(133) Investigating Actinidin as a Potential Collagenase on Human Peyronie’s Disease Cells
Authors: Feng K , Kiattiburut W , Hickling D , Burton J , Campbell J 
Published: February 2024

Abstract

Introduction
Peyronie's Disease (PD) is characterized by a connective tissue disorder in the penile tunica albuginea and the formation of collagen-rich plaques, leading to penile curvature, painful erections, and erectile dysfunction. Intralesional Collagenase Clostridium histolyticum injections are no longer available in Canada, making treatment options for PD limited. Actinidin is a soluble enzyme that has the ability to hydrolyze various types of collagen and fibrinogen. We have previously reported the efficacy of actinidin on reducing collagen in in vitro human PD models.

Objective
To determine the dose-response efficacy and cytotoxicity of actinidin in reducing the intercellular bound collagen in human PD plaque models.

Methods
Fibroblast cells are isolated from human PD tissue (N ≥ 3) and 2D models are cultured. These cells were subjected to various treatment groups including media-only control and 5 concentrations of actinidin treatment ranging from 0.5 mg/ml to 30 mg/ml. The collagenase treatment was prepared from Hayward kiwi by isolating actinidin by filtration and pH adjustment. A dose-response curve was generated at different time points at various actinidin concentrations using an MTT assay. After 24 hours of treatment, collagen extraction was performed and quantified using a Soluble Collagen Quantification Assay Kit and a fluorescent microplate reader. The effects of the treatments on the extracellular matrix were further investigated by staining the cells with DAPI for the nucleus and Phalloidin-conjugated FITC for actin filaments. A twelve-hour timelapse was conducted using a fluorescent confocal microscope to capture FITC and DAPI fluorescein images. The total fluorescent area of each well was measured, and actin signals were normalized by the DAPI signals.

Results
The dose-response curve revealed that the lethal concentration affecting 50% of the cells after a 48-hour period is approximately 15-20 mg/ml. Treatment of 1 mg/mL actinidin significantly reduced cellular collagen of human PD fibroblasts compared to the control group (P &lt; 0.001). The normalized stained β-actin signal was measured as the ratio between intracellular actin staining and nuclei count in each microscopic field. The high concentration actinidin treatment (30 mg/mL) exhibited a significantly higher normalized actin signal compared to other treatment groups (P &lt; 0.0001). These findings suggest that actinidin may possess mechanisms that affect the plasma membrane integrity.

Conclusions
Our continuation of preliminary results demonstrates the ability of actinidin to break down PD cells by compromising extracellular collagen and the cellular membrane. High doses of actinidin cause cell destruction and we suggest that treatment should not exceed 10 mg/ml. Further studies will investigate the molecular mechanism of actinin in hydrolyzing collagen and to evaluate the effects on an animal model. Given the absence of durable FDA-approved treatments for PD in Canada, this novel option holds future treatment potential.

Disclosure
Yes, this is sponsored by industry/sponsor: KiwiEnzyme.com Ltd.
Clarification: No industry support in study design or execution.