Gene Mapping Gives Rise To Drugs That Rebuild Tissue

The logical outcome of gene mapping is nigh. Biotechnology companies are using genetic information to design new drugs that may go beyond merely slowing or stopping a disease process to inducing the regeneration or repair of damaged tissue. Looking past the conventional drug targets of enzymes and gene-coupled receptors, researchers are seeking out molecules in the pathways along which biochemical signals are transferred. In some cases, their work has led to compounds--now in clinical trials or just released by major pharmaceutical companies--aimed at restoring functions damaged by disease or trauma. Potential beneficiaries are patients suffering from a variety of disorders of the metabolic, hematopoietic and central nervous systems, as well as various forms of cancer. The groundwork for these advances has been laid over recent years by genetic mapping--the arrangement of mutable sites on a chromosome as deduced from genetic recombination experiments. This mapping has revealed remarkable similarities, or conservation, in the genes of a few organisms up and down the evolutionary tree. By altering genes from conserved areas of the genome in such organisms as yeast, nematode worms, fruit flies, zebra fish, mice, and cats, developmental biologists are elucidating the biochemical pathways and are finding new points of intervention for drugs to treat humans. Photo: Exelixis FLY MODEL: Researchers at Exelixis Pharmaceuticals insert a human disease gene in the eye of a fruit fly to investigate the signaling pathway as a normal eye (left), degenerates into a "rough" eye. "Mass genetic mapping has put a library of just about all the known genes into the public domain," notes Geoffrey Duyk, chief scientific officer of Exelixis Pharmaceuticals Inc., a privately owned genomics company in South San Francisco. Duyk compares similar genes of the nematode and drosophila to words in French and Spanish, respectively --because both languages have the same roots in Latin. Continuing the metaphor, he adds, "Signaling pathways represent the grammar and syntax of organizing these words into sentences." The task of assembling those "sentences" has just begun. Humans have an estimated 70,000 genes and about 1,000 pathways. Although the fruit fly has only one-quarter as many genes as a human, it has about the same number of pathways, because humans are diploids (each type of chromosome is represented twice, except the sex chromosomes) and flies are octoploids. The nematode worm, with only 959 cells, will soon become the first animal to have its entire genome sequenced. Genes involved in Alzheimer's disease, cystic fibrosis, and various cancers are among human disorders for which homologous disease genes are found in the nematode. Other advantages of these lower organisms to researchers is that they reproduce and mature relatively quickly, can be handled by robots, can be frozen, and are inexpensive. Exelixis is a forerunner in using model organisms to define pathways involved in chronic human diseases. It has a patented technology for screening these pathways and processes and exclusive commercial rights to the entire Notch signaling cascade, which appears to be implicated in cancer, stroke, and dementia. "We live in an era of target-based growth discovery but in a sense, there are too many targets," Duyk notes. "We have all these biologically active molecules, but we don't know what their activity is. . . . More information is not necessarily better information." He compares the current situation to progress made in physics during the 1950s and 1960s, when the structure of the atom was being defined by deconstructing it. The knowledge gained from that work gave rise to contemporary theories about the birth of the universe. Similarly, he says, delineation of intracellular signaling cascades is leading to new knowledge concerning function of the whole organism. Duyk predicts the outcome: "At the end of the day, pharmaceutical companies will begin to diversify [their drug targets]." Exelixis Pharmaceuticals Inc. 260 Littlefield Ave. South San Francisco, CA 94080 (650) 825-2200 Fax: (650) 825-2205 http://www.exelixis.com Genentech Inc. 1 DNA Way South San Francisco, CA 94080-4990 (650) 225-1000 Fax: (650) 225-2021 http://www.gene.com Scios Inc. 2450 Bayshore Parkway Mountain View, CA 94043 (650) 966-1550 Fax: (650) 968-2438 http://www.sciosinc.com Philadelphia-based Wyeth-Ayerst Laboratories is among the first pharmaceutical firms already mounting such efforts. It now has Phase 2 and Phase 3 multicenter clinical trials under way in the United States and Europe for treatment of stroke using trafermin (trade name Fiblast), a recombinant basic fibroblast growth factor (bFGF). The normal function of bFGF, to promote growth of fibroblast cells that give rise to connective tissue, does not occur when trafermin is delivered intravenously to brain cells. Instead, it appears to substantially reduce the size of an infarct when administered shortly after a stroke, compared to stroke victims who do not receive the therapy. Studies led by neurologist Seth P. Finklestein of Massachusetts General Hospital in Boston and Harvard Medical School show that bFGF enhances functional recovery in rats following stroke, which may be due to stimulation of new neuronal growth (T. Kawamata et al., Proceedings of the National Academy of Sciences, 94:8179-84, 1997). Finklestein explained the mechanisms of growth factor treatment of stroke at "PharmacoGenesis: Postgenomic Drug Discovery Through Developmental Biology," a conference cosponsored in Boston on March 26 and 27 by Massachusetts General Hospital and the journal Nature Biotechnology. He said the amount of damaged tissue following an acute stroke can be reduced by polypeptide growth factors such as bFGF, which protect surrounding neurons from potentially toxic substances (thought to be glutamate) released by stroke-affected cells. The damaging effects of stroke, including neuronal deprivation of oxygen and glucose, also may be prevented or mitigated by bFGF, which appears to promote angiogenesis (see story on page 10). Last year, Finklestein's team collaborated on limited clinical trials of bFGF treatment of stroke with Scios Inc. of Mountain View, Calif., the biopharmaceutical company that cloned and produced trafermin. The Wyeth-Ayerst trials will include more than 1,800 patients, and if they are successful, registration filings for trafermin with the Food and Drug Administration could take place in the year 2000. "I'm hoping clinical science will catch up with preclinical science," Finklestein told the conference audience, "before we all get too discouraged with clinical trials." "One of the most difficult things about developing therapies for stroke is the lack of a good surrogate marker," states Elliott B. Grossbard, senior vice president of development for Scios. "It's difficult to ascertain any benefit beyond the principal benefit you're seeking." He explains that FDA requires evidence that a new compound will somehow improve the health of patients in a measurable way, compared to other therapies or no therapy at all. The severity of damage following stroke is not immediately apparent. Regardless of whether or not the patient is treated, recovery of motor function and speech may be substantial or not, and this "lacuna effect"--which can account for improvement of up to 30 percent of all patients--tends to dilute the impact of successful clinical trials. There also are technical hurdles to overcome, such as mitigating the damage from stroke by getting patients to the hospital quickly, evaluating them, and obtaining informed consent for participation in the trials. All this should be accomplished within 30 minutes (up to a maximum of about three to five hours) after the stroke for the trafermin to be effective. Stimulation of the development of collateral arteries--otherwise known as therapeutic angiogenesis--also appears to be achievable by manipulating vascular endothelial growth factor (VEGF). A protein typically inactive in adults, VEGF can be naturally activated for wound healing or in response to ischemia. Mutations are associated with disease processes involving vision loss and tumor formation. In a recent study led by cardiologist Jeffrey M. Isner at St. Elizabeth's Medical Center in Boston, the "naked" DNA of VEGF in a saline solution was injected directly into 10 limbs of nine patients with nonhealing ischemic ulcers due to peripheral arterial disease. Blood flow improved in eight of the limbs, and three patients avoided below-knee amputations (I. Baumgartner et al., Circulation, 97:1114-23, 1998). Scios has cloned recombinant VEGF and licensed it to privately held biotechnology company GenVec Inc. of Rockville, Md., which is collaborating with Warner-Lambert Co. of Morris Plains, N.J., to initiate clinical trials of the compound in an adenoviral vector to treat advanced cardiovascular disease. VEGF has been cloned in a slightly altered form by Genentech Inc., a publicly held biotechnology company headquartered in South San Francisco, which is in Phase 1 clinical trials of the compound to treat coronary arterial disease. Novel drug targets also have been identified for cancer patients receiving chemotherapy. They can sustain marrow damage from the chemotherapy, resulting in a depletion of large cells in the marrow called megakaryocytes. These cells are thought to be the source of platelets, the blood components that prevent bleeding and repair damaged blood vessels. Severe platelet deficiency, or thrombocytopenia, requires either transfusions or a reduction or halt to chemotherapy--actions that disrupt the treatment, may cause dangerous complications, and could allow malignant cells to spread. In late 1997, FDA approved oprelvekin (trade name Neumega), the first biologic to boost platelet production. Developed in Cambridge, Mass., by Genetics Institute, a biopharmaceutical subsidiary of Madison, N.J.-based American Home Products Corp., the compound is a recombinant human interleukin-11. The family of naturally produced interleukins is important in cell-mediated immunity. "Neumega stimulates the progenitor cells for megakaryocytes," explains Jack Morgan, vice president and general manager of Genetics Institute. On the heels of Genetics Institute, both Amgen Inc. of Thousand Oaks, Calif., and Genentech--in collaboration with the multinational Pharmacia & Upjohn, headquartered in England--also are investigating drugs to regulate platelet production. Their goal is to target thrombopoietin (TPO), which stimulates survival of megakaryocytes. "TPO clearly is a molecule that regulates, in normal physiological situations, the maturation of platelets," Morgan acknowledges. But he adds that during aggressive chemotherapy, the body naturally upregulates production of TPO, yet thrombocytopenia still develops. "It's not so clear to us that administering even more TPO to these patients is necessarily going to prove efficacious," he cautions. Sufferers of spinal cord injury also could benefit from chemical mechanisms that appear to be highly conserved in evolution. During embryonic development, when each neuron first sends the long "wire" of its axon toward target cells that will receive its nerve impulses, that migration is guided by molecules present in the embryo. The chemical mechanisms by which these molecules function--called "diffusable attractant and repellent cues"--are being studied in the embryos of model organisms to determine how human homologues might be manipulated to stimulate regeneration of neurons after an injury. In the peripheral nervous system, axons can sprout new growth after trauma, but such sprouting does not occur in the CNS. People with spinal cord injury currently have little or no prospect of recovery. Marc Tessier- Lavigne, professor of anatomy at the University of California, San Francisco, and Corey S. Goodman, head of the division of neurobiology in the department of molecular and cell biology at Howard Hughes Medical Institute, University of California, Berkeley, have described the mechanisms and molecules that direct axonal pathfinding (M. Tessier-Lavigne, C.S. Goodman, Science, 274:1123-33, 1996). Yet another group that might benefit from these advances in developmental biology are sufferers of peripheral neuropathy, a degenerative condition in the hands, legs, and feet that affects up to half of all diabetics and for which there currently is no corrective treatment. The condition can progress from uncomfortable sensations to foot ulcers or gangrene that requires amputation. In peripheral neuropathy, degeneration occurs in the fingerlike dendrites extending from the ends of axons to the target cells. This degeneration contributes to disruption of signals from sympathetic system neurons to small fiber sensory cells in the skin, impairing their ability to perceive pain, temperature, and touch. Among nine drugs Genentech currently has in Phase 3 trials is a recombinant nerve growth factor (NGF), a protein required for development and survival of the sympathetic nervous system and of "small fiber" sensory neurons. In collaboration with its majority owner, the Swiss multinational Roche Group, Genentech has enrolled 1,000 diabetic peripheral neuropathy patients in the U.S. and is recruiting 1,500 patients in Europe. During the PharmacoGenesis conference, John Kessler, vice chairman of the department of neurology at Albert Einstein College of Medicine, reported on NGF research in which his team has participated with Genentech. "I don't wish to be a Pollyanna," he told conference attendees, "but I've seen in my own patients--they get better." Cynthia Rask, senior clinical scientist at Genentech, led a Phase 2 trial of about 250 patients who were divided into three groups. One group received a placebo and the other two groups received different dosages of NGF, three times weekly. "The dosages were tiny," Rask notes, "0.1 or 0.3 micrograms per kilogram of body weight." After six months, a combined endpoint analysis was done that included neurological examination, the patient's self-evaluation, and performance on sensory tests. It indicated significant improvement for both groups using NGF compared to the placebo group. Some male patients who had been rendered impotent by the diabetes even announced improved sexual performance. The afflictions reported here are only some of those for which cross-species research offers new hope. "Human disease genes are remarkably valuable, because they offer recognizable targets," Exelixis's Duyk observes. But he is quick with the caveat: "If you're a pharmaceutical company, you have to be darned sure the target is validated." *

Gene Mapping Gives Rise To Drugs That Rebuild Tissue

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