Accumulation and nephrotoxicity of dextrin-colistin conjugates.

Infectious diseases account for millions of deaths worldwide annually, while bacterial resistance to antibiotic therapy is a major world health problem that is responsible for more than 700,000 of those deaths. Bacterial infection and the evolution of multi-drug resistance (MDR), therefore, represent an increasingly formidable challenge in the developed and developing world with a colossal economic, societal and environmental impact, posing a significant clinical challenge in patients with cystic fibrosis, burns and skin wounds. Increasing resistance to currently available antibiotics has been mirrored by decreases in the design/development of new antibiotic entities as the pharmaceutical industry has replaced antibiotic development with drug development in diseases with a better risk-reward ratio. These trends have led to the existence of organisms that are susceptible to just 1 or 2 antibiotics, and in some cases, none at all. To overcome these problems, clinicians are increasingly employing (as drugs of last resort) older antibiotics, such as colistin, which, despite the high incidence of toxicity and significant long-term complications, are effective. Use of colistin to fight infection is limited, as it is known to be toxic to the kidneys, despite the fact that it has been found to be effective against MDR bacteria.

This project will test a new method of drug delivery for the safe administration of antibiotics, currently rendered unusable due to toxicity issues. These so-called "polymer therapeutics" are capable of reducing the side effects of conventional drugs by shielding them in a polymer coat, but once they reach the site of disease, they can be triggered to release the drug by the body's own proteins. This approach enables drugs to specifically accumulate at sites of disease at a much higher concentration than normal, thus, patients require lower doses and less frequent dosing. This study will test a new nano-sized antibiotic system, called dextrin-colistin conjugates, in which the antibiotic, colistin, is chemically wrapped in a safe, water-soluble, naturally-occurring biodegradable polymer (dextrin).

Having previously demonstrated improvements in drug shielding, retention of antimicrobial activity and drug distribution in the body, this project will test whether dextrin attachment can effectively reduce colistin's harmful effects on the kidneys. We predict that dextrin-colistin conjugates will cause less damage to the kidneys than the unmodified antibiotic or the clinical formulation of colistin, CMS, due to reduced exposure of kidney cells to colistin. This study will use several different approaches to test this hypothesis, including:
1. Measure the growth and survival of cells grown in the laboratory in the presence of different drug treatments. These studies will use cells originating from the kidney as well as skin cells, for comparison.
2. Quantify the amount of drug that enters inside kidney and skin cells grown in the laboratory. Here, a compound that lights up under fluorescent light will be chemically attached to the drugs to allow visualisation under a microscope or in a laser beam.
3. Assess how drugs spread around the body of healthy rats compared to those with a bacterial infection. Damage to kidneys will be measured visually and by testing blood samples for specific proteins that are released when the kidneys are injured. These experiments will test different doses of antibiotic drugs to identify the maximum dose tolerated by the animals. This dose will then be used to test the ability of the drug treatments to cure a lung infection.

Ultimately, if this study is successful, not only will we have demonstrated the safety and efficacy of these new antibiotics, but there is great potential for the use of water-soluble natural polymers in a wide range of chronic human diseases of high public health impacts, including cystic fibrosis, spinal cord injury and diabetic foot ulcers.

This research will test a novel bio-triggered antibiotic delivery system, based on polymer therapeutics, that has previously shown antibacterial activity across a range of Gram-negative bacteria, reduced in vivo toxicity and prolonged plasma half-life. It is hypothesised that dextrin-colistin conjugates will be tolerated at much higher doses than the free drug, which would reduce the acquisition of antibiotic resistance and improve the efficacy of treatment.

This study will assess the mechanisms and extent of dextrin-colistin conjugates' nephrotoxicity, including renal cell toxicity and apoptosis, renal cell uptake and accumulation, and renal tissue distribution. Experiments will, first, use in vitro assays to assess cytotoxicity (MTT, LDH release, apoptosis) and cellular uptake, accumulation and localisation (fluorescence microscopy and flow cytometry) of 3 dextrin-colistin conjugates, compared to colistin sulfate and CMS, in a panel of human and rat kidney cell lines, compared to primary fibroblasts.

In vivo biodistribution and nephrotoxicity will be evaluated in healthy rats, then in a respiratory tract infection model of Klebsiella pneumoniae. Healthy animals will receive 2 doses (8 h apart) of dextrin-colistin conjugate, colistin sulfate or CMS and samples of blood and organs will be assessed for creatinine/ colistin content and cellular degeneration using histology. These studies will determine the maximum tolerated dose for the treatments, which will be used in subsequent in vivo efficacy studies. Observations and sampling will be performed as described previously, however, in addition, organs will be recovered and the antibacterial effects will be quantified as colony forming units (CFU)/g of lung tissue.

The results of this study are expected to demonstrate the improved toxicity profile and biodistribution of dextrin-colistin conjugates, to precede full-scale commercial development.

Who will benefit from this research?
This project will have a positive impact on several beneficiaries:
- Academic: Discussed in previous section.
- Pharmaceutical industries (including CROs and SMEs): They will benefit from increased scientific knowledge, improved nanomedicine legislation, strong UK research capacity and economic competitiveness, and availability of a highly skilled workforce in the field of nanomedicine. If this project is successful, Pharma may also benefit from commercial exploitation of this research.
- Public sector: The NHS will benefit from enhanced quality of life, reduced healthcare costs, formal links between clinicians and researchers, and tighter legislation relating to nanomedicines. Schools will benefit from educational opportunities, such as public lectures and access to the group's website articles.
- General public: Patients will benefit from enhanced quality of life, reduced healthcare spending and the availability of advanced therapies having reduced side effects and improved efficacy. Members of the public will benefit from clear use of public funding and greater understanding of science through public engagement activities.

How will they benefit?
- Enhancing quality of life: Technologies developed in this programme will offer the opportunity to develop effective, safe therapies to prevent and treat life-threatening infections and improve the care of the millions of individuals affected by infections caused by antibiotic-resistant bacteria annually. Ultimately, we hope that employing these novel polymer therapies for effective drug delivery and targeting will also provide a means of overcoming drug delivery challenges in a range of diseases, including cancer, arthritis and blindness.
- Workforce: The PDRA employed on this project will enhance their skills in a multitude of scientific disciplines, which will support their career development and develop expertise in the field of polymer therapeutics for further application and dissemination.
- Legislation: Nanomedicine challenges existing perceptions, dynamics and standards relating to ethics, safety and governance. The development of bioresponsive nanoantibiotics based on dextrin-colistin conjugates will usefully contribute to the regulation and development of specific new legislation (e.g. nanomedicines are currently regulated under legislation for medicinal products, devices and tissue engineering).
- Economic benefit: In addition to direct healthcare costs, lost productivity resulting from infections by MDR bacteria is estimated to cost more than 22EUR billion and results in >10 million days of lost work annually. Unless new means of treating escalating drug-resistance is developed, an increasing healthcare financial burden will arise, particularly in light of the ageing European and Worldwide population. Improving the healthcare of our ageing population is one of the most important grand societal challenges for the UK and the rest of the World. The development of polymer therapies for use in these diseases offers a significant opportunity to reduce direct treatment costs. Many major Pharmaceutical companies now have R&D programmes in Polymer Therapeutics and, in 2013, two polymer therapeutics featured in the Top 10 US pharmaceutical sales list. Encouraging results from these studies will trigger investment and further research in this field, as well as presenting new job opportunities, further benefiting the UK, EU and global economies; encouraging established industries to remain in the UK, while attracting relocation of established and new industries.
- Building UK Research Capacity: This interdisciplinary consortium comprises scientists from a range of disciplines including clinicians, material scientists, microbiologists, polymer chemists, pharmacists and staff from small enterprises with a common purpose of establishing the UK at the forefront of polymer therapeutics research.