Drug Makers on the Apoptotic Trail

Apoptosis, a key process in the development of embryonic tissue differentiation, later helps to regulate the normal cellular life cycle by destroying damaged cells. When something goes awry, too little apoptosis can make cancer cells resistant to chemotherapy and even death-defiant. At the other extreme, premature or excessive apoptosis has been linked to neurodegenerative diseases, such as Alzheimer's, and to nerve cell loss in strokes. Not surprisingly, many major pharmaceutical companies recognize the value of apoptosis research. Understanding and controlling programmed cell death is expected to yield new drug discoveries and therapeutics. Researchers are exploring possible targets through cell-cycle inhibitors and molecules known as apoptosis-inducing factors (AIF). Much of the pharmaceutical research involves caspases--a family of enzymes that cleave intracellular proteins leading to apoptosis--while other efforts focus on the Bcl-2 protein family. "There is an absolute explosion of research in apoptosis," says Sara Radcliffe, director of research for biologics, biotechnology, and preclinical affairs at Pharmaceutical Research and Manufacturers of America (PhRMA), the major drug company industry group. "It is most exciting because apoptosis has a wide range of applications, from cancer research to HIV to organ transplantation," she says. The research, also not surprisingly, is as varied as the apoptotic function itself. Abbott Laboratories, in Abbott Park, Ill., is searching for pro-apoptotic, small-molecule drugs using high-throughput screening. The firm is collaborating with Idun Pharmaceuticals Inc., a private biopharmaceutical startup in San Diego, to identify antagonists of Bcl-2 cell death inhibitors. Bcl-2 and other related proteins, such as Bcl-XL, are anti-apoptotic. Others proteins, such as Bak and Bax, trigger apoptosis by causing the mitochondrial membrane to release cytochrome c and other proteins that lead to caspase activation. Idun is developing primary molecular screening assays against targets in the apoptotic pathway. Abbott will be responsible for preclinical and clinical development and will own the rights to any drug leads emerging from the collaboration. Roche Holding Ltd., the pharmaceutical and diagnostic unit of Swiss-based Hoffmann-La Roche Ltd., is in Phase II multicountry clinical trials with a cell-cycle inhibitor compound called RO31-7453. "Unlike some other cell-cycle inhibitors that induced cytostasis, that is, induced a cell-cycle arrest and then just holds the cells at that stage of the cell cycle, this one causes a cell-cycle arrest in prometaphase and, separately, it induces apoptosis," says Steve Ritland, preclinical research leader at Roche. "Because we were able to induce apoptosis secondary to the cell-cycle arrest, we could actually cause tumors to regress and in some models get durable cures," he explains. Clinical trials are under way for cancers of the colon, lung, and breast. Roche claims it has more than 50 preclinical oncology drug discovery projects under way. "A fair number of these are directed at apoptotic pathways, Bcl-2, and caspase being some of the major players," Ritland says. As is the case with other pharmaceutical companies, Roche assesses all of its molecular-targeted oncology therapeutics for possible effects on apoptotic endpoints as part of the drug discovery process, explains Ritland. Maxim Pharmaceuticals, a late-stage pharmaceutical firm in San Diego, also is seeking small-molecule caspase inhibitors for drug development. In preclinical studies with mice, caspase inhibitor CV1013 protected liver cells from induced endotoxemia by inhibiting caspase-8, blocking the initial intracellular signal that would otherwise lead to apoptosis and liver failure.1 This year, Maxim received two patents for its proprietary caspase inhibitor technologies involving dipeptides. Millennium Pharmaceuticals Inc., Cambridge, Mass., is in Phase II clinical trials of LDP-341, a small molecule proteasome inhibitor that can induce apoptosis and inhibit cell growth, cellular adhesion molecule expression, and angiogenesis. Proteasomes are large complexes of enzymes that break down intracellular proteins, recognizing them by their ubiquitin molecular tags. In addition to degrading unwanted proteins, proteasomes are involved in regulating cellular signals that govern the cell division cycle, cell growth, and differentiation. In normal cells, the ubiquitin-proteasome pathway is responsible for the orderly breakdown of multiple proteins that helps define protein turnover rates. But in tumors, proteasome inhibition produces overwhelming cellular stress by stabilizing cell cycle regulatory proteins and disrupting cell proliferation, ultimately leading to apoptosis. LDP-341 has shown promise for multiple myeloma, which occurs when the immune system's plasma cells undergo abnormal uncontrolled growth and reproduction, preventing the marrow from forming normal plasma cells and other white blood cells important to the immune system. LDP-341 has also demonstrated effectiveness against a variety of human cancers, including hematologic malignancies and solid tumors of the breast, pancreas, prostate, lung, and colon. "We are encouraged by the Phase I data on LDP-341 in advanced stage cancers," says Lee R. Brettman, senior vice president for clinical development and medical affairs at Millennium. "Based on the early results seen in the Phase I trials, we are now initiating multicenter Phase II trials in multiple myeloma." Choosing a different pathway, Josef M. Penninger, an immunologist at the Amgen Institute in Toronto, and his colleagues are exploring mitochondrially regulated apoptosis. They have identified a molecule localized to AIF and have shown it to be a caspase-independent pathway.2 Overexpression of Bcl-2 prevents the release of AIF from the mitochondrion but does not affect the molecule's apoptogenic activity.3 "It is still early but [AIF] looks fairly promising for a second pathway for programmed cell death," Penninger says. "Caspase [inhibitors] are only blocking 40-50 percent of cell deaths. So you can interpret this either as the caspase inhibitors are not working well or there's actually a second pathway, which is what I am looking at." AIF appears to control the first wave of programmed cell death in morphogenesis, Penninger explains. "AIF is much older in evolution than caspases. You can find it in plants, slime molds, all over the place. We think AIF might actually be the primordial 'death trigger,'" he says. "Many organisms like plants and leaves and flowers undergo programmed cell death, but plants don't have any functional caspases. How can a plant cell undergo programmed cell death if this protein is actually not there?" Although AIF represents a major pathway of apoptosis, it is not a candidate for drug development because it is not an enzyme. "The whole field is probably a year away from using information" about AIF to find molecules to shut down the apoptosis pathways, Penninger says. Ted Agres (tagres@usa.net) is a freelance writer in Washington, D.C. References 1. H. Jaeschke et al., "Protection against TNF-induced parenchymal cell apoptosis during endotoxemia by a novel caspase inhibitor in mice," Toxicology and Applied Pharmacology, 169:77-83, 2000. 2. N. Joza et al., "Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death," Nature, 410:549-54, March 29, 2001. 3. S. Susin et al., "Molecular characterization of mitochondrial apoptosis-inducing factor," Nature, 397:441-6, 1999.

Drug Makers on the Apoptotic Trail

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