Carfilzomib: The Latest Triumph of Targeted Therapies Development

On July 20, 2012, the U.S. Food and Drug Administration granted accelerated approval of the drug carfilzomib, developed by Professor Craig Crews of Yale’s department of Molecular Cellular, and Developmental Biology. Carfilzomib, which goes by the name of Kyprolis, is approved for the treatment of patients with multiple myeloma who have received at least two prior therapies and have demonstrated disease progression on or within 60 days of the completion of their last therapy. The approval of carfilzomib represents 14 years of dedicated research and clinical trials, as well as another triumph of targeted therapies in the treatment of cancer.

Multiple Myeloma

Multiple myeloma is a cancer of plasma cells. Plasma cells are a type of white blood cell in the bone marrow that produce antibodies. These cells originate within the bone marrow, but differentiate into B lymphocytes (also known as B cells) by the time they enter the blood. From B-cells, they make their terminal differentiation into plasma cells. Plasma cells are an integral part of the immune system – they contribute to the immune response by producing antibodies that target foreign pathogens for destruction. Multiple myeloma accounts for 1% of all cancers, and 10% of all cancers of the bone marrow. The American Cancer Society estimates that over 20,000 new cases of multiple myeloma will be diagnosed in the United States each year.

Multiple myeloma cells (in purple), in a bone marrow smear from a patient with multiple myeloma, proliferate more quickly than normal cells. Courtesy of Craig Crews.

The exact cause of multiple myeloma is unknown, but it is believed to be the combined result of genetic defects and environmental toxins. Symptoms of multiple myeloma include abnormal bleeding, bone or back pain, anemia, and numbness or weakness in the limbs. Multiple myeloma usually affects people between 65 and 70 years of age and the prognosis is generally very poor: multiple myeloma is an incurable malignancy. The median survival time post-diagnosis is 3-4 years, although this number depends on the stage of cancer prior to diagnosis. According to the international staging system for Multiple Myeloma, which is based on the on the serum b-2 macroglobulin level and albumin level, patients at stage I have a median survival of 62 months (~5 years), patients at stage II have a median survival of 44 months (~3.5 years), and patients at stage III have a median survival of 29 months (~2.5 years).

Proteasome inhibitors as Multiple Myeloma drugs

Cancer is a disease causing uncontrolled growth and replication of cells. Thus, cancer treatments seek to slow the growth of or kill these mutant cells. The problem with many popular cancer therapy methods like chemotherapy and radiation treatments is that they are unable to distinguish between cancerous and non-cancerous cells and ultimately kill enough normal cells to make a patient very sick. These methods essentially try to cure the patient with a poison. The idea ofgtreating patients with drugs that have lesser side-effects is what makes newer, “targeted therapies” so appealing to researchers and physicians. The idea of targeted therapies is that the drug can be prescribed in a way more personalized to the patient’s specific disease, and should be able to selectively kill only cancer cells.

The nature of the plasma cells involved in multiple myeloma offers a unique way to target and kill these cells. One of the main functions of plasma cells is to produce large quantities of antibodies for the immune response. To produce the antibodies, the cell must synthesize the proteins the antibodies are composed of, ensure that they are folded properly, and then export them from the cell. The bulk of the synthesis and folding of antibodies occurs in the endoplasmic reticulum (ER), one of the cell’s main organelles. When proteins don’t fold properly and build up, the stress level of the ER increases. If the ER stress becomes too great, the ER will signal the cell to undergo apoptosis (programmed cell death). The proteasome can lower this stress. In simple terms, the proteasome is akin to a cellular garbage truck. It can break down proteins, and thus is the cell’s main tool in getting rid of unwanted or misfolded proteins. However, if the action of the proteasome is blocked or inhibited, it can no longer aid in lowering ER stress, and these cells are more likely to undergo apoptosis. Because cancerous plasma cells have a very high protein payload, they are much more susceptible to proteasome inhibitors. Thus, at the proper concentration, a proteasome inhibitor could selectively kill cancerous cells (by only targeting the sensitive, cancerous plasma cells).

Various depictions of the proteasome. (A) An electron micrograph of the proteasome. (B-C)Computer generated space filling models of the proteasome. Courtesy of Thomas Pollard.

A history of proteasome inhibitors

Epoxomicin, the linear peptide from which carfilzomib is derived, was one of the first identified proteasome inhibitors. It was isolated as a natural product produced by a fungus collected by a Japanese research group, Hanada et al, at the Bristol Meyers Squibb Research institute in Toyko in 1992. It was originally identified as an antitumor agent, but it was not until seven years later that the Crews lab determined that the method of antitumor action of epoxomicin was though the inhibition of the proteasome. The Crews lab also found that within epoxomicin, it is the a,ß-epoxyketone pharmacophore that is responsible for the selective inhibition of the proteasome. This selective inhibition occurs when expoxomicin forms an unusual six-membered morpholino ring with the amino terminal catalytic Thr-1 of the 20S proteasome. However, for such a compound to become a pharmaceutical, it would have to be even more specific in its binding selectivity, and would have to have a lower toxicity.

One of the most common drugs used currently for the treatment of multiple myeloma is a protease-specific inhibitor called bortezomib (known as Velcade). However, the drug has several undesirable side effects. One serious problem associated with bortezomib was the side effect of peripheral neuropathy, a condition in which damage to the peripheral nerves causes pain, numbness, and muscle problems. Another problem with bortezomib is that patients typically acquire resistance. These flaws led to the need for a second-generation proteasome inhibitor.

The system of targeting a protein to the proteasome for degradation. Ubiquitin is activated by the E1 and E2 ligases, and added to the protein to be targeted for destruction by the E3 ligase. The proteasome recognizes a chain of ubiquitin molecules attached to a protein, and then will proceed to degrade that protein. Courtesy of Thomas Pollard.

Drug development

For Crews, there was a great incentive to using natural products. Said Crews, “Evolution has selected for exquisitely potent and selective inhibitors. These are the ideal probes already, so the mystery was figuring out how they work. I’m interested in the mode of action.” Epoxomicin in particular interested Crews. The group at Bristol Meyers Squibb dropped the epoxomicin project because they did not know the mechanism of its antitumor activity. Crews, who was very much interested in mechanism-of-action studies, decided to pick up the study of this promising compound. As Crews explained, “Epoxomicin had already displayed potent antitumor activity. The fact that they were interested in it, but just didn’t know how it worked. Well, the challenge appealed to me.” He devised a total synthesis of the natural product, then, through biotin tagging experiments, the lab was able to determine that epoxomicin acts by binding the 20S subunit of the proteasome, effectively blocking it.

The lab then proceeded to alter the compound to try to get a more potent and specific binding and inhibition. According to Crews, “There are three catalytic activities in the proteasome that have different specificities. The natural product [epoxomicin] could target two of the three. The goal of our project was to develop an analog of this natural product that could be specific for just one.”

One of these derivatives, named YU101, had potent antitumor activity, and was able to inhibit the proteasome better and more specifically than epoxomicin. Indeed, it was able to inhibit the proteasome better than bortezomib, which at the time had just been approved for the treatment of multiple myeloma. Furthermore, Crews speculated that the peripheral neuropathy side effect of bortezomib was due to ths boronateygroup in the compound (a boron bonded to two hydroxyl groups and a hydrocarbon), as boronate is an unusual pharmacore. YU101 did not have a boronate moiety, and thus was not likely to cause peripheral neuropathy. It appeared that Crews might have a viable drug.

The structures of bortezomib and carfilzomib. The circled boronate moiety of bortezomib is essential to its activity, but unfortunately, is also likely responsible for its peripheral neuropathy side effect. Courtesy of Blood

Clinical trials and FDA approval

To start the long journey towards FDA approval, Crews started South San Francisco-based company, Proteolix, along with Caltech professor Raymond J. Deshaies. The company, despite trying many different variations of epoximicin, finally settled on YU101 as the best option, with one slight change. The final change was adding a morpholine ring to the end of compound opposite the epoxy ketone to boost its solubility. This compound is now known as carfilzomib.

With the support of investors, Proteolix was able to file a New Drug Application to start clinical trials. Carfilzomib defied the odds, successfully making its way through Phase I and II clinical trials. As predicted, carfilzomib did not produce the side effect of peripheral neuropathy. This lower toxicity is crucial. Crews explains that “the problem with Velcade is that the dose limiting toxicity prevents physicians from being able to achieve more than about 60% of proteasome inhibition. With Carfilzomib, because of the better side effect profile, physicians can dose to about 75% proteasome inhibition.”

After achieving this success in Phase I and II trials, in 2009, Onyx Pharmaceuticals acquired Proteolix and advanced the compound through a Phase IIb trial that led to the drug’s accelerated approval in July. Carfilzomib is also going through a Phase III trial to explore its efficacy in solid tumors.

The FDA approved Carfilzomib after 14 years of dedicated research and clinical trials. It is one of the triumphs of targeted therapies in the treatment of cancer. More than this though, carfilzomib is a triumph for academic labs. Pharmaceutical companies develop the vast majority of drugs, yet it was an academic lab that developed this drug. When the Crews lab picked up epoxomicin as a project, the goal was to understand its mechanism of action, rather than to develop a drug. However, using solid scientific methods and elegant experiments, they were able to capitalize on this academic endeavor and advance the field of cancer therapy.

Craig Crews, the Lewis B. Cullman Professor of Molecular, Cellular and Developmental Biology and executive director of the Yale Small Molecule Discovery Center. Courtesy of Craig Crews.

About the Author
Kaitlin McLean is a senior Molecular, Cellular and Developmental Biology major from Madison, Wisconsin. She has been working in the Crews Lab since her freshman year and will be conducting her intensive B.S. research project under the supervision of Professor Craig Crews.

I would like to profoundly thank Professor Crews for his time, effort, and passion.

Further Reading
Pingali, S. R., Haddad, R. Y., & Saad, A. (2012). Current concepts of clinical management of multiple myeloma.