Chronic Fatigue Syndrome: Targeting Serotonin and Insulin ... · Chronic fatigue syndrome (CFS) is still an enigmatic disorder. It is regarded as a complex disorder with tremendous

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Chronic Fatigue Syndrome: Targeting Serotonin and

Insulin-like Growth Factor

Gerard Kong Han The



The ondansetron clinical trial was supported by a grant of GlaxoSmithKline, The Netherlands.

The acclydine clinical trial was supported by a grant of Optipharma, Susteren, The Netherlands.

Financial support for the publication of this dissertation was kindly given by Van den Bogaert en partners.

ISBN

978-94-028-0262-7

Cover Ad Verstijnen, Boxtel, The Netherlands. Werk: 'Veerkracht'

Design/lay-out

Promotie In Zicht, Arnhem

Print

Ipskamp Printing, Enschede

Copyright © G.K.H. The, 2016

All rights are reserved.



Proefschrift

ter verkrijging van de graad van doctoraan de Radboud Universiteit Nijmegen

op gezag van de rector magnificus prof. dr. J.H.J.M. van Krieken,volgens besluit van het college van decanen

in het openbaar te verdedigen op dinsdag 1 november 2016 om 14:30 uur precies

door

Gerard Kong Han Thegeboren op 8 september 1974

te Nijmegen

Chronic Fatigue Syndrome: Targeting Serotonin and

Insulin-like Growth Factor



Promotoren Prof. dr. J.K. Buitelaar Prof. dr. J.W.M. van der Meer Prof. dr. G. Bleijenberg

Manuscriptcommissie Prof. dr. J.W.A. Smit, voorzitter Prof. dr. A.E.M. Speckens Prof. dr. A.M. van Hemert, Universiteit Leiden

Paranimfen drs. Roy Derikx dr. Jan Mathijs Schoffelen



Clouds come floating into my life,

no longer to carry rain or usher storm,

but to add color to my sunset sky.

Rabindranath Tagore (1861 - 1941)

voor mijn ouders





Contents

Chapter 1 General introduction and outline of the thesis 9

Chapter 2 The effect of Granisetron, a 5-HT3 receptor antagonist, in the treatment of chronic fatigue syndrome patients: a pilot study

27

Chapter 3 The effect of Ondansetron, a 5-HT3 receptor antagonist, in chronic fatigue syndrome: a randomised controlled trial

41

Chapter 4 Tryptophan depletion in chronic fatigue syndrome: a pilot cross-over study

57

Chapter 5 The effect of Acclydine in chronic fatigue syndrome: a randomised controlled trial

67

Chapter 6 General discussion 85

Summary

Samenvatting

List of publications

Dankwoord

Curriculum vitae

101

107

113

119

125





General introduction and outline of the thesis

1





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1

GENERAL INTRODUCTION AND OUTLINE OF THE THESIS

Introduction

Chronic fatigue syndrome (CFS) is still an enigmatic disorder. It is regarded as a complex disorder with tremendous impact on the lives of patients, their families and society in general. Full recovery without treatment is rare, with only 5% of the patients showing spontaneous recovery. The mean age of onset is between 29 and 35 years1. Women are more at risk than men and many patients have experienced a period in which they were bound to their homes. In 2005 the Dutch Health Council estimated a prevalence of 30,000 to 40,000 CFS patients in The Netherlands, based on prevalence studies in Dutch general practices2. A more recent study performed in the general population suggests there are probably about 80,000 CFS patients in The Netherlands3. The Expert Centre for Chronic Fatigue (ECCF) of the Radboud University Nijmegen Medical Centre is a multidisciplinary collaborative centre focussing on the assessment and management of patients with CFS. Since 1989, internists, virologists, neurologists, neuroscientists, psychiatrists and psychologists have been involved in CFS research in Nijmegen. During the years, the research has extended to chronic fatigue in several other conditions including rheumatoid arthritis, traumatic brain injury, myotonic dystrophy and cancer4,5,6,7.

Definition Chronic Fatigue Syndrome

Holmes et al. developed the first CFS working case definition8 in 1988. In 1994 and in 2003, the Expert Centre contributed to the development of international guidelines for the clinical evaluation and research of CFS, resulting in the 1994 case definition which is currently considered to be the standard (see panel)9,10. The 1994 case definition was primarily developed for research purposes, in which case specificity is important. In this thesis, the 1994 CDC criteria were used in all studies presented.

There is much debate among researchers and practitioners about the patho-physiology and definition of CFS11,12. Although the case definitions differ, all definitions identify severely fatigued CFS patients from healthy control subjects of the same age and sex. There are no pathognomonic signs or diagnostic test for CFS13. CFS is diagnosed by the presence of clinical symptoms, disability and by exclusion of certain psychiatric disorders and medical conditions explaining fatigue.



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CFS and psychiatric disorders

Psychiatric disorders play an important and complex role in chronic fatigue syndrome. First, as described previously, certain psychiatric diagnoses are exclusion criteria for CFS. The recommendations formulated by Fukuda et al. define current or past psychiatric disorders such as melancholic or psychotic depression, bipolar disorder, delusional disorder schizophrenia, anorexia nervosa or bulimia nervosa as exclusion criteria for CFS. It is difficult to interpret CFS symptoms in these psychiatric disorders, and care and treatment should focus on the psychiatric disorder.

In 2003, recommendations for a better application of the 1994 case definition were made and psychiatric exclusion criteria were further elaborated. If a major depressive disorder with psychotic or melancholic features, anorexia nervosa or

Panel: 1994 case definition for CFS from US Centers for Disease Control and Prevention

Characterised by persistent of relapsing unexplained chronic fatigue• Fatigue lasts for at least 6 months

• Fatigue is of new or definite onset

• Fatigue is not the result of ongoing exertion

• Fatigue is not the result of an organic disease

• Fatigue is not alleviated by rest

• Fatigue results in a substantial reduction in previous occupational,

educational, social and personal activities

• Four or more additional symptoms, concurrently present more than 6 months

- impaired memory or concentration - sore throat

- tender cervical or axillary lymph nodes - muscle pain

- new headaches - pain in several joints

- unrefreshing sleep - malaise after exertion

Exclusion criteria• Medical condition explaining fatigue

• Major depressive disorder (melancholic or with psychotic features) or bipolar disorder

• Schizophrenia, dementia, or delusional disorder

• Alcohol or other substance abuse within 2 years before onset

• Anorexia nervosa or bulimia nervosa

• Severe obesity (body mass index ≥ 40)



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bulimia have been resolved more than 5 years before the onset of chronic fatigue, these conditions should not be considered exclusionary.

Second, fatigue is a known symptom in various psychiatric disorders such as depression, dysthymia, panic disorder and posttraumatic disorder, and thus psychiatric disorders should be considered in the list of differential diagnoses of CFS. As there is an overlap between symptoms in CFS and psychiatric disorders, thorough assessment is required to minimise over and underdiagnoses of both CFS and psychiatric disorders. Devasahayam et al.14 explored the prevalence of alternative diagnosis in patients referred with a definite or provisional diagnosis of CFS to a specialised CFS clinic. The authors found that 49% of the referred patients had alternative diagnoses to CFS. The most common somatic diagnoses assessed were sleep disorders and the commonest alternative psychiatric diagnosis was depressive disorder. If fatigue is part of a current clinical axis-1 psychiatric disorder and the psychiatric disorder is a sufficient explanation for fatigue complaints, CFS cannot be diagnosed and a psychiatric disorder should be diagnosed and treated accordingly. For example: fatigue is a common symptom in depressive disorder. It is important to differentiate whether fatigue is a symptom of a depressive episode, or of CFS. In depressive disorder, depressed mood often precedes fatigue. A co-morbid depressive disorder can be diagnosed in 25% of CFS patients. In depressive disorder, anhedonia is a core symptom. CFS patients often report they want to be active, but are impaired as a consequence of their chronic fatigue.

Third, conditions like anxiety disorders and less severe forms of depression do not preclude the diagnosis of CFS.

There may be an association or co-morbidity between CFS and psychiatric disorders15, and patients with CFS and co-morbid psychiatric diagnoses have more symptoms and disability than CFS patients without psychiatric comorbidity16. Skapinakis et al. reported a high level of psychiatric co-morbidity, in particular depression and anxiety in CFS17. Cella et al. investigated the prevalence of depression and anxiety disorder in CFS. In a total of 640 CFS patients who were recruited into a treatment trial investigating the effect of pacing in CFS, 54% had a diagnosis of CFS and no depression or anxiety disorder, 14% had CFS and anxiety disorder, 14% had CFS and depressive disorder and 18% had CFS and both depression and anxiety disorder18. In 2003, our group investigated the prevalence of concomitant psychiatric disorders in 270 CFS patients participating in a randomised controlled trial



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CHAPTER 1

investigating the effect of Cognitive Behaviour Therapy (CBT) compared to support groups and natural course. A current psychiatric diagnosis was found in 32% of the CFS patients. One current psychiatric diagnosis was found in 21% and two or more diagnoses in 11%. It was investigated whether psychiatric co-morbidity has a negative effect on treatment outcome. In this study no differences were found in treatment outcome or withdrawal between CFS patients with and without psychiatric co-morbidity when treated with CBT19. It is not fully clear whether CFS is associated with elevated levels of personality disorders. While some studies have provided evidence for elevated rates of personality disorders in CFS20,21, other studies were unable to confirm a higher prevalence in CFS22,23. Courjaret et al. evaluated the prevalence of Diagnostic and Statistical Manual for Mental Disorders (DMS-IV-TR) personality disorders in CFS patients and in matched controls. They found no differences between the CFS sample and the healthy control group at the level of dimensional trait scores, number of personality disorder criteria and prevalence rates of personality disorders. The discrepant findings could be related to different methods used to assess personality disorders, research setting (primary, secondary, or tertiary setting, psychiatric care or rehabilitation setting) and use of different control groups23.

Evaluation of chronic fatigue

A systematic evaluation of fatigue symptoms was established for the diagnosis and treatment of patients with unexplained fatigue24,25,26,27. In the management of fatigue, three categories were described: 1) acute phase (fatigue up to one month after first consultation), 2) sub-acute phase (fatigue during one to six months) and 3) a chronic phase (fatigue lasting more than six months). In the acute phase the policy is to be expectative to one month, unless symptoms change or worsen during that time. In the sub-acute phase, laboratory testing is useful and health-promoting activities should be stimulated. In the clinical evaluation, somatic and psychiatric disorders should be taken into account in the differential diagnosis. If in the chronic phase no somatic or psychiatric diagnoses have been found, it is more likely that CFS can be diagnosed. It is important to diagnose CFS and to provide psycho-education to patients, since it is useless to keep searching for somatic causes. Moreover, patients with CFS can and should be offered evidence-based treatment28.



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Model of CFS

In the early days, most of the research was devoted to finding a unifying patho -genesis, i.e., one virus, one metabolic disorder and so on. These research efforts failed. The Expert Centre for Chronic Fatigue realised that it might be more fruitful to discern between predisposing factors, precipitating factors and perpetuating and prognostic factors. Predisposing factors are risk factors that make people vulnerable to develop CFS. Early life adverse experience (physical or emotional abuse or neglect), being female and physical inactivity have been reported as risk factors for CFS29,30. There is evidence that some genetic disposition exist for CFS29. Twin studies have supported modest genetic contributions to CFS31. Precipitating factors are somatic and psychological stressors that can trigger CFS symptoms. It was considered highly likely that various factors (a diversity of infectious agents, exposure to toxins, anaesthesia, trauma, lost of job or bereavement) could act as precipitating factors29. Concerning perpetuating factors, no satisfactory somatic factors could be identified over the years, but based on a rather large dataset it was possible to develop and test an empirically supported model of the perpetuating psychological factors in CFS (see Figure 1).

Figure 1 The model of perpetuating factors by Vercoulen et al. (1998) and Prins et al. (2004).

Sense ofcontrol FATIGUE

Somaticattribution

Physicalactivity Focusing on

symptoms

Social supportand reactions of others

IMPAIRMENT



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CHAPTER 1

According to this model, negative cognitions and unhealthy behaviour are the maintaining factors in CFS. Attributing CFS symptoms to a somatic cause lowers physical activity, which in turn increases fatigue and leads to impairment. A low sense of control and focusing on bodily symptoms have a negative influence on fatigue32. Prins et al. in 2004 identified a perceived lack of social support as another perpetuating factor of fatigue severity and impairment in CFS33.

CBT for CFS was developed based on the model of perpetuating factors. CBT is an effective and evidence-based treatment for CFS28,34,35. Wiborg et al. found that the effect of CBT for CFS was not mediated by a persistent increase in physical activity36, but mediated by a decrease in focusing on fatigue37.

Pathophysiology of CFS

The fact that psychological factors play a role and that CBT is effective does not preclude the importance of somatic factors and a somatic substrate. It should be noted that, despite many efforts, no drug treatment for CFS has proven efficacy35. Our research group therefore continues to investigate the pathophysiology of CFS, biological hypotheses and pharmacological treatment strategies. Many biological mechanisms have been postulated, but, as previously stated, none of these have been proven. More insight into the neurobiology of CFS is highly necessary to improve our understanding of this illness. CFS is probably a hetero - geneous condition. Neuroendocrine pathways and neurotransmitter metabolism have been important targets for CFS research38, and in particular disturbances in the growth hormone/ insulin-like growth factor-1 axis and in the serotonin system have been suggested to be relevant for CFS. The major challenge for this thesis is to explore the evidence for neurobiological disturbances in CFS by using pharmacological interventions. As a result, four studies are presented in which the role of serotonin and the growth hormone/ insulin-like growth factor-1 axis are investigated to elucidate the pathogenesis of CFS.

Serotonin and CFS

One pharmacological study, performed by our Nijmegen research group, concerned the effect of Fluoxetine, a serotonin (5-hydroxytryptamine, 5-HT) re-uptake inhibitor. In this randomised placebo-controlled clinical trial, CFS patients with and without a major depressive disorder were treated with Fluoxetine39. Fluoxetine is



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considered to be an effective antidepressant. It turned out, in contrast to expectations, that the clinical trial showed neither a beneficial effect on depression severity, nor on fatigue severity or on any CFS-related symptoms. Differences in underlying processes of depressive symptoms between CFS patients with depressive co-morbidity and patients with major depressive disorder were thought to explain these unexpected findings.

This pharmacological study was the first study performed by the Expert Centre involving the 5-HT system. The results did not provide support for a role of serotonin reuptake inhibition in the treatment of CFS. Serotonin plays a diverse and important role in the regulation of sleep and wakefulness, mood regulation, impulse control, memory, perception of pain and in behaviour that involves a high cognitive demand40. Serotonin pathways are widespread and project to the (frontal) cortex, striatum, hippocampus and the hypothalamus (see Figure 2).

Figure 2 Serotonin system.

Dumont G. Acute effecten van selectieve serotonine-heropnameremmers: een behandeling van het premenstrueel syndroom. 2013;1:36-39.



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CHAPTER 1

There is clinical and experimental evidence implicating enhanced serotonergic neurotransmission in fatigue states41,42,43. When the level of brain 5-HT is increased either by exercise or administration of the 5-HT precursor tryptophan, an increase of fatigue is observed44,45,46. In animals, tryptophan injection into the blood or directly into the central nervous system accelerates exhaustion during motor activity47. The time to exhaustion is decreased by administering a 5-HT agonist and increased by serotonin antagonists48. Accumulating evidence would support that 5-HT neurotransmission may be altered in CFS. 5-HT pathways from the dorsal raphe nuclei to the paraventricular nucleus of the hypothalamus are thought to bring about the secretion of hypo-thalamic-pituitary-releasing peptides involved in the release of prolactin and ACTH (adrenocorticotropic hormone) from the anterior pituitary.

Measuring prolactin and cortisol responses to 5-HT agonist drugs probably reflect hypothalamic-5-HT neurotransmitter function. Although it is well-established that the paraventricular nucleus of the hypothalamus is under serotonergic control, serotonin may have a direct action on the anterior pituitary and the adrenal cortex, thus directly stimulating both ACTH and cortisol release49. Neuroendocrine challenge studies have suggested a reduced function of the Hypothalamic- Pituitary-Adrenal (HPA) axis and an enhanced 5-HT function in CFS50.

Weaver et al.51 assessed serotonergic activity indirectly through assessment of the plasma prolactin response to intravenous tryptophan in patients with CFS with and without co-morbid fibromyalgia. Upregulated plasma prolactin responses were found only in women with CFS without co-morbid fibromyalgia. The authors stated that, despite syndromal overlap between the two conditions, fibromyalgia has an opposite, deficient central serotonergic regulation in contrast to an upregulated serotonergic activity in CFS. Consequently, the presence of co-morbid fibromyalgia may have confounded earlier serotonin challenge studies in CFS patients.

Besides serotonin challenge studies, many researchers have studied the HPA axis in CFS patients. One of the most consistent findings is a reduced basal glucocorticoid cortisol output (in conjunction with enhanced ACTH production) in at least some CFS patients, this is most consistently shown by 24-h urinary free cortisol measurement. No evidence of a primary dysfunction of the HPA axis is found in CFS38,52. It is likely that several other factors, including inactivity, medication and ongoing stress, influence the HPA-axis53. There is however some evidence that reduced cortisol levels are linked to symptom production or persistence.



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Roberts et al. investigated the effect of hypocortisolism on the effect of CBT in CFS. A weak correlation was found between low basal cortisol levels and higher disability scores in untreated CFS patients. Patients with a lower basal cortisol level responded less well to CBT54. Hypocortisolism can normalise after CBT in adolescents55 and adults54. There is a complex interaction between the HPA axis and the serotonin system. Studies have demonstrated that glucocorticoids may have an inhibitory effect on central serotonin function56. In depression, HPA system upregulation and 5-HT downregulation are prominent neuroendocrine hypotheses. Contrasting neuroendocrine responses have been found between depression and CFS: whereas CFS may be associated with reduced HPA activity and increased serotonin function, depression shows the reverse pattern50,57. Positron Emission Tomography studies58,59 and genetic studies60 have strengthened the hypothesis of an upregulated serotonin system in CFS.

Growth Hormone/ Insulin-like Growth Factor-1 and CFS

The integrity of the Growth Hormone/ insulin-like growth factor system has been an important area of study in CFS. An important reason is the known link between Growth Hormone (GH) deficiency and fatigue. GH deficiency in adults is associated with clinical symptoms such as fatigue and myalgia61. Insulin-like growth factor-1 (IGF-1), also known as somatomedin C, is synthesised in peripheral tissues and largely in the liver. More than 99% of IGF-1 that circulates in serum is protein-bound. The most important binding protein is IGF-binding-protein- number 3 (IGF-BP-3). IGF-BP-3 is the most abundant form in plasma with the highest affinity for IGF-1. IGF-1 and IGF-BP-3 plasma concentrations are influenced by growth hormone (GH), along with other factors such as age, gender, nutritional status and other hormones, including insulin. Unlike growth hormone, IGF-1 and IGF-BP-3 levels exhibit less daily fluctuation. Determining IGF-1 as well as IGFBP-3 levels is recognised as an effective test for assessing GH status. Abnormalities in the GH/ IGF-1 axis have been reported, but patient characteristics and data have not always been elaborately in these studies.

The small number of previously investigated CFS patients may also have played a role in interpreting results.

Baseline function, dynamic testing and treatment replacement studies have been performed. A few studies suggested low basal growth hormone and IGF-1 in CFS62,63. There is some evidence that basal growth hormone release is reduced nocturnally in CFS patients64. Di Giorgio et al. highlighted the lack of GH/ IGF-1



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studies adequately examining GH and IGF-1 levels across the circadian cycle with frequent sampling intervals65. In summary, reports investigating the integrity of the GH/ IGF-1 system in CFS have yielded conflicting results50.

Outline of this thesis

Given the controversies and uncertainties surrounding these potentially important mediators in CFS, we decided to investigate the role of tryptophan, 5-HT and IGF-1 in CFS in this thesis. Our studies aim to obtain a deeper insight into the pathophysiology of CFS by investigating the efficacy of three biological interventions for CFS.

The effect of Granisetron, a 5-HT3 receptor antagonist, in CFSIn chapter 2 we asked the question whether treatment with a 5-HT3 receptor antagonist is effective in CFS. Studies and clinical observations suggested a role for 5-HT3 receptor antagonism in the treatment of chronic fatigue associated with chronic liver disease66 and fibromyalgia67. The effect of 5-HT3 receptor antagonists in CFS patients had not been investigated in CFS patients before, hence a pilot study was carried out to investigate the effect of Granisetron.

The effect of Ondansetron, a 5-HT3 receptor antagonist, in CFSIn chapter 3 we addressed the same question based on positive findings found in the pilot study described in chapter 2. A randomised double-blind controlled trial was performed. Because granisetron was no longer available at that time, due to a merge of pharmaceutical manufacturer SmithKline Beecham with Glaxo Wellcome, the 5-HT3 antagonist Ondansetron (Zofran) was used to further investigate the effect of 5-HT3 receptor antagonism in CFS.

The effect of Acute Tryptophan Depletion in CFSBesides intervention with selective serotonin antagonists, we also investigated the effect of Acute Tryptophan Depletion (ATD), a well-known intervention in major depressive disorder. The results of a placebo-controlled cross-over ATD study are presented in chapter 4. Based on the hypothesis that CFS represents a hyperserotonergic state, we used ATD to decrease central serotonin availability, and assessed the effects on fatigue severity, concentration and mood.



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The effect of Acclydine in CFSIn chapter 5 we investigated whether CFS patients have lower IGF-1 levels than age, weight and gender matched neighbourhood controls. Based on the idea that the growth hormone metabolism is disrupted in CFS, De Meirleir in 2001 reported that treatment with Acclydine, a plant-sourced alkaloid, is effective in modifying growth hormone/ IGF-1 status in adult CFS patients and reverts complaints68. In 2002, the CFS patient organisation reported in their ‘MEdium Journal’ that the manufacturer of Acclydine had found that 75-80% of CFS patients have a growth hormone deficiency69. Double-blind randomised controlled trials were needed. Acclydine was an expensive treatment and is available on the internet without a prescription. It had been mentioned on the news that Acclydine was recommended for cancer patients with chronic fatigue, and that there were conflicts in Acclydine research70,71. Acclydine was administered to vulnerable patients, but the scientific evidence for efficacy was lacking. The effect of Acclydine, officially a food supplement, was thus evaluated in a randomised controlled clinical trial. We evaluated whether Acclydine is effective in CFS by increasing biologically active IGF-1 levels. Results of a randomised controlled trial are presented in this thesis.

Discussion and future prospectsIn chapter 6 I will discuss the main results and implications of the investigations performed, and present recommendations and prospects for future studies.



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activity during prolonged exercise to fatigue. J Appl Physiol 1993;74(6):3006-3012.42. Wilson WM, Maughan RJ. Evidence for a possible role of 5-hydroxytryptamine in the genesis of

fatigue in man: administration of paroxetine, a 5-HT reuptake inhibitor reduces the capacity to perform prolonged exercise. Exp Physiol 1992;77(6):921-924.

43. Cotel F, Exley R, Cragg SJ, Perrier JF. Serotonin spillover onto the axon initial segment of motoneurons induces central fatigue by inhibiting action potential initiation. Proc Nat Acad Sci 2013;110(12):4774-4779.

44. Fernstrom JD, Fernstrom MH. Exercise, serum free tryptophan, and central fatigue. J Nutr 2006; 136(2):553S-559S.



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45. Davis JM, Alderson NL, Welsh RS. Serotonin and central nervous system fatigue: nutritional considerations. Am J Clin Nutr 2000;72 (Suppl 2):573S-578S.

46. Blomstrand E. Amino acids and central fatigue. Amino Acids 2001;20(1):25-34. 47. Soares DD, Lima NR, Coimbra CC, Marubayashi U. Evidence that tryptophan reduces mechanical

efficiency and running performance in rats. Pharmacol Biochem Behav 2003;74(2):357-362.48. Bailey SP, Davis JM, Ahlborn EN. Serotonergic agonists and antagonists affect endurance

performance in the rat. Int J Sports Med 1993;14(6):330-333.49. Dinan TG. Serotonin and the regulation of hypothalamic-pituitary-adrenal axis function. Life Sci

1996;58(20):1683-1694.50. Parker AJ, Wessely S, Cleare AJ. The neuroendocrinology of chronic fatigue syndrome and

fibromyalgia. Psychol Med 2001;31(8):1331-1345.51. Weaver SA, Janal MN, Aktan N, Ottenweller JE, Natelson BH. Sex differences in plasma prolactin

response to tryptophan in chronic fatigue syndrome patients with and without comorbid fibromyalgia. J Womens Health 2010;19(5):951-958.

52. Papadopoulos AS, Cleare AJ. Hypothalamic-pituitary-adrenal axis dysfunction in chronic fatigue syndrome. Nat Rev Endocrinol 2011;8(1):22-32.

53. Cleare AJ. The HPA axis and the genesis of chronic fatigue syndrome. Trends Endocrinol Metab 2004;15(2):55-59.

54. Roberts AD. Charler ML, Papadopoulus A, Wessely S, Chalder T, Cleare AJ. Does hypocortisolism predict a poor response to cognitive behavioural therapy in chronic fatigue syndrome? Psych Med 2010;40:515-522.

55. Nijhof SL, Rutten JM, Uiterwaal CS, Bleijenberg G, Kimpen JL, Putte EM.The role of hypocortisolism in chronic fatigue syndrome. Psychoneuroendocrinology 2014;42:199-206.

56. McAllister-Williams, Ferrier IN, Young AH. Mood and neuropsychological function in depression: the role of corticosteroids and serotonin. Psychol Med 28(3):573-584.

57. Cleare AJ, Bearn J, Allain T, McGregor A, Wessely S, Murray RM, et al. Contrasting neuroendocrine responses in depression and chronic fatigue syndrome. J Affect Dis 1995;34(4):283-289.

58. Yamamoto S, Ouchi Y, Onoe H, Yoshikawa E, Tsukada H, Takahashi H, et al. Reduction of serotonin transporters of patients with chronic fatigue syndrome. Neuroreport 2004;15(17):2571-2574.

59. Cleare AJ, Messa C, Rabiner EA, Grasby PM. Brain 5-HT1a receptor binding in chronic fatigue syndrome measured using positron emission tomography and [11C]WAY-100635. Biol Psych 2005;57(3):239-246.

60. Smith AK, Dimulescu I, Falkenberg VR, Narasimhan S, Heim C, Vernon SD, et al. Genetic evaluation of the serotonergic system in chronic fatigue syndrome. Psychoneuroendocrinology 2008;33(2):188-197.

61. Wallymahmed ME, Foy P, MacFarlane IA. The quality of life of adults with growth hormone deficiency: comparison with diabetic patients and control subjects. Clin Endocr 1999;51(3):333-338.

62. Berwaerts J, Moorkens G, Abs R. Secretion of growth hormone in patients with chronic fatigue syndrome. Growth Horm IGF Res 1998;8(Suppl B):127-129.

63. Allain TJ, Bearn JA, Coskeran P, Jones J, Checkley A, Butler J, et al. Changes in growth hormone, insulin, insulin-like growth factors (IGFs), and IGF-binding protein-1 in chronic fatigue syndrome. Biol Psychiatry 1997;41(5):567-573.

64. Moorkens G, Berwaerts J, Wynants H, Abs R. Characterization of pituitary function with emphasis on GH secretion in the chronic fatigue syndrome. Clin Endocrinol 2000;53(1):99-106.

65. Di Giorgio A, Hudson M, Jerjes W, Cleare AJ. 24-hour pituitary and adrenal hormone profiles in chronic fatigue syndrome. Psychosom Med 2005;67(3):433-440.

66. Piche T, Vanbiervliet G, Cherikh F, Antoun Z, Huet PM, Gelsi E, et al. Effect of ondansetron, a 5-HT3 receptor antagonist, on fatigue in chronic hepatitis C: a randomised, double blind, placebo controlled study. Gut 2005;54(8):1169-1173.

67. Späth M, Current experience with 5-HT3 receptor antagonists in fibromyalgia. Rheum Dis Clin North Am 2002;28(2):319-328.



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68. De Becker P, Nijs J, McGregor N, De Meirleir K. A double-blind, placebo-controlled study of Acclydine in combination with amino acids in patients with Chronic Fatigue Syndrome. Clinical and Scientific Meeting Myalgic Encephalopathy/Chronic Fatigue Syndrome, “The Medical Practitioners’ Challenge. Sydney, Australia, Abstractbook 2001;45-46.

69. Doorduin T. Tekort aan groeihormoon speelt rol bij ME/ CVS. Medium Wetenschap 2002-1;15-19.70. C. claimde succes met aardappelkuur. De Limburger; oktober 1, 2003.71. De doofpot van de universiteit. De Limburger; september 25, 2005.





Gerard K.H. The, Judith B. Prins, Gijs Bleijenberg, Jos W.M. van der Meer.Netherlands Journal Medicine 2003; 61(9): 285-289

The effect of Granisetron, a 5-HT3 receptor antagonist,

in the treatment of chronic fatigue syndrome patients:

a pilot study

2



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Abstract

Objective: To explore the effect of granisetron, a 5-HT3 antagonist, on fatigue and functional impairment in patients with chronic fatigue syndrome (CFS).

Methods: Five female patients were eligible to receive oral granisetron for one month (1 mg a day for the first two weeks and 2 mg a day for the second two weeks). The patients had to be between 18 and 65 years of age and suffering from CFS according to the CDC criteria. The effect was assessed by pre- and post-testing, using validated instruments designed to assess the different dimensions of CFS. Treatment response was also evaluated by visual analogue scales (VAS) for fatigue. Analysis was based on intention to treat.

Results: Treatment with granisetron resulted in significant improvement in fatigue severity and functional impairment. Activity level showed no significant increase.

Conclusion: The promising results of this study have encouraged us to perform a placebo- controlled, double-blind study to evaluate the efficacy of 5-HT3 receptor antagonists in the treatment of CFS.



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THE EFFECT OF GRANISETRON

Introduction

Chronic fatigue syndrome (CFS) is a medically unexplained syndrome, characterised by severe disabling fatigue for a period of at least six months, which has led to considerable impairment in daily functioning.1 Various accompanying symptoms may be present, such as headache, joint and muscle pain, sore throat, and impaired memory and concentration. Of the many therapeutic interventions that have been undertaken so far, only cognitive behaviour therapy (CBT) and graded exercise therapy (GET) have met with succes.2,3

There is accumulating data in the literature supporting an important role for serotonin (5-hydroxytryptamine) in the neurobiology of CFS. Neuropharmacological studies point to an upregulated serotonin system.4-7

In a randomised controlled trial by our own research group, the selective serotonin reuptake inhibitor (SSRI) fluoxetine proved to be ineffective in CDC-diagnosed CFS subjects for the treatment of fatigue and depression,8 which is also in line with upregulation of the serotonin system. Positive reports of the use of serotonin inhibitors in the treatment of patients with fatigue (due to chronic hepatitis9 and to fibromyalgia10,11) support an effect. Based on these findings, we hypothesise that a serotonin antagonist could be effective in CFS. Therefore, we undertook this pilot study.

Materials and methods

PatientsFive female CDC-diagnosed patients with a high fatigue level and a substantial impairment in daily life, reflected by the Checklist Individual Strength (CIS)12,13 and the Sickness Impact Profile-8 (SIP-8),14,15 were treated with granisetron. The cut-off point of the CIS fatigue severity subscale was set at 40 and the weighted total score of the SIP-8 was set at 800.

In a CBT multicentre, randomised controlled trial by our research group, the CBT treatment protocol did not seem to be suitable for a group of CFS patients with low activity patterns.2 Therefore, we selected patients with a low activity pattern. Patients whose average daily physical activity scores stayed below the reference score in at least nine of the twelve assessment days could be included.16 The activity level was assessed prior to the treatment period with an actometer. We chose only female subjects, because in CFS the ratio male/female is



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CHAPTER 2

approximately 1:417 and combined with low activity as a disease characteristic, we created a homogeneous group.

Additional criteria were patients aged between 18 and 65 years, and no previous or current engagement in CFS research. Pregnant or lactating women and patients who were taking psychotropic medications were excluded. We received ethical clearance to perform a pilot study and obtained written informed consent from all patients.

Design and proceduresThere were four evaluation moments (E1-E4): E1 at baseline, E2+E3, in the middle and at the end of the treatment period and E4 at follow-up, two weeks after the treatment period. The treatment period was divided in two periods of two weeks. During the first period, the patients received an oral dose of granisetron of 1 mg a day. After two weeks the effect, compliance and side effects were evaluated. If the evaluation showed no significant improvement, the dose was increased to 2 mg a day.

Analysis was based on intention to treat. A linear model for repeated measures was used to analyse the effect of granisetron on the outcome measures CIS fatigue severity, CIS activity and SIP-8. The four evaluation moments were analysed as well as the three evaluation moments during the medication period. The visual analogue scales (VAS) actual fatigue scores were analysed by the Wilcoxon signed-rank test.

Assessments

Fatigue severityThe Checklist Individual Strength (CIS) is a reliable and validated self-report questionnaire. We used the subscale fatigue severity of the CIS (CIS fatigue severity).12,13 The score on this eight-item scale ranges from 8 (no fatigue) to 56 (maximally fatigued).

CIS fatigue severity analysis during the medication period (E1-E3) calculated a significant decline in time (p=0.046). The significant drop during the medication period (E1-E3) means that patients reported significantly lower fatigue levels after treatment with granisetron. Analysis over four measurements is significant in time as well (p=0.026). Follow-up (E4) showed an increase in fatigue severity after discontinuation of granisetron (figure 1).



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Visual analogue scales (VAS) (100 mm) are used to determine the actual fatigue severity. VAS ratings were used to evaluate the one-month treatment period. VAS assessments took place during the medication period (E1, E2 and E3).

The VAS actual fatigue showed a significant drop of 29% in the mean fatigue scores (p=0.042) during the treatment period (table 1).

Figure 1 Effect of granisetron in five patients on the outcome variable fatigue severity (CIS).

CIS

fat

igu

e sc

ore

s

Evaluation

0 1 2 3 4

60

30

40

50

Patient I

Patient IIIPatient II

Patient IVPatient VMean score

Table 1 Effect of granisetron in five patients on the outcome variable VAS actual fatigue severity (p=0.042)

Treatment VAS mean SD SEM Range mm

E1 – actual 76.4 13.7 6.1 59-95

E2 – actual 65.2 21.4 9.6 34-93

E3 – actual 54.4 28.1 12.6 22-92



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Functional impairmentThe sickness impact profile (SIP-8) measures the influence of symptoms on daily functioning, using the following eight subscales to rate both physical and psychological disability: home management, mobility, alertness behaviour, sleep/rest, ambulation, social interactions, work and recreation, and pastimes.14,15

SIP-8 analysis during the medication period (E1-E3) showed a significant decline in time (p=0.008). Patients reported significantly less functional impairment during the one-month medication period. Analysis over four measurements is significant in time as well (p=0.005). Follow-up (E4) showed an increase in functional impairment after discontinuation of granisetron. Within two weeks the mean SIP-8 score returned to the baseline level (figure 2).

Figure 2 Effect of granisetron in five patients on the outcome variable functional impairment (SIP-8).

Evaluation

0 1 2 3 4

Patient I

Patient IIIPatient II

Patient IVPatient VMean score

SIP

-8 s

core

s

2000

0

750

1500

250

1000

500

1250

1750



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Activity levelThe actometer is a small motion-sensing device that can register and quantify human physical activity.18 Three different activity patterns can be distinguished for patients with CFS: pervasively passive, fluctuating active and pervasively active. In an earlier study identifying physical activity patterns in CFS patients, we observed that healthy controls do not fall in the category of pervasively passive. Patients wore the actometer day and night for a two-week period. After the two-week period the average scores over 12 days were computed.16

Before treatment the mean actometer score was 44.6 (SD 22.2). During the last two weeks of the treatment period the mean actometer score was 46.2 (SD 20.3). Granisetron did not significantly change the mean actometer score (p=0.16).

The subscale activity of the CIS was used (CIS activity). The score on this three-item scale ranges from 3 (no activity) to 21 (maximally activity level).12,13

Analysis of the CIS subscale activity showed no significant improvement during the medication period (p=0.16). Analysis over four measurements is not significant in time either (p=0.191).

Results

The five women had a mean age of 34 years (range 23-44 years). Three of the five patients had a pervasively passive actometer pattern. Two patients had activity level scores lower than the mean CFS score for 9 out of the 12 days. All patients finished their study.

In the first two weeks the oral dose of 1 mg granisetron was well tolerated, but none of the patients showed significant improvement. In the second period all patients received 2 mg granisetron a day.

Four out of five patients reported marked improvement. One patient did not report any improvement on the outcome variables fatigue severity, activity level and functional impairment. Another patient complained of constipation as a side effect of granisetron during the last few days of the treatment.



34

CHAPTER 2

Discussion

In this pilot study we evaluated the effect of granisetron, a serotonin receptor antagonist, in chronic fatigue syndrome patients with low activity patterns. We found a substantial decrease in fatigue and functional impairment in four out of five patients as assessed by CIS fatigue severity, SIP-8 and visual analogue scale. That these changes in scores are clinically relevant can be deduced from our observations that these patients and their partners reported a remarkable improvement in fatigue and functional impairment at the end of the treatment period.

That granisetron, a serotonin antagonist, could have a favourable effect in CFS is not totally unexpected. First of all, there are reports in the literature pointing to a postsynaptic hyper-responsiveness in CFS.4,5 Also the challenge test with buspirone, a 5-HT agonist and D-fenfluramine, a serotonin reuptake inhibitor, met with exaggerated prolactine responses in CFS patients, consistent with a postsynaptic serotonergic hyper-responsiveness in CFS.6,7

Second, there are reports in the literature pointing to a favourable effect of serotonin antagonist in fatigued patients. Jones reported a positive effect on fatigue in a 35-year-old woman with profound fatigue associated with chronic hepatitis-C when treated with a 5-HT3 receptor antagonist.9 Positive results in fibromyalgia studies10,11 also support a favourable effect of serotonin antagonists in the treatment of fatigue.

A remarkable finding is that within two weeks after dis-continuation of granisetron, follow-up showed a marked increased mean fatigue level score and an increased functional impairment score. Within a few days granisetron is eliminated from the system (T1/2 elimination is 9-12 hours). The increase in symptoms within a short period after discontinuation of the medication supports the hypothesis that the intervention with granisetron is responsible for the reported improvement.

In this open study, a placebo effect cannot be excluded. However, in previous placebo-controlled studies8,19 we have not encountered remarkable changes in fatigue severity and functional impairment in the placebo group. The actometer activity level showed no significant improve-ment. A possible explanation is that a reduced level of fatigue will not immediately lead to an increased physical walking activity level, which is measured by the actometer. It is possible that CFS patients have quite structured daily routines. A reduced level of fatigue will probably not immediately lead to a change in the daily routines. We treated patients with low



35

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activity patterns. In addition there may be deconditioning of the patients, which is not reversible in the short term of one month. It might take more than four weeks to change the rather static activity patterns, despite a decreased level of fatigue. Perhaps a longer treatment protocol and a longer registration period could lead to (significantly) improved actometer activity levels. However, we do not know whether wearing off effects occur with prolonged treatment.

It is striking that the outcome measures showed an improvement of CFS-related symptoms after two weeks (figures 1 and 2). It is not clear whether the reported improvement after two weeks can be explained by a dose-dependent effect. A possible explanation is that time is a key factor in the reported improvement. It is possible that granisetron induced a postsynaptic receptor modification in time or decreased the sensitivity of the postsynaptic receptors.

Data in the literature point in the direction that CFS and depression are opposing disease entities with contrasting neuroendocrine responses and 5-HT functioning.20-22 The ‘serotonin hypothesis’ is the most widely accepted neural basis for depression. Drugs that preferentially increase serotonin activity by decreasing its reuptake (selective serotonin reuptake inhibitor, or SSRI) are effective in the treatment of depression23-25 and not in the treatment of CFS, even when the patients are depressed.8

It is worth noting that there is a therapeutic delay of two weeks in the treatment of depression with a SSRI. It is conceivable that a similar delay occurs with 5-HT receptor antagonism as applied in the presented study.

It is remarkable that one patient did not respond. This 23-year-old woman with a CFS history of three years was no different from the other patients with regard to her history, CDC criteria, CIS fatigue severity score and SIP-8 score.

Whether such nonresponsiveness has a serotonergic neuroendocrine basis has to be investigated in longer studies combined with serotonergic challenge tests and with serotonin receptor-status imaging studies. It is known that granisetron concentrations in blood may vary between subjects. A lower granisetron concentration might have caused the nonresponse in this individual. In this pilot study we did not measure blood granisetron concentrations. An interesting question is also whether further dose escalation would enhance the effect.

Our favourable results in this pilot study warrant a study with a randomised placebo- controlled, double-blind design.



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At the present time we are conducting such a randomised clinical trial with a longer treatment protocol and longer registration periods. In the future an interesting treatment concept might be a combination of a 5-HT3 receptor antagonist with CBT or GET.



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References

1. Fukuda K, Straus SE, Hickie I, Sharpe MC, Dobbins JG, Komaroff A. The Chronic Fatigue Syndrome Study: a comprehensive approach to its definition and study. International Chronic Fatigue Syndrome Study Group. Ann Intern Med 1994;121:953-9.

2. Prins JB, Bleijenberg G, Bazelmans E, et al. Cognitive behaviour therapy for chronic fatigue syndrome: a multicentre randomised controlled trial. Lancet 2001;357(9259):841-7.

3. Whiting S, Bagnall A, Sowden A, Cornell J, Mulrow C, Ramírez G. Interventions for the Treatment and Management of Chronic Fatigue Syndrome A Systematic Review. JAMA 2001;286:1360-8.

4. Bakheit A, Behan P, Dinan T, et al. Possible up-regulation of hypothalamic 5-hydroxytryptamine receptors in patients with post-viral fatigue syndrome. J Affect Disorder 1996;41:71-6.

5. Cleare AJ, Bearn J, Allein T, et al. Contrasting neuroendocrine responses in depression and chronic fatigue syndrome. Psychoneuroendocrinology 1995;35:283-9.

6. Sharpe M, Hawton K, Clements A, Cowen PJ. Increased prolactin response to buspirone in chronic fatigue syndrome. J Affect Disord 1996;41:71-6.

7. Sharpe M, Hawton K, Clements A, Cowen P. Increased brain serotonin function in men with chronic fatigue syndrome. BMJ 1997;315:164-5.

8. Vercoulen JHHM, Swanink CMA, Zitman FG, et al. Randomised, placebo-controlled trial of Fluoxetine in chronic fatigue syndrome. Lancet 1996;347:858-61.

9. Jones A. Relief from profound fatigue associated with chronic liver disease by long-term ondansetron therapy. Lancet 1999;354:397.

10. Färber L, Strats W, Brückle W, et al. Efficacy and tolerability of tropisetron in primary fibromyalgia – a highly selective and competitive 5-HT3 receptor antagonist.. Scan J Rheumatol 2000;29(suppl 113):49-54.

11. Papadopoulos IA, Georgiou PE, Katsimbri PP, Drosos AA. Treatment of fibromyalgia with tropisetron, a 5-HT3 serotonin antagonist: a pilot study. Clin Rheumatol 2000;19(1):6-8.

12. Vercoulen JHMM, Swanink CMA, Fennis JFM, Galama JMD, Meer JWM van der, Bleijenberg G. Dimensional assessment of chronic fatigue syndrome. J Psychosom Res 1994;38:383-92.

13. Vercoulen JHMM, Alberts M, Bleijenberg G. De Checklist Individual Strength (CIS). Gedragsthera-pie 1999;32:131-6.

14. Bergner M, Bobbit RA, Carter WB, Gilson BS. The Sickness Impact Profile: development and final revision of a health status measure. Med Care 1981;19:787-805.

15. Jacobs HM, Luttik A, Touw-Otten FW, Melker RA de. De sickness impact profile: resultaten van een valideringsonderoek van de Nederlandse versie. Ned Tijdschr Geneeskd 1990;134:1950-4.

16. Werf S van der, Prins J, Vercoulen J, Meer JWM van der, Bleijenberg G. Identifying physical activity patterns in chronic fatigue syndrome in chronic fatigue syndrome using actigraphic assessment. J Psychosom Res 2000;49:373-9.

17. Bazelmans E, Vercoulen J, Galama J, Weel C van, Meer JWM van der, Bleijenberg G. Prevalence of chronic fatigue syndrome and primary fibromyalgia syndrome in the Netherlands. Ned Tijdschr Geneenskd 1997;141:1520-3.

18. Tyron W. Activity measurement in psychology and medicine. New York: Plenum Press, 1991:47-4. 19. Brouwers FM, Werf S van der, Bleijenberg G, Zee L van der, Meer JWM van der. The effect of a

polynutrient supplement on fatigue and physical activity of patients with chronic fatigue syndrome: a double-blind randomized controlled trial. QJM 2002;95(10):677-83.

20. Cleare AJ, Bearn J, Allain T, et al. Contrasting neuroendocrine responses in depression and chronic fatigue syndrome. J Affect Disord 1995;34(4):283-9.

21. Parker AJR, Wessely S, Cleare AJ. The neuroendocrinology of chronic fatigue syndrome and fibromyalgia. Psychol Med 2001;31:1331-45.

22. Scott LV, Dinan TG. The neuroendocrinology of chronic fatigue syndrome: focus on the hypo-thalamic-pituitary-adrenal axis. Funct Neurol 1999;14:3-11.



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23. Dursun SM, Blackburn JR, Kutcher SP. An exploratory approach to the serotonergic hypothesis of depression: bridging the synaptic gap. Med Hypotheses 2001;56(2):235-43.

24. Owens MJ, Nemeroff CB. Role of serotonin in the pathophysiology of depression: focus on the serotonin transporter. Clin Chem 1994;40:288-95.

25. Smith KA, Fairburn CG, Cowen PJ. Relapse of depression after rapid depletion of tryptophan. Lancet 1997;349:915-9.

The, et al. Granisetron and chronic fatigue syndrome patients.



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Gerard K.H. The, Gijs Bleijenberg, Jan K. Buitelaar, Jos W.M. van der Meer.Journal Clinical Psychiatry 2010; 71(5): 528-533

The effect of Ondansetron, a 5-HT3 receptor antagonist, in chronic fatigue syndrome:

a randomised controlled trial

3



42

CHAPTER 3

Abstract

Background:Accumulating data support the in-volvement of the serotonin (5-hydroxytrypt-amine [5-HT]) system in the pathophysiology of chronic fatigue syndrome. Neu-ropharmacologic studies point to a hyperactive 5-HT system, and open-label treatment studies with 5-HT3 receptor antagonists have shown promising results. In this randomized controlled clinical trial, the effect of ondansetron, a 5-HT3 receptor antagonist, was assessed on fatigue severity and functional impairment in adult patients with chronic fatigue syndrome.

Method:A randomized, placebo-controlled, double-blind clinical trial was conducted at Radboud University Nijmegen Medical Centre, The Netherlands. Sixty-seven adult patients who fulfilled the US Centers for Disease Control and Prevention (CDC) criteria for chronic fatigue syndrome and who were free from current psychiatric comorbidity participated in the clinical trial. Participants received either ondansetron 16 mg per day or placebo for 10 weeks. The primary outcome variables were fatigue severity (Checklist Individual Strength fatigue severity subscale [CIS-fatigue]) and functional impairment (Sickness Impact Profile-8 [SIP-8]). The effect of ondansetron was assessed by analysis of covariance. Data were analyzed on an intention-to-treat basis. All patients were recruited between June 2003 and March 2006.

Results: Thirty-three patients were allocated to the ondansetron condition, 34 to the placebo condition. The 2 groups were well matched in terms of age, sex, fatigue severity, functional impairment, and CDC symptoms. Analysis of covariance showed no significant differences between the ondansetron- and placebo-treat-ed groups during the 10-week treatment period in fatigue severity and functional impairment.

Conclusions: This clinical trial demonstrates no benefit of ondansetron compared to placeboin the treatment of chronic fatigue syndrome.



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3

THE EFFECT OF ONDANSETRON

Background

Chronic fatigue syndrome is a medically unexplained syndrome, characterized by severe disabling fatigue for a period of at least 6 months that has led to considerableimpairment in daily functioning.1 Various accompanying symptoms may be present, such as headache, joint and muscle pain, sore throat, and impaired memory and concentration. In The Netherlands, a country with 16 million inhabitants, there are probably between 30,000 and 40,000 patients with chronic fatigue syndrome.2 Of the many therapeutic interventions that have been undertaken, so far only cognitive behavioral therapy and graded exercise therapy are of proven effectiveness.3,4

Neuroendocrinologic investigations have tried to elucidate the pathophysiology of chronic fatigue syndrome.5 Data in the literature support an important role for serotonin (5-hydroxytryptamine [5-HT]) in the pathophysiology of chronic fatigue syndrome.6–8 Serotonin plays a diverse and important role in the regulation of sleep and wakefulness, mood regulation, impulse control, memory, and perception of pain and in behavior that involves a high cognitive demand.9 Increased activity or metabolism of the 5-HT system have been implicated in the pathophysiology of chronic fatigue syndrome.10–13 An increased 5-HT metabolism in fatigue is supported by a rise in plasma tryptophan, the precursor of 5-HT during and after sustained exercise in healthy persons.14–16

Positive results of the use of 5 -HT3 inhibitors in the treatment of patients with chronic fatigue due to chronic hepatitis17,18 or fibromyalgia19,20 are in line with a role of serotonin in fatigue and support further research into this type of pharmacologic interventions.

Results of an open-label pilot study performed by our research group showed positive results during 1-month treatment with granisetron, a 5-HT3 receptor antagonist.21 The results of the pilot study encouraged us to investigate the effect of ondansetron in a well-defined chronic fatigue syndrome population using validated outcome measures in a randomized, double-blind, placebo-controlled design.

Method

The study was approved by the medical ethical committee of the Radboud University Nijmegen Medical Centre. Written informed consent was obtained from all participants prior to enrollment.



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ParticipantsPatients were recruited through the outpatient clinic of the Department of General Internal Medicine of the Radboud University Nijmegen Medical Centre. Furthermore, patients with chronic fatigue syndrome who were referred by general practitioners to the Nijmegen Expert Centre Chronic Fatigue for treatment were also asked to participate in the clinical trial. Patients were eligible for participation if they met the following inclusion criteria: aged between 18 and 65 years, satisfying the 1994 US Centers for Disease Control and Prevention (CDC) consensus criteria for chronic fatigue syndrome,1 and scoring above clinical cut-off on the Checklist Individual Strength fatigue severity subscale and the Sickness Impact Profile-8 (see below).

At the end of 2003, the International Chronic Fatigue Syndrome Study Group presented recommendations for better application of the 1994 case definition of chronic fatigue syndrome.22 The 1994 CDC criteria for defining chronic fatigue syndrome have been superseded by the revised 2003 CDC criteria. In our clinical trial, we did not use the 2003 CDC criteria because the inclusion of the study started before the publication of the revised 2003 CDC criteria. The Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-I)23 was performed by the investigator with a clinical background, who was trained in the SCID-I interview, to exclude patients with current psychiatric co-morbidity. Pregnant or lactating women were excluded, as were patients with lactose intolerance and patients taking psychotropic drugs or experimental medications.

InterventionsOndansetron (8-mg tablets) and an identical placebo were delivered by the manufacturer GlaxoSmithKline, The Netherlands. There was no difference in taste, appearance, or packaging between the active supplements and the placebo tablets. During 10 weeks, the chronic fatigue syndrome patients took either ondansetron (two 8 -mg ondansetron tablets) or 2 placebo tablets at night. The dose of 16 mg per day was based on the results of the pilot study performed before this clinical trial.21 In the pilot study, we observed a positive treatment effect with an equipotent dose of 16 mg ondansetron per day at night, and the patients tolerated the medication relatively well Späth et al24 reported positive effects in patients with chronic fatigue syndrome treated with 16 mg per day, as well.

Design and ProceduresThe study was a prospective, randomized, double-blind, placebo-controlled trial. The study was fully designed by the investigators and executed independently of the manufacturer of the study drugs.



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3


The outcome measures were assessed before the start of the pharmacologic interventions and at the end of the 10-week treatment period, when the patients were still taking the medication. All participants, investigators, and laboratory technicians were blinded to the treatment condition.

RandomizationBefore the start of the clinical trial, the hospital pharmacy prepared 70 treatment packages. Randomization and allocation to the treatment or placebo group was based on a patient’s study number. The pharmacy held the randomization list that correlated the study number with the treatment group. To maintain balance over time, the concealed randomization was done in blocks of 10. Treatments were generated randomly within the blocks using a computer program (Excel, Microsoft, Redmond, Washington, http:// www.microsoft.com). After acceptance of a patient by the junior researcher (G.K.H.T.) and the clinical psychologist (G.B.), the eligible patient received the lowest study number available (1–70).

Primary Outcome MeasuresFatigue severity The Checklist Individual Strength is a reliable and validated self-report questionnaire. We used the Checklist Individual Strength fatigue severity subscale (CIS-fatigue).4,25,26 The score on this 8-item scale ranges from 8 (no fatigue) to 56 (maximally fatigued).

The cut-off point for severe fatigue was set at 35.27 Patients who had chronic fatigue syndrome with a fatigue severity score of 35 or higher were included.

Functional impairment The Sickness Impact Profile-8 (SIP-8) measures the influence of symptoms on daily functioning, using the following 8 subscales to rate both physical and psychological disability: home management, mobility, alertness behavior, sleep/rest, ambulation, social interactions, work, and recreation and pastimes.4 A total score was calculated by addition of the weights of items. This widely used measure has good reliability and validity.28 Patients with chronic fatigue syndrome with substantial functional impairments, ie, a score of 800 or higher, were included.Patients had to fulfill both the fatigue severity and functional impairment criteria to participate in the study.



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Secondary Outcome MeasuresActivity levelBesides self-reported outcome measures, we measured physical activity with an actometer (Medical Instruments Services, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands).29 An actometer is a small motion-sensing device that can register and quantify human physical activity. The actometer is attached to the ankle; it was worn continuously for 12 consecutive days and nights during the assessment periods. It consists of a piezo-electric sensor that is sensitive in 3 directions, and it detects movements of the leg (eg, during walking or climbing stairs). Accelerations of the sensor above a predefined threshold are considered as activity and are stored into an internal memory of the actometer. Each second, the counter of the actometer is read and reset by the micro controller, which adds the value to the integration counter. The integration counter is set at 5 minutes, providing every 5 minutes an activity score that is stored into the internal memory of the actometer. A general physical activity score that expressed the mean activity level over the 12 days in the mean number of accelerations per 5-minute interval was calculated.30,31

Daily fatigue level During the 2-week actometer period, patients rated their fatigue levels in a diary. They rated their levels of fatigue 4 times a day on a 0 (no fatigue) to 4 (maximally fatigued) scale. The 4 scores for each day were summed to produce the daily observed fatigue (DOF) score, which ranged from 0 to 16.30 The mean of 12 consecutive DOF scores was used.

Statistical MethodsFor all analyses, SPSS 14.0 (SPSS Inc, Chicago, Illinois, http://www.spss.com) was used. Power calculations before the start of the trial showed that 30 persons were needed in each group to detect a difference of at least 1 standard deviation (SD) on the CIS-fatigue with a power of 90% and a 2-tailed significance level of 5%. Anticipating a dropout rate of 10%, 66 persons needed to be recruited. Analyses were performed on an intention-to-treat basis. Missing values were replaced by way of mean imputation.32

The effect of ondansetron was assessed by analysis of covariance (ANCOVA) of the posttreatment scores after 10 weeks as the dependent variable, the baseline scores as covariate, and condition as fixed factor.33 Analyses were completed before the code was broken.



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Results

All patients were recruited between June 2003 and March 2006. Figure 1 illustrates participant flow through the trial.

Figure 1 Participant flow.

Abbreviation: CFS = chronic fatigue syndrome

Burdon of study n = 39Did not want medication n = 20Inconvenient timing n = 5Other CFS treatment n = 5(planned) pregnancy n = 4

Did not believe in CFS researchwith ondansetron n = 2

Reason unknown n = 4

Current psychiatric comorbidity Depression n = 5 Anxiety disorder n = 1 Obsessive compulsive disorder n = 1 Current treatment for CFS n = 3

Current treatment for pain n = 2Age n = 1

Assessed for eligibility (n = 159)

Refused participation n = 79

Not eligible n = 13

Assigned (n = 34)Completers (n = 34)

Assigned (n = 33)Completers (n = 30)

Randomization (n = 67)

PlaceboOndansetron



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In total, 159 patients were given information about the study protocol; 79 persons refused to participate, and the main reason given for refusal was the intensity of the study. Thirteen patients did not meet the inclusion criteria. Seventy-four patients were screened with the SCID-I Interview. Of those, 7 were excluded due to current psychiatric comorbidity. A total of 67 patients, without current psychiatric comorbidity, were allocated randomly to the ondansetron group or to the placebo group. The ondansetron (n = 33) and placebo (n = 34) groups did not differ with respect to age, gender, fatigue severity, impairment, or number of CDC symptoms (Table 1).

During the screening process, we obtained data from all the patients who did not want to participate. There was no significant difference in age, number of CDC symptoms, fatigue severity, or functional impairment between the patients with chronic fatigue syndrome participating in the clinical trial and those who chose not to participate (data not shown). In the placebo arm, no participants dropped out. Three patients in the ondansetron arm dropped out within the first 2 weeks of the trial. One patient agreed to participate in the posttesting assessments, and 2 patients in the ondansetron group had missing values for the posttreatment measurements. The main reason for discontinuing the trial was an increased general feeling of malaise. Ondansetron and placebo treatments were relatively well tolerated. Four patients (3 ondansetron and 1 placebo) had complaints of constipation, and a laxative syrup was prescribed. No other important side effects were reported in either group.

Table 1 Patient Characteristicsa

Ondansetron Placebo Significance

Characteristic (n = 33) (n = 34) t P

Age, y 35.8 (9.9) 34.7 (9.4) … .64

Sex, female 67 74 … .54b

CIS-fatigue 49.4 (6.3) 50.0 (4.7) 0.424 .67c

SIP-8 1,375 (470) 1,359 (593.4) 0.117 .907c

CDC symptoms 7.4 (1.4) 6.8 (2.1) 1.34 .183c

aValues are means (SD) except for sex, which is given as percentage female.bChi-square test. cIndependent sample t test.Abbreviations: CDC = US Centers for Disease Control and Prevention, CIS-fatigue = Checklist Individual Strength fatigue severity subscale, SIP-8 = Sickness Impact Profile-8.



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Primary OutcomesEvaluation of fatigue severity and functional impairment posttreatment scores showed no significant differences between the ondansetron and placebo groups.

The posttreatment scores of both groups remained in the clinical range of severe fatigue and substantial functional impairment. Our primary analysis was to impute the missing values of the 2 patients using mean imputation. Moreover, we performed a sensitivity analysis testing 2 imputation methods: first by imputing the last observation carried forward and second by imputing the maximum score (worst case scenario) of the primary outcome measures. Both imputating methods had no significant impact on the conclusions of the primary analysis.

Table 2 Scores on Outcome Measures and ANCOVA Results for Patients With Chronic Fatigue Syndrome Treated With Ondansetron or Placeboa

Ondansetron Placebo ANCOVA

(n = 33) (n = 34) (df = 1,64)

Outcome Measure Mean SD Mean SD F P

Primary outcome measures

CIS-fatigue 0.116 .73

Pretreatment 49.4 6.3 50.0 4.7

Posttreatment 44.0 11.1 45.4 11.5

SIP-8 1.077 .30

Pretreatment 1,375 470.0 1,359 593.4

Posttreatment 1,063 525.5 1,172 694.6

Secondary outcome measures

Actometer 0.773 .38

Pretreatment 54.1 16.5 58.4 16.6

Posttreatment 55.0 15.5 60.6 17.9

DOF 0.015 .90

Pretreatment 8.5 2.0 8.4 2.0

Posttreatment 7.9 2.4 7.8 2.8

Abbreviations: ANCOVA = analysis of covariance, CIS-fatigue = Checklist Individual Strength fatigue severity subscale, DOF = daily observed fatigue, SIP-8 = Sickness Impact Profile-8.



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Secondary OutcomesPosttreatment actometer activity scores and DOF scores of the ondansetron and placebo group did not differ significantly. F-statistics and mean scores of the primary and secondary outcomes are shown in Table 2.

Discussion

This randomized, double-blind, controlled clinical trial investigated the therapeutic potential of the serotonin receptor antagonist ondansetron. We did not find significant differences between the ondansetron- and placebo-treated groups during the 10-week treatment period for any dimension of chronic fatigue syndrome.

This result was rather unexpected given the promising results in an earlier open study in which we tested the effect of granisetron, a compound similar to ondansetron. Four of 5 patients showed a remarkable improvement that reversed after cessation of the drug.21 Of course, such an open study is prone to observer bias, and a placebo effect cannot be excluded. However, in earlier studies in chronic fatigue syndrome performed by us34,35 and others,36 placebo effects were minimal. Besides pharmacologic and neuroendocrine studies implicating an increased 5-HT neurotransmission, positron emission tomography (PET) supports the hypothesis of an increased serotonergic state in chronic fatigue syndrome. Cleare et al37 found a widespread reduction in the number or affinity of 5-HT1A receptors, and the results of this study may be compatible with the neuroendocrine studies. A prolonged increased 5-HT state might result in a chronic 5-HT downregulation and, consequently, reduced 5-HT binding potential.37

On the basis of all of these previous findings, the question arises why our trial met with negative results. We have scrutinized our study regarding potential confounding factors, but could not identify any. The groups were well matched, and only well- diagnosed patients with chronic fatigue syndrome fulfilling the CDC criteria were included. Furthermore, we excluded patients with current psychiatric comorbidity. In this clinical study, we investigated the effect of serotonin receptor antagonism with ondansetron. We included patients from the outpatient clinic of the department of general internal medicine and patients who were referred by the general practitioner to the Expert Center Chronic Fatigue for treatment. Patients with chronic fatigue syndrome who were taking serotonergic medication were excluded from participating in the study. We also chose to exclude patients



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with current psychiatric comorbidity. Methodological differences and definition difficulties in the literature have produced conflicting results concerning the prevalence of psychiatric disorders in chronic fatigue syndrome and the impact of psychiatric disorders on the prognosis of chronic fatigue syndrome.38 Furthermore, evidence suggests an important role for the neurotransmitter serotonin in psychiatric disorders, such as in depression39,40 and in the pathophysiology and treatment of anxiety and panic attacks.41,42 In this way, we reduced the chance the results will be biased by current Axis I psychiatric disorders.

To our knowledge, ondansetron has no significant role in the treatment of depression, anxiety disorders, or obsessive-compulsive disorders.43 Therefore, we do not believe that excluding patients with current psychiatric comorbidity has reduced the likelihood of achieving response to ondansetron.

In our open-label study, we used granisetron, at that time marketed by SmithKline Beecham. When we were designing the present study, SmithKline Beecham merged with GlaxoWellcome and sold granisetron to Roche. Although this is a fully investigator-driven study, we were not able to obtain granisetron and placebo for the randomized controlled trial. Thus, we had to redesign the study and use ondansetron. Granisetron and ondansetron are both selective 5-HT3 receptor antagonists. To our knowledge, no differences in efficacy and side effects are described between the different 5-HT3 receptor antagonists. Given the similarities between the 2 drugs, we feel it is highly unlikely that the switch to ondansetron explains our negative results.

We did not monitor patient compliance on a daily basis. During the trial, patients had an appointment by telephone or at the outpatient clinic every 2 weeks. During these appointments, we assessed whether patients experienced side effects and whether the trial medication was taken as direct-ed. Although we have not rigorously checked compliance, we do not believe that lack of adherence can explain the negative findings.

We did not assess the baseline serotonin status of the patients with chronic fatigue syndrome. As mentioned previously, results from neuroendocrine challenge studies have suggested increased central 5-HT function in patients with chronic fatigue syndrome. Several different 5-HT agonists have been used to assess 5-HT function in chronic fatigue syndrome. Studies with buspirone10 and d-fenfluramine11,12 showed an enhanced prolactin response in patients with chronic fatigue syndrome compared to healthy controls and depressed subjects. Others showed a normal 5-HT activity.44,45 Appropriateness



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of matching and selection of patients with chronic fatigue syndrome with hetero- geneous psychiatric history could partly contribute to the inconsistent findings in the 5-HT challenge studies.5 One possible explanation is heterogeneity of the central serotonin bio-synthetic status within the patients with chronic fatigue syndrome, measured as the ratio of serum tryptophan to the sum of its competing large neutral amino acids.46 In our study, we do not know if the patients had a high serotonin status. If the patients in the ondansetron treatment group had a normal serotonin status, one could hypothesize that receptor antagonism could not be effective in these patients. However, we did not see any differences in the pattern of response between the placebo and the ondansetron group. For example, in both treatment groups, an equal number of 8 patients showed an improvement of more than 10 points in the CIS-fatigue severity.

In our clinical trial, we could detect changes over time in both groups. We have assessed the effects with validated instruments designed to assess different dimensions of chronic fatigue syndrome as well as treatment effects. In our trials on cognitive behavior therapy,4,26 those instruments were robust and reliable to show improvement at the group level as well as at the level of the individual patient. The negative findings in this randomized controlled trial cannot be explained by a power problem. In our opinion, it is very unlikely that a larger trial would detect a clinically relevant effect. The lack of significant differences on self-report outcome measures and physical activity strengthens our overall findings.

In conclusion, this randomized, placebo-controlled trial did not demonstrate any benefit in chronic fatigue syndrome-related outcome measures. Thus, the findings of this clinical trial do not support the use of 5-HT3 antagonism in treating chronic fatigue syndrome–related symptoms.

Drug names: buspirone (BuSpar and others), granisetron (Sancuso, Kytril, and others), ondansetron (Zofran and others).

Author affiliations: Department of General Internal Medicine, Nijmegen Expert Centre Chronic Fatigue (Drs The, Bleijenberg, and van der Meer); and Department of Psychiatry, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Centre (Drs The and Buitelaar), The Netherlands.



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Potential conflicts of interest: Dr van der Meer received grants from Optipharma BV Susteren, The Netherlands, and GlaxoSmithKline, Utrecht, The Netherlands, for research on chronic fatigue syndrome. Drs The, Bleijenberg, and Buitelaar have no additional personal affiliations or financial relationships with any commercial interest to disclose relative to the article.

Funding/support: This clinical trial was supported by a grant of GlaxoSmithKline, The Netherlands.

Acknowledgments: We thank all participants and the research assistants Harmen Vissers, MS, from the Department of General Internal Medicine, Nijmegen Expert Centre Chronic Fatigue, and Jan Leytens, from the Department of Psychiatry at the Radboud University Nijmegen Medical Centre, for assisting in data collection. Both acknowledged individuals have no conflicts of interest.



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References

1. Fukuda K, Straus SE, Hickie I, et al; International Chronic Fatigue Syndrome Study Group. The chronic fatigue syndrome: a comprehensive approach to its definition and study. Ann Intern Med. 1994;121(12):953–959.

2. Health Council of the Netherlands. Chronic Fatigue Syndrome. The Hague, The Netherlands: Health Council of The Netherlands; 2005; publication no. 2005/02E.

3. Chambers D, Bagnall AM, Hempel S, et al. Interventions for the treatment, management and rehabilitation of patients with chronic fatigue syndrome/myalgic encephalomyelitis: an updated systematic review. J R Soc Med. 2006;99(10):506–520.

4. Prins JB, Bleijenberg G, Bazelmans E, et al. Cognitive behaviour therapy for chronic fatigue syndrome: a multicentre randomised controlled trial. Lancet. 2001;357(9259):841–847.

5. Cleare AJ. The neuroendocrinology of chronic fatigue syndrome. Endocr Rev. 2003;24(2):236–252.

6. Parker AJ, Wessely S, Cleare AJ. The neuroendocrinology of chronic fatigue syndrome and fibromyalgia. Psychol Med. 2001;31(8):1331–1345.

7. Chaudhuri A, Behan PO. Fatigue in neurological disorders. Lancet. 2004;363(9413):978–988. 8. Georgiades E, Behan WM, Kilduff LP, et al. Chronic fatigue syndrome: new evidence for a central

fatigue disorder. Clin Sci (Lond). 2003;105(2):213–218. 9. Buhot MC. Serotonin receptors in cognitive behaviors. Curr Opin Neurobiol. 1997;7(2):243–254.10. Bakheit AM, Behan PO, Dinan TG, et al. Possible upregulation of hypo-thalamic 5-hydroxytrypt-

amine receptors in patients with postviral fatigue syndrome. BMJ. 1992;304(6833):1010–1012. 11. Cleare AJ, Bearn J, Allain T, et al. Contrasting neuroendocrine responses in depression and

chronic fatigue syndrome. J Affect Disord. 1995;34(4):283–289. 12. Sharpe M, Hawton K, Clements A, et al. Increased brain serotonin function in men with chronic

fatigue syndrome. BMJ. 1997;315(7101):164–165. 13. Cho HJ, Skowera A, Cleare AJ, et al. Chronic fatigue syndrome: an update focusing on

phenomenology and pathophysiology. Curr Opin Psychiatry. 2006;19(1):67–73.14. Davis JM, Bailey SP, Woods JA, et al. Effects of carbohydrate feedings on plasma free tryptophan

and branched-chain amino acids during prolonged cycling. Eur J Appl Physiol Occup Physiol. 1992;65(6):513–519.

15. Newsholme EA, Blomstrand E, Ekblom B. Physical and mental fatigue: metabolic mechanisms and importance of plasma amino acids. Br Med Bull. 1992;48(3):477–495.

16. Blomstrand E. Amino acids and central fatigue. Amino Acids. 2001; 20(1):25–34. 17. Jones EA. Relief from profound fatigue associated with chronic liver disease by long-term

ondansetron therapy. Lancet. 1999;354(9176):397. 18. Piche T, Vanbiervliet G, Cherikh F, et al. Effect of ondansetron, a 5-HT3 receptor antagonist, on

fatigue in chronic hepatitis C: a randomised, double blind, placebo controlled study. Gut. 2005;54(8):1169–1173.

19. Papadopoulos IA, Georgiou PE, Katsimbri PP, et al. Treatment of fibromyalgia with tropisetron, a 5HT3 serotonin antagonist: a pilot study. Clin Rheumatol. 2000;19(1):6–8.

20. Späth M. Current experience with 5-HT3 receptor antagonists in fibromyalgia. Rheum Dis Clin North Am. 2002;28(2):319–328.

21. The GKH, Prins JB, Bleijenberg G, et al. The effect of granisetron, a 5-HT3 receptor antagonist, in the treatment of chronic fatigue syndrome patients—a pilot study. Neth J Med. 2003;61(9):285–289.

22. Reeves WC, Lloyd A, Vernon SD, et al. International Chronic Fatigue Syndrome Study Group. Identification of ambiguities in the 1994 chronic fatigue syndrome research case definition and recommendations for resolution. BMC Health Serv Res. 2003;3(1):25.

23. First MB, Gibbon M, Spitzer RL, et al. User’s Guide for the Structured Clinical Interview for the DSM-IV Axis I Disorders – Research Version. New York, NY: Biometrics Research Department, New York State Psychiatric Institute; 1996.



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24. Späth M, Welzel D, Färber L. Treatment of chronic fatigue syndrome with 5-HT3 receptor antagonist: preliminary results. Scand J Rheumatol. 2000; (suppl 113):72–77.

25. Vercoulen JH, Swanink CM, Fennis JF, et al. Dimensional assessment of chronic fatigue syndrome. J Psychosom Res. 1994;38(5):383–392.

26. Stulemeijer M, de Jong LW, Fiselier TJ, et al. Cognitive behaviour therapy for adolescents with chronic fatigue syndrome: randomised controlled trial. BMJ. 2005;330(7481):14.

27. Servaes P, Verhagen S, Bleijenberg G. Determinants of chronic fatigue in disease-free breast cancer patients: a cross-sectional study. Ann Oncol. 2002;13(4):589–598.

28. Bergner M, Bobbitt RA, Carter WB, et al. The Sickness Impact Profile: development and final revision of a health status measure. Med Care. 1981;19(8):787–805.

29. Tyron WW. Activity Measurement in Psychology and Medicine. New York, NY: Plenum Press; 1991:47–49.

30. van der Werf SP, Prins JB, Vercoulen JH, et al. Identifying physical activity patterns in chronic fatigue syndrome using actigraphic assessment. J Psychosom Res. 2000;49(5):373–379.

31. Davis JM, Bailey SP, Woods JA, et al. Effects of carbohydrate feedings on plasma free tryptophan and branched-chain amino acids during pro-longed cycling. Eur J Appl Physiol Occup Physiol. 1992;65(6):513–519.

32. Newsholme EA, Blomstrand E, Ekblom B. Physical and mental fatigue: metabolic mechanisms and importance of plasma amino acids. Br Med Bull. 1992;48(3):477–495.

33. Blomstrand E. Amino acids and central fatigue. Amino Acids. 2001;20(1):25–34. 34. Jones EA. Relief from profound fatigue associated with chronic liver disease by long-term

ondansetron therapy. Lancet. 1999;354(9176):397. 35. Piche T, Vanbiervliet G, Cherikh F, et al. Effect of ondansetron, a 5-HT3 receptor antagonist, on

fatigue in chronic hepatitis C: a randomised, double blind, placebo controlled study. Gut. 2005;54(8):1169–1173.

36. Papadopoulos IA, Georgiou PE, Katsimbri PP, et al. Treatment of fibromyalgia with tropisetron, a 5HT3 serotonin antagonist: a pilot study. Clin Rheumatol. 2000;19(1):6–8.

37. Späth M. Current experience with 5-HT3 receptor antagonists in fibromyalgia. Rheum Dis Clin North Am. 2002;28(2):319–328.

38. The GKH, Prins JB, Bleijenberg G, et al. The effect of granisetron, a 5-HT3 receptor antagonist, in the treatment of chronic fatigue syndrome patients - a pilot study. Neth J Med. 2003;61(9):285–289.

39. Reeves WC, Lloyd A, Vernon SD, et al. International Chronic Fatigue Syndrome Study Group. Identification of ambiguities in the 1994 chronic fatigue syndrome research case definition and recommendations for resolution. BMC Health Serv Res. 2003;3(1):25.

41. First MB, Gibbon M, Spitzer RL, et al. User’s Guide for the Structured Clinical Interview for the DSM-IV Axis I Disorders–Research Version. New York, NY: Biometrics Research Department, New York State Psychiatric Institute; 1996.

41. Späth M, Welzel D, Färber L. Treatment of chronic fatigue syndrome with 5-HT3 receptor antagonist: preliminary results. Scand J Rheumatol. 2000; (suppl 113):72–77.

42. Vercoulen JH, Swanink CM, Fennis JF, et al. Dimensional assessment of chronic fatigue syndrome. J Psychosom Res. 1994;38(5):383–392.

43. Stulemeijer M, de Jong LW, Fiselier TJ, et al. Cognitive behaviour therapy for adolescents with chronic fatigue syndrome: randomised controlled trial. BMJ. 2005;330(7481):14.

44. Servaes P, Verhagen S, Bleijenberg G. Determinants of chronic fatigue in disease-free breast cancer patients: a cross-sectional study. Ann Oncol. 2002;13(4):589–598.

45. Bergner M, Bobbitt RA, Carter WB, et al. The Sickness Impact Profile: development and final revision of a health status measure. Med Care. 1981;19(8):787–805.

46. Tyron WW. Activity Measurement in Psychology and Medicine. New York, NY: Plenum Press; 1991:47–49.

47. van der Werf SP, Prins JB, Vercoulen JH, et al. Identifying physical activity patterns in chronic fatigue syndrome using actigraphic assessment. J Psychosom Res. 2000;49(5):373–379.





Gerard K.H. The, Robbert J. Verkes, Durk Fekkes, Gijs Bleijenberg,Jos W.M. van der Meer, Jan K. Buitelaar. BMC Research Notes 2014; 7: 650

Tryptophan depletion in chronic fatigue syndrome:

a pilot cross-over study

4



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Abstract

BackgroundChronic fatigue syndrome (CFS) is still an enigmatic disorder. CFS can be regarded as a complex disorder with tremendous impact on lives of CFS-patients. Full recovery without treatment is rare. A somatic explanation for the fatigue is lacking.

There is clinical and experimental evidence implicating enhanced serotonergic neurotransmission in CFS. Genetic studies and imaging studies support the hypothesis of upregulated serotonin system in CFS.In line with the hypothesis of an increased serotonergic state in CFS, we performed a randomised clinical trial investigated the effect of 5-HT3 receptor antagonism in CFS. No benefit was found of the 5-HT3 receptor antagonist ondansetron compared to placebo.

To further investigate the involvement of serotonin in CFS we performed a placebo controlled cross over pilot study investigating the effect of Acute Tryptophan Depletion.

Findings: Five female CFS-patients who met the US Center for Disease Control and Prevention criteria for CFS were recruited. There were two test days, one week apart. Each participant received placebo and ATD. To evaluate the efficacy of the ATD procedure tryptophan and the large neutral amino acids were measured. The outcome measures were fatigue severity, concentration and mood states.

ATD resulted in a significant plasma tryptophan to large neutral amino acid ratio reduction of 96%. There were no significant differences in fatigue-, depression and concentration between the placebo- and ATD condition.

Conclusions: These first five CFS-patients did not respond to the ATD procedure. However, a much larger sample size is needed to draw final conclusions on the hypothesis of an increased serotonergic state in the pathophysiology of CFS.

Trial registration: ISRCTN07518149

Keywords: Serotonin, Tryptophan, Depletion, Fatigue



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TRYPTOPHAN DEPLETION

Introduction

Chronic fatigue syndrome (CFS) is characterized by severe disabling fatigue of at least six months duration that has led to considerable impairment in daily functioning. A somatic explanation for the fatigue is lacking [1]. In the pathophysiology of CFS, several lines of evidence support the hypothesis of an underlying dysfunction of the serotonergic (5-hydroxytryptamine, 5-HT) system. In 1987 Newsholme inaugurated a possible link between an increased serotonin metabolism and chronic fatigue. In healthy persons exercise results in an increased brain 5-HT metabolism in response to an increased delivery of plasma tryptophan, the precursor of serotonin [2].

In CFS patients, neuroendocrine challenge studies have suggested both an increased [3] and normal [4] 5-HT function. These discrepancies might be explained by differences in methodology, composition of the patient cohort and the use of different 5-HT agonists [5]. Two positron emission tomography studies have investigated the serotonin system in CFS [6,7]. Both studies suggest an increased serotonin activity in CFS. A genetic study, which evaluated the 5-HT system in CFS, reported the 5-HT receptor subtype HTR2a −1438 A polymorphism to be associated with CFS. This variation in HTR2a may result in enhanced activity of the 5-HT system and contribute to the pathophysiology of CFS [8].

In line with the hypothesis of an increased serotonergic state in CSF, we investigated the effect of a 5-HT3 receptor antagonist in a small open label study in 5 CFS-patients and found positive effects on fatigue severity and on functional impairment [9]. In a subsequent randomized clinical trial in 67 patients with CFS, no benefit was found of the 5-HT3 receptor antagonist ondansetron compared to placebo [10]. An earlier placebo-controlled double-blind study with the serotonin reuptake inhibitor fluoxetine showed no beneficial effect on any characteristic of CFS, including depressive symptoms [11].

To further investigate the involvement of 5-HT in CFS, we performed a placebo controlled cross over pilot study investigating the effect of Acute Tryptophan Depletion (ATD).

ATD is a reliable and reversible challenge test for serotonin and has been widely used over the last decade to investigate the role of serotonin in a variety of psychiatric disorders [12]. We postulate that ATD would have a positive effect in CFS-related symptoms such as fatigue severity, concentration and mood.



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Methods

The study was approved by the medical ethical committee of the Radboud University Nijmegen Medical Centre. Written informed consent was obtained from the participants.

Five female outpatients (age mean 24.0, SD 3.5 year, range 19 to 29 year) who met the US Center for Disease Control and Prevention criteria for CFS, were recruited through the outpatient clinic of the Department of General Internal Medicine of the Radboud University Nijmegen Medical Centre. A structured Clinical interview for DSM-IV (SCID-I) was performed by the researcher, to exclude patients with current psychiatric co-morbidity. Exclusion criteria were: vegetarians, nursing women, pregnant women, use of psychotropic medication in the last month, previous or current engagement in CFS or psychiatric research. Patients were tested in the follicular phase.

We used ATD mixtures and placebo mixtures as described by Sobczak et al. 2002 [13]. There were two test days, one week apart. On each test day, patients were assessed after an overnight fast. 24 hours before the test days patients had a tryptophan low diet [12]. Each participant received placebo and ATD. Baseline assessments (T0) took place at 8.30 a.m and five hours after ingestion of the mixtures the second assessment (T1) took place. The hospital pharmacy prepared the mixtures and the order of treatment was balanced over two the test days. The participants, investigators and laboratory technicians were blinded to the treatment condition.

To evaluate the efficacy of the ATD procedure, at T0 and T1, total plasma Tryptophan and LNAAs (phenylalanine, tyrosine, isoleucine, leucine and valine) were measured simultaneously after completion of the study with high- performance liquid chromatography as described elsewhere [14].

Outcome measuresReliable and validated instruments were used. Current fatigue severity was measured by the Checklist Individual Strength, subscale fatigue severity [15]. The score on this eight-item scale ranges from eight (no fatigue) to 56 (maximally fatigued). Current concentration was measured by the subscale CIS-concentration. The subscale consists of 5 items; range five (no concentration problems) to 35 (severe concentration problems).



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The Dutch validated, shortened version of the Profile of Mood States (POMS), was used to assess mood changes during ATD. The subscale depression was used and current mood was assessed by a 5-point Likert Scale. The subscale depression consists of 8 items and ranges from 8 (no depression symptoms) to 40 (high level of depression symptoms). The Dutch POMS is considered a reliable instrument (Cronbach’s alpha subscale depression 0.87). Strong correlations with other mood state and measures of depression have been found and the validity of the Dutch POMS is considered good [16,17]. Current fatigue, concentration and mood were assessed at baseline (T0) and 5 hours after ingestion of the mixture (T1). The outcome measures will be presented by means and standard deviation. A GLM repeated measures analysis will be performed using subject factors; time and ATD/ placebo.

Results

All patients drank the ATD- and placebo mixtures within 15 minutes. Besides a little discomfort during and just after drinking the mixtures, the patients could tolerate the mixtures quite well. ATD resulted in a significant plasma tryptophan to large neutral amino acid ratio reduction of 96% (data not shown). There were no significant differences in fatigue-, depression and concentration between the placebo- and ATD condition. Also, at the individual level, none of the patients presented with clear clinical improvements in the depleted condition (Table 1).

Table 1 Outcome measures, Mean (sd), p-value

Outcome Treatment T0 Mean (sd) T1 Mean (sd) p-value

CIS-Fatigue Placebo 36.4 (11.9) 42.2 (7.2) 0.97 (F = 0.002, df = 1)

ATD 40.0 (7.2) 46.0 (3.9)

CIS-Concentration Placebo 20.0 (10.6) 23.2 (9.9) 0.26 (F = 1,751, df = 1)

ATD 22.4 (11.4) 23.0 (11.8)

POMS-depression Placebo 8.6 (0.6) 9.6 (0.6) 0.37 (F = 1, df = 1)

ATD 9.6 (2.5) 9.4 (2.6)



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Discussion

Different lines of evidence support the hypothesis of an increased serotonergic state in CFS. In this pilot study we evaluated the effect of an overall serotonin depletion in a cross-over design in CFS-patients. Serotonin depletion was achieved by acute depletion of the serotonin precursor tryptophan.

There is some evidence that there is a threshold for tryptophan depletion that needs to be exceeded before behavioral effects occur [18]. Although ATD resulted in a significant (96%) reduction of the tryptophan/LNAA ratio, no improvements were found in CFS-related symptoms. Since ATD very probably resulted in a transient serotonin depletion for a few hours, one could hypothesize that a more prolonged 5-HT depletion would be necessary to achieve neurobiological 5-HT changes and changes in fatigue severity in a chronic condition like CFS. A prolonged overall increased serotonergic synaptic neurotransmission could lead to a chronic downregulation of 5-HT1a receptors and a reduced binding potential [7]. Only a short and transient serotonin depletion would probably not lead to structural changes at the level of serotonin receptors. Furthermore, the most consistent finding of ATD is a mood-lowering response in patients recovered from depression [20]. To our knowledge, studies have not yet been performed investigating ATD effects on chronic fatigue in CFS patients; it is possible that ATD has a smaller impact in these patients. On the other hand, ATD studies have reported fast and significant effects on mood patients recovered from depression, particularly in those taking serotonergic antidepressants [20] and in patients with social anxiety disorder [20,21]. Assessment took place 5 hours after ingestion of the mixtures. Plasma tryptophan reaches its minimum level between 5 to 7 hours after ingestion of the ATD mixture. It can be understood that there is a delay from the time plasma tryptophan reaches the point of maximal depletion to the time of maximum serotonergic depletion in the brain. In this study it is not exactly known when maximum effects occur. The study was performed in a homogeneous group of medication-free female CFS-patients. To avoid confounding effects of current psychiatric conditions that can affect brain serotonergic function we recruited CFS patients without current axis-1 psychiatric comorbidity. There is a group of subjects who are vulnerable to the effects of ATD. Patients who had previous episodes of depression or people with a family history of depression are vulnerable [20].



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In our study we did not screen in detail the patients history or the family history. However any undetected vulnerability for depression would have increased the chance for finding ATD would lead to lowering of mood. In fact, in the study we did not observe a mood lowering effect of ATD. A major limitation of this study is the small sample size. Although there was no change in any of the outcome measures and robust improvements at the individual level were not detected either, further research in larger sample sizes is needed to allow a more definite answer, regarding the effect of ATD in CFS.

In conclusion, our earlier treatment study with a selective serotonin blocking agent as well as the present study with alimentary induction of significant ATD showed no positive results in any of the outcome measures in the treatment of CFS. Administering of ATD in a much larger sample size is needed to draw final conclusions on the hypothesis of an increased serotonergic state in the patho-physiology of CFS.

Abbreviations5-HT, 5-hydroxytryptamine; ATD, Acute tryptophan depletion; CFS, Chronic fatigue syndrome; SCID-1, Structured clinical interview for DSM-IV.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsGT carried out the pilot study, participated in the design of the study and drafted the paper. RV participated in the design of the study and helped to draft the manuscript. DF performed high-performance liquid chromatography analysis and helped to draft the manuscript. GB participated in the design of the study and helped to draft the manuscript. JM participated in the design of the study and helped to draft the manuscript. JB participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.

AcknowledgementThe authors would like to thank Armand van Oosterwijck for assisting in data collection.



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References

1. Fukuda K, Straus SE, Hickie I, et al: The Chronic Fatigue Syndrome Study: A comprehensive approach to its definition and study. International Chronic Fatigue Syndrome Study Group. Ann Intern Med 1994, 121:953–959.

2. Newsholme EA, Acworth II, Bloomstrand E: Amino acids, brain neurotransmitters and a functional link between muscle and brain that is important in sustained exercise. In Advances in myochemistry. Edited by Benzi G. London: John Libbey Eurotext; 1987:127–133.

3. Sharpe M, Hawton K, Clements A, et al: Increased brain serotonin function in men with chronic fatigue syndrome. BMJ 1997, 315:164–165.

4. Yatham LN, Morehouse RL, Chisholm BT, et al: Neuroendocrine assessment of serotonin (5-HT) function in chronic fatigue syndrome. Can J Psychiat 1995,40:93–96.

5. Parker AJ, Wessely S, Cleare AJ: The neuroendocrinology of chronic fatigue syndrome and fibromyalgia. Psych Med 2001, 31:1331–1345.

6. Yamamoto S, Ouchi Y, Onoe H, et al: Reduction of serotonin transporters of patients with chronic fatigue syndrome. Neuroreport 2004, 15:2571–2574.

7. Cleare AJ, Messa C, Rabiner EA, et al: Brain 5-HT1a receptor binding in chronic fatigue syndrome measured using positron emission tomography and [11C]WAY-100635. Biol Psych 2005, 57:239–246.

8. Smith AK, Dimulescu I, Falkenberg VR, et al: Genetic evaluation of the serotonergic system in chronic fatigue syndrome. Psychoneuroendocrinology 2008, 33(2):188–197.

9. The GK, Prins JB, Bleijenberg G, et al: The effect of granisetron, a 5-HT3 receptor antagonist, in the treatment of chronic fatigue syndrome patients – a pilot study. Neth J Med 2003, 61:285–289.

10. The GK, Bleijenberg G, Buitelaar JK, et al: The effect of ondansetron, a 5-HT3 receptor antagonist, in chronic fatigue syndrome: a randomized controlled trial. J Clin Psych 2010, 71(5):528–533.

11. Vercoulen JH, Swanink CM, Zitman FG, et al: Randomised, double-blind, placebo-controlled study of fluoxetine in chronic fatigue syndrome. Lancet 1996, 347(9005):858–861.

12. Hood SD, Bell CJ, Nutt DJ: Acute tryptophan depletion. Part I: rationale and methodology. Aust N Z J Psych 2005, 39(7):558–564.

13. Sobczak S, Honig A, Nicolson NA, et al: Effects of acute tryptophan depletion on mood and cortisol release in first degree relatives of type I and type II bipolar patients and healthy matched controls. Neuropsychopharm 2002, 27(5):834–842.

14. Fekkes D, Van Dalen A, Edelman M, et al: Validation of the determination of amino acids in plasma by high-performance liquid chromatography using automated pre-column derivatization with o-phthaldialdehyde. J Chromatogr 1995, 669(2):177–186.

15. Vercoulen JH, Swanink CM, Fennis JF, et al: Dimensional assessment of chronic fatigue syndrome. J Psychosom Res 1994, 38:383–392.

16. Wald FD, Mellenbergh GJ: Instrumenteel onderzoek: de verkorte versie van de Nederlandse vertaling van de Profile of Mood States( POMS). Ned Tijdschr Psychologie 1990, 45:86–90.

17. Van Hooff ML, Geurts SA, Kompier MA, et al: “How fatigued do you currently feel?” Convergent and discrimimant validity of a single-item fatigue measure. J Occup Health 2007, 49:224–234.

18. Van der Does AJ: The mood-lowering effect of tryptophan depletion: possible explanation for discrepant findings. Arch Gen Psych 2001, 58:200–202.

19. Smith KA, Fairburn CG, Cowen PJ: Relapse of depression after depletion of tryptophan. Lancet 1997, 349(9056):915–919.

20. Bell CJ, Hood SD, Nutt DJ: Acute tryptophan depletion. Part II: clinical effects and implications. Aust N Z J Psych 2005, 39(7):565–574.

21. Argyropoulos SV, Hood SD, Adrover M, et al: Tryptophan depletion reverses the therapeutic effect of selective serotonin reuptake inhibitors in social anxiety disorder. Biol Psych 2004, 56(7):503–509.



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Gerard K.H. The, Gijs Bleijenberg, Jos W.M. van der Meer.PLOS Clinical Trials 2007; 2(5): e19

The effect of Acclydine in chronic fatigue syndrome: a randomized controlled trial

5





69

5

THE EFFECT OF ACCLYDINE

Introduction

Chronic fatigue syndrome (CFS) is a medically unexplained syndrome, characterized by severe disabling fatigue for a period of at least 6 months that has led to considerable impairment in daily functioning1. Various accompanying symptoms may be present, such as headache, joint and muscle pain, sore throat, and impaired memory and concentration. Of the many therapeutic interventions that have been undertaken, so far only cognitive behavioral therapy and graded exercise therapy have met with success2.

Neuroendocrinological investigations have tried to elucidate the pathophysiology of CFS3. As CFS patients and adult patients with a growth hormone (GH) deficiency report similar symptoms such as fatigue, myalgia, a diminished sense of well-being, and reduced physical capacity4,5 and treatment of GH-deficient adults with GH has measurable effects on physical function and perception of fatigue6, GH status is a focus of this research. Changes in the GH/ insulin-like growth factor (IGF) 1 axis have been reported in CFS and fatigue-related disorders such as fibromyalgia7. IGF1 studies in CFS patients have yielded conflicting results: low8,9, normal10,11, and increased12 basal IGF1 levels have been found. Sample size, selection of controls, appropriateness of matching, and selection of CFS patients with psychiatric co-morbidity could explain the conflicting results.

Despite the conflicting publications concerning IGF1 status in CFS patients, in recent years there have been reports in patient organization newsletters that a new food supplement called Acclydine could be an effective treatment for CFS. The active ingredient of Acclydine is an alkaloid from Solanum dulcamara. It has been claimed that Acclydine is effective because it increases IGF1 concentrations in CFS patients by stimulating GH releasing hormone and, consequently, GH secretion. GH is converted to IGF1 in the liver. IGF1 activates tyrosine kinase and integrin receptors and stimulates intra-cellular lipid and glycogen synthesis.

Levels of IGF binding proteins (IGFBPs), particularly IGFBP3, modulate the biological activity of IGF1, and the ratio of IGFBP3 to IGF1 reflects IGF1 biological activity13. There are unpublished observations suggesting that Accly-dine could increase the IGF1 plasma concentrations in healthy humans and in CFS patients. An 8-wk controlled trial with Acclydine in combination with amino acid supplementation in 90 CFS patients has been reported to have a positive effect14.



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However, there are, to our knowledge, no published peer-reviewed studies investigating the effect of Acclydine. Double-blind randomized controlled trials are needed, for several reasons. First, this food supplement is available on the Internet without prescription and is thus easily accessible. Second, although the effects of Acclydine treatment have not been evaluated thoroughly, there are ongoing studies in other vulnerable patient categories, such as in cancer patients with chronic fatigue after treatment, but these studies are not listed in the international controlled trial registry Current Controlled Trials (http://www.controlled- trials.com).

For the reasons given above, we assessed the IGFBP3/IGF1 status in patients fulfilling the US Centers for Disease Control and Prevention (CDC) criteria for CFS1 and in healthy gender- and age-matched neighborhood controls, to evaluate whether there are intrinsic differences in IGFBP3/IGF1 status between the two groups. Furthermore, we investigated the effect of Acclydine on IGF1 concentration, IGFBP3/IGF1 ratio, and CFS-related symptoms in a well-defined CFS population using validated outcome measures and a random-ized double-blind placebo- controlled design.

Methods

The study was approved by the medical ethical committee of the Radboud University Nijmegen Medical Centre. Written informed consent was obtained from all participants prior to enrollment.

ParticipantsPatients were recruited through the outpatient clinic of the Department of General Internal Medicine of the Radboud University Nijmegen Medical Centre and through an advertisement in the newsletter of the Dutch CFS patient organization ME-Stichting (http://www.me-cvs-stichting.nl). Patients were eligible for participation if they fulfilled the following inclusion criteria: they had to be between 18 and 65 y of age, and they had to fulfill the CDC consensus criteria for CFS1. A Structured Clinical Interview for DSM-IV (SCID-1) was performed to exclude patients with current psychiatric co-morbidity and to ensure a homogeneous group of patients.

Pregnant or lactating women were excluded, as were patients with lactose intolerance and patients taking psycho-tropic drugs or experimental medications.



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An additional criterion to be met was an IGFBP3/IGF1 ratio greater than 2.5. We asked patients who could potentially participate in the trial, if possible, to bring a healthy neighborhood control individual of similar age, gender, body weight, and body height, to compare IGF status. Except for contraceptives, the control individuals were not allowed to take medication.

InterventionsAcclydine and an identical placebo were delivered by the manufacturer Optipharma. Each Acclydine capsule contained 250 mg of the alkaloid. Patients took a single daily dose on an empty stomach, with the following decreasing Acclydine dosage schedule over a total of 14 wk: weeks 1–2, 1,000 mg per day; weeks 3–6, 750 mg per day; weeks 7–8, 500 mg per day; weeks 9–10, 500 mg every 2 d; weeks 11–12, 250 mg per day; and weeks 13–14, 250 mg every 2 d.

Acclydine treatment was combined with amino acid supplements to provide sufficient essential and nonessential amino acid intake during treatment. Patients in the placebo group received placebo Acclydine and placebo amino acid supplements.

There was no difference in taste, appearance, or packaging between the active supplements and the placebo capsules.

Design and ProceduresThe study was a prospective, randomized, double-blind, placebo-controlled trial. The effect of Acclydine was assessed by pre- and post-trial testing. Post-trial testing was performed at the end of the 14-wk treatment period. During the post-trial assessments, the patients were still taking the trial supplements. All participants, investigators, and laboratory technicians were blinded to the treatment condition. IGF1 and IGFBP3 measurements were also done in a blinded fashion.

RandomizationBefore the start of the clinical trial, the pharmacy of the hospital prepared 57 treatment packages. Randomization and allocation to the treatment or placebo group was based on a patient’s study number. After acceptance of a patient by the junior researcher (G. K. H. T.) and the clinical psychologist (G. B.), the eligible patient received the lowest study number available (1–57). The pharmacy held the randomization list that correlated the study number with the treatment group. To maintain balance over time, the concealed randomization was done in blocks of ten. Treatments were generated randomly within the blocks using a computer program (Excel, Microsoft, http://www.microsoft.com).



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Primary Outcome MeasuresFatigue severityThe Checklist Individual Strength is a reliable and validated self-report questionnaire. We used the Checklist Individual Strength, subscale fatigue severity (CIS-fatigue)15,16. The score on this eight-item scale ranges from eight (no fatigue) to 56 (maximally fatigued). The cut-off point was set at 3517.

Functional impairmentThe Sickness Impact Profile-8 (SIP-8) measures the influence of symptoms on daily functioning, using the following eight subscales to rate both physical and psychological disability: home management, mobility, alertness behavior, sleep/rest, ambulation, social interactions, work, and recreation and pastimes18,19. CFS patients with substantial functional impairments were included. Patients with scores above the cut-off point of 800 were included.

Secondary Outcome MeasuresActivity levelBesides self-reported outcome measures, we measured physical activity with an actometer. An actometer is a small motion-sensing device attached to the ankle; it was worn continuously for 14 d during the assessment periods. After the 2-wk period, the average score over 12 d was computed20.

Daily fatigue levelDuring the 2-wk actometer period, patients rated their fatigue level in a complaint diary. They rated their level of fatigue four times a day on a zero (no fatigue) to four (maximally fatigued) scale. The four scores for each day were summed to produce the daily observed fatigue (DOF) score, which ranged from 0 to 1621. The mean of 12 consecutive DOF scores was used.

Blood SamplesBlood samples of the patients and the matched neighborhood controls were taken at the same time, and the paired blinded samples were handled in an identical fashion. Serum IGF1 was measured by an immune radiometric assay (Diagnostic Systems Laboratories, http://www.dslabs.com). For IGF1, the inter- and intra-assay coefficients of variation were 6.25% and 4.93%, respectively. IGFBP3 was also assessed by an immune radiometric assay (Immulite, DPC, http://dpcweb. com). For IGFBP3, the inter- and intra-assay coefficients of variation were 4.25% and 1.3%, respectively.



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Statistical MethodsFor all analyses SPSS 12.01 (SPSS, http://www.spss.com) was used. Power calculations before the start of the trial showed that 22 persons were needed in each group to detect a difference of at least one standard deviation (SD) on the CIS-fatigue with a power of 90% and a two-tailed significance level of 5%. Anticipating a dropout rate of 10%, at least 49 persons needed to be recruited. Analyses were performed on an intention-to-treat basis. Missing values were replaced by way of the last observation carried forward. Independent sample t-tests were performed on the change scores, defined as the difference between baseline scores and the post-treatment scores after 14 wk. IGF data are given as mean ± SD. The hormonal measurements were analyzed by independent sample t-test.

Results

All patients were recruited between February 2003 and April 2005. In total, 22 patients were able to bring a neighborhood control individual (four men and 18 women). The results of the hormonal assessments are shown in Table 1. Hormonal values including the CFS patients of the outpatient clinic and patients who did not meet the hormonal inclusion criteria for participation in the clinical trial are reported. No significant differences were found in the IGF status of the CFS patients versus the controls.

Figure 1 illustrates participant flow through the trial. In total, 112 patients were given information about the study protocol; 31 persons refused to participate, and the main reason given for refusal was the intensity of the study. Data obtained from 26 of the 31 persons who chose not to participate showed no significant

Table 1 Hormonal Blood Levels

Hormone Patients (n = 22) Controls (n = 22) p-Value (95% CI)Mean (SD) Range (nm/l) Mean (SD) Range (nm/l)

IGF1 (nm/l) 37.0 (19.7) 18.6-96.1 36.9 (14.1) 18.6-76.1 0.98 (-10.3 to 10.6)

IGFBP3 (nm/l) 117.6 (60.1) 51.1-224.7 114.5 (54.7) 56.0-240.8 0.86 (-31.8 to 38.1)

Ratio IGFBP3/IGF1

3.7 (2.3) 0.89-9.24 3.5 (2.3) 1.37-9.84 0.82 (-1.2 to 1.5)



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difference in age, number of CDC symptoms, fatigue severity, or functional impairment between the CFS patients participating in the clinical trial and those who chose not to participate (data not shown). Twenty-four patients did not meet the inclusion criteria. A total of 57 patients were randomized in the clinical trial: 15 patients were recruited from the outpatient clinic, and 42 patients came in response to the advertisement in the patient organization newsletter. One patient in each arm dropped out after randomization, and as a consequence, in each arm, baseline data for one patient was carried forward, and a sensitivity analysis was not required.

Figure 1 CONSORT Flowchart.

Reasons:Burdon (n = 18)Starting other therapy (4)Research with placebo (3)Acclydine not feasible in CFS treatment (3)Unknown reason (3)

Reasons:IGFBP3/IGF1 ratio < 2.5 (8)Psychiatric co-morbidity (4)Not fulfilling fatique or impairment criteria (4)Not fulfilling the CDC criteria (4)No CFS diagnosis (3)Pregnancy (1)

Assessed for eligibility (n = 112)

Refusal n = 31

Not eligible n = 24

Placebo (n = 27)Completers (n = 26)

Supplement (n = 30)Completers (n = 29)

Randomization (n = 57)

AssignedAssigned



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The Acclydine and placebo treatments were well tolerated. No important side effects were reported in either group. The Acclydine and placebo groups did not differ with respect to age, gender, fatigue severity, impairment, or number of CDC symptoms (Table 2).

Primary OutcomesNo significant differences in change scores were found between the treatment and placebo groups on the primary outcome measures (Table 3). The CFS patients treated with Acclydine did not show a significant decrease in fatigue severity (CIS-fatigue +1.1 [95% CI -4.4 to +6.5, p=0.70]) or functional impairment (SIP-8 +59.1 [95% CI -201.7 to +319.8, p=0.65]) compared to the placebo group.

Secondary OutcomesActometer activity scores did not show significant differences between the two groups. Analysis of fatigue severity rated with the DOF did not show significant differences either.

No significant differences were found between the treat-ment and placebo groups in IGF1 blood level, IBFBP3 blood level, or IGFBP3/IGF1 ratio at baseline; after 4, 8, or 14 wk of treatment; or during follow-up (Figure 2).

Table 2 Patient Characteristics

Charateristic Accydine Group (n = 30) Placebo Group (n = 27)

Age, years 40.9 (9.4) 43.4 (11.2)

Female 77% 59%

CIS-fatigue 46.5 (7.4) 46.2 (7.9)

SIP-8 1,484 (520.4) 1,317 (481.7)

CDC symptoms 7.6 (1.4) 7.5 (1.3)



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Tab

le 3

Tre

atm

ent e

ffect

Ou

tco

mes

Eva

luat

ion

Gro

up

a

Bas

elin

eb

At

14 w

kbTr

eatm

ent

Eff

ect

(95%

CI)

p

-Val

ue

Prim

ary

CIS

-fat

igue

Acc

lyd

ine

4

6.5

(7.4

)

42

.4 (1

1.6)

1.1

(-4

.4 to

6.5

)0.

70

Pla

ceb

o

46.

2 (7

.9)

43.0

(12.

6)

SIP

-8A

ccly

din

e1,

484.

0 (5

20.4

) 1

,228

.1 (

619.

7)

59

.1 (

-201

.7 to

319

.8)

0.65

Pla

ceb

o1,

137.

0 (4

81.7

) 1

,120

.2 (

543.

0)

Sec

ond

ary

Act

omet

erA

ccly

din

e

60.

8 (2

0.5)

64.9

(23

.4)

4.1

(-5

.9 to

14.

0)0.

42

Pla

ceb

o

64.

8 (2

5.2)

64.9

(23

.5)

DO

FA

ccly

din

e

8

.6 (

2.3)

8.0

(2.

8)

-

0.2

(-1.2

to 0

.90.

76

Pla

ceb

o

7

.7 (

2.2)

7.0

(2.

6)

IGF1

Acc

lyd

ine

4

3.2

(32.

1)

37

.7 (

22.0

)

-

4.6

(-17.

7 to

8.6

)0.

49

Pla

ceb

o

37.

3 (1

8.0)

36.3

(18.

8)

IGFB

P3

Acc

lyd

ine

16

3.4

(34.

1)

160

.6 (

32.9

)

-1

.7 (

-17.

5 to

14.

0)0.

83

Pla

ceb

o

168.

9 (3

9.6)

1

64.3

(29

.5)

IGFB

P3/

IGF1

rat

ioA

ccly

din

e

5

.3 (

3.7)

5.7

(3.

2)

-

0.5

(-2.

8 to

1.7

)0.

63

Pla

ceb

o

5

.6 (

3.1)

5.4

(2.

1)

aA

ccly

din

e g

roup

, n =

30;

pla

ceb

o g

roup

, n =

27.

b Val

ues

are

mea

n (S

D).

A d

eclin

e in

CIS

-fat

igue

, S

IP-8

, DO

F sc

ore

s, IG

FB

P3

sco

res

or

the

IGF

BP

3/IG

F1 is

co

nsid

ered

a p

osi

tive

outc

om

e, i.

e., a

n im

pro

vem

ent.

A d

eclin

e in

act

om

eter

, IG

F1, i

s co

nsid

ered

a n

egat

ive

outc

om

e.Th

e tr

eatm

ent e

ffec

t is

cons

ider

ed p

osi

tive

whe

n th

e im

pro

vem

ent i

n th

e A

ccly

din

e g

roup

is la

rger

than

that

of t

he p

lace

bo

gro

up.



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5


Figure 2 Hormone Data of the Two Treatment Groups.

(A) IGF-1 concentration, mean (sd)

(B) IGF-BP3 concentration, mean (sd)

Accydine (n = 30)

Placebo (n = 27)

(C) Ratio IGF-BP3 / IGF-1, mean (sd)



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Discussion

InterpretationIn this randomized controlled blinded clinical trial, no therapeutic effect of Acclydine in CFS was found. In this study we also addressed the issue of a deficiency of bioactive IGF1. To control for unwanted stress effects and other confounders, the blood samples were taken simultaneously from the patients and their matched neighborhood controls. For each pair of patient and control, the measurements of IGF1 and IGFBP3 were performed in the same run. In addition, we detected no differences in IGF1 or IGFBP3 concentrations in CFS patients without psychiatric co-morbidity versus closely matched healthy neighborhood controls. Although the sample size is relatively small, the results do not point to a different IGFBP3/IGF1 status in CFS patients compared to controls. These results do not support the hypothesis that changes in IGF1 and IGFBP3 metabolism are involved in the pathophysiology of CFS.

GeneralizabilityIn this study we enrolled a representative sample of adult referred and non-referred CFS patients who fulfilled the international CDC consensus criteria for CFS1. In contrast to previous intervention studies with cognitive behavioral therapy22 or with a polynutrient supplement23 conducted by our research group, we excluded patients with psychiatric co-morbidity to exclude hormonal influences caused by current psychiatric co-morbidity. The mean fatigue severity and functional impairment scores in this study represent high fatigue severity levels and high functional impairment in the CFS patients. Compared to the previously mentioned intervention studies, the patients in this clinical trial reported slightly lower fatigue and functional impairment scores. Selection of CFS patients without current psychiatric co-morbidity could explain this difference.

Overall EvidenceTo our knowledge, this is the first published report of a randomized controlled trial evaluating the effect of the food supplement Acclydine in CFS patients. The effect was assessed by pre- and post-trial testing with validated instruments designed to assess different dimensions of CFS as well as treatment effects. Treatment with Acclydine was not more effective than placebo in changing self-reported outcome measures, hormonal blood levels, or physical activity.

The lack of significant differences on any of the dimensions of fatigue or the hormonal assessments strengthens our overall findings.



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Previous unpublished studies by others on file at the manufacturer claimed that treatment with Acclydine would be more effective in CFS patients with a higher IGFBP3/IGF1 ratio. Based on this claim, we did a subset analysis for patients with a ratio greater than 4.5:19 CFS patients in the Acclydine group and 20 in the placebo group had a ratio greater than 4.5. No significant differences in change scores were seen between the subgroups in any of the outcome measures.

A second claim was that Acclydine treatment works by increasing biologically active IGF1. However, assessment of IGF1 concentration and IGFBP3/IGF1 values in the Acclydine group at baseline, 4 wk, and 8 wk during treatment, at the end of treatment, and at follow-up (1 mo after the treatment period) did not show a significant change.

The power of our study was sufficient to detect changes in time in both groups. Thus, the negative findings found in this randomized controlled trial cannot be explained by a power problem. Considering the reported treatment effect on the primary outcome measures, we believe it is very unlikely that a larger trial would detect a clinically meaningful effect.

It is of interest to note that we detected a minor placebo effect in this study. This is in accordance with observations from earlier randomized controlled clinical trials in CFS23,24,25. However, the minimal decline in fatigue severity and functional impairment during pre- and post-trial testing in both treatment groups could also be partly explained by the phenomenon regression to the mean or a Hawthorne effect.

We did not monitor patient compliance on a daily basis. During the trial, patients had an appointment by telephone or at the outpatient clinic every 2 wk. During these appointments, we assessed whether the capsules were taken as directed, and the treatment protocol for the next 2 wk was discussed with the patient. Although adherence could not be verified fully, there is no reason to believe that lack of adherence can explain the negative findings.

In conclusion, this randomized controlled trial did not demonstrate an effect of Acclydine on biologically active IGF1, nor did it demonstrate any benefit in CFS- related outcome measures. Thus, the findings of this clinical trial do not support the use of Acclydine in the treatment for CFS.



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We feel that the negative results of this trial are important for two reasons: Acclydine is expensive, so patients might be spending a lot of money on an ineffective treatment, and it is also available without prescription on the Internet, making it available to patients potentially without a doctor’s oversight.

AcknowledgmentsHormonal measurements were performed by Centrum voor Medische Analyse, Herentals, Belgium. We thank all participants and the research assistants Ilja Klabbers-Helsper, Jetske Oostelbos, and Harmen Vissers for assisting in data collection, and Jan-Mathijs Schoffelen for helpful comments.

Author ContributionsGKHT, GB, and JWMvdM designed the study, enrolled patients, participated in the statistical analysis and contributed to the writing of the paper. GKHT drafted the paper. JWMvdM dealt with the manufacturer of the drug. The grant from Optipharma was used to perform the present study.



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References

1. Fukuda K, Straus SE, Hickie I, Sharpe MC, Dobbins JG, et al. The Chronic Fatigue Syndrome Study: A comprehensive approach to its definition and study. International Chronic Fatigue Syndrome Study Group. Ann Intern Med 2004;121:953–959.

2. Chambers D, Bagnall AM, Hempel S, Forbes C. Interventions for the treatment, management and rehabilitation of patients with chronic fatigue syndrome/myalgic encephalomyelitis: An updated systematic review. J R Soc Med 2006;99:506–520.

3. Cleare AJ. The neuroendocrinology of chronic fatigue syndrome. Endocr Rev 2003;24:236–2524. de Boer H, Blok GJ, Van der Veen EA. Clinical aspects of growth hormone deficiency in adults.

Endocr Rev 1995;16:63–86. 5. Wallymahmed ME, Foy P, MacFarlane IA. The quality of life of adults with growth hormone

deficiency: Comparison with diabetic patients and control subjects. Clin Endocrinol 1999;51:333–338.

6. Carroll PV, Christ ER, Bengtsson BA, Carlsson L, Christiansen JS, et al. Growth hormone deficiency in adulthood and the effects of growth hormone replacement: A review. Growth Hormone Research Society Scientific Committee. J Clin Endocrinol Metab 1998;83:382–395.

7. Landis CA, Lentz MJ, Rothermel J, Riffle SC, Chapman D, et al. Decreased nocturnal levels of prolactin and growth hormone in women with fibromyalgia. J Clin Endocrinol Metab 2001;86:1672–1678.

8. Berwaerts J, Moorkens G, Abs R. Secretion of growth hormone in patients with chronic fatigue syndrome. Growth Horm IGF Res 1998;8:127–129.

9. Allain TJ, Bearn JA, Coskeran P, Jones J, Checkley A, et al. Changes in growth hormone, insulin, insulin-like growth factors (IGFs), and IGF-binding protein-1 in chronic fatigue syndrome. Biol Psychiatry 1997;41:567– 573.

10. Cleare AJ, Sookdeo SS, Jones J, O’Keane V, Meill JP. Integrity of the GH/IGF system is maintained in patients with chronic fatigue syndrome. J Clin Endocrinol Metab 2000;85:1433–1439.

11. Buchwald D, Umalli J, Stene M. Insulin-like growth factor-1 (somatomedin C) levels in chronic fatigue syndrome and fibromyalgia. J Rheumatol 1996;23:739–742.

12. Bennett AL, Mayes DM, Fagioli LR, Guerriero R, Komaroff AL. Somatomedin C (insulin-like growth factor 1) levels in patients with chronic fatigue syndrome. J Psychiat Res 1997;31:91–96.

13. Kostecka Z, Blahovec J. Insulin-like growth factor binding proteins and their functions. Endocr Regul 1999;33: 90–94.

14. De Becker P, Nijs J, Van Hoof E, McGregor N, De Meirleir K (2001) A double-blind, placebo controlled study of Acclydine in combination with amino acids in patients with Chronic Fatigue Syndrome [abstract]. Alison Hunter Memorial Foundation Third International Clinical and Scienti-fic Meeting on ME/CFS; Sydney, Australia; 30 November to 3 December 2001.

15. Vercoulen JH, Swanink CM, Fennis JF, Galama JM, Van der Meer JW, et al. Dimensional assessment of chronic fatigue syndrome. J Psychosom Res 2004;38: 383–392.

16. Beurskens AJ, Bu¨ltmann U, Kant IJ, Vercoulen JH, Bleijenberg G, et al. Fatigue among working people: Validity of a questionnaire measure. Occup Environ Med 2000;57:353–357.

17. Servaes P, Verhagen S, Bleijenberg G. Determinants of chronic fatigue in disease-free breast cancer patients: A cross-sectional study. Ann Oncol 2002;13: 589–598.

18. Bergner M, Bobbitt RA, Carter WB, Gilson BS. The Sickness Impact Profile: Development and final revision of a health status measure. Med Care 1981;19:787–805.

19. Jacobs HM, Luttik A, Touw-Otten FW, De Melker RA. De sickness impact profile: Resultaten van een valideringsonderzoek van de Nederlandse versie. Ned Tijdschr Geneeskd 1990;134:1950–1954.

20. van der Werf S, Prins JB, Vercoulen JH, van der Meer JW, Bleijenberg G. Identifying physical activity patterns in chronic fatigue syndrome using actigraphic assessment. J Psychosom Res 2000;49:373–379.



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21. Vercoulen JH, Bazelmans E, Swanink CM, Fennis JF, Galama JM, et al. Physical activity in chronic fatigue syndrome: Assessment and its role in fatigue. J Psychiatr Res 1997;31:661–673.

22. Prins JB, Bleijenberg G, Bazelmans E, Elving LD, de Boo TM, et al. (2001) Cognitive behaviour therapy for chronic fatigue syndrome: A multicentre randomised controlled trial. Lancet 357: 841–847.

23. Brouwers FM, Van der Werf S, Bleijenberg G, Van der Zee L, Van der Meer JW. The effect of a polynutrient supplement on fatigue and physical activity of patients with chronic fatigue syndrome: A double-blind randomized controlled trial. QJM 2002;95:677–683.

24. Vercoulen JH, Swanink CM, Zitman FG, Vreden SG, Hoofs MP, et al. Randomised, double-blind, placebo-controlled study of fluoxetine in chronic fatigue syndrome. Lancet 1996;347:858–861.

15. Cho HJ, Hotopf M, Wessely S. The placebo response in the treatment of chronic fatigue syndrome: A systematic review and meta-analysis. Psychosom Med 2005;67:301–313



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General discussion

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In this thesis, I have presented the results of four studies in chronic fatigue syndrome. These trials were performed to seek evidence for disturbances in Insulin-like Growth Factor and in the serotonin metabolism. In this final chapter, I will summarise the results of these studies, place them into perspective of the existing CFS literature, and discuss scientific and clinical implications. I will furthermore provide suggestions for future research.

Insulin-like Growth Factor Regarding a potential role of Insulin-like growth factor (IGF), two hypotheses were tested. The first hypothesis was that basal IGF concentrations are low in CFS. To that end, basal IGF concentrations in adult CFS patients were compared with those in matched neighbourhood controls. The second hypothesis tested was that Acclydine, a food supplement, is an effective treatment in CFS. This was in accordance with a claim of the manufacturer of this compound and the assumption that it would enhance IGF concentrations. A randomised placebo-controlled trial was performed to assess the efficacy of Acclydine treatment during 14 weeks. First of all, there was no difference in basal serum IGF-1 and IGF-BP3 levels between the CFS patients and age and gender-matched neighbourhood controls. With regard to the second hypothesis, treatment with Acclydine was ineffective in CFS. No differences between the treatment and placebo groups were found in any of the outcome measures. In addition, Acclydine did not alter IGF metabolism in the treatment group.

The Acclydine RCT was published in 2007. Until 2007, Acclydine was promoted and sold on the internet. In addition, an unpublished study from Belgium was presented at the Australian CFS/ME Conference in 2001, claiming a significant increase in IGF-1 levels in CFS patients and a significant improvement in CFS symptoms. The fact that many patients and insurance companies asked our opinion prompted us to carry out this investigation. No studies have been published investigating the effect of Acclydine after 2007. In our opinion, further Acclydine research is not needed and an expensive Acclydine treatment is not warranted. CFS studies investigating insulin-like growth factor status were mainly published between 1996 and 2005. A more recent cross sectional study found no statistically significant changes of IGF in chronic fatigue, chronic widespread pain, irritable bowel syndrome, and chronic orofacial pain1. There is no convincing evidence that IGF dysfunction plays a role in CFS2,3,4.



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The main reason for performing the Acclydine clinical trial was to assess its efficacy for the aforementioned reasons. The trial was not designed to thoroughly investigate the insulin-like growth factor axis, and focusing on one pathway obviously has its limitations as neuroendocrine pathways and neurotransmitter systems interact in a complex manner. The search for a single cause or one specific disruption in a specific neuroendocrine system or one neurotransmitter system in CFS has not led to a specific explanation of CFS symptoms.

SerotoninWe performed three intervention studies to pursue the question on whether serotonin metabolism is upregulated in CFS. Although our first open label pilot study with the 5-HT3 receptor antagonist granisetron met with promising results (chapter 2), no therapeutic benefits were found in the larger placebo-controlled double-blind study using ondansetron (chapter 3). In this study we used ondansetron, another 5-HT3 receptor antagonist because we could not obtain granisetron after the merger of two large pharmaceutical companies. The differences between granisetron and ondansetron are discussed in more detail below.

In a next study (reported in chapter 4), the effect of acute tryptophan depletion was assessed in a placebo-controlled design. We can conclude that all three serotonin interventions performed had negative results. Though results regarding the efficacy of selective serotonin reuptake Inhibitors (SSRI’s) in CFS are mixed5, our Nijmegen group previously found in a randomised placebo controlled double-blind study that the SSRI fluoxetine is ineffective in CFS patients with depression and without depression6. Blocking serotonin with ondansetron and lowering brain serotonin levels with acute tryptophan depletion are therefore ineffective in CFS treatment.

How can these negative results be explained? A negative clinical trial may be due to lack of a pharmacological effect, though other possibilities should also be taken into account.



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Ondansetron clinical trial

Could pharmacological differences between granisetron and ondansetron be an explanation for the difference in effect between granisetron and ondansetron?

PharmacodynamicsOndansetron has proven anti-emetic properties and is used in chemotherapy and postoperative induced nausea and vomiting7. Many studies of ondansetron have been published since its introduction in the early Nineties. The vomiting reflex after chemotherapy is probably caused by a chemo-therapy-induced increase of serotonin release. Chemotherapy-induced increase of serotonin is treated with serotonin-blocking agents like ondansetron. There is a parallel with our hypothesis of an increased serotonin metabolism in CFS and investigating the effect of serotonin antagonism. Ondansetron is effective by blocking serotonin receptors from the enterochromaffin cells and by blocking serotonin at the level of the central nervous system8. Ondansetron is considered the prototypical drug in the class of 5-HT3 receptor antagonists. Differences among the 5-HT3 antagonists are mainly attributed to chemical structures, pharmacokinetics and binding properties9. The binding potential of several 5-HT3-receptor antagonists has been evaluated in radioligand binding studies in animals10. The affinity of granisetron and ondansetron for the 5-HT3 receptor in rats are >600 and 184 times that of serotonin. Both antagonists are highly effective in competing with serotonin for the 5-HT3 receptor11. Although both are considered effective 5-HT3 antagonists, they may differ in their binding potential to centrally located 5-HT3 receptors. For example, preclinical pharmacodynamic studies have shown that granisetron exhibits little or no binding to centrally located 5-HT3 receptors12,13. So this would not explain the observed difference in effect in CFS of the two drugs. However, there is evidence that receptor blockade produced by ondansetron is much shorter than that produced by granisetron14, and there are observations that ondansetron penetrates the blood-brain barrier slowly15. Although we used an equipotent dosage of ondansetron compared to 2 mg granisetron used in the pilot study, it is not fully clear if binding profile differences explain the lack of an ondansetron effect.

PharmacogeneticsIt has in recent years been recognised that genetic polymorphisms contribute to drug responsiveness. The science of pharmacogenetics is developing and differences in the genetic makeup of the CFS patient participating in our clinical trial could influence the response to ondansetron. Potential factors influencing



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response include polymorphisms associated with drug transport, the serotonin receptor and drug metabolism via cytochromes P450.

Transporter polymorphismDrug transporters play a key role in the passage of drugs across the blood brain barrier, thus determining the disposition of drugs and the drug concentration in the central nervous system. The transporter adenosine triphosphate-binding cassette subfamily B member 1 (ABCB1) has been identified in playing an important role in the ondansetron pharmacokinetics16. The ABCB1 transporter functions as an efflux pump17 and its highly polymorphic and genetic variance could influence the efficacy of Ondansetron. For example, Chio et al. found a strong association between the polymorphisms (2677G>T/A) and 3435C>T/A of the ABCB1 transporter and the response to ondansetron in post-operative nausea and vomiting patients. Patients were treated with ondansetron and the incidence of postoperative nausea and vomiting was lower in patients with the 2677G>T/A and 3435C>T/A polymorphism18 . Patients with homozygous 2677TT and 3435TT genotypes responded better to ondansetron treatment. The authors hypothesise that it is likely that a higher concentration of ondansetron in the central nervous system due to the ABCB1 polymorphisms and a reduced transporter activity could explain a better efficacy of ondansetron. The two polymorphisms have shown a high inter-ethnic variability. It is estimated that approximately 25% of the Caucasian population19,20 are homozygous carriers of 3435TT polymorphism and 16% of the Caucasian population are homozygous carriers of 2677TT polymorphism21. Many single-nucleotide polymorphisms in the ABCB1 transporter have been identified and the clinical relevance should be further established22. Since these polymorphisms are found in a substantial percentage of Caucasians, it cannot be ruled out, given the sample size of our trial (n=67), that transporter polymorphisms could explain the negative findings.

5-HT3 receptor polymorphismThe 5-HT3 receptor is a ligand-gated ion channel. Until now five different 5-HT3 receptor subunits (HT3A - HT3E) have been identified. Ondansetron specifically binds to the 5-HT3A and 5-HT3B receptor complex. The 5-HT3B subunit has shown to be the most important for its functionality23. Tremblay et al. found that cancer patients who were homozygous for the 100_-102AAG deletion polymorphism of the 5-HT3 gene were associated with a higher incidence of chemotherapy-induced nausea and vomiting in cancer patients24. However, Rueffert et al. could not confirm this specific result in patients with post-operative nausea and vomiting25. In 2015, Kim and colleagues found the



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-100_-102AAG deletion polymorphisms of the 5-HT3b gene to be associated with a higher incidence of post operative nausea and vomiting and with ondansetron responsiveness26. There is still limited data on the 5-HT3B gene frequency status and allele frequency in different populations. Further research is necessary to assess the relationship between the genotype of the 5-HT3 receptor and drug response to 5-HT3 receptor antagonists. Research in the treatment of post-operative nausea and vomiting with 5-HT3 antagonists demonstrates that we should consider the possible influence of polymorphisms of the 5-HT3 receptor.

PharmacokineticsDifferent studies point out that, unlike the other 5-HT3 receptor antagonist, granisetron’s principal route of metabolism is mediated by P450 CYP3A4 isoenzyme27,28. Granisetron metabolism does not rely on CYP2D629. Ondansetron is also metabolised by the cytochrome P450 CYP2D6 and is subject to potential genetic polymorphisms9. Candiotti et al. found Ondansetron to be less effective in ultra rapid metabolisers in postoperative vomiting30. In a more recent study, Trammel et al. report less efficacy of ondansetron in acute chemotherapy- induced and vomiting in ultra rapid metabolisers29. The frequency of ultra rapid metabolisers differs widely among ethnic groups. Bernard et al. report high frequencies of ultra rapid metabolisers in Saudi Arabian patients (21%), Ethiopian (29%), Spanish (10%) and Turkish patients (8.7%). Ultra rapid metabolisers are present in less than 5% in Caucasians31. In our clinical trial, CYP2D6 polymorphism could be another factor to consider when reflecting on the negative results found in our randomised clinical trial. Although we did not specifically check the ethnicity in our randomised clinical trial, the patients were mainly Caucasians and it is highly unlikely that differences in 2D6 metabolism could explain the negative results in the ondansetron trial. In conclusion, the study design of our ondansetron clinical trial did not take into account genetic polymorphisms. This factor should be included in the future trial designs.

Could non-adherence be an explanation?

Patients were asked in the RCT if the medication had been taken consequently every two weeks. Our publication states that we do not believe that lack of adherence can explain the negative findings. Regular assessment of ondansetron blood concentration levels would be the appropriate method for checking compliance, but this would have increased both the burden for already incapacitated patients and the investigator-initiated trial costs.



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We noted that the CFS-patients participating in the burdensome clinical trial were highly motivated, and the very low withdrawal rate in the clinical trial (3 out of 33 patients in the ondansetron group and no withdrawal in the placebo group) could be a reflection of this high motivation and good compliance.

Is the placebo response similar to that in other CFS trials?

The ondansetron trial is a study oriented towards a somatic (‘biological’) disease mechanism. CFS patients have somatic attributions and somatic attributions are considered to be perpetuating factors32. Such a somatically-oriented study would meet with less scepticism and perhaps with higher expectations, beliefs of a positive result and high motivation in the CFS patients participating in the trial. These factors could influence the placebo effect. Cho et al. investigated the placebo effect in CFS. The placebo response, which was defined as the proportion of responders in a placebo arm, was investigated. The authors found a low (19.6%) pooled placebo response in CFS. Psychological-psychiatric interventions were shown to have the lowest placebo response (14%) and interventions based on the assumption of physical causality (infectious or immunological) were shown to have the highest placebo response (24%)33. In the placebo group in our study, we found a placebo response of 23%. Eight out of the 34 patients in the placebo group showed a significant improvement in fatigue severity. Thus the placebo response in our ondansetron clinical trial seems similar to placebo response found in the literature for studies directed towards physical disease mechanisms.

Could strict inclusion criteria have reduced the likelihood of achieving response to ondansetron?

A sizeable percentage of CFS patients have current psychiatric co-morbidity. The presence of current psychiatric disorders was an important exclusion criterion in our clinical trial. High fatigue severity scores and high impairment levels were also part of the inclusion criteria, and patients had to be able to visit the research facility. It is important to take into account, when there are too many inclusion and exclusion criteria or if the criteria are too strict, the risk that patients participating in a trial are not representative of the patients seen in clinical practice, and the sample could be considered as a select sample with a low external validity. The characteristics of the population enrolled in our trial did not differ from other studies of groups of CFS patients studied in our centre: age, gender, fatigue severity and CDC symptoms were similar34,35,36. No other trials have been published investigating the effect of serotonin-blocking strategies after the ondansetron publication in 2010. Performing analysis based



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on pooled data and larger sample sizes is therefore still not possible. Despite our study’s limitations, we will assume that the absence of an effect in the larger ondansetron trial reflects a lack of a pathophysiological role of serotonin in CFS.

Tryptophan DepletionAcute tryptophan depletion was not effective in the treatment of CFS. Acute tryptophan depletion is an accepted method to lower central serotonin levels. The pilot study was performed in five CFS patients. A major concern is the lack of power in our study and larger trials are required to assess the effect more thoroughly.

Could heterogeneity at the serotonin level explain the negative findings?

Badawy et al. reported that two subgroups of CFS patients could be identified based on their serotonin status. They proposed criteria for defining a high serotonin status. Free serum tryptophan, total serum tryptophan and the ratios (free serum tryptophan/BCAA and total serum tryptophan/BCAA) should lie outside the 2 SD range37. We compared the total tryptophan/BCAA ratios of the 5 CFS patients participating in our ATD study with the ratios of 5 healthy female controls of the same age from the same laboratory. The ratios of the CFS patients and the healthy controls were similar. We found no evidence for a high serotonin status in the CFS patients. One could hypothesise that acute tryptophan depletion or treatment with serotonin antagonism could only be effective in CFS patients with a high serotonin status. We did not find evidence for an increased serotonin status in CFS patients in this study. Our conclusion was based on a small intervention study only and what can be concluded is that the negative results did not yield much hope of more extensive studies greatly changing our insight. But an integrated approach investigating serotonin metabolism in CFS may certainly yield more fundamental insight into serotonin status in CFS. Besides peripheral assessment of serotonin status with blood serum and urine measurements, serotonin status could also be determined with central parameters. Use of positron emission tomography (PET) and single photon emission computed tomography (SPECT) with 5-HT-specific radioligands will allow to eventual 5-HT(1A) and 5-HT(2A) receptor and 5-HT transporter alterations in CSF. In this way, correlations between central and peripheral outcome measures, as well as changes after CBT could be investigated. As previously stated, we did not have high expectations from such studies, and it is uncertain if more insight in the serotonin status will lead to clinical relevant findings for treatment. More importantly, funding for these studies was not



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available. Based on the intervention studies presented in this thesis, I do not think that further exploration of the serotonin status in CFS should have a high priority.

New developments

Definitions of chronic fatigue syndromeThe studies presented in this thesis were published between 2003 and 2014. Other important recommendations and guidelines have been published since 2003. In 2007, the CFS NICE clinical guideline was published38. The CFS report of the Health Council of the Netherlands was published in 200539, and the development of a Dutch CFS guideline was recommended. In 2013 the Dutch CFS guideline was published40. All publications emphasise the necessity of further scientific research. The international case definition (CDC 1994 criteria), which was primarily formulated for research purposes, is still considered to be the standard, and we applied this in all our studies41,42 (Fukuda 1994, Reeves 2003). Another definition has recently been proposed. The prestigious Institute of Medicine in the United States established a committee on CFS in 2015. This committee released its report ‘Beyond Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome: Redefining an Illness’. The committee proposes to change the name Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome to Systemic Exertion Intolerance Disease (SEID). In this definition, the core feature of the disorder is post-exertional malaise instead of persistent or relapsing unexplained chronic fatigue in the 1994 case definition.

The scientific community has received this proposal with astonishment and disagreement. Most scientists and doctors agree that the name Myalgic Enceph-alomyelitis is a misnomer, but there is little reason to change a name when there is not a profound change in the understanding of the pathophysiology of the disorder. Secondly, to change from ‘syndrome’ to ‘disease’ also suggests that it concerns a homogeneous disease entity and that we understand the patho-physiology.

A third reason regards the proposed name of Systemic Exertion Intolerance Disease. Systemic suggests that we know it is a systemic disorder. The evidence for this is virtually absent. Most data point to the brain as the site of the substrate, and all other symptoms can been seen as secondary to that. Using ‘exertion intolerance’ in the name cannot be seen as an improvement. The fatigue and the accompanying symptoms are not a result of exertion: patients wake up fatigued, sleep does not refresh; exercise and exertion are not critical for the symptomatology.



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Finally, the diagnostic criteria lack a sound scientific basis. Sensitivity and specificity are not taken into account. Though the 3 mandatory symptoms are reasonable, but – like in the CDC criteria – no effort has been undertaken to try and quantify the degree of impairment. The choice of the two ‘minor’ manifestations, i.e., cognitive impairment and orthostatic intolerance also gives rise to concern. Cognitive impairment in CFS most often cannot be substantiated. Orthostatic intolerance is also relatively rare, as became apparent in three studies from our group43,44.

Future research priorities

It is clear that the IOM report is not a good starting point for further research. So which research priorities do I envisage? In this respect, it is of interest that the National Institute of Mental Health (NIMH) recently updated its strategic plan and defined research priorities and launched the Research Domain Criteria Initiative (RDoC)45 (see https://www.nimh.nih.gov/research-priorities/rdoc/index.shtml). Two objectives may be relevant for future CFS research. Firstly, based on the understanding of brain disorders, the NIMH states that mental illnesses are the late signs of changes in brain circuits. A more refined understanding of underlying mechanisms of perception, cognition and motivation is essential. The answers will come from hypothesis testing and discovery-based studies examining the biological mechanisms underlying symptoms and mental processes.

Next, the NIMH states that the best time to address a mental illness is before the appearance of symptoms that disrupt daily life. Research will be supported to develop biomarkers and assessment tools to predict illness onset, course and intervention response. Critical questions furthermore include how biological mechanisms are linked with environmental factors such as stress and experience such as trauma (NIMH strategic plan)46,47. One can conclude that, in line with NIMH mental health research priorities, the challenge in CFS research is investigating biological, psychological and environmental factors in a multidisciplinary and integrated approach. To study CFS in an early stage is a relevant suggestion. This would mean to study patients within 6 months and 1 year of illness should be a research priority. The ambition to study CFS patients before symptoms appear is more difficult, given the rarity of CFS. Studies of this kind have been performed in patients with acute Epstein Barr virus infection48,49,50,51,52.



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Other infections that are regularly followed by CFS or symptoms that resemble CFS are Q fever and Lyme disease. Recently Keijmel et al. published the first study comparing CFS patients and Q fever fatigue syndrome (QFS) patients53. Investigating differences and similarities between CFS and QFS will yield a better understanding of the pathogenesis of chronic fatigue that may complicate Q fever infection.

As stated above, I would not prioritise further research on the role of serotonin in the pathogenesis of CFS. The Nijmegen Expert Centre Chronic Fatigue is at present carrying out research on the potential role of the cytokine interleukin-1 by doing a controlled clinical trial with the specific inhibitor anakinra (Il-1 receptor antagonist). Such an intervention is based on the hypothesis that there is (low-grade) inflammation in the brain (Clinicaltrials.gov: registry: NCT02108210 and BMC clinical trials 2015,16:439).

Exploring the inflammatory hypothesis is definitely an important next step. Accumulating evidence from neuroimaging studies demonstrate neural abnormalities in CFS patients54. Other studies currently undertaken deal with the structure and functioning of the brain, using MRI scanning. Patients with CFS were found to have reduced grey matter volumes that increased following treatment with CBT55. Furthermore, a randomised controlled trial has begun aiming to replicate these effects of CBT on grey matter volumes. Also the mechanisms that underlie the effects of CBT are investigated by means of task-dependent functional MRI and immune and endocrine biomarkers56. These studies are much needed and are, in my opinion, a good direction to choose.



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28. Janicki PK. Cytochrome P450 2D6 metabolism and 5-hydroxytryptamine type 3 receptor antago-nists for postoperative nausea and vomiting. Med Sci Monit 2005;11(10):322-328.

29. Trammel M, Roederer M, Patel J, McLeod H. Does pharmacogenomics account for variability in con-trol of acute chemotherapy-induced nausea and vomiting with 5-hydroxytryptamine type 3 receptor antagonists? Curr Oncol Rep 2013;15(3): 276-285.

30. Candiotti KA, Birnbach DJ, Lubarsky DA, Nhuch F, Kamat A, Koch WH, et al. The impact of pharma-cogenomics on postoperative nausea and vomiting: do CYP2D6 allele copy number and polymor-phisms affect the success or failure of ondansetron prophylaxis? Anesth 2005;102(3):543-549.

31. Bernard S, Neville KA, Nguyen AT, Flockhart DA. Interethnic differences in genetic polymorphisms of CYP2D6 in the U.S. population: clinical implications. Oncologist 2006:11(2):126-135.

32. Vercoulen JH, Swanink CM, Galama JM, Fennis JF, Jongen PJ, Hommes OR, et al. The persistence of fatigue in chronic fatigue syndrome and multiple sclerosis: development of a model. J Psychosom Res 1998;45(6):507-517.

33. Cho HJ, Hotoph M, Wessely S. The placebo response in the treatment of chronic fatigue syndrome: a systematic review and meta-analysis. Psychosom Med 2005;67(2):301-313.

34. Brouwers FM, van der Werf S, Bleijenberg G, van der Zee L, van der Meer JW. The effect of a polynutrient supplement on fatigue and physical activity of patients with chronic fatigue syndrome: a double-blind randomized controlled trial. QJM 2002;95(10):677-683.

35. Prins JB, Bleijenberg G, Bazelmans E, Elving LD, de Boo TM, Severens JL, et al. Cognitive behaviour therapy for chronic fatigue syndrome: a multicentre randomised controlled trial. Lancet 2001;357(9259):841-847.

36. Tummers M, Knoop H, Bleijenberg G. Effectiveness of stepped care for chronic fatigue syndrome: a randomized noninferiority trial. J Consult Clin Psychol 2010;78(5):724-731.

37. Badawy AA, Morgen CJ, Llewelyn MB, Albuquerque SR, Farmer A. Heterogeneity of serum tryptophan concentration and availability to the brain in patients with the chronic fatigue syndrome. J Psychopharmacol 2005;19(4):385-391.

38. Turnbull N, Shaw EJ, Baker R, Dunsdon S, Costin N, Britton G, et al. Chronic fatigue syndrome/myalgic encephalomyelitis (or encephalopathy): diagnosis and management of chronic fatigue syndrome/myalgic encephalomyelitis) in adults and children. Royal Coll Gen Pract 2007. (NICE Clinical Guidelines, No. 53.).

39. Gezondheidsraad. Het chronische-vermoeidheidssyndroom [Health Council. Chronic fatigue syndrome]. Den Haag, 2005, publicatie nr 2005/02.

40. CBO. Richtlijn Diagnose, behandeling, begeleiding en beoordeling van patiënten met het chronisch vermoeidheidssyndroom (CVS). Utrecht, februari 2013.



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41. Fukuda K, Straus SE, Hickie I, Sharpe MC, Dobbins JG, Komaroff A. The Chronic Fatigue Syndrome Study: A comprehensive approach to its definition and study. International Chronic Fatigue Syndrome Study Group. Ann Intern Med 1994;121:953–959.

42. Reeves WC, Lloyd A, Vernon SD, Klimas N, Jason LA, Bleijenberg G, et al. Identification of ambiguities in the 1994 chronic fatigue syndrome research case definition and recommendations for resolution. International Chronic Fatigue Syndrome Study Group. BMC Health Serv Res 2003;3(1):25.

43. Soetekouw PM, Lenders JW, Bleijenberg G, Thien T, van der Meer JW. Autonomic function in patients with chronic fatigue syndrome. Clin Auton Res 1999;9(6):334-340.

44. Timmers HJ, Wieling W, Soetekouw PM, Bleijenberg G, van der meer JW, Lenders JW. Hemodynamic and neurohumoral responses to head-up tilt in patients with chronic fatigue syndrome. Clin Auton Res 2002;12(4):273-280.

45. Insel TR, Cuthbert BN, Garvey M, Heinssen R, Pine DS, Quinn K, et al. Research domain criteria (RDoC): toward a new classification framework for research on mental disorders. Am J Psychiatry 2010;167(7):748-751.

46. Cuthbert BN, Insel TR. Toward the future of psychiatric diagnosis: the seven pillars of RDoC. BMC Med 2013;11:126.

47. Insel TR. The NIMH Research Domain Criteria (RDoC) Project: precision medicine for psychiatry. Am J Psychiatry. 2014;171(4):395-397.

48. Buchwald DS, Rea TD, Katon WJ, Russo JE, Ashley RL. Acute infectious mononucleosis: character-istics of patients who report failure to recover. Am J Med 2000;109(7):531-537.

49. White PD, Thomas JM, Sullivan PF, Buchwald DS. The nosology of sub-acute and chronic fatigue syndromes that follow infectious mononucleosis. Psychol Med 2004;34(3):499-507.

50. Vernon SD, Whistler T, Cameron B, Hickie IB, Reeves WC, Lloyd A. Preliminary evidence of mito-chondrial dysfunction associated with post-infective fatigue after acute infection with Epstein Barr virus. BMC Infect Dis 2006;6:15.

51. Cameron B, Bharadwaj M, Burrows J, Fazou C, Wakefield D, Hickie I, et al. Dubbo Infection Outcomes Study. Prolonged illness after infectious mononucleosis is associated with altered immunity but not with increased viral load. J Infect Dis 2006;193(5):664-671.

52. Loebel M, Strohschein K, Giannini C, Koelsch U, Bauer S, Doebis C, et al. Deficient EBV-specific B- and T-cell response in patients with chronic fatigue syndrome. PLoS ONE 2014;9(1):e85387.

53. Keijmel SP, Saxe J, van der Meer JW, Nikolaus S, Netea MG, Bleijenberg G, et al. A comparison of patients with Q fever fatigue syndrome and patients with chronic fatigue syndrome with a focus on inflammatory markers and possible fatigue perpetuating cognitions and behaviour. J Psychosom Res 2015;79(4):295-302.

54. Browning M, Fletcher P. Sharp M. Can neuroimaging help us to understand and classify somatoform disorders? A systematic and critical review. Psychosom Med 2011;73(2):173-184.

55. De Lange FP, Koers A, Kalkman JS, Bleijenberg G, Hagoort P, van der meer JW, et al. Increase in prefrontal cortical volume following cognitive behavioural therapy in patients with chronic fatigue syndrome. Brain 2008;131:2172-2180.

56. Van der Schaaf ME, Schmits IC, Roerink M, Geurts DE, Toni I, Roelofs K, et al. Investigating neural mechanisms of change of cognitive behavioural therapy for chronic fatigue syndrome: a randomized controlled trial. BMC Psychiatry 2015;15:144.





Summary





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Summary

The title of this thesis is: ‘Chronic Fatigue Syndrome; Targeting Serotonin and Insulin-like Growth Factor’. This dissertation reports on four studies, in which the role of serotonin and Insulin-like growth factor are investigated to elucidate the pathogenesis of chronic fatigue syndrome (CFS).

Patients with CFS experience severe and medically unexplained chronic fatigue that results in tremendous impact on the lives of patients and their families.

Cognitive behavioural therapy (CBT) has proven to be a successful intervention leading to a reduction in fatigue and impairment. CBT can result in a full recovery. Unfortunately some patients do not profit from CBT and more insight into the neurobiology of CFS is highly necessary to improve our understanding of this illness.

Chapter 1In the introduction the outline of this thesis is presented. The diagnostic criteria for CFS and the evaluation of chronic fatigue are described and the complex role of psychiatric disorders in CFS is discussed. Furthermore the hypothesis of an upregulated serotonin system and the hypothesis of deficient insulin-like growth factor hormone in the pathophysiology of CFS are explained.

This thesis comprises three studies targeting serotonin and one study targeting insulin-like growth factor in the pathophysiology of chronic fatigue syndrome.

Chapter 2The goal of chapter 2 was to investigate the therapeutic potential of the serotonin receptor antagonist granisetron in adult CFS-patients. This was the first CFS study investigating the hypothesis of an up-regulated serotonin system by means of serotonin antagonism. A pilot study was performed in 5 CFS-patients, with low activity levels. The effect of 1 mg granisetron for two weeks and 2 mg for the second two weeks was evaluated. Four out of the five patients reported significant improvement in fatigue severity and functional impairment. Treatment did not result in improved physical activity. We concluded that performing a randomised placebo-controlled clinical trial was warranted.



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Chapter 3 In chapter 3, the results of a double-blind clinical trial are presented. We were interested if the positive results of the pilot study would be replicated in a clinical trial design. Sixty seven adult CFS patients were treated with the 5-HT3 receptor antagonist ondansetron, 16 mg per day. Patients were treated for a period of 10 weeks with an equipotent dose of 2 mg granisetron. The primary outcome measures were fatigue severity and functional impairment. Secondary outcome measures were physical activity level and daily fatigue level. We did not find significant differences between the ondansetron- and placebo group in any of the outcome measures. The findings of this clinical trial do not support the use of 5-HT3 receptor antagonism in the treatment of CFS.

Chapter 4In chapter 4, serotonin metabolism in CFS was investigated further. In a small proof of principle study the effect of transient acute tryptophan depletion (ATD) was assessed. Serotonin metabolism was significant reduced by the paradigm ATD. The effect of ATD was assessed in a double-blind cross-over design. We found no significant differences between the ATD- and the placebo group.Further research with larger sample sizes is needed to allow a more definite answer.

Chapter 5In chapter 5, the hypothesis of a deficient insulin-like growth factor is elaborated.The results of a randomised clinical trial with Acclydine, a food supplement, are presented. Acclydine was promoted as an effective treatment for CFS. Evidence for efficacy is lacking. We found no differences in insulin-like growth factor status in CFS-patients compared to healthy matched neighbourhood controls. Treatment with Acclydine during 14 weeks was not effective and no differences were found between insulin-like growth factor status and CFS related symptoms between the Accyline- and the placebogroup.

Chapter 6Finally, in chapter 6 the findings of the four studies were discussed and were put into perspective. The 2015 proposed new definition of CFS by the Institute of Medicine is discussed and possible directions for future research were formulated.



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Samenvatting





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Samenvatting

In dit proefschrift getiteld: ‘Chronic Fatigue Syndrome; Targeting Serotonin and Insulin-like Growth Factor’, worden de resultaten van 4 studies beschreven waarin de rol van serotonine en Insulin-like growth factor zijn onderzocht met als doel meer inzicht te verkrijgen in de pathogenese van het chronisch vermoeidheids-syndroom (CVS). CVS wordt gekenmerkt door ernstige aanhoudende vermoeidheid. Er is geen lichamelijke verklaring voor de vermoeidheid en CVS heeft grote gevolgen voor de patiënten en hun familie.

Cognitieve gedragstherapie (CGT) is een effectieve behandeling die leidt tot een afname van de vermoeidheid en beperkingen. Volledig herstel van CVS na CGT is mogelijk, echter behandeling met CGT is niet bij alle patiënten succesvol. Meer kennis van de neurobiologie van CVS is nodig.

Hoofdstuk 1In de introductie wordt een algemene inleiding gegeven over CVS. De diagnostische criteria en uitgangspunten voor de analyse van patiënten met chronische vermoeidheidsklachten en de complexe rol van psychiatrische stoornissen worden beschreven. De hypotheses van een ‘overgevoelig serotonine systeem’ en een ‘deficient insulin-like growth factor hormoon’ systeem bij CVS worden uiteengezet.

Dit proefschrift beschrijft drie studies die het serotonine systeem onderzoeken en een studie die zich richt op het insulin-like growth factor hormoon bij CVS- patiënten.

Hoofdstuk 2Het doel van hoofdstuk 2 is het effect van granisetron, een serotonine receptor antagonist, te onderzoeken bij volwassen CVS-patiënten. Dit was het eerste CVS onderzoek dat de hypothese van een ‘overgevoelig serotonine systeem’ onderzocht door middel van een medicamenteuze interventie met een serotonine antagonist. Een pilot onderzoek werd verricht bij 5 CVS-patiënten met een lager niveau van fysieke activiteiten. Het effect van 1 mg granisetron gedurende twee weken en 2 mg gedurende de opeenvolgende twee weken werd onderzocht. Vier van de vijf patiënten lieten een significante verbetering in de ernst van de vermoeidheid en in de ernst van beperkingen zien. Behandeling met granisetron leidde niet tot een



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verbetering van het fysieke activiteiten niveau. Gezien de positieve resultaten van de pilot studie werd besloten een gerandomiseerde dubbebblinde clinical trial uit te voeren.

Hoofdstuk 3In hoofdstuk 3 worden de resultaten van de dubbelblinde clinical trial beschreven. De vraag was of de positieve resultaten van de pilot studie gerepliceerd konden worden. Zevenenzestig volwassen CVS-patiënten werden behandeld met 16 mg ondansetron, een 5-HT3 receptor antagonist. De patiënten werden gedurende 10 weken behandeld met een equipotente dosering van 2 mg granisetron. De primaire uitkomstmaten waren ernst van de vermoeidheid en beperkingen. De secundaire uitkomstmaten waren fysiek activiteiten niveau en dagelijks ervaren vermoeidheid. Er werd geen enkel significant verschil gevonden in alle uitkomstmaten tussen de ondansetron- en placebo groep. De resultaten geven geen onderbouwing CVS-patiënten te behandelen met een 5-HT3 receptor antagonist.

Hoofdstuk 4In hoofdstuk 4 wordt het serotonine systeem verder onderzocht. In een klein ‘proof of principle’ onderzoek werd het effect van Acute Tryptofaan Depletie (ATD) onderzocht. Door middel van ATD werd het serotonine niveau significant verlaagd. Door middel van dubbel-blind cross-over onderzoek werd het effect van ATD bekeken. Er werd geen significant verschil gevonden tussen de ATD- en placebo groep. Verder onderzoek met grotere aantallen is nodig om tot een meer definitief antwoord te komen.

Hoofdstuk 5In hoofdstuk 5 wordt de ‘deficient insulin-like growth factor hormoon’ hypothese bij CVS toegelicht. De resultaten van een gerandomiseerd clinical trial met Acclydine, een voedingssupplement, worden beschreven. Acclydine werd gepromoot als een effectieve CVS-behandeling, hoewel hier geen wetenschappelijk bewijs voor was. Er werd geen verschil gevonden in insulin-like growth hormoon status tussen CVS-patiënten en gezonde gematchte buurtcontroles. Behandeling met Acclydine gedurende 14 weken bleek niet effectief. Er werden geen verschillen gevonden in insulin-like growth hormoon status en CVS gerelateerde uitkomstmaten tussen de Acclydine- en placebogroep.



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Hoofdstuk 6Ten slotte, in hoofdstuk 6 worden de resultaten van de vier onderzoeken in breder perspectief geplaatst. In 2015 stelde de Institute of Medicine een nieuwe definitie voor CVS voor. Deze nieuwe definitie wordt bediscussieerd en aanbevelingen voor toekomstig onderzoek worden gedaan.





List of publications





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Publications

The GKH, Verkes RJ, Fekkes D, Bleijenberg G, van der Meer JWM, Buitelaar JK.Tryptophan depletion in chronic fatigue syndrome, a pilot cross-over study.BMC Res Notes 2014;7:650.

The GKH, Bleijenberg G, Buitelaar JK, van der Meer JWM.The effect of ondansetron, a 5-HT3 receptor antagonist, in chronic fatigue syndrome: a randomized controlled trial. J Clin Psych 2010;71(5):528-533.

Groen WB, The GKH, Pop-Purceleanu M, Lagro-Janssen T, Fortuyn HA, Voshaar RC. Pseudologica fantastica. Ned Tijdschr Geneeskd 2009;153:A139.

The GKH, Bleijenberg G, van der Meer JWM. The effect of acclydine in chronic fatigue syndrome: a randomized controlled trial. PLoS Clin Trials. 2007 May 18;2(5):e19.

The GKH, Elving LD, Bleijenberg G, van der Meer JWM. Hoe ver moet men gaan bij vermoeidheid e.c.i.? Internisten vademecum.Jaargang 10 nr. 1 - 3 januari 2005.

The GKH, Elving LD, Bleijenberg G, van der Meer JWM. Wat is de zin van vitamine B12 injecties bij ME? Vademecum permanente nascholing huisartsen; interne ziekten. Jaargang 22 nr. 48 – 23 november 2004.

The GKH, Elving LD, Bleijenberg G, van der Meer JWM. Hoe ver moet men gaan bij vermoeidheid e.c.i.? Reumatologen vademecum. Jaargang 7 nr. 5 - 10 maart 2004.

The GKH, Prins JB, Bleijenberg G, van der Meer JWM. The effect of granisetron, a 5-HT3 receptor antagonist in the treatment of chronic fatigue syndrome patients, a pilot study. Neth J Med. 2003;61(9):285-289.

Van der Meer JWM, Van de Kerkhof R, The GKH, Boers GHJ. Hemopyrrollactamurie (HPU), van vlek naar pseudoziekte? Ned Tijdschr Geneesk 2003;147(36):1720-1721.



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The GKH. Zwangerschap lijkt geen ongunstige gevolgen te hebben bij patiënten met chronisch vermoeidheidssyndroom. Ned Tijdschr Geneesk 2004;148(41):2042.

Van der Meer JWM, Van de Kerkhof R, The GKH, Boers GHJ. Hemopyrrollactamurie (HPU), van vlek naar pseudoziekte? Ned Tijschr Geneesk 2003;147(59):2601.

Contributions to books

The GKH, Bleijenberg G, van der Meer JWM. De differentiële diagnose van chronische vermoeidheid. In: W.D. Reitsma, J.W.F. Elte, D. Overbosch (red). Differentiële diagnostiek in de interne genees - kunde. Bohn Stafleu van Loghum Houten 2005, 450-455 [ISBN 7890 31 342822].

The GKH, Bleijenberg G, van der Meer JWM. De differentiële diagnose van chronische vermoeidheid. In: W.D. Reitsma, J.W.F. Elte, D. Overbosch (red). Compendium Differentiële diagnostiek in de interne geneeskunde. Bohn Stafleu van Loghum Houten 2005, 232-233 [ISBN 7890 31 342815].

Abstracts

The GKH, Bleijenberg G, Buitelaar JK, van der Meer JWM. The effect of Ondansetron in chronic fatigue syndrome: a randomized controlled trial.Travel award European College Neuropsychopharmacology, 21st ECNP Congress, Barcelona Spanje. European Neuropsychopharmacology 2008;21 (suppl 4), S569.

The GKH, Prins JB, Bleijenberg G, van der Meer JWM. 34e Voorjaarscongres Groningen, NVVP. Samenvattingen april 2006:209.Het effect van Granisetron, een 5-HT3 receptor antagonist, op vermoeidheid en beperkingen bij patiënten met het chronisch vermoeidheidssyndroom.



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Dankwoord

Na vele jaren kan ik nu de laatste bladzijdes van mijn proefschrift schrijven.

Allereerst gaat mijn dank uit naar alle patiënten die hebben deelgenomen aan het onderzoek beschreven in dit proefschrift. Op en neer reizen, invullen van vragen- lijsten en bloedprikken zijn een grote belasting. De contacten met de patiënten en hen gedurende het onderzoek begeleiden gaven mij veel voldoening. Ik ben hen dankbaar voor alle inspanningen en moeite, terwijl de patiënten zelf alle energie nodig hebben.

Mijn promotoren Jos van der Meer, Gijs Bleijenberg en Jan Buitelaar wil ik bedanken dat ze mij de kans hebben gegeven te promoveren. Zonder jullie expertise, enorme enthousiasme voor de wetenschap, jullie begeleiding en vasthoudendheid was het mij niet gelukt onderzoek te verrichten en een proefschrift te schrijven. Dank voor jullie geduld en alle hulp!

Een speciale dank gaat uit naar de psychologisch medewerkers Carel Kruip, Harmen Vissers, Jetske Oostelbos, Armand van Oisterwijck, Ilja Helsper, Ria te Winkel † en Tini Fasotti voor de hartelijke samenwerking en dataverzameling. De verpleegkundigen van de poli algemeen interne geneeskunde wil ik bedanken voor de bloedafnames. De medewerkers van het laboratorium interne geneeskunde en Johanna van der Ven wil ik bedanken voor de fijne opvang en alle uitleg.

De paranimfen:Beste Roy, 4 jaar hebben we zeer intensief samengewerkt en we zijn fruits de mer specialisten geworden in Zeeland. Ik kon altijd op je bouwen, zeker in die tijd toen het niet goed ging met Annick en Lao. Dank!

Beste Thijs, dank voor al jouw hulp. Je was altijd bereid wat gemopper aan te horen en mee te denken. Al jaren praten we over naar Rome te gaan. Wellicht kunnen we in 2017 daar een cola light drinken?

Dear Magda, Jeremy, Daniel, Alyssa, Cecilia, Greg, Thierry, Chiara and Isabella.Although we are living on other sides of the world, you always feel close and you are part of our lives. Thank you for your support and the great family holidays we enjoy together.



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Lieve papa en mama, dank voor al jullie goede zorgen en het kompas dat jullie mij hebben meegegeven. Papa, helaas heb je deze promotie niet meer mogen meemaken. Regelmatig denk ik, had ik het maar sneller afgerond. Jij zou luchtig zeggen: zo is het leven.

Tot slot, lieve Boukje, Annick, Lao en Mila. Wat zijn we gelukkig samen. Boukje, al 27 jaar zijn we samen en ik ben blij met jou de mooie en de soms minder leuke dingen te kunnen delen. Dank je wel voor alles. De tijd van om de dag dienst en geen weekenden vrij, zijn verleden tijd. Kids, jullie humor en vrolijkheid geven mij energie en laten mij zien wat echt belangrijk is in het leven. We gaan samen mooie avonturen beleven, waar ook ter wereld.



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Curriculum vitae





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Curriculum Vitae

Gerard The werd op 8 september 1974 geboren te Nijmegen. In 1993 behaalde hij zijn VWO diploma. Na 2 jaar biomedische gezondheidswetenschappen te hebben gestudeerd werd hij ingeloot voor de studie geneeskunde aan de Katholieke Universiteit Nijmegen. In 2002 behaalde hij zijn artsexamen en in datzelfde jaar startte hij zijn promotieonderzoek bij de afdeling Algemeen Interne Geneeskunde/ Nijmeegs Kenniscentrum Chronische Vermoeidheid. Vanaf 2003 werden onderzoek en de opleiding tot psychiater in de tijd afgewisseld.

In mei 2009 registreerde hij zich als psychiater en werkte hij tot september 2015 als psychiater in het Admiraal de Ruyter Ziekenhuis in Zeeland. Vanaf oktober 2015 werkt hij als psychiater in het Amphia Ziekenhuis Breda.

Gerard The woont samen met Boukje van der Staak. In april 2010 ging een koningswens in vervulling en kregen ze de tweeling Annick en Lao. In 2014 werd Mila geboren. De wens is om enige tijd te werken en met het gezin te wonen in Nieuw Zeeland.



Documents

Chronic Fatigue Syndrome: Targeting Serotonin and Insulin ... · Chronic fatigue syndrome (CFS) is still an enigmatic disorder. It is regarded as a complex disorder with tremendous