Cationic amphiphilic non-hemolytic synthetic polymers as potential agents to combat bacteria with antibiotics resistance

A.Punia, E. He, K. Lee and N.-L. Yang

Center for Engineered Polymeric Materials and Chemistry Ph.D. Program. City University of New York ,N.Y. USA

*E-mail: Nanloh.yang-cepm@csi.cuny.edu Professor Nan-Loh Yang

CEPM Diretor & Chair Ph.D Program in Polymer ChemistyCity University of New York, CUNY

Nanloh.yang-cepm@csi.cuny.edu, 1 718 982 3926the New York State Office of Science, Technology, and Academic Research

Dec 15, 2016

Nulastin

CEPM Industrial Sponsors

23,000 people killed every year in United Stateswww.nih.gov

Nearly half of E. coli infections in India are practically untreatablewww.abc.net.au

2 million people infected each year with drug resistant bacteria nationwidewww.nih.gov

Common surgeries will soon become impossible to perform

~ 11,000 deaths/year from methicillin resistant S. aureuswww.nih.gov

~450,000 new cases of MDR-TB each year – 1/3rd

diewww.who.int

Beforeitsnews.com

www.sciencephoto.com

floridamedicalmalpracticelaws.com

www.examiner.com

D. Jabes, Curr. Opin. Microbiol. 2011, 14, 1; www.cdc.gov; www.who.int; www.reuters.com.

The superbug, carbapenem-resistant enterobacteriaceae (CRE), was found after researchers swabbed the floors and walls of the farm’s pens and collected fecal samples during a five-month period in 2015. Journal Antimicrobial Agents and Chemotherapy. Dec. 5, 2016, by Ohio State

antibiotic drugs targeting The Ribosome

the

The ribosome, one of the ubiquitous molecular machines in living cells, is responsible for the critical task of translating the genetic code into functional proteins ( U of Ill)

The story of Magainins..Michael ZasloffProc. Natl. Acad. Sci. USAVol. 84, 1987, 5449-5453

African clawed frog Xenopus laeviso After performing surgery under non-

sterile conditions, the frogs were beingplaced in microbially contaminatedwater tanks

o Surgical wounds never developedinfections

o Normal healing of scar always occurred

o Potent antimicrobial peptides werepurified from frog’s skin and named“Magainins” http://www.upenn.edu/gazette

Surgery

No infection!!!

Positive Charge and facialamphiphilicity being the commonfeature of AMPs

AMPs generally manifest onehydrophobic & one hydrophilic facealong their backbones

Direct action against bacterialmembrane

Development of bacterial resistance isimprobable

α-Helical Cationic AMP

Cationic

Hydrophobic

Membrane rupturePore formation

Leakage of cellular componentMembrane potentialbreakdown

Natural Antimicrobial Peptides (AMPs)

e.g. Magainin (African clawed frog) LL-37 (humans)Cecropins (Silk moth)

Bacterial CellDeath

M. Zasloff, Nature, 2002, 415, 389.K. Kuroda, G. A. Caputo, WIREs: Nanomed. Nanobiotechnol., 2012, 5(1), 49

Chem. Eur. J. 2009, 15, 11784 – 11800

E. coli (ampicillin resistant) E. coli after polymer treatment

Figures of Merits from Biological AvtivitityMeasurement

1) MIC (µg/mL) Minimum Inhibitory Concentration MIC is expressed as the minimum polymers concentration that resulted in

100% inhibition of bacterial growth after an incubation period of 18 hours.

2) HC50 (µg/mL) , RBCsHemolytic activities of polymers were determined in terms of hemolytic concentration-50% (HC50), the minimum polymer concentration resulting in 50% lyses of mouse RBCs within an incubation period of 1 hour.

Selectivity : HC50/MIC; MIC /MIC’

Focus on the effects of topological placement of hydrophilic and lipophilic structure segments. Various structural parameters examined included: amphiphilic balance, spatial arrangement of cationic and hydrophobic groups, random copolymer and homopolymer architecture, and cationic charge density

Structural Isomers

Structural Isomers

Why Synthetic Polymers Large scale implementations of AMPs is a challenge

1) Large scale isolation or manufacturing of complex AMPs would be extremely costly and time consuming

2) Decreased activity of AMPs, once introduced into complex serum milieu

3) Efficient digestion of protein/peptide material by gastrointestinal tract leads to lower oral availability of AMPs

4) Limited broad availability will result from intravenous delivery

Synthetic mimics of antimicrobial peptides??

Spacer arm design strategyPalermo, E. F.; Vemparala, S.; Kuroda, K. Biomacromolecules2012, 13(5)

OO

NH3

OO

NH3

OO

NH3

2-ca

rbon

6-ca

rbon

4-ca

rbon

MIC (E. coli) 1000 2000 10 MIC (S. aureus) 63 250 7.8

HC50 >2000 >2000 2.6

Highly antibacterial Highly hemolytic

All values in µ/mL)

S

O

O

OO

NH3

q

Effect of spacer arm length (Kuroda & Co-workers)

S

O

O

OO

NH3

OO

MIC MIC HC50 E. coli S. aureus RBCs(µg/mL) (µg/mL) (µg/mL)

10 7.8 2.6

7.8 7.8 2.8

6.5 10 2.3

Antibacterial but extremely hemolytic

27% Ethyl

40% Ethyl

Palermo, E. F.; Vemparala, S.; Kuroda, K. Biomacromolecules 2012, 13(5)

HONH2

(Boc)2O, room temp.

1M NaOH : THF (1:1)24 hrs

HO

HN

Boc

OCl

Triethyl amine, CH2Cl20 oC

room temp, 24 h

O

O

NH

Boc M6

HO

HN

HON

Boc

(Boc)2O, 34 oC

H2O, 3 hrs

O

O

N

Boc

OCl

N,N-diisopropylethyl amine0 oC

room temp, CH2Cl224 h

M2

a) Monomer Synthesis

Experimental

1

10

100

1000

10000

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

MIC

(µg/

mL

)

Mole % of M6 Monomer

Polymer Avg-MIC(µg/mL)

PM6-0% 1428

PM6-10% 809

PM6-20% 250

PM6-30% 125

PM6-40% 62

PM6-50% 52

PM6-60% 41

PM6-70% 26

PM6-80% 15

PM6-90% 7.8

PM6-100% 5.8

S

O

O

OO

NH3

q

Antibacterial activity towards E. coli *

Increasing antibacterial activity

*Values reported here are the averages of three separate experiments performed on different days. Error bars represent standard deviation.

S

OO

NH2

p

O

O

S

O

O

OO

NH3

OO

NH2

q1-q

1

10

100

1000

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

MIC

(µg/

mL

)

Mole % of M6 Monomer

Polymer Avg-MIC(µg/mL)

PM6-0% 104

PM6-10% 62

PM6-20% 62

PM6-30% 52

PM6-40% 52

PM6-50% 52

PM6-60% 31

PM6-70% 31

PM6-80% 26

PM6-90% 15

PM6-100% 15

S

O

O

OO

NH3

q

Antibacterial activity towards S. aureus

S

OO

NH2

p

O

O S

O

O

OO

NH3

OO

NH2

q1-q

1

10

100

1000

10000

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

HC

50

(µg/

mL

)

Mole % of M6 Monomer

Polymer HC-50(µg/mL)

PM6-0% >2000

PM6-10% >2000

PM6-20% >2000

PM6-30% >2000

PM6-40% 1619

PM6-50% >2000

PM6-60% >2000

PM6-70% >2000

PM6-80% >2000

PM6-90% 1619

PM6-100% <1.9

S

O

O

OO

NH3

q

Hemolytic activity towards mouse Red Blood Cells*

S

OO

NH2

p

O

O

S

O

O

OO

NH3

OO

NH2

q1-q

* Values reported here are the averages of 3 experiments. Error bar represents standard deviation. RBCs were obtained from freshly drawn mouse blood.

1

10

100

1000

10000

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

MIC

or H

C50

(µg/

mL)

Mole % of M6 Monomer

E. coli

S. aureus

HC50

PolymerSelectivityHC50/MIC

E. coli S. aureus

PM6-0%

PM6-10%

PM6-20%

PM6-30%

PM6-40%

PM6-50%

PM6-60%

PM6-70%

PM6-80%

PM6-90%

PM6-100%

>1.4

>2.5

>8

>16

26

>38

>49

>80

>133

208

<0.33

>19

>32

>32

>38

31

>38

>64

>64

>80

108

<0.13

Biological activity of polymers: Highly selective polymers

S. aureus > E. coli

E. coli ~ S. aureus

Doubly selective polymers: Bacteria over RBCsBacteria A over Bacteria B (switchable) Punia, A.; Yang, N. U.S. Provisional Patent 61889401, 2013

Punia, A.; He, H.; Lee, K.; Banerjee, P.; Yang, N-L., Chem. Commun. 2014, 50, 7071-7074

0

20

40

60

80

100

120

0 2 4 6 8 10

Frac

tiona

l Cel

l Sur

viva

l (%

)

Time (h)

1×MIC

2×MIC

a b c d

e f g h

Time (h)

E. coli

1 × MIC 2 × MIC Control

0

1

2

4

6

8

6.0 × 104

3.0 × 104

4.7 × 103

6 × 102

nil

nil

2.3 × 104

3.5 × 103

2 × 102

nil

nil

nil

0.9 × 105

1.5 × 105

4.8 × 105

4.8 × 106

1.1 × 108

3.6 × 108

Time dependent killing efficiency study

positive control and is shown in a) at 1 h, b) at 6 h time interval. E. coli treated withPM6-90% at c) 0 h, d) 1h, e) 2 h, f) 4 h, g) 6 h, and h) 8 h time interval

Viable cells (CFU/mL) are shown after incubating with PM6-90% or without PM6-90% (control), for various time intervals

S

O

O

OO

NH3

OO

NH2

0.90.1

PM6-90%

N

Cetyltrimethylammonium bromide

N

Cetylpyridinium chloride

NR

Alkyl dimethylbenzyl ammonium chloride(C: 12 to 18)

HC50 = 0.192 µg/mLMIC = 25 µg/mL (gram negative E. coli)R =12

MIC = 4 µg/mL (E. coli)

HC50 ~ 0.23 µg/mL

Quaternary Ammonium Compounds (QACs)Vs

Amphiphilic Polymers

MIC = 7.8 µg/mL (E. coli)HC50 > 1619 µg/mL

S

O

O

OO

NH3

OO

NH2

0.90.1

>7000 times less hemolyticthan Cetyltrimethylammoniumbromide

Terpolymer

•

Time-Kill for Terpolymer

0

20

40

60

80

100

120

0 1 2 3 4 5 6Time (h)

E. coli Pe

rcen

tcel

l sur

vica

l 1XMIC (PolyE-12%)

2XMIC (PolyE-12%)

c)

0

20

40

60

80

100

120

0 2 4 6 8 10

Frac

tiona

l Cel

l Sur

viva

l (%

)

Time (h)

1×MIC (PM6-70-300peg)

2×MIC (PM6-70-300peg)

1×MIC (PM6-90-950peg)

2×MIC (PM6-90-950peg)

0

20

40

60

80

100

120

0 2 4 6 8 10

Frac

tiona

l Cel

l Sur

viva

l (%

)

Time (h)

1×MIC (PM6-70-300peg)

2×MIC (PM6-70-300peg)

1×MIC (PM6-90-950peg)

2×MIC (PM6-90-950peg)

Time dependent killing efficiency study

E. coli

S. aureus

Confirmation of bactericidal activity of PM6-90-PEG950 at various time intervals using Agarplates. The final polymers concentration was equal to MIC value. E. coli without polymertreatment was used as positive control and is shown in a) at 1 h, b) at 6 h time interval. E. colitreated with PM6-90-PEG950 at c) 0 h, d) 1h, e) 2 h, f) 4 h, g) 6 h, and h) 8 h time interval

PM6-90-PEG950 at 1×MIC against E. coli

a b c d

e f g h

PM6-90-PEG950 at 2×MIC against S. aureus

Confirmation of bactericidal activity of PM6-90-PEG950 against S. aureus at various timeintervals using Agar plates. The final polymers concentration was equal to 2×MIC value. S.aureus without polymer treatment was used as positive control and is shown in a) at 1 h, b) at 8 htime interval. E. coli treated with PM6-90-PEG950 at c) 0 h, d) 1h, e) 2 h, f) 4 h, g) 6 h, and h) 8 htime interval

2014-2015 - Our Publications and Non-Provisional Patents on

Cationic amphiphilic non-hemolytic polyacrylates with superior antibacterial activity

Royal Society of Chemistry, Chem. Commun., 2014,50, 7071-7074American Chemical Society , Macro Letters 2015, 4, 426 – 430. Royal Society of Chemistry Advances. 2015, 5, 80318-80324. Royal Society of ChemistryRSC Advances 2015, DOI: 10.1039/C5RA17875DRoyal Society of Chemistry Advances, 2015,5, 95300 - 95306International Journal of Molecular Sciences 2015, 16(10), 23867-23880.

US non-provisional Patent App. No.14/510,686 YANG and PUNIA

Polymer Killing Action

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