Interviews

Interview with Prof. Dr. Joachim Reidl

Due to our special interest in microbial behavior of our system in the human guts, we talked to University Professor Joachim Reidl from the Karl Franzens University, Graz about our project. Professor Reidl is working at the Institute of Molecular Biosciences and his research field especially includes Medical Microbiology.
Regarding our project, he told us, that it will be very interesting to observe the differences in terms of regulation of your quorum sense based system in E. coli Nissle 1917, due to the big difference to ordinary lab strains like E. coli BL-21. He also mentioned that it would be interesting to find out to what extend the intestinal produced dopamine really goes into the blood stream and subsequently how much of it reaches the brain. A final comparison of how much dopamine is actually produced in vivo in comparison to a standard lab reactor experiment has to be drawn to.
Furthermore, this would be especially important to discriminate the exact amount of bacteria that has to be brought and cultivated in the human gut. According to web resources [1], medication such as “Levodopa” is usually administrated to patients in doses from 500 mg to 6000 mg a day. In previous work done on L-DOPA metabolic engineering, researchers developed E. coli strains that were able to produce up to 1.51 g/l of L-DOPA in 50 hours in batch reactors [2], meaning that we would have to improve these results significantly to actually develop a sufficient production strain that can be administrated directly to the human guts.
Another interesting aspect is the molecular mechanism of how bacteria would keep the information of Dopamine/L-DOPA synthesis. In the intestine, different to the fermenter where an antibiotic can be added, selection pressure would not be present. The latter would mean that the engineered bacteria eliminate their plasmids. To counteract this problem, integration into the bacterial chromosome could be approached or selection pressure with knocked out essential E. coli genes could be applied. Still, this is no guarantee for the integrated DNA or the plasmid to be kept intact by the bacteria because of possible cut out or mutation of the foreign DNA.

The main point we were able to take back to the lab from this discussion was that we will most probably not have to worry about our developed strain overgrowing the human gut composition. Actually there is a possibility that the strain may have difficulties reaching the required L-DOPA levels, this is a problem we will have to work on in the future.

Interview with Dr Monty Silverdale

Dr Monty Silverdale is a consultant neurologist, honorary lecturer in Neuroscience at the Institute of Brain Behaviour and Mental Health at The University of Manchester and member of the Association of British Neurologists. Dr Silverdae’s research is about PD and pain – a nationwide study of polymorphism of genes that may be responsible for pain in PD, a study involving more than 18 hundred people across the UK. Dr Silverdale shared his valuable experience in treating PD, gave comprehensive answers to our questions and provided us with useful and specific feedback on out project on August 7th 2015 in Wolfson Molecular Imaging Centre in Withington.

Dr Silverdale said that patients with motor fluctuations, they have three options: duodopa, surgery (deep brain stimulation) and apomorphine. In duodopa there are issues with the tubes: they get infected, they block and move out of the jejunum to the stomach, and back, they can even move of the stomach to the parteneum [0:41] and start effusing stuff in there and that is not good. The second issue is the main cost, so patients and physicians ask the question “Why is it to expensive?” However, they duodopa is much better than giving levodopa orally.

Apomorphine, which is a dopamine agonist, so it works a bit differently form levodopa. As a dopamine agonist it targets D1 and D2 receptors, whereas most of the oral agonists target only D2. Thus it is more effective, but it is not absorbed orally, so it has to be given subcutaneously: by subcutaneous infusion. Apomorphine works quite well, but the pump the patient has to carry around for the subcutaneous infusion makes the patient something [2:21] a bit, it is more likely to promote psychiatric problems that [2:23] is levodopa, so it is not ideal either.

Dr Silverdale mentioned that he was involved in research of non-motor symptoms in PD with pain being one of them. Everything about pain could go for any other non-motor symptoms like depression, autonomic symptoms like posture hypertension, blood symptoms, bowel symptoms, sleep disturbance – there are hundreds of them. All non-motor fluctuations are not always necessarily caught on sight so you can have doctors in clinic who may say that these patients are absolutely fine, because they are completely mobile, and yet they have depression fluctuating through the day, pain fluctuating through the day and sleep problems and bladder problems and etc.

The fluctuations are not always synced with medication concentration, someone can take a peak dose of medication and just have switch on at all, and someone can have no medication for several hours and be good. Having a good level of dopamine in the blood stream is not the deal end of having an anti-PD response because there is something going in the striatum as well. We do not exactly know what happens but, for example, one theory is that the receptors in the striatum are cycling in and out and the dopamine the receptors are either in the synaptic membrane where they can be stimulated or internalized where they cannot be stimulated. If you give a high dose of dopamine, for example, you stimulate, lets say you get a bit peak and it stimulates, because in PD the peaks are 50 times higher than in a normal situation and the throughs are much-much lower so you can get a big peak differences and these big peaks as well as causing an anti-PD, might actually internalize the dopamine receptors and that internalization might last for several hours – so no matter that you do after you cannot stimulate this. We do not understand it completely, but probably, if you cover a more continuous level of dopamine stimulation, you could gradually avoid that sort of things. Continuous dopa is a big buzz in PD treatment, you need to treat motor symptoms normally.

Dr Silverdale also commented on controversy of GMO in patients’ guts. He thinks that there will not be patient acceptability issues. The point is that treatment of PD with human embryotic stem cells was accepted, the developed therapeutic strain is likely to be accepted as well.

Dr Silverdale also suggested some improvements to our project. The DopaDoser should not biosynthesise dopamine, because it makes people vomit. At the early stages of the project, the DopaDoser should biosynthesise L-DOPA at constant levels. However, at the later stages L-DOPA levels should be auto-regulated. This can be done clinically by monitoring dopamine concentration in the brain, developing badges that would record and signal motor fluctuations and incorporation of a dopamine receptor in the therapeutic strain to make the self-regulatory system that will be activated by neurotransmitter imbalance.

This interview showed:

• Current treatment (duodopa) is invasive and not effective
• Non-motor symptoms are often underestimated and not treated
• Peaks and throughs in medication conentraion should be minimized, because they can cause internalization of dopamine receptors
• DopaDoser will alleviate pain that is induced by fluctuations in levodopa concentration and treat not noticed non-motor fluctuations like depression, blood and bowel conditions, sleep etc.
• DopaDoser is cheaper and safer and less invasive
• Patients will accept DopaDoser
• DopaDoser should not synthesize dopamine
• DopaDoser is may be improved by a dopamine receptor and MAO-B and COMT inhibitors

Interview with Prof. Dr. Christoph Högenauer

In order to find out more about human gut bacteria composition we met Prof. Dr. Christoph Högenauer from the Institute of Gastroenterology and Hepatology at the Medical University of Graz.
We found out that composition of gastrointestinal colonization is not only different from person to person but also depends on geographic location, genetic disposition, possible disease and current eating habits. Quantification of these bacteria strains is quite difficult as some might only be abundant in very low concentrations. For actual quantification, q-PCR is the method of choice. However, Escherichia coli makes up 0,1-1% off all gut bacteria but up to 50% during enteritis [1].
Our selected probiotic E. coli strain Nissle1917 is currently sold under the trademark Mutaflor ®. If applied to the patient only once it is observed that the strain does not stay in the gut permanently but has a lifetime of only a few days and is not detectable after a week. Regular administration is still required.

Interestingly during our interview, Dr Högenauer also told us that he was also working with Parkinson’s patients, preparing them for a therapy form called Duodopa ® from Abbott [2]. This therapy especially serves patients that cannot take L-DOPA pills for varying reasons (intolerance or too high tolerance to oral L-DOPA). It is a special way for administration of L-DOPA (Levodopa ®) directly into the human jejunum through a PEG-PEJ tube. This approach allows for a continuous and stable dosage of L-DOPA over the whole day. However some major problems of this approach are high costs (around 700€ per week plus costs for placement of the tube and aftercare) and wear out of the tube every 2 years. Also the system needs to be rinsed on a daily basis and during the night medication has to be taken oral anyways. This ads up to 100.000 € per year per patient.

Obviously this is exactly where our project comes into play! Our idea allows for continuous administration of L-DOPA straight into the gastrointestinal trace which does not only get rid of tubes going into your body but also allows for a much smaller concentration of L-DOPA administration. Levodopa is currently applied through Duodopa® in concentrations of 20-200mg per hour, in contrast to previously mentioned pills in the multi gram range. Gastrointestinal production of L-DOPA would not only allow for a better lifestyle for patients but also discharge health insurance funds.

Interview with Prof. Angela Tod

Professor Angela Tod is the Florence Nightingale Foundation Chair of Clinical Nursing Practice Research at the Faculty of Medical and Human Sciences at the University of Manchester. Her research focuses primarily on access to health services as well as health inequalities during which she has worked with Parkinson’s patients and Parkinson’s UK. [1]

Support of the project

• Two things creating a lot of care needs for patients and also impact on their quality of life are the unpredictable nature and the fluctuating nature of the disease. Peaks and troughs in the day make the condition very challenging – DopaDoser will improve life quality of patients
• Parkinson’s UK campaigns are abut taking tablets in tie – may be supported by Parkinson;s UK, less money on campaigns and DopaDoser with less doses required will be beneficial
• NHS funding depends on inequalities in the patient groups – gives information that it is possible that DopaDoser will be beneficial for the national health
• In the scenario, when patients have some excruciating pain and they think that they are going to die. In this sort of scenario, people go for the treatment, because they are suffering. “the unpredictable nature of it and the fluctuating nature of the condition destroys people’s lives. That fact that you can actually level things out, the benefit of that would make people make a lot of trade offs.”– proves patients acceptability
• “If it does work there is not a massive cost to the patient in terms of what they have to give up” – informs that
• Incrementally over years their life just gets smaller and smaller, because of the increased burden of the condition. If you can prevent that happening then that is fantastic – positive comment on the project
• DopaDoser has the potential to ameliorate some inequalities people face and also prevent problems that happen later on in the condition
• DopaDoser might reduce the need for care and support in the sense that less support would be required to help take medication etc.

Improvements:

• Monitoring – would patients needs to be monitored on our treatment? Ongoing requirements?. Need to test whether bacteria establish depending on personal gut flora. Adjust dose accordingly. Individual variation – personalised medicine. Monitoring previously to treatment and after. Patients would be concerned about monitoring, how much monitoring, what is it I will be taking and for how long
• Antibiotics could impact on levels – recommendations would be issued further down the line
• Project – someone is being offered the therapy, is there anything that would compromise somebody in complying with it or being able to tolerate it? Where they would be more disadvantaged if they came from a low income household and lower level of education. Religious reasons for acceptance? – need to consider social determinants of health
• Need to understand the reasons for refusal and research the sources general public trusts and why?

Interview with Dr. Mariella Kögl-Wallner

To get some more information on the medical aspects on Parkinson’s we visited Dr. Mariella Kögl-Wallner, an expert in therapy of Parkinson’s patients at the Institute of Neurology at the Medical University of Graz. We mainly talked to her about the daily burden of people with Parkinson’s disease. Since medications against Parkinson’s disease have become well established, patients do not have any reduced life expectancy. Still, the treatment means a lot of change in every day life of a patient. Levodopa® has to be taken dependent on the status of the disease several times a day at least 30 minutes before a meal and can result in peaks and shortages of L-Dopa. Those “off” times (periods with no symptomatic relief) [2] and dyskinesia [1] often cause a more progressive pathology. Compared to the traditional Levodopa® therapy, the application of Duodopa® already has some advantages over the oral medications. With this therapy method, a mostly constant L-Dopa level can be reached in the blood, meals do not have to be considered and no blockers like MAO-B (monoaminooxidase blockers) have to be taken.

Dr. Mariella Kögl-Wallner told us that her patients are usually very keen to take part in the testing of potentially new drugs. Furthermore, she confirmed that none of her patients would reject a therapy that includes genetically engineered bacteria as long as they see a positive effect about it. Not only the patients but also the doctors always try to stay on top of the latest research results about Parkinson’s disease, to find new ways how Parkinson’s might be treated or some day even be cured. Regarding diagnostics, Dr. Kögl-Wallner told us that they always sequence a few specific genes at locations where most of the patients show mutations. Still, there are groups of patients where those locations do not show any conspicuity. Those patients might have mutations in other locations of the human genome that have not been studied yet.

All in all, there are still many gaps of knowledge when it comes to Parkinson’s disease. A lot of research is done in this particular area. But ever since 2004 where Duodopa® was approved in Europe no other breakthroughs have occurred. Transplantable dopamine neurons produced from stem cells [3] or gene therapeutic approaches might one day become the therapy of choice for Parkinson’s patients.