A Novel Anabolic Agent: A Simvastatin Analogue without HMG-CoA

Aug 13, 2014 - HMG-CoA reductase (HMGR) is primarily distributed in the liver for lipid biosynthesis, and we propose that compounds with high affinity...
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A Novel Anabolic Agent: A Simvastatin Analogue without HMG-CoA Reductase Inhibitory Activity Kuang-Chan Hsieh,† Chai-Lin Kao,‡,∇ Chien-Wei Feng,○ Zhi-Hong Wen,◆ Hsin-Fang Chang,§ Shu-Chun Chuang,∥ Gwo-Jaw Wang,∥,¶ Mei-Ling Ho,∥,⊥ Shou-Mei Wu,*,† Je-Ken Chang,*,∥,# and Hui-Ting Chen*,∥,§ †

School of Pharmacy, ‡Department of Medicinal and Applied Chemistry, §Department of Fragrance and Cosmetic Science, Orthopedic Research Center, ⊥Department of Physiology, and #Department of Orthopedics, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan ∇ Department of Chemistry and ◆Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan ○ Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Kaohsiung, Taiwan ¶ Department of Orthopedic Surgery, University of Virginia, Charlottesville, Virginia United States ∥

S Supporting Information *

ABSTRACT: For the first time, structural information regarding the role of simvastatin in bone anabolism is described, and a bone-specific statin is introduced. Polyaspartate-conjugated simvastatin was synthesized by solidphase synthesis with the assistance of microwave irradiation. It displays significant bone targeting and bone formation with less toxicity than simvastatin.

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are beneficial for treatment in bone have not been reported. This fact has hindered the development of statins as anabolic agents. Accordingly, we propose an improvement in bone specificity to enhance the distribution of simvastatin derivatives in the bone microenvironment and to prevent their accumulation in the liver. HMG-CoA reductase (HMGR) is primarily distributed in the liver for lipid biosynthesis, and we propose that compounds with high affinity to HMGR will therefore be trapped in the liver. Consequently, decreasing the inhibitory activity of HMGR may allow such compounds to escape from the liver. Three approaches were used to reach these criteria: (1) enhanced hydrophilicity to increase distribution into extrahepatic tissue,14 (2) decreased HMGR inhibitory activity to prevent deposition in the liver,15 and (3) increased bone affinity to enhance mineral formation.16 The regulation of bone resorption by the MVA pathway has been documented,17 but statin-induced bone formation was shown to occur through alternative pathways.18 Currently, the role of the MVA pathway in bone formation is still unclear. In this study, we synthesized the first bone-targeted statin derivative 1 (Figure 1), and we suggest the structural requirements for the use of statins as anabolic agents. The benefits of statins in rodent bones do not correspond to bone mass enhancement in humans;19 therefore, a zebrafish model was used to evaluate bone formation capacity.20 Zebrafish biomineralization charac-

tatins have been used as practical cholesterol lowering agents for more than four decades. In addition to this known effect, statins also exhibit lipid-independent effects, or pleiotropic effects,1 including bone anabolism.2 Recent investigations have indicated that simvastatin acts as an osteoclastogenesis inhibitor and an osteogenesis enhancer, thereby assisting in bone formation and increasing bone mass. In addition, simvastatin promotes mineral formation in rodents.3,4 Accordingly, statinrelated pharmaceutics are developed for the treatment of bone disease.5 In terms of clinical potential, however, the effective dosage in rat models is exceptionally high.6 In human cell studies, simvastatin-induced osscification7 and demonstrated a clear anabolic effect when locally administered in humans,8 but the results of clinical studies on systemic administration in humans remain controversial.9 Regarding the clinical biochemistry of lipid reduction, simvastatin is a structural mimic of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) with its hydroxyl and acid moieties masked to form a hydrophobic lactone prodrug. Because of its hepatoselectivity,10 90% of orally administered simvastatin is distributed to the liver and subsequently interrupts lipid biosynthesis by blocking the mevalonate (MVA) pathway.11 A small portion of administered simvastatin is delivered to other tissues; for example, less than 5% of administered simvastatin is distributed in the bone. Therefore, a high dosage is necessary to induce bone activity, but this high dosage is accompanied by toxicity.12 Hence, systemic uses of simvastatin in clinical applications are unfeasible, and the discovery of bone-specific statins is pursued.13 The structural characteristics of statins that © XXXX American Chemical Society

Received: June 9, 2014

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amide linkage (3). Several typical and thermal conditions of solid-phase peptide synthesis failed, and thus, microwave irradiation was required for this addition. Under 200 W irradiation at 105 °C, the conjugation was successfully induced after 100 min. Remarkably, the α-dimethylbutyryl ester motif on SVA was not altered by amine attack during the microwave synthesis or by amino acid coupling during peptide synthesis. In the presence of hydrazine, the 1-(4,4-dimethyl-2,6dioxocyclohexylidene)-3-methylbutyl (ivDde) group on lysine was removed to free the δ-amino group, and the amine was iteratively coupled with aspartic acid using the Fmoc protection approach to give the hexa-aspartate (Asp6) analogue 4. For further functionalization, an additional lysine was incorporated into Asp6 to yield 5. After removal of Fmoc from the N-terminal amine of lysine, PEG500 was conjugated through p-nitrophenyl chloroformate activation. The ivDde group was subsequently removed by treatment with hydrazine to free the δ-amino group of lysine for the introduction of the FITC fluorescence tag, affording 1-F. An acetyl group was used instead of FITC in 1. Compound 2-F, which is an analogue of 1-F without simvastatin acid, was synthesized as a reference compound. A lysine residue was conjugated with the Asp6 peptide 7 to introduce α- and δ-amino groups for PEG and FITC conjugation, respectively. Simvastatin and 1-F were subjected to HMGR inhibitory experiments. Compound 1-F (21 μM) exhibited 60% inhibition; however, a loss of inhibitory activity against HMGR (IC50 > 30 μM) was indicated because it is a much weaker inhibitor than simvastatin (IC50 = 50.3 nM)24 (Figure 2). To investigate the binding affinity of the synthesized compounds to Ca2+ ions, isothermal titration calorimetry (ITC) was performed and showed that 1 strongly binds Ca2+ (Kb = 278.1 kM−1) (Figure 3c). In addition to soluble Ca2+ ions, a hydroxylapatite (HAP) particle (20 μm) was used as a model of bone tissue to examine the heterogeneous affinity. In this

Figure 1. Structure of target compounds.

teristics are similar to biomineralization in humans, and zebrafish bone composition is similar to bone composition in mammals.21 The benefit of the simvastatin derivative 1 for bone anabolism was improved by increasing its water solubility via linkage with poly(ethylene glycol), and mineral binding was enhanced by the addition of polyaspartate hexapeptide.22 In addition, to visualize bone targeting ability, compound 1 and polyaspartate with fluorescent probe (denoted by 1-F and 2-F) were also prepared. We proposed that the HMG mimic moiety is not necessary for bone anabolism. Thus, in 1 and 1-F, the hexapeptide attacks the lactone in the simvastatin and forms an amide bond to link with the simvastatin acid (SVA), thus destroying the HMGR inhibitory activity.23 Additionally, the amide form of the simvastatin derivative has a lower level of consumption in the liver. The target compounds were prepared by solid-phase peptide synthesis (SPPS) (Scheme 1). To prevent potential steric hindrance, lysine was introduced to glycine-preloaded Wang resin. SVA was conjugated to the α-amino group of lysine by hydrolyzing the lactone in statin with a peptide amine to form an

Scheme 1. Preparation of Target Compounds by Solid-Phase Peptide Synthesis (SPPS)

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indicated that 1-F exhibits substantial bone anabolic activity, enhancing (150%) bone formation at a nontoxic level (0.32 μM), whereas 2-F and simvastatin exhibited no bone formation effect (Figure 4). Fleming also reported that simvastatin did not produce a significant benefit in zebrafish bone,25 which may explain the controversial results in human studies.

Figure 2. HMGR inhibition of simvastatin (closed circle, IC50 = 50.3 ± 1.4 nM) and 1-F (open circle, IC50 > 30 μM). Experiments were performed in triplicate.

Figure 4. Effects of the test compounds in zebrafish. The bone targeting abilities of each substance tested are presented in panels a (calcein) and b (2-F without calcein). Panel c presents the effects of test compounds on the bone formation of fish, and the formation zone is labeled with calcein. Experiments were performed in triplicate and data present as mean ± SEM. *: P < 0.05 vs control.

The results demonstrate that amide conjugation of simvastatin acid with a polyaspartate peptide enhances both bone selectivity and bone anabolic capacity as well as significantly attenuating toxicity. Compound 1 exhibited excellent bone targeting and formation activities. The bone anabolism capacity of statins also was improved. Additionally, 1 elicited a substantial bone formation effect in a human skeleton model in mature and larval zebrafish. This strategy may be potentially useful for generating new anabolic agents for the treatment of human maladies. However, compound 1 exhibits poor HMG-CoA reductase inhibition compared to simvastatin, which reinforces the idea that anabolism is independent of the MVA pathway. This report is the first to suggest that HMGR inhibition is not essential for bone formation.

Figure 3. Binding of 1-F (10.0 μM) monitored by HAP particles and Ca2+: (a) bright field, (b) fluorescence field, (c) ITC binding curves of 1 in combination with calcium ions.

experiment, the attachment of the fluorophore-labeled analog 1F (Figure 3a,b) to HAP was visualized by fluorescence microscopy. The presence of multiple carboxylate groups in 1 was expected to produce high affinity with Ca2+ in either the solution or solid phase. The stimulation of cell osteogenesis by simvastatin and 1 was evaluated by determining calcium deposition in rat bone marrow stem cells (D1 cells). The result suggests that both simvastatin and 1 induce mineralization at concentrations up to 2 μM. However, simvastatin caused cytotoxicity at concentrations higher than 4 μM. Conversely, no cytotoxicity was observed in 8 μM of 1. Thus, 1 is as potent as simvastatin in inducing cell mineralization but exhibits lower cytotoxicity (Figure S10, Supporting Information). The effects of 1-F and 2-F on bone selectivity, bone development, and formation were further examined in zebrafish. Zebrafish larvae were treated with fluorophore-labeled peptide 2F and calcein (3.2 μM). The resulting fluorescent images indicated that 2-F effectively binds to the larval bone after 15 min of administration. In addition, toxicity studies in which the same dose was administered for 5 days showed that 1-F did not harm the development of zebrafish, while simvastatin resulted in larval death. Bone mass was also measured by calcein staining and

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ASSOCIATED CONTENT

* Supporting Information S

Detailed experimental section, methods, and data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (for identification). *E-mail: [email protected] (for bone formation). *E-mail: [email protected] (for synthesis). Fax: +886-73210683. Tel: +886-7-3121101 ext 2803. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Science Council of the Republic of China (98-2113-M-037-003-MY2), the Ministry of Economic Affairs of the Republic of China (98EC-17-A-S1-041), and Kaohsiung Medical University (KMUM102001 and NSYSUKMU103-I 008). We acknowledge the C

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Zhang, J.; He, G.; Jiang, B.; Wang, Q.; Chen, Z.; Pan, J.; Li, Y.; Guo, L. Bioorg. Med. Chem. 2011, 19, 3750. (23) (a) Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160. (b) Stokker, G. E.; Hoffman, W. F.; Alberts, A. W.; Cragoe, E. J., Jr.; Deana, A. A.; Gilfillan, J. L.; Huff, J. W.; Novello, F. C.; Prugh, J. D.; Smith, R. L.; Willardt, A. K. J. Med. Chem. 1985, 28, 347. (24) Carbonell, T.; Freire, E. Biochemistry 2005, 44, 11741. (25) Fleming, A.; Sato, M.; Goldsmith, P. J. Biomol. Screen 2005, 10, 823.

Center for Research Resources and Development in KMU and Prof. Shih-Hsiung Wu for ITC support.

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