Safe and effective subcutaneous adipolysis in minipigs by a collagenase derivative

Autoři: Fuding Chen aff001;  Gang Du aff001;  Meishu Shih aff002;  Hongjiang Yuan aff002;  Peng Bao aff001;  Sheng Shi aff001;  Yong Cang aff001;  Zhen Zhang aff001
Působiště autorů: Research and Development Division, Rejuven Dermaceutical Co., Ltd., Hangzhou, China aff001;  PharmaLegacy Laboratories (Shanghai) Co., Ltd., Shanghai, China aff002
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article


Adipocytes attached to the extracellular matrix (ECM) mainly consist of collagen in adipose tissues, while the degradation of ECM by collagenase induces the apoptosis of adipocytes, leading to a decrease in local subcutaneous adipose. To achieve this goal, we are developing a mutant collagenase H (ColH) to remove local subcutaneous fat such as submental fat (SMF). Three vectors were constructed for expressing rColH(FM, mutant for fat melting, with 6xHis tag), rColH(WT, wild-type, with 6xHis tag), and rColH(E451D, E451D mutant, without 6xHis tag) in Escherichia coli. rColH(FM) & rColH(WT) were purified by Ni Sepharose on a laboratory scale, while rColH(E451D) was purified by five chromatography purification steps on a large scale. Then, the stability of rColH(FM) and rColH(WT) was tested by SDS-PAGE to investigate the influence of the E451D mutation on stability. Afterwards, the enzyme kinetics of ColH (mutant or wild-type, with or without His tag) were investigated and compared. Finally, the adipolysis of rColH(E451D) at various doses was tested in vitro and in vivo. The ultrasound results in minipigs suggested that effective adipolysis was induced by rColH(E451D) compared with the negative control, and the histological results suggest dose-dependent fibrosis, necrosis, inflammation and cholesterol cleft formation. These findings indicate the possibility of rColH(E451D) becoming a new injectable drug to safely remove subcutaneous adipose.

Klíčová slova:

Adipocytes – Collagenases – Collagens – Enzyme kinetics – Fibrosis – Lipolysis – Pharmacodynamics – Ultrasound imaging


1. Mandl I, Maclennan JD, Howes EL. Isolation and characterization of proteinase and collagenase from Cl. histolyticum. J Clin Invest 1953; 32:1323–9. doi: 10.1172/JCI102861 13109000

2. Bond MD, Van Wart HE. Purification and separation of individual collagenases of Clostridium histolyticum using red dye ligand chromatography. Biochemistry 1984; 23:3077–3085. doi: 10.1021/bi00308a035 6087887

3. Bond MD, Van Wart HE. Characterization of the individual collagenases from Clostridium histolyticum. Biochemistry 1984; 23:3085–3091. doi: 10.1021/bi00308a036 6087888

4. Bond MD, Van Wart HE. Relationship between the individual collagenases of Clostridium histolyticum: evidence for evolution by gene duplication. Biochemistry 1984; 23:3092–3099. doi: 10.1021/bi00308a037 6087889

5. Matsushita O, Okabe A. Clostridial hydrolytic enzymes degrading extracellular components. Toxicon 2001; 39:1769–80. doi: 10.1016/s0041-0101(01)00163-5 11595639

6. Gelbard MK, Walsh R, Kaufman JJ. Collagenase for Peyronie’s disease experimental studies. Urol Res. 1982; 10:135–40. doi: 10.1007/bf00255956 6291216

7. Starkweather KD, Lattuga S, Hurst LC, Badalamente MA, Guilak F, Sampson SP, et al. Collagenase in the treatment of Dupuytren's disease: an in vitro study. J Hand Surg Am 1996; 21:490–5. doi: 10.1016/S0363-5023(96)80368-6 8724485

8. Watanabe K. Collagenolytic proteases from bacteria. Appl Microbiol Biotechnol 2004; 63:520–6. doi: 10.1007/s00253-003-1442-0 14556041

9. Duarte AS, Correia A, Esteves AC. Bacterial collagenases—A review. Critical Reviews in Microbiology 2016; 42:106–126. doi: 10.3109/1040841X.2014.904270 24754251

10. Alipour H, Raz A, Zakeri S, et al. Therapeutic applications of collagenase (metalloproteases): A review. Asian Pac J Trop Biomed 2016; 6:975–981.

11. Fujio A, Murayama K, Yamagata Y, Watanabe K, Imura T, Inagaki A, et al. Collagenase H Is crucial for isolation of rat pancreatic islets. Cell Transplant 2014; 23:1187–98. doi: 10.3727/096368913X668654 23768818

12. Rodbell M. Metabolism of isolated fat cells I. Effects of hormones on glucose metabolism and lipolysis. J Biol Chem 1964; 239:375–80. 14169133

13. Tchoukalova YD, Harteneck DA, Karwoski RA, Tarara J, Jensen MD. A quick, reliable, and automated method for fat cell sizing. J Lipid Res 2003; 44:1795–801. doi: 10.1194/jlr.D300001-JLR200 12777477

14. Estes BT, Diekman BO, Gimble JM, Guilak F. Isolation of adipose-derived stem cells and their induction to a chondrogenic phenotype. Nat Protoc 2010; 5:1294–311. doi: 10.1038/nprot.2010.81 20595958

15. Moscatello DK, Schiavi J, Marquart JD, Lawrence N. Collagenase-assisted fat dissociation for autologous fat transfer. Dermatol Surg 2008; 34:1314–1322. doi: 10.1111/j.1524-4725.2008.34282.x 18665845

16. Youn S, Shin JI, Kim JD, Kim JT, Kim YH. Correction of infraorbital dark circles using collagenase digested fat cell grafts. Dermatol Surg 2013; 39:766–772. doi: 10.1111/dsu.12140 23437990

17. Schlessinger J, Weiss SR, Jewell M, Narurkar V, Weinkle S, Gold MH, et al. Perceptions and practices in submental fat treatment: a survey of physicians and patients. Skinmed 2013; 11:27–31. 23540074

18. Deeks Emma D. Deoxycholic acid: A review in submental fat contouring. Am J Clin Dermatol 2016; 17:701–707. doi: 10.1007/s40257-016-0231-3 27785706

19. Katz B1, McBean J. Laser-assisted lipolysis: a report on complications. J Cosmet Laser Ther 2008; 10:231–3. doi: 10.1080/14764170802524437 19016103

20. Keramidas E, Rodopoulou S. Radiofrequency-assisted liposuction for neck and lower face adipodermal remodeling and contouring. Plast Reconstr Surg Glob Open 2016; 4: e850. doi: 10.1097/GOX.0000000000000809 27622118

21. Manstein D, Laubach H, Watanabe K, et al. Selective cryolysis: a novel method of non-invasive fat removal. Lasers Surg Med 2008; 40:595–604. doi: 10.1002/lsm.20719 18951424

22. Jung CM, Matsushita O, Katayama S, Minami J, Sakurai J, and Akinobu O. Identification of metal ligands in the Clostridium histolyticum ColH collagenase. J Bacteriol 1999; 181:2816–2822. 10217773

23. U.S. Pharmacopeia. Available from biologics /resources/gc89.2_collagenase.pdf

24. Giglione C, Boularot A, Meinnel T. Protein N-terminal methionine excision. Cell. Mol. Life Sci 2004; 61:1455–1474. doi: 10.1007/s00018-004-3466-8 15197470

25. Syed F, Thomas AN, Singh S, et al. In vitro study of novel collagenase (XIAFLEX®) on Dupuytren's Disease fibroblasts displays unique drug related properties. PLOS ONE 2012; 7:e31430. doi: 10.1371/journal.pone.0031430 22384021

26. Datta R, Podolsky MJ, Atabai K. Fat fibrosis: friend or foe? JCI Insight 2018; 3: pii: 122289. doi: 10.1172/jci.insight.122289 30282827

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2019 Číslo 12
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