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VERY month, Brian Bingham, a young American PhD student, packed his bags and
headed for the Florida Keys. There in the mangroves around a tiny sea-scoured
island were some of the finest colonies of sea squirts a marine biologist could
wish for. The colonies thrived on the dangling roots of the mangrove trees, and
were just perfect for studying the rise and fall of sea squirt populations.
Bingham carefully marked a number of roots with fluorescent pink tape so he
could find them on his next visit. And, each month, he photographed the roots,
recording any changes in their burdens of sea squirts.

Then, one day in the late 1980s, as Bingham pulled up in his boat, he found a
group of divers busily scooping sea squirts into bags. “We marked the roots
clearly . . . I thought,” says Bingham. “They said they hadn’t noticed. They got
back into their boat and we never heard any more.”

All of a sudden, Ecteinascidia turbinata, this unremarkable little
sea squirt, was in demand by the drug prospectors. It had produced what the
prospectors call a “hit”. Thanks to automation in the lab, chemists looking for
potentially useful compounds can screen extracts from animals and plants a
thousand at a time. And Ecteinascidia had come up trumps. This watery
sac of jelly proved to contain substances that can kill human cancer cells.

Now, under the direction of the Spanish company PharmaMar, one of those
chemicals—ecteinascidin-743—is in the first phase of clinical trials
in several European countries. At the moment, researchers are testing the
compound on terminally ill cancer patients to see how well people tolerate it.
It could be years before we know whether it really works, but already fears are
growing about the impact of the research on sea squirt populations. According to
one chemist in the field, divers have so far bagged about a tonne of sea squirts
from around the Florida Keys, and another two tonnes from the Caribbean. “When
we started the study we had no idea it would become the hot animal it did,” says
Bingham, now professor of environmental studies at Western Washington
University.

Nor is Ecteinascidia turbinata a lone target. Drugs companies are
turning to the sea in droves after years of scouring the rainforests for novel
chemicals. In recent years, prospectors have identified three other potential
stars in cancer research, a sea hare, a sponge and a bryozoan. In each case, the
animal produces a compound that has stayed the course through to clinical or
advanced pre-clinical trials for cancer. Most likely leads fall at the first or
second hurdles.

With new technologies enabling scientists to collect marine organisms from
ever more remote places and scrutinise the biological effects of the chemicals
they contain ever more closely, the number of “hits” is likely to increase in
future. Indeed other potential stars are already in the pipeline (see The
next big thing?). This will fuel concerns about over-exploitation, but
moves are afoot to find alternatives to the wild populations of such organisms.
Some researchers are designing ways of culturing medically valuable marine
organisms, opening the way to aquafarms for drugs. Others aim to produce the
chemicals artificially, using genetically engineered bacteria or cultures of
cells taken from the marine organisms. “People are starting to get these
substances into the clinic, so we need to think about their sustainable use
now,” says Amy Wright, a chemist at Harbor Branch Oceanographic Institution in
Fort Pierce, Florida.

In most cases demand will exceed what natural populations can provide. “The
ocean is an incredible resource for novel chemical substances,” says Bill
Fenical of the Scripps Institution of Oceanography in La Jolla, California. One
reason for this is that many invertebrate animals live fixed to a rock or a reef
and must defend themselves against both predators and rivals for a firm
foothold. In the main, they rely on potent poisons to put off their enemies.
Even those creatures that can swim around look like easy pickings for predators:
they are slow, soft bodied and often advertise their presence with spectacularly
bright colouring. Again, the reason why they are not snapped up immediately is
generally because they produce a toxic chemical.

But what a predator finds off-putting can be very attractive to the drugs
prospectors. Most of today’s anticancer drugs are toxic, working on the
principle that they will kill the fast-growing tumour cells before doing too
much damage to healthy cells. But drug researchers are always looking out for
compounds that kill cancer cells more specifically and so have fewer unpleasant
side effects. Researchers hope ecteinascidin-743 will prove to be one such
compound.

The problem is that most of the chemicals are present in minute quantities.
In the case of Ecteinascidia, it takes 1 tonne of animals to isolate 1
gram of ecteinascidin-743. And you probably need 5 grams to see you through
clinical trials, says Ken Rinehart of the University of Illinois at
Urbana-Champaign, who with Tom Holt isolated the compound in the 1980s.

If ecteinascidin-743 is eventually approved as a drug, will the wild
populations be able to satisfy demand? “Up to now Ecteinascidia has
been collected on a sustainable basis,” says Rinehart. “You can get three crops
a year from the mangrove swamps of the Caribbean. They do grow back.” Not
everyone shares his optimism. If collectors pick the sea squirts off carefully,
leaving the root-like “stolons” behind, then sea squirts will grow back and go
on to reproduce sexually as adults, says Craig Young, an expert on these animals
at Harbor Branch. But if collectors continue to cut off colony-bearing
mangrove roots or strip them clean, recovery is unlikely, he says.

What everyone wants is a renewable source of the chemicals. Leaving aside the
ethical and ecological problems of plundering the oceans, it would be unwise to
rely on populations that are at the mercy of nature. A population that is
thriving one year could be wiped out by predators or succumb to disease the
next. In many cases, too, the cost of collecting the raw material is
prohibitive, particularly if the animal comes from the deep sea. “People can’t
ride around in a submersible all the time collecting stuff,” says Wright.

Salmon and oysters

In an ideal world, chemists would simply make the drug in the laboratory. But
the unusual characteristics that make these chemicals so interesting also make
them difficult to manufacture. Chemists have discovered how to synthesise
dolassatin from the sea hare, but other marine compounds elude them. “If you
can’t synthesise it, you need to show drug companies how you could supply it,”
says Wright.

One strategy is to farm the animals like so many salmon or oysters.
Aquaculture has obvious advantages over harvesting natural populations. With
some species, collecting from the wild can be wasteful because not all
populations contain the target chemical. With aquaculture, growers can select
animals known to make the chemical, and even pick a particularly high-yielding
population as a starting point.

Aquaculture also provides opportunities to manipulate the animals to push up
yields. If, for instance, a sponge secretes a toxic chemical to ward off algae
that might smother it, bright lights that would encourage the growth of algae
should stimulate it to crank out more of the chemical.

In practice, though, simply raising the animals is proving a challenge. Most
marine creatures live in highly specialised microhabitats and are fussy about
the conditions they will grow in. “Trying to do invertebrate aquaculture is a
nightmare,” says Wright. Her colleague John Scarpa is trying to discover what
will induce the sea squirt to grow and reproduce in culture. Scarpa has reared
adults from larvae, but has not so far persuaded them to breed. In the meantime,
he is trying other tricks to encourage the animals to make more ecteinascidin.
“We are working on supplements to see if we can alter their chemistry to get a
better yield,” he says. “If you can persuade the animal to make 1 gram of drug
per kilogram of organisms instead of 1 microgram, it becomes more cost effective
to isolate the chemical.”

One future strategy might be to extract the chemicals not from the sea
squirts but from flatworms that eat them. The tiger worm, Pseudoceros
crozieri, concentrates ecteinascidins in its tissue. This would make
extraction of the chemical much easier than from the watery sea squirt.

While the aquaculturists have yet to solve the problems of rearing
Ecteinascidia, they have had more luck with two other species. Along the
coast of southern California, CalBioMarine Technologies, a seven-person company
in Carlsbad, is growing “fields” of Bugula neritina, a stringy, brown
bryozoan normally found fouling piers and piles. Bugula makes
bryostatin-1, a complex lactone currently in clinical trials in cancer patients
in the US.

CalBioMarine began by growing the bryozoans in tanks on shore, but is now
concentrating on “ranching” them in the sea, hugely reducing food bills for the
creatures. “It’s the sane way to go,” says Dominick Mendola, president of the
company. “In the sea, nature feeds the colonies.” The first step is to sow the
Bugula larvae like seeds onto perforated plastic plates. Once the
larvae are firmly attached, the plates are taken out to sea and fixed to trays
stacked from top to bottom of a submerged tower. The company harvested its first
crop this summer. After five months in the sea the bryozoans had grown as well
as natural colonies in the same area.

A second success story comes from across the Pacific. Around the coast of New
Zealand, researchers from the National Institute of Water and Atmospheric
Research are growing another “hit” animal, a sponge that produces halichondrin
B, an anticancer agent currently in pre-clinical trials in the US. The sponge, a
newly discovered species of Lissodendoryx, grows on rocky outcrops at a
depth of between 100 and 300 metres on a small section of reef on the edge of
the Kaikoura Canyon, South Island, New Zealand. “We estimate that there is only
about 300 tonnes in existence,” says Chris Battershill, leader of the group.

Sponges are notoriously tricky to grow, and each species is exacting about
conditions. Battershill and his team have tried rearing Lissodendoryx
at various depths and on several types of structure around the coast of New
Zealand, looking for a combination that produces the best yield of halichondrin.
Get it right, says Battershill, and you can grow the sponges in quite artificial
conditions. Lissodendoryx, for example, comes from deep water in the
open sea but is now growing quite happily in Wellington Harbour at a depth of
just three metres. “We are now looking at techniques for enhancing biosynthesis
of halichondrin immediately before harvest,” says Battershill.

Nasty habit

However successful sea ranching is, whole animals have drawbacks as chemical
factories. They don’t always do exactly what you want. They have a nasty habit
of contracting diseases or refusing to breed. And there is a lot of wastage.
Culturing only those cells that make the drug would help here.

At Florida’s Harbor Branch, Shirley Pomponi of the biomedical marine research
division is trying to identify which sponge or sea-squirt cells make the
interesting chemicals and then culture them. Just like the whole animals, cells
are choosy about growing conditions. And in general, invertebrate cells are even
harder to culture than cells from mammals. Nevertheless, with the help of
various growth factors and chemicals, Pomponi’s team has persuaded sponge cells
to divide several times and produce the desired chemicals. Mysteriously, these
primitive invertebrates even seem to respond to growth factors and hormones from
mammals. Nobody knows why, but sponge cells will, for example, respond to
insulin in much the same way as mammalian cells, synthesising the proteins and
DNA that are needed for cell division.

Persuading sponge cells to carry on dividing is proving difficult, however.
“After a certain number of divisions they stop,” says Pomponi. “And they only
produce the chemicals you want when they are active.” Once she has beaten this
problem, Pomponi hopes to culture cells from other marine species which might
produce chemicals new to science.

In some cases, the animals may be redundant altogether. Although research is
at an early stage, it is becoming clear that some of the interesting chemicals
found in extracts from marine invertebrates are actually made by bacteria that
live in or on the animals. Marine bacteria are quite different from their
terrestrial counterparts. Many contain bromine or other halogens which are not
available to land organisms. These allow for some creative chemistry and explain
why the marine microorganisms produce so many novel molecules with structures
unlike any seen before.

But marine bacteria are even less cooperative than their invertebrate hosts.
Most have proved impossible to culture, making the job of screening them for
interesting chemicals a tough proposition. “We’ve had to start from scratch in
learning how to grow them,” says Fenical. “The concepts of how you grow bacteria
are all based on work with medical microbes.”

One approach, which the team at
Harbor Branch is exploring with One Cell Systems, a company based in
Massachusetts, is to encapsulate individual bacteria inside tiny spheres made of
sugary polymers, mimicking their natural conditions. With the bacterium locked
inside its microsphere, it is possible to manipulate its environment to see if
it secretes anything interesting, says Pat McGrath, of One Cell.

If it proves impossible to culture the bacteria there are other options. At
ChromaXome, a small biotechnology company in San Diego, researchers have snipped
out the biosynthetic pathways of marine microorganisms and spliced them into
easy-to-grow bacteria such as Escherichia coli and
Streptomyces or a fungus such as Aspergillus. “Effectively you
capture the genetic pathway of the chemical,” says Michael Dickman, president of
the company. The host organisms then do the work of manufacturing the chemical.
At this stage, the company is using its techniques mainly to speed up the
screening process. ChromaXome’s genetic engineers take the DNA from hundreds of
marine microorganisms, stitch them into a range of new hosts, and screen the
chemicals they produce.

ChromaXome is also using what it calls “combinatorial biology”, to mix and
match genes from different species of marine microorganism. The enzymes produced
by those genes never normally meet in nature, but here are thrown together
inside an E.coli cell. “This provides the opportunity to find truly
unique chemistry,” says Dickman.

The drawback with most of these strategies is that they lag a long way behind
the drug prospectors. While work such as ChromaXome’s is in demand by drugs
companies looking for a faster way to screen microorganisms, few companies are
prepared to invest in studies of invertebrate animals—at least until they
can be sure they have a winning product. If bryostatin-1 makes it onto the drug
market it could be worth up to $1 billion a year. But the “if” is a big
one. A prospective drug might reach the final phase of clinical trials and then
side effects start to turn up. Or it might be a perfectly good drug but offer no
advantage over existing therapies.

If the companies are unwilling to pay, then most researchers think the job
falls to government agencies such as the National Institutes of Health. The
National Cancer Institute and the US Department of Agriculture have funded a few
small studies once a chemical shows real promise. In the meantime, who knows
what damage the massive collections of an animal like Ecteinascidia
have done? “We know very little about the populations that are being heavily
exploited,” says Bingham.

The risk of course, is that the drug lives up to
its promise before chemists have cracked its synthesis or aquaculturists have
learnt to grow it. “What if ecteinascidins do make it?” asks Wright. “I’d be the
first to want to use it if I had cancer.” If the animals have gone, that might
not be an option.

* * *

The next big thing?

IT STARTED out as a promising new immune suppressant. Now it is being touted
as an anticancer drug that could be better even than taxol. Discodermalide is a
polyhydroxylated lactone isolated from Discodermia dissoluta, a sponge
that grows at a depth of around 200 metres in the Caribbean.

Early experiments in the 1990s showed that discodermalide was very efficient
at stopping the proliferation of white blood cells, halting the body’s attack on
transplanted organs or bone marrow. But when a more discriminating compound
isolated from a fungus came on the scene, interest in discodermalide waned.

At Harbor Branch Oceanographic Institution in Fort Pierce, Florida, Ross
Longley and his colleagues kept their interest in discodermalide. They wanted to
discover how it stopped white blood cells from proliferating. By 1993, they had
found that the compound was not killing the cells but stopping them in their
tracks as they were about to divide.

With Billy Day at the University of Pittsburgh and Ernie Hamel at the
National Cancer Institute near Washington DC, Longley went on to show that if
you treat breast cancer cells with discodermalide, it also halts them at a
specific point in the normal cycle of cell division, after the nucleus has
divided but before the cytoplasm splits to form separate cells. The chemical
interferes with the process that softens up the cytoplasm ready for
division.

Just before a cell splits it must dismantle its cytoskeleton, a structure of
microscopic tubules made mainly of the protein tubulin. These tubules are broken
down before division and then reform in the newly created cells. But in the
presence of discodermalide, instead of performing the normal “meltdown” trick,
Hamel and Day found the tubules rearranged themselves into large, unbending
bundles. Under the microscope the change in the cells was quite spectacular,
with star-like accumulations of tubules where no structure should be visible at
all. With the cell skeleton frozen in time, the cells were stuck at this point
of the cycle, and died within 24 hours.

“This is the exact mechanism by which taxol works,” says Longley. Taxol, a
compound originally extracted from the bark of the Pacific yew tree, is now an
approved treatment for breast cancer. Only two other compounds-—isolated
from slime moulds—are known to perform the trick of halting cell division
by preventing the breakdown of the cytoskeleton.

Pre-clinical tests show that discodermalide is at least as potent as taxol
against breast cancer cells and possibly more potent against lung cancer cells.
But, says an excited Longley, it is eighty times as potent against leukaemia
cells. And the good news for the sponge is that when the compound was discovered
a decade ago, chemists at once began working on ways to make it in the lab.
There are now at least three methods in the works.