Elsevier

Journal of Neuroimmunology

Volume 293, 15 April 2016, Pages 82-85
Journal of Neuroimmunology

Short communication
A study of the utility of azathioprine metabolite testing in myasthenia gravis

https://doi.org/10.1016/j.jneuroim.2016.02.015Get rights and content

Highlights

  • Azathioprine remains a first line broad acting immunosuppressant for myasthenia.

  • Azathioprine metabolites in myasthenia gravis have not been investigated before.

  • Correlations between symptoms, 6-TGN and lymphocytes were identified.

  • Azathioprine metabolites may be considered in patients with treatment complications.

Abstract

Myasthenia gravis (MG) is an autoimmune neuromuscular disorder characterised by fatigable voluntary skeletal muscle weakness. The underlying pathogenesis is complex involving adaptive autoimmune responses. Azathioprine remains a first line broad acting immunosuppressant for MG. Due to varied clinical responses to azathioprine we aimed to investigate the relationship between azathioprine metabolites and symptom control. Mild correlations between Quantitative Myasthenia Gravis Score (QMG) vs. 6-thioguanine nucleotides (R =  0.317 p = 0.186) and QMG vs. lymphocyte count (R = 0.402 p = 0.08) were found. Azathioprine metabolite measurement should be considered in MG patients with; pancytopenia, deranged liver function or recurrent infections.

Introduction

Myasthenia gravis (MG) is an autoimmune neuromuscular disorder characterised by fatigable voluntary skeletal muscle weakness often caused by anti-nicotinic acetylcholine receptor autoantibodies. The underlying pathogenesis of MG is complex and involves a combination of both autoimmune adaptive humoral and cellular responses with loss of tolerance to several components of the neuromuscular junction as often implicated by the presence of specific autoantibodies (Gilhus, NE, et al., 2011, Vincent, A and Willcox, N, 1999). Considering the uncertainties surrounding the pathogenesis and clinical heterogeneity of MG, patients are frequently treated with broad acting immunosuppressive regimes including corticosteroids and steroid-sparing drugs to control symptoms.

Azathioprine is an immunosuppressant drug with randomised control trial evidence of efficacy in myasthenia gravis (Bromberg, MB, et al., 1997, Palace, J, et al., 1998). The thiopurine drug remains the first line steroid sparing immunosuppressant for MG (Gilhus and Verschuuren, 2015). The immunosuppressive action of azathioprine is due to the generation of 6-thioguanine nucleotides (6-TGN). 6-TGN exerts immunosuppressive actions in two ways; the intracellular accumulation of 6-TGN in lymphocytes leads to incorporation of these foreign exogenous nucleotides into the cell's DNA causing cell cycle arrest and apoptosis. Secondly, the thioguanine nucleotide; 6-thioguanine-5′-triphosphate inhibits Rac1-GTPase dependent CD28 co-stimulation of T cells leading to T cell apoptosis (Tiede et al., 2003). Excessive levels of 6-TGN lead to myelosuppression via inhibition of the mitotic active of rapidly dividing haematopoietic stem cells.

6-methylmercaptopurine (6-MMP) is an inactive by-product of azathioprine metabolism by thiopurine S-methyltransferase (TPMT). Variation in the enzymatic active of TPMT within the population is common and due to frequent genetic polymorphisms (Gisbert et al., 2007). Low TPMT activity results in increased 6-TGN production and greater risk of myelosuppression. In contrast, high TPMT enzymatic activity may cause clinical non-response due to preferential metabolism to inactive 6-MMPN and a lack of immunosuppressive 6-TGN production in vivo. It is good practice to check TPMT enzymatic function or genotype in patients prior to commencing azathioprine as elevated 6-MMPN levels can cause hepatotoxicity and high 6-TGN can lead to myelosuppression.

The utility of routine measuring of the azathioprine metabolites 6-TGN and 6-MMPN in cases of inflammatory bowel disease has reported in many studies with conflicting recommendations (Amin, J, et al., 2015, Wright, S, et al., 2004). Azathioprine is typically prescribed at a dose of 2–2.5 mg/kg/daily for MG patients, with regular monitoring of full blood count to check for leucopenia, mean corpuscular volume and liver function tests (Heerasing et al., 2015). With the observation of varying efficacy of azathioprine in MG we aimed to investigate azathioprine metabolites in MG, which has not been reported previously. The purpose of this study was to investigate the relationship between azathioprine metabolite profiles and clinical symptom control in MG patients and provide power calculations for larger studies. Patients were clinically assessed using the Quantitative Myasthenia Gravis Score (QMG) (Hart et al., 2007) (Barnett et al., 2012) and had blood taken for azathioprine metabolites and routine blood parameters during the same clinic visit.

Section snippets

Subjects

Nineteen patients with MG were enrolled into the study. Participants had not been exposed to immunosuppressive agent other than azathioprine and prednisolone.

Inclusion criteria included:

  • 1.

    A clinical diagnosis of MG with fatigable muscle weakness and

    Either

    Antibodies to anti-acetylcholine receptor and/or anti-muscle specific kinase autoantibodies.

    Or

    Single fibre electromyography demonstrating diagnostic jitter, with/without supportive progressive decline in compound muscle action potential on

Results

The MG patient cohort demographics were typical for patients attending the MG clinic with a mean age of 59.5 years, heterozygous mix of gender and generalised or ocular MG. 13/19 participants had seropositive MG. 5/19 of participants had undergone thymectomy due to thymoma. No participant had undergone thymectomy in the absence of radiological evidence of a thymoma pre-operatively (Table 1). The mean azathioprine dosage use was 180 mg/day (range 150–225 mg) and mean prednisolone dosage was 6 mg

Discussion

In this study the most significant correlation observed was the between clinical symptom control (as assessed by QMG) and lymphocyte count. This raises some interesting questions as to the underlying pathological mechanism of MG as well as whether future treatment strategies should be directed towards the adaptive cellular immune response (Dalakas, 2015). A lower total lymphocyte count and increased MCV have mild correlations with improved symptom control in MG patients treated with

Conclusion

In summary, on the basis of this study we would advocate the testing of TPMT activity or genotype, in MG patients prior to commencing azathioprine (Lennard, 2014). We suggest that the measurement of azathioprine metabolites in selected individuals with MG who suffer pancytopenia, deranged liver function tests or recurrent infections whilst on azathioprine can be of benefit. It currently remains unclear whether azathioprine metabolite monitoring is of use in clinical non-responders. The

Conflicts of interest

The authors report no conflicts of interest.

Acknowledgements

We thank the patients for their kind participation and support for this study. We also thank Myaware for their support with funding the study and supporting Myasthenia patients, their families and carers and for promoting research into myasthenia. We acknowledge Lisa Joyce, Gabriella Howard, the Trust Wide Research Team at University Hospital Southampton NHS Foundation Trust, and the National Institute for Health Research (NIHR)/Wellcome Trust Clinical Research Facility at University Hospital

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