Citation

Can Chen, Yiyan Li, Xiaoping Yu, Qinglin Jiang, Xiaohong Xu, Qian Yang, Zhiyong Qian. Bone-targeting melphalan prodrug with tumor-microenvironment sensitivity: Synthesis, in vitro and in vivo evaluation[J]. Chin. Chem. Lett., 2018, 29(11): 1609-1612  

Can Chen^a, Yiyan Li^b, Xiaoping Yu^c, Qinglin Jiang^b, Xiaohong Xu^b, Qian Yang^b, Zhiyong Qian^d    

Bone tumors were classified as "primary tumors", which originate in bone or from bone-derived cells and tissues, and the "secondary tumors" which initiate in other tissues and spread to the skeleton [1]. The main treatments of bone tumor are highly dependent on the type of tumors, however, mainly concentrated on surgery, chemotherapy, radiotherapy and hormone therapy. In chemotherapy, bisphosphonates (BPs) possessed effective accumulation in bone tissue, preferentially at the metastatic bone lesions, were used herein to as front line treatment for malignant bone disease [2-5]. To date, several bisphosphonates which vary central oxygen groups with different carbon units and adhesion to calcium hydroxyapatite are in clinical use [6].

During the treatment of bone metastasis, BPs are potent therapeutic agents for clinical used in spite of a dose-dependent side effects [7, 8]. Beyond their therapeutic effect, bisphosphonates are currently used as promising targeting agents in chemotherapy due to their bone selectivity [9]. Salerno et al. reported a novel bone-seeking PLGA-alendronate nanoparticles (NPs), which showed directly targeting effect for the site of tumor-induced osteolysis by systemic administration [10]. Another study employed a similar strategy demonstrated that the paclitaxelPEG-alendronate conjugated NPs exhibited an improved binding affinity to the bone mineral hydroxyapatite (HA) in vitro, and a comparable cellular proliferation inhibition to that of the combination of free drugs in the PC3 human prostate adenocarcinoma cells [11]. A covalent conjugation of 5-FdU and bisphosphonate alendronate demonstrated the growth inhibition of Lewis lung carcinoma and murine macrophages due to the high specificity for bone tissue [12]. The water-soluble doxorubicin prodrugs, which incorporated a bisphosphonate as the bone targeting moiety and an environment sensitive linkage cleaved at an acidic pH value or enzymatically by cathepsin B, was reported [6]. Therefor the strategy of the tissue specific conjugated drug delivery strategy was proved enhancing the therapeutic effect of chemo-drugs and reducing their toxicity.

Since 1990s, matrix metalloproteinases (MMPs) are known to play a crucial role in the invasion and metastasis of tumors [13]. The MMP-2 cleavable peptides (Pro-Val-Gly-Leu-Ala-Gly), characterized by the Langer group [14], were employed as the linage to conjugated chemotherapeautic drugs with carriers[15-18].Herein, wesetoutto develop a novel mephalan-bisphosphonate prodrug that exploit the bone-targeting properties of the bisphosphonate fragment to ensure a selective release and accumulation of anticancer agent at the desired site of action (Scheme 1). In this work, we describe the synthesis of the mephalan-bisphosphonate prodrug which could effectively cleaved in enzyme-sensitive manner by MMP and demonstrate binding properties to bone tissue in vitro and in vivo. This prodrug with bone microenvironment specificity would provide the potential to increase the therapeutic effects and decrease the off-target toxicity (Fig. 1).

Scheme 1

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Scheme 1. General design of novel melphalan-bisphosphonate prodrug.

Figure 1

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Fig. 1. Scheme of chemical synthesis of prodrugs L1 and FITC-labeled L1.

In this study, based on the MMP-responded prodrug synthesis, we designed and synthesized a novel mephalan-bisphosphonate prodrug L₁ which could be effectively cleaved in enzyme-sensitive manner by MMP and be accumulated at the bone tissue. Firstly, commercial tetraethyl methylenebisphosphonate was functionalized with amino group, and then a MMP-2 sensitive hexapeptide (Pro-Leu-Gly-Leu-Ala-Gly) with Boc-protected was added for conjugation with bisphosphonate. After deprotection of the Boc group, Cbz-protected Melphalan was used for reaction with the obtained hexapeptide bisphosphonate. At last, the target compound L₁ was obtained after deprotection. The detail methods for prodrug synthesis has been listed in Supporting information (The structure was confirmed by ¹H NMR and HRMS). The prodrug exhibited excellent solubility in aqueous solutions and showed desirable binding affinity to bone tissue. In addition, in order to further study the affinity to bone tissue through fluorescence imaging in vitro and in vivo, L₁ was conjugated with fluorescein isothiocyanate isomer I (FITC), which formed FITC-labeled L₁.

The hydroxyapatite-binding assay provided information on the interaction between melphalan-bisphosphonate prodrug L₁ and hydroxyapatite (HA), which represents the mineral components of the bone tissue [19]. L₁ and free melphalan of 10 μg/mL were incubated with 50 equiv of HA at pH 7.4 and 37 ℃, respectively. Only very weak binding was observed (lower than 10%, Fig. 2A), even when melphalan was incubated with HA for 24 h. In contrast, the prodrug L₁ demonstrated an ability to bind the bone tissue more than 8 times greater, compared to the native free melphalan. The binding affinity of prodrug showed significantly increase with time, almost completely binding of L₁ and HA within 2 h were observed (Fig. 2A). Not unexpectedly, after the initially rapid binding, binding percentage of the prodrug L₁ to HA showed slowly decrease, which would contribute to the cleavage of prodrug L₁.

Figure 2

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Fig. 2. (A) Binding of melphalan and prodrugs L1 to hydroxyapatite (HA) at pH 7.4 and 37 ℃; (B) Release of melphalan for prodrug L1 with or without MMP-2 at pH 7.4 and 37 ℃; (C) The magnification of the release profiles in the initial 24 h of B.

The release profiles of mephalan from conjugated prodrug with or without MMP-2 were investigated by the dialysis method. Prodrug L₁ (1.0 mg) was dissolved and then incubated in release buffer (150 μmol/L NaCl, 10 μmol/L CaCl₂, 40 μmol/L Tris, pH 7.4), with or without MMP-2 containing Tween 80 (0.5 wt%) at 37 ℃. At given time points, 1 mL of medium was withdrawn and replaced with same volume of fresh buffer. To calculate cumulative amount of released mephalan, the samples were analyzed by HPLC. The prodrug was cleaved to melphalan quickly under the conditions chosen (MMP-2: 50 nmol/L), the half-life for L₁ was about 75 min, the prodrug was almost completely released after 6–8 h. Under the same conditions without MMP-2, cleavage was slower, with melphalan already being released about 75% after six days (Fig. 2B). The results indicated that the in vitro drug release of prodrug L₁ was responsive to MMP-2.

Following the study of drug release behavior, the cleavage properties of prodrug L₁ was studied before its further application. The prodrug L₁ with or without BB-94 were incubated with human osteosarcoma MG-63 cells over a period of 24 h, respectively. Free FITC was also incubated with MG-63 cells serving as the negative control. Then the culture medium in each well was discarded and washed with phosphate buffered solution (PBS) (pH 7.4, 0.01 mol/L) twice, subsequently incubated with DAPI for another 0.5 h. Finally, the fluorescence images of intracellular uptake performance were taken by a fluorescence microscope (Olympus, Japan) to evaluate the uptake efficiency of sensitive prodrug. Under the microenvironment of MG-63 cells with high MMP-2 expression, most of the prodrug L₁ was enzymatically cleaved to melphalan, which resulted in low uptake within the same incubation time, so the fluorescent intensity was observed as lower as the free FITC treated group did. However, with the additional MMPs inhibitor BB-94 incubation, the prodrug L₁ still maintained hydrophilic molecular structure with the segments of BPs and MMP-2 cleavable peptides, which was easily uptake by cancer cell, and the fluorescent intensity of FITC-labeled L₁ treated group was significantly stronger than that of free FITC, as expected (Fig. 3A).

Figure 3

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Fig. 3. (A) The cellular uptake of FITC-labed L1 with or without MMPs inhibitor BB-94; (B) IC50 of free melphalan and prodrug L1 on the MC3T3-E1, MG63, U-2OS, SAOS-2, MCF-7, HepG2 and A549 cell lines.

In vitro cytotoxicity of L₁ and free melphalan were determined by cell viability assay on human sarcoma MG63, U-2OS and SAOS-2 cells, and the results were shown in Fig. 3B. Free melphalan showed slightly higher cytotoxicity than L₁. The mean concentration of 50% cell inhibition (IC₅₀) of L₁ was increased to 0.79-1.58 μmol/L compared with 0.68–1.35 μmol/L of free melphalan. Moreover, free melphalan displayed higher cytotoxicity on MCF-7, HepG2 and A549 cells than that of L₁, which suggested that the selectivity of prodrug L₁ was better that free melphalan. And both free melphalan and L₁ showed moderate to low cytotoxicity on MC3T3-E1 preosteoblast cell, with the IC₅₀ values of 6.34 μmol/L and 8.75 μmol/L, respectively.

In vitro hydroxyapatite binding assay and cellular proliferation assay have been listed in Supporting information.

The in vivo targeting affinity of the mephalan-bisphosphonate prodrugs was conducted by the rat models, which received of FITC-labeled mephalan-bisphosphonate prodrugs L₁ (1 mg/kg) and equivalent amount of the free FITC intravenously, respectively. After 24 h, the rats were sacrificed and thigh bones and shin bones were immediately eviscerated, as well as the main organs. Fluorescence imaging of each sample was performed in macro imaging system LT-9 equipped with illume tool dual light system LT-99D2 (Lightools Research, Encinitas, CA, USA). As shown in Fig. 4, FITC-labeled melphalan-bisphosphonate prodrug L₁ showed a greater bone tissue selectivity than the mixture of free FITC and melphala did at the investigated biodistribution times. After 24 h injection with FITC-labeled L₁ and free FITC showed a significantly greater accumulation in thigh bone and shin bone tissues (Fig. 4, upper row). Conversely, the administration of the mixture of free FITC resulted in a non-specific body-wide distribution of fluorescence (Fig. 4, lower row). These results suggested that the bone tissue specificity of bisphosphonate melphalan prodrug L₁ was indeed attributed to the bisphosphonate fragment, which would be employed as a promising candidate for the targeting bone cancer therapy.

Figure 4

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Fig. 4. The tissue distribution of FITC-labeled prodrug L1, the mixture of free FITC and melphalan.

In summary, novel MMP-2 enzyme sensitive melphalanbisphosphonate prodrug was designed and synthesized, and the in vitro affinity for bone tissue was primarily confirmed by hydroxyapatite binding assay. The cellular uptake and drug release results demonstrated that melphalan-bisphosphonate prodrug mainly depended on the bisphosphonate moiety linked to melphalan via the MMP-2 cleavable peptides. The in vitro cytotoxicity study proved the efficient tumor cell growth inhibition of Prodrug L₁. Moreover, in vivo biodistribution studies further demonstrated that specific bone accumulation of L₁, compared with the free melphalan. The findings of our study represent the advancing use of bisphosphonate, enzyme-sensitive peptide and chemotherapeutics as potent strategy for bone tumor treatment.

This work was financially supported by the National Natural Science Foundation of China (Nos. 31600811, 81573154, 81773432), the Application Fundamental Research Foundation of Sichuan Province Science and Technology Department, China (Nos. 2016JY0157, 2017JY0123), and Scientific Research Foundation of the Health and Family Planning Commission of Sichuan Province, China (Nos. 17PJ556, 17PJ563).

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.cclet.2018.02.010.