Fighting AMR of hard-to-reach microbial pathogens by repurposing antibiotics using a targeted liposomal delivery strategy: A Helicobacter pilot study

Background: Antimicrobial resistance (AMR) is a major threat to global health, particularly in low-income countries, where access to antibiotics is less restricted, leading to frequent overuse. This in turn leads to increasing failure of previously efficacious antibiotics to eradicate infection. H. pylori (Hp) is a bacterium infecting 80% of adults and 70% of children In Vietnam. In comparison, prevalence is <15% in many parts of the UK. The increasing prevalence of AMR strains is the crucial cause for failure in controlling Hp, leading to higher medical costs, and increased mortality and morbidity from related diseases. A 2015 study in Vietnam found 42.4% strains resistant to clarithromycin, 41.3% to levofloxacin, and 76.1% to metronidazole.

ODA compliance: Hp is the main cause of gastrointestinal ulcers and stomach cancer. The high prevalence of disease and a high rate of reinfection in Vietnam is thought to depend on the low socioeconomic status, e.g. crowded living and poor hygienic conditions. There is now a huge discrepancy between low income and affluent countries in terms of Hp prevalence and related diseases, such as gastric cancer. The latter is the 4th most common type of cancer in Vietnam, but only the 16th in the UK. The impact of Hp is particularly strong on the more vulnerable sections of the population; cancer (e.g. gastric cancer caused by Hp) is a significant cause of impoverishment associated with the costs of its treatment in vulnerable sections of the Vietnamese population, pushing many households into poverty. Therefore, our project, which is designed to train Vietnamese scientists and clinicians in cutting-edge advanced drug delivery, empowering the Vietnamese health system to develop and ultimately make available this new treatment to the general population. Hence it is likely to lead to long term socioeconomic benefits to poorer sections of the Vietnamese population, in line with official development assistance principles.

One cause underlying the failure of antimicrobial treatment to eradicate Hp infection is the low accessibility of the drug to the bacterium, underneath the thick mucus and in crypts. Further factors are the short retention times of drugs in the stomach, and the susceptibility of some antibiotics to stomach acid. As a result, some antibiotics which are highly effective against Hp in vitro have failed in vivo. Solving these two problems, by increasing the gastric retention time and protecting the antibiotics from the stomach acid, would increase efficacy of treatment in eradicating Hp infection while allowing the use of existing antibiotics which currently cannot be used. Repurposing of antibiotics is a very cost-effective strategy to fast-track the drug development process.

Our long term aim is to contribute to improve the health of the population in Vietnam by reducing the prevalence of GI ulcers and gastric cancer. We suggest solving the problem of short gastric retention times and gastric acid inactivation by using functionalized liposomes. These will adhere to the gastric epithelium and mucus due to the addition of two Hp adhesins (BabA and LabA) on their surface. The increased gastric retention of drugs encapsulated in the functionalized liposomes will be verified in vivo using NanoSPECT-CT imaging, which we have successfully tested in mice. Binding of functionalized liposomes to epithelial cells will be assessed in vitro by measuring binding to human gastric cell line AGS stably transfected with human MUC5AC, a mucin which contains the physiological ligands of BabA and LabA used for liposome functionalization. Screening of ~600 clinical Hp isolates from both the UK and Vietnam will allow us to identify various levels of AMR and allow us to test the efficacy and safety (e.g. cell toxicity) of our functionalized liposomes in vitro. Finally, the ability of this formulation to eradicate Hp will be assessed in vivo using am established murine infection model.

This project has 4 main experimental aims.

Aim 1) Antimicrobial susceptibility testing of ~600 available H. pylori (Hp) isolates against standard first and second line drugs (amoxicillin, clarithromycin, levofloxacin, tetracycline, metronidazole and rifampicin) as recommended by the British Society for Antimicrobial Chemotherapy, using Etest strips (bioMérieux SA) on Mueller-Hinton agar with 10% horse blood. The strains will also be tested against antibiotics not traditionally used for Hp, but efficacious in vitro (erythromycin, cefixime and tigecycline).

Aim 2) Production of liposomes with extruder technology using established techniques. Once liposomes have been produced, they will functionalized using copper-free 'click' chemistry (conjugation of lipids containing a reactive bicyclo[6.1.0]nonyne (BCN) cyclo-octyne group with an azide). The latter will be added recombinantly to the C-terminal of the H. pylori adhesins LabA and BabA as p-azido-L-phenylalanine, using the pEVOL-pAzF plasmid (Addgene).

Aim 3) In vitro binding of functionalized liposomes: The properties of functionalized liposomes will be investigated in vitro using protocols established in our lab. We will use a human gastric epithelial cell line stably transfected with human MUC5AC (a known ligand for BabA), using untransfected AGS cells and BabA binding site mutants as a control.

Aim 4) In the final year of the grant, we will test the optimal formulations developed as described above using our C57BL/6 mouse model under an existing project licence. Tc-99m NanoSPECT-CT imaging, which we have previously established, will be used A) to verify increased gastric retention of liposomes functionalized with LabA in the murine stomach, and B) determining the increased efficacy of gastric targeted liposome-formulated antibiotics in vivo, by comparing the effectiveness of our liposomal formulation with the standard triple therapy in eradicating a highly-colonising SS1 strain of Hp in mice.

1. Impact through publicisation of findings

Initially, we will informally update members of the public and scientific community on our progress via our webpages. As the project evolves, the website will detail our progress in discovery and will be linked with e.g. the University twitter account and Press Office releases in an effort to engage more rapidly with the wider health and sciences communities. Falcone has a 'News from the lab' section on the lab webpages which is regularly updated (see http://tiny.cc/w9akxx). In addition, the University and UKRC operate a policy of open-access publishing. Publication in the most relevant journals such as Helicobacter or The Journal of Infectious Diseases is not only in line with this policy, but also highly visible and possesses greater impact in terms of traditional academic indicators of impact, e.g. UK research excellence framework REF2014. Using open-access publication (CC BY 4.0 license) also will increase the audience that our work will reach, enabling researchers in Vietnam and other low income countries most negatively affected by H. pylori infection to benefit from this work. Further opportunities for publicisation are available through Robinson, who is Scientific Conferences Officer and Council member for the Microbiology Society (MicrobioSoc), and Director of Doctoral Programmes for the School of Medicine (SoM). Tran will organise a local meeting and a Training School in Vietnam, targeting Vietnamese Clinicians and Researchers working in the area of gastrointestinal infections.

The University of Nottingham currently possesses a very active and visible press office that interacts with national and international media to promote research and research impacts wherever possible. All UK applicants on this proposal have had experience in dealing with the national press via the press office for publicisation of their research outputs. Using these resources, national and international media can be alerted to any future publications, findings or presentations. Our research has received coverage not only by scientific media, but also in the national and international press (see e.g. a short highlight of our H. pylori work in the daily Telegraph on August 15th 2015). Attendance at cross-theme international meetings, primarily towards the end of the grant lifetime, will enable us to promote our findings and highlight the importance of implementing our therapeutic strategy to combat AMR. Attendance at these meetings and the theme of the meetings will serve as a milestone in our effort to maximise impact but will also be integral to the goals of the project itself.

2. Impact through promotion of research tools

To help with development of tools against AMR, a repository of the fully characterised clinical isolates will be made available to the scientific research community, who will be able to obtain such strains on the basis of an MTA. This will provide additional research strands and greater impact.

3. Economic and societal impacts

Fostering global economic performance, and economic competitiveness in Vietnam. To the best of our knowledge, there are no public health studies addressing the socioeconomic impact of H. pylori infection and associated diseases (gastrointestinal ulcers and cancer) on Public Health in Vietnam, but this is likely to be high, as gastric cancer is the 4th most common cause of cancer and the prognosis after diagnosis is usually unfavourable. Existing studies have looked at the economic benefit in other countries, such as Harvey RF (Aliment Pharmacol Ther. 2010; 32:394-400; Bristol Helicobacter Project) suggesting cost effectiveness of a community H. pylori eradication program, particularly in populations with a high prevalence of H. pylori infection. The fabrication of liposomes is relatively simple and does not require sophisticated systems, so could be done in hospital pharmacy if needed.