Mar.Drugs 2014,12 1071 observed that a sponge lays alone in the habitat,and around it there is a clean area,most probably,its metabolites inhibit the growth of competing organisms [50].Unfortunately,these sampling facilities are very expensive and only a small number of laboratories have access to them,a drawback that is difficult to overcome,especially if we think that the majority of biological diversity is located in underdeveloped countries from the tropical and subtropical regions [51].This is just one of the reasons why international collaboration is so important in this research field.However,the access to biodiversity on natural resources is now under the host of the Convention on Biological Diversity (CBD).Unfortunately.the different levels of Nagova protocol implementation (which clarifies the scope of the CBD)in different regions,and the increasing difficulties to work under a still unclear regulatory framework on biodiversity access may push current industries out of the NP arena The lack of taxonomic knowledge for marine species,and the still large number of unidentified species and strains,is alsoa major blockage faced by marine natural products programs.The selection for pharmacological purposes.of macro or microorganisms.either terrestrial or marine.must be grounded on a correct taxonomic identification and classification.An incorrect classification of a species may compromise an entire drug discovery project,not only because it is impossible to reproduce the isolation in the event of a bioactive extract and/or metabolite.but also because it can mislead the dereplication process-the process by which the bioactives are identified.Approaches to classification of marine macroorganisms (algae and invertebrates)and microorganisms(fungi and bacteria)are quite different.For the majority of marine macroorganisms taxonomic knowledge is still insufficient to enable unambiguous species classification [52].Macroinvertebrates are especially challenging.not only due the fact that there are still many undescribed species.but also because many related species must be distinguished based on subtle morphological characteristics [531 Following the process of target identification and validation,the next step of a drug discovery process is the development of the screening assays.A variety of screening paradigms exist to identify hit molecules [54]being HTS the most widely used in the case of NP.The success key to apply HTS methodology to NP is constructing high quality libraries.Researchers at Pfizer proposed that the output from HTS is dependent on the interrelationships between the quality of the compound library. the target and the screening process [55].Ideally,the library itself should be composed by crud extracts.simplified extract fractions and pure compounds for a well-balanced natural product discovery program [56].Crude extract libraries are easier and cheaper to construct,have moderate overall size and a high degree of diversity,but have major disadvantages when compared with pure compounds libraries.Crude extracts are complex mixtures of several compounds that may have synergistic interaction.a fact that accounts for the disappearance of the bioactivity in purified fractions and,ultimately,in final pure compounds [57].On the other hand,false negative readouts may also be obtained.either because an active metabolite is present in a small percentage in the crude extract.or because of the interference of compounds such as tannins [56]that bind to other metabolites masking its activity.Due to these reasons.in a recent past this approach was discouraged in drug discovery programs [56].Screening pre-fractionated libraries is an effective strategy to avoid these problems [3]. Depending on the method used for pre-fractionation and on the number of compounds in the original crude extract,the resulting fractions can vary widely in complexity from a mixture of multiple compounds to a single major compound of%purity.Pre-fractionation can eliminate several undesired compounds and facilitate hit identification
Mar. Drugs 2014, 12 1071 observed that a sponge lays alone in the habitat, and around it there is a clean area, most probably, its metabolites inhibit the growth of competing organisms [50]. Unfortunately, these sampling facilities are very expensive and only a small number of laboratories have access to them, a drawback that is difficult to overcome, especially if we think that the majority of biological diversity is located in underdeveloped countries from the tropical and subtropical regions [51]. This is just one of the reasons why international collaboration is so important in this research field. However, the access to biodiversity on natural resources is now under the host of the Convention on Biological Diversity (CBD). Unfortunately, the different levels of Nagoya protocol implementation (which clarifies the scope of the CBD) in different regions, and the increasing difficulties to work under a still unclear regulatory framework on biodiversity access may push current industries out of the NP arena. The lack of taxonomic knowledge for marine species, and the still large number of unidentified species and strains, is also a major blockage faced by marine natural products programs. The selection, for pharmacological purposes, of macro or microorganisms, either terrestrial or marine, must be grounded on a correct taxonomic identification and classification. An incorrect classification of a species may compromise an entire drug discovery project, not only because it is impossible to reproduce the isolation in the event of a bioactive extract and/or metabolite, but also because it can mislead the dereplication process—the process by which the bioactives are identified. Approaches to classification of marine macroorganisms (algae and invertebrates) and microorganisms (fungi and bacteria) are quite different. For the majority of marine macroorganisms taxonomic knowledge is still insufficient to enable unambiguous species classification [52]. Macroinvertebrates are especially challenging, not only due the fact that there are still many undescribed species, but also because many related species must be distinguished based on subtle morphological characteristics [53]. Following the process of target identification and validation, the next step of a drug discovery process is the development of the screening assays. A variety of screening paradigms exist to identify hit molecules [54] being HTS the most widely used in the case of NP. The success key to apply HTS methodology to NP is constructing high quality libraries. Researchers at Pfizer proposed that the output from HTS is dependent on the interrelationships between the quality of the compound library, the target and the screening process [55]. Ideally, the library itself should be composed by crude extracts, simplified extract fractions and pure compounds for a well-balanced natural product discovery program [56]. Crude extract libraries are easier and cheaper to construct, have moderate overall size and a high degree of diversity, but have major disadvantages when compared with pure compounds libraries. Crude extracts are complex mixtures of several compounds that may have synergistic interaction, a fact that accounts for the disappearance of the bioactivity in purified fractions and, ultimately, in final pure compounds [57]. On the other hand, false negative readouts may also be obtained, either because an active metabolite is present in a small percentage in the crude extract, or because of the interference of compounds such as tannins [56] that bind to other metabolites masking its activity. Due to these reasons, in a recent past this approach was discouraged in drug discovery programs [56]. Screening pre-fractionated libraries is an effective strategy to avoid these problems [3]. Depending on the method used for pre-fractionation and on the number of compounds in the original crude extract, the resulting fractions can vary widely in complexity from a mixture of multiple compounds to a single major compound of >90% purity. Pre-fractionation can eliminate several undesired compounds and facilitate hit identification
Mar.Drugs 2014.12 1072 All in all,the association of modern HTS methods and robust NP libraries,representative of a wide biodiversity,is a powerful tool to streamline cosmeceutical and therapeutic lead discovery programs 3.2.Supply and Technical Challenges Several different problems are associated with supply and technical issues.The first one is related to the variability of the organism itself.For instance,taking the example of sponges,the high frequency of their bioactive metabolites is interpreted as chemical defense against environmental stress factors such as predation,overgrowth by fouling organisms or competition for space.The highest incidence of toxic or deterrent sponge metabolites is found in habitats such as coral reefs that are characterized by intense competition and feeding pressure.Because these environmental conditions are not static,it is likely that a resupply of the same organism does not provide the same metabolite.Also, in the case of marine invertebrates another challenge is the fact the microorganisms are sometimes the actual producers of the bioactives Once a particular natural product has been isolated and identified as a lead compound,the issue of its sustainable supply is faced.Most of the times,the compound of interest is present only in low amounts and/or can be very difficult to isolate [17].In the case of tissues of marine invertebrates, which present unique extraction-related problems due to their high water and salt content,this problem can be even more challenging.Whatever the use of the compound (drug cosmetic,rc.).several grams to hundreds of grams are required for preclinical development,multikilogram quantities are needed for clinical phases and tons for cosmetic uses. Mariculture(favoring by farming the growth of the organism in its natural milieu)and aquaculture (culture of the organism under artificial conditions)have been attempted in order to solve the problem of sustainable supply of macroorganisms.However,the unique and sometimesexclusive.conditions of the sea make cultivation or maintenance of the isolated samples very difficult and often impossible. For example,sponges and their microbiota are generally not suitable for cultivation,hence,the compound of interest may need to be extracted and purified from the specimens collected in the wild [47].These constraints lead to the loss of a major portion of the available marine biodiversity and represent a major bottleneck in the sustainable supply of the desired natural compound. This lack of sustainable supply of substances has stopped further development of several highly promising marine compounds,and attempts have been made to overcome this barrier by developing synthetic or hemisynthesic analogues,derivatives with more manageable properties.or by design of a pharmacophore of reduced complexity which can then be synthesized [301.However.it is worth noting.that these approaches embrace themselves their own challenges.Total synthesis is by no means an easy undertaking task,and chemistry still has a very long way to go before it can make any molecule in a practical manner.NP are complex and exquisite molecules possessing,almost always, one or several stereocenters,a fact that renders their synthesis hard to achieve,since enanteo or diastereoselective synthetic or purification processes are difficult to perform.Hemisynthesis may be.in some cases,a good solution for compound's supply.This process involves harvesting a biosynthetic intermediate from the natural source,rather than the lead itself,and converting it into the lead.This approach has two advantages.First,the intermediate may be more easily extracted in a higher yield than the final product itself.Second,it may allow the syntheses of analogues of the final product
Mar. Drugs 2014, 12 1072 All in all, the association of modern HTS methods and robust NP libraries, representative of a wide biodiversity, is a powerful tool to streamline cosmeceutical and therapeutic lead discovery programs. 3.2. Supply and Technical Challenges Several different problems are associated with supply and technical issues. The first one is related to the variability of the organism itself. For instance, taking the example of sponges, the high frequency of their bioactive metabolites is interpreted as chemical defense against environmental stress factors such as predation, overgrowth by fouling organisms or competition for space. The highest incidence of toxic or deterrent sponge metabolites is found in habitats such as coral reefs that are characterized by intense competition and feeding pressure. Because these environmental conditions are not static, it is likely that a resupply of the same organism does not provide the same metabolite. Also, in the case of marine invertebrates another challenge is the fact the microorganisms are sometimes the actual producers of the bioactives. Once a particular natural product has been isolated and identified as a lead compound, the issue of its sustainable supply is faced. Most of the times, the compound of interest is present only in low amounts and/or can be very difficult to isolate [17]. In the case of tissues of marine invertebrates, which present unique extraction-related problems due to their high water and salt content, this problem can be even more challenging. Whatever the use of the compound (drug, cosmetic, etc.), several grams to hundreds of grams are required for preclinical development, multikilogram quantities are needed for clinical phases and tons for cosmetic uses. Mariculture (favoring by farming the growth of the organism in its natural milieu) and aquaculture (culture of the organism under artificial conditions) have been attempted in order to solve the problem of sustainable supply of macroorganisms. However, the unique and sometimes exclusive, conditions of the sea make cultivation or maintenance of the isolated samples very difficult and often impossible. For example, sponges and their microbiota are generally not suitable for cultivation, hence, the compound of interest may need to be extracted and purified from the specimens collected in the wild [47]. These constraints lead to the loss of a major portion of the available marine biodiversity and represent a major bottleneck in the sustainable supply of the desired natural compound. This lack of sustainable supply of substances has stopped further development of several highly promising marine compounds, and attempts have been made to overcome this barrier by developing synthetic or hemisynthesic analogues, derivatives with more manageable properties, or by design of a pharmacophore of reduced complexity which can then be synthesized [30]. However, it is worth noting, that these approaches embrace themselves their own challenges. Total synthesis is by no means an easy undertaking task, and chemistry still has a very long way to go before it can make any molecule in a practical manner. NP are complex and exquisite molecules possessing, almost always, one or several stereocenters, a fact that renders their synthesis hard to achieve, since enanteo or diastereoselective synthetic or purification processes are difficult to perform. Hemisynthesis may be, in some cases, a good solution for compound‘s supply. This process involves harvesting a biosynthetic intermediate from the natural source, rather than the lead itself, and converting it into the lead. This approach has two advantages. First, the intermediate may be more easily extracted in a higher yield than the final product itself. Second, it may allow the syntheses of analogues of the final product
Mar.Drugs 2014,12 1073 Additionally,the synthesis or hemisynthesis of a bioactive natural compound must be supported by a correct identification of the compound isolated from the biological source.Despite the fact that modern methodologies of struetural elucidation are well advanced,errors can never be completely ruled out.In fact,there are numerous structural revisions reported in literature,even of recently elucidated NP.In average,per 5-year period,369 NP and 135 MNP are misassigned [58].The structural complexity of the isolated compounds and the small amount of samples,especially in the case of compounds from marine sources.can contribute to misassignments which can be divided in several categories:incorrect formula,constitution (planar connectivity),double bond configuration, absolute configuration,and one or several stereocenters assigned incorrectly [59]. To avoid"rediscovery of the known"more specialized and effective dereplication strategies need to be employed.With over 150,000 small molecules characterized from natural sources,previously known natural compounds are often re-isolated during bioassay-guided fractionation and that should be avoided [29].Hyphenated technics such as liquid chromatography with ultraviolet detection (LC-UV),liquid chromatography-mass spectrometry (LC-MS,LC-MS/MS)or LC-NMR are valuable toos for the dereplication process,especially if used early in the prefractionation step [60].Access to suitable databases is essential for the rapid dereplication of crude extracts in natural product research. Several commercial databases are available to implement the dereplication process,from which the most comprehensive ones are:Chemical abstracts,including NAPRALERT,Beilstein,AntiBase [61] (>40,000 natural compounds from micro-organisms and higher fungi),MarinLit (-24,000 marine compounds isolated from approximately 6000 species)[62]Chapman&Hall's Dictionary of Natural Products (~170.000 compounds from both marine and terrestrial organisms)63]and NAPROC-13 ('CNMR spectral information of over 6000 natural compounds)[641. When using pure natural compound libraries,virtual screening is also a possibility that must be stressed out.Virtual screening can be used for browsing databases in the quest for molecules fitting either an established pharmacophore model or a three dimensional structure of a macromolecular target.The advantages of this approach overinro sereening are obvious:higher capacity,no need for physically isolating the compounds,less time-consuming and expensive and theoretically interactions of all compounds to all structurally defined targets can be calculated and predicted Additionally early evaluation of absorption.distribution.metabolism.and excretion/toxicity in pharmacokinetics(ADMET)properties is also possible [65].But,because virtual screening is only a predictive tool,in the case of NP it is important the integration with traditional avenues,gathering information from bioassay guided fractionation,on-line analytical activity an optimization of drug lead discovery 65. Finally.upon the identification of a lead it is necessary to understand its mode of action against the specific target.This includes secondary testing in which molecular and cellular techniques are normally applied.This identification constitutes a major challenge but is becoming more and more compulsory in both pharmaceutical and cosmeceutical industries
Mar. Drugs 2014, 12 1073 Additionally, the synthesis or hemisynthesis of a bioactive natural compound must be supported by a correct identification of the compound isolated from the biological source. Despite the fact that modern methodologies of structural elucidation are well advanced, errors can never be completely ruled out. In fact, there are numerous structural revisions reported in literature, even of recently elucidated NP. In average, per 5-year period, 369 NP and 135 MNP are misassigned [58]. The structural complexity of the isolated compounds and the small amount of samples, especially in the case of compounds from marine sources, can contribute to misassignments which can be divided in several categories: incorrect formula, constitution (planar connectivity), double bond configuration, absolute configuration, and one or several stereocenters assigned incorrectly [59]. To avoid ―rediscovery of the known‖ more specialized and effective dereplication strategies need to be employed. With over 150,000 small molecules characterized from natural sources, previously known natural compounds are often re-isolated during bioassay-guided fractionation and that should be avoided [29]. Hyphenated technics such as liquid chromatography with ultraviolet detection (LC-UV), liquid chromatography-mass spectrometry (LC-MS, LC-MS/MS) or LC-NMR are valuable tools for the dereplication process, especially if used early in the prefractionation step [60]. Access to suitable databases is essential for the rapid dereplication of crude extracts in natural product research. Several commercial databases are available to implement the dereplication process, from which the most comprehensive ones are: Chemical abstracts, including NAPRALERT, Beilstein, AntiBase [61] (>40,000 natural compounds from micro-organisms and higher fungi), MarinLit (~24,000 marine compounds isolated from approximately 6000 species) [62], Chapman & Hall‘s Dictionary of Natural Products (~170,000 compounds from both marine and terrestrial organisms) [63] and NAPROC-13 ( 13CNMR spectral information of over 6000 natural compounds) [64]. When using pure natural compound libraries, virtual screening is also a possibility that must be stressed out. Virtual screening can be used for browsing databases in the quest for molecules fitting either an established pharmacophore model or a three dimensional structure of a macromolecular target. The advantages of this approach over in vitro screening are obvious: higher capacity, no need for physically isolating the compounds, less time-consuming and expensive and theoretically, interactions of all compounds to all structurally defined targets can be calculated and predicted. Additionally early evaluation of absorption, distribution, metabolism, and excretion/toxicity in pharmacokinetics (ADMET) properties is also possible [65]. But, because virtual screening is only a predictive tool, in the case of NP it is important the integration of in silico screening with traditional avenues, gathering information from bioassay guided fractionation, on-line analytical activity profiling, ethnopharmacological screening, if it is the case, and chemoinformatics, in order to achieve an optimization of drug lead discovery [65]. Finally, upon the identification of a lead it is necessary to understand its mode of action against the specific target. This includes secondary testing in which molecular and cellular techniques are normally applied. This identification constitutes a major challenge but is becoming more and more compulsory in both pharmaceutical and cosmeceutical industries
Mar.Drugs 2014,12 1074 3.3.Market Challenges Finally the commercial and market issues are very relevant and most of the times disregarded in the natural product development programs.Since the very early stages of the development programs. several very important questions must be addressed by the researchers or the companies:(i)what are the potential industry applications and the market need of that particular activity;(ii)what is the target price/kg of the final bioactive (i)what is the formulation desired and the route of administration (iv)what is the manufacturing process and how sustainable is the supply:(v)how can the product reach the market value chain.The high number of NP hits and leads coming out of the HTS technologies has stressed out the need for a focused strategy on this field.Small and Medium Enterprises (SME's)have a commercialization goal and,therefore,introduce very early on their discovery and development programs the issues indicated earlier.It is crucial for them to have a clearly defined strategy,otherwise the risk of failing and running out of cash fast is high.It is important to be aware that the cost of technology and manufacturing processes,sometimes with poo yields,raises the market cost per kilogram and may render these products economically unviable.This is particularly true in personal care industry where recombinant technologies are not acceptable and the profit margins are too small to introduce very expensive ingredients per pack.Recently,academia has started to be more aware of the "market issues as it became obvious that most of the discoveries on the NP pipeline where barely reaching the market and consumers.This has been partially achieved by serious encouragement of industry-academia partnerships,both at national and transnational levels that went beyond the traditional funding by the industry of small research projects of academia.These alliances benefit both partners that work side-by-side,with a common set of goals and in a win-win collaboration system.Academia gets knowledge.publications and funding and industry gets new NP with higher probabilities of market success. Bearing in mind all the challenges just pointed out,NP developers can thrive to find better models of development to surpass or minimize their impact. 4.Marketed Marine Natural Products.Examples of Success Stories The Marine Board of the European Science Foundation has published a position paper in which it provides a roadmap for European research in Marine Biotechnology and sets out an ambitious scienc and policy agenda for the next decade [66].Development of novel drugs,treatments and health and personal care products,is one of the five research areas prioritized in this document that can greatly contribute to key societal challenges An overview of marine drugs and cosmeceuticals that successfully reached the market is the focus of next section.Hopefully,the analysis of the issues related with their development will allow a deeper understanding of the key factors behind their success 4.1.Pharmaceutical Applications As pointed earlier,natural product screening remains one of the most useful avenues for bioactive discovery.In the past decades,studies on MNP have been focused mainly on macroorganisms,i.e., sponges,corals and other marine invertebrates,although significant developments have been made in
Mar. Drugs 2014, 12 1074 3.3. Market Challenges Finally the commercial and market issues are very relevant and most of the times disregarded in the natural product development programs. Since the very early stages of the development programs, several very important questions must be addressed by the researchers or the companies: (i) what are the potential industry applications and the market need of that particular activity; (ii) what is the target price/kg of the final bioactive; (iii) what is the formulation desired and the route of administration; (iv) what is the manufacturing process and how sustainable is the supply; (v) how can the product reach the market value chain. The high number of NP hits and leads coming out of the HTS technologies has stressed out the need for a focused strategy on this field. Small and Medium Enterprises (SME‘s) have a commercialization goal and, therefore, introduce very early on their discovery and development programs the issues indicated earlier. It is crucial for them to have a clearly defined strategy, otherwise the risk of failing and running out of cash fast is high. It is important to be aware that the cost of technology and manufacturing processes, sometimes with poor yields, raises the market cost per kilogram and may render these products economically unviable. This is particularly true in personal care industry where recombinant technologies are not acceptable and the profit margins are too small to introduce very expensive ingredients per pack. Recently, academia has started to be more aware of the ―market issues‖ as it became obvious that most of the discoveries on the NP pipeline where barely reaching the market and consumers. This has been partially achieved by serious encouragement of industry-academia partnerships, both at national and transnational levels that went beyond the traditional funding by the industry of small research projects of academia. These alliances benefit both partners that work side-by-side, with a common set of goals and in a win-win collaboration system. Academia gets knowledge, publications and funding and industry gets new NP with higher probabilities of market success. Bearing in mind all the challenges just pointed out, NP developers can thrive to find better models of development to surpass or minimize their impact. 4. Marketed Marine Natural Products. Examples of Success Stories The Marine Board of the European Science Foundation has published a position paper in which it provides a roadmap for European research in Marine Biotechnology and sets out an ambitious science and policy agenda for the next decade [66]. Development of novel drugs, treatments and health and personal care products, is one of the five research areas prioritized in this document that can greatly contribute to key societal challenges. An overview of marine drugs and cosmeceuticals that successfully reached the market is the focus of next section. Hopefully, the analysis of the issues related with their development will allow a deeper understanding of the key factors behind their success. 4.1. Pharmaceutical Applications As pointed earlier, natural product screening remains one of the most useful avenues for bioactive discovery. In the past decades, studies on MNP have been focused mainly on macroorganisms, i.e., sponges, corals and other marine invertebrates, although significant developments have been made in
Mar.Drugs 2014,12 1075 the microorganisms area.However,despite the large number of NCEs isolated from marine organisms many of them with pronounced biological activity,the great majority does not surpass the pharmaceutical pre-clinical trials and only a very few have been marketed as pharmaceutical products. Besides the usual drawbacks in any drug discovery process,the industrial development of many promising MNP was hampered by additional difficulties such as sustainable source and issues related to structural complexity and scale up.Nevertheless,the global marine pharmaceutical pipeline remains very active and includes,at the moment,eight Food and Drug Admnistration(FDA)or European Medicines Agency (EMEA)approved drugs and several compounds in different phases of the clinical pipeline [67].From the eight compounds currently on the market (Figure 1),only three (Prialt while the rest of them suffered lead optimization,in different steps of their development.Overall,from lead discovery to the entry in the market it took 20 to 30 years.Ensuring natural product supply on an industrial scale.optimization of formulation and ADMET properties were the main blockades faced by pharmaceutical companies.Optimization of NP by structural modifications,synthetic supply of unmodified natural molecule or immunoconjugation of NP was the strategy behind these successful stories.The history of the development of the market drugs will be discussed in this subsection with a focus on the approaches tackled by the pharmaceutical companies that succeeded in marketing their products Figure 1.Chemical structures of marine drugs on the market divided by therapeutic area. Cance N-C ypertriglyceridemi
Mar. Drugs 2014, 12 1075 the microorganisms area. However, despite the large number of NCEs isolated from marine organisms, many of them with pronounced biological activity, the great majority does not surpass the pharmaceutical pre-clinical trials and only a very few have been marketed as pharmaceutical products. Besides the usual drawbacks in any drug discovery process, the industrial development of many promising MNP was hampered by additional difficulties such as sustainable source and issues related to structural complexity and scale up. Nevertheless, the global marine pharmaceutical pipeline remains very active and includes, at the moment, eight Food and Drug Admnistration (FDA) or European Medicines Agency (EMEA) approved drugs and several compounds in different phases of the clinical pipeline [67]. From the eight compounds currently on the market (Figure 1), only three (Prialt® , Yondelis® and Carragelose® ), became drugs without any modification of the original natural molecule, while the rest of them suffered lead optimization, in different steps of their development. Overall, from lead discovery to the entry in the market it took 20 to 30 years. Ensuring natural product supply on an industrial scale, optimization of formulation and ADMET properties were the main blockades faced by pharmaceutical companies. Optimization of NP by structural modifications, synthetic supply of unmodified natural molecule or immunoconjugation of NP was the strategy behind these successful stories. The history of the development of the market drugs will be discussed in this subsection with a focus on the approaches tackled by the pharmaceutical companies that succeeded in marketing their products. Figure 1. Chemical structures of marine drugs on the market divided by therapeutic area