Investigation into methods for giving early warning and prevention of urinary catheter blockage
Lead Research Organisation:
University of Bath
Department Name: Chemistry
Abstract
Catheter-associated urinary tract infections have been identified as a priority area for the NHS, with healthcare-associated infections costing some £1bn per year. As well as the economic burden and a concern for patient outcomes, the NHS has a commitment to reducing the burden of antimicrobial resistance (AMR), and infection-prevention is a key part of this. The objective of the project is to develop a low-cost treatment for urease positive catheter associated urinary tract infections. Whereby a small molecule will be delivered through the catheter balloon to treat at site of infection.
This fits directly to the EPSRC remit in healthcare technologies specifically in the areas of (1) developing future therapies; (2) optimising treatment; and (3) transforming community health and care.
Why do urinary catheters block?
Proteus mirabilis is a Gram-negative bacterium which is commonly found in the bladder of patients who are long term catheterised. P. mirabilis forms biofilms on the catheter and secretes the enzyme urease. Urease, allows P. mirabilis to exploit urea as a nitrogen source, allowing the bacteria to grow. Urea hydrolysis results in the production of carbonic acid and two molecules of ammonia. Ammonia raises the urinary pH in the bladder to pH 7.5-9, compared to healthy, acidic urine pH of 5.5-6.5. Consequently, local supersaturation and precipitation of struvite MgNH4PO4.6H2O and apatite, Ca10(PO4CO3OH)6(OH)2) into the catheter lumen causes abrasive crystalline deposits to become incorporated onto the catheter lumen external and internal surfaces. Total occlusion of the catheter lumen may follow blocking the catheter in as little as 16 hours from placement of catheter into an infected bladder.
The Project
2-Mercaptoacetamide (2-MA) has been identified as a unique anti-urease small molecule. This molecule is cheap and easy to synthesise, but importantly has shown to be more effective at urease enzyme inhibition than any other urease inhibitor used clinically such as Acetohydroxamic acid (AHA), which is marketed as Lithostat in the USA, and Uronefrex in Europe. The principal problem with AHA is that it is intensely toxic and is rarely used in Europe.
Preliminary in-vitro studies have shown very promising results, with 2-MA being far more effective than AHA, as well as being less toxic.
The Ph.D programme
Part 1: Synthesis of 2-MA and analogues
2-MA is one of a family of related compounds, all with the same chemical 'warhead'. A library of compounds based on 2-MA will be synthesised and tested for their urease inhibition properties, (part 2) toxicity (part 3) and delivery through the catheter balloon (part 2).
Part 2: Testing of 2-MA and analogues in bladder model
Using Scarlet's established test protocols the compound library made in part 1 will be tested in our in-vitro bladder with inhibition to catheter blockage, reduction in P. mirablis biofilm formation, urinary pH, reduction in P. mirablis viable cells all measured as outcomes. From this work, around 3-4 lead compounds will be taken through for toxicity testing in phase 3.
Part 3: Toxicity testing
Toxicity testing using blood haemolysis and eukaryotic cell viability assays will be used to assess toxicity of lead compounds, benchmarked against the commercial urease inhibitor, AHA.
Part 5: testing of delivery through the catheter balloon
Urinary catheters are kept in place in the bladder via the 'inflation' of a balloon with water. The concept for delivery of our urease inhibitors will be to use the balloon to deliver the drug directly into the bladder. This will rely on the small molecules crossing the ultrathin silicone balloon. We will investigate adding excipients to assist in drug transfer.
Part 5: Preparation for clinical study
Once lead compounds have been identified and tested (above) we will work closely with Mr Ed Jefferies, consultant urologist at the Royal United Hospital i
This fits directly to the EPSRC remit in healthcare technologies specifically in the areas of (1) developing future therapies; (2) optimising treatment; and (3) transforming community health and care.
Why do urinary catheters block?
Proteus mirabilis is a Gram-negative bacterium which is commonly found in the bladder of patients who are long term catheterised. P. mirabilis forms biofilms on the catheter and secretes the enzyme urease. Urease, allows P. mirabilis to exploit urea as a nitrogen source, allowing the bacteria to grow. Urea hydrolysis results in the production of carbonic acid and two molecules of ammonia. Ammonia raises the urinary pH in the bladder to pH 7.5-9, compared to healthy, acidic urine pH of 5.5-6.5. Consequently, local supersaturation and precipitation of struvite MgNH4PO4.6H2O and apatite, Ca10(PO4CO3OH)6(OH)2) into the catheter lumen causes abrasive crystalline deposits to become incorporated onto the catheter lumen external and internal surfaces. Total occlusion of the catheter lumen may follow blocking the catheter in as little as 16 hours from placement of catheter into an infected bladder.
The Project
2-Mercaptoacetamide (2-MA) has been identified as a unique anti-urease small molecule. This molecule is cheap and easy to synthesise, but importantly has shown to be more effective at urease enzyme inhibition than any other urease inhibitor used clinically such as Acetohydroxamic acid (AHA), which is marketed as Lithostat in the USA, and Uronefrex in Europe. The principal problem with AHA is that it is intensely toxic and is rarely used in Europe.
Preliminary in-vitro studies have shown very promising results, with 2-MA being far more effective than AHA, as well as being less toxic.
The Ph.D programme
Part 1: Synthesis of 2-MA and analogues
2-MA is one of a family of related compounds, all with the same chemical 'warhead'. A library of compounds based on 2-MA will be synthesised and tested for their urease inhibition properties, (part 2) toxicity (part 3) and delivery through the catheter balloon (part 2).
Part 2: Testing of 2-MA and analogues in bladder model
Using Scarlet's established test protocols the compound library made in part 1 will be tested in our in-vitro bladder with inhibition to catheter blockage, reduction in P. mirablis biofilm formation, urinary pH, reduction in P. mirablis viable cells all measured as outcomes. From this work, around 3-4 lead compounds will be taken through for toxicity testing in phase 3.
Part 3: Toxicity testing
Toxicity testing using blood haemolysis and eukaryotic cell viability assays will be used to assess toxicity of lead compounds, benchmarked against the commercial urease inhibitor, AHA.
Part 5: testing of delivery through the catheter balloon
Urinary catheters are kept in place in the bladder via the 'inflation' of a balloon with water. The concept for delivery of our urease inhibitors will be to use the balloon to deliver the drug directly into the bladder. This will rely on the small molecules crossing the ultrathin silicone balloon. We will investigate adding excipients to assist in drug transfer.
Part 5: Preparation for clinical study
Once lead compounds have been identified and tested (above) we will work closely with Mr Ed Jefferies, consultant urologist at the Royal United Hospital i
| Description | The award funded an optimization of 2-mercaptoacetamide (2-MA) as a urease inhibitor and optimization of the diagnostic lozenge to predict and treat urinary catheter blockage of long-term catheter users. The initial work was covered by the Jenkins group and has been published.1,2 Initially, in silico computational docking of 2-MA was completed, to examine the inhibitory affect of 2-MA against the virulence factor, urease. Urease is an enzyme that metabolizes urea to ammonia, causing an increase in pH and subsequent blockage of urinary catheters. Certain microorganisms contain urease, and these are often found in long-term users of urinary catheters, for example, Proteus mirabilis. To optimize 2-MA, a computational screen of known urease inhibitors was completed. We describe this drug discovery technique as 'in silico rational drug design', from the compounds screened we picked 3 compounds that could be purchased. These compounds were tested against purified Canavalia ensiformis's urease and whole-cell P. mirabilis. This identified a new inhibitor which showed 100-fold greater potency compared to our control compounds of acetohydroxamic acid (a known urease inhibitors, licensed under the Lithostat), and 2-MA. Therefore, we have optimized 2-MA and identified a new compound that could be used a urease inhibitor. The second aim of the award was to optimize the diagnostic lozenge. The lozenge is placed within the drainage bag of the catheter and gives a warning of impending catheter blockage. The lozenge was initially designed as dip coated PVA polymer containing a pH dependent fluorescent dye, and coated in a pH sensitive polymer, however this was fragile and difficult to manufacture. The newly improved lozenge consists of a solid tablet containing sodium fluorescence, which is drum-coated by the pH sensitive polymer. The optimization of the lozenge has been published.3 The lozenge has been tested in a small-scale clinical trial, using patients who have long-term urinary catheters. This trial is currently ongoing. 1 S. Milo, R. A. Heylen, J. Glancy, G. T. Williams, B. L. Patenall, H. J. Hathaway, N. T. Thet, S. L. Allinson, M. Laabei and A. T. A. Jenkins, Sci. Rep., 2021, 11, 1-15. 2 S. Milo, F. B. Acosta, H. J. Hathaway, L. A. Wallace, N. T. Thet and A. T. A. Jenkins, ACS Sensors, 2018, 3, 612-617. 3 R. A. Heylen, M. Branson, L. Gwynne, B. L. Patenall, N. Hauschildt, J. Urie, J. Mercer-, N. T. Thet, M. Laabei and A. T. A. Jenkins, Biosens. Bioelectron., 1-11. |
| Exploitation Route | The outcomes of this funding can be taken forward by other researchers, further optimization of the urease inhibitors and a larger-scale drug screen will be required. As well, as in vitro assessment of the drugs using artificial bladder models and cytotoxicity testing. The results of the lozenge clinical trial require analyzing and a large-scale randomized trial will need to be planned and conducted. For this to occur the lozenge will have to be manufactured and deposited within the drainage bags which will be given to long-term catheter users to assess lozenge performance. |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |