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Translational studies in Zellweger spectrum disorders
Berendse, K.
Publication date
2016
Document Version
Final published version
Link to publication
Citation for published version (APA):
Berendse, K. (2016). Translational studies in Zellweger spectrum disorders.
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Chapter 8
Summary, general discussion, future research and
implications for clinical practice
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Chapter 8 Summary and general discussion
Summary of the thesis and general discussion
This chapter provides a general discussion of the results described in this thesis, placing it into the context of the field of peroxisomal disease research, and discussing the priorities for further research.
Current concepts on disease course and biochemistry of the Zellweger spectrum disorders (ZSDs) are introduced in part I, Chapter 1 and 2, of this thesis. The estimated incidence of the disease is 1:50.000 in the United States 1 and is probably similar in
Europe although no specific data for the European Union (EU) is available. Recently, X-ALD was implemented in the newborn screening program in New York, USA
2. The screening will also be implemented in the newborn screening program of
the Netherlands but only for male neonates, since only male patients benefit from early diagnosis. Since the screening involves measurement of the levels of C26:0-lysoPC in dried bloodspots, male patients with a ZSD or ACOX1- and HSD17B4-deficiency will also be identified. The inclusion of this test in the newborn screening programs will provide more accurate data on the incidence of ZSDs. Similar as previously observed for other disorders included in newborn screening programs, it is likely that the incidence will be higher than currently estimated, due to earlier diagnosis of milder cases. The identification of (pre-symptomatic) newborns with ZSDs requires that clinical guidelines for follow-up will be developed. No curative or disease modifying treatment is available for patients with a ZSD. However, patients are at risk to develop complications like vitamin K deficiency or adrenal insufficiency. To optimize supportive care, recommendations for management are reviewed in Chapter 2.
Despite extensive research using different model systems, such as skin fibroblasts and knockout mouse models, no effective treatment is available to restore peroxisomal function in patients with a ZSD. In Chapter 3, we describe the effect of (L-)arginine as a compound that can (partially) restore peroxisomal function in skin fibroblasts of a subset of ZSD patients with specific mutations in PEX1, PEX6 and PEX12. Although high concentrations of arginine were needed to increase residual function (i.e. 20mM in culture medium), it would be of interest to test this compound in patients. In addition, we tested other promising compounds for the ability to increase peroxisomal proliferation in skin fibroblasts, determined with peroxisomal catalase immunofluorescence microscopy and increase in processing of peroxisomal thiolase (PTS-2 protein). For example, bezafibrate 3, 4-phenylbutyrate 4, resveratrol
and celastrol (unpublished data) were tested, but no positive effect on peroxisomal function was observed. However, incubation of cells with similar concentrations of (L-)homoarginine resulted in a more effective recovery of peroxisomal function compared to arginine. The mechanism by which (homo)arginine recovers peroxisomal function is unclear and currently under investigation. L-arginine is the precursor of
nitric oxide 5. Nitric oxide is reported to induce expression of heat shock proteins 6 7. Heat shock proteins are molecular chaperones that facilitate folding of proteins
and assist in refolding denatured proteins both in normal and stressed conditions
8. We thus hypothesize that arginine activates the nitric oxide-mediated pathway,
thereby inducing the expression of heat shock proteins and improving folding of proteins, i.e. the defective PEX1, PEX6 and PEX12 protein. It is also possible that the known in vitro chemical chaperone arginine, corrects protein folding directly
9 10. Another hypothesis is that arginine inhibits pexophagy 11. This will increase/
rescue the peroxisomes with the unstable and/or incorrectly folded PEX protein which are rapidly degraded in the normal situation, leading to (partly) restoration of peroxisomal function. This hypothesis is supported by the observation of Angcajas et al that nitric oxide act as a signalling molecule in the arginine-mediated autophagy pathway 11. However, in our experiments we could not find evidence for increased
production of nitric oxide by measuring the major metabolites of nitric oxide (i.e. nitrite and nitrate 1213) in the culture medium of human PEX1 fibroblasts nor in Pex1
mouse embryonic fibroblasts treated with arginine. In contrast, when cells were treated with similar concentrations of D-arginine, an inactive form of L-arginine and not involved in the nitric oxide pathway, the positive effect was diminished. We also tested the effect of NG-nitro-L-arginine methyl ester (L-NAME), a known inhibitor of nitric oxide synthesis 14, on arginine treated ZSD skin fibroblasts. Although the
positive results diminished, interpretation of these results was not reliable due to the toxic effect of L-NAME on the cells and this needs to be addressed in future studies. Recently, more articles were published that showed a positive effect of arginine in restoring residual function in other diseases with a defect in protein folding 15 16,
suggesting a more general effect not specific for ZSDs. Future studies have to be conducted to optimize these experiments and to elucidate the exact mechanism by which arginine exhibits its effect. Currently, microarray data of ZSD skin fibroblasts treated with arginine and homoarginine is being analysed. The results will also be compared to microarray data of cells cultured at different temperatures (i.e. culturing at 30°C and 40°C) and related changes in peroxisomal function (as described in Chapter 3). It is important to note that novel compounds, which show promising results in vitro, first need to be tested in a more complex system, such as a mouse model, before a human trial can be initiated.
One of the factors that hampers the research for effective therapies in ZSDs, is the lack of a good model for the most common milder ZSD phenotype. Until now, only severe mouse models or mouse models with selective peroxisomal inactivation in different organs were created. Much knowledge of peroxisomal dysfunction and consequences at organ levels is gained from this type of research with special emphasis on the work of Prof. Dr. M. Baes, Professor at the University of Leuven and her team. In Chapter 4, the results of the characterisation of the Pex1 mouse model are described. Because the model harbours the most frequent mutation found in
8
human ZSD patients (i.e. homozygous PEX1-p.G843D 17), which is associated with
a relatively mild phenotype 18, the model is of special interest. These mice develop
the characteristic liver features found in ZSD patients, can survive into adulthood and can be used to study the effect of different treatments. Moreover, this mouse model provides an excellent opportunity to study the exact pathogenesis of symptoms at the organ level. In the USA, a similar model was created but with different and milder clinical symptoms 19, possibly related to the mixed background of these mice. It
would be of interest to study knockout mice with different backgrounds, since survival was better in the Swiss background.20. If Pex1 mice also develop a neurological
phenotype, similar as ZSD patients, remains to be determined. Unfortunately, during this PhD study the AMC mice breeding facility had to be closed due to infection by the highly contagious mice hepatitis virus, which has halted additional studies with this mouse model. As of now, the opening of the facility is foreseen in spring 2017, after which new Pex1 mice rederived by Janvier Labs, France will be imported. It may well be possible that these mice have a different phenotype, as the microbiome in the gut will be different. This may affect several important metabolic processes, including composition of bile 2122, and thus could affect the mouse phenotype.
In Chapter 5, we describe the effect of cholic acid supplementation in a large cohort of ZSD patients. Cholic acid inhibits bile acid synthesis through negative feedback, thereby lowering the concentrations of toxic C27-bile acid intermediates. We show that cholic acid supplementation leads to partial suppression of the bile acid synthesis in the majority of patients, but it can be harmful for patients with advanced liver disease. The treatment period of nine months was too short to evaluate relevant clinical endpoints, such as neurological deterioration. Long-term studies in ZSD patients without severe liver disease, have to be conducted to resolve whether or not the disease stabilizes.
The clinical spectrum of ZSDs is much broader than originally assumed and includes patients without characteristic biochemical abnormalities in plasma and with milder phenotypes. A latest example is the addition of patients with Heimler syndrome to the ZSD spectrum 2324. Also older patients without some of the classical symptoms
have been described 25. The old phenotypic classification (i.e. ZS, NALD and IRD) is
therefore obsolete and needs to be revised. We have proposed a new classification based on age of presentation in Chapter 2. In Chapter 6, we have described a large cohort of ZSD patients with survival into adulthood. It is clear that the markers for peroxisomal dysfunction in plasma decline and eventually can normalise with age in some patients. This phenomenon was also confirmed in a pilot study (performed by K. Berendse and Prof. Dr. Poll-The) in a large cohort of patients in whom the various peroxisomal biomarkers have been followed over a long period of time. In this pilot study, we also found a correlation between the level of several abnormal peroxisomal metabolites (e.g. bile acid intermediates) and phenotypic severity in
these patients (i.e. high concentrations in severely affected patients). When patients survive into adulthood, there is great phenotypic variability. For example, some patients are completely care dependent while others have normal cognition and are able to attend secondary school. When patients reach adulthood, the main symptom is a peripheral neuropathy and pyramidal tract signs. The disease can remain stable for several years, but eventually the disease progresses, albeit slowly. Lastly, we describe a high prevalence (29%) of adrenal insufficiency in ZSD patients in Chapter 7. Because some patients were asymptomatic, the results of this study have important clinical implications, as the resulting Addison’s disease can be lethal, when remained untreated.
Future research
Recently, arginine supplementation in a ZSD patient was shown to have a positive effect on markers of peroxisomal function in plasma, although plasma concentrations of arginine were low (in contrast to the in vitro experiments) 26. Larger clinical trials
have to be initiated to determine efficacy of arginine on peroxisomal function in ZSDs. Arginine supplementation is reported to be safe, not only in this patient, but also in patients suffering from MELAS and urea cycle defects 2728 . A disadvantage is
that arginine has a low bioavailability (30 – 70% after oral administration 29) because
it is metabolized by arginase in the gut and liver to urea and ornithine. In plasma, arginine has a half-life of approximately only one hour 29. In addition, suppletion
of arginine increases arginase expression and activity, reducing the concentrations of arginine even further 30. Homoarginine appears a promising alternative because
it has higher efficacy at lower concentrations. However, pharmacokinetic data of oral homoarginine supplementation is lacking and studies in humans and mice are currently underway (clinicaltrials.gov identifier: NCT02675660) 31. (L-)Citrulline
is a precursor of arginine 32 and is not metabolized in the gut or liver. Moreover, it
inhibits arginase activity 30. Supplementation of citrulline was shown to increase
levels of arginine in plasma compared to administration of arginine. Treatment with citrulline could therefore be a strategy to reach higher plasma levels of arginine. However, care must be taken as exposure to high levels of arginine can lead to overproduction of nitric oxide that can lead to hyponatraemia, vasodilation and subsequent hypotension 2833.
When a novel compound that can rescue peroxisomal function in vitro is discovered, it needs to be tested in an animal model before a clinical trial in humans can be initiated. However, when a compound is already used in other diseases and pharmacodynamics are known (as is the case for arginine) a clinical trial can be initiated directly. An example is a drug named betaine that was tested directly in patients with a ZSD (clinicaltrials.gov identifier: NCT0183894). Trials to determine
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Chapter 8 Summary and general discussion
efficacy need robust clinical endpoints and long follow-up. This is a challenge for rare diseases with a highly variable disease course. Although these trials are costly and challenging, they remain necessary. Compounds can be effective and promising in vitro but may be ineffective when tested in patients (e.g. lovastatin in X-ALD 34).
There are a few pitfalls when designing and performing clinical trials in ZSDs. A great challenge is the highly variable clinical course of ZSD. This requires the addition of control groups, always a particular challenge in the field of rare disease. In addition, to determine an effect of treatment, reliable clinical endpoints are necessary. At this time there are no specific clinical severity scores for ZSDs, neither is there natural history data from large prospective studies. A severity score for ZSD is currently being developed at the Department of Paediatric Neurology of the AMC. Before this score can be used in clinical practice, a good international validation is necessary. We and others (clinicaltrails.gov: NCT01668186) are currently collecting prospective data to determine the natural history of ZSDs in a large cohort of patients. Surrogate outcome parameters (i.e. peroxisomal metabolites in plasma) are often used. In Chapter 6 it is reported that the levels of these metabolites often normalize in older patients, which implies that a decrease of these parameters cannot always be attributed to the therapeutic intervention involved. Studies solely based on these surrogate outcome parameters in plasma have to be interpreted with caution. Furthermore, biochemical parameters can never be a substitute for clinical endpoints. In fact, improvement in biochemical parameters does not necessarily correlate with clinical benefit. It is possible that some biochemical parameters are usable as surrogate outcomes, for instance the novel markers C26:0-lysoPC or C26:0-carnitine in bloodspots 35
(e.g. less fluctuations over time), but additional research with both long-term follow-up and measurements in different age grofollow-ups is necessary. To date, few patients have been identified with normal levels of the C26:0-lysoPC (Prof. Dr. Waterham, personal communication). Therefore, there is a need to develop new biochemical markers. New lipodomics and metabolic studies are currently underway. Plasma or bloodspot samples might not be sufficient to diagnose the more milder phenotypes and novel diagnostic techniques are necessary, such as analysis of markers for peroxisomal dysfunction in leukocytes.
Research requires funding. Obtaining adequate funding is always challenging, even more so in the field of rare diseases, as these are often considered of less priority than for instance diabetes or heart disease. Although understandable, the study of rare diseases can lead to fundamental insights that are applicable to other diseases. A good example is Alzheimer’s disease, in which peroxisomal alterations are reported to contribute to progression of the Alzheimer pathology 3637. Peroxisomes may also
play a role in aging 38. Models or patients with peroxisomal dysfunction and research
therein provide unique opportunities to study the pathophysiology of these diseases in more detail.
To stimulate funding for research on orphan diseases and development of orphan drugs, the Orphan Drug Act was introduced in the USA in 1983. In 2000, the European Commission implemented new legislation, the Orphan Medicinal Products (OMPs), in order to stimulate the development of orphan medicinal products (Regulation Number 141/2000) in the EU. A disease is considered orphan in the EU when no more than 5 in 10,000 people are affected. Because it is difficult to initiate randomized controlled trials, due to the limited number of patients, data from case reports and/or in vitro data is acceptable when applying for market authorization. Moreover as an incentive, the EU also provides protocol assistance and community research programmes. When market authorization is received, 10-year exclusive marketing is granted within the EU. Within 10 years, already 60 orphan drugs have received marketing authorization in Europe 39, making
this new legislation successful. However, the flip side of the OMP is that several Orphan drugs may be ineffective due to relatively low standards of evidence to obtain market authorization and cause false hope for patients 40. The legislation
is misused by some pharmaceutical companies, as they market orphan drugs at exorbitant prices 41. Some compounds are already prescribed for dozens of years
for several diseases, so compared to expensive development of novel drugs, no additional research was necessary (e.g. pharmacodynamics and safety studies). Exorbitant costs for the treatment of one adolescent patient of approximately $50,000 each month is unfortunately no exception. This cost versus benefit analysis is absolutely disproportionate and will raise public concern on the OMPs and will question the orphan drug legislation in its present form. It should also be noted that the pharmaceutical industry already influences evidence based production, clinical practice guidelines and eventually health care professionals 42, and that through
the European Orphanet Drug Act, these companies are at risk of becoming too influential. In order to prevent possible conflicts of interest, an international policy regarding scientific integrity and independence is necessary as stated recently by Hollak et al for the set-up of European Reference Networks 43.
In recent years, the legislation regarding animal welfare in animal scientific research has become more strict. In 2010 the EU adopted new legislation, based on the principle of the Three R’s, to Replace, Reduce and Refine the use of animals for scientific purposes (Directive 2010/63/EU). This new legislation widens the scope and includes for example also foetuses of mammalian species in their last trimester of development, as before this type of research was not considered as animal scientific research. For research groups within the EU (e.g. The Netherlands) more accessible and primitive organisms can be used as an alternative. For ZSD research, models such as C. elegans 44 (with various different Pex mutations), Drosophila 45
and zebrafish 46 are available 47. In the near future these models will probably be
8
In conclusion
ZSDs are extremely heterogeneous in clinical presentation, disease progression and metabolic profile. The expanded newborn screening program will provide the opportunity to diagnose patients at an early age and provides opportunities for early intervention.
Besides the development of new treatments, improving supportive care for these patients is currently the most important. Despite the enormous progress that is being made since the discovery of peroxisomes in 1966, on knowledge, symptomatology of peroxisomal disease and peroxisome biology, there are still numerous questions to be answered. It is important to centralize rare diseases nationwide (at least within the EU) to allow experts to work together (e.g. laboratory and outpatient clinic) and to provide the opportunity to test treatments in proper designed clinical trials. International collaborations for orphan diseases are therefore crucial, in order to improve quality of life for these groups of patients and eventually to find a cure for this often devastating disease.
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