Screening for natural products from marine microorganisms – from research to commercialisation

Ulrike Lindequist Institut für Pharmazie und

Institut für Marine Biotechnologie e.V. lindequi@uni-greifswald.de

http://pharm1.pharmazie.uni-greifswald.de

Rendez Vous de Concarneau, October 2013

Number of marine natural compounds by groups of organisms

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

Presently 16-18% of all published novel marine compounds are of microbiological origin (Blunt et al., Nat. Prod. Rep. 2010, 27, 165-237)

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 • 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 • Genetically guided („Genome mining“) Search for genes and gene clusters coding for interesting biosynthetic proteins, e.g. polyketide synthases Risk: long way, high risk

Biological Screening

Antibacterial/antifungal Antiviral Antitumor

Bone cells Keratinocytes (Osteoporosis) (Wound healing, UV protection, cosmetics)

Groups of marine microorganisms

• Marine bacteria, mainly actinomycetes • Marine fungi, mainly ascomycetes • Marine cyanobacteria H

N

O

O

O

ClH2C

HHO

Salinosporamid A

Cyanobacteria

• Gram-negative photoautotrophic prokaryotes • Free-living or living in invertebrates • Cause of toxic water blooms • Important producents: Lyngbya sp. • Focus on cytostatic, antimicrobial and enzyme-inhibiting activities • Many cyclic or linear peptides often containing lipophilic side chains

or macrolides • Mixed biosynthetic pathways (polyketide synthase/nonribosomal

peptide synthetase)

• Till now of some importance for food/feed industry, cosmetic and technical purposes

Cyanobacteria: examples

Carbamidocyclophan A: MIC St. aureus: 0.1 mmol IC 50 MCF 7 cells : 0.8 µmol IC 50 FL cells: 3.1 µmol

HO OH

OCH3

H2N

O

Cl

Cl

Cl

HO OH

OCH3

NH2

O

Cl

Carbamidocyclophanes from Nostoc sp. CAVN 10

Ha T. N. Bui et al.: J Nat Prod 70, 499 (2007), Dissertation Michael Preisitsch

OH

O

NH

CH3

O

N

O

OH

NH

O

NH

ONH

CH3

O

NO

CH3

CH3

CH3O

NH

O

OHOH

CH3

NH2

O

Tyr

Ahp

Val

N-Me-Phe

IleGln

PAA

Thr

O

NH

CH3

O

N

O

OH

NH

O

NH

ONH

CH3

O

NO

CH3

CH3O

NH

O

OHOH

CH3

CH3CH3

CH3

O

NH2

Leu

Ahp

Ile

N-Me-Phe

ValThr

PAA

Asn

Ichthyopeptin A

Ichthyopeptin B

IC 50 Influenza Virus A: 12.5 µg/ml

Zainuddin E et al.: J. Nat. Prod. 70, 1084 (2007)

Microcystis ichthyoblabe

Cyanobacteria: examples

Problem: Multiresistant bacteria (MRSA, VRE, ESBL etc.)

*European Antimicrobial Resistance Surveillance Network (EARS-Net), 2010

What can we do? •Better hygiene •New antibiotics •Effective antiseptics

•Prevention of colonisation: an example for commercialisation

Prevention of colonisation - Screening of about 120 different marine microbial strains (biological screening) - Selection of microalgal strain Bio 33, isolated from the Baltic Sea - Microencapsulation of the cultivated biomass into Maresome® (patent protected) - Preparation of Maresome® ointments - In vitro assays - In vivo assays

Bio 33

Bio33-Maresome®*

• Microparticles consisting of the whole biomass of cultivated microalgae strain • No additives • Prepared by microencapsulation using the lipids of the microalgae Multifactorial mode of action: • Stabile system in aqueous dispersion with negative zeta potential –

repulsion of bacteria • Increased surface: better release kinetics and bioavailability of bioactive

compounds (antibacterial fatty acids etc.) • Multiresistant strains more sensitive because of higher extracellular lipase

activity of MRSA and possibly other factors • Positive effects on skin

Prevention of contamination of the skin by multiresistant pathogenic microorganisms (MRSA etc.)

Lukowski et al.: PCT-03/00747

*in the following declared as Maresome® Trade name Maresome® refers to principle of preparation.

0100200300400500600700800900

Number of colonies

Control Donator Acceptor

Prevention of transfer of MRSA from a donator to an acceptor

Influence of Maresome ® on the colonisation of MRSA „North German Epidemic Strain“ in the model „Mouse ear”

Lukowski et al.: Skin pharmacology and physiology 21, 98-105, 2008

0

200

400

600

800

1000

1200

number of colonies

control Chlorella Spirulina Bio 33

Influence of Maresome® on the colonisation of MRSA „North German Epidemic Strain“ in the model „Cow udder teat”

Comparison to other microalgae strains

Lukowski et al.: Skin pharmacology and physiology 21, 98-105, 2008

0

500

1000

1500

number of colonies

Control Col Mu 50 N315 Nord. Epe.Strain

Influence on the colonization of several MRSA strains

Influence of Maresome® on the colonisation of several MRSA strains in the model „Cow udder teat”

Lukowski et al.: Skin pharmacology and physiology 21, 98-105, 2008

0

200

400

600

800

1000

1200

Control Algae ointment

colo

ny fo

rmin

g un

itsInfluence on the colonization of Pseudomonas aeruginosa

Influence of Maresome® on the colonisation of P. aeruginosa in the model „Cow udder teat”

0200

400600

8001000

12001400

16001800

Donor Acceptor Control

colo

ny fo

rmin

g un

itsInfluence on the transfer of Candida maltosa

Influence of Maresome® on the transfer of C. albicans in the model „Cow udder teat”

Clinical observation

Before treatment Up to 3 days after treatment

Pathogenic skin flora (Cfu/cm2) S. aureus 570 0

P. aeruginosa 60 0

S. agalactiae 50 0

Physiologic skin flora (Cfu/cm2)

Coagulase neg. Staphylococci

3 x 103 102

Patient with phlebolipolymph edema and erysipel

G. Lukowski , Lindequist U, Mundt Jülich WD, Jünger M, Daeschlein,G, COSMODERM XVI – ESCAD, 2010

Clinical observation

Patient: 82y, ulcus cruris venosum left lower limb d1

Control (cfu/ plate)

d2 with ointment (cfu/ plate)

d3, d4, d5 with ointment (cfu/ plate)

S. aureus 4 0 0

Pseudomonas aeruginosa

10 0 0

Klebsiella oxytoca 2 0 0

Application 1 times per day

Comparison with positive and negative controls

Effects of a 30min and 120min treatment on MSSA on healthy skin

3,1 4,8 1,2 0,2

51,3

31,8

0,0

20,0

40,0

60,0

80,0

100,0

120,0

30min 120min

%

Gentamicin

Maresome Base Control

Effect on dermal barrier function

TEWL

15 139,7

14,5518,2

13,512,3 11,1 11,2

05

101520

Algae-O

intmen

t

Betamethaso...

untreate

d

Med

ian

Day 1 Day 3 Day 8

TEWL = transepidermal water loss (g/m2*h)

Cosmetic trial with atopic patients

skin care improvement

0

10

20

30

40

50

60

70

80

strongly improved improved not improved

% o

f all

resp

onds

after 4 Weeks after 8 Weeks

Interview with 104 patients, ambulant, 2011

Summary Bio33-Maresome ®

– Protection against bacterial (re)infections

– Prevention of (re)colonization with pathogens (MRSA etc.)

– Preservation of physiological skin flora

– Prevention of nosocomial infections and in cosmetic procedures

– Ideal skin care for patients with atopic dermatitis, eczema, superficial folliculitis, diabetic and phlebologic problems

Maresome ®: from research to commercialisation: Research

• Successful screening interesting biological effect

• Meeting high medicinal need

• First clinical results

• Patent protected formulation • Protected brand name (Maresome®)

• Search for possible industrial collaborators

Maresome ®: from research to commercialisation: Commercialisation: Present situation

• Licensation for use as cosmetics to a company

• In the company: Scale up of production Formulation Packing Commercialisation • Sale as cosmetic product in Germany and and Mexico

Maresome ®: from research to commercialisation

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

Challenges on the way from research to commercialisation

• Sustainable production + • Scale up + • Patent protection + • Money - • Clinical trials - • Regulatory aspects, licensation -

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 • Finances

Contact: Prof. Dr. Ulrike Lindequist E-mail: lindequi@uni-greifswald.de

Thanks to all colleagues, especially PD Dr. Sabine Mundt and her group, students and collaborators Thank you for your attention!