Investigating Nature's Mysteries for Drug Development
Living in the competitive environment of a coral reef, marine invertebrates, such as delicate sea squirts and immobile sponges, must find creative ways to protect themselves. For some organisms, having an arsenal of toxic chemicals is what keeps them alive. Scientists in NCI-Frederick's Natural Products Branch (NPB) are exploring ways to harness these chemicals, and those from other plant, animal, and microbial sources, for drug discovery.
Interest in natural products for drug development has waxed and waned, but there's no denying their importance in oncology, according to NPB Chief Dr. David Newman. Today, over half of the drugs approved to treat cancer come from a natural product or a natural product prototype.
At NCI-Frederick, researchers look for active toxic compounds from natural sources, explained Dr. Newman, then modify those compounds, if necessary, to maintain their potency while simplifying their complex molecular structures. NPB works with other NCI programs, collaborators in academia and the pharmaceutical industry, and governments of countries supplying natural product sources, and may join in the development of products with these collaborators at any stage of development.
Biological Detective Work
NPB, part of NCI's Developmental Therapeutics Program (DTP), has existed for more than 50 years. The collection and compound extraction process was systematized more than 25 years ago and has essentially stayed the same since. (See the accompanying video.)
Organisms—marine invertebrates, plants, and microorganisms—collected from around the world are sent to NPB, where researchers look for compounds that show potential anticancer activity when screened against the NCI-60 panel of human cancer cell lines. That project, also managed by DTP, uses 60 different human tumor cell lines, representing leukemia, melanoma, and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney to screen both synthetic compounds and natural product samples.
The first step of the extraction process begins with grinding up the organism and sending it to an extraction lab. Solvents are then added and two extracts are produced; one contains compounds that are soluble in water, and the other contains materials that are insoluble in water. Dr. Newman explained that an extract is like a library; each extract can have anywhere from 50 to more than 100 compounds, like a library with different books.
"We don't know which book refers to which target, or which book tells a story," Dr. Newman said. The extracts are tested against the NCI-60 panel of cells lines in the laboratory, and then the most promising are tested in animal models of a variety of tumor types to look for anticancer properties.
When they find a "hit," the extract is sent to the isolation lab to identify which book, or books, in the library of compounds is responsible for the biological activity of the extract. The researchers separate individual chemical compounds from the extract through a process known as bioactivity-driven chromatography. Once in a pure state, the compounds are studied to define their chemical structures using state-of-the-art instrumentation. These include mass spectrometry for determining the size or molecular weight of a compound, and nuclear magnetic spectroscopy, which reveals how atoms within a molecule are linked together.
Unraveling the Complexities of Natural Compounds
Compounds from nature are often quite complex structurally. Until the advent of new chromatography-based analytical techniques roughly 10 years ago, it could take anywhere from 6 months to a year to isolate and identify a compound. Natural products research was criticized for that slow pace, Dr. Newman explained. Now researchers can often find an answer in 1 to 2 weeks. And because of these improvements, materials found today can be developed into testable treatments faster.
Another hurdle is finding enough of the source material to sustain the drug development process. The need to find sustainable sources in nature led researchers to shift their focus from macro-organisms, such as marine invertebrates, to micro-organisms that can be cultured in the lab and induced to make compounds in quantities that will provide a continuous source.
"That helped with the supply issue, but it still didn't address the problem of these compounds being really complex," said Dr. Chris M. Ireland, a long-time researcher in the field and dean of the University of Utah's College of Pharmacy.
Some chemists thought that, when combinatorial chemistry took off in industry in the 1990s, that it could be used to create "libraries" of molecules that would be similar to those obtained from natural products. Combinatorial chemistry enables researchers to create a large number of different but structurally similar compounds. However, it has yet to showcase the structural complexity and diversity that can be found in natural products. "Where combinatorial chemistry is magnificent, though, is taking an active structure and modifying it," Dr. Newman said.
"That's exactly what Harvard and [the pharmaceutical company] Eisai did with halichondrin B," a natural compound from deep-sea sponges that had shown promising antitumor properties, he continued.
Deep Sea Sponge Inspires Cancer Drug
The cancer drug eribulin mesylate (Halaven) was modeled after halichondrin B. Eribulin interferes with the assembly and disassembly of microtubules, cellular structures that help move chromosomes during cell division. Other cancer drugs, including vinca alkaloids and taxanes, also disrupt microtubules. But eribulin has unique tubulin interactions, and in early animal studies it was found to work better against breast and lung cancer than either its parent natural compound, halichondrin B, or the commonly used drug paclitaxel.
The eribulin story, which spans nearly three decades of research, is based on basic research in synthetic organic chemistry. In 1982, when Japanese researchers initially isolated halichondrin B, the prospect of developing a synthetic version of such a complex compound seemed impossible with the technology available. But in 1987, Dr. Yoshito Kishi, a researcher in the Department of Chemistry and Chemical Biology at Harvard, and his colleagues decided they were up to the challenge.
Dr. Kishi considered the compound a good candidate to showcase the potential of a new carbon–carbon bond-forming reaction he and his team had discovered. Additionally, halichondrin B had demonstrated antitumor activity in mouse models of some human tumors.
By 1992, the Harvard team had completed total synthesis of the complex molecule. "By chance we found the right half of halichondrin B had the [antitumor] properties we were looking for, so I applied for a patent," Dr. Kishi said.
Dr. Kishi was working with chemists and biologists at Eisai to convert his synthesis into the molecule that would eventually become eribulin. In the process, they made over 200 derivatives. "Because of its unprecedentedly complex structure, there were internal and external concerns for Eisai to develop eribulin further," he explained.
Researchers from NCI-Frederick's NPB, meanwhile, were working independently with very small amounts of material from the sponges that produce halichondrin B to isolate the active compound, and the compound was subsequently approved for initial preclinical evaluation.
In 1998, NPB and Eisai combined efforts to evaluate Eisai's two best synthetic compounds and the purified natural halichondrin B compound to see which one performed the best in animal models.
"One of Eisai's compounds trumped the others, and so NCI and Eisai came up with a Cooperative Research and Development Agreement, and a phase I trial began in 2001," Dr. Newman recounted. The promising results of the trial led Eisai to begin full-scale clinical development.
November 2010, the FDA approved eribulin to treat late-stage metastatic breast cancer in patients who have completed two or more rounds of chemotherapy.
A Bright Future for Marine-Sourced Medicines
Dr. Newman and Dr. Ireland agree that, due to halichondrin B's structural complexity, no synthetic chemist would, "in their wildest imagination," ever have dreamed of creating the compound that inspired eribulin, or, for that matter, paclitaxel (which was originally developed from the Pacific Yew tree). Eribulin is now being tested for its potential use in the treatment of other solid tumors, including non-small cell lung cancer, prostate cancer, and bladder cancer.
However, eribulin is only the second cancer drug from a marine source that has been approved. The other is trabectedin (Yondelis), which was approved by the European Union in 2007 to treat soft tissue sarcomas. "The fact that these … drugs have come out within the past few years is huge," Dr. Ireland said. "The future looks good for marine organisms as a source.
"There is the realization that nature provides us with unique chemicals," Dr. Ireland said. "And now the tools are available to take those molecules that nature gives us and optimize those structures to make drugs to treat ailments that have been around for centuries."