Methotrexate is an established treatment for inflammatory bowel disease, however it is commonly only used as second-line therapy due to concerns over side effects. This article reviews the evidence for using methotrexate polyglutamate levels in the management of rheumatoid arthritis and psoriasis in addition to inflammatory bowel disease with a view to optimizing treatment and helping to prevent toxicity.
by Dr E. L. Johnston, Dr S. C. Fong, Dr A. M. Marinaki, Dr M. Arenas-Hernandez and Dr J. D. Sanderson
Introduction
Methotrexate (MTX) is a folate analogue. It was first used in the 1950s to induce remission in childhood leukemias. Since then its clinical benefit has been widely utilized in the treatment of several inflammatory conditions, including rheumatoid arthritis (RA) and psoriasis, and more recently, inflammatory bowel disease (IBD).
Crohn’s disease (CD) and ulcerative colitis (UC) are chronic inflammatory conditions affecting the gastrointestinal tract, collectively known as IBD. MTX is not as commonly used in the treatment of IBD as other immune modulators, particularly thiopurines. This centres around concerns regarding toxicity and side effects, although in the RA population MTX is frequently used and is considered safe and effective. Monitoring methotrexate, by means of measuring red-cell methotrexate-polyglutamate (MTX-PG) levels, offers the potential to assess adherence along with optimizing dose. However, MTX-PG levels are currently underused because of conflicting evidence regarding interpretation of levels.
Inflammatory bowel disease and methotrexate
The use of methotrexate as a treatment in IBD was initially postulated in the late 1980s when a small study showed an improvement in disease activity indexes, and some histological improvement in the CD cohort, in patients with refractory IBD [1]. Since then, MTX has increasingly been used as a second-line treatment, particularly in those when thiopurine or anti-TNF therapy has failed or not been tolerated.
The European Crohn’s and Colitis Organisation (ECCO) guidelines on the management of CD [2] advise that methotrexate 25 mg/week can be used to treat active CD as an alternative to thiopurines. This is based on a randomized control trial (RCT) in 1995 [3] that showed a significant benefit in taking 25 mg/week of intramuscular (IM) MTX compared with placebo following withdrawal from steroids (39% vs. 19%). It is commonly prescribed orally which is easier for administration and favoured by patients. However, a small study [4] comparing oral to subcutaneous (SC) MTX showed the bioavailability of the oral preparation was variable, despite folic acid use, and favoured SC delivery.
There have been no large studies comparing thiopurines and methotrexate to treat CD and the largest RCT to date looking at the use of MTX as a concomitant immunosuppressant when combined with infliximab, compared to infliximab as monotherapy, showed no benefit in steroid free remission [5].
The evidence to support MTX use in inducing and maintaining remission in patients with UC is less robust with very few good quality RCTs. These studies have shown no benefit over placebo and, therefore, a recent Cochrane review did not support its use [6]. However, two large international RCTs (METEOR and MERIT-UC) looking at the use of MTX for active UC are ongoing.
When MTX is being considered as a treatment option for IBD there are often concerns over the safety of the drug. MTX use requires careful monitoring, particularly of liver function tests because of the risk of hepatotoxicity. However, a retrospective study of its use in CD found it was safe and well tolerated [7]. The commonest side effect was nausea in 22% (17 patients) with only 10% of patients experiencing abnormal liver function tests, resulting in 6% having to stop MTX.
Methotrexate polyglutamate levels
MTX is taken weekly and is commonly administered orally but can be used SC or IM. Despite a stable dose and route of administration there is significant interpatient variability in clinical response and the prevalence of side effects, which is a major drawback of therapy. It has, therefore, long been hypothesized that measuring MTX drug levels could be both a predictor of drug efficacy and a marker of potential toxicity.
MTX levels peak within hours of oral ingestion and are detectable for less than 24 hours in the serum. Weekly dosing offers no steady-state concentration and, therefore, serum levels are of no clinical benefit. Once in the serum, MTX is transported intracellularly by a reduced folate carrier (RFC) and is changed into a polyglutamated form (MTX-PG1). Further glutamic acid residues (GLUT) are added resulting in up to seven polyglutamates (MTX-PG1–7). This is show in Figure 1.
By using high-performance liquid chromatography it is possible to quantify the seven glutamic residue species in red blood cells [8]. This was first used in children with acute lymphoblastic leukemia [9] and has subsequently been found to correlate with disease activity in other chronic inflammatory conditions. However, MTX-PG6–7 have not previously been detected in RA patients taking MTX [14]; therefore, commonly only MTX-PG1–5 are measured.
Early data suggested that MTX-PG1–2 correlated poorly with drug efficacy in RA; however, the total long-chain polyglutamates (MTX-PG3–5) better reflected the drug effect [8]. MTX-PG3 is the predominant polyglutamate species in red blood cells and is useful to calculate the total long-chain concentrations [10].
Clinical use of methotrexate polyglutamate levels
MTX is widely prescribed for the treatment of RA. Dervieux et al. [10] first looked at the clinical use of MTX-PG measurements in the RA population. In 108 patients who had been on MTX over 3 months, higher MTX-PG levels were associated with a better clinical response to the drug. In particular, patients with a total MTX-PG1–5 that was >60 nmol/L were found to have less tender and swollen joints. The same group expanded their cohort and once again showed that patients with MTX-PG1–5 <60 nmol/L were four times more likely to have a poor response to MTX than those with MTX-PG1–5 >60 nmol/L [11].
Stamp et al. [12] noted large interpatient variability in MTX-PG levels and set out to identify factors that influence levels. Using univariate analysis they found that increased age, impaired renal function, longer duration of treatment and the use of prednisolone resulted in higher MTX-PG levels, whereas smokers generally had lower MTX-PG levels. In contrast to the studies by Dervieux et al., they also surprisingly found that higher doses of MTX were associated with higher MTX-PG levels and increased disease activity [13]. In addition there was no association between MTX-PG levels and adverse effects.
The same group looked at the timing of MTX-PG blood levels and time to steady state [14]. MTX-PG1 was detected 1–2 weeks after first ingestion; however, MTX-PG5 was detected after a median of 7 weeks (range 1–28 weeks). In addition the median time for MTX-PG1–5 to reach steady-state concentration was 27.5 weeks and the median time for MTX-PG1–5 to become undetectable after the last dose was 15 weeks. This highlights that MTX may take up to 6 months to achieve full clinical benefit, which is important to consider when using the levels to assess compliance or to guide dose alteration.
The main trial to be done outside the field of rheumatology was a 55-patient, prospective study into using MTX-PG levels to assess clinical response and compliance in patients with psoriasis [15]. This found the time to steady state of MTX-PG1–5 was between 12–24 weeks, and there was no significant correlation between MTX-PG levels and disease activity.
Methotrexate polyglutamate levels and inflammatory bowel disease
There have been only two studies addressing the potential use of MTX-PG levels in IBD. Egan et al. looked at the total levels when addressing the question of the optimal dose of MTX needed to induce remission in steroid-requiring IBD [16]. They found that subcutaneous initial doses of 15 and 25 mg/week in 32 patients were equally efficacious. In this cohort MTX-PG concentration reached a plateau at around 6–8 weeks after the initiation of therapy and no statistical difference was found between the levels across both doses of the drug. In addition the levels did not correlate with active disease or drug toxicity and did not change significantly after change in MTX dose.
A more recent prospective study from Brooks et al. looked specifically at MTX-PG concentrations in 18 patients with IBD that were on stable doses of MTX [8]. MTX-PG were measured on three occasions and compared to disease activity and reports of toxic side effects. MTX-PG were detected in all the patients and there was little variability in the levels over the study period. Similar to the Stamp et al. RA study [13], higher MTX-PG4&5 were associated with worse disease activity as well as higher toxic effects.
The cohort was small and heterogeneous with different doses of MTX prescribed (median 20 mg/week) and varied administration methods (oral, subcutaneous and via percutaneous endoscopic gastrostomy tube), which is likely to have had a bearing on the results. The data from a similar cohort was presented at Digestive Diseases Week in 2014 [17], which concluded that MTX-PG could be useful in assessing adherence. A non-significant trend showed higher concentrations were associated with active disease, but this may be due to higher doses of MTX being used in those with active disease.
Summary
Methotrexate is an established treatment for IBD. It is an efficacious and well tolerated therapeutic option in CD, particularly when administered SC. More studies are ongoing in the UC population. Measuring MTX-PG levels in RBC has the potential to not only monitor compliance but also correlate with disease activity and toxicity. Two large studies in patients with RA have produced conflicting results but in the small, IBD trials, higher MTX-PG levels, particularly MTX-PG4&5 correlated with increased disease activity and toxicity. It is important, however, to be aware that MTX-PG are influenced by other factors, particularly age and renal function, and may take up to 6 months to reach steady state.
Future trends and developments
Measuring drug levels plays an important role in the management of patients with IBD, as demonstrated by the monitoring of thioguanine nucleotides in those prescribed azathioprine [18]. Measuring MTX-PG offers an exciting step towards individualizing drug treatment and reducing toxicity in those taking MTX. However, at the moment there is a lack of substantial evidence to support the use of measuring MTX-PG levels in IBD, aside from monitoring compliance [19]. A large, prospective trial is warranted to determine clinical benefit before widespread use in the IBD population is advocated.
References
1. Kozarek RA, Patterson DJ, Gelfand MD, et al. methotrexate induces clinical and histological remission in patients with refractory inflammatory bowel disease. Ann Intern Med. 1989; 110: 353–356.
2. Dignass A, Van Assche G, Lindsay JO, et al. The second European evidence-based consensus on the diagnosis and management of Crohn’s disease: Current management. J Crohn’s Colitis 2010; 4: 28–62.
3. Feagan BG, Rochon J, Fedorak RN, et al. Methotrexate in the treatment of Crohn’s disease. New England Journal of Medicine. 1995; 332: 292–297.
4. Kurnik D, Loebstein R, Fishbein E, et al. Bioavailability of oral vs. subcutaneous low-dose methotrexate in patients with Crohn’s disease. Aliment Pharmacol Ther. 2003; 18(1): 57–63.
5. Feagan BG, McDonald JW, Panaccione R, et al. Methotrexate in combination with infliximab is no more effective than infliximab alone in patients with Crohn’s disease. Gastroenterology 2014; 146(3): 681–688.
6. Chande N, Wang Y, MacDonald JK, et al. Methotrexate for induction of remission in ulcerative colitis. Cochrane Database Syst Rev. 2014; 8.
7. Chande N, Abdelgadir I, Gregor J. The safety and tolerability of methotrexate for treating patients with Crohn’s disease. J Clin Gastroenterol. 2011; 45: 599–601.
8. Brooks A, Begg E, Zhang M, et al. Red blood cell methotrexate polyglutamate concentrations in inflammatory bowel disease. Ther Drug Monit. 2007; 29: 619–625.
9. Lena N, Imbert AM, Brunet P, et al. Kinetics of methotrexate and its metabolites in red blood cells. Cancer Drug Deliv. 1987; 4(2): 119–127.
10. Dervieux T, Furst D, Lein DO, et al. Polyglutamation of methotrexate with common polymorphisms in reduced folate carrier, aminoimidazole carboxamide ribonucleotide transformylase, and thymidylate synthase are associated with methotrexate effects in rheumatoid arthritis. Arthritis Rheum. 2004; 50(9): 2766–2774.
11. Dervieux T, Furst D, Lein DO, et al. Pharmacogenetic and metabolite measurements are associated with clinical status in patients with rheumatoid arthritis treated with methotrexate: results of a multicentred cross sectional observational study. Ann Rheum Dis. 2005; 64: 1180–1185.
12. Stamp LK, O’Donnell JL, Chapman PT, et al. Determinants of red blood cell methotrexate polyglutamate concentrations in rheumatoid arthritis patients receiving long-term methotrexate treatment. Arthritis Rheum. 2009; 60(8): 2248–2256.
13. Stamp LK, O’Donnell JL, Chapman PT, et al. Methotrexate polyglutamate concentrations are not associated with disease control in rheumatoid arthritis patients receiving long-term methotrexate therapy. Arthritis Rheum. 2010; 62(2): 359–368.
14. Dalrymple JM, Stamp LK, O’Donnell JL, et al. Pharmacokinetics of oral methotrexate in patients with rheumatoid arthritis. Arthritis Rheum. 2008; 58(11): 3299–3308.
15. Woolf RT, West SL, Arenas-Hernandez M, et al. Methotrexate polyglutamates as a marker of patient compliance and clinical response in psoriasis: a single-centre prospective study. Br J Dermatol. 2012; 167: 165–173.
16. Egan LJ, Sandborn WJ, Tremaine WJ, et al. A randomised dose-response and pharmacokinetic study of methotrexate for refractory inflammatory Crohn’s disease and ulcerative colitis. Aliment Pharmacol and Ther. 1999; 13: 1597–1604.
17. Ward MG, Fong S, Nasr I, et al. Higher red blood cell methotrexate polyglutamates correlate with increased disease activity, and are useful in assessing adherence. Abstract presented at Digestive Disease Week 2014.
18. Smith M, Blaker, P, Patel C, et al. The impact of introducing thioguanine nucleotide monitoring into an inflammatory bowel disease clinic. Int J Clin Pract. 2013; 67(2): 161–169.
19. Bruns T, Stallmach A. Drug monitoring in inflammatory bowel disease: helpful of dispensable? Dig Dis. 2009; 27: 394–403.
The authors
Emma L. Johnston1 MBBS BSc MRCP, Steven C. Fong1 MBBS MRCP, Anthony M. Marinaki2 PhD, Monica Arenas-Hernandez2 PhD, Jeremy D. Sanderson*1 MD FRCP
1Inflammatory Bowel Disease Centre, Dept of Gastroenterology, Guy’s and St Thomas’ NHS Foundation Trust, London, UK.
2Purine Research Laboratory, Viapath, Guy’s & St. Thomas’ NHS Foundation Trust, London, UK.
*Corresponding author
E-mail: jeremy.sanderson@kcl.ac.uk
Monitoring methotrexate polyglutamate levels in inflammatory bowel disease: where do we stand?
, /in Featured Articles /by 3wmediaMethotrexate is an established treatment for inflammatory bowel disease, however it is commonly only used as second-line therapy due to concerns over side effects. This article reviews the evidence for using methotrexate polyglutamate levels in the management of rheumatoid arthritis and psoriasis in addition to inflammatory bowel disease with a view to optimizing treatment and helping to prevent toxicity.
by Dr E. L. Johnston, Dr S. C. Fong, Dr A. M. Marinaki, Dr M. Arenas-Hernandez and Dr J. D. Sanderson
Introduction
Methotrexate (MTX) is a folate analogue. It was first used in the 1950s to induce remission in childhood leukemias. Since then its clinical benefit has been widely utilized in the treatment of several inflammatory conditions, including rheumatoid arthritis (RA) and psoriasis, and more recently, inflammatory bowel disease (IBD).
Crohn’s disease (CD) and ulcerative colitis (UC) are chronic inflammatory conditions affecting the gastrointestinal tract, collectively known as IBD. MTX is not as commonly used in the treatment of IBD as other immune modulators, particularly thiopurines. This centres around concerns regarding toxicity and side effects, although in the RA population MTX is frequently used and is considered safe and effective. Monitoring methotrexate, by means of measuring red-cell methotrexate-polyglutamate (MTX-PG) levels, offers the potential to assess adherence along with optimizing dose. However, MTX-PG levels are currently underused because of conflicting evidence regarding interpretation of levels.
Inflammatory bowel disease and methotrexate
The use of methotrexate as a treatment in IBD was initially postulated in the late 1980s when a small study showed an improvement in disease activity indexes, and some histological improvement in the CD cohort, in patients with refractory IBD [1]. Since then, MTX has increasingly been used as a second-line treatment, particularly in those when thiopurine or anti-TNF therapy has failed or not been tolerated.
The European Crohn’s and Colitis Organisation (ECCO) guidelines on the management of CD [2] advise that methotrexate 25 mg/week can be used to treat active CD as an alternative to thiopurines. This is based on a randomized control trial (RCT) in 1995 [3] that showed a significant benefit in taking 25 mg/week of intramuscular (IM) MTX compared with placebo following withdrawal from steroids (39% vs. 19%). It is commonly prescribed orally which is easier for administration and favoured by patients. However, a small study [4] comparing oral to subcutaneous (SC) MTX showed the bioavailability of the oral preparation was variable, despite folic acid use, and favoured SC delivery.
There have been no large studies comparing thiopurines and methotrexate to treat CD and the largest RCT to date looking at the use of MTX as a concomitant immunosuppressant when combined with infliximab, compared to infliximab as monotherapy, showed no benefit in steroid free remission [5].
The evidence to support MTX use in inducing and maintaining remission in patients with UC is less robust with very few good quality RCTs. These studies have shown no benefit over placebo and, therefore, a recent Cochrane review did not support its use [6]. However, two large international RCTs (METEOR and MERIT-UC) looking at the use of MTX for active UC are ongoing.
When MTX is being considered as a treatment option for IBD there are often concerns over the safety of the drug. MTX use requires careful monitoring, particularly of liver function tests because of the risk of hepatotoxicity. However, a retrospective study of its use in CD found it was safe and well tolerated [7]. The commonest side effect was nausea in 22% (17 patients) with only 10% of patients experiencing abnormal liver function tests, resulting in 6% having to stop MTX.
Methotrexate polyglutamate levels
MTX is taken weekly and is commonly administered orally but can be used SC or IM. Despite a stable dose and route of administration there is significant interpatient variability in clinical response and the prevalence of side effects, which is a major drawback of therapy. It has, therefore, long been hypothesized that measuring MTX drug levels could be both a predictor of drug efficacy and a marker of potential toxicity.
MTX levels peak within hours of oral ingestion and are detectable for less than 24 hours in the serum. Weekly dosing offers no steady-state concentration and, therefore, serum levels are of no clinical benefit. Once in the serum, MTX is transported intracellularly by a reduced folate carrier (RFC) and is changed into a polyglutamated form (MTX-PG1). Further glutamic acid residues (GLUT) are added resulting in up to seven polyglutamates (MTX-PG1–7). This is show in Figure 1.
By using high-performance liquid chromatography it is possible to quantify the seven glutamic residue species in red blood cells [8]. This was first used in children with acute lymphoblastic leukemia [9] and has subsequently been found to correlate with disease activity in other chronic inflammatory conditions. However, MTX-PG6–7 have not previously been detected in RA patients taking MTX [14]; therefore, commonly only MTX-PG1–5 are measured.
Early data suggested that MTX-PG1–2 correlated poorly with drug efficacy in RA; however, the total long-chain polyglutamates (MTX-PG3–5) better reflected the drug effect [8]. MTX-PG3 is the predominant polyglutamate species in red blood cells and is useful to calculate the total long-chain concentrations [10].
Clinical use of methotrexate polyglutamate levels
MTX is widely prescribed for the treatment of RA. Dervieux et al. [10] first looked at the clinical use of MTX-PG measurements in the RA population. In 108 patients who had been on MTX over 3 months, higher MTX-PG levels were associated with a better clinical response to the drug. In particular, patients with a total MTX-PG1–5 that was >60 nmol/L were found to have less tender and swollen joints. The same group expanded their cohort and once again showed that patients with MTX-PG1–5 <60 nmol/L were four times more likely to have a poor response to MTX than those with MTX-PG1–5 >60 nmol/L [11].
Stamp et al. [12] noted large interpatient variability in MTX-PG levels and set out to identify factors that influence levels. Using univariate analysis they found that increased age, impaired renal function, longer duration of treatment and the use of prednisolone resulted in higher MTX-PG levels, whereas smokers generally had lower MTX-PG levels. In contrast to the studies by Dervieux et al., they also surprisingly found that higher doses of MTX were associated with higher MTX-PG levels and increased disease activity [13]. In addition there was no association between MTX-PG levels and adverse effects.
The same group looked at the timing of MTX-PG blood levels and time to steady state [14]. MTX-PG1 was detected 1–2 weeks after first ingestion; however, MTX-PG5 was detected after a median of 7 weeks (range 1–28 weeks). In addition the median time for MTX-PG1–5 to reach steady-state concentration was 27.5 weeks and the median time for MTX-PG1–5 to become undetectable after the last dose was 15 weeks. This highlights that MTX may take up to 6 months to achieve full clinical benefit, which is important to consider when using the levels to assess compliance or to guide dose alteration.
The main trial to be done outside the field of rheumatology was a 55-patient, prospective study into using MTX-PG levels to assess clinical response and compliance in patients with psoriasis [15]. This found the time to steady state of MTX-PG1–5 was between 12–24 weeks, and there was no significant correlation between MTX-PG levels and disease activity.
Methotrexate polyglutamate levels and inflammatory bowel disease
There have been only two studies addressing the potential use of MTX-PG levels in IBD. Egan et al. looked at the total levels when addressing the question of the optimal dose of MTX needed to induce remission in steroid-requiring IBD [16]. They found that subcutaneous initial doses of 15 and 25 mg/week in 32 patients were equally efficacious. In this cohort MTX-PG concentration reached a plateau at around 6–8 weeks after the initiation of therapy and no statistical difference was found between the levels across both doses of the drug. In addition the levels did not correlate with active disease or drug toxicity and did not change significantly after change in MTX dose.
A more recent prospective study from Brooks et al. looked specifically at MTX-PG concentrations in 18 patients with IBD that were on stable doses of MTX [8]. MTX-PG were measured on three occasions and compared to disease activity and reports of toxic side effects. MTX-PG were detected in all the patients and there was little variability in the levels over the study period. Similar to the Stamp et al. RA study [13], higher MTX-PG4&5 were associated with worse disease activity as well as higher toxic effects.
The cohort was small and heterogeneous with different doses of MTX prescribed (median 20 mg/week) and varied administration methods (oral, subcutaneous and via percutaneous endoscopic gastrostomy tube), which is likely to have had a bearing on the results. The data from a similar cohort was presented at Digestive Diseases Week in 2014 [17], which concluded that MTX-PG could be useful in assessing adherence. A non-significant trend showed higher concentrations were associated with active disease, but this may be due to higher doses of MTX being used in those with active disease.
Summary
Methotrexate is an established treatment for IBD. It is an efficacious and well tolerated therapeutic option in CD, particularly when administered SC. More studies are ongoing in the UC population. Measuring MTX-PG levels in RBC has the potential to not only monitor compliance but also correlate with disease activity and toxicity. Two large studies in patients with RA have produced conflicting results but in the small, IBD trials, higher MTX-PG levels, particularly MTX-PG4&5 correlated with increased disease activity and toxicity. It is important, however, to be aware that MTX-PG are influenced by other factors, particularly age and renal function, and may take up to 6 months to reach steady state.
Future trends and developments
Measuring drug levels plays an important role in the management of patients with IBD, as demonstrated by the monitoring of thioguanine nucleotides in those prescribed azathioprine [18]. Measuring MTX-PG offers an exciting step towards individualizing drug treatment and reducing toxicity in those taking MTX. However, at the moment there is a lack of substantial evidence to support the use of measuring MTX-PG levels in IBD, aside from monitoring compliance [19]. A large, prospective trial is warranted to determine clinical benefit before widespread use in the IBD population is advocated.
References
1. Kozarek RA, Patterson DJ, Gelfand MD, et al. methotrexate induces clinical and histological remission in patients with refractory inflammatory bowel disease. Ann Intern Med. 1989; 110: 353–356.
2. Dignass A, Van Assche G, Lindsay JO, et al. The second European evidence-based consensus on the diagnosis and management of Crohn’s disease: Current management. J Crohn’s Colitis 2010; 4: 28–62.
3. Feagan BG, Rochon J, Fedorak RN, et al. Methotrexate in the treatment of Crohn’s disease. New England Journal of Medicine. 1995; 332: 292–297.
4. Kurnik D, Loebstein R, Fishbein E, et al. Bioavailability of oral vs. subcutaneous low-dose methotrexate in patients with Crohn’s disease. Aliment Pharmacol Ther. 2003; 18(1): 57–63.
5. Feagan BG, McDonald JW, Panaccione R, et al. Methotrexate in combination with infliximab is no more effective than infliximab alone in patients with Crohn’s disease. Gastroenterology 2014; 146(3): 681–688.
6. Chande N, Wang Y, MacDonald JK, et al. Methotrexate for induction of remission in ulcerative colitis. Cochrane Database Syst Rev. 2014; 8.
7. Chande N, Abdelgadir I, Gregor J. The safety and tolerability of methotrexate for treating patients with Crohn’s disease. J Clin Gastroenterol. 2011; 45: 599–601.
8. Brooks A, Begg E, Zhang M, et al. Red blood cell methotrexate polyglutamate concentrations in inflammatory bowel disease. Ther Drug Monit. 2007; 29: 619–625.
9. Lena N, Imbert AM, Brunet P, et al. Kinetics of methotrexate and its metabolites in red blood cells. Cancer Drug Deliv. 1987; 4(2): 119–127.
10. Dervieux T, Furst D, Lein DO, et al. Polyglutamation of methotrexate with common polymorphisms in reduced folate carrier, aminoimidazole carboxamide ribonucleotide transformylase, and thymidylate synthase are associated with methotrexate effects in rheumatoid arthritis. Arthritis Rheum. 2004; 50(9): 2766–2774.
11. Dervieux T, Furst D, Lein DO, et al. Pharmacogenetic and metabolite measurements are associated with clinical status in patients with rheumatoid arthritis treated with methotrexate: results of a multicentred cross sectional observational study. Ann Rheum Dis. 2005; 64: 1180–1185.
12. Stamp LK, O’Donnell JL, Chapman PT, et al. Determinants of red blood cell methotrexate polyglutamate concentrations in rheumatoid arthritis patients receiving long-term methotrexate treatment. Arthritis Rheum. 2009; 60(8): 2248–2256.
13. Stamp LK, O’Donnell JL, Chapman PT, et al. Methotrexate polyglutamate concentrations are not associated with disease control in rheumatoid arthritis patients receiving long-term methotrexate therapy. Arthritis Rheum. 2010; 62(2): 359–368.
14. Dalrymple JM, Stamp LK, O’Donnell JL, et al. Pharmacokinetics of oral methotrexate in patients with rheumatoid arthritis. Arthritis Rheum. 2008; 58(11): 3299–3308.
15. Woolf RT, West SL, Arenas-Hernandez M, et al. Methotrexate polyglutamates as a marker of patient compliance and clinical response in psoriasis: a single-centre prospective study. Br J Dermatol. 2012; 167: 165–173.
16. Egan LJ, Sandborn WJ, Tremaine WJ, et al. A randomised dose-response and pharmacokinetic study of methotrexate for refractory inflammatory Crohn’s disease and ulcerative colitis. Aliment Pharmacol and Ther. 1999; 13: 1597–1604.
17. Ward MG, Fong S, Nasr I, et al. Higher red blood cell methotrexate polyglutamates correlate with increased disease activity, and are useful in assessing adherence. Abstract presented at Digestive Disease Week 2014.
18. Smith M, Blaker, P, Patel C, et al. The impact of introducing thioguanine nucleotide monitoring into an inflammatory bowel disease clinic. Int J Clin Pract. 2013; 67(2): 161–169.
19. Bruns T, Stallmach A. Drug monitoring in inflammatory bowel disease: helpful of dispensable? Dig Dis. 2009; 27: 394–403.
The authors
Emma L. Johnston1 MBBS BSc MRCP, Steven C. Fong1 MBBS MRCP, Anthony M. Marinaki2 PhD, Monica Arenas-Hernandez2 PhD, Jeremy D. Sanderson*1 MD FRCP
1Inflammatory Bowel Disease Centre, Dept of Gastroenterology, Guy’s and St Thomas’ NHS Foundation Trust, London, UK.
2Purine Research Laboratory, Viapath, Guy’s & St. Thomas’ NHS Foundation Trust, London, UK.
*Corresponding author
E-mail: jeremy.sanderson@kcl.ac.uk
Determination of methylphenidate and ritalinic acid in serum and saliva of patients with ADHD
, /in Featured Articles /by 3wmediaby Sophie Studer, Hans-Willi Clement, Christian Fleischhaker, Eberhard Schulz
Attention deficit hyperactivity disorder (ADHD)
What do fidgets and Johnny Head-in-the-Clouds (a fictional character from a German tale) have in common with Alexander the Great, Winston Churchill or Benjamin Franklin? For all of these, a diagnosis of ADHD would be made today [1]. The first indications of behavioural abnormalities in childhood date back to the mid-19th century. However, clear descriptions of the medical condition were first found in 1902 in the notes of the English pediatrician George Still. The characteristics he described were extreme motor unrest and “the abnormal inability to maintain concentration”, which led to failure to achieve at school. In 1932, two neurologists at the Berlin Charité Hospital, Kramer and Pollnow, described the symptoms of an illness they termed a “hyperkinetic disease”, which included the inability to appreciate danger, to follow rules, to control impulses and a lack of planning skills [2], as well as being easily distracted and showing motor hyperactivity. This was the first description of the leading symptoms of ADHD in German language, which is still valid – hyperactivity, inattentiveness and impulse control disorder. In order to be able to evaluate these characteristics and to investigate hyperactivity symptoms in a standardized way, Conners developed parent and teacher questionnaires at the end of the 60s that are still used today [3].
Most scientific papers are merely limited to attempts to explain the origin and course of the disease portrayed. Whereas these hyperactivity symptoms are actually seen to be the interaction of morphological changes already present at birth with external factors that affect the organism.
Methylphenidate (Ritalin®)
Today, stimulants such as Ritalin® in combination with psychotherapy and psychoeducation represent the method of choice for the treatment of hyperkinetic disorders. When a definite diagnosis has been made, pharmacotherapy is always indicated if the ADHD symptoms are marked, occur in many situations and when the effectiveness or practicability of psychoeducative and behavioural therapy measures are lacking. In addition, no contraindications for the individual psychostimulants must exist.
Methylphenidate (MPH) demonstrably improves the core symptoms of ADHD [4] and is one of the best-researched pediatric psychopharmaceuticals with long-term clinical experience. Nevertheless, the “pill for the troublemaker” is one of the most controversially discussed pharmacological products. A frequently mentioned point is the possible addiction potential of Ritalin®, for which reason the drug is also subject to the German controlled substances act. However, one must differentiate here between oral administration in therapeutic doses and “snorting” or intravenous application in excessive amounts.
The story of Ritalin® begins at the Swiss company Ciba, where the psychostimulant was successfully synthesized and the effectiveness of the substance proven in a self-experiment. When the drug was taken by Leandro Panizzon’s wife Marguerite (“Rita”), she made considerable progress. Ritalin®, probably the best-known MPH today, is named after her. Ciba introduced it to the market in 1954, 10 years after its development, for the treatment of psychoses, chronic tiredness and lethargy [5]. A short time later, meta-analyses became possible based on numerous study results. A distinct alleviation of symptoms was shown in about 75 % of all children treated with Ritalin® for ADHD. Alongside the reduction of hyperactivity and impulsiveness in the mentioned group, the ability for concentration and attentiveness increased considerably, also manifested in improved school achievements [6-8].
Structure and metabolism of methylphenidate
The fundamental structure of MPH is based on the phenylethylamine skeleton (Fig. 1a) and exhibits no hydroxyl group on the phenyl ring, facilitating diffusion into the central nervous system. It exhibits two chiral centres, consequently there are four configuration isomers (Fig. 1b). In practice, only the D- and L-threo forms find use in the treatment of ADHD. In the USA and in Switzerland the pure D- threo dextromethylphenidate isomer (Focalin®) is approved, and is regarded as the main pharmacologically active form. In comparison, the original Ritalin® consists of a mixture of the enantiomeric D- and L-threo forms. MPH is always manufactured in the protonated form as the hydrochloride salt [5].
The oral bioavailability of MPH is about 30 % (D-enantiomer > L-enantiomer), whereby foodstuffs have no relevant influence on the resorption. Generally available preparations reach their maximum plasma level within 1.5-2 hours. The effect is already shown after 15-30 minutes and reaches its highest level after 2-3 hours. In contrast, retard preparations such as Concerta® have a considerably longer duration of effect, which can be around 10-12 hours.
MPH is rapidly metabolized renally by carboxylesterase CES1A1 to pharmacologically inactive 2-phenyl-2-(piperidin-2-yl) acetic acid (ritalinic acid, RA). The maximum plasma level of the metabolite is 30-50 times greater than that of the original drug and the half-life is about twice as long. However, as RA possesses only a small pharmacodynamic activity, or none at all, this fact is of minor significance.
TDM of Ritalin® in children
For monitoring pharmacotherapy through concentration measurements, the collection of blood has so far been unavoidable. However, invasive methods present a compliance obstacle, particularly for children. Therefore, to ensure a high degree of drug safety, a method based on alternative body fluids for TDM is desirable. Saliva is becoming increasingly significant in this respect and is already being investigated routinely in immunology and infectious serology diagnostics, in drug and drug-abuse screening and for determining levels of the hormone cortisol [9].
The research group of Marchei et al. has already successfully developed saliva diagnostics for MPH and RA using LC-MS/MS [10]. Further investigations demonstrated almost parallel changes in the MPH and RA concentrations over the time in serum and saliva [11].
These facts, and the availability of the MassTox® TDM Series A Kit from Chromsystems, which permits the determination of the psychostimulant methylphenidate and its metabolites in serum/plasma, were the starting point for an investigation into an LC MS/MS method from Chromsystems for the determination of these analytes in saliva.
For this, serum and saliva samples from 19 ADHD patients (nine children, one adolescent and nine adults) being treated with MPH were collected and investigated. The study participants mainly took long-acting retard products, such as Medikinet retard® or Ritalin LA®. The daily intake ranged from 5 to 60 mg of MPH, corresponding to a dosage of 0.11 to 1.43 mg MPH per kilogram body weight.
As part of the routine follow-up investigations, serum was obtained by blood collection using a serum Monovette, two hours after administration of the drug where possible. In parallel, saliva samples were obtained from the patients using the Salivette system. For this, the subjects chewed on a cotton swab for 2-5 minutes during blood collection. The samples were centrifuged immediately afterwards, aliquoted and then shock frozen in liquid nitrogen to avoid degradation of the substances to be analysed.
Materials and methods
Kit for LC-MS/MS analysis: MassTox® TDM Antidepressants 2/Psychostimulants (atomoxetine, methylphenidate, mianserin, reboxetine, ritalinic acid, trazodone; (Chromsystems GmbH), methylphenidate hydrochloride C-II (Sigma-Aldrich), saliva (IBL Hamburg).
After a brief storage at -80°C, the serum and saliva samples were processed using the parameter set for Antidepressants 2/Psychostimulants for LC-MS/MS analysis and following the manufacturer’s instructions (Table 1). The calibrators and control materials for the determination of MPH in serum/plasma were also from Chromsystems. To produce a series of MPH standards in saliva, saliva (IBL Hamburg) was spiked with MPH hydrochloride (Sigma-Aldrich).
After sample preparation, the eluates obtained were separated chromatographically in an analytical column at a flow rate of 0.6 ml/min (MasterColumn® A, Chromsystems) and then quantified in a mass spectrometer (Thermo TSQ Quantum Ultra) according to their mass-to-charge ratio (Fig. 2).
The Chromsystems test is approved for the determination of psychostimulants in serum/plasma. Figure 3A shows the chromatogram of a patient who has taken Medikinet adult® at a dosage of 30 mg per day. The determination in serum gave a value of 5.5 ng/ml for MPH and 195 ng/ml for RA. As was to be expected from data in the literature, the values for the determination of MHP in saliva were considerably higher – in this case by a factor of 4 – whereas considerably lower values were determined for RA (Fig. 3B) [12].
A comprehensive verification of the determination of MPH and its acid metabolite is still to be performed. Nevertheless, initial experiments to determine the variance within a preliminary inter-assay study have already been carried out. The results are summarized in Table 2.
The values measured for saliva were only slightly poorer than the values for MPH in serum, also determined with the
MassTox® TDM Parameter Set Antidepressants 2/ Psychostimulants.
Conclusions
In order to carry out an effective pharmacotherapy with few side effects, it is necessary to establish less-invasive TDM methods. This applies to sensitive patient groups, such as children and adolescents who display distinctly different pharmacokinetic characteristics, indicating the need for a much tighter monitoring of compliance [13]. A similar situation in respect of altered metabolic characteristics can be found in patients with liver or kidney failure, who would also benefit from a less-invasive sample collection method. In summary, the data described here have shown the methodological and analytical suitability of the MassTox® TDM Series A – PARAMETER Set Antidepressants 2/
Psychostimulants in serum/plasma (Chromsystems) – for the determination of MPH and its metabolite RA by LC-MS/MS in both serum/plasma and in saliva. Thus facilitating a much more simplified way of drug monitoring in this special case of pharmacotherapy.
References
1. Krause J, Krause KH. ADHS im Erwachsenenalter. Die Aufmerksamkeitsdefizit-/ Hyperaktivitätsstörung bei Erwachsenen. 3. Aufl, Schattauer Verlag Stuttgart (2009).
2. Kramer F, Pollnow H. (1932) Über eine hyperkinetische Erkrankung im Kindesalter. Monatsschrift für Psychiatrie und Neurologie 82(1-2): 1-40.
3. Steinhausen HC. Der Verlauf hyperkinetischer Störungen. In: Steinhausen HC (Hrsg). Hyperkinetische Störungen im Kindes- und Jugendalter. Kohlhammer Verlag Stuttgart (1995).
4. Riederer P, Batra A. Neuro-Psychopharmaka. Ein Therapie-Handbuch. 2. neu bearbeitete Aufl, Springerverlag Berlin, Heidelberg (2006).
5. Kappeler T. (2007) Methylphenidat: Basics für die Apotheke. pharmaJournal 10: 4-7.
6. Kavale K. (1982) The efficacy of stimulant drug treatment for hyperactivity: a meta-analysis. J Learn Disabil 15(5): 280-9.
7. Schachter HM, Pham B, King J, Langford S. (2001) How efficacious and safe is short-acting methylphenidate for the treatment of attention-deficit. CMAJ 165(11): 1475-88.
8. Spencer T, Biederman J, Wilens T, Harding M, O’Donnell D. (1996) Pharmacotherapy of attention-deficit hyperactivity disorder across the life cycle. J Am Acad Child Adolesc Psychiatry 35(4): 409-32.
9. Chiappin S, Antonelli G, Gatti R, De Palo EF. (2007) Saliva specimen: A new laboratory tool for diagnostic and basic investigation. Clin Chim Acta 383(1-2): 30-40.
10. Marchei E, Farrè M, Pellegrini M, Rossi S, García-Algar Ó, Vall O, Pichini S. (2009) Liquid chromatography–electrospray ionization mass spectrometry determination of methylphenidate and ritalinic acid in conventional and non-conventional biological matrices. J Pharm Biomed Anal 49(2): 434-9.
11. Marchei E, Farrè M, Garcia-Algar O, Pardo R, Pellegrini M. (2010a) Correlation between methylphenidate and ritalinic acid concentrations in oral fluid and plasma. Clin Chem 56(4): 585-92.
12. Marchei E, Farrè M, Pellegrini M, Rossi S, García-Algar Ó, Vall O, Pacifici R, Pichini S. (2010b) Pharmacokinetics of methylphenidate in oral fluid and sweat of a pediatric subject. Forensic Sci Int 196(1-3): 59-63.
13. van den Anker JN, Schwab M, Kearns GL. (2011) Developmental pharmacokinetics. Handbook of experimental pharmacology 205: 51-75.
The authors
Sophie Studer, Hans-Willi Clement, Christian Fleischhaker, Eberhard Schulz
University Hospital Freiburg, Department of Child and Adolescent Psychiatry, Psychotherapy and Psychosomatics, Neuropharmacological Research Laboratory, Freiburg, Germany
Warning: over-diagnosis can seriously damage your health!
, /in Featured Articles /by 3wmediaWhilst in many less developed countries there is a paucity of diagnostic testing and appropriate therapies, we in the West are suffering from the ‘modern epidemic’ of over-diagnosis and over-treatment. Today’s highly sensitive biomarker and imaging tests increasingly identify asymptomatic or very mild conditions that if left untreated would not cause symptoms or reduce longevity. A recent report on mammography screening in the UK suggested that 19% of breast cancers were over-diagnosed, and a US task force concluded that PSA-based prostate cancer screening over-diagnosed up to 50% of tumours. Other over-diagnosed and over-treated conditions include thyroid cancers as well as a range of cardiovascular diseases, chronic kidney disease and ADHD. At best treating such subjects is an imprudent use of health service funds; at worst ‘patients’ suffer both psychological and physical harm from their diagnosis and subsequent treatment. Of course effective screening for cancer and other serious conditions is vital, but how can the problem of over-diagnosis be at least alleviated when tests (and cut-off values) must be sensitive enough to detect pathologies that really require treatment?
When diagnostic tests are evaluated for accuracy the average sensitivity and specificity are reported. But of course individuals vary, and diseases have stages of severity. What is needed is the identification of those patients for whom treatment will do more good than harm. Similarly average results in therapeutic trials may be positive, so negative effects in some patient groups are not evident, but again the potential benefit of a treatment should be weighed against possible harm according to disease severity. And subjects being screened should surely be informed about the risk of over-diagnosis. Yet in a recent random sample of 500 Australians, only 10% of the women who had had mammography, and 18% of the men who had had prostate cancer screening reported that they had been told about the limitations of these tests.
There is also an urgent need to scrutinize the panels of medical professionals setting disease definitions. Diagnostic thresholds are frequently lowered without considering the balance between good and harm of treating the additional patient group who have a lower risk or milder symptoms. And although it may sound cynical, panels with three quarters of the members having multiple ties to pharmaceutical companies – some of which will directly benefit from an increased number of patients with the disease under discussion – surely can’t be unbiased!
Hopefully appropriate action can be taken before the seemingly inexorable trend towards over-diagnosis makes patients of us all!
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