Isolating and purifying new
therapeutic agents from plants and marine sources is
essential to the continued success of the
pharmaceutical industry. One out of four
prescription drugs sold in the United States
contains active ingredients extracted or derived
from plants. 50% of the top selling drug products
are derived from natural products. More than 40
different plant species are used, and yet less than
1% of the world’s flora has been screened for
biochemical activity, with even less of the marine
species explored.
In the pharmaceutical industry
the value of an organism, whether of plant or marine
origin lies in its potential to contain a unique
biologically active compound that can be fully
synthesized in the laboratory. Signs of biochemical
activity in a natural product extract must be
followed by a long period of testing to isolate and
characterize the active compound. The dominant
strategy in the pharmaceutical industry is to
maximize the number of diverse compounds screened in
order to maximize the probability of discovering
novel compounds for drug development. Chemical
libraries, which are collections of chemical
structures that have either been synthesized in the
laboratory or isolated from natural sources are
assembled for creating new lead drug compounds.
Natural products chemistry encompasses isolation of
a desired compound, use of purification technologies
to get as pure a product as possible from the
initial sample and subsequent analytical and
structural analysis of the compound using
spectroscopic and nuclear magnetic resonance
techniques. The key role for the separation
scientist is to isolate novel structures from
natural products which can then serve as blueprints,
or starting material for the biochemical synthesis
of new drugs. Bioactive natural products serve as
lead structures which are then optimized through
classical medicinal chemistry techniques or
combinatorial synthesis methods to yield new drug
compounds having either superior activity or less
toxicity.
Drug discovery offers a bright
future for marine biotechnology research.
Biochemicals produced by marine invertebrates, algae
and bacteria are different than those from
terrestrial organisms and offers the potential for
new classes of medicines. Many of the marine derived
natural products which are being isolated exhibit
exceptional levels of biological activity, combined
with unique modes of action. The oceans cover more
than 70% of the earth’s surface and in places are
more than 5 miles deep. There are over 200,000
invertebrate and algal species, with nearly 150,000
species of algae (sea weed) alone. Other types of
marine organisms include sponges (Porifera)
cnidarians or coelenterates (corals, octocorals
(including sea fans), hydroids and sea anemones),
nemerteans (worms), ascidians (including sea
squirts), mollusks (sea snails and sea slugs) and
echinoderms (brittlestars, sea urchins, starfish
ands sea cucumbers). Sponges are very simple animals
that live permanently attached to a location in the
water and they are sessile, being permanently
attached to an underlying substrate and unable to
move on their own. Presently, more than 35% of
useful medical compounds from the sea are isolated
from sponges. Important compounds for drug discovery
come from soft bodied or slow moving marine
creatures which do not have protective structures
such as spines or protective shells and which
produce toxic natural products as their means to
fight off potential predators. The harsh marine
environment which they inhabit, together with their
lack of physical defenses has required these
organisms to develop chemical deterrents in order to
survive.
In addition to new medicines,
other uses for marine derived compounds include
cosmetics (from algae, crustacean and sea fan
compounds), nutritional supplements (algae and fish
compounds), artificial bone (corals), and industrial
compounds (including fluorescent compounds from
jellyfish, novel glues from mussels, and heat
resistant enzymes from deep sea bacteria).
Marine organisms have been used
for the development of pharmaceutical compounds for
more than 50 years. Natural products tend to have
higher molecular weight than synthetic counterparts,
contain more rings and are sterically complex. This
makes synthesis of these compounds challenging to
medicinal chemists who attempt to synthesize the
active agents. The first drugs were developed for
the treatment of viral infections and cancer. The
Caribbean sponge Cryptotethya crypta yielded
arabinose nucleosides which were the lead compounds
for the synthesis of analogs ara A (vidarabin) and
ara C (cystosine arabinoside). The fungus
Cephalospirium was isolated from sea water collected
from Cagliari, Italy which led to the isolation of
cephalosporins, which remain a most useful class of
antibiotic agents. Large quantities of
prostaglandins found in the gorgonian Plexaura
homomalla has become the take off point for a
systemic investigation of marine environments as
sources of novel biologically active compounds.
New drug agents are being
isolated from marine organisms and finding their way
into clinical practice. Ziconotide (Prialt) is a
novel non-opioid, non local anesthetic, developed
for the treatment of severe chronic pain. Ziconotide
is the synthetic form of a 25 amino acid peptide
isolated from the venum of the marine snail Conus
magus. The drug works through a unique mechanism of
action, in that it binds to N type calcium channels
in the spinal cord and blocks the ability to
transmit pain signals to the brain. Its action is
not blocked by opioid antagonists. In December 2004
the Food and Drug Administration approved ziconotide
for use as an infusion into the cerebrospinal fluid
using an intrathecal pump system. Trabectedin is an
anti-tumor drug which was isolated from an extract
from the sea squirt Ecteinascidia turbinata.. Sea
squirts are so named as many species expel streams
of water through a siphon. It is sold by Johnson and
Johnson and Zeltia Yondelis and is currently
approved for use in Europe, Russia and South Korea
for the treatment of advanced soft tissue sarcoma..
It is also undergoing clinical trials for the
treatment of breast, prostate, and pediatric m.
The European Commission and the
US Food & Drug Administration have granted orphan
drug status to trabectedin for soft tissue sarcomas
and ovarian cancer. The mechanism of action for the
drug is believed to involve the production of
superoxide near the DNA strand, resulting in DNA
backbone cleavage and cell apoptosis. It is being
used for patients who have failed treatment with
anthracycline drugs and ifosphamide, the most
commonly used agents for the treatment of sarcomas.
Many compounds isolated from the
marine environment display anti-cancer activity.
Some appear to work as microtubule stabilizing
agents, disrupting the formation and maintenance of
microtubules in cells thus suppressing cell
division. Much interest has been generated in
examining the marine environment for such compounds
because of the clinical success in the isolation of
taxol from the plant species Taxus from the Pacific
Yew tree. Taxol is the most powerful drug in the
treatment of breast cancer, has significant activity
in the treatment of ovarian tumors as well as having
significant activity in the treatment of other
squamous cancers and adenocarcinomas. Furthermore,
it is used as an agent coating vascular stents to
inhibit the regrowth of endothelial cells and is
coated onto stents inserted during angioplasty
procedures.
The marine natural product
laulimalide has a similar mechanism of action as
that of taxol. Although laulimalide is only 1/5th as
potent as taxol in drug sensitivity laboratory cell
lines, it is as much as 100x more potent than taxol
in multi drug resistant cell lines. This compound
was isolated from the Okinowan Ocean sponge
Cacospongia mycofijensis and has proven effective in
the treatment of breast and ovarian cancers which
become resistant to taxol. Furthermore, the drug has
now been completely synthetically synthesized.
Multiple analogs of laulimalide have been isolated
which initiate an increased density of interphase
microtubules, aberrant mitotic spindles, and
ultimately apoptosis or programmed cell death, which
is the method of destruction of cells. Taxol and
laulimalide bind at different sites on tubulin
polymer and appear to have synergistic cytotoxic
activity.
There are difficulties working
with marine organisms as a natural source for drug
development. Marine invertebrates cannot be easily
cultured, cannot be collected on a large scale and
compounds from marine compounds often cannot be
synthesized. Preclinical and clinical developments
are often hampered by the limited supply from the
natural source. To that end, the goal of medicinal
chemists is total synthesis of materials derived
from the sea. Total synthesis allows for the ready
production of synthetic analogs and for the
elucidation of structure activity relationships and
the design of more active or less toxic molecules.
Analog design for synthetic compounds is based upon
the assumption that only certain structural features
are involved in discrete interactions with the
biological target.
Marine sampling starts with
freeze drying of the material until taken back to
the laboratory. The samples are frozen at – 20 C
until they are ready to be analyzed. Next the sample
extract is fractionated by liquid-liquid
partitioning followed by the separation and
isolation of the individual components using
chromatographic separation techniques including thin
layer chromatography (TLC), vacuum liquid
chromatography, column chromatography and
preparative high performance reversed phase liquid
chromatography. Isolation of bioactive secondary
metabolites is usually monitored by bioactive and
cytotoxicity assays. Structural elucidation of
desired active compounds is studied using
spectroscopic techniques especially 2D nuclear
magnetic resonance and mass spectrometry.
Extracts from marine organisms
may possess antibacterial, antifungal and cytotoxic
activities. Bioassays rely on determining the
biological activity of crude extracts for numerous
target specific assays such as enzyme assays and
receptor binding assays. Antimicrobial activity is
analyzed by testing the crude extracts against the
standard strains of gram positive Bacillus subtilis,
gram negative Escherichia coli, the yeast
Saccaromyces cerevisiae and the fungal strains
Cladosporium herarum and Cladosporium cucmerinum.
Protein kinase screening assays
are also performed to determine inhibition of the
serine/threonine kinases, which control the
transmission between the successive stages of the
cell cycle. These are high volume, automated
screening processes which may allow thousands of
samples to be studied per week in larger
pharmaceutical houses.
Complimenting these bioassays
the separation techniques of TLC, column
chromatography, UV and MS are used to isolate the
chemically most interesting substances. In general,
the more hydrophilic metabolites may best be
isolated using ion exchange chromatography, reversed
phase silica gel chromatography or size exclusion
chromatography on polysaccharide resins. The more
lipophilic metabolites can be further purified by
chromatography on normal phase silica gel, florisil,
alumina or lipophilic size exclusion resins such as
Sephadex LH-20. Due to its amphoteric properties
alumina appears to be the superior method for clean
up purification. Further separation techniques are
beyond the scope of this paper. However the benefits
of using alumina for column chromatographic analysis
of marine organisms appears to parallel the benefits
of its use in the isolation and purification of
plant alkaloids.
