MARINE DRUGS IN CLINICAL USE

Marine-derived compounds are more bioactive than those of terrestrial origin, especially in terms of cytotoxicity. It is no surprise, therefore, that marine natural products have their stronghold in the area of anticancer chemotherapy, as indicated by the list of compounds that have already made it to the market or are currently under clinical investigation. To date, the global marine pharmaceutical pipeline consists of seven approved pharmaceuticals in clinical use, four of which are anticancer drugs (Figure 1). The first marine-derived anticancer agent to be developed for clinical use, cytarabine or Ara-C, is a synthetic analogue of a C-nucleoside from the Caribbean sponge, Cryptothethya crypta, approved in 1969 and still in use today to treat acute myelocytic leukaemia and non-Hodgkin's lymphoma (Sagar et al., Reference Sagar, Kaur and Minneman2010). Almost 20 years later in 2007 the next anticancer agent from the tunicate Ecteinascidia turbinata would be approved, trabectedin (Yondelis), for the treatment of soft tissue sarcomas and ovarian cancer (Schoffski et al., Reference Schoffski, Dumez, Wolter, Stefan, Wozniak, Jimeno and Van Oosterom2008). A third marine anticancer success story was the discovery of the polyether metabolite halichondrin A from the sponge Halichondria okadai (Hirata & Uemura, Reference Hirata and Uemura1986). A simpler structure containing the pharmacophore obtained synthetically gave rise to Eribulin (Halaven), the most complex synthetic drug ever made, approved for the treatment of drug refractory breast cancer in 2010 (Menis & Twelves, Reference Menis and Twelves2011). The most recently approved anticancer compound from the marine environment is brentuximab vedotin (Adcetris), a chimeric antibody attached through a protease-cleavable linker to a derivative of the potent antitubulin agent dolastatin 10 (Katz et al., Reference Katz, Janik and Younes2011) used for the treatment of Hodgkin's lymphoma and anaplastic large cell lymphoma.

Fig. 1. Pharmaceutical drugs in commercial use.

Marine natural products have also found applications as antiviral drugs used to treat herpes simplex infections. Vidarabine or Ara-A is a synthetic analogue of spongouridine with improved antiviral activity, originally isolated in 1950 from the sponge Tethya crypta (Sagar et al., Reference Sagar, Kaur and Minneman2010), but currently obtained from fermentation cultures of the bacterium Streptomyces antibioticus. Another important marine natural product, ziconotide (commercial name Prialt; Klotz, Reference Klotz2006), is a synthetic calcium-channel binding conotoxin from Conus magus, the Magician's cone snail, which has proved effective in patients with intractable severe pain who either do not respond to or cannot tolerate other drugs. Additional conotoxins are in clinical development with potential applications in pain management and are widely employed tool compounds in neurotoxin research (Daly & Craik, Reference Daly and Craik2011). Finally, marine products obtained from fish oils, typically from oily fish such as mackerel and anchovy, have found applications as a lipid-regulating agent (commercial name Lovaza) to reduce severe elevations of triglycerides associated with conditions such as obesity, insulin resistance, diabetes mellitus and other factors that contribute to the risk of atherosclerosis or hardening of the arteries responsible for coronary artery disease. According to Gerwick & Moore (Reference Gerwick and Moore2012) the current success rate of discovery from the marine world, namely seven clinically useful and approved drugs from 28,175 discovered molecular entities (e.g. one drug per 4025 natural products described) is approximately 1.2- to 2.5-fold better than the industry average (1 in 5000–10,000 tested compounds) (http://www.pharma.org/sites/default/files/159/rd_brochure_022307.pdf).

NEW DRUGS IN DEVELOPMENT

Currently there are about 26 natural products in Phase I to Phase III clinical trials, 23 as anticancer cancer agents, two for schizophrenia and Alzheimer's, and one for chronic pain (http://marinepharmacology.midwestern.edu/clinPipeline.htm). Thus, the pipeline of promising marine derived compounds is very strong, and several of these agents are likely to reach the market in the coming years (Mayer et al., Reference Mayer, Glaser, Cuevas, Jacobs, Kem, Little, McIntosh, Newman, Potts and Shuster2010). Some of these new marine drugs are discussed briefly in the next section.

MARINE NUTRACEUTICALS AND COSMECEUTICALS

Currently there is also great interest in marine-derived products as nutraceuticals and cosmeceuticals because of their beneficial effects on human health. These often have drug-like properties (hence the term -ceutical) and contain active ingredients such as vitamins, phytochemicals, enzymes, antioxidants and essential oils which are finding uses as natural additives in foods, as nutritional supplements including colour additives and antioxidants, and as vitamins, oils and cofactors which enhance general well-being. Compared with high-value pharmaceuticals, these medium-value products have a rapid route to market and many companies have chosen to go along the functional product route as it offers lower risk and a quicker potential return on investment than the high-risk high-reward pharmaceutical market.

The main products of primary interest for marine nutraceuticals include omega-3 fish/algal oil, phospholipids (bound omega 3-fatty acids), micro/macro algal nutrition supplements, fish proteins and peptides, hydrolysates, shellfish chitin, fish collagen and mineral supplements (Figure 2). Polyunsaturated ω−3 fatty acids are purported to have a range of beneficial effects including improved heart health and reduced inflammation. Several population studies report that dietary ω−3 fatty acids or fish oil may also reduce the risk of developing breast, colon or prostate cancer. In addition to the traditional sources of the ω−3-fish oils, krill oil has also become very popular. This oil is different from the traditional fish oils because it contains three active components: ω−3 fatty acids, phospholipids and the carotenoid astaxanthin, a potent antioxidant. Marine microalgae are also rich in ω−3 oils. Made from various species of microalgae, this new omega-3 oil is reported to contain the same lipid type as fish oil, triglycerides. The advantage in this case is that production from this source is more eco-sustainable and less damaging to the environment than production of fish and krill oils.

Fig. 2. Marine nutraceuticals and their health benefits.

Current nutraceutical markets from marine organisms are also focused on chemicals such as carotenoids due to their high market value, projected to reach € 1.27 billion by 2019 (http://www.marketsandmarkets.com/PressReleases/carotenoid.asp). Carotenoids have much potential as food colourants, feed supplements, nutraceuticals, and for cosmetic and pharmaceutical purposes. There are well over 600 known carotenoids, with beta-carotene, alpha-carotene, lutein, zeaxanthin, lycopene, fucoxanthin and astaxanthin being the most common. Astaxanthin is one of the better known in terms of its status as a ‘supernutrient,’ as it is the only one to easily cross the blood–brain barrier and other bio-membranes which makes it more easily absorbed and transported to all tissues and organs in the body. Research suggests that astaxanthin is an optimum ingredient choice for eye protection, skin health, anti-ageing, anti-fatigue or any condition where protection from free radicals is needed (Fiedor & Burda, Reference Fiedor and Burda2014).

Another carotenoid, fucoxanthin, may play a role in reducing obesity (Gammone & D'Orazio, Reference Gammone and D'Orazio2015). So far, only animal studies have been conducted but these show that fucoxanthin, found in edible brown seaweed and microalgae such as the diatoms Phaeodactylum tricornutum or Cylindrotheca closterium, promotes the loss of abdominal fat in obese mice and rats. Although it is not fully understood how fucoxanthin works, it appears to target protein UCP1 that increases the rate at which abdominal fat is burned. Fucoxanthin has also been found in animal studies to decrease insulin and blood glucose levels. Researchers hypothesize that fucoxanthin may have anti-diabetes effects because it stimulates the formation of ω−3 fatty acids which are thought to increase insulin sensitivity, improve triglycerides and reduce LDL (‘bad’) cholesterol.

Together with the pharmaceutical and nutraceutical industries, the cosmeceutical industry is increasingly turning to the sea in the search for new molecules. One of the most common molecular classes of compounds used in the personal care bioactive ingredients sector is the exopolysaccharides (EPS). Various microorganisms produce EPS, including proteobacteria, cyanobacteria and archaea. Abyssine by Lucas Meyers is an Alteromonas ferment extract containing the EPS HYD657, named deepsane, produced and secreted by the strain Alteromonas macleodii subsp. fijiensis biovar deepsane (Cambon-Bonavita et al., Reference Cambon-Bonavita, Raguenes, Jean, Vincent and Guezennec2002). Deepsane is commercially available under the name of Abyssine for soothing and reducing irritation of sensitive skin against chemical, mechanical and ultraviolet B aggression (Martins et al., Reference Martins, Vieira, Gaspar and Santos2014). A ferment of the deep sea hydrothermal vent bacterium Thermus thermophilus (Venuceane^™ produced by Sederma Cosmetics) is a potent antioxidant that inhibits damage from reactive oxygen species, protects natural defence enzymes, shields from UV damage, and restores barrier lipids.

Resilience is a line of skincare products from the Estée Lauder Company that contains a special extracellular extract from the Caribbean Sea whip (gorgonian) Pseudopterogorgia elisabethae. This extract is mainly composed by pseudopterosins, which are potent anti-inflammatory and analgesic agents that inhibit eicosanoid biosynthesis by inhibition of both phospholipase A2 and 5-lipoxygenase. Additionally, a derivative of a natural pseudopterosin, methopterosin, has completed phase I and II clinical trials as a wound-healing agent.

Microalgae are the source of some of the most innovative skincare products today. Some examples include Dermochlorella DG, XCELL-30, Alguronic Acid and Alguard. Dermochlorella DG from CODIF Recherche et Nature (Brittany, France) is a Chlorella sp. extract containing oligopeptides that increases firmness and skin tone. XCELL-30 from Greensea (Mèze, France) is developed from microalgae endemic to Madagascar, and specifically acts on cellular turnover in the basal layer of the epidermis, thus allowing the preservation of the youthful characteristics of the skin. Alguronic Acid from Algenist (San Francisco, CA, USA) is an undetermined mix of polysaccharides produced by microalgae with significant anti-ageing properties, helping to rejuvenate the skin for a more youthful appearance. Alguard is a natural sulphated polysaccharide compound isolated from a single red microalga (Porphyridium sp.) that protects against photo damage, ageing and micro-abrasion of the skin.

PHARMASEA: A CASE STUDY OF A DRUG DISCOVERY PROJECT

Marine natural products constitute a strategic research area with potentially enormous economic and social revenues. According to EuroOcean (http://www.eurocean.org/np4/2502.html) there are over 590 European marine projects that have been funded under both FP6 and FP7 programmes (see also Martins et al., Reference Martins, Vieira, Gaspar and Santos2014), which shows the engagement of academia and industry in bringing more marine bioactives into the market.

An example is the EU PharmaSea project, an SME-academia-driven project initiated by 24 partners in 2012 to discover novel products for the treatment of bacterial infections, inflammation and neurodegenerative conditions such as Alzheimer's disease. The novelty of the project is that it searches for new microbial biodiversity to treat these diseases in some of the deepest, coldest and hottest places on the planet. By choosing deep and cold marine environments scientists hope to tap novel diversity not seen before. Deep ocean trenches are islands of diversity in which evolution may have progressed differently. The work on microorganisms from deep trenches shows they are indeed different. The chemistry derived from these, though limited in scope, shows high novelty.

Why study these diseases? In the case of antibiotics, the pipeline for new antibiotics has paradoxically experienced a long-term decline. Antibiotics are being developed, but not ones targeting the most urgent needs, and not in the diverse portfolio required to combat the rise of bacterial resistance. No new class of antibiotics has been discovered since 1987 but a new infection emerges on an almost yearly basis (Bérdy, Reference Bérdy2012). Diseases such as MRSA have become major problems in hospitals, evolving to become resistant to the limited number of antibiotics available. Hence the urgent need to discover new antimicrobials in many EU drug discovery projects (e.g. MicoB3, MACUMBA and others).

PharmaSea is also looking for (a) anti-inflammatory compounds targeting diseases such as atherosclerosis and Alzheimer's, and (b) neuroactive compounds targeting CNS disorders such as epilepsy. An estimated 44 million people worldwide suffer from dementia and Alzheimer's disease is the biggest cause, but the available treatments are inadequate and only improve some of the symptoms. There are also an estimated 70 million epilepsy patients worldwide, and 30% of these are pharmacoresistant, creating an urgent need for novel anti-epileptic drugs. With the growing and ageing population worldwide, and without the effective cures, it is anticipated that the number of patients with these diseases will increase dramatically in the coming decades. To avert the worst of this crisis, and to secure a sustainable source of more effective drugs, bottlenecks in the drug discovery process must be overcome.

Having its main focus on the discovery of new compounds bioactive in the above-mentioned therapeutic areas, PharmaSea also aims to overcome some of the bottlenecks currently found in the biodiscovery process, leading to (1) improvements in the quality of marine resources available for biotechnological exploitation, (2) to shorten time to market, and (3) develop sustainable modes of supply of raw materials for industry. The first challenge is being targeted by exploring the biotechnological potential of microorganisms such as bacteria, fungi and microalgae to overcome problems associated with supply of bioactives since microorganisms are generally more amenable to mass cultivation in large-scale enclosed bioreactors, as already demonstrated for the biofuel and nutritional supplement industries. The two last challenges centre on enabling activities to clarify legal aspects to facilitate access to marine resources, their sustainable use, and their secure exploitation; and second, to create an improved framework for accessing marine biotechnology data and research materials. To achieve these goals, PharmaSea brings together complementary and world-leading experts, integrating biology, genomics, natural product chemistry, bioactivity testing, industrial bioprocessing, legal aspects, market analysis and knowledge exchange.

Furthermore, to address the issue of inaccessible biodiversity, innovative technologies are being developed to allow more frequent and cost efficient retrieval of, for example, deep-sea microbes. Selection of new habitats such as the deep-sea has increased the number of novel species, which are obtained using selective isolation techniques. Phylogenetic and genomic strategies have further guided selection of strains based on biosynthetic capacity. These high-quality strains are being cultured in varying conditions to elicit their biosynthetic repertoire while those recalcitrant to culture are being exploited using a metagenomic approach.

To date the PharmaSea project has cultivated and extracted more than 1400 microbial strains from extreme marine environments, with the majority being fungi and actinobacteria (Figure 3A). These have been cultivated in a number of different media with different stresses applied to elicit production of secondary metabolites. The strains investigated so far have yielded a total of over 15,000 extracts and fractions which have been subjected to a broad range of antimicrobial and central nervous system disease assays (Figure 3B). The pathogenic bacteria and fungi used in this assay panel include Gram-positive (methicillin resistant Staphylococcus aureus, MRSA) and Gram-negative bacteria (Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli), fungi (Candida albicans and Aspergillus fumigatus) (Audoin et al., Reference Audoin, Bonhomme, Ivanisevic, de la Cruz, Cautain, Monteiro, Reyes, Rios, Perez and Thomas2013), and two surrogate (avirulent) strains of Mycobacterium, namely Mycobacterium tuberculosis H37Ra and Mycobacterium bovis BCG. Additionally, all samples are also tested in an assay to detect compounds potentiating the effect of the β-lactam antibiotic imipenem against P. aeruginosa, with extracts and fractions tested against the pathogen in the presence or absence of a sub-lethal concentration of the antibiotic. Assay incubation times range from overnight in the antibacterial and antifungal assays to 7-day incubations in assays against Mycobacterium strains. In order to reduce the high number of hits in anti-infective screens (Figure 3C) we are using a state of the art LC/MS based dereplication approach. This immediately excludes extracts containing known antibiotics and identifies those containing chemical novelty (Lacret et al., Reference Lacret, Oves-Costales, Gómez, Díaz, de la Cruz, Pérez-Victoria, Vicente, Genilloud and Reyes2015). These extracts are being subjected to further work to identify and chemically characterize the active components.

Fig. 3. Pie diagrams showing composition of the PharmaSea strain library (A) and extract library (B) for different groups of microorganisms. Total number of anti-infective (C) and other (D) screening events up to June 2015.

Neuroactive extracts and fractions, for potential applications to treat epilepsy and other CNS disorders, are tested using high-throughput behavioural fingerprinting and seizure assays in zebrafish larvae (e.g. VanHook, Reference VanHook2010; Cunliffe, Reference Cunliffe2015). Zebrafish-based behavioural fingerprinting provides initial data as to the putative mechanism of action of neuroactive molecules, and provides preliminary mechanism of action information for isolated compounds, thereby helping to rapidly prioritize those with novel mechanisms of action. In addition to anti-infective and neuroactivity screens, anti-inflammatory and toxicity screens are also carried out (Figure 3C). Figure 3D further shows the different assays used to evaluate the neuroactivity and anti-inflammatory activity in the primary screen. It also shows the percentage of screening events performed to evaluate toxicity.

Simultaneous with the screening process, extracts are being dereplicated at an early stage through the use of innovative chemometric methods. Extracts identified as validated actives in a selected assay and which are non-toxic have also been evaluated for their likelihood of containing novel compounds using chemometrics. Extracts identified using this protocol are purified using targeted chromatographic techniques followed by pioneering compound dereplication strategies leading directly into an accelerated workflow for full structure determination. This is only possible through the involvement of one of the world's largest chemical databases and a chemical software company, which has enabled the prediction of NMR and MS spectra of most known marine and microbial natural products.

The global aim of PharmaSea is to produce two compounds at a larger scale and advance them to pre-clinical evaluation. Assay cascades are used as the first step in a protocol to decide which extracts to progress. Those showing good in vitro potency, low toxicity and high probability of containing novel compounds are taken forward to isolate the active compounds as potential pharmaceutical candidates. Scale-up fermentation in saline media is being used to generate adequate amounts of target compounds. In addition, identified novel gene clusters are being expressed in heterologous hosts. Extracts that show interesting bioactivity e.g. low cytotoxicity and/or dual activity from cell based screens e.g. anti-inflammatory and antioxidant, will be subjected to additional screens to identify their potential value as novel applications of marine natural products in personal care and nutrition as part of follow-on projects.

Bioactive compounds will be developed by SME partners and with larger pharmaceutical companies, if appropriate. To address relevant challenges in marine biodiscovery related to policy and legal issues, PharmaSea has brought together practitioners, legal experts, policy advisors/makers and other stakeholders, focusing on the feasibility of harmonizing, aligning and complementing current legal frameworks with recommendations and ready to use solutions tailored to marine biodiscovery (Lallier et al., Reference Lallier, McMeel, Greiber, Vanagt, Dobson and Jaspars2014). This work will also generate a toolkit to enable users to explore the existing difficulties of access and benefit sharing under different legal regimes and find practical guidance to secure access to marine genetic resources. Uptake of products derived from marine bioresources will be improved via a number of mechanisms making PharmaSea data/IP available under licence to interested end-users.

CONCLUDING REMARKS

Discovery of natural bioactive molecules is, of course, only the first step in the biodiscovery pipeline. The hardest challenge is turning these products into useful medicines because the costs of bringing a new drug to market from discovery through clinical trials to approval is currently $2558 million, according to a new study by the Tufts Center for the Study of Drug Development (http://csdd.tufts.edu/news/complete_story/pr_tufts_csdd_2014_cost_study), costs that have more than doubled in the last decade. The same Center also reports that only 11.8% of drug compounds entering clinical testing are eventually approved. Another major challenge is to reduce the time to market, which is currently ~10 years. Both factors are discouraging pharmaceutical industries which also have to cope with the fact that the marine environment is largely unexplored and does not offer certainty on the successful outcome of investment. This is complicated by the large investment required to organize oceanographic cruises and to sample in extreme environments where chances of finding greater biodiversity are higher. A further complication regards the legal aspects to access and utilization of marine natural products (or genetic resources) that have been formalized in a series of agreements such as the Convention on Biological Diversity (CBD) and its Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization (Nagoya Protocol, as well as the United Nations Convention on the Law of the Sea (UNCLOS)). The Nagoya protocol, expected to enter into force in 2015, will enable stakeholders to better understand and comply with national access and benefit-sharing procedures. For example, when the genetic material is accessed from an ‘ex situ’ collection such as a biorepository then the access provision of the CBD and the Nagoya protocol apply. If the genetic material is collected ‘in situ’ during an expedition then UNCLOS provisions will also apply (for further details on legal framework to access and benefit sharing see Lallier et al., Reference Lallier, McMeel, Greiber, Vanagt, Dobson and Jaspars2014 and references therein). Notwithstanding these difficulties, the discovery of new ocean medicines is one of the most promising new directions of marine science today. Novel initiatives including partnering between governmental organizations and industry are being developed to meet the difficulties incurred in the drug discovery pipeline. Projects such as PharmaSea represent an example of such a partnership, as will future programmes being organized within the framework of Horizon 2020.

The oceans can therefore provide us with many invaluable benefits and services, including some of the medicines we use to cure human disease. However, the oceans are under serious threat due to pollution and global change. We even contaminate the ocean and its inhabitants by dumping chemicals from drug manufacturing into the world's waterways, polluting villages, cities and aquatic ecosystems around the world, eventually contaminating the fish we eat (Larsson, Reference Larsson2014) and the very organisms that could provide us with new cures for human diseases in the future. With the global population projected to increase from 7 to 9 billion people in the next few decades, there is an urgent need to secure a healthy and productive global ocean. A healthier ocean that is better managed could provide more food and employment. It could also ensure that the benefits from the exploitation of ocean resources can be sustainably managed and equitably shared. Knowledge on the effects of pollution and global change on marine organisms are limited and still relatively unexplored, leaving critical knowledge gaps for those seeking to develop effective policies for sustainable use of marine resources and environmental and human health protection (European Marine Board, 2013). A better understanding of the potential health benefits from marine organisms, the compounds they produce, and the environmental conditions affecting their production will allow for the better management and sustainable development of these valuable marine resources in the future. By pursuing the medical promises hidden within the ocean, while also managing the dangers to human health found in this new frontier, ocean scientists can make a major contribution to improving human health in the 21st century and beyond.