Marine Drugs and Cosmeceuticals:

From Lab to Market

Prof. (em.) Ulrike Lindequist

University Greifswald
Institute for Pharmacy
lindequi@uni-greifswald.de
Bogor, September 2019 1
Content
• Introduction
• Approved marine drugs
• The potential of seaweeds
• The potential of marine fungi
• What is to do to explore the potential?
 Specialities of Marine Environment

Water
Temperature - 1.5 - 350 o C
Pressure 1 - > 1000 atm
Salt
Light
Number of species
Close communities of different species

This means: Marine organisms need special mechanisms

for adaption to their environment. They produce
other secondary metabolites than terrestric organisms.
 Number of Marine Species
Estimated: 3 Mio till 500 Mio
Known: Plants: 40.000 species
3900 red algae
1500 brown algae
900 green algae
45 flowering plants
Animals: 33 of 34 known Phyla (15 only in the Sea)
157.000 benthic
5.000 sponges
9.000 cnidarian
8.000 echinoderms
Microorganisms???
 Marine natural products

By organism By region

(Source: Blunt et al., Nat. Prod. Rep., 2009, 26, 170–244)

 Comparison Marine (MNP) – Terrestrial Natural
Products (TNP) by Cheminformatics (Shang et al. 2018)
MNP TNP

Lower solubility in water Higher solubility in water

Often larger

Contain often ester bonds connected Contain often more stable ring
to 10-membered rings systems and bond types

More nitrogen and halogen atoms

Fewer oxygen atoms

More diverse biosynthetic pathways? (Shang J et al.: J Chem Inf Mod 58(6), 1182-1193, 2018
 Types of Products
• Pharmaceutical product = pharmaceutical = medicinal product = medication =
medicine = drug:
a drug used to diagnose, cure, treat or prevent disease

• Food supplement: any food the purpose of which is to supplement the normal diet
and which is concentrated source of a vitamin or mineral or other substances with a
nutritional or physiological effect, alone or in combination and is sold in dose form
(EU law); between food and drugs

• Food

• Cosmetic: a preparation applied to the body, especially the face, to improve its
appearance
8
 Types of Products
Cosmeceutical:
- „hybrid“ product between a cosmetic and a pharmaceutical

- to provide desired esthetical effects (immediate) and

- to treat dermatological conditions (prolonged)

- anti-aging, anti-acne, solar-protective, wound healing, skin whithening

activities

Regulation: in most countries not a separate category from cosmetics

(exceptions: Japan, Korea: = functional cosmetics)

But: Safety and efficacy have to be ensured.

 FDA-
approved: 9*

Clinical Phase III: 7

Clinical Phase II: 14

Clinical Phase I: 10

Pipeline of marine Drugs 2019

* 1 only in Australia (Aplidin) http://marine pharmacology.midwestern.edu/
 Approved Marine Drugs
Virostatika: Vidarabin Vidarabin 3% Thilo ®

Cancerostatics: Cytarabin Alexan®,

ARA-Cell ® etc.
Trabectedin Yondelis®
Eribulin Mesylat Halaven®
Brentuximab Vedotin Adcetris®
Polatuzumab Vedotin Polivy ®
Plitidepsin Aplidin® *Australia

Analgetics : Ziconotid Prialt®

Dietetics: Omega-3-acid-ethyl esters

 Nucleosides from Sponges
O
O N NH
HO
O
HO OH

Spongouridin
O www.spongeguide.org

O N NH Tethya crypta (Karibik)

HO
O Arabinose instead of Ribose!
HO OH

Spongothymidin Models for Vidarabin and Cytarabin

 NH2
Vidarabin N
N

Mode of action: Transformation into Arabinosidtriphosphate, HO N N

O
Inhibition of virale (HSV) DNA-Polymerase HO

and DNA-Synthesis
OH

Indication: as eye ointment (3%) for treatment of acute

Keratoconjunctivitis and recurring
epithelial Keratitis, caused by
HSV I oder II

Today mostly replaced by Acyclovir

 Cytarabin, Ara-C, Alexan®, ARA-cell®
NH2
Cytosinarabinosid
N
Transformation to Cytosinarabinosidtriphosphate HO
N O
O
Mode of action: Inhibition of DNA Polymerase by HO
competition with the physiological substrate
Deoxycytidintriphosphate OH

Indication:
Acute lymphatic and non-lymphatic leukemia

Chronic myeloic leukemia

 Trabectedin, Yondelis ®

Tetrahydroisochinolinalkaloid = ET 743

Produced by partial synthesis; starting substance Cyanosafracin B (from

Pseudomonas fluorescens)
 Trabectedin, Yondelis ®
• Occurence in Tunicata
• Animals with a mantle (tunica) as a
coat outside the epidermis
• Benthic
• about 2000 species

Bertelsmann Lexikon

Ecteinascidia turbinata,
Living on corals in the Mediterranean, looks like grapes

Photo: Courtesy PharmaMar

 Trabectedin, Yondelis ®

Mode of action: Binding at small groove of DNA,

Inhibition of cell proliferation

Indication:
Advanced soft tissue sarcom
Ovarian carcinom (recurrent, platin sensitive)

Approval 2007 (2001 as orphan drug),

Producer: PharmaMar
 Trabectedin, Yondelis ®
• Randomisied study with 266 patients iiposarkom or leiomyosarkom, Progress
of disease though pretreatment with anthracyclins and ifosamid
1,5 mg/m2 body surface 24 h i.v. each 3 weeks:
after 1 Jahr: survival of > 60% of patients,
average survival time: 14 month

• Open multicenter study with 672 patients with recurring ovarial carcinom
Combination of trabectedin and pegylated liposomal doxorubicin
significant prolongation of survival time in comparison to doxorubicin alone
no additional impairment of life quality
 Eribulin Mesylat, Halaven ®

Synthetic Analogon of Halichondrin B

Makrolid, Polyketid
 Halichondrin B

Halichondria okadai,

(Pacific)

Lyssodendoryx sp.
http://vitalsignsme.org/observation/species-

H H H
O O O
H
O H
O O O
O O O
HO H H H H
O
H H O O
HO O
O

O H
HO
 Eribulin Mesylat, Halaven ®
Mode of action: Reaction with Mikrotubuli (other binding
place than taxanes and Vinca alkaloids)

Indication: metastasized breast cancer after

two earlier ineffective chemotherapies

Approval 2011

Producer: Eisai Co., Japan

 Brentuximab Vedotin, Adcetris ®
Conjugat consisting of
• Antibody against CD30
• Linking molecule and
• the cytostatic Auristatin
( Monomethyl-Auristatin E, MMAE)

Conjugat stable in blood

After internalisation into

CD30 carrying tumour cells release of the
cytostatic Auristatin

22
 Auristatin

CH3
H3C
O
H H
N N
N N
O CH3 O O CH3
O
H 3C
H3C
O NH
Teuscher/Lindequist: Biogene Gifte

Synthetic derivate of Dolastatin 10, isolated from Dolabella auricularia

(Mollusc, Gastropoda),
Produced by Cyanobacteria, genus Symploca
Peptid
Because of high toxicity direct application not possible
 Brentuximab Vedotin Adcetris®

Mode of action: Attack at Tubulin

Indication: Advanced Morbus Hodgkin

(Marker CD30)
recurrent anaplastic lymphom

Approval: November 2012

Producer: Takeda, Japan

 Polatuzumab Vedotin Polivy®

The same principle like Brentuximab Vedotin

Antibody against CD79b – Linker – Monomethylauristatin E

Indication: therapy resistent or recurrent diffuse large cellular B cell lymphom

Approval: June 2019 (FDA)

Producer: Roche
 ®
Ziconotid, Prialt
10
_
E C C N P A C GR H Y S C NH2 a-Conotoxin GI
10 20
R D C C T P P K K C K D R Q C K P Q R C C A _ NH2 m-Conotoxin GIIIA
10 20
C K S P G S S C S P T S Y N C C R S C N P Y T K R C Y _ NH2 v-Conotoxin GVIA
10
_
G E g g L Q g N Q g L I R g K S N NH2 Sleeper-Peptid
10 20 30
A C S G R G S R C P P Q C C M G L R C G R G N P Q K C I G A H g D V _ NH2 Conotoxin GS

(intrachenare S-S-Brücken vorhanden)

P = L-Hydroxyprolin, g =g -Carboxy-L -Glutaminsäure

Peptid, consisting of 25 amino acids with 3 disulfide bridges, basic,

Model: Ω-Conotoxin
 Ziconotid, Prialt ®

Conus spec.
Cone snails

Teuscher/Lindequist: Biogene Gifte

Mebs „Gifttiere",
1992
 Hunting strategy of cone snails
- Snail hidden in the soil
- Fishes recognize the red
pharynx tube of the snail as
„food“
- poisonous file comes out
and stings the fish
- Paralysis in 1-2 sec
- paralysed fish is engulfed
by the snail

Olivera, B.M. (1985) Science 230:1338-

1334
LD 50: 0.82 mg/kg i.v.
 Ziconotid, Prialt®: Mode of Action

-Selective Inhibition of N Typ-Ca-channels

-Inhibition of voltage sensitive Calcium influx into primary nozizeptive afferente neurons

-Inhibition of release of excitatoric neurotransmitters from praesynaptic vesicels

-Prevention of transmission
of pain signal Aus Putzier und Frings, 2002
 Ziconotid, Prialt®: Application
Long lasting infusion through intrathekal catheter
by implanted infusion pump

Treatment of nozizeptive and neuropathic pain

1000x stronger active than morphine

No dependance

Tolerance development unprobably

108 patients with malign,
255 with non malign pain
Treatment for 10-12 d
Increased risk for suicide?
Wissenschaftliche Broschüre Ziconotid
 Recently approved: Pegvaliase (Palynzig ®)
- Enzym Phenylalanin-Ammoniak-Lyase (PAL) from the cyanobacterium
Anabaena variabilis

- Recombinant produced in Escherichia coli

- Pegylated

- Metabolizes the amino acid phenylalanine

- As Orphan Drug for patients with phenylketonuria (PKU, cannot metabolize

phenylalanine, neurological, cognitive and other disturbances)
Further marine substances with medicinal importance

Auxiliary products:
Agar, Carrageenan, Alginic acid, Chitin, Chitosan

Diagnostics: LAL, GFP, Phycoerythrin, Taq Polymerase

COOH
H 3C

Eicosapentaensäure (Omega - 3 - Fettsäure)

Food: PUFAs, Iod COOH

H3 C

Cosmetics: Docosahexaensäure (Omega-3-Fettsäure)

Compatible Solutes, Pseudopterosins, Sun protecting

Agents

Wellness: Thalasso
 Seaweeds
• = Macroalgae
• Macroscopic organisms in the marine ecosystem
• Three main phyla associated with different pigments:
Chlorophyta (green algae) – chlorophyll Enteromorpha compressa
Rhodophyta (red algae) – phycobilins
Phaeophyta (brown algae) – fucoxanthin
• Good source of carbohydrates, dietary fiber, proteins, vitamins,
PUFAs, minerals

33
 COOH

Seaweeds: Biopolymers CH2 OR O H

CH 3 C

O CH
2
O

HO H
HO O O H
H O H H O H
H CH2 HO O - H
HO
CH2OSO 3H O
H H H O H H H O
H H H OH H
• Rhodophyceae: Agar, Carrageenan
OH

• Phaeophyceae: Alginic acid D-Galaktose (R=H)

6-0-Methyl-D-
galaktose(R=CH 3 )
3,6-Anhydro
L-galaktose
4,6-0-(1-Carb-
oxyethyliden)
D-galaktose
L-Galaktose -
6-sulfat

• Wound healing materials, against

gastritis, as dietary agent, as gelling Agar
agent, to stop bleeding
 Seaweeds: Cosmeceuticals
• Photo-protective compounds:
-mycosporine-like amino acids
-carotinoids O
O
C
H
-photolyase 3
H
O
H
O
N
H
• Skin care and protection C
O
O
H

Mycosporine glycin
O
OH

HO La mer med
O Couperose Creme
Astaxanthin
35
 Seaweeds: Pharmaceuticals (research)
• Along the last five decades more than 3,000 NPs have been discovered from algae,
most of them with cytotoxic activities (Review Alves C et al.: Frontiers in Pharmacol, August 2018)

• Cancer: Isoprenoids, e.g. halomon (halogenated monoterpene), dictyolactone (diterpene), dimeric

sesquiterpenes of the cycloaurane-type; fucoxanthin; alkaloids, e.g. caulerpin
• Cardio-vascular diseases: Polyphenols (green and red algae: bromophenols, phenolic acids,
flavonoids, brown algae: phlorotannins)
• Disturbances in blood coagulation: Sulfated polysaccharides
• Inflammation: Polyphenols
• Diabetes: Polyphenols
• Infectious diseases (human and animal, e.g. aquaculture): Sesquiterpenes
• Prebiotic/dietetics: Dietary fibers (Review O´Sullivan L et al. Mar Drugs 8, 2038-2069, 2010)

36
 Seaweeds: Pharmaceuticals (examples)

Eckol, a phlorotannin from Halomon, a monoterpen from Chamigranepoxid, a sesquiterpen

Ecklonia spec. Portieria hornemannii from Laurencia glomerata
antioxidative cytotoxic cytotoxic

37
 Seaweeds: Pharmaceuticals (examples)

C
l

B
r B
r
B
r

H
O H
O

Halogenated sesquiterpenes from Laurencia chondrioides with activity

against human and fish pathogenic bacteria

Bansemir A et al.: Chem Biodiv 1, 463-467, 2004

38
 Marine Fungi
• Ecological diverse group which belong to different phyla, mainly Ascomycota
• Grow on numerous substrata such as decaying wood and leaves, algae, corals etc.
• Found in sand, muds, soils, sediments
• Play a substantial components role in nutrient cycling (www.marinefungi.org)
• Up to 10,000 species of maine fungi estimated
• > 1,100 species are documented (Jones MD et al. Nature 474, 200-203, 2011)

• Occur often as endophytes

• Among the 272 new compounds discovered from marine fungi till 2002, 85% of
them are produced by epi/endophytes (Zhang Y et al., Mar Drugs 7, 97-112, 2009)

39
 Marine Fungi: Cosmeceuticals
• Photo-protective compounds: Mycosporine-glutaminol-glucoside
• Anti-aging products: Polysaccharides EPS, PUFAs
• Antioxidant compounds: Mycosporine like amino acids (MAAs) , carotenoids,
diketopiperazine alkaloids, dioxopiperazine alkaloids
• Skin-whitening products: kojic acid and derivatives, pyron derivatives, thalassothalic
acids etc.
• Additives (preservatives, surfactants, emulsifier, thickener, stabilizers, moistourizing):
polysaccharides (chitin, chitosan and derivatives), glycolipids, lipopeptides
(Review Espinosa-Leal CA et al.: Planta med 85, 535-551, 2019)

40
 Marine Fungi: Pharmaceuticals (research)
• Antimicrobial: about 50% of test extracts contain antimicrobial activity; peptides,
alkaloids, pyridines, diketopiperazines, steroids, terpenoids, polyketides (Review Xu L et al.:
Mar Drugs 13, 3479-3513, 2015)
• Cytotoxic (anticancer?): Alkaloids (Review Gomes NGM et al.: Mar Drugs 13, 3950-3991, 2015)
• First cephalosporin: Cephalosporin C from Acremonium chrysogenum
O
H O
N S
HO
NH2 O O O
O
O HO
O OH

Cephalosporin C Helicascolide C from a fungus (KT32)

isolated from a Gracilaria spec.
active against phytophathogenic fungi
Tarman K et al.:Planta med. 76, 1246, 201041
 Marine Fungi: Pharmaceuticals (Examples)

Neoechinulin B, isolated from Eurotium rubrum, Stachybotrin D, isolated from Stachybotrys

prenylated indole diketopiperazine alkaloid chartarum, a phenylspirodriman,
inhibits H1N1 virus and drug-resistant influenza inhibits NNRTI-resistant HIV strains
clinical isolates

(Abdelmohsen et al.: Lancet Infect Dis 17(2), E30-E41,

2017) 42
 What is to do to explore this potential?
Discovery
• Collection, identification and, if possible, cultivation of organisms
• Screening
• Isolation and identification of active compounds

Product development
• Establishment of sustainable manufacturing processes
• Development of suitable application forms (Formulation)
• Preclinical and clinical assays

Authorization and Marketing

• Authorization and commerzialization as pharmaceutical
• Commerzialization as cosmetic/cosmeceutical
• Commerzialization as food/food supplement
 Phases of Drug Development
(Example Antibiotic)

Small company

Basic research: -Screening for antibiotic materials

(mushrooms, plants, synthetic compounds…)
-Identification of antibiotic material
-Production in small amounts 44
 Screening strategies
• Biologically guided
Search for selected biological activities (antibacterial, cytostatic, enzyme inhibiting etc.) of
an extract mostly by in vitro methods
Fractionation of extracts according to the observed activities
Structure elucidation of the isolated bioactive compounds
Risk: compounds are already known
 Screening strategies
• Chemically guided
Search for unknown chemical structures in an extract by NMR, MS etc.
Isolation and structure elucidation of the novel compounds
Tests for biological activities
Risk: compounds without interesting biological activities
 Screening strategies
• Genetically guided („Genome mining“)
Search for genes and gene clusters coding for interesting biosynthetic pathways,
e.g. polyketide synthases
Activation of silent genes
Risk: long way, high risk

OSMAC concept (One Strain – Many Compounds)

Changes in cultivation conditions (composition of growth media, temperature, aeration, co-
cultivation etc.)

Expression of silent genes

Production of different (novel) compounds
Main criteria for a pharmaceutical product

• Pharmaceutical Quality
• Safety
• Efficacy

48
 Pharmaceutical quality: Identity

Is it really the declared material?

• Taxonomy, strain differences
• Confusion due to the similar popular names use of
scientific names
• Voucher specimen
• Content of substrate or other residues?

Methods:
Taxonomic, chemotaxonomic, macroscopic, organoleptic,
microscopic, chromatographic, DNA analysis

49
 Pharmaceutical quality: Purity
Are there any unallowed impurities, adulterations etc.?
• Microbiological quality
• Limits for residues of pesticides, fumigation agents, toxic
metals, radioactivity, possibly toxic components etc.
• Adulterations by foreign drugs, pure drugs (e.g.
, corticosteroids, hormones…), cheap material (starch…) etc.
• Contents of ash, water

Methods:
Visuell, microscopic, microbiologic, chromatographic, NMR, AAS,
specific methods

50
 Pharmaceutical quality: Content
Contains the product the active components or analytical lead
compounds in the right amount?

• Assays of constituents with known therapeutic activity, responsible

for the main effect of the product
• Assays of marker substances (where responsible active substances
are not known)
• Content must be indicated the lowest, possible tolerance

Methods
HPLC, physico-chemical methods, chemical methods,
determination of biological value

51
 Efficacy and Safety
• Pharmacodynamics: Study of pharmacological actions on
living systems, including the reactions with and binding to cell
constituents, and the biochemical and physiological
consequences of these actions (IUPAC)

• Pharmacokinetics: Process of the uptake of drugs by the

body, the biotransformation they undergo, the distribution of
the drugs and their metabolites in the tissues, and the
elimination of the drugs and their metabolites from the body
over a period of time (IUPAC)

52
 Preclinical studies: Efficacy

• In vitro assays
spectrum of activities
determination of main activity
mode of action

• Animal assays
justification of main activity
selectivity
bioavailability
pharmacokinetics

53
 Preclinical studies: Safety

• In vitro assays + animal assays (one rodent + one non rodent

species)
• Acute, subacute and chronic toxicity
• Genotoxicity (mutagenic potential, carcinogenic potential)
• Reproductive Toxicity

• Observations during clinical studies and further application

54
 Clinical Studies

• First stage (I)

small group of healthy adult people (10-50)
safety and pharmakokinetics
• Second stage (II)
small group of patients with the relevant indication (100-
300)
efficacy and pharmacokinetics
• Third stage (III)
controlled, randomized and multicentric study with
higher number of patients
• Fourth stage (IV)
observation of treated patients after licensation
55
 Criteria for a Good Clinical Study

• Good Clinical Practice (GCP)

• Allowance and supervision by appropriate regulatory
authorities
• Clinical trial protocol
• Statistical power
The larger the number of participants, the greater the
statistical power and the greater the costs.
• Informed consent
• Ethical aspects
• Regarding the types of studies interventional studies are
higher evaluated than observational studies and case reports

56
 Authorization of pharmaceuticals
• The regulation varies greatly between countries and global
regions.

European Union:
• National authorization procedure
• Centralized
• Decentralized (mutual recognition procedure)

Authorization as well established drugs or as traditional drugs.

57
 Alternatives

The development of a pharmaceutical for EBM is very strongly

regulated, very expensive and needs a lot of time. In the case of
natural products patent protection is often impossible.

A way out can be food supplements. Marine derived food supplements

can represent an easier way to enable the consumer access to
promising products.

Another way out can be cosmetics / cosmeceuticals.

58
 Cosmetics/Cosmeceuticals

-Global market for cosmetics and cosmeceuticals is forecasted to grow at a

rate of 4.3 % by 2022 with a value of USD 430 billion.

-Anti-aging market: is expected to grow at 7.5% from 2016 to 2021.

-Photo-protective, skin-care and hair-care products drive this trend in

increasing demand.

-Consumers´demand is turning to natural products given health concerns and

popular trends. (Corinaldesi et al. 2017)
Cosmeceuticals: Development

Fig. 1 Espinosa
 Cosmeceuticals: Bioactivity analysis
Anti-aging
Anti-collagenase test
Anti-elastase test
Hyaluronidase activity
Vitality and proliferation of cultivated fibroblasts and keratinocytes
Anti-oxidative tests

Anti-acne
Agar disk-diffusion method
Minimum inhibitory concentration
Anti-inflammatory activity

Solar protection
SPF measurement
UVA-PF
 Cosmeceuticals: Bioactivity analysis

Skin whitening
Tyrosinase inhibition

Wound healing
in vitro: scratch assay, co-culture of keratinocytes and fibroblasts

in vivo: rodents (skin differs strongly from human skin), rabbits

excision wound model: measurement of wound area and
hydroxyproline content
 Cosmeceuticals: Safety
Acute toxicity QSAR, predict based on chemical structure

Skin corrosion or irritation Cell models

Eye irritation Cell models

Skin sensitization Cell models

Genotoxicity Micronucleus test

Carcinogenicity Cell models

Phototoxicity Cell models

 Cosmeceuticals: New trends

• Formulation: Use of delivery systems: liposomes, microsomes,

transferosomes, lipid nanoparticles, polymeric microparticles, nanoparticles
to facilitate penetration of the skin barrier by the active ingredients to reach
appropriate sites

• Eco-friendly: use of less polluting solvents or solvent-free processes, like

microwave irradiation, for extraction of active ingredients

• Restriction of in vivo tests: creation of new in vitro methods (safety, cell

models for skin penetration)
 Example Maresome®
• A preparation consisting of microparticles of a defined cyanobacterial strain
• Protects human skin against colonization by MRSA
• Developed at the University Greifswald in collaboration with the IMaB e.V.
• Patent protection
• Licensation for use as cosmetics to a company
900

• In the company:
800
700
600
Number of 500

Scale up of production colonies 400

300
200

Formulation 100
0
Control Donator Acceptor

Packing
Commercialisation as cosmetic
 Example Maresome ®
Can we be satisfied with the present situation? No!
This product deserves application in hospitals, nursing homes etc.

Necessary are:
Licensation of the patent for use in hospital hygiene

Broader clinical trials

Licensation as medical product

 Problems, open questions
• Not enough starting material
• Real producer of the active metabolites?
• Often only in vitro assays
• Often high toxicity
• Lack of good clinical assays
• High risk
• High financial need
What can be done if the organisms cannot be cultivated?
• Collection in greater amounts (sustainability!)

• Synthesis of responsible biologically active compounds

• Partial synthesis of responsible biologically active compounds

• Isolation of genetic information and expression in

cells/organisms which can be cultivated more easily
(increasing importance of metagenomic assays)
 What is necessary in general?
• Expanding the knowledge on marine life and ecological relationships

• Sustainable production in accordance with ecology: biotechnology, gentechnology, synthesis

• Improvement of teaching and education

• Close interactions between basic and applied research and between research and industry

• Development of intelligent management and application strategies, patent protection

• Finances
Thank you very much for your attention!
Greifswald / Germany

Greifswald