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Periodic Report Summary 2 - PNEUMONP (Nanotherapeutics to Treat Antibiotic Resistant Gram-Negative Pneumonia Infections)

Project Context and Objectives:
Antimicrobial resistance (AMR) is a global public health concern and the EU Commission considers the development of new effective antimicrobials or alternatives for treatment a current priority. For this reason, an Action Plan to fight against AMR has been set up.
The number of antibiotic resistant bacteria strains is increasing and new types of antibiotic-based therapy are urgently required. Gram-negative bacteria that cause pneumonia are one of the main sources of nosocomial infections, mainly in people with a weakened immune system. Apart from pneumonia, they can cause bacteraemia and other infections. Early detection of the infection source combined with the development of effective therapies to treat multi-drug resistant (MDR) bacteria caused infections will definitely radically improve the healing process of patients and avoid complications in the hospital.
In this context, the use of nanomaterials, i.e. chemical systems that are 10,000 times smaller than the diameter of a human hair and that show special properties due to their size, can assist to find innovative solutions in the development of new diagnostic systems and novel efficient therapies against diseases. The medical application of nanomaterials is part of the so-called “Nanomedicine” and it is expected to gain importance to address biomedical problems. Currently, several nanopharmaceuticals are available on the market, especially for cancer treatment. However the full range of possibilities opened up by the use of nanoparticles in health care systems has not yet been fully realized. The nanoparticles can be manipulated to generate enormously versatile drug-carrying systems with the following characteristics:
i) Targeted delivery of drugs to their specific site of action in the human body, decreasing the risk of side-effects and increasing the therapeutic effect;
ii) Improved solubility and bioavailability of the drug;
iii) Facilitation of drug internalization in infected cells;
iv) Simultaneous loading of different drugs and imaging agents to combine synergetic therapy with diagnostic accuracy.
PneumoNP aims at the development of a theragnostic system for the treatment of resistant Gram-negative bacteria infections of the lung, with a focus on Klebsiella pneumonia-caused infections.
The main objectives of PneumoNP are:
1) Development of a new nanotherapy via inhalation:
For the treatment a nanotherapeutic-based inhalable antibiotic will be developed. The therapeutic nanosystem will be based on a nanocarrier (NC) combined with an antimicrobial peptide (AMP). Three different types of NCs will be tested with two AMPs to obtain a novel effective inhalable antimicrobial nanosystem (NS). Nanotherapeutics offers many advantages in pulmonary drug-delivery, due to the huge surface area available in the lungs and their potential to achieve uniform distribution of drug dose among the alveoli. To improve this delivery to the lungs, an aerosol system will also be developed.
2) Development of a diagnostic kit and an efficiency-efficacy test:
A diagnostic kit will be developed (a) to enable a rapid and precise identification of the bacteria strain causing the infection and to avoid the use of wide spectrum antibiotics and (b) to monitor the efficacy and efficiency of the therapy.
Once this treatment is proved to be effective, it could then be applied to any Gram-negative lung bacterial infection.
In an effort to promote collaborative research for bringing new antimicrobials to patients and to disseminate the results to the general audience and the industry, PneumoNP is part of a cluster with 2 other consortiums working on antimicrobial resistance: FORMAMP (Innovative Nanoformulation of Antimicrobial Peptides to Treat Bacterial Infectious Diseases) and NAREB (Nanotherapeutics for antibiotic resistant emerging bacterial pathogens).

Project Results:
PneumoNP consists of five technical Work Packages (WPs) and three cross-functional WPs.

WP1 focuses on the preparation and characterization of nanocarriers (NCs) based on single chain polymer nanoparticles (SCPNs) and liposomes, as well as antimicrobial peptides (AMPs) as therapeutic agents.
Two types of polymeric-based NCs have been generated: SCPNs based on poly(methacrylic acid) (PMAAc) and based on dextran (DXT). In addition, liposomes of different sizes and lipid compositions have been prepared and a solid-phase synthesis of two different AMPs has been set up. All the NCs have been characterized by different techniques (including NMR, DLS, TEM, IR, and GPC) to fulfill the requirements of quality control established at the beginning of the project. Selected NCs have also been functionalized with fluorescent dyes for in vitro studies and with chelating agents for further radio-labelling and in vivo studies. Biocompatibility and cytotoxicity studies of NCs have been performed (see WP3).
Regarding the AMPs, the project is focused on the use of M33 and Arenicin-3. These peptides have standard synthetic approaches in automated synthesizer using solid phase synthesis and both synthetic processes have been established, using HPLC for purification and MALDI-ToF for characterization. Peptides already come from a selection of stability and workability, then scale up is expected as straightforward.
Work in WP2 focused on the generation of nanosystems (NSs) combining a nanocarrier with a therapeutic agent, as well as formulation of the NSs for aerosol inhalation through an aerosol prototype.
The SCPNs have been loaded with AMPs and the antibiotic Meropenem (control system) using three different strategies: electrostatic interactions, covalent coupling or trapping during nanoparticles formation. Regarding liposomes, passive and remote loading strategies have been used. Studies of stability, characteristics and loading ratios of the polymeric and liposomal NSs have been successfully performed in aqueous media. Importantly, in vitro efficiency tests (WP3) have been performed to start the selection of the most promising NSs that will be tested in vivo (WP5).
The development of an aerosol prototype system has started and will be tested in vivo. The system includes a nebulisation system and a mist chamber, for the delivery of NSs to lungs by inhalation both in biodistribution assays with healthy animals and in therapeutic/toxic tests with infected rats. The construction and validation of the different components were carried out. Preliminary tests of nebulisation in the presence and absence of NCs were successfully performed: the mean drop sizes during nebulisation do not change significantly at the concentration tested.
WP3 is concentrating on in vitro studies to evaluate the antibacterial efficiency of NSs in different Klebsiella pneumoniae strains.
Fifty geographically diverse K. pneumoniae isolates from individual patients recovered from a variety of clinical samples have been completed. Antibiotics from various classes including Meropenem (MEM) were tested with six K. pneumoniae strains to check the antimicrobial drug susceptibility by minimum inhibitory concentration (MIC) and time-kill kinetics (TKK) assays. Peptides AA139 and M33 showed strong concentration-dependent activity, whereas MEM is not active towards the ESBL+KPC strain.
In addition, an ex vivo test system using Alveolar Exudates from rats with K.pneumoniae-ESBL pneumonia was set up. This ex vivo test system simulating the natural environment at the site of infection where the NSs will end up after inhalation will be used to screen the potency of the NSs.
5 NS have been selected for further in vitro and in vivo studies at this time of the project.
In WP4, the development of a viable real-time PCR assay has started to discriminate between live and dead bacteria and find out the efficacy/efficiency of the NSs. A prototype multiplex assay for K. pneumoniae resistance markers has also been developed. The latter one is able to currently detect 21 of the 30 proposed resistance markers present in K. pneumoniae strains using 2SMARTFinder-based technology.
Selected systems are being evaluated in vivo in WP5.
Radionuclides have been incorporated into NCs as well as AMPs in order to perform nuclear imaging for future biodistribution studies with NSs. Dual labelling will enable the tracking of both components in vivo.
Two K. pneumoniae infected rat models have been generated (with two different resistant strains).
The in vivo Maximun Tolerated Dose (MTD), Early bactericidal activity (EBA) and Potential Toxic side effects (PTSE) for those 5 selected NSs have been determined.
For the purpose of informing the scientific community and wider public about PneumoNP, a visual identity kit, flyers, and brochures and a video have been produced The project’s public website was set up and contains plenty of information on the project.

Potential Impact:
PneumoNP aims at generating the following four results: a new inhalable drug system composed of a nanotherapeutic system combining an antimicrobial peptide and a nanocarrier; a new aerosol technology specifically developed for the nanotherapeutic system; an innovative efficiency-efficacy test to follow-up the treatment; a new diagnostic kit for the rapid and multiplex identification of bacteria causing respiratory infections.
Main impacts
1. New therapy with novel administration route
PneumoNP aims at the utilization of NCs to deliver antimicrobial peptides (AMPs) to the lungs, clearly impelling the nanotechnology use in medicine. The use of nanovehicles for pulmonary delivery is an essential part of the project because the AMPs would not be able to reach the infected region via inhalation on their own. For this reason, in this project nanomedicine is expected to assist in topic administration to the lower respiratory tract.
Regarding the inhalation route proposed in this project and compared to actual nebulizers that can only be used as co-adjuvants due to their low efficacy, the nanotechnology-based inhalation system will include NSs capable of better diffusing the drug into the lungs. The main goal is to obtain a direct action on the affected area, enhancing the targeting and allowing accumulation of the drug where the bacteria are located. As a consequence the antibiotic dose and the frequency of its administration can be reduced, decreasing side effects and accelerating the recovery of the patients. This inhalation route has been not much used so far and seems convenient for nanosystems.
2. Combining therapy and diagnostics
A radical improvement is expected in the field of diagnosis, since patients will be rapidly diagnosed with the kit developed in PneumoNP, enabling the identification of specific bacterial strains and allowing immediate therapeutic intervention. This will reduce mortality and the risk of generating antibiotic resistant strains, avoiding the utilization of wide spectrum activity antibiotics.
3. Research close to market
The pharmaceutical sector has experienced major changes due to patent expiring. In order to maintain their competitiveness pharma companies need innovation. In this sense, nanomedicine is a key component which may offer novel therapeutic approaches and profitable drugs against generic competition.
Although in the early technology readiness level (TRL), the PneumoNP project has been structured since the beginning to accomplish good laboratory practice (GLP) and take into account that the compounds should eventually be produced in good manufacturing practice (GMP) conditions. This is of outmost importance in order to fulfill the requirements of pharmaceutical companies which could be interested in the results and products of PneumoNP.
4. Socio-economic impact
Nanomedicine accounts for around 80 marketed products worldwide and the nanomedicine market is expected to grow dramatically in the next ten years.
The urgent need of new antibacterials to treat multidrug resistant (MDR) bacterial infections and the lack of new agents reaching the market indicates that not only human, but also economic consequences related to antimicrobial resistance are very serious. For this reason the development of novel NSs to treat infections caused by resistant bacteria could avoid enormous costs and obtain great benefits for the European pharmas.
Use and exploitation of the results
At the end of the PneumoNP project, three different products are expected to be able to be commercialized by the corresponding SME. In case of the most promising NS, its use as potential nanotherapy will still be far from the market and for that reason the best business model to exploit the outcome needs to be analyzed in the consortium. As some of the partners already have their products finishing preclinical studies and starting clinical trials, their experience will help in the regulatory aspects and exploitation model.
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