The objective of this Workshop was to identify and discuss the nature of current activities, including sustainability, limits to growth, timelines, regulatory requirements and potential environmental consequences. The programme and participants are listed in FAO (2005) together with a more comprehensive meeting summary.
The Workshop addressed a number of important pre-identified questions.
Biological Research & Technology:
Law & Policy:
In this context the workshop examined existing policies and their use as exemplars for others.
There were divergent interpretations of the terms “bioprospecting” and “high seas” and it found important to define how the terms were used. It was noted that there is already considerable bio-discovery in high seas areas with the potential to expand and yield valuable products. However, marine research is expensive and technological challenges limit the industry. Except in general terms, high-seas bio-discovery and bioprospecting are unregulated with no clear legal regime for management, benefit sharing (who “owns” the resources?) and access.
There appeared to be no evidence that bio-discovery and bioprospecting have greater impact on the marine environment than any other marine scientific research and fishing and mining appear to have a greater effect. The need for regionally and globally consistent approaches to access and benefits, sustainable sample collection and environmental impact assessment were identified. It was concluded that the high seas are a global commons and its biodiversity could be considered a “common heritage of mankind”.
Antartic Climate & Ecosystems Cooperative Research Centre
Institute of Antartic & Southern Ocean Studies, University of Tasmania
Private Bag 77
Hobart 7001, Tasmania, Australia
A group of experts met at the University of Otago, Dunedin, New Zealand, 27–29November 2003 to discuss bioprospecting in the high seas. The objective of the meeting was to identify and discuss the nature of current activities, including sustainability, limits to growth, timelines, regulatory requirements and potential environmental consequences.
1. OVERVIEW OF PRESENTATIONS
Research scientists working on sponges, microorganisms and fish gave an overview of their experiences with regard to sample collection, laboratory investigation, findings and knowledge of the bioprospecting industry.
The group heard that the oceans are the largest ecosystems on earth with immense biodiversity already known and thousands of new species being discovered as marine scientific research intensifies. Novel marine biodiversity is concentrated most specifically in four areas or hot spots: coral and temperate reefs, seamounts, hydrothermal vents and abyssal slopes and plains. These concentrations of biodiversity are largely untouched, despite being highly sought after by scientists, governments and companies that have speculated about the immeasurable pharmaceutical potential of novel structures. However, each of the hot spots also has idiosyncrasies that make them particularly vulnerable to other ocean uses such as trawl fishing.
Case studies of work in progress highlighted the nature of some current activities. A compound, IPL576,092, based on the sponge steroid contignasterol completed US Phase I trials as an asthma drug in 2000. Cytotoxins from deep-water sponges found on the Chatham Rise 400 km off the New Zealand coast are also under investigation. Other work in progress involves the Conus venoms (the source of the first of the modern marine-based drugs and cytotoxic organic extracts); cold adapted enzymes from deep-sea microbial extremophiles in the Southern Ocean and deep-sea extreme environments such as hydrothermal vents; and genes for “anti-freeze” proteins from fish (Southern Ocean). The difficulties with assay and the long time frame of investigation of potential leads were explained. In the case of fish proteins, for example, it was noted that the proteins could be replicated from genetically modified organisms and did not require the direct harvesting of fish. In a similar fashion, most bacteria can be cultured. Sponges have historically been harvested, but it is also possible to culture them under certain conditions in a natural environment.
Potential applications from marine-sourced material include:
A study of small-molecule new chemicals introduced globally as drugs between 1981–2002 showed that 61 per cent can be traced to, or were inspired by, natural products (Newman, Cragg and Snader 2003). This figure rose to 80 per cent in the year 2002–2003. Compounds from natural products are considered to be more agreeable to consumers and two-thirds of the anti-cancer drugs, for example, are derived from both terrestrial and marine natural products. Marine-sourced material (e.g. from sea water and marine sediments) has a higher chance of a successful commercial ‘hit’ because of its mega-diversity (using the assumption: samples x biodiversity x assays = probability of a hit).
The USA National Cancer Institute (NCI) was one of the first organisations to begin systematic large scale collection of marine invertebrates and in the mid-1980s formal collection programmes were initiated to protect access to the original material (Newman, Cragg and Snader 2003). The cost of sample collection, laboratory investigation and further downstream processing is high, and there is only an estimated 1:50 chance of successfully producing a marketable product beyond a pre-clinical lead. For example, one kilogram of shallow water marine invertebrate collected, prepared for sampling, identified and transported costs approximately US$1000 a sample. From the one-kilogram sample, only approximately 20–50 g of liquid and 4–15g of organic material will be extracted, costing approximately US$200 a sample. Subsequent testing (in the 60 cell line screen, for example) may cost as much as US$300 a sample. If all associated costs (laboratory staff and equipment) are included, the total rises to tens of thousands of dollars a sample. However, only about 10 per cent of samples are eventually determined to be 'active'. These figures refer to shallow water collections (Newman, Cragg and Snader 2003).
Sampling from shallow water is economically more viable than from the deep-sea, from which specimens may be difficult to retrieve. Early NCI collection programmes used submersibles and remote operated vehicles (ROV), but the cost was too high and their deep-sea programme was suspended. Others have had more success. Harbour Branch Oceanographic Institution uses manned submersibles and has successfully synthesized a molecule, discodermolide, from a previously undescribed deep-sea sponge. Another compound, halichondrin B, has also been isolated from a sponge species by a New Zealand joint venture. In the latter case, one tonne of sponge was harvested that yielded 300 mg of pure halichondrin B. This process cost approximately US$500 000 (Newman, Cragg and Snader 2003). The scheme shown in Figure 1 represents the NCI approach to the processes of biological prospecting.
It is important to note that current US legislation prohibits government institutions from “encumbering a future invention” (Newman, Cragg and Snader 2003) therefore in terms of sharing benefits, they are prohibited from entering into royalty agreements in the phases of sample collection and testing. This may bring the government institutions into contravention of the Convention on Biological Diversity (CBD) if the US Government ratifies it. NCI approaches benefit sharing in a novel way, it began prior to the CBD, but in many ways in conformity with the principles contained therein. It involves a 'letter of collection' agreement, which requires absolutely that any licencee of an NCI patent must involve the country of origin in the further development of the compound (Newman, Cragg and Snader 2003). Despite the argument that the acts of collection and routine testing of extracts are not inventions in themselves, institutions such as NCI cannot infringe US law by collecting in some countries where the CBD (and its royalty provisions) would apply. Another significant point is the fact that no sample collected by an NCI collector may be analysed by other researchers.
NCI schema of bioprospecting process (from Newman and Crag 2005)
Ongoing access to material (i.e. because it cannot be replicated in a laboratory or because further samples are sought) is of primary importance. Aquaculture and mariculture have both been used successfully in some cases (e.g. shallow-water sponges).
Industry presentations at the Workshop placed great emphasis on the odds of success, with a figure of approximately only one to two percent of preclinical candidates actually becoming commercially produced.
1.3 Law and policy
Presentations were made giving an overview of international law and international obligations, protecting the biodiversity of hydrothermal vents and the unique situation in the Southern Ocean. Relevant laws include intellectual property laws, environmental protection and biodiversity conservation laws, the United Nations Convention on the Law of the Sea (LOSC) and Antarctic-specific laws.
It was noted regarding patentable inventions (i.e. products and processes that provide a technical solution to a technical problem) that patenting involves elements of novelty, an inventive step and industrial applicability (or utility, i.e. it can be commercialized). The patenting of living organisms and products of nature is a poorly defined area in the law. While products of nature are currently excluded, even minor modification that introduces the elements noted above may allow patenting to proceed. Patentable biotechnological inventions may include genetically modified plants, animals, and microorganisms, and isolated, synthetically produced, cells, proteins and genes of known function. Important points for discussion were the potential for conflict between sovereign rights over resources and patent rights over inventions; bioprospecting and biopiracy, traditional knowledge and novelty (e.g.does traditional knowledge compromise the element of novelty?); and equitable access and benefit sharing (consistent with the Convention on Biological Diversity but see earlier comments regarding US legislation).
The applicable legal regime, if any, to monitor activities and provide protection and regulation of hydrothermal vents will depend on their location. If the vents are located within territorial waters and exclusive economic zones (EEZs), coastal state jurisdiction prevails over access to, and use of, genetic resources. If they are located on the continental shelf beyond the EEZ, the coastal state can only regulate access to sedentary species. If hydrothermal vents are located outside national jurisdiction, access is largely open and unregulated except where states regulate the activities of their nationals, consistent with the Convention on Biological Diversity and other international law (Leary 2005). Discussion ranged across broad areas of potential international regulation, including expanding the mandate of the International Seabed Authority to include the superjacent waters above the area.
It was acknowledged that the Southern Ocean is a special case because of the overlap of international law and Antarctic-specific law as well as the unproven nature of sovereignty over the continent and, thus, the marine areas. This complex case highlights how the traditional freedoms of the sea have been modified in the Antarctic. A regional fishery body - the Commission for the Conservation of Antarctic Marine Living Resources - regulates the conservation and rational use of all Antarctic marine living resources (but excluding whales and seals). Furthermore, an environmental protocol - the Madrid Protocol to the Antarctic Treaty - requires environmental evaluation of all activities in the Antarctic Treaty Area (i.e. south of 60° South) prior to the operation being undertaken. Activities in this case include marine scientific research. The initial phase of bioprospecting (sample collection) would be unlikely to breach either of these arrangements, but large-scale collection through harvesting would require closer scrutiny.
Consideration was also given to the Southern Ocean as a global commons and its resources, the “common heritage of mankind”, not unlike the situation with the deep-sea bed of the high seas.
1.4 Case studies
The first case study described the extent of some of the leads discovered by one institution - the Australian Institute of Marine Science (AIMS). The collection housed by AIMS includes 10000species of marine bacteria, fungi and microalgae and 12 000 species of invertebrate macroorganisms. The presentation also introduced new terminology and a new concept: a dichotomy between biodiscovery (primary collection to find leads) and bioprospecting (looking for more of the lead material - re-collection).
Biodiscovery was considered to have the following practical applications in addition to the ones listed above: seafood toxin testing, antifoulants, bioremediation, environmental monitoring and as research tools. The latter is a lucrative application with some marine natural products valued extremely highly, e.g. Neosaxitoxin derived inter alia from dinoflagellates, blue-green algae and toxic shellfish is valued at US$21400 per milligram. In terms of supply, however, AIMS (citing Garson 1994) noted the following quantities of original material required to yield relative quantities of lead material.
|Original material||Quantity yielded|
|450 kg acorn worms||1 mg cephalostatin|
|1 600 kg sea hares||10 mg dolastatin|
|2 400 kg sponge||<1 mg spongistatin|
|847 kg moray eel livers||.35 mg ciguatoxin|
This table highlights the importance of sustainable methods of wild harvest, chemical synthesis, aquaculture, cell and tissue culture and genetic splicing.
The AIMS presentation also considered Australian policy. Prior to 1994 the AIMS collections were undertaken in conformity with a scientific research permit and no benefit sharing was applied. Subsequent collections were subject to new permit conditions, which meant that new permits became more difficult to obtain in some jurisdictions and doubt was cast over the legal certainty of some existing collections. In addition, permit conditions restricted use. Benefit sharing also became difficult with questions arising about a lack of process and legislative basis, who should be beneficiaries and what exactly are the benefits? As a result, AIMS put in place best practice guidelines on these issues. In addition, the Queensland government (the Australian state in which AIMS is located) is introducing a Biodiscovery Bill into Parliament, which will provide greater clarity as to the legal obligations in this area.
The presentation on environmental aspects of bioprospecting acknowledged that many agencies expect environmental impact to occur with bioprospecting activities because historically, extracting resources from the oceans (especially fishing) has had environmental consequences. Conversely, the proponents would be inclined to see bioprospecting as posing no, or only slight, risk to the environment. It is likely that the proponents see their activities this way, e.g. because they are comparing their level of activity with hyper-extractive fishing. It was considered, however, that this generation of bioprospectors represent only the artisanal stage of the activity. All human activities related to ocean usage have impact. Those relating to bioprospecting will be relative to the location; the modes of transport, support and sample retrieval; the discard of unwanted material; and the nature of the target (i.e. compare microorganisms with fish). It was noted that the presumption that extraction of target taxa will have a negligible impact is only a presumption.
There are considerable legal obligations arising from, inter alia, the LOSC and CBD for the protection and preservation of the marine environment, including the conducting of environmental evaluation of proposed activities. The final message from the Workshop was that the juridical situation is complex and is still evolving.
2. CRITICAL POINTS AND CONCLUSIONS EMERGING FROM GENERAL DISCUSSION
It became apparent from the outset that there were divergent interpretations of the critical words “bioprospecting” and “high seas” and, therefore, it was important to define the way in which the terms were used throughout the meeting.
2.2 Level of activity and future potential
There is already a considerable amount of marine scientific research conducted in high-seas areas, including biodiscovery, and this has the potential to expand into more substantial bioprospecting activities in the future. Biodiscovery activity can be both targeted (e.g. at locations such as hydrothermal vents and seamounts, or at events such as the death and decay of marine mammals) and the serendipitous (e.g. curiosity-driven marine scientific research, bycatch, etc.).
The rich biological diversity of the high seas has the potential to yield biological products of broad ranging applicability. In particular there are unique mega-diverse areas where the biodiversity is relatively untouched. Significantly, the ratio of potentially pharmaceutically useful compounds to compounds screened is higher in marine-sourced materials. There is, therefore, a higher probability of commercial success. However, marine research is expensive and the high cost together with difficult technological challenges of retrieving material from the deep ocean impose significant limitations on the industry.
Spin-offs include the dedicated technology that is required to assist in biodiscovery. It is important to note that technology developed from high-seas experiences has much wider application. Bioresource spin offs include:
2.4 Legal status
Except in the general terms prescribed in the LOSC and the CBD, biodiscovery and bioprospecting in the high seas are largely unregulated. Specifically there is no clear legal regime for:
Patenting is the main avenue for securing economic benefit as a return for investment. But there is a dividing line between biodiscovery, bioprospecting and the requirement to share benefits from commercialisation.
2.5 Environmental vulnerability
There is at present no evidence that biodiscovery and bioprospecting are having any greater impact on the marine environment than any other form of marine scientific research. Currently there are greater threats to high-seas biodiversity from other activities such as various technical aspects of fishing and mining. However, a precautionary approach was agreed as necessary.
3. CONCLUDING REMARKS
In conclusion, three ways forward were advanced.
The high seas is a global commons and it was considered that its biodiversity could, therefore, be considered “common heritage of mankind” in similar fashion to the mineral resources of the deep-sea bed.
This work was supported by the Australian Government's Cooperative Research Centres Programme through the Antarctic Climate and Ecosystems Cooperative Research Centre (ACE CRC) and the University of Tasmania's Institute of Antarctic and Southern Ocean Studies, Centre for Law and Genetics and Tasmanian Institute of Agricultural Research.
5. LITERATURE CITED
Leary, D. 2005. Bioprospecting and the genetic resources of hydrothermal vents on the high seas: What is the existing legal position? Where are we heading and what are our options? In Shotton,R. (Ed) 2005. Deep Sea 2003: Conference on the Governance and Management of Deep-sea Fisheries, Conference Poster Papers (1–5 December 2003, Queenstown) and Workshop Papers (27–29 November 2003, Dunedin), New Zealand. FAO Fish. Proc. 3/2. FAO, Rome. pp. 455–487.
Newman, D.J., G.M. Cragg and K.M. Snader 2003. Natural Products as Sources of New Drugs over the Period 1981–2002. J. Nat. Prod. (Review) 66(7):1022–1037.
Newman, D.J. and G.M. Cragg 2004. Political, Legal, Scientific and Financial Aspects of Marine Biodiscovery Programmes. In Shotton, R. (Ed) 2005. Deep Sea 2003: Conference on the Governance and Management of Deep-sea Fisheries, Conference Poster Papers (1–5 December 2003, Queenstown) and Workshop Papers (27–29 November, 2003 Dunedin), New Zealand. FAO Fish. Proc. 3/2. FAO, Rome. pp. 440–447.
BIODIVERSITY, BIOPROSPECTING AND THE HIGH SEAS
J. Blunt, A. Cole and M. Munro
University of Canterbury
Private Bag 4800
Christchurch, New Zealand
Loosely defined biodiversity is “the variety of species and ecosystems in any given area” and is a measure of the abundance of life at that site. When biodiversity is coupled with research that is looking for a useful application or product from nature the term bioprospecting is often used. Typically, bioprospecting is a search for useful organic compounds from microorganisms, fungi and terrestrial and marine macroorganisms. Bioprospecting is nothing new.In fact, people have been bioprospecting since the dawn of civilization.Much of the bioprospecting that is taking place in the marine environment is focused on discovery of pharmaceuticals. But, is this the best way to discover new pharmacueuticals? Three methods are used: the traditional approach based on natural products (bioprospecting), the empirical approach based on rational design and more recently the molecular approach based on the better understanding now possible of the molecular target. Each approach has its strengths and weaknesses and each has found favour from time to time.
The role and the strengths of the biodiversity approach to drug discovery will be addressed and illustrated with relevant examples including current work on drug discovery from Antarctic organisms.
MICROBIAL BIOPROSPECTING IN THE HIGH SEAS THE PROSPECTS FROM HOT TO COLD
K. Sanderson and D.S. Nichols
Centre for Food Safety and Quality, University of Tasmania
Private Bag 54
Hobart 7001 Tasmania, Australia
Microbial bioprospecting may be considered as the search for valuable chemical compounds and genetic material from microorganisms. In the High Seas, those areas falling outside zones of exclusive economic interest, microbial communities exist in both the free oceanic environment, the deep ocean and sediment. This presentation will address the case of bioprospecting from both bacterial and archaeal communities (prokaryotes) from deep-sea ecosystems. In global oceanic waters below 200m, prokaryotic abundance is estimated at 6.5 × 1028 cells. The large population size of prokaryotes may imply that events considered as relatively rare (e.g. genetic mutations) could occur relatively frequently. Hence marine prokaryotes have a large potential to accumulate unique genetic and metabolic diversity. Two specific examples where this appears to be the case are: 1. abyssal sediments which harbour bacterial populations highly adapted to low temperature and high pressure 2. hydrothermal vents which harbour prokaryotic populations adapted to high temperature and unique energy metabolism. Examples of bioprospecting from these habitats will be discussed and potential biotechnological outcomes highlighted.
SOUTHERN OCEAN BIOPROSPECTING
Julia Jabour Green
Institute of Antarctic & Southern Ocean Studies
Antarctic Climate & Ecosystems CRC
University of Tasmania
Private Bag 77
Hobart 7001, Tasmania Australia
The extreme Antarctic environment has led to the evolution of a range of novel physiological adaptations in the local biological species, drawing bioprospectors to the region. Indigenous microorganisms, bacteria, krill and fish are all seen as potentially rich sources of raw material for the pharmaceutical industry, for example. However, the Antarctic is not a “normal” place. Nobody “owns” the continent, although seven countries, including Australia and New Zealand, claim parts of it. Nevertheless, the whole of the Southern Ocean is considered high seas. The “government” consists of a group of 45 countries that have ratified the Antarctic Treaty. There is no indigenous population and therefore no indigenous knowledge or folk-lore to protect. This situation raises questions of a very different kind; notwithstanding, Antarctica and the Southern Ocean are possibly more interesting than they are contentious, at this stage in the development of bioprospecting industries in the region. However, it is likely that the Treaty Parties will want to address the possibility that in the future the bioprospecting industries will pay more serious attention to the living resources of the region than they do now. Because the Parties already regulate all marine living resources activity, they may also decide to regulate bioprospecting.
PLUMBING THE DEPTHS OF MARINE BIODISCOVERY
C. Battershill and E. Evans-Illidge
Australian Institute of Marine Science
PMB 3, Townsville MC 4810
Can we have it all: drugs and other products from the deep sea, royalties, ownership, international investment, sustainability, and conservation outcomes? The answer is an emphatic Yes! if we heed some of the legislative and policy progress made in recent times associated with biodiscovery. Advances in areas such as access and benefit sharing policy linked to state and federal legislation and the CBD; clarity of jurisdictional responsibility; streamlining environmental compliance while permitting biodiscovery; non-exclusivity; and stakeholder identification, can provide a blueprint for the future. The glue that binds this, is robust science.
Drawing on recent, albeit continental shelf, examples of biodiscovery research activities from around Australia and New Zealand we will describe how national and international industrial investment and success can be harnessed in harmony with sound biodiscovery ethic and beneficial return to the country, state and/or region of origin of any biochemical discovery. We will describe why rigorous scientific strategies, at the earliest stages of discovery, are essential to successful commercialisation and conservation of resources.
ENVIRONMENTAL IMPACTS AND HIGH SEAS BIOPROSPECTING
A.D. Hemmings and E. Fellow
Gateway Antarctica Centre for Antarctic Studies and Research
Private Bag 4800
University of Canterbury
Christchurch 8020, New Zealand
Compared with historic and contemporary levels and types of harvesting on the high seas, bioprospecting may be seen as posing no, or far less, environmental risk. This may or may not be true - the present state of development of the activity makes it too early to know. But human attitudes to, and the environmental consequences of, exploitation of the high seas have usually been problematical. History in this area hardly offers reassurance. It is therefore appropriate to consider the potential to create adverse environmental impact through bioprospecting activity and this paper offers a preliminary examination of some of the issues.
These include a background of standards and expectations of environmental protection in the marine environment almost everywhere lower, and environmental management regimes less developed, than in terrestrial environments. That standard tool of scrutiny, the Environmental Impact Assessment, is not evident in the marine environment. The least developed regulatory system in the marine environment is that applying to the high seas. We still have only limited scientific knowledge about ecosystem functioning and particular taxa in this area. The basis for modern governance of the high seas, UNCLOS and associated instruments, essentially predate the emergence of bioprospecting as a viable activity, and reflect the norms of the 1970s.
The possible economic benefits of bioprospecting, and the likely relative high costs of acquiring those benefits, will occur in a poorly developed but complex international legal and policy context. This may pose challenges to wise management of any environmental impacts arising through bioprospecting.
BIOPROSPECTING FROM POLAR FISH
P.W. Wilson1 and A.D.J. Haymet2
1Department of Physiology, Otago Medical School
PO Box 913, Dunedin, New Zealand
2CSIRO Marine Research
Castray Esplanade, Hobart 7000, Tasmania, Australia
Will polar fishes be the source of interesting and unusual proteins for applications? It is well known that most Antarctic fishes have so-called “antifreeze” proteins in their blood to stop them from freezing in the sub-zero temperatures. Only very small numbers of these fish are currently being sacrificed for scientific study of the proteins and their method of action. Many possible uses have been suggested for the novel, ice-active properties of these proteins, including preservation of human organs, transgenic salmon, making smoother ice cream, de-icing of airplane wings, making crops frost-tolerant, freezing beef with less drip-loss and many more. Most of these suggestions have been tried to some extent and little success is evident to date.
Biosynthesis of some of the classes of these molecules has been successful and may be scaled up. However, some classes remain elusive and aquaculture or other harvesting of fish to extract proteins from the blood is not impossible.
Higher forms of life living under extreme conditions, such as polar fish, may have other molecules of interest, such as myoglobin and heat shock proteins optimized to work in other than temperate conditions.
THE ROLE OF LAW IN BIOPROSPECTING
Centre for Law and Genetics, Law Faculty , University of Tasmania
Private Bag 89, Hobart 7001
One of the key debates in relation to bioprospecting is the relationship between sovereign rights over biological resources and intellectual property rights in inventions developed from those resources. The Agreement on Trade-related Aspects of Intellectual Property requires that members of the World Trade Organisation must allow patents to be granted for inventions in all areas of technology, aside from a few specific exclusions. At the same time, the Convention on Biological Diversity and Bonn Guidelines establish the principal international legal regime for regulating access to biological resources in areas of national jurisdiction. The situation is more complex in regions of the world where sovereign rights are disputed or absent. One such region is the deep sea bed and another is the Antarctic. In this talk I examine the roles played by the various international legal regimes in these areas, particularly focusing on the interplay between freedom of scientific research and commercially sponsored bioprospecting.
Of themselves, patents may not impinge too greatly on freedom of scientific research, provided that they only claim rights over pharmaceutical and other downstream applications. However, the situation becomes more problematic where patent rights are claimed at the boundary between discoveries and inventions, for such things as genes and proteins. Freedom of scientific research and free exchange of observations and results may be further constrained by confidentiality and non-publication obligations required by commercial partners. For these and other reasons, it is timely to examine the need for regulation of bioprospecting in areas outside national jurisdiction.
D.J. Newman and G.M. Cragg
National Cancer Institute, Natural Products Branch
Developmental Therapeutics Programme
Suite 206, Fairview Center, P.O. Box B
Frederick, MD 21701, USA
1. THE NATIONAL CANCER INSTITUTE (NCI) COLLECTION PROGRAMMES
The NCI has had a programme for over 35 years that is designed to investigate the potential of Mother Nature's pharmacopoeia as a source of antitumor and, for a significant time, anti-HIV agents. The original collections, which could be best described as being “opportunity-based”, were made by various groups of botanists, marine biologists and microbiologists as they saw fit. Thus the plant collections that led to the discovery of paclitaxel (Taxol®) and camptothecin were made by USDA botanists as part of a programme of economic botany and resulted totally as a matter of happenstance. In a similar fashion, the marine invertebrates were collected as bycatch in some cases and by small collection programmes in others and the microbial samples were either from pharmaceutical houses who wanted their fermentations checked for anti-tumor activity or were simply “cupboard cleaning” of assembled extracts and organisms when programmes altered course in pharmaceutical houses.
The NCI offered screening in animals and the ability to follow activities in cell lines derived from the animals (originally mouse leukemia lines), which then developed into screening against mouse lines and follow-up in immunologically compromised mice with implanted human cell lines. From these relatively simple screens the vast majority of the well known agents (the Vinca alkaoids, the anthracyclines, bleomycin, etc.) were discovered and developed further.
However, by the late 1970s to the early 1980s it was realized in 20/20 hind sight that the agents now in use were active against fast growing tumors, but not as potent against the solid tumors, which was not surprising when one realized that the test systems were themselves fast-growing cell lines, the leukemias. Thus, there was an element of “shoot the messengers” and stop the natural product collection and assay programmes. So for around five years, no direct NCI collections were made. It should be pointed out that these collections were all made under the then current world political, social and governmental systems that did not consider any “rights” of the producing countries.
In the years between 1981 and 1986, one man, Michael Boyd, a physician and pharmacologist at the NCI, had the idea that if the assay systems could be modified to look for selectivity against a tumor type in a non-animal model as the primary screen, and followed up with suitable animal models utilizing the same cell lines as xenografts in nu nu or SCID mice, then one should be able to screen only compounds that demonstrated a “biochemical selectivity” from the beginning.
It took over four years to finalize the process and to develop the original NCI 60 cell line screen as a (then) high throughput assay that would permit both the screening of synthetic compounds, and most importantly, natural product extracts from all sources. Luckily, Michael Boyd was (and still is) a proponent of natural product sources as a source of test agents for antitumor use. It did not hurt that at the time that this screening system was coming on line, that paclitaxel was demonstrating its unique mechanism of action and potential utility in ovarian cancer and that derivatives of camptothecin were also demonstrating activity in pre-and early clinical trials.
At the same time (middle 1980s), the Developmental Therapeutics Programme of the NCI (which Boyd led), began the process of setting up formal collection programmes in marine and plant areas, together with small programmes looking at cyanobacteria and other sources such as fungi, micro, and macroalgae that could be cultivated. The fundamental reason for the formal collection process (and it applied to all collections that occurred thereafter) was that we had learned from our earlier programme, in particular the collection that led to paclitaxel, that we had to be able to generate enough of the initial extract to be able to follow through to chemical identity from the original sample. This was particularly evident in the earlier studies with the bryostatins, interesting agents isolated from certain “strains” of Bugula neritina in vanishingly small amounts, and was replicated in the studies with the dolastatins, isolated originally from the nudibranch, Dolabella auricularia.
Thus, NCI set up a series of competitive collection contracts that required the collection, taxonomic identification, site identity and suitable photographs of all samples of marine invertebrates (initially between the tropics), with concomitant processes for terrestrial plants. These samples would then be shipped to NCI, processed and screened, using the 60 cell line assay. If of interest, the compound(s) then would be isolated using bioactivity-driven isolation processes and then further investigated.
2. THE NCI'S LETTEROF COLLECTION
At the same time that the collection programmes were announced as being open for competition, NCI started to consider what could be done to persuade countries to permit what would now be large-scale collecting programmes operating on their lands and, or, from their seas. From these discussions within NCI, and also involving some source country personnel, the first NCI Letter of Collection (LOC) was formulated and signed by Madagascar in 1989. This was a full three years before the Convention on Biodiversity (CBD) and the LOC had as part of its non-negotiable requirement, that a “best effort” should be made to make certain that any company or organization that licensed any product from an NCI collection would have to involve the source country in the further development of that agent.
Over the years, with significant input from countries with extensive biodiversity (in particular, the State of Sarawak in Malaysia), the document has been refined and now, fifteen years after the first agreement was signed, the document has an absolute requirement that any licensee of an NCI patent must involve the country of origin it the further development of the compound.
There is one major difference between the LOC and the CBD. The CBD implies that royalty statements should be made when permission is granted for collections and this particular statement has led to major differences in perception between the collectors and the countries. We often use the term “green gold” to denote the misunderstanding that every collection has a new and very powerful drug within it. Nothing could be further from the truth. In a later section, we will give some odds on a discovery versus the size of the collection.
We, being a US government institution, have yet another problem related to royalties and incorrect perceptions. By US law, we are forbidden from “encumbering a future invention”. Or to use the vernacular, “if it ain't been invented yet, we cannot list royalties”. Since the simple act of collection is not an invention, and neither is the routine testing of extracts, then to establish a royalty stream in advance would infringe US law. This has caused immense difficulties with some countries, and we have in fact, ceased collections in a number following the CBD and the slight differences between the LOC and the CBD.
Another oft-held perception that is totally incorrect can be expressed as “if it is patented then it will be a drug whose sales will be measured in hundreds of millions of US dollars”. Nothing could be further from the truth. What a patent is, is a licence to sue somebody who infringes it, thus protecting your intellectual property, but only if you patent in the country where the infringement is taking place. If you simply go back 10 or so years, and look at the number of patents issued to companies such as Merck, Glaxo SmithKline, Pfizer etc., and then look forward 8 to 10 years at the drugs that have been approved for sale for any disease, you can see the vast disparity between the International Patient (IP) protected drugs and the drugs subsequently developed from these agents and approved for sale. The discrepancy between number of patents approved and drugs approved from those patents is measured in orders of magnitude. However, the perceptions as a result of totally incorrect information often disseminated by the media and some NGOs, still causes major disruption in totally beneficial collection programmes that take into account all of the tenets of the CBD.
3. SHALLOW WATERMARINE COLLECTIONS
The US government, through a variety of funding mechanisms has effectively paid (directly or indirectly) for a substantial number of all marine natural products that are currently in clinical trials, or in the earlier pipeline, for a substantial number of diseases, not just cancer. However, the figures that we will discuss are those from our anticancer and the coincident anti-HIV programme.
If we use current US dollar figures, then the collection of 1 kg wet weight of a marine invertebrate from anywhere on the globe (the programme was expanded to all seas a couple of years ago) consisting of collection, voucher sample preparation, identification, and shipping to NCI-Fredrick is approximately $1000 a sample. From this 1 kg (and we require that better than 75 percent of samples be of this weight), we will obtain around 20–50 g of an aqueous extract and 4–15 g of an organic extract, depending upon the phylum. The cost of extraction and storage at -20°C is ~ $200 a sample initially. Then, depending upon the type of storage container, costs vary.
Subsequent testing of a sample in the 60 cell-line screen in a two-step process is between $30 for those that “fail” and $300 for those that “pass”. We usually see around a 10–15 percent pass rate, though in earlier years, the costs were significantly higher due to differences in the prescreen (60 cell lines versus the current three).
The rate-limiting-step in reduction to a compound (and hence a possible inventive step if utility and novelty criteria are met) is the number of chemists and associated biologists that can be assigned to the task. Suffice to say that we are now approaching figures in the many tens of thousand dollars per new compound, particularly when the costs of the associated equipment are included.
We realized many years ago that these collections were an invaluable resource for mankind and in 1991, we began to permit groups outside of the US government to access these materials under strictly controlled conditions. This was originally known as the “Open Repository” programme and used only those extracts that we had decided did not show utility in our screens. However, they could and did, show activity in other screens with different modus operandi. All recipients had to agree, a priori, and in writing that if anything commercially valuable came from their work, the country of origin must be involved in its development, and most importantly, that the raw material should come from their waters (lands). We also notified countries that their materials were tested but in a way that did not infringe on IP rights. There was also one other important requirement. If the recipient needed more material, then they had to negotiate with the source country for permission to recollect. At this stage, not being the US government, the recipient could write royalty statements.
This programme was successful and from the middle of 1996, the programme was expanded to include the approximately 10 percent of samples that were in the “Active” category. Subsequently, the requirements were significantly relaxed as to who could receive materials and now they can be used for investigating any disease of interest to the NIH and foreign investigators are also permitted to access materials. These are all provided simply for the payment of the cost of shipping though there are restrictions on the number of samples that can be held at any one time depending upon their initial activity in the NCI screen.
There were two important tenets that we invoked from the beginning. The first was that if the source country wished, they could have access to the extracts from materials collected in their waters and lands. There have been materials sent back to some countries where they have the technical capabilities to screen and isolate materials. Secondly, we also have a clause that permits suitably trained scientists from the source countries to come to the US at NCI's expense for up to one year to learn the processes involved, and if they prefer, to work not only at NCI but at other laboratories in the US where they may learn different techniques.
4. DEEPER WATER COLLECTIONS
In the early collection programme, we had a “deepwater collection” system whereby manned submersibles and also some remote-operated vehicles (ROV-based) collections were made. However, these turned out to be extremely expensive and were mutually discontinued after about 15 months. Subsequently, we have permitted some dredging sub-programmes usually as a part of a bycatch opportunity when we know that we can go back if necessary. These are almost always performed in a particular country's EEZ. We have also had some experimental collections to 100–120 m using mixed gas rebreathers in Palau and a successful demonstration of the use of the “Deep Rover” one-man submersible, again in Palau, where due to the safety considerations of only having access to one submersible, a 400 m limit tether was used. The cost of these particular experiments was absorbed in the overall collection programme .
To give an idea of the costs involved in manned submersible work (and these are from a few years ago). The Harbor Branch Oceanographic Institutions (HBOI) charges used to be around $12 000 a day for ship transit time, plus ~$4500 a day for diving with the submersible. This would approximate to one major dive (or two shallower ones) for around 4–5 hours of collection time. Since a significant part of the cost of diving in the tropics away from their base in Florida would be actual transit time, one can perhaps understand why a large amount of the collection work by HBOI is in the Caribbean, which is on their front door step.
However, the use of submersibles of one type or another has led to discoveries that have significant drug potential. Thus, the HBOI were able to isolate the interesting molecule, discodermolide (Figure 1) from the previously undescribed sponge Discodermia dissolut, and this compound is now in Phase I clinical trials for cancer because of its similarity in mechanism of action to Taxol® as a drug that “freezes” the microtubule system in tumor cells. We should add however, that discodermolide, unlike Taxol® is now made by total synthesis, a tribute to the skill of the synthetic organic chemist once shown a “chemical mountain to conquer”.
Similarly, the compound known as halichondrinB (Figure2), which was originally discovered from a shallow water sponge off Japan in small amounts, and then was rediscovered by the University of Canterbury group of Munro and Blunt from a deepwater Lyssodendorix sp. off the Kaikoura shelf at >100 metres, was recovered by dredging and following a series of complex political and legal discussions, a joint venture between the University of Canterbury, NIWA (and indirectly the NZ government) with the NCI enabled a tonne of sponge to be collected by dredging, from which 300mg of pure halichondrin B was recovered by chemical purification. The total costs of this operation were over $500000 (with 50 percent from the NCI in direct payments to the joint-venture).
Concomitantly, NCI funded both the synthesis of halichondrinB via its grants mechanism and the aquaculture work performed by Battershill (then at NIWA) on growth in shallow waters. All of these endeavors were successful and have led to the trials of the modified halichondrin, E 7389 (Figure 3) which was developed by Eisai from the synthetic work performed by the NCI-funded group of Kishi at Harvard. No composition of matter patents could be taken out on halichondrinB as the structure had been published previously, but the Eisai company was able to patent the “half-hali” and Harvard had the patent on the synthesis, which was licensed to Eisai.
5. DRUG DISCOVERY AND MARINE SOURCES
The potential for novel active structures from accessible waters is well documented with many hundreds of publications, particularly the annual reports written by the late John Faulkner for 17 of the past 18 years (and now carried on by a consortia of New Zealand marine natural products chemists led by Blunt and Munro) showing the production of either completely unknown structural classes or of compounds that have striking similarities to those produced by terrestrial microbes. The link between microbes and marine natural products had long been hypothesized by marine natural products chemists and marine microbiologists as the phylum Porifera, which has provided close to 50 percent of all reported structures, can be considered to be a solid-state fermentor operating in a saline environment, as in a large number of cases, sponges contain massive amounts of all classes of microbial life.
The “smoking gun” was finally discovered by a consortium from the Center for Marine Biotechnology (COMB) in Baltimore and the Pharmacy Department at the University of Mississippi, when work by Hill at COMB on the isolation of a Micromonospora from an Indonesian sponge that produced the manzamine alkaloids (well known from a number of sponges) also produced the basic manzamine and a derivative when cultured in some media but did not produce it when cultured in others. The microbe is now being further investigated for the producing gene clusters and the compounds are being developed as anti-tuberculosis compounds at this moment.
The basic problem from this area of science has always been “supply of materials” when a successful structure has been discovered. Initially, the objectives were to produce enough material by aquaculture or mariculture (and this is a successful strategy in a number of cases), and can be utilized in areas of the world where the infrastructure is not optimal for more advanced studies. However, the current paradigm might be expressed as a continuum as shown in Scheme 1.
Thus the marine invertebrate (from whatever depth) is collected, an extract is made and a new, active structure determined. This is a patentable invention as there is novelty and utility, but usually it is not patented at this point as a stronger case can be made if there is in vivo data. To obtain enough material for further work in animals and to establish what other pharmacological possibilities there are, one has to consider all types of “production”.
Nowdays, the route is often concurrent with attempts at synthesis (either complete or the putative pharmacophore), investigation of the invertebrate's microbial flora by both classical isolation and by searching for putative gene clusters that might be involved in the biosynthesis of the metabolite, or potentially cell culture of the invertebrate cells if a suitable line from the source organism can be established. The speed with which synthetic and biosynthetic and genetic experiments can be performed nowdays is orders of magnitude faster than even five years ago. Examples are in the chemical and biochemical literature almost weekly, so the initial rate-limiting step (of further supply) that significantly held up marine drug discovery in the past is now not as much of a problem; but the real rate limiter is the actual acquisition of enough viable sample to proceed.
Collection and evaluation process
6. LEGAL ISSUES OF COLLECTIONS
Shallow water collections (and this could include waters out to the 200 nautical mile EEZ), though usually one only considers territorial waters (12 nautical miles), are easily covered by the NCI's letter of collection or by simple agreements between collectors and the relevant agencies in the country.
When one considers the seabed beyond the EEZ, or even within the EEZ, but at depths below 200 m, then the legal statues are highly debatable. The 1982 Convention on the Law of the Sea (LOSC) has been signed by the vast majority of countries in the world, but the USA is notable in that it has only ratified the LOSC provisions covering migratory fish stocks, though the Department of State did present testimony at the United Nations on 21 November 2001 stating that the Bush administration was recommending acceptance of the modified 1994 agreement on deep seabed mining. Due to the fact that the US Senate must ratify any such agreement, even though the President has signed such a document, as yet, the agreement has not been ratified.
Recently the Department of State has notified US-funded researchers that they need to consider the CBD when collections are being made, particularly from a genetic perspective and they point to the CBD secretariat's website as a source of further information.
Where marine bioprospecting in the deep-sea falls (if it even does) in the LOSC is debatable as it is not deep-sea mining. Only three or four countries have the technical capability to perform such collection programmes, Japan, The Russian Federation, the USA and perhaps France to some extent if one considers manned exploration. Other methods such as ROV explorations and deep dredging with some recovery do not require such levels of technical expertise. However, when it comes to investigation of the samples and then particularly the further development to a drug, there is only one country, the USA, where the government funds materials through to clinical trials (and then only in the case of cancer and AIDS). In all other cases, industrial involvement is necessary and, again, only a few groups can go all the way without involving others.
Thus, the legal waters are indeed deep and cloudy in these respects. Suggestions have been made that if materials are recovered from international waters and ultimately lead to a commercial drug, some form of international trust fund could be set up and administered by a suitable body. However, this suggestion is fraught with political problems, but might be solvable with a minimum of legal entanglements and a maximum of scientific input. For example, a fund that deals with “orphan diseases”, such as malaria or tuberculosis and under WHO auspices, might be possible and the CBD has a biodiversity trust fund that is also a possibility, but it would have to be modified as it is “country-biased”.
The search for novel structures that can be used to produce compounds that are active against diseases of interest to man is a laborious process. The marine invertebrates (and their associated microbial flora) have definitely demonstrated their potential to produce novel structures that frequently have no comparison from terrestrial sources. However, it would be remiss of us not to put some form of odds on finding a novel drug. Although one can only do these calculations in their entirety once a drug has become a commercial product, there are enough data in the public domain to be able to make the following generalizations across all disease states.
For every 100 preclinical candidates (defined below), only ten will reach Phase I clinical trials and for every ten that enter clinical trials, one or two will become commercial drugs. A preclinical lead is a compound that has novelty and efficacy and does not effect any biological activity other than the one desired; it can also be produced in sufficient quantity to be tested in the preclinical and clinical processes.
If asked how many compounds and extracts have to be tested to get one preclinical lead, the answer is from greater than 1 000 to less than a 1 000 000 in most cases. These figures are extremely difficult to obtain as they are in the archives of pharmaceutical companies, but from our experience it is usually somewhere in excess of 10000. Thus the process can best be described as a “numbers game” but with high rewards if one is successful. Obviously, the more screens that a given extract or compound can be assayed in, the better the overall odds of success.