1. Introduction
Around 70% of the Earth’s surface is covered by the oceans which occupy 90% of the biosphere. Marine species make up around half of the total global biodiversity and they have been extensively explored in the last decades for potential sources of novel bioactive natural products. Because of the difficulties in exploring deep water habitats, many bioactive natural products have yet to be isolated, identified and characterized, thus the oceans constitute a rich source of novel compounds. Peptides are important bioactive natural products which are present in many marine species and extensive research has been conducted on them [
1]. In most cases, the origins and roles of bioactive peptides in marine species are uncertain. Their strong bioactivities do not relate to their
in situ roles, such as antitumor, antidiabetic, neroprotective and cardioprotective actions [
2]. The discovery of these bioregulatory roles together with elucidation of the mechanisms of action of the marine peptides would advocate the peptides to be used as potential drugs for cancer, diabetes or hypertension treatment.
Bioactive peptides were first discovered and isolated in marine species as neurotoxin [
3], cardiotonic peptide [
4], antiviral and antitumor peptide [
5], cardiotoxin [
6] and antimicrobial peptide [
7]. Since then, the investigations on marine bioactive peptides have continued with intent to also ascertain their applications. The broad bioactivity spectrum of marine peptides has high potential nutraceutical and medicinal values which attract the attention of the pharmaceutical and nutraceutical industry, hoping that they can be used in treatment or prevention of various diseases. Nutraceutical is a word formed by the combination of “nutrition” and “pharmaceutical”. In recent years, substantial research efforts were dedicated to this area and it was projected that the global nutraceutical market would reach USD 250 billion by 2018 [
8]. Nutraceuticals or health promoting products are food-derived components (naturally occurring or enzymatically generated) that, in addition to their nutritional value exert a physiological effect on the body [
9]. They are usually claimed to prevent chronic diseases, enhance the immune system, manage stressful conditions, control body weight, regulates the blood glucose level, improve cognitive function, delay the aging process or increase life expectancy,
etc. Bioactive peptides are commonly incorporated into nutraceutical products. They are transformed into the active form after gastrointestinal digestion, absorbed through the intestine, and transported to the circulatory system to exert their diverse bioactivities [
10].
There are two representative marine peptide-derived pharmaceutical products, ziconotide (see
Table 1) and brentuximab vedotin, which are a natural marine peptide and a peptide derivative, respectively, have reached the market. Both of them are manufactured by chemical synthesis. Ziconotide (Prialt
®), a peptide found in marine cone snail, was the first marine peptide approved by FDA in 2004 for analgesic use [
11]. Then, in 2011, another marine peptide-derived drug (Adcetris
®) from sea hare was approved by FDA for cancer treatment. Several other marine peptide-derived compounds are currently being assessed in different phases of clinical trials in the United States and Europe. They included plitidepsin (see
Table 1) and glembatumumab vedotin which is use for treatment of various cancers [
12]. Dermochlorella
® DG in which its active ingredient is oligopeptide purified from algae. It came into cosmetic market as skin firmer and toner [
13]. Katsuobushi oligopeptide (see
Table 1) is a linear pentapeptide isolated from dried bonito (katsuobushi) was found to exhibit angiotensin-I converting enzyme inhibitory activity. Some antihypertensive capsules which are sold as nutraceuticals contain the peptide as one of the components [
14]. Gelatin is a polypeptide obtained from animal collagen by hydrolytic degradation. It usually serves as a protein supplement in nutraceutical and food industries. Gelatin is conventionally extracted from the skin and bone collagens of cows and pigs. Consumers raised concern about its safety especially when there was an outbreak of mad cow disease in the 1980s. Besides, from the perspectives of ethics and religion, fish gelatin was promoted as a better alternative to traditional gelatin products made from cows and pigs. It is a protein-rich source and usually included in muscle-building diets [
15]. Another related product containing a mixture of tripeptides, dipeptides and free amino acids obtained from the collagen hydrolysate is marketed as nutraceuticals for the maintenance of healthy bones [
16]. It is quite common for people in USA and Europe to take nutraceuticals or dietary supplements with anxiolytic properties. They contain marine-derived hydrolysates which contain opioid-like peptides as the active ingredients [
17]. There are several commercial products available in Europe and the USA such as Stabilium
®, Gabolysat
® PC60, Protizen
® and Procalm
®. However, there are only a few clinical studies about the opioid-like effect and anxiolytic properties of these marine-protein hydrolysates. Marine bioactive peptides can be released from protein by fermentation. A commercially available fermented fish protein concentrate Seacure
® was found to modulate the mucosal immune response and induce biological repair-promoting response in the murine gut model [
18]. The two products from marine protein hydrolysates, Nutripeptin
® and Hydro MN Peptide
®, were capable of lowering the postprandial blood glucose level and alleviating type II diabetes symptoms. Nutripeptin
® is manufactured by enzymatic hydrolysis of fish fillet or fish muscle protein [
19]. One of the components of Hydro MN Peptide
® is Peptide N
® which is a marine protein hydrolysate [
20]. Both products have also been claimed to prevent body fat deposition and thus aid in weight management [
19,
20].
Table 1 summarizes the structures or amino acid sequences of selected marine peptides with different bioactivities.
With the advancement of marine peptides to the current preclinical and clinical pipeline, their contribution to the future pharmacopeia seems to be promising. New technologies and close collaborations between institutional and industrial investigators will be crucial to ensure the future success of marine peptides as novel therapeutics that can make a vital contribution to the treatment or prevention of various diseases [
21].
Table 1.
Structures or amino acid sequences of selected marine peptides with different bioactivities.
2. Procedure for Isolating Marine Peptides
In a typical procedure for discovery of marine bioactive (linear or cyclic) peptides, the peptides are firstly extracted from the sources. The extract is screened for a specific bioactivity, fractionated using a bioassay-guided fractionation procedure, and finally purified to yield a single bioactive peptide. In general, organic solvents like methanol or ethyl acetate are usually used in first step of cyclic peptide extraction. Then the methanol or ethyl acetate extract is concentrated and partitioned with other solvents like hexane, carbon tetrachloride or dichloromethane. The partially purified extract is subjected to silica gel or size exclusion chromatography and the product is eluted with solvents of increasing polarity. Reversed phase C18 HPLC is used in the final purification step [
22,
23]. For linear peptides, the general procedure usually includes size exclusion chromatography, reverse-phase HPLC and ion exchange chromatography. The aqueous extract is first size-fractionated using a size exclusion chromatography column. The target fraction is concentrated, applied on a cation/anion exchange chromatography column and eluted in accordance with the basicity of the peptide. Reverse phase C18 HPLC with an additional step of rechromatography on the same column for excluding impurities will be used for final purification [
24,
25].
Bioactive peptides could also be obtained from digestion of proteins of the marine organisms. The most commonly used digestive enzymes are pepsin, trypsin, α-chymotrypsin, papain and some commercial protease cocktails [
26]. The hydrolysates are screened for various bioactivities after digestion and fractionated according to size by ultrafiltration [
27]. The fraction that shows the highest bioactivity is then further resolved to individual peptides using reverse phase high performance liquid chromatography or size exclusion chromatography. Finally the individual peptide fractions are identified using the combined techniques of mass spectrometry and protein sequencing [
28].
The results of some isolations from marine origins showed that the crude extracts or purified fractions contained structurally uncharacterized compounds (probably peptides) exhibited promising
in vitro or
in vivo bioactivities and they still deserve further investigations [
29].
Bioactive peptides or protein hydrolysates can be extracted and isolated from the protein of the marine species by various methods in industrial-scale production. Organic solvent extraction method was used traditionally, but it is a time-consuming, expensive and environmental unfriendly technique. Nowadays, better extraction techniques like supercritical fluid extraction, pressurized solvent extraction, microwave-assisted extraction, ultrasound-assisted extraction, pulsed electric field-assisted extraction and enzyme-assisted extraction are preferred [
30]. After the extraction procedure, the proteins are subjected to hydrolysis by which the proteins are hydrolyzed into bioactive peptides. Enzymatic hydrolysis is preferred in the nutraceutical and pharmaceutical industries in order to avoid harsh chemical and physical treatment [
31] and preserve the functionality and nutritive values [
32]. The hydrolysis products, bioactive peptides, are further concentrated and separated according to their different molecular weights by membrane filtration. Usually an ultrafiltration membrane with a molecular weight cutoff (MWCO) at 20 kDa or above is used to separate peptides from unhydrolyzed proteins. Membranes with a MWCO at 4–8 kDa are suitable for fractionation of bioactive peptides with desired molecular weights. Membranes with a low MWCO at around 0.2 kDa are used to concentrate the peptide. Diafiltration using a nanofiltration membrane is employed to desalt, deodorize and decolorize the peptide solution [
33,
34]. A novel ultrafiltration membrane bioreactor technology has recently emerged. It is possible to obtain sequential enzymatic digestions from the marine protein in a system by multistep recycling membrane reactor combined with an ultrafiltration membrane system to fractionate marine hydrolysates according to different molecular weight ranges [
35]. Finally, more sophisticated techniques mentioned in the previous paragraphs can be used for further purification and characterization of the bioactive peptide.
4. Marine Peptide Products in Market and Clinical Trials
As described above in this review, many bioactive marine peptides have been proven to have different bioactivities. Their original or modified form could be utilized as the lead structures for potential nutraceutical and pharmaceutical uses. There is a repertoire of peptides isolated from marine species but only a small portion has been approved for evaluation in the clinical phases and even fewer have managed to reach the market.
Table 2 highlights the status of marine peptide products in market and clinical trials. Successful examples comprise ziconotide and brentuximab vedotin, other commercialized examples belong to cosmetics and nutraceuticals. Some peptides are currently being evaluated in different phases in clinical trials such as plitidepsin and CDX011.Some marine peptide candidates that have yielded promising
in vitro results were subjected to clinical trials, but the investigations have been terminated or discontinued due to lack of efficacy, absence of objective responses or adverse effects experienced in patients. In the following section, some selected examples are used to demonstrate the novel marine peptides discovery, their development and progression to clinical trials as original forms or derivatives.
Ziconotide is a 25-amino acid peptide derived from the ω-conotoxin toxin of
Conus magus (cone snail found in tropical water) [
140]. The fish-hunting snails produce a few conotoxin peptides that act synergistically by targeting the neuromuscular system to immobilize the prey [
141]. The ω-conotoxin blocks N-type voltage-sensitive calcium channels and inhibits the pain-related release of neurotransmitters. The nerve signal conduction is inhibited thus resulting in pain relief [
142]. As the marine sources cannot provide enough amounts for large-scale production, ziconotide was manufactured by peptide synthesis. The approval by FDA of ziconotide, a synthetic form of ω-conotoxin, as an analgesic agent for amelioration of chronic pain, prompted investigations on other conotoxins, like contulakin G (CGX-1160), for potential therapeutic and clinical usage [
143,
144].
Table 2.
The status of marine peptide products in market and clinical trials.
Table 2.
The status of marine peptide products in market and clinical trials.
Compound | Natural Product/Derivatives | Source | Applications | Status |
---|
ziconotide | natural product | ω-conotoxin toxin from Conus magus | analgesics | FDA approved |
brentuximab vedotin | derivatives | dolastatin 10 from Dolabella auricularia | cancer treatment | FDA approved |
glembatumumab vedotin | derivatives | dolastatin 10 from Dolabella auricularia | cancer treatment | phase I/II clinical study |
katsuobushi oligopeptide | natural product | pentapeptide from hydrolysate of dried bonito | antihypertensive | sold as nutraceuticals |
Dermochlorella® | natural product | oligopeptide extract from Chlorella vulgaris | Skin toner and firmer | sold as skin care product |
plitidepsin | natural product | cyclic depsipeptide from Aplidium albicans | cancer treatment | phase I/II clinical study |
HTI-286 | derivatives | hemiasterlin from Hemiasterella minor | cancer treatment | preclinical study |
kahalalide F | natural product | cyclic tridecapeptide from Elysia rufescens | cancer treatment | phase I clinical study |
elisidepsin | derivatives | kahalalide F from Elysia rufescens | cancer treatment | phase I clinical study |
fish gelatin | natural product | hydrolysate of fish collagen and gelatin | nutrient supplements and bone health | sold as nutraceuticals |
Gabolysat PC60®/Stabilium®/Protizen®/Procalm® | natural product | hydrolysate of fish protein | anxiolytic | sold as nutraceuticals |
Seacure® | natural product | hydrolysate of fish protein | intestinal health | sold as nutraceuticals |
Nutripeptin®/Hydro MN Peptide® | natural product | hydrolysate of fish protein | postprandial blood glucose control | sold as nutraceuticals |
Brentuximab vedotin is an antibody-drug conjugate which targets on cell membrane protein CD30. It consists of (1) a chimeric monoclonal anti-CD30 antibody, brentuximab, linked to (2) a cathepsin-cleavable linker; (3) a para-aminobenzylcarbamate spacer and (4) monomethyl auristatin E (MMAE) which is a strong antimitotic agent. MMAE binds in the vicinity of the vinca peptide site, blocks microtubule assembly and tubulin polymerization [
55]. It was approved by FDA in 2011 for treatment of Hodgkin and systemic anaplastic large cell lymphoma. The MMAE is a synthetic analog of dolastatin 10 isolated from
Dolabella auricularia (sea hare found in the Indian Ocean). The extract from
Dolabella auricularia was found to be highly efficacious against cancer cells as early as 1972. However, it was not until 1987 that the most potent constituent, dolastatin 10, was isolated and characterized. It is a linear pentapeptide with four distinctive amino acids and exhibited potent inhibitory activity against a battery of human cancer cell lines [
145]. However, when it entered clinical trials, the results were unsatisfactory due to lack of efficacy. These disappointing results did not justify further endeavors and consequently dolastatin 10 was withdrawn from antitumor clinical trials [
146]. Nevertheless, its synthetic derivatives, MMAE, showed clinically significant activity when it is linked with an antibody that targets CD30 protein [
147,
148]. The antibody portion attaches to CD30 present on the surface of Hodgkin’s lymphoma cells, delivering MMAE selectively to the tumor cells. Clinical study has started for a similar drug CDX011 which is also known as glembatumumab vedotin, for the treatment of advanced, refractory or resistant breast cancer [
149,
150]. Its structure contains MMAE links with glembatumumab which targets transmembrane glycoprotein-expressing cancer cells. These similar antibody-drug conjugates, SGN-75, ASG-5ME and soblidotin derived from dolastatin 10 and synthadotin and tasidotin (ILX-651) derived from dolastatin 15, have advanced to Phase I/II/III clinical trials for treatment of cancer, but the developments were suspended by the pharmaceutical company due to “strategic” reasons.
Katsuobushi oligopeptide, a linear pentapeptide from dried bonito (katsuobushi, a traditional Japanese food), was derived from katsuobushi by thermolysin hydrolysis. It manifested ACE inhibitory activity when transformed to the active form in the human digestive system [
151]. Katsuobushi oligopeptide exerted antihypertensive activity in patients with hypertension and borderline hypertension in a small-scale clinical trial. Official approval of katsuobushi oligopeptide as foods for specified health use was issued in 1999 by Ministry of Health and Welfare in Japan [
152]. Subsequent reports indicated that the peptide brought about relaxation of vascular smooth muscle [
153]. They are incorporated in blood pressure-lowering capsules which are sold as nutraceutical [
14].
Dermochlorella
® is a skin care product which contains
Chlorella vulgaris (green algae) extract with oligopeptides as the active ingredient. It helps to firm the skin and reduce the colour of the stretch marks. Dermochlorella increases the expression of collagen, elastin, laminin and the inhibitors (elafin and tissue inhibitors of metalloproteinase) of the enzymes that degrade the extracellular matrix and restore skin elasticity [
13].
Plitidepsin (also known as dehydrodidemnin B or Aplidin
®) is a cyclic depsipeptide isolated from
Aplidium albicans (a tunicate found in Mediterranean Sea) in 1991. It is classified as a didemnin member and its chemical structure closely resembles that of didemnin B which has been submitted to clinical trials for various cancer treatments [
154,
155,
156,
157]. Yet, the clinical trials were stopped due to severe fatigue and anaphylaxis experienced in patients [
156,
158]. Plitidepsin showed comparable levels as didemnin B on the
in vitro antitumor activity to tumor cell lines [
158,
159]. Plitidepsin was demonstrated to elicit apoptosis in a cell type- and dose-dependent fashion. These actions are associated with the triggering of early oxidative stress, activation of Rac1 GTPase and suppression of protein phosphatases, which altogether contribute to the sustained activation of JNK and p38 mitogen-activated protein kinases [
160]. The natural resource of plitidepsin is limited because of the difficulties in collecting the sacred
Aplidium albicans and the lack of possible aquaculture or mariculture conditions. The constituent amino acids of plitidepsin are unnatural and heterologus expression is not feasible, and thus they are manufactured by multi-step total synthesis. Phase I/II clinical trials of plitidepsin yielded promising results of antitumor activity in patients with advanced melanoma [
161], multiple myeloma [
162], non-Hodgkin’s lymphoma [
163], advanced medullary thyroid carcinoma [
164] and urothelium carcinoma [
165].
Hemiasterlin is a linear tripeptide isolated from the sponge
Hemiasterella minor in 1994. It contains two unique amino acids and exhibited strong cytotoxic activity on the P388 leukaemia cell line [
166]. It induces mitotic arrest and apoptosis by blocking mitotic spindle formation and causes tubulin depolymerization [
54]. HTI-286 (also known as SPA-110 or taltobulin), a synthetic analogue of hemiasterlin, shows more potent cytotoxic activity than hemiasterlin on human cell lines. The mechanisms of action of both compounds are similar. Preclinical studies showed that HTI-286 inhibited the growth of human tumor xenografts in mice [
167]. It has been submitted to phase I clinical trial but unfortunately the results were unsatisfactory. There were no objective responses in patients with advanced solid tumors, besides neutropaenia, nausea, alopecia and pain were observed and further trials were terminated [
168]. However, some researchers are still working on this peptide. The combination of antisense oligonucleotides OGX427 and HTI-286 used in therapy intravesically was shown to effectively inhibit orthotopic tumor growth without toxic side effects. The results gave hope to a potential treatment method for bladder cancer [
169].
Kahalalide F is a cyclic tridecapeptide that contains several atypical amino-acid residues which showed antitumor activity. It was isolated from
Elysia rufescens (a sea slug found in Hawaiian water) in 1993 [
170]. However, later, it was found that the compound originated from the green alga
Bryopsis sp., present in the sea slug’s diet [
171]. It was active against human cancer cell lines [
170,
171] and several mechanisms of action have been proposed including an action on lysosomal membrane [
172], inhibition of erbB2 tyrosine kinase activity [
173], oncosis induction [
174], effect on cell membrane permeability [
175] and induction of necrosis-like cell death [
176], yet no conclusive mechanism has been elucidated. Kahalalide F has entered into clinical trials for the treatment of patients with solid tumors including melanoma, non-small lung cancer and hepatocellular carcinoma [
177,
178], but the trials came to an end due to lack of antitumor activity. Yet, there is still interest in kahalalide F given that report of a recent investigation in advanced solid tumors therapy in phase I clinical trial has appeared [
179]. Elisidepsin (PM02734 or known as Irvalec
®) is a synthetic kahalalide F derivative which has similar action as kahalalide F. It manifests
in vitro activity against a variety of tumor cell lines such as breast, colon, pancreas, prostate and lung [
180,
181,
182] as well as
in vivo activity in xenografted human tumors [
183]. These results were promising and gave a rational basis for further investigations such as clinical studies of cancer treatment. A few clinical trials were submitted, they were the study of elisidepsin in patients with advanced solid tumor [
184] and the study of combination therapy with erlotinib in patients with advanced malignant solid tumors. The two studies have been completed and the pharmaceutical company decided to suspend the development of elisidepsin in 2012 in view of commercial unviability of this product. However, a phase I study of combination therapy with carboplatin or gemcitabine in patients with advanced malignancies was initiated recently [
185]. However the results did not show clinically significant antitumor activity, thus its development program may probably be deferred.
Fish gelatin is derived from hydrolytic degradation of collagen which is generated from seafood processing waste such as skin, bones, scales and fins from fish [
15]. It is a valuable protein source and usually incorporated into protein drinks, protein energy bars, muscle-building food and nutritional formula. A similar product, known as collagen peptides, is the protein hydrolysate of collagen or gelatin with a molecular weight around 1–5 kDa. They contain tripeptides, dipeptides and free amino acids that are easily absorbed in the body. Currently, they are marketed as nutraceuticals for the maintenance of normal bone and tendon integrity, improving joint health, brittle nail treatment and scalp hair nourishment [
186]. Clinical studies showed that collagen peptides are involved in cartilage matrix synthesis and treatment with the peptides reduced pain in osteoarthritic patients. So they are potential therapeutic agents for osteoarthritis and osteoporosis [
16].
Several nutraceuticals or dietary supplements that are marine protein hydrolysates in nature claimed to have anxiolytic properties were commercially available. Gabolysat PC60
®, a ling fish protein hydrolysate, exhibited diazepam-like effects on stress responsiveness of the rat pituitary-adrenal axis and the sympathoadrenal activity [
17]. Stabilium
® is another fish protein hydrolysate obtained from enzymatic autolysis of blue ling viscera. It accounted for reducing anxiety in humans and improving memory and concentration in stressed rats [
108,
109]. Protizen
® (a pollock hydrolysate) and Procalm
® (a veterinary drug) were reported for similar actions.
Seacure
® is a commercial dietary supplement product obtained from proteolysis of Pacific whiting proteins by fermentative yeast. They are packaged as “intestinal health promoting” product. They work directly on animal gastric-damaging models and induce epidermal growth factor-like responses,
i.e., stimulate the proliferation of epidermal and epithelial tissues [
187]. In a small scale clinical trial, [
18] described its prevention and treatment of nonsteroidal anti-inflammatory drugs-induced and other gastrointestinal injurious conditions.
Two commercial products, Nutripeptin
® and Hydro MN Peptide
®, were sold as nutraceuticals for lowering of postprandial blood glucose [
19,
20]. Nutripeptin
® is enzymatic fish protein hydroylsates which can lower and stabilize blood glucose, increases fat burning, and extend the satiety sensation after meals [
19]. Its addition to sport drinks has been suggested for improving energy recovery and performance [
188]. One of the components of Hydro MN Peptide
® is Peptide N
® which is a marine protein hydrolysate. Clinical studies found that Peptide N
® supplementation facilitated blood glucose stabilization, alleviated obesity risk, mitigated type II diabetes symptoms and induced satiety feeling through its action on metabolic hormone [
20].
We have mentioned the large-scale production of protein hydrolysates in the previous section. They are manufactured by protease digestion of the muscles, skin, bones, scales and fins from fish or other marine species in the nutraceutical industry. For the process of manufacture of peptide-based drugs, the drugs are pure compounds rather than a mixture of peptides as in the case of protein hydrolysates. Usually, minute amounts of the marine peptides are present in the marine organisms, and it is difficult to extract enough quantities for commercial use. It is impractical to rely on marine sources to provide the sustainable supply for commercialization [
189]. As a result, chemists developed total synthesis approach for production of peptide drugs (solid-phase peptide synthesis of ziconotide [
190] and convergent total synthesis of dolastatin 10 [
191] and MMAE [
192]).
To increase the chance of success in commercializing marine peptides, the best way is to develop close collaborations between academic and industrial partners at the initial stage. Academic institutions can get industrial sponsorship at the early development stage. This approach merges the expertise of academics with knowledge of marine life, pharmacology, analytical and synthetic techniques and the industrial partners with market awareness and business expertise. Starting from the beginning of the marine peptide discovery and development project, like accessibility to marine resources, bioactivity screening, study of the mechanism of action, production of the peptides and recruitment for clinical trials and studies, to the final phase like financial arrangement and marketing strategies, the joint academic-industry venture can push the research project forward, bridge the innovation gap and unlock the potential of the under-exploited ocean habitats. The joint venture benefits both partners that the academia gets knowledge, publications and funding and industry gets new marketable products [
13].