Novel bone targeted drug delivery system based on specific bisphosphonate

Abstract

The invention discloses application of a novel bone targeted drug delivery system based on specific bisphosphonate in new drug development and clinical application. The invention relates to an osteogenesis targeted drug conjugate, which is characterized in that the osteogenesis targeted drug conjugate BP-Btz is prepared by combining bisphosphonate (BP) without anti-bone resorption activity with a proteasome inhibitor bortezomib (Btz). The conjugate realizes local release of Btz in a bone microenvironment through the bone targeting ability of BP, the Btz level in non-bone tissues is very low, the curative effect is improved, and systemic side effects are reduced. In-vitro research shows that BP-Btz has similar effects to Btz in the aspects of inhibiting osteoclast formation, reducing bone resorption and stimulating osteoblast differentiation. In-vivo research shows that BP-Btz is more effective than Btz in the aspect of increasing bone volume, and side effects are reduced at the same time. In the aspect of pharmacodynamics, BP-Btz can be combined with a bone matrix and release Btz under the condition that the pH is 4-5, so that the Btz concentration in bone is higher, the half-life period is longer, and the drug resistance of Btz is effectively overcome. Therefore, the bone targeted drug delivery system can provide a new method for innovative application of drugs, and has important clinical application prospects and commercial potential.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a novel bone-targeted drug delivery system of specific bisphosphonates, the preparation of new drugs and the application thereof in skeletal muscle system diseases. Technical Background

[0002] The drug delivery system (DDS) is a drug delivery method that integrates multiple disciplines such as clinical medicine, pharmacy, and chemical materials science. It delivers drugs precisely to the target location by building a drug-loading platform.

[0003] With the development of population aging, bone-related diseases have become one of the factors that seriously affect people's quality of life. Systemic drug administration for the treatment of bone-related diseases has the disadvantages of poor targeting, large side effects, and low drug utilization. Bone-targeted drug delivery system can effectively overcome the defects of traditional drug administration methods for the treatment of bone-related diseases. Functional modification of drug carriers with bone-targeting groups can effectively improve drug efficacy, reduce costs, and reduce adverse reactions. For example, osteoporotic fractures are a serious health problem for the elderly, which will reduce the quality of life and increase medical expenses. Developing new treatments to increase bone mass and bone strength is a key clinical need. Studies have found that regulating the ubiquitin-proteasome system can increase the differentiation of mesenchymal stem cells into osteoblasts, thereby promoting fracture healing. The proteasome inhibitor bortezomib (Btz) is a proteasome inhibitor approved by the FDA for the treatment of multiple myeloma. It also increases bone volume by promoting osteoblast differentiation and inhibiting osteoclastogenesis in mice. However, Btz has serious systemic side effects, which will limit its use as a bone anabolic agent. Here, we designed and synthesized a bone-targeted form of Btz by coupling it to bisphosphonates (BPs) without antiresorptive activity. Bisphosphonate (BP) molecules are a class of drugs widely used to treat osteoporosis and other bone metabolic diseases. They inhibit the activity of osteoclasts by binding to calcium on the surface of hydroxyapatite, reducing bone resorption, increasing bone density and strength, and providing more matrix for bone formation, thereby promoting the fracture healing process. However, long-term use of bisphosphonates may cause some side effects, such as osteonecrosis of the jaw and atypical femoral fractures. These side effects may be related to the long-term accumulation of drugs in the bone. Therefore, there is an urgent need to develop new drug delivery routes to solve this problem.

[0004] There are three main types of drug delivery systems: liposome nano-delivery system, GalNAc conjugated delivery system, and other delivery systems. Bone-targeted delivery system is a drug delivery strategy that aims to deliver drugs or therapeutic agents specifically to bone tissue to increase local concentrations for better therapeutic effects and reduce side effects on other tissues. This strategy is suitable for the treatment of bone-related diseases such as fractures, bone cancer, osteoporosis, and other bone metabolic diseases. Currently, bone-targeted strategies can be achieved through several methods, such as drug modification, nanotechnology, bioactive molecules, gene therapy, etc.

[0005] In vitro studies have confirmed that BP has a very high affinity for hydroxyapatite and can therefore bind tightly to mineralized surfaces, which has been demonstrated in the literature. This property makes BP an ideal bone-targeted carrier to effectively deliver other drugs or therapeutic molecules to the bone. Therefore, a new bone-targeted drug delivery system for bisphosphonates was designed, which is characterized by the fact that BP has no anti-bone resorptive bioactivity and that a new drug structure "BP-Btz" is formed by combining Btz with BP, which is locally released in the bone, resulting in higher Btz concentrations in the bone and a longer half-life. Since Btz is locally released, the level of Btz in non-bone tissues is very low, so the adverse effects on the whole body are relatively small. Summary of the invention

[0006] In view of the shortcomings of current systemic drug administration for the treatment of bone-related diseases, such as poor targeting, large side effects, and low drug utilization, the present invention studies and designs a new bone-targeted drug delivery system of bisphosphonates, which is a bisphosphonate without anti-bone resorption activity coupled with bortezomib to form BP-Btz. BP-Btz utilizes the bone-targeting properties of BP to release Btz locally in the bone, increase its concentration and half-life in the bone, thereby improving the efficacy and reducing systemic adverse reactions.

[0007] In order to achieve the purpose of the invention, the present invention achieves the above purpose through the following technical solutions:

[0008] On the one hand, a bone-targeted drug delivery system, called BP-Btz, was designed by coupling bortezomib (Btz) with a phosphonate (BP) without antiresorptive activity.

[0009] Further evaluation of the bone metabolism regulation of BP-Btz in vitro and in vivo and comparison with Btz were conducted to explore whether BP-Btz has better bone selectivity and fewer systemic adverse reactions compared with Btz.

[0010] Research Methods

[0011] In vitro experiments. Bone marrow cell culture experiments were used to evaluate the effects of BP-Btz on osteoblast differentiation, osteoclast differentiation and bone resorption. Bone marrow cells from transgenic UbG76V-GFP mice were used to detect the inhibitory effect of Btz on the ubiquitin-proteasome system.

[0012] In vivo experiments. C57BL / 6J female mice were used for in vivo experiments. Three-month-old C57BL / 6J female mice were used to study the effects of BP-Btz on basal bone volume and OB differentiation. In addition, three-month-old C57BL / 6J female mice were subjected to ovariectomy (OVX) to observe the effects of BP-Btz on OVX-induced bone loss and OB differentiation. Tibial fracture models were established in aged C57BL / 6J mice (18 months old, male) to observe the effects of BP-Btz and Btz on bone mass, fracture healing, and systemic adverse reactions in these models.

[0013] Detection methods: Micro-CT analysis of bone tissue microstructure parameters, biomechanical testing analysis of bone strength, and histological staining for complete bone tissue morphology analysis of long bones.

[0014] Compared with existing products, the present invention has the following beneficial results: BP-Btz can achieve local release of Btz in the bone microenvironment through BP's bone targeting ability. The level of Btz in non-bone tissue is very low, which improves the efficacy and reduces systemic side effects. BP-Btz has similar effects to Btz in inhibiting osteoclast formation, reducing bone resorption, and stimulating osteoblast differentiation; it is more effective than Btz in increasing bone volume; BP-Btz can achieve higher Btz concentrations in bones, longer half-life, and effectively overcome Btz resistance. In summary, BP-Btz has more advantages than existing Btz, may benefit more in clinical practice, and has certain innovation and clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The invention discloses the chemical structure and molecular formula of the BP-Btz conjugate and the principle of releasing Btz on the bone surface, and also includes the molecular weight and molar dosage parameters of Btz and BP-Btz used in the invention.

[0016] Figure 2 BP-Btz has the function of inhibiting the formation of osteoclasts (OCs) and stimulating the differentiation of osteoblasts (OBs) by binding to bone.

[0017] Figure 3 In vivo experiments on mice showed that Bp-Btz has a stronger bone anabolic effect than Btz.

[0018] Figure 4BP-Btz restored OVX-induced bone loss more effectively than Btz.

[0019] Figure 5 BP-Btz promotes fracture repair more effectively than Btz in aged mice

[0020] Figure 6 The systemic side effects of BP-Btz on C57BL / 6J mice were lower than those of Btz.

[0021] Figure 7 The effect of injection of BP-Btz analogs with different release rates on tibial MM lesions in mice bearing 5TMG1-GFP myeloma cells.

[0022] Figure 8 BP-Btz acts as a drug release depot on the bone surface.

[0023] Fig. 9 This is a diagram demonstrating the principle of bone targeting effect of BP-Btz. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below by specific implementation methods. The embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0025] Example 1 Design and synthesis of targeted bortezomib analogs.

[0026] The bisphosphonate-bortezomib (BP-Btz) conjugate was designed using aminomethylene diphosphonic acid. In the BP analog, the amino group was directly connected to the BP part to make it lose its biological activity, and then diethanolamine was connected and coupled with Btz to synthesize 1,3,2-dioxazaboryl compound. It can specifically release BP and has no active effect ( Figure 1 A). Next, Btz was linked to the above BP analog using boronate ( Figure 1 B). The basic principle of this method is that BP slowly releases Btz from the most active part of bone metabolism into the acidic environment of the bone resorption cavity produced by active osteoclasts (OCs), resulting in an increase in local Btz concentration and a prolonged half-life, which can reduce or eliminate systemic adverse reactions of Btz ( Figure 1 C). The molecular weight of BP with a linker structure is 366.06, the molecular weight of Btz is 384.34, and the molecular weight of BP-Btz is 795.32. Because the BP part of BP-Btz has no absorption activity, we speculate that the biological activity of BP-Btz is likely to come from the proteasome. In order to compare the efficacy of BP-Btz with Btz, we selected a mouse experimental model to compare the efficacy of BP-Btz with Btz at the same dose ( Figure 1 D).

[0027] The following three mouse models were selected for the following experimental verification.

[0028] All animal experiments in the present invention were approved by the Animal Care and Use Committee. (1) Three-month-old UbG76V-GFP mice were used for bone marrow (BM) cell isolation and in vitro experiments to test the inhibitory effect of Btz on the degradation of UbG76V-GFP protein via the ubiquitin-proteasome pathway. (2) Three-month-old C57BL / 6J female mice (WT mice). (a) Mice (n=4 / group) were given the same dose (0.6 mg / kg / ip) of PBS, BP, Btz or BP-Btz every day for 3 weeks to determine the effect of BP-Btz on normal bone volume and OB differentiation, and to detect the side effects after BP-Btz treatment. (b) The model (OVX) was established by surgically removing the bilateral ovaries of WT mice. Mice (n=8 / group) were given equal doses of BP, Btz or BP-Btz 4 weeks after surgery, where Btz and BP were 0.6 mg / kg and BP-Btz was 1.2 mg / kg. The effects of BP-Btz on OVX-induced bone loss and osteoblast differentiation were determined after 4 weeks of treatment. (3) C57BL / 6J mice (18 months old, male) were treated with open tibial fracture surgery. Surgical steps: After anesthesia, a 6 mm incision was made on the skin of the anterior tibia of the mouse, and a 27G sterile steel needle was inserted into the bone matrix cavity through the proximal articular surface of the tibia. The steel needle was then withdrawn, the mid-section was transected with a scalpel, and the steel needle was reinserted to stabilize the fracture. The success of the fracture surgery was confirmed by X-ray. The surgical incision of the mouse was sutured with sutures, and 0.5 mg / kg buprenorphine was given postoperatively for analgesia. All mice were treated on days 1, 3, and 5 after fracture surgery. The experiment was divided into 5 groups (n = 6–8 / group). Mice in group 1 were injected with PBS solution. Mice in groups 2 to 4 were given equal doses of BP, Btz, and BP-Btz. Mice in group 5 were given a third lower dose of BP-Btz.

[0029] BP-Btz inhibits osteoclast formation

[0030] (1) OC preparation: BM cells (5×10 4 OCs were cultured in 96-well plates containing M-CSF and RANKL. After multinucleated cells were observed under a microscope, the cells were fixed and TRAP staining was performed to identify OCs.

[0032] (2) Bone resorption: BM cells (1×10 5) were inoculated on bone slices and cultured for 10 to 12 days. Then, OCs expressing GFP on bone slices were observed using an LSM 510 confocal fluorescence microscope. OCs were quantified by measuring the area and fluorescence intensity of GFP staining in the entire bone slice using Image ProPlus6.0 software. Finally, the bone slices were fixed and TRAP stained to co-label OCs.

[0031] (3) OB differentiation: Mouse bone-derived mesenchymal stem cells (MSCs) were isolated and cultured in 60 mm culture dishes at a rate of 2 × 10 6 The cells were cultured at a density of 10 cells / dish in α-MEM medium containing 10% FBS, and 50 μg / mL ascorbic acid and 10 mM β-glycerophosphate were added. The medium was changed every 4 days for a total of 12 days. At the end of the culture period, the cells were fixed in formalin and the number and area of ​​positive cells were calculated after ALP staining.

[0032] The effects of different doses of Btz and BP-Btz on OC formation in culture dishes were examined using BM cells from WT mice. The results showed that Btz and BP-Btz inhibited OC formation at the same concentration ( Figure 2 A).

[0033] Study whether BP-Btz can bind bone mineral and inhibit bone resorption

[0034] BM cells from mice with a ubiquitin / protease system reporter gene (UbG76V-GFP) were used, in which the Ub-GFP protein was found to cause proteasome degradation in the absence of proteasome inhibitors. Bone slices were pre-cultured overnight with 1 mL of BP, Btz, and BP-Btz, the drug solution was removed, and the bone slices were washed thoroughly with PBS. Then, BM cells were inoculated onto bone slices and a bone resorption assay was performed. It was found that Ub-GFP green fluorescent protein was degraded when Btz or BP was pre-incubated and cultured on bone slices. When cells were pre-incubated with BP-Btz and cultured on bone slices, OC formation and bone resorption were inhibited ( Figure 2 B).

[0035] The stimulatory effects of BP-Btz and Btz on OB differentiation were compared.

[0036] Mouse BM and MPCs ( Figure 2 C) or human bone marrow mesenchymal stem cells ( Figure 2 D), and both were found to promote OB differentiation with similar effects, as assessed by ALP staining. The experiments showed that BP-Btz, when added directly to the culture medium as a complete conjugate, had similar biological effects on OBs and OCs as Btz. BP-Btz was able to exert its anti-OC activity after binding to bone slices.

[0037] BP-Btz had a higher bone anabolic effect than Btz in WT mice.

[0038] A key difference between BP-Btz and Btz is that BP-Btz can be targeted for release to specific local bone locations in the body, increasing local drug concentrations and thus exerting its greatest degree of biological activity. In specific local bone treatments, Btz usually exerts systemic biological activity. It was hypothesized that BP-Btz would be more effective than Btz in increasing bone volume and stimulating osteoblast differentiation. To test this, 7-week-old female C57BL / 6JWT mice were treated with equal doses of BP (0.6 mg / kg), Btz (0.6 mg / kg), or BP-Btz (0.6 mg / kg) daily for 3 weeks ( Figure 3 A).

[0039] Micro-CT: Fractured tibiae were dissected, fixed overnight in 10% formalin, and scanned at high resolution (10.5 μm) on a VivaCT40 μCT scanner (Scanco Medical, Bruttisellen, Switzerland) using 300 ms integration time, 55 kVp energy, and 145 μa intensity. Analysis showed that BP-Btz-treated mice had higher tibia volume than BP-treated mice, and this value was significantly higher than that of Btz-treated mice ( Figure 3 B).

[0040] Histomorphometric analysis of H&E-stained sections confirmed that BP-Btz-treated mice had the highest bone volume and bone surface area ( Figure 3 C). TRAP-stained sections showed that both Btz and BP-Btz treatments reduced OC parameters to similar extents ( Figure 3 C, d). Interestingly, the bone formation rate of mice treated with BP-Btz was significantly higher than that of mice receiving Btz. Other bone formation parameters of mice treated with BP-Btz were higher than those of mice treated with Btz ( Figure 3 D).

[0041] BP-Btz was more effective than Btz in preventing ovariectomy-induced bone loss.

[0042] OVX is the most commonly used mouse model for evaluating and developing new drugs for the treatment of postmenopausal osteoporosis. Since the dose of BP-Btz is half that of Btz when increasing the same bone volume, it is necessary to study whether BP-Btz can prevent OVX-mediated bone loss through a less frequent dosing schedule than Btz ( Figure 4A). OVX mice were given Btz daily as a positive control, and BP-Btz or Btz+BP were given twice a week as a control group. We used equimolar concentrations of BP-Btz (1.2 mg / kg) and Btz (0.6 mg / kg, the same dose of Btz used in other mouse studies). The results showed that BP-Btz was more effective than Btz in restoring OVX-induced bone loss and promoting OB differentiation.

[0043] Histomorphometric analysis of H&E-stained and TRAP-stained sections showed that BP-Btz-treated OVX mice had significantly reduced bone volume, higher OB and OC parameters, and more bone marrow fat compared with negative control mice (non-ovariectomized). Figure 4 B, C). In the positive control Btz group, daily Btz supplementation reversed the reduction in bone volume, OCs, and marrow fat, returning them to the levels of the sham group. Btz increased OBs. Interestingly, compared with mice treated with Btz+BP twice a week, mice in the BP-Btz (twice a week) group showed increased bone volume and OBs levels and decreased OCs and marrow fat, which was similar to the Btz daily treatment group ( Figure 4 B, C).

[0044] BP-Btz promotes fracture repair in aged mice more effectively than Btz.

[0045] To test whether BP-Btz has similar or better efficacy than Btz in enhancing fracture repair in aged mice, aged mice (18 months old) were treated with Veh or equal doses of BP, Btz, and VEh on days 1, 3, and 5 after fracture surgery. or BP-Btz. A low-dose group (one-third of the BP-Btz group) was also set up. Fracture healing was examined by Micro-CT and histology on day 14, and by biomechanical testing on day 28 ( Figure 5 A). Micro-CT analysis showed that BP-Btz and Btz increased callus bone volume compared with Veh or BP-treated mice ( Figure 5 B). Histomorphometric analysis of ABH-stained sections showed increased areas of woven bone and cartilage ( Figure 5 C), biomechanical testing showed that the bone strength of BP-Btz and Btz-treated mice was improved ( Figure 5D). Importantly, low doses (one-third) of BP-Btz promoted fracture repair in these old mice to a similar degree as Btz. We treated young mice (3-month-old, C57BL / 6J male mice) using the same regimen as old mice and found that BP-Btz also increased callus bone volume. The magnitude of the increase was significantly greater than that in old mice (153% ± 1.2% in young mice and 131% ± 4.5% in old mice, P = 0.0231). These data suggest that the bones of young mice may absorb more BP-Btz than the bones of old mice.

[0046] BP-Btz has less systemic side effects than Btz in C57BL / 6J wild-type mice

[0047] A key difference between BP-Btz and Btz is that BP directs Btz to specific bone sites in the body, thereby promoting its local bioactive effects, whereas Btz acts systemically in the body. In patients, peripheral neuropathy and thrombocytopenia are the main side effects of Btz.

[0048] In animals, Btz primarily causes thymocyte death, thrombocytopenia, and dorsal root ganglion (DRG) damage. Therefore, changes in thymus, platelet counts, and DRGs were used as surrogates to assess the systemic toxicity of BP-Btz in mice. We treated 7-week-old C57BL / 6J WT mice with equal doses of BP, Btz, and BP-Btz once daily for 3 weeks. Body weight was measured weekly, and thymus, DRG, and megakaryocytes ( Figure 6 A). No differences in body weight were detected among BP, Btz, and BP-Btz-treated mice. However, thymus weight and thymocyte number were significantly lower in Btz-treated mice than in BP-treated mice, whereas these parameters were normal in BP-treated mice.

[0049] At the same time, flow cytometry was used to detect that CD3 + T cells and CD19 + The number of B cells was also reduced, but not in BP-Btz-treated mice ( Figure 6 B). More importantly, transmission electron microscopy showed that myelin thickness and axon diameter in DRG of Btz-treated mice were significantly lower than those of BP control mice, whereas these parameters were normal in BP-Btz-treated mice ( Figure 6 C, D).

[0050] Due to its targeting effect, BP-Btz may cause BM toxicity and lead to more severe thrombocytopenia than Btz. To test this possibility, the megakaryocyte counts in the BM sections of the above mice showed that neither Btz nor BP-Btz affected the number of megakaryocytes ( Figure 6E).

[0051] Effects of injection of BP-Btz analogs with different release rates on tibia MM lesions in mice bearing 5TMG1-GFP myeloma cells.

[0052] In order to determine whether BP-Btz can improve its anti-myeloma efficacy by adjusting its release rate, two new BP-Btz analogs were designed and synthesized, BP-Btz-fast (BP-Btz-F) ​​and BP-Btz-slow (BP-Bts-S). By changing the structure of the connecting unit, its release rate will be faster or slower than the initial compound (BP-Btz). BP-Btz-F or BP-Btz-S intervened in myeloma cells carrying 5TMG1-GFP (=5T) mice, and it was found that their killing response curves to myeloma cells were similar to those of the initial compound BP-Btz ( Figure 7 A).

[0053] 5T myeloma cells were injected into recipient mice and scanned with IVIS invoimaging 3 weeks later to assess the intensity of GFP-loaded tumor cells. Mice were randomly divided into groups according to their GFP intensity (mean radiative efficiency) and given equal doses of BP-Btz, BP-Btz-F, or BP-Btz-S by intravenous injection 3 days / 1 for a total of 6 cycles ( Figure 7 B). Results BP-Btz treatment significantly reduced tumor cell burden, as shown by GFP intensity ( Figure 7 C) and GFP+ area ( Figure 7 D). X-ray image analysis showed that the area of ​​osteolytic lesions in the tibia of mice treated with BP-Btz was significantly reduced ( Figure 7 E). H&E stained sections showed an increase in bone volume in mice treated with BP-Btz ( Figure 7 F). The analogs BP-Btz-F and BP-Btz-S with modulated release rates were less effective than the lead candidate BP-Btz in all outcome measures ( Figure 7 ). Therefore, BP-Btz should be selected for further research.

[0054] BP-Btz acts as a drug release depot on the bone surface.

[0055] It was hypothesized that Btz is released near the bone surface under acidic conditions but not under physiological normal pH conditions. Therefore, it was questioned whether Btz could be observed to be released from bone-bound BP-Btz under low pH conditions. To test this possibility, a bone-binding in vitro assay was first used to determine whether more myeloma cells were killed when they were cultured on bone slices preincubated with BP-Btz than when they were cultured on bone slices preincubated with Btz ( Figure 8A and B).

[0056] A release assay was established in which BP-Btz-bound bone slices were incubated at pH 5 or 7.4, the conditioned medium (CM) was collected, and the effects of the CM on myeloma cells were examined. The resulting reaction was based on the assumption that Btz would be released from BP-Btz bound to the bone slices after incubation at pH 5 and then enter the CM to subsequently kill myeloma cells. In contrast, CM extracted from BP-Btz-bound slices after incubation at pH 7.4 did not kill myeloma cells because Btz cannot be released at neutral pH ( Figure 8 C). It was also found that BP-Btz pre-incubated at pH 5 killed myeloma cells, while BP-Btz pre-incubated at pH 7.4 did not affect the survival of myeloma cells in CM of bone slices ( Figure 8 D). Using LC-MS / MS, Btz was detected in CM collected from BP-Btz-bound bone slices pre-incubated at pH = 5, consistent with the release of Btz at this pH ( Figure 8 E and F).

Claims

1. Application of bone-targeted drug delivery systems represented by BP-Btz in new drug development / clinical musculoskeletal system diseases.

2. Bone-targeted drug conjugates represented by BP-Btz have bone-targeting capabilities, achieving local release of Btz in the bone microenvironment. The level of Btz in non-bone tissues is very low, which improves efficacy and reduces systemic side effects.

3. It is mainly reflected in the bone-targeted drug conjugate BP-Btz, which is a combination of non-anti-bone resorption bisphosphonate (BP) and proteasome inhibitor bortezomib (Btz).

4. Development of a new drug with a mechanism of bone-targeted drug delivery system without antiresorptive BP-targeted drugs.

5. Development of a new drug involving a bone-targeted drug delivery system without antiresorptive BP-targeted drugs.

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