Safe and effective subcutaneous adipolysis in minipigs by a collagenase derivative

PLoS One. 2019 Dec 31;14(12):e0227202. doi: 10.1371/journal.pone.0227202. eCollection 2019.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adipocytes / drug effects*
  • Adipocytes / pathology
  • Animals
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / pharmacology*
  • Collagen / metabolism*
  • Collagenases / isolation & purification
  • Collagenases / pharmacology*
  • Escherichia coli / genetics
  • Lipolysis / drug effects*
  • Mice
  • Mice, Obese
  • Obesity / drug therapy
  • Subcutaneous Fat / drug effects*
  • Subcutaneous Fat / pathology
  • Swine
  • Swine, Miniature

Substances

  • Bacterial Proteins
  • Collagen
  • ColH protein, Clostridium histolyticum
  • Collagenases

Grants and funding

This work was funded by Rejuven Dermaceutical Co., Ltd. (http://www.irejuven.com/). The funder provided support in the form of salaries for authors F.C., G.D., M.S., H.Y., P.B., S.S., Y.C., Z.Z., but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.