ANTIBACTERIAL POLYMYXIN B ANALOGS

Programa Cooperación Farma-Biotech
Jornada 1-2012: Áreas Terapéuticas de Inflamación, Infección y Respiratorio

Antibacterial Polymyxin B analogs
Francesc Rabanal Anglada

A project of:

Managed by:

Barcelona, 14 de marzo de 2012


Content
1. The Research Group
2. The Product
a) Target indications
b) Innovative mechanisms of action
c) Differential features facing the market
d) Current status of development
e) IPR protection
f) Pitfalls & Risks to be considered
3. Partnering Opportunities


Research Group - Team and collaborators

Dr. Francesc Rabanal (PI) (Dept. Organic Chemistry, UB)
Design and Synthesis

Dr. Yolanda Cajal (Dept. Physical Chemistry, UB)
Biophysical Studies MoA

Antimicrobial activity evaluation (Dept. Microbiology, UB)
Dr. Ángeles Manresa

Antimicrobial activity in MDR bacteria (UB, CRESIB, CEK, Hospital Clínic)
Dr. Jordi Vila

In vivo studies (CERETOX/ UTOX-UB, Parc Científic de Barcelona)
Dr. Miquel Borràs


The Product. Target Indication
Need of new antibiotics
▫ Infectious diseases  Second cause of death in the world (third in the
developed world)
▫ Inadequate use of antibiotics  Increase in resistant bacteria 
Antibiotics loose effectiveness
▫ Reduced pipeline of new antibiotics


AntiMicrobial Peptides (AMP) - Innovative mechanism of action

• New antibiotics with novel MoA
• AMPs: act on bacterial membrane (no enzymes)
 lower risk of resistance (or reversible resistance)

Zasloff, Nature, 2002


...facing de market
• Need of new antibiotics
• Remergence of Polymyxin


Objectives and differential features
• Design and synthesis new antibiotics: lipopeptides based on the
structure of polymyxin/colistin

• Looking for...
 Activity in resistant and multi-resistant bacteria
 Lower toxicity
 Broader spectrum of activity
 Chemical synthesis designed for optimal scale up

Target Lead
Target ID Validation Pre-Clinical
Optimization

Clinical Trials Commercialization

Drug Discovery Animal Test


Objectives and differential features
H2 N
O
O
N
H
OH O NH HN

O
H
N
O
N
H
O
H
N
O
N
H
S
O NH Design
S O NH2
HN
NH2 NH2 O
N
H
O
NH2 NH2

MoA Synthesis

In vitro
Efficacy

In vitro
Design & Synthesis MoA IN VIVO IN VIVO
Efficacy
Of New Compounds SAR TOX. (LD50) EFFICACY
(MIC)

In vitro TOX


Design Rational…

Polymyxin B


H2 N

Analogs Design Polymyxin O

N
O
H
OH O NH HN
O O
H H
N N N N O NH
H H
O O
Designed lipopeptide HN O
HN
NH2
NH2 NH2 O
O N
H
HO
NH2

H2 N
O
O
N
H
OH O NH HN
O O
H H
N N N N O NH
H H
O O S
S O NH2
HN
NH2 NH2 O
N
H
O
NH2 NH2


Antibacterial Activity – in vitro
(measured as Minimum Inhibitory Concentration, MIC, in μg/mL)
Gram+ Gram-
Nº Peptide sequence
S. aureus P. aeruginosa E. coli
pxb CH3-octanoyl-Dab-Thr-Dab-Dab-Dab-Phe-Leu-Dab-Dab-Thr >32 2 1
0 nonanoyl-Dab-Thr-Dab-Cys-Dab-Phe-Leu-Dab-Dab-Cys >32 8 4
1 nonanoyl-Arg-Thr-Dab-Cys-Dab-Phe-Leu-Arg-Dab-Cys 32 16 8
2 nonanoyl-Arg-Thr-Arg-Cys-Dab-Phe-Leu-Arg-Dab-Cys 8 8 4
3 nonanoyl-Dab-Thr-Dab-Cys-Dab-Phe-Met-Dab-Dab-Cys 32 16 4
34 nonanoyl-Arg-Thr-Dab-Cys-Dab-Trp-Leu-Arg-Dab-Cys 8 8 4
79 decanoyl-Arg-Thr-Dab-Cys-Dab-Phe-Leu-Arg-Dab-Cys 16 8 8
85 dodecanoyl-Arg-Thr-Dab-Cys-Dab-Phe-Leu-Arg-Dab-Cys 32 8 8
16 nonanoyl-Arg-Thr-Dab-Cys-Dab-Phe-Leu-Arg-Dab-Cys 16 8 4
93 decanoyl-Lys-Thr-Arg-Cys-Lys-Trp-Leu-Arg-Lys-Cys 16 >64 64
100 Up to 90 analogs synthesized and tested
4
decanoyl-Arg-Thr-Arg-Cys-Dab-Trp-Nle-Arg-Dab-Cys 64 8
101 nonanoyl-Dab-Thr-Arg-Cys-Dab-Phe-Leu-Arg-Dab-Cys 8 16 2
103 ------------------------------------------------------------------ 4 2 1
104 ------------------------------------------------------------------ 4 1 4
105 ------------------------------------------------------------------ 4 1 2


(measured as Minimum Inhibitory concentration, MIC, in μg/mL)

Comparison with reference marketed products.
PxB
Analog Analog Analog Vancomycin Daptomycin
Species G(-)
103 104 105 G(+) control G(+) control
control
Pseudomonas aeruginosa 2 1 1 2 - >32
GRAM -
Escherichia coli 1 4 4 1 - >32

Mycobacterium phlei 4 4 2 16-32 - >32
GRAM
+
Staphylococcus aureus 4 4 4 >32 1 2



In multi-drug resistant bacteria.
Analog Analog Analog
Resistant and multi-resistant Gram negative bacteria
103 104 105
40b Carbapenem-resistant strain 4 2 4
P. aeruginosa
38a Highly-resistant strain 0,5 1 16
MAC 21a Intermediate resistance to quinolones 0,5 1 2
VAL 10 Intermediate resistance to quinolones 0,5 0,5 1
E. coli
VAL 5 Intermediate resistance to quinolones 0,5 0,5 1
NDM Highly-resistant strain 0,5 0,5 1

• Resistance panel:
▫ 40b (clinical isolate): resistance to IMP
▫ 38a (clinical isolate): resistance to Ceftazidime; Ciprofloxacin; Imipenem ; Piperacillin-tazobactam
▫ NMD, New Delhi metallo-beta-lactamase: Amoxicillin 256 mg/L; Amoxicillin clavulanate 32 mg/L ;
Piperacillin/tazobactam 256 mg/L; Cefoxitin 256 mg/L; Cefotaxime 256 mg/L; Ceftazidime 256 mg/L;
Cefepime 256 mg/L; Imipenem 8 mg/L; Meropenem 16 mg/L; Doripenem 6 mg/L; Ertapenem 24 mg/L;
Aztreonam 256 mg/L; Gentamicin 8 mg/L; Amikacin 32 mg/L, Tobramycin 8 mg/L; Ciproﬂoxacin 32 mg/L



In Colistin resistant a. baumannii strains.

Analog Analog Analog
Colistin-Resistant Acinetobacter baumannii Colistin
103 104 105

ATCC-wt Low resistance strain 1 8 2 4

In vitro mutant of ATCC with resistance to
ATCC 256 64 16 32
colistin

A. Baumannii In vitro mutant of a clinical strain with resistance
77778 256 256 128 128
to colistin

Ab 10 Clinical isolate resistant to colistin 512 32 16 8

Ab 19 Clinical isolate resistant to colistin 512 32 16 16

Moderate antibacterial activity in colistin-resistant Acinetobacter baumannii
suggests an alternative mechanism of action


Mechanism of action

• By Flow cytometry

Propidium iodide Bis-(1,3-Dibutylbarbituric Acid)Trimethine Oxonol
DIBAC4(3)


Mechanism of action results

CONTROL, 120 min PXB, 120 min PEPT. 100, 120 min PEPT. 103, 120 min

E. coli

S. aureus

Flow-cytometric tests show a different behaviour
of lipopeptides in front Gram+ and Gram- bacteria


In vivo Toxicity

• Acute toxicity test in mice for
peptide 103

• LD50 = 283 mg/Kg
(subcutaneous)
Polymyxin LD50 = 59,5 mg/Kg

Protocol OECD 425.
5 reversals in 6 consecutive animals. Animals (3) administered at 200 mg/kg
survived after 14 days; Necropsy indicated no visual damage in vital organs. Mice
(3) administered at 400 mg/Kg died


Current status of development

In vitro
Design & Synthesis MoA IN VIVO IN VIVO
Efficacy
Of New Compounds SAR TOX. (LD50) EFFICACY
(MIC)

In vitro TOX
H2 N
O
O
N
H
OH O NH HN
O O
H H
N N N N O NH
H H
O O S
S O NH2
HN
NH2 NH2 O
N
H
O
NH2 NH2
Design

MoA Synthesis

In vitro
Efficacy


IPR Protection

• 2 different patents

• ES 200802626 Granted

• ES 2.374.779 A1 Filed - Priority March 2010
PCT/ES2011/070153 Published as WO2011110716 - ISR Positive

• Ownership
– University of Barcelona 100%


Pitfalls & Risks to be considered

Threats
• Efficacy in in vivo model.
• PK/PD & ADME development.
• Additional Toxicology development
• Patent going to National phases in Sep. 2012

Weaknesses
• No Oral Administration (not confirmed)
• Early stage project. Few in vivo tests performed.
• Need of partner to accelerate development


Pitfalls & Risks to be considered
Strengths
• Good antibacterial activity front both pathogen and multi-drug
resistant bacteria
• Better in vivo and in vitro tox. profile than PxB
• MoA independent of membrane receptors
• Easy Synthesis
• Intl. patent file WO2011110716. Priority march 2010- Positive ISR.
• Ownership 100% UB

Opportunities
• Market Needs of new antibacterial compounds with wide spectrum /
low toxicity profile.
• In vivo efficacy tests in 2012


Market competitors
PolyMedix, for S. aureus
Several products in preclinical studies for indications such as tuberculosis, malaria,
bacterial infections. PMX-30006 is a potent broad spectrum antibiotic, which is active
against a number of bacterial strains. PMX-30006 is being developed for the treatment of
bacterial infections.

Cubist ; CB-182804, macrolactam PxB
CB182804 is a lipopeptide which interacts with cell membranes and rapidly shuts down
bacterial DNA, RNA and protein synthesis. CB182804 was under development as
intravenous injection for the treatment of serious infections caused by multi-drug resistant
(MDR) gram-negative bacteria.
– Discontinued in phase I in 2009.

Northern Antibiotics (complex synthesis: macrolactam)
NAB 7061 is a polymyxin derivative that sensitizes target bacteria to other antibiotics
such as rifampin and clarithromycin. NAB 7061 is being developed for the treatment of
serious gram-negative hospital infections.


Partnering Opportunities

The project is available to licensing out through a
collaboration and license agreement.

Contact details

Salvador Mena
Project Manager
Tel: +34 93 403 97 95
smena@fbg.ub.edu


Acknowledgments

Funding has been provided by:

▫ Ministerio de Ciencia e Innovación (CTQ2008-06200)
▫ Generalitat de Catalunya (VALTEC 08-1-0016, ACC10)
▫ Fundació Bosch i Gimpera (UB)


THANK YOU FOR YOUR ATTENTION!


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Distaclor 53.40 54.00
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Summary:

-Proof of concept in vitro for EC, PA and SA

-Activity in resistant and MDR bacteria

-In vivo toxicity (acute, sc) better than PxB

-Optimized chemical synthesis and scale up
(i. e, octreotride, lanreotide)

-Next step: proof of concept in vivo (efficacy)


Optimized chemical synthesis and scale up
(i. e, octreotide, lanreotide) OH

O H
N
O NH HN
O
H2 N NH
N O NH2
H
S
S O
HN O
N
H
O
NH
Lanreotide:
O H-D-2-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2
H2 N
Lanreotide is a synthetic analog of somatostatin with
properties similar to octreotide
H2 N
O
O
N
H
OH O NH HN
O O
H H
N N N N O NH
H H
O O S
S O NH2
HN
NH2 NH2 O
N
H
O
NH2 NH2

ANTIBACTERIAL POLYMYXIN B ANALOGS

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