From modelling to synthesis to

formulation to microbe: A multi-disciplinary approach to developing treatment for

multi-drug resistant respiratory infection

Antimicrobial resistance (AMR)- a global threat

Source: The Review on Antimicrobial Resistance, Jim O’Neill, 2014

Increasing incidence of respiratory infection-associated mortality

Source: Troeger, C et al, 2017. Estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in 195 countries: a systematic analysis for the Global Burden of Disease Study 2015. The Lancet Infectious Diseases, 17(11), pp.1133-1161.

Antimicrobial resistance (AMR) and respiratory infection

Respiratory infections with increasing AMR incidence

include:

• Hospital-acquired pneumonia 1

• Escherichia coli

• Acinetobacter baumanii

• Cystic fibrosis-associated respiratory infection2

• Pseudomonas aeruginosa

• Staphylococcus aureus

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What is needed to tackle the problem of MDR-respiratory infection?

1. Discovery of new antimicrobial molecules

• Desperately need new antimicrobial drugs

• Should be designed to tackle the cellular

mechanisms of MDR

• Must demonstrate activity against MDR-pathogens

2. Effective way of delivering new compounds to the infected lung

• Inhalation – enables higher local concentration of drug in the lung

• Residence time is important – “right drug, right time, right dose” 3

• Formulation approaches (e.g. liposomal encapsulation) have been demonstrated to facilitate longer retention and

improved clinical outcomes for antimicrobials 4

Project rationale

• Interdisciplinary approach:

ChemistryComputational

Synthesis

PharmaceuticsFormulation design,

preparation and characterisation

MicrobiologyAbility to test drug and

formulation against clinically isolated MDR-pathogens

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Development, characterisation and biological testing of a liposomal

formulation of a novel lead candidate antimicrobial, AB1 which would be

suitable for inhalation

PROJECT AIM:

Ready for pre-clinical testing as an inhaled treatment for MDR-respiratory infection

Methods: Design of AB1 (ARB-drug hybrid)

• Low molecular weight -

typically <150 dalton

ARB Fragments criteria:

• In silico study must show greater affinity and stronger interaction

with the binding pocket of the transporter than the unmodified

antibiotic

• Must retain the intrinsic mechanism of action of the antibiotic class

ARB-Antibiotic Hybrids criteria:

Source: Laws, M., Jamshidi, S., Nahar, K., Sutton, M. and Rahman, K. M. (2017), Antibiotic Resistance Breakers,UK Patent Application Number: GB1708606.6, PCT Patent Application No. PCT/GB2018/051468

AB1 – designed by Dr Miraz Rahman AMR Research Group (KCL)

ARB: Antibiotic Resistance Breaker

Methods: Synthesis of AB1 and the ARB compounds

Example synthetic strategy: ARB derivatives of ciprofloxacin

Lead candidate (AB1)• Improved binding to bacterial target • Improved antimicrobial activity

against MDR-strains• BUT increased hydrophobicity• Challenge for inhalation rapid

absorption

Synthesise on small scale (20-200 mg)

Test antimicrobial activity

Methods: Development of liposomal formulation

Objective 1: Preparation of liposome carrier

Analysis: Size and polydispersity analysed using dynamic light scattering (DLS) (Zetasizer NanoSeries, Malvern Panalytical)

Method: Thin lipid film hydration, extrusion and ammonium sulphate gradient loading 5

Results: Preparation of liposomal carrier

Outcome 1: Extrusion produces a good quality liposome suspension with diameter ~150 nm and

narrow polydispersity (P.d.I.<0.1)

Outcome 2: Liposomes remain stable at 37°C for 7 days, and 4°C for up to 4 weeks

Methods and Results: Drug loading of liposomal formulation

Objective 2: Load the liposome carrier with AB1 and characterise drug loading

Statistical optimisation: • Two variables (incubation time, mins, and temperature, °C), 3 levels• Nine combinations

Analysis: (1) Extract drug using acetonitrile. Quantify using HPLC (2) Size measured using DLS

Outcome 3:Optimised loading conditions:• Drug +

liposomes at 1:2 ratio

• Incubated at 55°C for 70 mins

Results:

Results: Drug loading of liposomal formulation

• Outcome 4:

• Drug loading capacity (%DLC) of 20-25% achieved

• Comparable to levofloxacin (control)

• Outcome 5:

• Drug loading does not affect liposome size

Methods: Investigation of controlled release properties

In vitro release assay:

Sample:Liposomal AB1ORFree AB1

Drug release media:150 mM NaCl at 37°C

Samples removed over 24h

Analysis:Drug release

quantified using HPLC

Objective 3: Assess the controlled release profile of AB1 from liposomal formulation vs free drug

For fluoroquinilone-type molecules like AB1, rate of drug release across dialysis membrane has been shown to correlate with lung absorption in vivo 6

Hypothesis: Liposomal AB1 will have reduced release rate compared to free drug

Results: Investigation of controlled release properties

Liposomal AB1

Free AB1

AB1 drug release profile Levofloxacin drug release profile

Liposomal levo

Free levo

Sample T50 (h)

Free AB1 4.7

Liposomal AB1 13.8

Sample T50 (h)

Free levo 5.7

Liposomal levo 27.5

• Outcome 6: AB1 demonstrated controlled release kinetics from liposomal formulation vs free drug

Methods: Biological testing of antimicrobial activity

• Objective 4: Test antimicrobial activity of liposomal AB1 compared to free drug

• Collaboration with Research and Development Institute, National Infections Service, Public

Health England

• Antimicrobial susceptibility to AB1 in free and liposomal formulation tested by Minimum

Inhibitory Concentration (MIC) testing using the broth microdilution method

• Bacterial panel used:

Strain ID Species Antimicrobial

susceptibility profile

NCTC 6571 S. aureus Susceptible

NCTC 13616 S. aureus Resistant

NCTC 13277 S. aureus Resistant

NCTC 775 E. faecalis Susceptible

NCTC 12201 E. faecalis Resistant

NCTC 12204 E. faecium Resistant

Results: Biological testing of antimicrobial activity

• Outcome 7: Liposomal AB1 retains activity compared to free drug

• Modest improvement (MIC>2 fold reduction*) for liposomal AB1 compared to levofloxacin

liposomes in S. aureus and E. faecalis (both associated with MDR-respiratory infection7,8)

MIC (µg/mL)

Strain ID Species Antimicrobial

susceptibility

profile

Free AB1 AB1

liposomes

Free

levofloxacin

Levofloxacin

liposomes

NCTC 6571 S. aureus Susceptible 0.125 0.03125-

0.125

0.125 0.25

NCTC 13616 S. aureus Resistant 4 2* 8 16

NCTC 13277 S. aureus Resistant 4 2* 8 16

NCTC 775 E. faecalis Susceptible 0.5 0.25 * 2 2

NCTC 12201 E. faecalis Resistant 0.25 0.125 * 1 1

NCTC 12204 E. faecium Resistant 4 2 2 4

Future directions:

Project output:

Liposomal formulation of a novel antimicrobial

Stable

High Drug loading capacity

Controlled drug release profile

Retained/improved antimicrobial efficacy against drug-

resistant respiratory pathogens

Next directions:

In vitro biocompatibilityToxicity and pro-inflammatory

potentialFree drug and liposomal

formulation

Formulation developmentFreeze-dried formulation

optimisation (in progress)Stability to aerosolization (in

progress)

Pre-clinical testingProof-of-concept study

Safety and efficacy of liposomal AB1 against S. aureus lung infection in mouse model

Conclusions

• A stable liposomal formulation of a novel antimicrobial lead candidate, AB1, has been developed

which shows controlled release properties and retains antimicrobial efficacy

• The formulation will progress to pre-clinical testing and further formulation development for

inhaled use

• The multi-disciplinary approach combining computational and synthetic chemists, formulation

scientists and microbiologists has enabled rapid development of this formulation for use in

inhaled AMR-respiratory infection treatment

• This study has shown the benefits of an interdisciplinary approach to tackle one of the biggest

challenges facing global healthcare, antimicrobial resistance, and should serve as a model for

greater holistic research approaches to this considerable challenge

Acknowledgements

King’s College London

Medicines Discovery Group

Mark Laws

Dr Kazi Nahar

Dr Shirin Jamshidi

Dr Khondaker Miraz Rahman

Public Health England

Research and Development Institute, National

Infections Service, Public Health England

Dr Charlie Hind

Melanie Clifford

Dr Mark Sutton

Funding:

Dr Arcadia Woods is a holder of the CW Maplethorpe Fellowship in Pharmacy Research and Teaching

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