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Matrix Metalloproteinase Responsive Delivery of Myostatin Inhibitors

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Abstract

Purpose

The inhibition of myostatin - a member of the transforming growth factor (TGF–β) family - drives regeneration of functional skeletal muscle tissue. We developed a bioresponsive drug delivery system (DDS) linking release of a myostatin inhibitor (MI) to inflammatory flares of myositis to provide self-regulated MI concentration gradients within tissues of need.

Methods

A protease cleavable linker (PCL) – responding to MMP upregulation – is attached to the MI and site-specifically immobilized on microparticle surfaces.

Results

The PCL disintegrated in a matrix metalloproteinase (MMP) 1, 8, and particularly MMP-9 concentration dependent manner, with MMP-9 being an effective surrogate biomarker correlating with the activity of myositis. The bioactivity of particle-surface bound as well as released MI was confirmed by luciferase suppression in stably transfected HEK293 cells responding to myostatin induced SMAD phosphorylation.

Conclusions

We developed a MMP-responsive DDS for MI delivery responding to inflammatory flare of a diseased muscle matching the kinetics of MMP-9 upregulation, with MMP-9 kinetics matching (patho-) physiological myostatin levels.

ᅟ

Schematic illustration of the matrix metalloproteinase responsive delivery system responding to inflammatory flares of muscle disease. The protease cleavable linker readily disintegrates upon entry into the diseased tissue, therby releasing the mystatin inhibitor.

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Abbreviations

ActRIIB:

Activin receptor IIB

au:

Arbitrary units

BCA:

Bicinchoninic acid

BSA:

Bovine serum albumin

CuAAC:

Copper(I)-catalyzed azide-alkyne cycloaddition

DBCO:

Dibenzocyclooctyne

DDS:

Drug delivery system

DM:

Differentiation medium

DMD:

Duchenne muscular dystrophy

ECM:

Extracellular matrix

EDC:

1-Ethyl-3-(3dimethylaminopropyl)carbodiimide

Fmoc:

N-α-(9-Fluorenylmethyloxycarbonyl)

GDF-8:

Growth differentiation factor 8

HPLC:

High-performance liquid chromatography

IGF-I:

Insulin-like growth factor I

IL-1β:

Interleukin 1β

MALDI-MS:

Matrix-assisted laser desorption ionization mass spectrometry

MI:

Myostatin inhibitor

MMP:

Matrix metalloproteinase

MSTN:

Myostatin

MyHC:

Myosin heavy chain

NF-kB:

Nuclear factor-kappa B

NHS:

N-hydroxysuccinimide

PCL:

Protease cleavable linker

PEG:

Polyethylene glycol

PMMA:

Poly(methyl methacrylate)

RLU:

Relative light unit

RT-PCR:

Real time polymerase chain reaction

SBE:

SMAD binding element

SC:

Satellite cell

SPAAC:

Strain-promoted azide-alkyne cycloaddition

SPPS:

Solid phase peptide synthesis

TG:

Transglutaminase (human, fXIIIa)

TGF–β:

Transforming growth factor beta

THPTA:

Tris(3-hydroxypropyltriazolylmethyl)amine

TNF-α:

Tumor necrosis factor α

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ACKNOWLEDGMENTS AND DISCLOSURES

We thank Petra Knaus (Freie Universität Berlin, Germany) for providing us with the pGl3ti-SBE constructs and Melanie Krug for excellent technical assistance. We thank Dr. Joachim Nickel for providing C2C12 myoblast cells. The financial support of the Bavarian research foundation (grant # AZ-1044-12 ‘FORMOsA’) and the Deutsche Forschungsgemeinschaft (DFG; ME 3820/3-1) are gratefully acknowledged. H.G. is full time associate of Gilyos GmbH.

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Authors and Affiliations

  1. Institute for Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, DE-97074, Würzburg, Germany

    Alexandra C. Braun, Marcus Gutmann, Tessa Lühmann & Lorenz Meinel

  2. Orthopedic Center for Musculoskeletal Research, University of Würzburg, Friedrich-Bergius-Ring 15, 97076, Würzburg, Germany

    Regina Ebert & Franz Jakob

  3. GILYOS GmbH, Friedrich-Bergius-Ring 15, 97076, Würzburg, Germany

    Henning Gieseler

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  1. Alexandra C. Braun
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Correspondence to Lorenz Meinel.

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Braun, A.C., Gutmann, M., Ebert, R. et al. Matrix Metalloproteinase Responsive Delivery of Myostatin Inhibitors. Pharm Res 34, 58–72 (2017). https://doi.org/10.1007/s11095-016-2038-6

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  • DOI: https://doi.org/10.1007/s11095-016-2038-6

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