October 15, 2005 (Vol. 25, No. 18)
Susan Aldridge, Ph.D.
Prospecting for Commercial Opportunities in Marine Environments
Norway has considerable experience with fishing and related industries and is now using its close relationship with the sea to develop a thriving marine biotechnology sector. Several new initiatives around the country promise to help “blue biotech” discover drugs, novel enzymes, and other useful compounds, while exploring the genetic diversity of the marine ecosystem.
Another key focus of blue biotech for Norway is developing aquaculturefish health, breeding, and feeding.
The Norwegian University of Science and Technology (NTNU; Trondheim) has been pursuing a program in marine bioprospectingthat is, searching and screening for significant biomolecules in marine sourcessince July 2003.
Researchers at NTNU’s department of biotechnology are searching the Trondheim fjord for bacteria that produce novel antibiotics, carotenoid and other dyes, and fatty acids such as DHA. The fjord has many rich ecological niches such as the deep sediments and the microlayer (10 micrometers deep) on the surface.
The latter has unique properties, such as strong exposure to ultra violet light, which may favor organisms containing protective compounds, including the carotenoids. Meanwhile, the low temperatures of the water favor organisms whose enzymes are cold-adapted.
The latest expedition collected various species of sponge, which filter millions of liters of water every day and are believed to harbor bacteria that produce novel anti-infectives.
“We are already finding production organisms for all the products we are looking for and are now screening these for industrial potential,” says Trond Ellingsen, Ph.D., research director at SINTEF (Foundation for Scientific and Industrial Research at the Norwegian Institute of Technology), who is confident that the biotech department’s expertise in strain development, process development, and high throughput screening is more than equal to the task.
“Marine environments have a big potential that has not really been investigated,” adds Svein Valla, Ph.D., associate professor at the department of biotechnology. Since 90% or more of the organisms isolated from nature cannot be cultured, Dr. Valla and his team are creating a “metagenome” library from the surface layer of the fjord.
They isolate large DNA fragmentsin the hope of finding clusters of genes encoding complex processesfrom a pool of organisms, clone them into vectors, and then transform them in the E. coli. They are using unique vectors that can be cloned into a broad host range to increase the screening potential of the project because genes expressed in one host may not be expressed in another.
“The more hosts we screen in, the more we will get from the same gene library,” adds Dr. Valla. So far, this ambitious project has revealed an enormous number of different species and huge genetic variability within species gathered from the surface layer of water. Moreover, these species and strains are present in very unequal numbers that may vary over time.
Actinomycetes
Meanwhile, Sergei Zotchev, Ph.D., associate professor at the department of biotechnology, and his colleagues are collecting from the deep sediment of the fjord, concentrating on actinomycetes. They carry out selective isolation of these bacteriato exclude the streptomyces, whose products are already well known.
“Rare and unusual actinomycetes are often producers of genuinely novel antibiotics,” says Dr. Zotchev. Robotized screening of the marine extracts is followed by LC/MS-TOF, and the mass is then matched through a database, which will reveal whether or not the molecule is new.
“The key of this project is to identify novel compounds. We already have several very good hits in our antifungal program and are now going to antibacterials.” The next stage is molecular biology to look at mode of action followed by animal studies. Dr. Zotchev is also CSO of Biosergen, a drug discovery start-up that is using metabolic engineering to obtain new antifungals from Streptomyces noursei.
The Northern Norway area is another important center for marine biotechnology. It has around 20 biotech SMEs with 300 employees. Scientists at the University of Troms are involved in bioprospecting for enzymes, enzyme inhibitors, antioxidants, and immune modulators from species such as sea anemones, starfish, sponges, sea urchins, and spider crabs.
“These species use chemicals to protect themselves from parasites, such as viruses,” says Trond Jrgensen, Ph.D., professor at the department of marine biotechnology at the University. They are also working in fish health and breeding as well as in marine byproducts such as lipids including DHA, fish feed, and polymers.
“The marine environment is less explored than the terrestrial one. There are many future opportunities for drugs, antivirals, and pigments,” he says. What is more, these species contain molecules, such as enzymes, which are adapted to low temperatures. “Their specific activity may be higher in the cold sea than in warmer places like the Mediterranean,” adds Dr. Jrgensen.
Troms is the location of Marbank, a marine biobank that will collect whole specimens, extracts, and DNA/RNA samples for management, scientific, and commercial purposes and is funded by the Norwegian Ministry of Fisheries and Coastal Affairs.
It will operate in close synergy with another project, Marbio, which is a high throughput screening platform for bioactivity financed by the Research Council of Norway, the University of Troms, and industry. Funding for both projects totals about s5 million and data generated from them will feed into Norstruct, the national databank of protein stucture.
Marine Bioactivity
Another new initiative is MabCent, a research program focused on marine bioactivity from the Arctic. Potentially valuable extracts are to be screened versus tumor and bacteria for immune modulatory, enzyme, and enzyme inhibitor activity. A new kind of high throughput assay will be developed, and lead compounds will be further developed.
MabCent’s partnerstwo Norwegian companies and two international, so farwill get one to 12 months exclusive access to any extracts and first right of refusal to hits generated.
The University is giving 25% of a total of about a2.5 million of funding to the eight-year program. “The University is really pushing for this kind of activity,” says Dr. Jrgensen.
Meanwhile, Mabit is a networking program that links academia, industry, and local authority in the Troms area. It has funded various projects including a process/pilot production lab and a research vessel for bioprospecting.
There are several companies in Norway with products on the market that have come from blue biotech. One is the pharmaceutical company Navamedic (Lysaker), which manufactures glucosamine hydrochloride for osteoarthritis from shrimp shells. Glucomed was recently registered in Sweden, which opens the door to approval elsewhere. The shells are processed into chitin powder, from which glucosamine is produced.
Glucosamine has long been used as a food supplement for arthritis but has recently been reclassified as a medicine in several European countries, in light of recent clinical evidence showing its efficacy versus placebo in osteoarthritis and its lack of side effects. Data from a large clinical trialthe glucosamine and chondroitin arthritis intervention trial will be presented in November.
This opens up new opportunities for companies like Navamedic that can manufacture glucosamine to a high standard. “The biggest growth in the glucosamine market is in the pharma segment,” comments yvind Brekke, Navamedic’s CEO.
“What differentiates Navamedic from the other players is that we aim to cover the whole value chain.” A key issue is assuring the supply of raw materials. “Demand is growing faster than the availability of shrimp shell. We think it is critical to control our supply to offer stable pricing and the volume needed.”
Accordingly, Navamedic has secured long-term contracts with shell suppliers and is currently looking at the shrimp supply in different locations, including the north of Iceland, while deciding on a site for full-scale GMP manufacturing of glucosamine by 2008.
Marine Enzymes
Biotec Pharmacon (Troms) is one of the key players in the Norwegian biotech industry, with interests in marine enzymes and soluble beta glucan (SBG), a complex carbohydrate being developed as an immunomodulator and anticancer therapy.
Biotec Pharmacon began by looking for enzymes in fish waste, such as the water used to wash shrimps, a strategy that generated cash from day one. These enzymes were used in seafood processing, such as in scaling fish and skinning squid. But this side of the business was given up because of lack of control over raw materials.
The company turned to the research and diagnostics market for their cold-adapted enzymes such as shrimp alkaline phosphatase, shrimp nuclease, and cod uracil DNA glycosylase. These are now being made in recombinant systems.
The cold-adapted enzymes have new propertiesfor instance, shrimp nuclease has a specificity for double-stranded DNAleaving single stranded alone, which is useful in PCR and other molecular biology applications.
The enzymes are a source of cash, but Biotec Pharmacon also wants to make drugs. “We are interested in bioprospecting for pipeline products for pharmaceutical development, including finding novel genes for antimicrobial peptides,” says Jan Raa, Ph.D., vp research and development.
The company already has a promising product in SBG, an immunostimulant used in fish feed to decrease mortality. The properties of SBG first became apparent when fish receiving it showed unexpectedly high survival rates in an incident where tanks were contaminated by seawater.
“We saw immediately that this could be an important commercial product. We were looking for something to increase immunity generally, because you can’t vaccinate against everything,” says Dr. Raa.
SBG stimulates macrophages, switching on genes that control key immune functions. It acts on the mucosal immune system and transmits signals to other parts of the body.
There are many forms of glucan, however, and the biological effects of SBG depend upon it having the correct length of side chain, which fits into the macrophage receptors.
Biotec Pharmacon’s R&D efforts have led to a way of manufacturing a high-quality version of SBG from yeast. In humans, SBG can reportedly enhance the effect of monoclonal antibodies, and trials with cancer patients are ongoing at Memorial Sloan-Kettering Institute in a proof-of-concept study.
If SBG can synergize with the number of monoclonals both on the market and in the pipeline, then it has true blockbusting potential.
There are several other ongoing trials involving SBG. For example, SBG can enhance the effect of nasal flu vaccine and another trial is looking at SBG in diabetic ulcers, where it can help normalize dysfunctional macrophages.
In other experiments, rats with experimental periodontitis were treated with SBG in their drinking water, delaying the onset of gingivitis, a finding that has been replicated in Phase I/II studies.
In a Phase II study at the Royal Marsden Hospital, London, SBG was beneficial in mucositis caused by chemotherapy and in animals, it can protect from LPS-induced septic shock. “A lot of things happen when these molecules are administered to mucosal surfaces,” says Dr. Raa.
Biopolymers
Novamatrix (Oslo), a business unit of FMC BioPolymer, is another company operating in the complex carbohydrate sector. They have developed ultrapure versions of chitosan (Protasan), alginate (Pronova), andhyaluronan. These biopolymers are already well known but Novamatrix is finding new applications for them. The alginate is being used in cell encapsulation to make biofactories.
In an animal trial, an alginate-encapsulated endostatin was able to prevent re-growth of glioblastoma multiforme. Chitosan can improve the pharmacokinetics of morphine by allowing penetration of the tight junctions formed by cells in nasal epithelium.
It can also be applied as a non-viral gene delivery system in gene therapy; experiments show that chitosan can effectively transfect cells in vivo.
Novamatrix has also developed a nonanimal (bacterial) form of hyaluronan, which is found in Restylane, a dermal filler used in cosmetic surgery. The alginates can also be extracted from various seaweeds found off the coast of Norway and have wound-healing properties that allow them to be used as dressings.
Alginate is a bioactive therapy that stimulates an immune response, restoring chemokine/ cytokine balance, for instance, and balancing the action of proteolytic enzymes.
Other key Norwegian companies include Lytix Biopharma (Troms), which is developing novel bioactive peptides as anti-infectives with a mode of action avoiding the development of resistance.
Algeta (Oslo) has promising clinical data on its alpha particle-emitting therapeutic, Alpharadin, for bony metastases in breast and prostate cancer.
Spermatech (Oslo) is developing a nonhormonal male contraception based upon a unique target that regulates sperm motility. Finally, Avexxin (Trondheim) is looking at developing anti-inflammatories for the treatment of psoriasis.
