Researchers at The University of Alabama at Birmingham have identified idiopathic pulmonary fibrosis as a repurposing opportunity for fasudil, a Rho kinase inhibitor that Asahi Kasei Pharma Corp. markets as Eril to treat aneurysm. The key observation was that the drug acts on a pathway that converts biochemical or biomechanical stimuli into fibrogenic signals that sustain myofibroblast activation and survival.1

The team now needs to develop a formulation of fasudil or other Rho kinase inhibitors that works only in the lung and thus avoids systemic side effects.

In normal wound healing, fibroblasts that reside in tissues are activated and transform into myofibroblasts, which are characterized by high contractile activity that results from actin a2 smooth aorta muscle (ACTA2; a-SMA) accumulation in stress fibers. The myofibroblasts help remodel and repair tissue by secreting extracellular matrix components, such as collagen and fibronectin.

Once the wound is re-epithelialized, the myofibroblasts undergo apoptosis.2

In fibrotic disease, apoptotic-resistant myofibroblasts persist in injured tissues and can lead to excessive accumulation of fibrous connective tissue and tissue stiffening. This accumulation can result in permanent scarring, organ malfunction and possibly death, as seen in end-stage idiopathic pulmonary fibrosis (IPF).

Fibrogenic cytokines and growth factors, such as transforming growth factor-b1 (TGFB1), are known promoters of fibroblast-to-myofibroblast differentiation, but the Alabama team has been searching for other contributors.

In 2012, the group showed that biomechanical properties of the extracellular matrix contribute to differentiation. The researchers reported that mouse lung fibroblasts cultured on a stiff matrix showed greater actin cytoskeletal reorganization and myofibroblast differentiation than those cultured on soft matrix.3

However, inhibiting Rho kinase activity prevented those changes. Rho kinases-including Rho-associated coiled-coil containing protein kinase 1 (ROCK1) and ROCK2-are involved in regulating cell contractility, actin cytoskeletal organization, stress fiber formation and focal adhesion assembly.

Now, the team has probed the underpinnings of the pathway that converts biomechanical stimuli into biochemical fibrogenic signals.

For biochemical stimuli the group used healthy human lung fibroblasts treated with TGFB1, and for biomechanical stimuli the group cultured the fibroblasts on a stiff matrix. In both cases, fasudil blocked the fibroblast-to-myofibroblast differentiation and myofibroblast contractility that was seen in untreated cells.

The team concluded that the drug targets both biochemical and biomechanical signals that mediate myofibroblast differentiation.

The Alabama team also showed that fasudil led to apoptosis of a-SMA+ lung myofibroblasts but not normal fibroblasts.

Notably, fasudil showed antifibrotic effects even when given to mice with established fibrosis.

Further work with fasudil-treated IPF myofibroblasts provided a clear mechanistic picture of what was occurring. Specifically, the drug impeded differentiation and induced apoptosis through blocking nuclear localization of myocardin-related transcription factor A (MKL1; MAL; MRTF-A) and downregulating B cell lymphoma 2 (BCL-2; BCL2) and a-SMA (see "Myofibroblast dynamics" and "TGFB1 and Rho kinase inhibitors").

"This new work very elegantly shows the mechanistic details to unravel how biochemical and biomechanical stimulus leads to remodeling and especially differentiated apoptosis of myofibroblasts versus resident fibroblasts," said Dirk Leysen, CSO and founder of Amakem N.V. "Many people are working on the various triggers and processes that lead to fibrosis, including persistent inflammation. This work focuses on the postinflammatory fibrotic phase, which is much more useful for late-stage fibrosis such as that seen in IPF. What they find suggests that not only could Rho kinase inhibition stop fibrosis from progressing but might also be able to reverse fibrosis."

The new work shows that "cellular sensing of and response to tissue stiffness almost certainly plays a significant role in the progression of fibrosis," said Scott Seiwert, SVP of research and technical development at InterMune Inc.

InterMune markets Esbriet pirfenidone to treat IPF in Europe and Canada. Esbriet is a small molecule inhibitor of proinflammatory cytokines such as tumor necrosis factor-a (TNF-a) and IL-1b as well as profibrotic cytokines, including platelet derived growth factor (PDGF) and TGFB (TGFb).

Making it work

Seiwert cautioned that "fasudil is a rather nonspecific kinase inhibitor, and so its ability to probe the role of Rho kinases is limited."

Regardless, Martin Kolb, research director of the Firestone Institute for Respiratory Health and associate professor of medicine, pathology and molecular medicine at McMaster University, ultimately wanted to see studies of pirfenidone plus fasudil.

Kolb is collaborating with Genoa Pharmaceuticals Inc. to develop an aerosol formulation of pirfenidone for IPF to avoid the drug's nausea side effect. With aerosol delivery, the hope is the drug would be concentrated in the lungs and avoid high concentrations systemically, which can lead to nausea.

Seiwert was also interested in seeing the effects of that combination. "Pirfenidone reduces TGFB1 levels in dozens of animal models of fibrosis across several organ systems," he said. "Since pirfenidone lowers TGFB1 levels and fasudil disrupts TGFB1-induced myofibroblast differentiation, the two compounds could potentially display additive or synergistic antifibrotic effects in vivo."

Prior to any combination studies, there was consensus that fasudil needs to be reformulated for IPF.

"Fasudil could not be used to treat pulmonary fibrosis as is," said Leysen. "Rho kinase activity is needed for vascular smooth muscle homeostasis, so systemic exposure to Rho kinase inhibitors will lead to dangerous side effects such as extremely low blood pressure. One way around this would be to apply a very specific lung delivery technology."

An alternative, he said, would be using Amakem's approach of chemically modifying Rho kinase inhibitors "so that they are only active in the tissue they intend to treat and degraded once they reach systemic circulation."

Amakem's AMA0076 is in Phase II trials for glaucoma and ocular hypertension.

Although Asahi's Eril is the only Rho kinase inhibitor on the market and is used to treat aneurysm, multiple companies are pursuing the target for glaucoma, neurodegeneration, pain and solid tumors.

Asahi declined requests for interviews.

The Alabama team has filed an IP disclosure with the UAB Research Foundation, but the work is currently unpatented. The team is looking to find a partner that is currently working on formulating Rho kinase inhibitors for cancer or other clinical indications to start Phase II studies of Rho kinase inhibitors in patients with IPF.

Baas, T. SciBX 6(11); doi:10.1038/scibx.2013.255 Published online March 21, 2013


1.   Zhou, Y. et al. J. Clin. Invest.; published online Feb. 22, 2013; doi:10.1172/JCI66700 Contact: Victor J. Thannickal, The University of Alabama at Birmingham, Birmingham, Ala. e-mail: Contact: Yong Zhou, same affiliation as above e-mail:

2.   Wynn, T.A. & Ramalingam, T.R. Nat. Med. 18, 1028-1040 (2012)

3.   Huang, X. et al. Am. J. Respir. Cell Mol. Biol. 47, 340-348 (2012)


Amakem N.V., Diepenbeek, Belgium

Asahi Kasei Pharma Corp., Tokyo, Japan

Firestone Institute for Respiratory Health, Hamilton, Ontario, Canada

Genoa Pharmaceuticals Inc., San Diego, Calif.

InterMune Inc. (NASDAQ:ITMN), Brisbane, Calif.

McMaster University, Hamilton, Ontario, Canada

UAB Research Foundation, Birmingham, Ala.

The University of Alabama at Birmingham, Birmingham, Ala.