Cartilage Regeneration by the Chondrogenic Small Molecule PRO1 during Osteoarthritis

Funding Type: 
Early Translational II
Grant Number: 
Award Value: 
Disease Focus: 
Bone or Cartilage Disease
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Public Abstract: 

The ability to direct the differentiation of resident mesenchymal stem cells (MSCs) towards the cartilage lineage offers considerable promise for the regeneration of articular cartilage after traumatic joint injury or age-related osteoarthritis (OA). MSCs can be stimulated in vitro to form new functional cartilage. In the OA-affected joint, the repair is insufficient, leaving a damaged matrix, suggesting that key factors are missing to properly direct the regenerative process. Molecules that activate the chondrogenic potential of cartilage stem cells may potentially prevent further cartilage destruction and stimulate repair of cartilage lesions.

Currently there are no disease-modifying therapeutics available for the 40 million Americans suffering from OA. Therapeutic options are limited to oral and intra-articularly injected pain medications and joint replacement surgery. The primary objective of this project is to develop a non-invasive, therapeutic for the regeneration of cartilage in OA. This new therapy will target the resident MSCs in the joint, stimulate production of new cartilage matrix, promote repair and thus limit additional joint damage and improve joint pain and function.

To provide a proof-of-concept for our strategy, a cell-based screen of a diverse small molecule library led to compounds capable of enhancing the formation of articular cartilage (chondrogenesis) from MSCs in vitro. In secondary assays, molecules were assessed for protection of the existing cartilage against induced tissue damage. Through these approaches, the lead low molecular weight small molecule PRO1 was identified which promotes cartilage differentiation and protects cartilage from damage. PRO1 reproducibly demonstrated in vivo efficacy in two animal models of OA (surgical and enzyme-induced). OA-associated pain was reduced and the architecture of the cartilage was restored. PRO1 therefore appears to activate the regenerative potential of the resident cartilage stem cells.

Statement of Benefit to California: 

Osteoarthritis (OA) is the most prevalent musculoskeletal disease and globally the 4th leading cause of Years Lost to Disease (YLD). OA affects over 40 million Americans and the magnitude of the problem is predicted to increase even further with the obesity epidemic and aging of the baby boomer generation. It is estimated that 80% of the population will have radiographic evidence of OA by age 65 years. The annual economic impact of arthritis in the U.S. is estimated at over $100 billion, representing more than 2% of the gross domestic product. OA accounts for 25% of visits to primary care physicians. In 2004 OA patients received 650,000 knee and hip replacements at a cost of $26 billion. Without change in treatment options 1.8 million joint replacements will be performed in 2015.

OA is a painful, degenerative type of arthritis; physical activity can become difficult or impossible. Some patients with osteoarthritis are forced to stop working because their condition becomes so limiting. OA can interfere with a patient's ability to even perform routine daily activities, resulting in a decrease in quality of life. The goals of osteoarthritis treatment are to relieve pain and other symptoms, preserve or improve joint function, and reduce physical disability. Current therapeutic options are limited to pain medications and joint replacement for patients with advanced disease. No disease-modifying OA drugs are approved for clinical use. OA is thus a major unmet medical need with a huge clinical and socioeconomic impact and a complete absence of effective therapies. Clearly the development of a new therapeutic that is both symptom and disease modifying would have a significant impact on the well-being of Californians and reduce the negative economic impact on the state resulting from this highly prevalent disease.

Progress Report: 

We have carried out a structure-activity relationship study to identify highly potent analogues of kartogenin with chondrogenic and chondroprotective activities. Over 150 analogues were synthesized with structurally diverse elements and assessed for chondrogenic activity (ability to induce mesenchymal stem cells to differentiate into cartilage producing chondrocytes) on human and rodent mesenchymal stem cells. A number of highly potent lead compounds were identified which will next be assessed in chondroprotective assays, cell-based selectively and toxicity assays, pharmacokinetic assays and in vivo rodent efficacy models. At the same time a number of assays were developed and used to assess the chondrocyte protective effects, joint retention, and proliferative activity on human chondrocytes of the parent compound, kartogenin. Kartogenin was found to: (1) have long term human and rodent chondrogenic activity; (2) possess chondroprotective activity in bovine chondrocytes (i.e., protects against degradative activities in the joint); (3) minimally induce chondrocytes proliferation (an undesired activity that could lead to fibrotic and immune responses); (4) have good joint retention (compound retained in the intra-articular space at the site of action); and (5) is subject to rapid systemic clearance (a desirable property to minimize systemic adverse effects).

We also identified the mechanism by which the compound functions. In contrast to other drugs in development for osteoarthritis, kartogenin does not target extracellular enzymes involved in joint cartilage degradation. Rather it appears to act directly on an endogenous stem cell population and induce chondrocyte formation. The molecule binds selectively to an intracellular protein filamin A, a protein involved in regulating the cell’s cytoskeletal network (structural elements inside the cell). Rather than modulating the interaction of filamin A with other structural proteins, kartogenin blocks its interaction with the protein CBFβ (core binding factor β subunit, a subunit of a transcription complex with the runt-related transcription factor (RUNX) family). The result is an increase in CBFβ levels in the nucleus where it binds and activates transcription of RUNX dependent genes. In particular CBFβ activates RUNX1 dependent transcription of genes that play key roles in chondrogenesis. Thus this molecule acts by a novel mechanism directly and selectively on gene transcription to induce the selective differentiation of mesenchymal stem cells to chondrocytes. Importantly molecules that act by this method should complement the activity of drugs in clinical trials aimed at blocking degradative enzymes.

We have made excellent progress toward the identification of a preclinical candidate for the treatment of osteoarthritis. A large structure-activity relationship study was carried out with the chemical synthesis of over 250 analogues of the original lead compound. We have identified molecules with improved activity in cell culture and in relevant preclinical in vivo models. Based on these efforts we are synthesizing a final series of molecules which we will profile with respect to in vitro and in vivo chondrogenesis activity, pharmacokinetics and safety. We expect to choose the final preclinical candidate from this series in the third year of the grant.

Osteoarthritis (OA) is the most prevalent musculoskeletal disease affecting about 27 million people in the United States, and is the leading cause of chronic disability in the United States. Current therapeutic options are limited to pain or symptom-modifying drugs and joint replacement surgery; no disease-modifying drugs are approved for clinical use. OA is characterized by progressive degeneration of the articular cartilage, resulting from abnormal activation, differentiation and death of cartilage cells. A unique and unexplored therapeutic opportunity exists to induce somatic stem cells to regenerate the damaged tissue and reverse the chronic destructive process. Because limited joints are affected in most OA patients, intra-articular (IA) drug injection is an attractive treatment approach that allows high local drug concentration with limited systemic exposure. Targeting resident stem cells pharmacologically also avoids the risks and costs associated with cell-based approaches.

Cartilage contains resident mesenchymal stem cells (MSCs) that can be differentiated in vitro to form chondrocytes. This observation suggests that intra-articular injection of a small molecule that promotes chondrogenesis in vivo will preserve and regenerate cartilage in OA-affected joints. Using an image-based screen, we identified a drug-like small molecule, kartogenin (KGN), that promotes efficient and selective chondrocyte differentiation from MSCs in vitro. Intra-articular injection of KGN also shows beneficial effects in surgery-induced acute and enzyme-induced chronic cartilage injury models in rodents, as well as positive effects in incapacitance pain models. This project is aimed at the development of new lead compounds with improved biological activity, the demonstration of efficacy of the lead compounds in rodent and dog OA models and the elucidation of the cellular mechanisms underlying the cartilage regeneration activities of KGN and its analogs.

Through medicinal chemistry efforts, we have designed and synthesized over 400 analogs of KGN. Using cell culture based assays, we assessed the chondrocyte differentiation activity of these analogs and identified 17 compounds exhibiting improved potency compared to KGN (EC50 < 100 nM). These compounds showed no obvious cytotoxicity at high concentrations (100 μM) when incubated with a variety of cells present in the joints including MSCs, chondrocytes, osteoblasts and synoviocytes. Up to date, we have assessed the efficacy of 7 compounds using a rat OA model (medial meniscal tear). Two of the tested compounds showed significantly improved cartilage repair at the end of the study. At the same time, no adverse effects, such as body weight loss, pain or impaired motor functions, were observed in any compound treated animals. We are currently studying the effects of another 10 analogs using the same OA model, which is expected to conclude within two to three months. Next, we will assess the efficacy of active compounds in a canine OA model (partial meniscectomy using beagles). Furthermore, full rodent pharmacokinetics and non-GLP toxicology studies will be performed for the lead compounds.

To study the underlying mechanisms of KGN induced chondrogenesis, we designed and synthesized an affinity probe with biological activities comparable to that of KGN. Through affinity-based methods, we identified protein filamin A (FLNA) as the target of KGN. In MSCs, KGN binds to FLNA and disrupts its interaction with core binding factor β (CBFβ), which leads to the nuclear translocalization of CBFβ, subsequent activation of the RUNX1-CBFβ transcription program and, as a result, chondrocyte differentiation. This mechanism has been confirmed using cell biological methods including RNAi mediated gene silencing and cDNA overexpression of relevant genes such as FLNA, CBFβ and RUNX1. These studies have been published in the journal Science.

We have demonstrated efficacy in preclinical in vivo models of a potential drug candidate for the treatment of osteoarthritis. The small molecule functions by selectively differentiating meschenchymal stem cells to chondrocytes to repair damaged cartilage .