toxic side effects of HA-DOX. Moreover, the area under the curve (AUC) of HA-DOX (2.201 ± 0.905 (mgh)/mL) was higher than that of DOX (2.061 ± 0.824 (mgh)/mL), which proved sustained release profile and long circulation time of HA-DOX. The HA-DOX conjugate exhibited 3.2-folds longer mean residence time than free DOX, which could minimize administration frequency and provide convenience for patients. In conclusion, high drug loading and solubility, sustained release and low toxicity all contributed to the development of HA-DOX.
2)
2.2. HA-anticancer drug bioconjugates without linker The coupling reaction of HA and drugs could be realized without linkers, when the reactive groups of anticancer drug were exposed enough for direct reaction. Bioconjugates without linker are illustrated under-mentioned at length. 2.2.1. HA-Curcumin bioconjugate Curcumin (Cur) has drawn growing attention due to a lot of physiological activities of this substrate, such as inhibiting the growth of cancer cells, resisting oxidant, anti-inflammatory and anti-HIV, preventing fatty liver and protect kidney [41e46]. However, many shortcomings such as low solubility, low permeability and weak stability accompanied with the administration of Cur, which led to low bioavailability [47]. Cur was bonded to hydrophilic biomaterials could improve solubility, enhance stability and increase therapeutic efficacy of the drug. HA-Cur conjugate was prepared and the conjugate self-assembled to form micelles [48]
Fig. 2. Schematic representation of HAeCur conjugate and the formation of HAeCur micelles.
(Fig. 2). After carboxylic group of HA was activated by DCC and DMAP, Cur was added and reacted with the activated HA for 6 h at 60e65 C to obtain HA-Cur conjugate. Then the conjugate was dissolved in aqueous solution to self-assembly form micelles. Evaluation results showed that the HA-Cur conjugate displayed favorable drug loading (appropriately 1.3%), high solubility (7.5 mg/ mL) and strong stability (over 8 h). Only 13 mg of Cur in HA-Cur could lead to nearly 80% cells deaths, which showed high inhibition rate on cancer cells. The micelle formed by HA-Cur conjugate presented a mean diameter of 287 nm with obvious core shell structure, zeta potential in the range of 25 mV to 75 mV at different pH conditions and low critical aggregation concentration of 33.4 mg/mL. Considering the existence of HA in the conjugate, the micelle would be endowed with potential active targeting capacity, but there was no concrete evidence to prove in this study. All results suggested that this developed HA-Cur conjugate successfully overcame the low solubility and permeability and weak stability of Cur and provided a reference for further research. 2.2.2. HA-cisplatin bioconjugate Commonly, once primary breast tumor cells spread, breast cancers will migrate to regional lymph nodes. The surgery or radiation therapy brings painful lymphedema [49]. It has been certified that combined application of cisplatin and taxanes and trastuzumab displayed great therapeutic efficacy on breast cancer. Owing to the existence of platinum, cisplatin also showed some adverse effects [50,51]. HA may be suitable carrier material for delivering cisplatin, which would achieve accumulation and retention in lymphatic location and lower the toxicity of platinum. Cisplatin conjugated to HA could be used for intralymphatic chemotherapy [52] (Fig. 3). First, HA and cisplatin were dissolved in water and activated by silver nitrate for 18 h to obtain HA-cisplatin conjugate. Cisplatin content in this conjugate was up to 25% and half-life release lasted for 42 h in water and 10 h in physiological condition. In cell toxicity, the conjugate had equal IC50 (7 mg/mL) with pure cisplatin on human breast cancer cell lines. And the AUC of the conjugate (776.0 mg h/g) was 1.74 times higher than that of native cisplatin (446.0 mg h/g) in draining lymph node basin. There was no distinct difference between the AUC of conjugate and native drug in non-draining lymph node yet. The result illustrated that the strong affinity of HA and receptor favored more drugs into tumor cells. In addition, the conjugate showed longer release than control group, which accounted for decreased organ toxicity and targeting accumulation on lymph node. The results verified that this study realized the intra-lymphatic delivery of cisplatin and HA would be a promising carrier for drug delivery. 2.2.3. HA-Butyrate bioconjugate Butyric acid (BTA), a short-chain fatty acid, is a potential inhibitor on the growth of various tumor cells such as breast cancer cells [53,54]. Owing to the short half-life (only 5 min), BTA was provided insufficient therapeutic concentrations in vivo, so it failed to use in practice [55,56]. HA-BTA pro-drug could prevent BTA from degradation or excretion and prolong drug exposure time. After vast tries and improvements, HA-BTA conjugate was prepared to confirm the guess [57]. HA was transformed into acidic form firstly.
Fig. 3. Schematic representation of Cisplatin and HA conjugate (a) and cross-linking form (b).
H. Zhang et al. / European Journal of Medicinal Chemistry 86 (2014) 310e317
Butyric anhydride and HA were catalyzed by pyridine for 24 h to form HA butyric esters. Based upon cell experiment, the results demonstrated that HA-BTA conjugate enhanced therapeutic effect of original drug. HA-BTA with 19% substitution degrees of BTA exhibited IC50 of 0.48 mg/mL. It has been proved that MAb J173 was an inhibitor on affinity of HA and CD44 receptors. So, after MCF-7 cells were incubated with MAb J173, the uptake of HA-BTA conjugate by the cancer cells decreased obviously [58]. The enhanced absorption of this conjugate might closely attribute to the affinity effect of HA to CD44 receptors. HA-BTA conjugate also could transfer more drugs into tumor cells to play anti-proliferative and anti-tumor activity. Besides, this study confirmed that the molecular weight of HA had no evident effect on the activity of the conjugate, which was consistent with previous reports [59]. These evidences proved that HA-BTA conjugate would be a potential targeted delivery system for BTA. 3. HA-anti-inflammatory drug bioconjugates 3.1. HA-anti-inflammatory drug bioconjugates with linker Some anti-inflammatory drugs like methotrexate, dexamethasone, suffering from stereo-hindrance effect also need the spacers for the covalently combination of HA and drugs. 3.1.1. HA-methotrexate bioconjugate It is generally known that osteoarthritis (OA) might suffer the risk of the occurrence of synovial inflammation [60e62]. Owing to natural viscoelasticity and lubricity of polysaccharide, HA could weaken the pain of injection administration during treating OA as drug delivery carrier. But HA had no curative effect on inflammation [63]. Methotrexate (MTX) is effective to resist rheumatoid arthritis, while some adverse sides such as lung, liver and marrow toxicity also occurred along with administration [64,65]. The combination of MTX and HA may be a good solution [66]. A series of safe and effective HA-MTX conjugates were prepared to fight osteoarthritis [67]. In this study, four HA-MTX conjugates were designed with polypeptide and 4,7,10-trioxa-1,13-tridec-anediamine (PEG13) as spacers and showed in Fig. 4. The function of these compounds was tested. Conjugate 1, 2, 3 were linked by aPEG13, g-PEG13, GlyePheeLeueGly peptide chain and PEG13, respectively. Conjugate 4 was linked by PEG13 and AsnePheePhe peptide chain. The test result displayed that conjugate 4 had better
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activity than other conjugates (conjugate 1e3) and MTX or physical mixture of HA and MTX. In the study of antiproliferative action, conjugate 3 and 4 had strong inhibiting effect on human fibroblastlike synoviocytes (HFLS), which could be explained that the peptide chain was digested and broke by cathepsin and then mass drugs were released from conjugates into cancer cells to kill these cells rapidly. Conjugate 1 and 2 without peptide showed no inhibition on HFLS. Based on the in vivo assay on antigen-induced arthritis rats, all these conjugates had stronger anti-inflammatory function than pure MTX, HA or physical mixture. For simple and controlled industrial production, the conjugate 4 was further optimized in terms of the amount of amino acids, molecular weight of HA and substitution degree of MTX [68]. Linker, molecular weight and substitution degree were investigated and screened on the base of degraded fragmentations of peptide chain and inhibition efficiency on HFLS proliferation and therapeutic effect on knee swelling. PheePhe peptide chain and ethylenediamine were decided as the optimal linker. The optimized HA-MTX conjugate showed a favorable drug loading of 1.9e2.1% and molecular weight of 1860e2180 kDa. This conjugate succeeded to weaken pain, reduce side effects and inhibit HFLS proliferation and knee swelling, which proved that HA-MTX conjugate would be a feasible and great drug delivery strategy. 3.1.2. HA-dexamethasone bioconjugate Because of inflammation, most intra-abdominal adhesions occur in abdominal surgeries, which may lead to pains of patients, bowel obstruction and even sterility [69e71]. HA could be applied into the peritoneum to relieve abdominal adhesions [72]. Dexamethasone (DEX), a sort of glucocorticoid drugs, has powerful antiinflammatory activity. Many formulations like liposome, hydrogel, conjugate, nanoparticle have been studied and developed [71,73,74]. Therefore, DEX was combined to HA and the conjugate further cross-linking to form hydrogel to resist inflammatory of peritoneal adhesions [75] (Fig. 5). DEX and succinic anhydride (suc) were incubated in presence of DMAP overnight and activated by NHS and DCC to obtain DEX-suc-NHS ester (DEX-suc-NHS). DEXsuc-NHS solution was dropped into HA-ADH solution to get HADEX conjugate. Then the conjugate was dissolved in water and injected into a rubber mold to obtain HA-DEX hydrogel. In addition, HA-aldehyde (HA-ALD) conjugate was also synthesized. HA-DEX was cross-linked with HA-ALD to prepare HAX-DEX hydrogels. Besides, HA-ADH was also cross-linked with HA-ALD to prepare
Fig. 4. Schematic view of HA-MTX linked by PEG13 (a, b), PEG13 and GlyePheeLeueGly peptide chain (c), PEG13 and AsnePheePhe peptide chain (d), EI and PheePhe peptide chain(e).
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Fig. 5. The schematic representation of cross-linked hydrogel composed by HA-ALD and HA-DEX.
HAX hydrogels as control group. MTT test results demonstrated that HA-DEX had a much higher cytotoxicity than HA, HA-ADH or HA-ALD. In the release experiment, approximately 20% DEX was broken from the HA-DEX conjugate in cell culture media after 8 days. When inflammation occurred, cells would release tumor necrosis factor-alpha (TNF-a) and inter leukin-1 (IL-1) and IL-6). These cytokines could reduce the activity of plasminogen activator and weaken the degradation action of fibrin, which caused adhesion and inflammation [76,77]. In the test of antiinflammatory, HAX-DEX showed more obvious suppression on production of IL-6 or TNF-a than HAX. It was concluded that this HAX-DEX conjugate hydrogel showed controlled release, strong anti-inflammatory effect and biocompatibility of DEX.
3.2. HA-methylprednisolone bioconjugate without linker Methylprednisolone (MPL), a synthetic glucocorticoid, has strong anti-inflammatory effects and could be used for the treatment of joint disease [78]. But some efforts need to be made to achieve long retention time, low toxicity to cartilage matrix and great biocompatibility. MPL could be grafted to HA by esterification reaction between the extended hydroxyl groups of MPL in C-21 position and the carboxyl groups of HA to prepare HA-MPL conjugate [79]. The release in vitro and in vivo of MPL from HA-MPL conjugate films and microspheres were compared [80]. HA-MPL conjugate in the form of films or microspheres showed much longer t1/2 (71 ± 27 h and 96 ± 12 h) than corresponding control groups (2.6 ± 0.4 h and 17 ± 5 h). And the conjugate showed a longer sustained release time (nearly 8 h) than the control groups (3 h) in tear fluid. When the HA-MPL conjugate was incubated in buffer solution under different pH conditions, the drug release amount was gradually increased along with the rising pH varying from 6.5 to 8.0 [81]. This release also accelerated along with the temperature rising from 20 C to 37 C. The aggregation behavior of HA-MPL conjugate was further assessed in aqueous solution [82]. Probably owing to hydrogen bond network being destroyed to some extent, high substitution degree of MPL in the conjugate resulted in low viscosity. Based on continuous research and
evaluation, HA-MPL conjugate would be further studied and developed and achieved greater breakthrough. 4. Conclusion Based on many distinct advantages of HA like inherent viscoelastic properties, strong water-binding ability, biocompatibility and non-toxicity, HA-drug conjugates strategy could be used to overcome some disadvantages of coupled drugs to realize broaden applications. Through numerous trials and attempts, many HAdrug bioconjugates have been studied and developed. These conjugates showed superior characteristics such as improved solubility and permeability, strong stabilization, controlled release profiles, enhanced therapeutic effect, less side effects and targeting ability. All results illustrated that HA-drug bioconjugates were potential in delivering drug as reference for further research and development. Acknowledgments This work was partly supported by ChinaeAustralia Centre for Health Sciences Research of Shandong University, China (2014GJ09) and the Natural Science Foundation of Shandong Province, China (No. ZR2011 HM026). References [1] E.J. Oh, K. Park, K.S. Kim, J. Kim, J.A. Yang, J.H. Kong, M.Y. Lee, A.S. Hoffman, S.K. Hahn, J. Control Release 141 (2010) 2e12. [2] V.O. Rousseau, Eur. J. Cancer 46 (2010) 1271e1277. [3] M. Slevin, J. Krupinski, J. Gaffney, S. Matou, D. West, H. Delisser, R.C. Savani, S. Kumar, Matrix Biol. 26 (2007) 58e68. [4] J.D. Kahmann, R. O'Brien, J.M. Werner, D. Heinegard, J.E. Ladbury, I.D. Campbell, A.J. Day, Structure 8 (2000) 763e774. [5] D.G. Jackson, Trends Cardiovas. Med. 13 (2003) 1e7. [6] P.H. Weigel, C. McGary, B. Zhou, J.A. Weigel, Hyaluronan 1 (2002) 401e410. [7] K. Kakehi, M. Kinoshita, S. Yasueda, J. Chromatogr. B 797 (2003) 347e355. , G. Zuber, C. Herlin, T.F. Vandamme, Carbohyd. Polym. 85 (2011) [8] C.E. Schante 469e489. [9] R. Stern, A. Asari, K. Sugahara, Eur. J. Cell. Biol. 85 (2006) 699e715. [10] C.B. Knudson, W. Knudson, Semin. Cell. Dev. Biol. 12 (2001) 69e78. [11] H. Lee, H. Mok, S. Lee, Y.K. Oh, T.G. Park, J. Control Release 119 (2007) 245e252.
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Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Invited review
Current research on hyaluronic acid-drug bioconjugates Haiqun Zhang a, Siling Huang b, Xiaoye Yang a, Guangxi Zhai a, * a b
Department of Pharmaceutics, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua Xilu, Jinan 250012, China Bloomage Freda Biopharm Co., Ltd., Jinan 250101, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 December 2013 Received in revised form 4 August 2014 Accepted 25 August 2014 Available online 27 August 2014
Hyaluronic acid (HA) is a mucopolysaccharide acid composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine. Based on numerous characteristics such as viscoelastic properties, water-binding ability, biocompatibility and non-immunogenicity, HA has been approved by FDA for biological and medical applications. In addition, multifarious receptors of HA like CD44, RHAMM and TSG6 are over-expressed on the surface of malignant cells, which play important roles in targeting ability. Bioconjugates linking drugs to HA could improve solubility, prolong half-life, provide active targeting capability and then increase the bioavailability of these coupled drugs by pro-drug strategy. Therefore, a large number of HA-drug bioconjugates have been studied. The purpose of this review was to summarize these HA-drug bioconjugates and further discuss synthetic methods and the relevant application in pharmaceuticals. © 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Hyaluronic acid Pro-drug Bioconjugates Synthesis Evaluation
1. Introduction Based on biocompatibility, biodegradability, nonimmunogenicity, water-bonding property and receptors such as CD44, RHAMM, TSG6, HA has drawn lots of attention in pharmacy [1e6]. In addition, HA held many significant biological functions such as stabilizing and organizing extracellular matrix, regulating cell adhesion and motility [7e9]. Therefore, HA and its derivatives have been studied widely [10]. In the biopharmaceutics classification system, there is a kind of drugs suffering from low bioavailability because of the low solubility and low permeability. A prodrug strategy of HA-drug conjugates could make full use of the superiorities of HA to make up for many deficiencies of these drugs. In the past few decades, investigators have devoted numerous efforts to develop HA-drug bioconjugates to enhance targeting ability, improve bioavailability and weaken adverse effects [11,12]. For example, HA-Exendin 4 and HA-Insulin bioconjugates were studied for the treatment of diabetics [13e19], which were prepared by peptide covalently combined with HA to prolong the short half-life, enhance stability and therapeutical effect [14,20e24]. Through vast trials and attempts, great achievements have been made. The aim of this paper was to present an overview of synthesis and evaluation of HA-drug bioconjugates. The structural formulas
* Corresponding author. E-mail addresses: [email protected] (H. Zhang), [email protected] (G. Zhai). http://dx.doi.org/10.1016/j.ejmech.2014.08.067 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.
and corresponding thumbnails of HA and all drugs were showed in Table 1 and structural formulas of linkers in HA-drugs bioconjugates were showed in Table 2. Detailed examples were illustrated and discussed as reference for future research and application. 2. HA-anticancer drug bioconjugates 2.1. HA-anticancer drug bioconjugates with linker Owing to steric hindrance, HA and anticancer drugs could not be directly combined covalently. Therefore, the bioconjugates need linkers the process needs of synthesis, which are discussed as follows in details. 2.1.1. HA-paclitaxel bioconjugate Paclitaxel (PTX), initially extracted from the bark of pacific yew, is a powerful anticancer drug [25,26]. However, many disadvantages such as poor solubility, toxic side effects and drug-resistance limited the application of PTX. A lot of investigations have been conducted to overcome these disadvantages [27]. Luo et al. prepared HA-PTX conjugate by the dihydrazide method [28] (Fig. 1A). PTX was combined to HA with anhydride and hydrazide as spacers. HA was activated by 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and reacted with adipic dihydrazide (ADH) to obtain HA-ADH. PTX was reacted with succinic anhydride (Suc) in the presence of pyridine for 3 days at room temperature to obtain the 20 -hemisuccinate PTX derivative (PTX-Suc) [29]. Then the PTX-Suc
H. Zhang et al. / European Journal of Medicinal Chemistry 86 (2014) 310e317 Table 1 Structural formulas and thumbnail of HA and drugs.
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was activated by carbodiimide coupling reagent composed of diphenylphosphoryl chloride, NHS and triethylamine, and reacted with N-hydroxysuccinimide (NHS) to obtain PTX-NHS ester [30]. HA-ADH and PTX-NHS eater were mixed at pH 6.5 for 24 h to obtain HA-PTX conjugate. In the study of cell toxicity, the HA-PTX conjugate showed the strong toxicity on ovarian cancer, breast cancer, and colon tumor cells, which could over-express receptors of HA. While the conjugate had no effect on mouse fibroblast cells without these receptors [31]. In the cell uptake assays, the result suggested that the HA-PTX conjugate entered into the cell within several minutes and then gradually appeared in the nucleus of ovarian cancer, breast cancer, and colon tumor cells. Conversely, a slow uptake process was showed in fibroblast cells group. The conjugate had lower IC50, e.g. the half inhibitory concentration, than free Taxol and HA-ADH. In addition, the conjugate exhibited high drug loading (up to 15%) and solubility, strong cytotoxicity and targeting capability. Moreover, HA-PTX conjugates were prepared and investigated by other synthesis methods. Rosato and colleagues prepared HAPTX conjugate with halogenated acid as the linker for fighting superficial bladder cancer [32] (Fig. 1B). PTX was activated by EDC and reacted with 4-bromobutyric acid (4-BBa). The intermediate product mixed with HA-thiobarbituric acid under anhydrous conditions for 7 days to obtain HA-PTX conjugate. Besides, this conjugate was prepared by Suc and polyamine as the spacer [33] (Fig. 1C). A 20 -hemisuccinate derivative of PTX was prepared and activated by NHS and dicyclohexylcarbodiimide (DCC) to get the activated ester. HA was activated by EDC and linked to ethyleneimine (EI) at pH 5.0 to obtain an amine-modified HA. The two
intermediate products were reacted to form HA-PTX conjugate. Lee et al. further developed HA-PTX conjugate [34] (Fig. 1D). HA was desalted by dialysis primarily. HA and poly(ethylene glycol) dimethyl ether (dmPEG) were mixed in deionized water to prepare HA/dmPEG. The HA/dmPEG was dialyzed and freeze-dried to get complex powder. This powder was used to improve the solubility of HA in organic solvents such as DMSO/DMF. Then, this HA/dmPEG complex powder was activated by DCC and 4dimethylaminopyridine (DMAP) and reacted with PTX in anhydrous DMSO to obtain the final HA-PTX conjugate. Based on a series of evaluations on these conjugates, HA-PTX conjugates showed many improvements such as improved solubility, biocompatibility, active targeting ability, high antitumor activity and cell uptake ability. 2.1.2. HA-Doxorubicin bioconjugate Doxorubicin (DOX), an anthracycline antibiotic, is an important anticancer agent with extensive biological activities [35,36]. However, the occurrence of renal toxicity, hepatic toxicity, cardiactoxicity and foaming phenomenon limited the application of DOX [37]. Bioconjugates of DOX and biocompatible materials like HA could decrease or compromise the toxicity and had active targeting ability [38,39]. Hence, these superiorities might provide a platform for HA-DOX conjugate to deliver DOX selectively to breast cancer cells and lymphatic cells. HA-DOX conjugate was made for treating breast cancers [40]. HA and ADH was dissolved in deionized water by activation of EDC to prepare HA-ADH. Then HA-ADH was reacted with DOX at pH 6.5 in sodium phosphate buffer for 2 h. The HADOX conjugate solution was dialyzed until no change of color.
H. Zhang et al. / European Journal of Medicinal Chemistry 86 (2014) 310e317 Table 2 Structural formulas of linker in HA-drugs bioconjugates.
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H. Zhang et al. / European Journal of Medicinal Chemistry 86 (2014) 310e317
HA
COOH
+ Suc +EI
4-BBa+ADH
Suc +ADH
+
PTX
PTX
OCO
Suc+ADH PTX
NH
OCO
CO
No linker
OH
HA
4-BBa +ADH
PTX
PTX NH
CO
OCO
OCO
HA
HA Suc +EI
NH
CO
HA
Fig. 1. Schematic view of HA linking with PTX by Suc and ADH (A), 4-bromobutyric acid and ADH (B), Suc and EI(C), direct link (D).
Properties of this conjugate were further evaluated. The half-life (t1/ of DOX releasing from the conjugate lasted for 172 h at pH 7.4, 110 h at pH 6.0 and 45 h at pH 5.0, respectively, which indicated the pH-sensitivity of linker of the HA-DOX conjugate. The Cmax value, peak concentration of drug, of HA-DOX (0.130 ± 0.020 mg/mL within 30 min) was lower than that of free DOX (2.580 ± 0.670 mg/mL within 5 min), which revealed lower toxic side effects of HA-DOX. Moreover, the area under the curve (AUC) of HA-DOX (2.201 ± 0.905 (mgh)/mL) was higher than that of DOX (2.061 ± 0.824 (mgh)/mL), which proved sustained release profile and long circulation time of HA-DOX. The HA-DOX conjugate exhibited 3.2-folds longer mean residence time than free DOX, which could minimize administration frequency and provide convenience for patients. In conclusion, high drug loading and solubility, sustained release and low toxicity all contributed to the development of HA-DOX.
2)
2.2. HA-anticancer drug bioconjugates without linker The coupling reaction of HA and drugs could be realized without linkers, when the reactive groups of anticancer drug were exposed enough for direct reaction. Bioconjugates without linker are illustrated under-mentioned at length. 2.2.1. HA-Curcumin bioconjugate Curcumin (Cur) has drawn growing attention due to a lot of physiological activities of this substrate, such as inhibiting the growth of cancer cells, resisting oxidant, anti-inflammatory and anti-HIV, preventing fatty liver and protect kidney [41e46]. However, many shortcomings such as low solubility, low permeability and weak stability accompanied with the administration of Cur, which led to low bioavailability [47]. Cur was bonded to hydrophilic biomaterials could improve solubility, enhance stability and increase therapeutic efficacy of the drug. HA-Cur conjugate was prepared and the conjugate self-assembled to form micelles [48]
Fig. 2. Schematic representation of HAeCur conjugate and the formation of HAeCur micelles.
(Fig. 2). After carboxylic group of HA was activated by DCC and DMAP, Cur was added and reacted with the activated HA for 6 h at 60e65 C to obtain HA-Cur conjugate. Then the conjugate was dissolved in aqueous solution to self-assembly form micelles. Evaluation results showed that the HA-Cur conjugate displayed favorable drug loading (appropriately 1.3%), high solubility (7.5 mg/ mL) and strong stability (over 8 h). Only 13 mg of Cur in HA-Cur could lead to nearly 80% cells deaths, which showed high inhibition rate on cancer cells. The micelle formed by HA-Cur conjugate presented a mean diameter of 287 nm with obvious core shell structure, zeta potential in the range of 25 mV to 75 mV at different pH conditions and low critical aggregation concentration of 33.4 mg/mL. Considering the existence of HA in the conjugate, the micelle would be endowed with potential active targeting capacity, but there was no concrete evidence to prove in this study. All results suggested that this developed HA-Cur conjugate successfully overcame the low solubility and permeability and weak stability of Cur and provided a reference for further research. 2.2.2. HA-cisplatin bioconjugate Commonly, once primary breast tumor cells spread, breast cancers will migrate to regional lymph nodes. The surgery or radiation therapy brings painful lymphedema [49]. It has been certified that combined application of cisplatin and taxanes and trastuzumab displayed great therapeutic efficacy on breast cancer. Owing to the existence of platinum, cisplatin also showed some adverse effects [50,51]. HA may be suitable carrier material for delivering cisplatin, which would achieve accumulation and retention in lymphatic location and lower the toxicity of platinum. Cisplatin conjugated to HA could be used for intralymphatic chemotherapy [52] (Fig. 3). First, HA and cisplatin were dissolved in water and activated by silver nitrate for 18 h to obtain HA-cisplatin conjugate. Cisplatin content in this conjugate was up to 25% and half-life release lasted for 42 h in water and 10 h in physiological condition. In cell toxicity, the conjugate had equal IC50 (7 mg/mL) with pure cisplatin on human breast cancer cell lines. And the AUC of the conjugate (776.0 mg h/g) was 1.74 times higher than that of native cisplatin (446.0 mg h/g) in draining lymph node basin. There was no distinct difference between the AUC of conjugate and native drug in non-draining lymph node yet. The result illustrated that the strong affinity of HA and receptor favored more drugs into tumor cells. In addition, the conjugate showed longer release than control group, which accounted for decreased organ toxicity and targeting accumulation on lymph node. The results verified that this study realized the intra-lymphatic delivery of cisplatin and HA would be a promising carrier for drug delivery. 2.2.3. HA-Butyrate bioconjugate Butyric acid (BTA), a short-chain fatty acid, is a potential inhibitor on the growth of various tumor cells such as breast cancer cells [53,54]. Owing to the short half-life (only 5 min), BTA was provided insufficient therapeutic concentrations in vivo, so it failed to use in practice [55,56]. HA-BTA pro-drug could prevent BTA from degradation or excretion and prolong drug exposure time. After vast tries and improvements, HA-BTA conjugate was prepared to confirm the guess [57]. HA was transformed into acidic form firstly.
Fig. 3. Schematic representation of Cisplatin and HA conjugate (a) and cross-linking form (b).
H. Zhang et al. / European Journal of Medicinal Chemistry 86 (2014) 310e317
Butyric anhydride and HA were catalyzed by pyridine for 24 h to form HA butyric esters. Based upon cell experiment, the results demonstrated that HA-BTA conjugate enhanced therapeutic effect of original drug. HA-BTA with 19% substitution degrees of BTA exhibited IC50 of 0.48 mg/mL. It has been proved that MAb J173 was an inhibitor on affinity of HA and CD44 receptors. So, after MCF-7 cells were incubated with MAb J173, the uptake of HA-BTA conjugate by the cancer cells decreased obviously [58]. The enhanced absorption of this conjugate might closely attribute to the affinity effect of HA to CD44 receptors. HA-BTA conjugate also could transfer more drugs into tumor cells to play anti-proliferative and anti-tumor activity. Besides, this study confirmed that the molecular weight of HA had no evident effect on the activity of the conjugate, which was consistent with previous reports [59]. These evidences proved that HA-BTA conjugate would be a potential targeted delivery system for BTA. 3. HA-anti-inflammatory drug bioconjugates 3.1. HA-anti-inflammatory drug bioconjugates with linker Some anti-inflammatory drugs like methotrexate, dexamethasone, suffering from stereo-hindrance effect also need the spacers for the covalently combination of HA and drugs. 3.1.1. HA-methotrexate bioconjugate It is generally known that osteoarthritis (OA) might suffer the risk of the occurrence of synovial inflammation [60e62]. Owing to natural viscoelasticity and lubricity of polysaccharide, HA could weaken the pain of injection administration during treating OA as drug delivery carrier. But HA had no curative effect on inflammation [63]. Methotrexate (MTX) is effective to resist rheumatoid arthritis, while some adverse sides such as lung, liver and marrow toxicity also occurred along with administration [64,65]. The combination of MTX and HA may be a good solution [66]. A series of safe and effective HA-MTX conjugates were prepared to fight osteoarthritis [67]. In this study, four HA-MTX conjugates were designed with polypeptide and 4,7,10-trioxa-1,13-tridec-anediamine (PEG13) as spacers and showed in Fig. 4. The function of these compounds was tested. Conjugate 1, 2, 3 were linked by aPEG13, g-PEG13, GlyePheeLeueGly peptide chain and PEG13, respectively. Conjugate 4 was linked by PEG13 and AsnePheePhe peptide chain. The test result displayed that conjugate 4 had better
315
activity than other conjugates (conjugate 1e3) and MTX or physical mixture of HA and MTX. In the study of antiproliferative action, conjugate 3 and 4 had strong inhibiting effect on human fibroblastlike synoviocytes (HFLS), which could be explained that the peptide chain was digested and broke by cathepsin and then mass drugs were released from conjugates into cancer cells to kill these cells rapidly. Conjugate 1 and 2 without peptide showed no inhibition on HFLS. Based on the in vivo assay on antigen-induced arthritis rats, all these conjugates had stronger anti-inflammatory function than pure MTX, HA or physical mixture. For simple and controlled industrial production, the conjugate 4 was further optimized in terms of the amount of amino acids, molecular weight of HA and substitution degree of MTX [68]. Linker, molecular weight and substitution degree were investigated and screened on the base of degraded fragmentations of peptide chain and inhibition efficiency on HFLS proliferation and therapeutic effect on knee swelling. PheePhe peptide chain and ethylenediamine were decided as the optimal linker. The optimized HA-MTX conjugate showed a favorable drug loading of 1.9e2.1% and molecular weight of 1860e2180 kDa. This conjugate succeeded to weaken pain, reduce side effects and inhibit HFLS proliferation and knee swelling, which proved that HA-MTX conjugate would be a feasible and great drug delivery strategy. 3.1.2. HA-dexamethasone bioconjugate Because of inflammation, most intra-abdominal adhesions occur in abdominal surgeries, which may lead to pains of patients, bowel obstruction and even sterility [69e71]. HA could be applied into the peritoneum to relieve abdominal adhesions [72]. Dexamethasone (DEX), a sort of glucocorticoid drugs, has powerful antiinflammatory activity. Many formulations like liposome, hydrogel, conjugate, nanoparticle have been studied and developed [71,73,74]. Therefore, DEX was combined to HA and the conjugate further cross-linking to form hydrogel to resist inflammatory of peritoneal adhesions [75] (Fig. 5). DEX and succinic anhydride (suc) were incubated in presence of DMAP overnight and activated by NHS and DCC to obtain DEX-suc-NHS ester (DEX-suc-NHS). DEXsuc-NHS solution was dropped into HA-ADH solution to get HADEX conjugate. Then the conjugate was dissolved in water and injected into a rubber mold to obtain HA-DEX hydrogel. In addition, HA-aldehyde (HA-ALD) conjugate was also synthesized. HA-DEX was cross-linked with HA-ALD to prepare HAX-DEX hydrogels. Besides, HA-ADH was also cross-linked with HA-ALD to prepare
Fig. 4. Schematic view of HA-MTX linked by PEG13 (a, b), PEG13 and GlyePheeLeueGly peptide chain (c), PEG13 and AsnePheePhe peptide chain (d), EI and PheePhe peptide chain(e).
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Fig. 5. The schematic representation of cross-linked hydrogel composed by HA-ALD and HA-DEX.
HAX hydrogels as control group. MTT test results demonstrated that HA-DEX had a much higher cytotoxicity than HA, HA-ADH or HA-ALD. In the release experiment, approximately 20% DEX was broken from the HA-DEX conjugate in cell culture media after 8 days. When inflammation occurred, cells would release tumor necrosis factor-alpha (TNF-a) and inter leukin-1 (IL-1) and IL-6). These cytokines could reduce the activity of plasminogen activator and weaken the degradation action of fibrin, which caused adhesion and inflammation [76,77]. In the test of antiinflammatory, HAX-DEX showed more obvious suppression on production of IL-6 or TNF-a than HAX. It was concluded that this HAX-DEX conjugate hydrogel showed controlled release, strong anti-inflammatory effect and biocompatibility of DEX.
3.2. HA-methylprednisolone bioconjugate without linker Methylprednisolone (MPL), a synthetic glucocorticoid, has strong anti-inflammatory effects and could be used for the treatment of joint disease [78]. But some efforts need to be made to achieve long retention time, low toxicity to cartilage matrix and great biocompatibility. MPL could be grafted to HA by esterification reaction between the extended hydroxyl groups of MPL in C-21 position and the carboxyl groups of HA to prepare HA-MPL conjugate [79]. The release in vitro and in vivo of MPL from HA-MPL conjugate films and microspheres were compared [80]. HA-MPL conjugate in the form of films or microspheres showed much longer t1/2 (71 ± 27 h and 96 ± 12 h) than corresponding control groups (2.6 ± 0.4 h and 17 ± 5 h). And the conjugate showed a longer sustained release time (nearly 8 h) than the control groups (3 h) in tear fluid. When the HA-MPL conjugate was incubated in buffer solution under different pH conditions, the drug release amount was gradually increased along with the rising pH varying from 6.5 to 8.0 [81]. This release also accelerated along with the temperature rising from 20 C to 37 C. The aggregation behavior of HA-MPL conjugate was further assessed in aqueous solution [82]. Probably owing to hydrogen bond network being destroyed to some extent, high substitution degree of MPL in the conjugate resulted in low viscosity. Based on continuous research and
evaluation, HA-MPL conjugate would be further studied and developed and achieved greater breakthrough. 4. Conclusion Based on many distinct advantages of HA like inherent viscoelastic properties, strong water-binding ability, biocompatibility and non-toxicity, HA-drug conjugates strategy could be used to overcome some disadvantages of coupled drugs to realize broaden applications. Through numerous trials and attempts, many HAdrug bioconjugates have been studied and developed. These conjugates showed superior characteristics such as improved solubility and permeability, strong stabilization, controlled release profiles, enhanced therapeutic effect, less side effects and targeting ability. All results illustrated that HA-drug bioconjugates were potential in delivering drug as reference for further research and development. Acknowledgments This work was partly supported by ChinaeAustralia Centre for Health Sciences Research of Shandong University, China (2014GJ09) and the Natural Science Foundation of Shandong Province, China (No. ZR2011 HM026). References [1] E.J. Oh, K. Park, K.S. Kim, J. Kim, J.A. Yang, J.H. Kong, M.Y. Lee, A.S. Hoffman, S.K. Hahn, J. Control Release 141 (2010) 2e12. [2] V.O. Rousseau, Eur. J. Cancer 46 (2010) 1271e1277. [3] M. Slevin, J. Krupinski, J. Gaffney, S. Matou, D. West, H. Delisser, R.C. Savani, S. Kumar, Matrix Biol. 26 (2007) 58e68. [4] J.D. Kahmann, R. O'Brien, J.M. Werner, D. Heinegard, J.E. Ladbury, I.D. Campbell, A.J. Day, Structure 8 (2000) 763e774. [5] D.G. Jackson, Trends Cardiovas. Med. 13 (2003) 1e7. [6] P.H. Weigel, C. McGary, B. Zhou, J.A. Weigel, Hyaluronan 1 (2002) 401e410. [7] K. Kakehi, M. Kinoshita, S. Yasueda, J. Chromatogr. B 797 (2003) 347e355. , G. Zuber, C. Herlin, T.F. Vandamme, Carbohyd. Polym. 85 (2011) [8] C.E. Schante 469e489. [9] R. Stern, A. Asari, K. Sugahara, Eur. J. Cell. Biol. 85 (2006) 699e715. [10] C.B. Knudson, W. Knudson, Semin. Cell. Dev. Biol. 12 (2001) 69e78. [11] H. Lee, H. Mok, S. Lee, Y.K. Oh, T.G. Park, J. Control Release 119 (2007) 245e252.
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