The purpose of this article is to review the 2003-4 primary literature on pharmacological studies with marine natural products using the same format as in our previous reviews of the marine pharmacology peer-reviewed literature (Mayer and Lehmann, 2000), (Mayer and Hamann, 2002, 2004, 2005). Consistent with our previous reviews, only those articles reporting on the bioactivity or pharmacology of 166 marine chemicals whose structures have been established are included in the present review. As in our previous reviews, we have used Schmitz's chemical classification (Schmitz et al., 1993) to assign each marine compound to a major chemical class, namely, polyketides, terpenes, nitrogen-containing compounds or polysaccharides. Those publications reporting anthelminthic, antibacterial, anticoagulant, antifungal, antimalarial, antiplatelet, antiprotozoal, antituberculosis or antiviral properties of 67 marine chemicals have been tabulated in Table 1 with the corresponding structures shown in Fig.1. The articles reporting on 45 marine compounds affecting the cardiovascular, immune and nervous systems, as well as those with anti-inflammatory effects are grouped in Table 2 and the structures presented in Fig. 2. Finally 54 marine compounds targeting a number of distinct cellular and molecular targets and mechanisms are shown in Table 3 and their structures depicted in Fig. 3. Publications on the biological or pharmacological activity of marine extracts or as yet structurally uncharacterized marine compounds have been excluded from the present review, although several promising reports were published during 2003-4: a specific inhibitor of a thyrotropin releasing hormone-specific peptidase (Pascual et al., 2004); antimicrobial activity in sub-Arctic marine invertebrates (Lippert et al., 2003); antifilarial activity of the red alga Botryocladia leptopoda (Lakshmi et al., 2004); antiviral effects of a sulfated exopolysaccharide from the marine microalga Gyrodinium impudicum (Yim et al., 2004) and Sargassuum patens (Zhu et al., 2004); a polyhydroxylated fucophlorethol isolated from the marine brown alga Fucus vesiculosus shown to be bactericidal towards selected Gram-positive and Gram-negative bacteria in vitro (Sandsdalen et al., 2003); and an improvement of “current cytokine-based therapies” by sulphated polysaccharides purified from the green alga Codium fragile, as well as fucoidan and carrageenan, isolated from brown and red algae, respectively (Nika et al., 2003).

Table 1 Marine pharmacology in 2003-4: Marine Compounds with Anthelmintic, Antibacterial, Anticoagulant, Antifungal, Antimalarial, Antiplatelet, Antiprotozoal, Antituberculosis, and Antiviral Activities.

Figure 1

Table 2 Marine pharmacology in 2003-4: Marine Compounds with Anti-inflammatory activity, and affecting the Cardiovascular, Immune and Nervous Systems.

Figure 2

Table 3 Marine pharmacology in 2003-4: Marine Compounds with Miscellaneous Mechanisms of Action.

Figure 3

Table 1 summarizes new pharmacological findings reported during 2003-4 on the preclinical anthelmintic, antibacterial, anticoagulant, antifungal, antimalarial, antiplatelet, antiprotozoal, antituberculosis, and antiviral pharmacology of the 67 marine natural products shown in Fig. 1.

In view of the fact that resistance to current antibiotics remains a significant challenge for pathogenic bacterial infections, during 2003-4, 19 studies contributed to the search for novel antibacterial marine natural products, an increase from 1998-2002 (Mayer and Lehmann, 2000; Mayer and Hamann 2002, 2004, 2005). Four studies reported on the mechanism of action of novel marine antibacterial agents (2-6; Fig. 1). Bugni et al., (Bugni et al., 2004) investigated a series of kalihinols, diterpenes isolated from the Philippine marine sponge Acanthella cavernosa, as potential bacterial folate biosynthesis inhibitors. The investigators reported that the pyranyl-type kalihinols Y (5) and X (6), although potent antibacterials (MIC=1.56 μg/mL), were however less selective inhibitors of bacterial folate biosynthesis than the furanyl type kalihinols, with the “C-10 position important for potency”. Isnansetyo and Kamei (Isnansetyo and Kamei, 2003) reported that a bactericidal compound named MC21-A (7), a 3,3′,5,5′-tetrabromo-2,2′-biphenyldiol, from the new marine bacterium Pseudoalteromonas phenolica sp. nov. MC21-A was bactericidal (MIC=1-2 μg/mL) against 10 clinical isolates of methicillin-resistant Staphylococcus aureus, and displayed comparable bioactivity to vancomycin (MIC=0.25-2 μg/mL). The mechanism of action of MC21-A involved permeabilizing bacterial cell membranes, and thus “might be a useful compound” because of a mode of action that differs from vancomycin. A new dimeric bromopyrrole alkaloid, nagelamide G (8) was isolated from the Okinawan marine sponge Agelas sp. (Endo et al., 2004). Nagelamide G exhibited antibacterial activity against M. luteus, B. subtilis and E. coli, but weakly inhibited protein phosphatase 2A (IC₅₀=13 μM), thus suggesting that this enzyme may not be the main molecular target responsible for the antibacterial activity of this compound. Tincu et al. (Tincu et al., 2003) reported a new antimicrobial octapeptide plicatamide (9) from the hemocytes of the marine tunicate Styela plicata. In an extensive and detailed mechanistic study these investigators discovered that despite its small size, the octapeptide plicatamide proved to be a potent, rapidly acting and broad spectrum antimicrobial. The fact that both wild type and methicilin-resistant S. aureus responded to plicatamide with a massive and rapid potassium efflux is “consistent with an antimicrobial mechanism that targets their cell membrane”.

Although additional novel marine antibacterials were reported in 2003-4, no mechanism of action studies were reported for compounds (10-29). Nevertheless, these studies highlight the fact that novel antibiotics are present in marine bacteria, tunicates, sea hares, soft corals, algae, sponges, worms, and fish. Two papers reported on antibacterial activity in compounds isolated from marine sponges: Namikoshi et al. (Namikoshi et al., 2004) reported the isolation of several manoalide derivatives (10) from a Luffariella sp. sponge collected in Palau, which were active against S. aureus at 5-10 μg/disk. The investigators noted that the presence of an “OH group at the C-25 position (hemiacetal moiety) is important for antibacterial activity.” Wang et al. (Wang et al., 2003) reported thirteen novel tetramic acids isolated from the marine sponge Melophlus sarassinorum. Interestingly, only melophlin C (11) displayed “pronounced antibacterial activity” against B. subtilis and S. aureus. One paper reported on new antimicrobial compounds isolated from marine tunicates: Schupp et al. (Schupp et al., 2003) discovered that the β-carboline eudistomin X (12), isolated from the Micronesian ascidian Eudistoma sp. was active against B. subtilis, S. aureus and E. coli. One paper reported on a new antimicrobial peptide isolated from sea hares: Iijima et al. (Iijima et al., 2003) reported a novel 33 amino acid antimicrobial peptide dolabellanin B2 (13) from the sea hare Dolabella auricularia. One hundred percent inhibition of growth of B. subtilis, H. influenza and Vibrio vulnificus was reported with 2.5-5 μg/mL dolabellanin B2. Two papers reported on new antimicrobial peptides isolated from marine soft corals: Ata et al. (Ata et al., 2004) reported two new diterpenes, pseudopterosin X and Y (14-15) from the soft coral Pseudopterogorgia elisabethae which showed antibacterial activity against Gram-positive bacteria Streptococcus pyogenes, S. aureus, and Enterococcus faecalis, while being inactive against Gram-negative bacteria. Dmitrenok et al. (Dmitrenok et al., 2003) reported several sphingolipids and glycolipids (16-18) from soft corals of the Andaman Islands (Indian Ocean). Although the MIC were not reported, “preliminary tests for antibacterial activity of lipids” demonstrated that these compounds inhibited the growth of E. coli, P. aeruginosa, B. subtilis and B. pumilus on solid agar. One paper reported on the presence of antibacterial compounds in marine algae: Xu et al. (Xu et al., 2003) reported that among 5 bromophenols isolated from the marine red alga Rhodomela confervoides, the known compound bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (19) showed antibacterial activity against S. aureus (MIC=70 μg/mL), Staphylococcus epidermidis and Pseudomonas aeruginosa (MIC=70 μg/mL). Additional antibacterial marine natural products were isolated from sponges: Pettit et al. (Pettit et al., 2004) reported the antibacterial activity of a novel nitrogen heterocyclic compound cribrostatin 6 (20) isolated from the dark-blue marine Cribochalina sp. sponge. Cribrostatin 6 showed antibacterial activity against Gram-positive bacteria, and it was most active against S. pneumoniae (MIC= 0.5 μg/mL), a leading cause of infection and mortality worldwide. Goud et al. (Goud et al., 2003b) reported a novel purpuramine L (21) from the Indian marine sponge Psammaplysilla purpurea which was active against S. aureus, B. subtilis and C. violaceum. A new nitrogenous sesquiterpene germacrane (22) was isolated from an Axinyssa n. sp. sponge that demonstrated strong antimicrobial activity against S. aureus and B. subtilis (Satitpatipan and Suwanborirux, 2004). Yang et al. (Yang et al., 2003c) isolated a new bicyciclic guanidine alkaloid (23) from the marine sponge Ptilocaulis spiculifer, contributing a new member to the crambescin A class of compounds. Interestingly, 50 μg of the guanidine alkaloid was as potent as 10 μg gentamicin. Two diterpenes membranolides C and D (24, 25) derived from an Antarctic cactus sponge, displayed “modest yet broad spectrum” Gram-negative antibiotic activity (Ankisetty, S. et al. 2004). Two novel antibacterial peptides were isolated from marine worms: Ovchinnikova et al. (Ovchinnikova et al., 2004) purified and characterized two small 21-residue peptides arenicin-1 and -2 (26-27), from the coelomocytes of the marine lugworm Arenicola marina. Both arenicins were active against Gram-positive L. monocytogenes, Gram-negative E. coli and the fungus C. albicans. Pan et al. (Pan et al., 2004) isolated and characterized a 51-amino acid highly basic and hydrophobic peptide perinerin (28), from the marine clamworm Perinereis aibuhitensis, an organism that is extensively used as bait in fisheries and aquaculture. Perinerin, a peptide that is constitutively present in the marine worm and whose sequence appears to be novel among all know antimicrobial peptides, was active against Gram-negative and Gram-positive bacteria as well as fungi. Patrzykat et al. (Patrzykat et al., 2003b) reported active novel antimicrobials peptides by screening both genomic and mRNA transcripts from a number of different species of flatfish. The most active peptide coded as NRC-13 (29) which was derived from the American plaice Hippoglossoides platessoides Frabricius, “rapidly (5 to 10 min) and efficiently (95-100%)” killed antibiotic-resistant P. aeruginosa, methicillin-resistant S. aureus and C. albicans.

Several novel marine antifungals (34-38) were isolated from marine sponges. Yang et al. (Yang et al., 2003b) reported a new sterol sulfate (34) isolated from a deep-water marine sponge of the family Astroscleridae, which exhibited antifungal activity against “supersensitive” Saccharomyces cerevisiae (MIC=15 μg/ml). As part of an ongoing project to discover potential new drugs to treat resistant opportunistic fungal infections, Jacob et al. (Jacob et al., 2003) reinvestigated the antifungal properties of a previously described sterol (35) isolated from the marine sponge Dysidea arenaria. Interestingly, they observed a reversal of fluconazole resistance from 300 to 8.5 μM when combined with 3.8 μM of the Dysidea arenaria sterol, putatively as a result of inhibition of the MDR1-type efflux pump in multidrug-resistant C. albicans. With the purpose of finding more selective antifungal agents, Nishimura et al. (Nishimura et al., 2003) focused their research efforts in identifying inhibitors of the pathogenic fungus C. albicans geranylgeranyltransferase (GGTase), an enzyme that shares only 30% amino acid sequence homology with the human GGTase. Bioassay-guided fractionation resulted in the isolation of a novel alkaloid massadine (36) from the marine sponge Stylissa aff. massa, which inhibited fungal GGTase (IC₅₀=3.9 μM). One novel imidazole alkaloid, naamine G (37) was reported from the Indonesian marine sponge Leucetta chagosensis that exhibited strong antifungal activity against the phytopathogenic fungus Cladosporium herbarum (Hassan et al., 2004). It remains to be determined if this compound will also be effective against fungi that infect mammalian hosts. Rifai et al. (Rifai et al., 2004) reported that untenospongin B (38), isolated from the Moroccan marine sponge Hippospongia communis, was more potent than amphotericin B, a clinically used antifungal agent, against Candida tropicalis (MIC=4-8 μg/mL) and Fusarium oxysporum (MIC=2-4 μg/mL). Further studies are required to determine the toxicity of untenospongin B in vivo as well as its molecular mechanism of action.

Kossuga et al. (Kossuga et al., 2004) reported a new antifungal agent polyketide, (2S, 3R)-2-aminododecan-3-ol (39), isolated from the Brazilian ascidian Clavelina oblonga, which was very active against C. albicans (MIC=0.7± 0.05 μg/mL). Although the mechanism of action of this compound remains undetermined its bioactivity was comparable to the clinically used antifungal agents nystatin (MIC=1-4 μg/mL) and ketoconazole (MIC=1.0-4.0 μg/ml).

Three compounds were shown to possess antiprotozoal activity. Nakao et al. (Nakao et al., 2004) reported the isolation of renieramycin A (50) a new compound from the Japanese sponge Neopetrosia sp. that dose-dependently inhibited recombinant Leishmania amazonensis proliferation (IC₅₀=0.2 μg/mL) while showing cytotoxicity at “ten times higher concentration (IC₅₀=2.2 μg/mL)”. Savoia et al. (Savoia et al., 2004) examined the activity of the sesquiterpene euplotin C (51), isolated from the marine ciliate Euplotes crassus on pathogenic protozoa Leishmania major and Leishmania infantum. Because a significant leishmanicidal activity was noted against both Leishmania species (LD₅₀=4.6-8.1 μg/mL), the authors proposed evaluation of this natural product as “synergistic compound(s) for current antiprotozoon chemotherapeutics”. Roch et al. (Roch et al., 2004) reported that novel non-cytotoxic variant analogues of truncated defensins isolated from the Mediterranean mussel Mytilus galloprovincialis were antiprotozoal. Two defensin fragments, designated D (Sequence CGGWKRKRC) and P (Sequence CGGYCGGWKRKRCTSYRCG), killed both the African trypanosome Trypanosoma brucei (ID₅₀=4-12 μM), which causes sleeping sickness, and the causative agent of cutaneous leishmaniasis, namely Leishmania major (ID₅₀=12-45 μM), in a time- and concentration-dependent manner. The mechanism may involve binding of the defensin fragments “to parasite membranes”, perhaps affecting membrane fluidity.

Seven novel compounds (47, 52-57; Fig. 1) were contributed to the search for novel antituberculosis agents. Rodriguez et al. (Rodriguez and Rodriguez, 2003) reported that a novel diterpene alkaloid homopseudopteroxazole (52), isolated from the Caribbean sea plume Pseudopterogorgia elisabethae, inhibited growth of Mycobacterium tuberculosis H₃₇Rv (MIC=12.5 μg/mL). As part of a manzamine alkaloid structure-activity relationship study, Rao et al. (Rao et al., 2003) noted that (+)-8-hydroxymanzamine A (47) was very potent against M. tuberculosis (H₃₇Rv, MIC=0.91 μg/mL), comparing favorably with rifampin (MIC=0.5 μg/mL). De Oliveira et al. (de Oliveira et al., 2004) reported a new alkaloid ingenamine G (53) that demonstrated activity against Mycobacterium tuberculosis H37Rv at 8μg/ml. A new scalarane-type bioactive sesterterpene, 12-deacetoxyscalarin 19-acetate (54), which was purified from the Thai sponge Brachiaster sp (Wonganuchitmeta et al., 2004), inhibited growth of a nonvirulent strain of Mycobacterium tuberculosis by 50% at MIC= 4 μM, comparing favorably with kanamycin sulfate (MIC= 3.5-8.5 μM). As a result of a research program designed to identify marine natural products that inhibit the mycothiol-S-conjugate amidase, a mycobacterial detoxification enzyme, Nicholas et al. (Nicholas et al., 2003) reported several active compounds: a mixture of 1,3 pyridinium polymers (55) isolated from the marine sponge Amphimedon sp., IC₅₀=0.1 μM; an Oceanapiside sp.-derived bromotyrosine compound (56), IC₅₀=3 μM; and the glycosphingolipid oceanapiside (57), IC₅₀=10 μM. Oceanapiside, was observed to be a “simple non-competitive inhibitor” of the mycothiol-S-conjugate amidase enzyme.

Two studies contributed to antiplatelet pharmacology of marine natural products during 2003-4. Pimentel et al. (Pimentel et al., 2003) using a new microplate assay for Ca²⁺-induced platelet aggregation, determined that xestospongin A (58), isolated from the marine sponge Xestospongia sp., inhibited both collagen-and epinephrine-induced platelet aggregation more potently than aspirin. Villar et al. (Villar et al., 2003) evaluated the effects of several zoanthamine-type alkaloids isolated from the zoanthids Zoanthus numphaeus and Zoanthus sp. on the aggregation of human platelets: 11-hydroxyzoanthamine (59) demonstrated strong inhibition of thrombin-, collagen- and arachidonic acid-induced platelet aggregation which appeared related to the hydroxyl group at C-11; in contrast, aromatization in ring A was probably responsible for the selectivity of zoanthenol (60) towards collagen- induced aggregation.

Table 2 summarizes the preclinical pharmacological research completed during 2003-2004 with the 45 marine chemicals shown in Fig. 2.

Ohgami et al. (Ohgami et al., 2003) reported the effect of astaxanthin (68), a carotenoid found in crustacean cells, salmon and sea stars on lipopolysaccharide-induced uveitis in rats both in vitro and in vivo. The investigators observed that astaxanthin, at 100 mg/kg, suppressed development of uveitis and was as potent as 10 mg/kg prednisolone. The mechanism of action determined for astaxanthin probably involved inhibition of nitric oxide, prostaglandin E₂ and TNF-α generation. Lucas et al. (Lucas et al., 2003b) described a detailed mechanistic study on the modulatory effect of bolinaquinone (69), a sesquiterpenoid isolated form a Dysidea sp. sponge, in several models of acute and chronic inflammation. The observation that bolinaquinone significantly inhibited cytokine, iNOS expression and eicosanoid (LTB₄, PGE₂) generation in vitro and in vivo in several models of inflammation through secretory PLA₂ inhibition led the authors to propose that bolinaquinone is a marine compound of “potential interest in the search for new anti-inflammatory agents”. Posadas et al. (Posadas et al., 2003a) extended the cellular and molecular pharmacology of the sesterterpene cacospongionolide B (70) isolated from the Mediterranean sponge Fasciospongia cavernosa, and previously shown to be an inhibitor of secretory phospholipase A₂. In mouse peritoneal macrophages in vitro, as well as in an in vivo model of inflammation, cacospongionolide B decreased nitric oxide, prostaglandin E₂ and TNF-α generation as well as the corresponding gene expression. At the molecular level, cacospongionolide B inhibited nuclear factor-κ-DNA binding activity and enhanced IκB-α expression. Keyzers et al. (Keyzers et al., 2003) contributed a novel anti-inflammatory sterol, clathriol B (71) from the New Zealand sponge Clathria lissosclera. Clathriol B was shown to moderately inhibit production of superoxide anion from agonist-stimulated human peripheral blood neutrophils. A novel indole alkaloid histamine antagonist, conicamin (72), was isolated from the Mediterranean tunicate Aplidium conicum (Aiello et al., 2003). Ex vivo studies with guinea pig ileum demonstrated a concentration-dependent reduction of histamine-induced contractions, probably by a non-competitive mechanism. Lucas et al. (Lucas et al., 2003a) examined the effects of a series of 6 new cycloamphilectenes isolated from Vanuatu sponge Axinella sp. on murine macrophage and human neutrophil functions. All compounds tested reduced nitric oxide (NO) production in the submicromolar range. Interestingly, cycloamphilectene 2 (73), which reduced NO production and elastase release without affecting TNF-α release, inhibited the nuclear factor-κB pathway and also exhibited in vivo activity. One novel iododinated plakohypaphorine (74) with antihistamine activity was isolated from the Caribbean sponge Plakortis simplex (Borrelli et al., 2004). The authors noted that the antihistamine activity appeared to be “connected to the number and nature of the halogen atoms”, because replacement of the iodine atom by chlorine resulted in a loss of the antihistamine property of this compound. Three novel diterpenes, namely pourewic acid A (75), methylpourewate B (76) and cadlinolide C (77), were purified from the New Zealand sponge Chelonaplysilla violacea (Keyzers et al., 2004). The three diterpenes moderately inhibited production of the inflammatory superoxide anion from human peripheral blood neutrophils stimulated with phorbol myristate acetate or N-formyl-methionine-leucine-phenylalanine. Hong et al. (Hong et al., 2003) investigated the anti-inflammatory properties of petrocortyne A (78), a C₄₆ polyacetylenic alcohol isolated from the marine sponge Petrosia sp.. Petrocortyne A inhibited release of both TNF-α (IC₅₀=2.35 μM) and nitric oxide from macrophages and induced homotypic aggregation of U937 human leukemic monocytes, a process that appears to involve phosphorylation of several intracellular signaling molecules. The molecular pharmacology of petrosaspongiolide M (79) was characterized by Posadas et al. (Posadas et al., 2003b), who determined that this marine sesterterpenoid inhibited production of nitrite, prostaglandin E₂ and TNF-α both in vitro and in vivo while concomitantly inhibiting NF-κB signaling. The authors' results suggested that petrosaspongiolide M had “potentially (a) wide therapeutic spectrum” in inflammatory conditions. Monti et al. (Monti et al., 2004) extended the molecular pharmacology of the marine anti-inflammatory petrosaspongiolides M-R (79-83), bioactive sesterterpenes isolated from the marine sponge Petrosaspongia nigra. The irreversible inhibition of bee-venom phospholipase A₂ (PLA₂) was investigated by mass spectrometry and molecular modelling approaches and demonstrated that petrosapongiolides N, O, P and R shared the same inhibition mechanism and covalent binding site already reported for petrosaspongiolide M. Ata et al. (Ata et al., 2003) reported two new diterpenes pseudopterosin N (84) and seco-pseudopterosin E (85) and the hydroxyquinone elisabethadione (86) from the marine gorgonian Pseudopterogorgia elisabethae with in vivo anti-inflammatory activity. Interestingly all three compounds inhibited edema (inflammation) in a mouse ear anti-inflammatory assay more potently than “the well characterized pseudopterosins A and E”. With the purpose of contributing to the search of novel agents to treat neuroinflammation, several novel diterpene glycoside pseudopterosins and seco-pseudopterosins from the Caribbean sea whip Pseudopterogorgia elisabethae were evaluated in an in vitro anti-neuroinflammatory assay (Rodriguez et al., 2004). Pseudopterosin R (87) was observed to be the most promising compound because it significantly inhibited the generation of thromboxane B₂ (IC₅₀=4.7 μM) from activated rat brain macrophages. Although the molecular mechanism of action of pseudopterosin R remains currently undetermined, the authors concluded that it “could become an anti-inflammatory lead compound” for anti-neuroinflammatory drug design if its inhibitory effect on thromboxane B₂ release was further enhanced.

Perez et al. (Perez et al., 2003) investigated the inhibitory effect of the proline-containing cyclic peptide aplidine (95), isolated from the tunicate Aplidium albican, on the in vitro aggregation of peptide PrP 106-126, a fraction of the prion protein which has been hypothesized to be involved in fatal neurodegenerative diseases and which can kill neuronal cells in culture. The fact that aplidine was observed to be a strong inhibitor of the aggregation of PrP 106-126 into β-sheet fibrils at a 1:1 molar ratio prompted these investigators to propose that it may be “a leading compound for drug development efforts”.

Induction of neurite outgrowth in vitro was reported for the marine natural compounds aspermytin A, labuanine A, linckosides C-E, parguerol and isoparguerol, ganglioside species SJG-2 and sargaquinoic acid. Tsukamoto et al., (Tsukamoto et al., 2004a) isolated a new polyketide, aspermytin A (96) from a cultured marine fungus, Aspergillus sp. that induced neurite outgrowth at 50 μM in more than 50% of rat pheochromocytoma (PC-12) cells after two days of in vitro treatment, an effect comparable to that of 50 ng/mL of nerve growth factor. Aoki et al. (Aoki et al., 2003) purified a novel pyridoacridine alkaloid labuanine A (97) from the Indonesian marine sponge Biemna fortis that induced multipolar type neurite outgrowth in Neuro 2A neuroblastoma cells (1-3 μM) by a putative mechanism that “may relate with inhibition of topoisomerase II”, a hypothesis currently under investigation. Qi et al. (Qi et al., 2004) contributed three new bioactive steroid glycosides linckosides C-E (98-100) from the Okinawan marine sea star Linckia laevigata, which potently induced neurite outgrowth in rat PC12 cells, as well as synergistically enhanced the neuritogenic activity of nerve growth factor, a chemical that has been shown to be essential for neuronal outgrowth, survival, function maintenance and prevention of aging in the central and peripheral nervous system. Bioassay-guided fractionation lead to the isolation of parguerol (101) and isoparguerol (102) from the sea hare Aplysia kurodai (Tsukamoto et al., 2004b). Both compounds, the first neurotrophic compounds reported from sea hare metabolites, stimulated neurite outgrowth in PC-12 cells treated with either 25 or 50 μg/mL of the compounds for two days. Further pharmacological research will be required to determine the molecular mechanism leading to morphological changes in PC-12 cells. Two studies were reported on the pharmacology of the low molecular weight quinonic compound sargaquinoic acid (103) isolated from the marine brown alga Sargassum macrocarpum, and previously noted to possess a novel nerve growth factor-dependent neurite outgrowth promoting activity at the nanogram range. Kamei and Tsang (Kamei and Tsang, 2003) investigated the signaling pathways involved using a pharmacological approach and concluded that sargaquinoic acid enhanced neurite outgrowth in PC12 neuronal cells by involving both TrkA-mitogen-activated protein kinase and adenylate cyclase-protein kinase A signal transduction pathways. In a subsequent study, the neuroprotective effect of sargaquinoic acid was shown to be independent of nerve growth factor and phosphatidylinositol 3-kinase, a key signaling molecule (Tsang and Kamei, 2004). Kaneko et al. (Kaneko et al., 2003) reported the structure of a novel ganglioside SJG-2 (104) isolated from the sea cucumber Stichopus japonicus which appears to be the “first ganglioside containing either the branched sugar chain moiety or the N-acetylgalactosamine residue”. While SJG-2 only displayed neuritogenic activity in the presence of nerve growth factor, it was more potent than the GM1 mammalian ganglioside.

Four marine compounds (105-108; Fig. 2) were shown to target receptors present in the nervous system. Sakai et al., (Sakai et al., 2003) contributed to the search for novel ionotropic glutamate receptor ligands by reporting the isolation of the novel amino acid cribronic acid (105) from the marine sponge Cribrochalina olemda. Cribronic acid induced potent convulsive behavior in mice upon intracerebroventricular injection (ED₅₀= 29±3.0 pmol/mouse), as well as inhibited binding of an N-methyl-d-aspartic acid (NMDA) receptor ligand (IC₅₀=83±15 nM). In 2004, Sakai et al. (Sakai et al., 2004) isolated two novel cyclopropane amino acids dysibetaine CPa (106) and dysibetaine CPb (107) from the marine sponge Dysidea herbacea collected in Micronesia, that showed weak affinity toward the NMDA-type and the kainic acid-type ionotropic glutamate receptors in a radioligand binding assay. The investigators concluded that the binding of these compounds to the glutamate receptor was of interest because they both lacked “a glutamate equivalent structure in the molecules”. A screening program for bioactive compounds from marine cyanobacteria led to the isolation of the novel lipopeptides jamaicamide A (108), B and C (Edwards et al., 2004), compounds that exhibited sodium channel blocking activity at 5 μM, producing about half the response of saxitoxin applied at 0.15 μM.

During 2003-4, additional marine compounds (109-112; Fig. 2) were reported to exhibit pharmacological effects on the nervous system. Bioassay-guided fractionation of the marine sponge Raspailia sp. yielded the known synthetic compound esmodil (109), reported in the patent literature in 1935 (Capon et al., 2004a). More than six decades ago, esmodil, an acetylcholine mimic, was shown to be potentially “useful in treating retention of urine in humans and as influencing the parasympathetic nervous system in cats, rabbits and the Indian buffalo”. Hu et al. (Hu et al., 2004) reported interactions of an acidic oligosaccharide sugar chain (AOSC) from the brown alga Echlonia kurome with the amyloid beta protein. AOSC was observed to inhibit the toxicity induced by amyloid beta protein in both rat cortical cells as well as human neuroblastoma cells by a mechanism that involved inhibition of apoptosis, reduction of intracellular Ca²⁺ and the generation of reactive oxygen species (ROS). Thus the authors concluded that AOSC “might be a potentially therapeutic compound for Alzheimer's disease”. Capasso et al. (Capasso et al., 2003) reported that petrosaspongiolide M (79) reduced morphine withdrawal in an in vitro model. Although the mechanism underlying petrosaspongiolide M-induced inhibition of morphine withdrawal remains undetermined, one possibility raised by the investigators was the putative inhibition of extracellular type II phospholipase A₂ by this potent anti-inflammatory marine sesterterpene.

Extensive research on the preclinical and clinical pharmacology of conotoxin molecules resulted in the synthetic equivalent of ω-conopeptide MVIIA (110), a 25-amino-acid polybasic peptide derived from the marine snail Conus magus, receiving approval from the Food and Drug Administration on December 23, 2004. Currently, Ziconotide (Prialt®), which is marketed by Elan Biopharmaceuticals, Inc., constitutes the third drug in the U.S. Pharmacopeia which has been derived from marine chemicals. Staats et al. (Staats et al., 2004) reported a double-blind, placebo-controlled, randomized trial conducted from 1996-1998 in 32 study centers in the United States, Australia and the Netherlands which assessed the safety and efficacy of intrathecal ziconotide. Ziconotide selectively binds to the N-type voltage-sensitive calcium channels located in neurons thereby blocking neurotransmission and resulting in significant analgesia. In this clinical study, intrathecal ziconotide provided clinically and statistically significant analgesia in 111 patients with pain from cancer or AIDS. As part of a program to explore the diversity of conotoxins produced by Conus marine snails found off the Indian coast, Sudarslal et al. (Sudarslal et al., 2003) reported the isolation and characterization of a novel 26 peptide δ-conotoxin (111) isolated and purified from the venom of the marine snail Conus amadis, collected in the Bay of Bengal. The observation that this novel δ-conotoxin inhibited the inactivation of sodium current in cloned rat brain IIA α-subunit channels led the investigators to conclude that “conotoxins from some molluscivorous snails may also be active on mammalian Na⁺ channels”. Sharpe et al. (Sharpe et al., 2003) extended the molecular pharmacology of χ-conopeptide MrIA (112), a 13-residue peptide isolated form the venom of the marine snail Conus marmoreus. The investigators reported that the χ-conopeptide MrIA inhibited the norepinephrine transporter by a non-competitive mechanism that possibly involve a binding site “predicted to be distinct from the substrate binding site but to share some commonality with the antidepressant binding site”.

Table 3 lists marine compounds with miscellaneous pharmacological mechanisms of action, with their respective structures presented in Fig. 3. Interestingly, and in contrast with the 109 chemicals included in Tables 1 and 2, this third group of 54 marine compounds which was isolated from a variety of marine organisms, includes not only nitrogen-containing compounds (i.e. proteins, peptides), but also terpenes and polyketides.

As shown in Table 3, for a limited number of these marine natural products, namely acylspermidine D & E (113,114), ageladine A (115), alkylpyridinium (117,118), Atriolum robustum nucleoside (121), calyculin A (123), Cell-III, meridianin E (139), Psammocinia spp. diterpenes (143-147), punaglandins (148-152) and strobilin-felixinin (163,164), both the pharmacological activity and a molecular mechanism of action have been investigated and reported. In contrast, for all the other compounds shown in Table 3, although a pharmacological activity was reported, no additional information was available on the molecular mechanism of action of these chemicals during 2003-2004.

Specific areas of marine pharmacology research benefited from a number of excellent reviews published during 2003-4: (a) general marine pharmacology: natural products as sources of new drugs over the period 1981-2002 (Newman et al., 2003); marine natural products from marine invertebrates and sponge-associated fungi (Proksch et al. 2003b; Proksch et al., 2003a); the biopotential of marine sponges from China oceans (Zhang et al., 2003); marine natural products as therapeutic agents (patents): part 2 (Frenz et al., 2004); natural-product diversity of New Caledonia: a pharmacologically oriented view (Laurent and Pietra, 2004); (b) antimicrobial marine pharmacology: genomic screening to identify novel marine antimicrobial peptides (Patrzykat and Douglas, 2003a); marine natural products as anti-infective agents (Donia and Hamann, 2003); mining marine microorganisms as a source of new antimicrobials and antifungals (Bernan et al., 2004); antimicrobial peptides from marine invertebrates (Tincu and Taylor, 2004); bioactive peptides from marine sources: pharmacological properties and isolation procedures (Aneiros and Garateix, 2004); (c) anticoagulant marine pharmacology: recent advances in marine algal anticoagulants (Matsubara, 2004); the use of algae and invertebrate sulfated fucans as anticoagulant and antithrombotic agents (Mourao, 2004); (d) antituberculosis, antimalarial and antifungal marine pharmacology: antimycobacterial natural products (Copp, 2003); naturally occurring peroxides from marine sponges with antimalarial and antifungal activities (Jung al., 2003); antifungal compounds from marine organisms (Molinski, 2004); (e) antiviral marine pharmacology: algae as a potential source of antiviral agents (Luescher-Mattli, 2003); marine natural products as lead anti-HIV agents (Gochfeld et al., 2003); anti-HIV activity from marine organisms (Tziveleka et al., 2003); proteoglycans from sponges as tools to develop new agents for AIDS and Alzheimer's disease (Fernandez-Busquets and Burger, 2003); antiviral marine natural products (Gustafson et al., 2004); (f) anti-inflammatory marine pharmacology: anti-inflammatory metabolites from marine sponges (Keyzers and Davies-Coleman, 2005); (g) nervous system marine pharmacology: conotoxins as drug leads for neuropathic pain and other neurological conditions (Alonso et al., 2003); conotoxins and structural biology: a prospective paradigm for drug discovery (Grant et al., 2004); drugs from the sea: conopeptides as potential therapeutics (Livett et al., 2004); ziconotide: neuronal calcium channel blocker for treating severe chronic pain (Miljanich, 2004).

During 2003-4, and for the first time in many decades, a marine natural product, namely ziconotide (Prialt®) (110) was approved for patient care by the U.S. Food and Drug Administration for the management of severe chronic pain in patients for whom intrathecal therapy is necessary, and who appear to be intolerant of or refractory to other treatments, such as systemic analgesics, adjunctive therapies or intrathecal morphine. Although research into the non-antitumor and cytotoxic pharmacology of marine natural products remained concentrated in the specific areas we have highlighted in our present review, this contribution together with our previous ones (Mayer and Lehmann, 2000; Mayer and Hamann, 2002, 2004, 2005), demonstrates that marine pharmacology research continued to proceed at a very active pace in 2003-4, involving natural product chemists and pharmacologists from 28 foreign countries and the United States. Thus, if the rate of preclinical and clinical pharmacological research continues to be sustained over time, additional marine natural products will probably become available as novel therapeutic agents to treat multiple disease categories.

This publication was made possible by grant number 1R15 ES012654, from the National Institute of Environmental Health Sciences, NIH, to AMSM, and 1R01A136596, from the National Institute of Allergy and Infectious Diseases, NIH, and the Medicines for Malaria Venture (MMV), to MTH. Its content is solely the responsibility of the authors and does not necessarily represent the official views of the NIEHS, NIH. Additional financial support by Midwestern University is gratefully acknowledged. Jennifer Allman is acknowledged for her assistance with the preparation of Fig. 1 (MTH). The excellent support for literature searches in PubMed, Marinlit, Current Contents^® and Chemical Abstracts^®, as well as article retrieval by library staff members as well as medical and pharmacy students of Midwestern University is most gratefully acknowledged. The authors specially thank Ms. Mary Hall for carefully reviewing this manuscript.