“The Universe is full of magical things patiently waiting for our wits to grow sharper.” Eden Phillipotts, A Shadow passes
The legend says that a long time ago, in the part of the world that is now known as Peru, a raging storm a felled giant tree that came to rest in a pool of stagnant water. Eventually, a native passed that way. He was extremely ill, burning with fever, having what we call today malaria. His fever had caused intense thirst and he drank from the pond. Shortly after, a miracle occurred and his fever vanished.
From the dawn of history, people turned to nature in the attempt to cure diseases. Primitive people discovered that cinchona bark cured intermittent fever. The coca leaves numbed the tongue and reduced the appetite and the latex from the capsule of the opium poppy allayed pain. Extensive lists of these natural products have survived from antiquity and they are of considerable interest, because of the many well-known products they contain. The Chinese Pen Ts’ao, written in 2800 B.C. lists 366 plant drugs, among them the familiar Ephedra. The Egyptian Papyrus Ebers, dating from 1550 B.C., mentions opium and aloes, among others. The economy of these ancient cultures depended on the commerce with natural products, being considered ones of the most expensive products at that time. The Greek and Romans traded them widely and the European colonial expansion was influenced by the discovery of rich territories in natural products.
For more than fourteen centuries, the natural products extracted from plants reigned supremely in medicine, but then a Swiss pharmacist, known as Paracelsus (around year 1500) introduced a new dimension to drug therapy. He advocated the use of chemical therapy and he challenged the alchemists to prepare medicines, not gold. Although the field was slow to develop, medicinal chemistry was born with Paracelsus.
Conventional therapy with the plant drugs continued almost unchanged for three more centuries. Then, around year 1800, a small-town pharmacist in Germany attempted and succeeded the isolation of the active principle of opium, which he called morphine after Morpheus, the Greek god of dreams. He found the chemical alkaline in character, the first of a compound class later called alkaloids. The search for alkaloids continued well into the twentieth century. The most famous compounds extracted from plants in the treatment of tumors are vinca alkaloids (Vincristine), from the Catharanthus roseus (Madagascar periwinkle)[1],[2] and taxane (Taxol), isolated the compound from the bark of the Pacific yew tree Taxus brevifolia.[3],[4]
It was not until 1870s, however, that Tyndall, Pasteur and Roberts separately observed the antagonistic effects of one microbe upon another. Pasteur, with his characteristic foresight, suggested the therapeutic potential of the phenomenon. For the next half-century, various microbial preparations were tested as medicines, but they were either too toxic or inactive. Finally, in 1929, Fleming published his historic observation that a contaminating mold, identified as Penicillium notatum killed his bacterial culture of Staphylococcus aureus. He named the active substance Penicillin. The importance of Fleming’s discovery was that it led to the first successful chemotherapeutic agent produced by a microbe, thus initiating the golden age of antibiotics. Soil microbiologists succeeded in isolating many new antibiotics from soil-inhabiting bacteria, i.e. actinomycetes, of which the best known is Streptomycin, the first active antibiotic against tuberculosis.
By 2002, over 22.000 bioactive compounds have been discovered from microbes. Of the actinomycete antibiotics about 80% are made by members of the genus Streptomyces. Two of the compounds with anti tumor activity, isolated from Streptomyces and involved in clinical trials are amrubicin 3, isolated from Streptomyces peucetius, and geldanamycin 4, isolated from Streptomyces hygroscopicus.[5]
Soil has the largest population of microbes of any habitat, but only about 0.3% of soil microbes are cultivable with current techniques. Cultured soil microbes have been an incredibly productive source of drugs for cancer chemotherapeutics. Unfortunately, the current yield of new drugs of soil microbes is low due to repeated cultivation of the same small fraction of cultivable microbes.[6]
This problem is being studied and some success has been achieved by using one or more of the following strategies:
1. very low nutrient concentration,
2. signaling molecules,
3. inhibitors of undesired microbes,
4. long periods of incubation,
5. growth conditions resembling the natural environment,
6. protection of cells from exogenous peroxides,
7. addition of humic acid,
8. hypoxic or anoxic atmospheres,
9. encapsulation of cells in gel microdroplets and detection of microcolonies by flow cytometry,
10. high CO2 concentration along with high throughput polymerase chain reaction technology.
Chemists on a worldwide basis have turned their attention toward the potential of marine microorganisms as an alternative source for isolation of novel metabolites with interesting biological and pharmaceutical properties. The world oceans do indeed represent a microbial broad and microbiologically diverse resource of huge dimension but about which we know relatively little. As it is estimated that less than 5% of marine bacterial and fungal species are known, it is clear that the microbial diversity of oceans is still poorly understood. In the area of natural products chemistry, only a few early reviews have covered the small number of metabolites derived from marine fungi.[7]
Marine microorganisms continue to be the subject of vigorous chemical investigation, although the studies of marine bacteria might be decreasing in comparison with those of other microorganisms. Studies of marine fungi appear to be expanding at a much faster rate than those of other unicellular organisms.[8]
A statistic analysis of the National Cancer Institute shows that marine flora prove to be a very effective source for cytotoxic compounds. Their percentage is with 2% significantly higher than terrestrial plants (approximatively 0.3%), although the latter have been explored three times more.[9]
With several marine-derived now in clinical trials and others on the way, ocean products may be outstripping land-based plants and microorganisms as promising sources of potential anti tumor drugs.[10]
The explanation for the fact that the marine habitat is the place for interesting bioactive materials is very simple: the conditions that dominate the aquatic environment differentiate themselves seriously from the terrestrial counterpart. The influence of the seawater conditions – density, viscosity and high specific heat, pressure, salt content (specific pressure), lighting conditions (high irradiation at the surface, obscurity in the depth create different properties in the marine organisms. The oceans cover more than two thirds of the earth’s surface, include 90% of the biosphere and therefore represent the biggest habitat of our planet, with a high level of composition diversity. This diversity includes a high number of sessile invertebrate organisms, for example sponges (poriferans), corals (cnidaria), moss organisms (bryozoans). Their lifestyle is facilitated by the marine currents that supply them with all necessary nutrients, still being subject to a competitive rivalry, including shortage of space, nutrition, natural cover and predators. Having a sedentary and an impossibility of changing their location, they have developed effective chemical defense mechanisms that lead to the desired marine secondary metabolites with their multitude of pharmacological properties, among which the cytotoxic ones have a special meaning. Because of the diluting effect of seawater, the effect has to be exquisitely potent.
This enormous interest in potential cytostatics is based on the fact that one from three persons of the first world would eventually develop cancer at one point in their lives. Taking the family and friends in consideration, the number of people affected increases dramatically. Although not every case leads to death, still in year 2000 alone there were about 10 million incidences of malign tumors of which 6.2 million fell prey to it. In Europe, the percentage is about a third, meaning 2.8 millions new patients and 1.7 millions deaths. Taking in consideration the current trend of an aging population, the unhealthy lifestyle, the World Health Organization estimated that in the next 20 years the figures would increase with 50%, meaning 10 million people. In the last couple of years in some developed countries, the mortality rate decreased due to a better diagnostic and medication. In the search for new effective and innovative active substances, the marine biological resources have been strongly taken into consideration. With the development of modern analysis methods, thousands of substances from the entire flora and fauna have been tested for pharmacological activity, by high-throughput screening. When an active substance is identified by screening and proved to meet the expectations, bigger quantities of the compound are necessary. This aspect is associated with significant problems, because of the small concentration in the host organism and often the extremely complicated chemical structure. Serious shortcomings can be encountered on the market launch, despite successful medical studies. For the approval of a medicine, besides the successful medical studies, a sufficient provision of the active substance is necessary (approximatively one kilogram is necessary before any medical studies, depending on the effectiveness of the substance). In order to overcome this obstacle, an economically meaningful and efficient method of fabrication or isolation has to be developed. In the industrial production, the following possibilities are conceivable in principle:
1. The exploitation of the natural resources,
2. the cultivation of marine organisms and microorganisms, respectively (aquaculture, fermentation),
3. in vitro in cell cultures of the organism,
4. production by genetically modified third organisms like, for example, E. coli or giant hamster cells,
5. synthesis of bioactive molecules in the laboratory.
The advantages for each of these methods preponderate differently. For a conclusion, exact knowledge of population dynamic and ecology are necessary. The cultivation in the laboratory is problematic for many organisms that often produce the desired active agent only under certain conditions. An open aquaculture in offshore-waters is connected with a total loss risk. Both pure biotechnological methods are still in the incipient phase of development and still struggling with children’s diseases that will be illustrated in the following examples. A real alternative for the biological and biotechnical methods is offered by the chemical synthesis. The more complicated the molecule is, the more unlikely is a synthesis to be proven valuable in the laboratory. An additional possibility is the synthesis of analogues with the same efficacy spectrum, simpler in their structure, with a better pharmacological profile in the same time, translated in a higher efficacy and reduction of side effects.[11]
Recently, the attention is oriented not only at the microorganism, but also at the genes within it that make a specific compound. For example, the genes responsible for the synthesis of patellamides (cytotoxic cyclic peptides from a tunicate) have been cloned. To produce these peptides, the researchers inserted the cloned patellamide genes into E. coli. As E. coli reproduced, it also produced Patellamide. Developing genetic engineering approaches may be the next step in moving drug discovery and development forward. Chemists may be able to use these genetic techniques in tandem with chemical synthesis to make complicated molecules with fewer steps.[10]
The first modern marine-derived drugs date back more than 50 years. Among the first bioactive compounds from marine sources, spongouridine and spongothymidine from the Carribean sponge (Cryptotheca crypta) were isolated serendipitously in the early 1950s.[11]
They were approved as an anti-cancer drug (cytosine arabinoside, Ara-C) and an anti-viral drug (adenine arabinoside, Ara-A), respectively, 15 years later. Ara-C was approved for the treatment of certain leukemia kinds in 1969, making it the first such approved marine-derived drug for use in cancer chemotherapy. The secondary metabolites of marine organisms have been studied extensively over the past 30 years. Drug discovery research from marine organisms has been accelerating and now involves interdisciplinary research including biochemistry, biology, ecology, organic chemistry and pharmacology.[13], [14]
Another example is ecteinascidin 743 (ET-743, 6)[15] (Scheme 3) that was isolated from a Carribean mangrove tunicate species, Ectenaiscidia turbinata. The relative abundance and the ease of collection of E. turbinata also played a role in selection of this natural product as a drug development candidate. The special advantage of this compound is the absence of the common side effects in chemotherapy. ET-743 is currently obtained through aquaculture of the tunicates and semi-synthetically. A gram of pure substance can be isolated from a ton of wet mass containing the active substance.
Many times there is the dispute between natural versus synthetic drugs in curing a disease like cancer. Nature and laboratory have always coexisted, in this matter. The border between natural and synthetic is not completely delimited. Synthetic drugs have been always inspired from the miracles the nature provides. Nature still blows Man’s mind away.
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[3] I. Ojima, J. Med. Chem. (Book Review) 1996, 39, 807-807.
[4] C. Kaiser, J. Med. Chem. (Book Review) 2001, 44, 3335-3336.
[5] Y. Chin, M. J. Balunas, H. B. Chai, A. D. Kinghorn, The AAPS Journal 2006, 8, 239-253.
[6] R. K. Pettit, Cancer Chemother. Pharmacol. 2004, 54, 1-6.
[7] M. A. Farooq Biabani, H. Laatsch, J. prakt. Chem. 1998, 340, 589-607.
[8] D. J. Faulker, Nat. Prod. Rep. 2001, 18, 1-49.
[9] M. H. G. Munro, J. W. Blunt, E. J. Dumdei, S. J. H. Hickfort, R. E. Lill, L. Shangxiao, C. N. Battershill, A. R. Duckworth, J. Biotechnol. 1999, 70, 15-25.
[10] L. H. O’Hanlon, Journal of National Cancer Institute 2006, 98, 662-663.
[11] N. Cramer, Dissertation, Universitt Stuttgart, 2005.
[12] D. J. Newman, G. M. Craigg, Curr. Med. Chem. 2004, 11, 1693-1713.
[13] D. J. Newman, G. M. Craigg, J. Nat. Prod. 2004, 11, 1693-1713.
[14] R. J. Carpon, Eur. J. Org. Chem. 2001, 633-645.
[15] K. L. Rinehart, T. G. Holt, N. L. Fregeau, P. A. Kiefer, G. R. Wilson, T. J. Perun Jr., R. Sakai, A. G. Thompson, J. G. Sthroh, L. S. Shield, D. S. Seigler, L. H. Li, D. G. Martin, C. J. P. Grimmelikhuijzen, G. J. Gde, J. Nat. Prod. 1990, 53, 771-792.
