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
Ultra Fast Mass Spectrometry
, /in Featured Articles /by 3wmediaThe Aptima HIV-1 Quand Dx assay
, /in Featured Articles /by 3wmediaClinical response biomarkers in ovarian cancer: current challenges and future directions
, /in Featured Articles /by 3wmediaEffective screening strategies have not yet been developed for the early detection of ovarian cancer. The serum biomarker CA125, routinely used to aid diagnosis and monitor treatment response, is not informative in all patients. Recent analytical developments have prioritized promising candidate novel biomarkers or multi-biomarker panels for future clinical evaluation.
by E. L. Joseph, Dr M. J. Ferguson and Dr G. Smith
Introduction to ovarian cancer
Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy and the fifth leading cause of cancer related death among women with around 140 000 annual deaths worldwide. EOC can develop as one of four histotypes with the serous histotype being the most common and most aggressive. The remaining three non-serous histotypes, endometrioid, clear cell and mucinous cancers present less frequently. High grade serous ovarian cancer, heterogeneous in nature and rapidly progressive, has a poor prognosis, where a major contributing factor is the lack of ability to diagnose the disease at a sufficiently early stage to facilitate curative surgery. The 5-year survival rate is less than 30% for patients presenting with advanced disease spread beyond the ovaries (FIGO Stage 3/4), but if detected earlier combination therapy of cytoreductive surgery and adjuvant or neo-adjuvant chemotherapy with platinum and taxane-based drugs has the potential to cure 90% of patients [1]. Consequently the identification of biomarkers capable of detecting ovarian cancer at the earliest stages and monitoring disease progression are inherently important in tackling this lethal disease. Due to its prevalence, the ideal biomarker for detecting early stage ovarian cancer requires an extremely high specificity (>99%) and a minimum sensitivity of 75% [2]. Despite extensive research, no optimal ovarian cancer biomarker has yet been identified and such high specificity is unlikely to be met by a single agent. Many promising candidate biomarkers are however currently undergoing evaluation in clinical trials.
CA125
The only ovarian cancer biomarker routinely used in the clinic is cancer antigen 125 (CA125; mucin 16) currently considered the ‘gold standard’ cancer biomarker despite its limitations. In the majority of patients with EOC, expression of the CA125 glycoprotein is raised above the normal reference range (>35 U/ml blood), but it only has a sensitivity of 50% to 60% with a specificity of 90% in early stage postmenopausal patients [3]. Several factors, however, limit the utility of CA125 in routine population screening: it is not expressed in 20% of ovarian cancers, is only significantly elevated in 47% of early stage ovarian cancers (although increasing to 80–90% in advanced stage cancers) and can be raised in many benign conditions including endometriosis and peritonitis. Variability in CA125 expression throughout the menstrual cycle and in pregnancy is also a common confounding issue. A major clinical utility of CA125, however, is related to its ability to commonly reflect clinical response following chemotherapy treatment and as such is often successfully used to monitor a patient’s progress through chemotherapy. A reduction in CA125 expression during treatment is considered a positive prognostic outcome for the patient and serial serum measurements are currently used to predict therapeutic outcomes and estimate stability of the disease (Fig. 1).
Biomarkers under evaluation
There have now been multiple attempts to identify novel ovarian cancer biomarkers with varying success. The most promising serum biomarkers include HE4 and mesothelin.
HE4
Human epididymis protein 4 (HE4) has been shown to be consistently elevated above the normal level (151 pM) in ovarian cancers, with sensitivity of 95% and specificity of 73%. HE4 is differentially expressed in specific subtypes of ovarian cancer, potentially allowing clinicians to distinguish histotypes to aid treatment; HE4 was found to be overexpressed in 100% of endometrioid cancers, 93% of serous cancers but only 50% of clear cell cancers [4]. Unlike CA125, it is less likely to produce false positives in benign masses and it has also been proposed to be the best candidate biomarker for early detection of Stage I disease despite sensitivity and specificity of 46% and 95% respectively [2]. HE4 has recently obtained FDA approval in the USA for monitoring recurrence or progression of EOC and, in comparative tests, has been found to be superior to CA125 in classifying benign and borderline ovarian cancers.
Mesothelin
Mesothelin is a glycoprotein expressed by mesothelial cells, the expression of which has been found to be raised in mesothelioma, pancreatic and ovarian cancers.
It can be easily measured in both urine and serum, highlighting its potential as a non-invasive biomarker. Serum mesothelin levels were found to be increased in approximately 60% of ovarian cancers with 98% specificity.
One study found elevation of mesothelin in 42% of urine assays as opposed to 12% serum assays of early stage EOCs at 95% specificity which reinforces the potential of this glycoprotein as an early detection biomarker and the use of urine in preference to serum [2]. Higher levels of mesothelin were also found to be associated with poorer overall survival in patients following optimal debulking surgery or who have advanced stage ovarian cancer. A recent study, however, revealed that lifestyle choices such as smoking and BMI can affect mesothelin levels, which also often increase with age.
Identification of new candidate biomarkers
Due to an urgent need for better biomarkers for early detection of ovarian cancer and reliable biomarkers to monitor clinical response, ongoing efforts are focused on the application of state of the art technologies e.g. mass spectrometry and quantitative proteomic analysis to identify novel biomarkers [5]. These approaches however often generate multiple candidate biomarkers for further investigation, prioritization and clinical evaluation of which is an ongoing challenge. These methods allow comparison of multiplex biomarker panels and identification of novel differentially expressed proteins not previously linked to ovarian cancer.
Another powerful technology is microarray-based mRNA analysis which allows genome wide expression studies which have already enhanced the understanding of the genes and pathways which influence ovarian cancer progression, chemotherapy response and survival. For example, the candidate biomarkers osteopontin and kallikrein (Table 1) were discovered by this method.
Our own studies have revealed significant differences in the expression of fibroblast growth factor 1 (FGF1) and additional FGF pathway genes in ovarian cancers of different histologies (Fig. 2A) and in paired sensitive and resistant ovarian cancer cell lines (Fig. 2B). We have additionally shown that FGF1 expression is significantly inversely correlated with both progression-free (Fig. 2C) and overall survival in ovarian cancer patients [6]. We are therefore currently recruiting patients to longitudinal clinical studies to investigate whether FGF1 or additional related growth factors can predict disease progression and/or the development of treatment-limiting drug resistance.
MicroRNAs (miRNAs) are small non-coding RNAs (19–25 nucleotides) that regulate gene expression by binding to mRNA target sequences and disrupting translation [7]. MiRNAs have great potential as diagnostic and clinical response biomarkers in ovarian and additional cancers as miRNA expression can now routinely be quantitatively assessed in small biopsies and in formalin-fixed material. For example, approximately 30 miRNAs (including miR-21, miR-141, miR-203, miR-205 and miR-214) are differentially expressed in ovarian cancer [8], while miRNAs including miR-200a, miR-200b and miR-429 have also been associated with cancer recurrence and have been shown to predict survival. For example, high expression of miR-200, miR-141, miR-18a and low expression of let-7b, and miR-199a were found to predict poor survival in a cohort of 20 ovarian cancer patients [9]. Meanwhile, recent data from our own laboratory has identified multiple miRNAs including miR-125b and miR-130 associated with the development of platinum resistance. MiRNAs are particularly promising candidate biomarkers due to their stability, and abundant expression in solid cancers, whole blood and routinely collected plasma and serum samples.
Future directions
Due to the challenges of finding a single biomarker that can encompass the complexity and heterogeneity of ovarian cancer it is logical that optimization of a multi-biomarker panel may be the most practical approach, for example combining HE4 and mesothelin with CA125 to augment both sensitivity and specificity. This type of approach has recently been proposed in algorithms such as the Risk of Ovarian Malignancy Algorithm or ROMA which combines CA125 and HE4 levels with a sensitivity of 94% and specificity of 75%. [4]. Combinations of CA125 and mesothelin have also been found to detect more cancers than each biomarker alone. Several current studies have, however, suggested that combination biomarker analysis significantly increases the predictive power of CA125, but also unfortunately appears to decrease specificity. Ongoing studies therefore aim to develop improved biomarker panels suitable both for early detection and treatment guidance of ovarian cancer (Table 2). All of these results still require validation but they are indicative of the possible power of using a multi-biomarker panel in diagnostic tests and for monitoring the clinical responses of ovarian cancer.
Concluding remarks
An ideal biomarker for ovarian cancer will have a high enough sensitivity to correctly diagnose women with the disease and be specific enough to avoid false positive results. With ongoing efforts to identify biomarkers which match this ideal, hundreds of candidates with clinical relevance have been found but still require much validation before having a routine place in the clinic. It is expected that the future of ovarian cancer detection will be based on panels of combination serum-based biomarkers alongside biological imaging techniques to improve diagnosis, treatment and disease management.
References
1. Shapira I, Oswald M, Lovecchio J, Khalili H, Menzin A, Whyte J, Dos Santos L, Liang S, Bhuiya T, Keogh M, Mason C, Sultan K, Budman D, Gregersen PK, Lee AT. Circulating biomarkers for detection of ovarian cancer and predicting cancer outcomes. Br J Cancer 2014; 110: 976–983.
2. Nguyen L, Cardenas-Goicoechea SJ, Gordon P, Curtin C, Momeni M, Chuang L, Fishman D. Biomarkers for early detection of ovarian cancer. Women’s Health 2013; 9: 171–185; quiz 186–187.
3. Sarojini S, Tamir A, Lim H, LI S, Zhang S, Goy A, Pecora A, Suh KS. Early detection biomarkers for ovarian cancer. J Oncol. 2012; 15.
4. Jordan SM, Bristow RE. Ovarian cancer biomarkers as diagnostic triage tests. Current Biomarker Findings 2013; 3: 35–42.
5. Zhang B, Barekati Z, Kohler C, Radpour R, Asadollahi R, Holzgreve W, Zhong XY. Proteomics and biomarkers for ovarian cancer diagnosis. Ann Clin Lab Sci. 2010; 40: 218–225.
6. Smith G, NG MT, Shepherd L, Herrington CS, Gourley C, Ferguson MJ, Wolf CR. Individuality in Fgf1 expression significantly influences platinum resistance and progression-free survival in ovarian cancer. Br J Cancer 2012; 107: 1327–1336.
7. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116: 281–297.
8. Zhang B, Cai FF, Zhong XY. An overview of biomarkers for the ovarian cancer diagnosis. Eur J Obstet Gynecol Reprod Biol. 2011; 158: 119–123.
9. Nam EJ, Yoon H, Kim SW, Kim H, Kim YT, Kim JH, Kim JW, Kim S. MicroRNA expression profiles in serous ovarian carcinoma. Clin Cancer Res. 2008; 14: 2690–2695.
10. Yurkovetsky Z, Skates S, Lomakin A, Nolen B, Pulsipher T, Modugno F, Marks J, Godwin A, Gorelik E, Jacobs I, Menon U, LU K, Badgwell D, Bast RC, JR, Lokshin AE. Development of a multimarker assay for early detection of ovarian cancer. J Clin Oncol. 2010; 28: 2159–2166.
11. SU F, Lang J, Kumar A, NG C, Hsieh B, Suchard MA, Reddy ST, Farias-Eisner R. Validation of candidate serum ovarian cancer biomarkers for early detection. Biomark Insights 2007; 2: 369–375.
12. Zhang Z, YU Y, XU F, Berchuck A, Van Haaften-Day C, Havrilesky LJ, de Bruijn HW, van der Zee AG, Woolas RP, Jacobs IJ, Skates S, Chan DW, Bast RC, Jr. Combining multiple serum tumor markers improves detection of stage I epithelial ovarian cancer. Gynecol Oncol. 2007; 107: 526–531.
13. Gorelik E, Landsittel DP, Marrangoni AM, Modugno F, Velikokhatnaya L, Winans MT, Bigbee WL, Herberman RB, Lokshin AE. Multiplexed immunobead-based cytokine profiling for early detection of ovarian cancer. Cancer Epidemiol Biomarkers Prev. 2005; 14: 981–987.
14. Lokshin AE, Winans M, Landsittel D, Marrangoni AM, Velikokhatnaya L, Modugno F, Nolen BM, Gorelik E. Circulating IL-8 and anti-IL-8 autoantibody in patients with ovarian cancer. Gynecol Oncol. 2006; 102: 244–251.
The authors
Emma L. Joseph1 BSc; Michelle J. Ferguson2 MBChB, MD; and Gillian Smith1* PhD
1Division of Cancer Research, Medical Research Institute, University of Dundee, Dundee UK
2Tayside Cancer Centre, Ninewells Hospital & Medical School, Dundee UK
*Corresponding author
E-mail: g.smith@dundee.ac.uk
Unanswered questions about testing for low testosterone in men
, /in Featured Articles /by 3wmediaTestosterone is a steroid hormone that develops and maintains the primary and secondary sex characteristics in men. In recent years, there has been an explosive increase in prescriptions for testosterone replacement therapy (TRT) in adult men who are thought to have adult-onset hyopgonadism. This increase has been fueled by changing demographics and by increased public awareness of so-called “low-T” syndrome. Despite recent controversies about risks for cardiovascular complications in men receiving TRT, the trend of increased testing and treatment for low-T is likely to continue. This article explores current controversies and unanswered questions regarding testing for low-T in men. Topics covered include variations in reference intervals for testosterone and thresholds for interpretation of results. Controversies and questions surrounding testing for free (unbound) testosterone will also be explored. Finally, the emerging evidence regarding the roles of dihydrotestosterone and estradiol will be discussed in the context of testing for low-T.
by Dr Michael Samoszuk
Testosterone metabolites include estradiol (produced by the aromatase enzyme found in fat and other tissues such as testes) and dihydrotestosterone (DHT)-an androgenic hormone that is approximately three- to ten-times more potent than testosterone. DHT is produced from testosterone by 5-alpha reductase, an enzyme that is found primarily in hair follicles, prostate, testes, and adrenal glands but not in skeletal muscle.
In recent years, there has been an explosive increase in prescriptions (Figure 1) for testosterone replacement therapy (TRT) in men who are thought to have adult onset hypo-gonadism, a condition that is often referred to as “low-T”. This condition is characterized by a variety of signs and symptoms, including loss of body hair, accumulation of visceral and body fat, loss of skeletal muscle, anemia, mood disturbances, loss of libido, and erectile dysfunction. Because of changing demographics and increased awareness of low-T due to marketing campaigns, it is likely that testing for (and treatment of) low-T will continue to increase significantly in the next five years. This increased testing and treatment for low-T will probably occur despite recent controversies about the cardiovascular risks that may be associated with TRT.
How should total testosterone levels be interpreted in men being tested for low-T?
There is considerable variation in the reference intervals for total testosterone assays that are produced by various manufacturers of in vitro diagnostics (Table 1). It is notable that the reference intervals are based on the range of values between the 5th-95th percentiles of men of various ages. Because the populations of men that were used to derive these reference intervals are poorly defined with respect to age distribution and possible symptoms of low-T, it is unclear if these reference intervals provide a reliable basis for interpreting test results from men being tested for low-T. Of particular concern are the lower limits of the reference intervals, which may be too low to identify the significant proportion of men who are truly hypo-gonadal but whose total testosterone levels fall above the 5th percentile of the reference range.
Reference intervals for total testosterone levels reported by reference laboratories also have considerable variation (Table 2). The variation is of particular concern at the low end of the reference interval because many clinicians use this value to determine whether or not a man should be diagnosed as having low-T. Unanswered questions regarding the use of reference intervals that are reported by clinical laboratories include:
An emerging way to interpret total testosterone levels in men is to use a clinical threshold (cut-off) value for the level. Although there are significant differences in the definitions of the clinical thresholds (Table 3), it appears that the clinical threshold for diagnosing low-T probably lies somewhere between 300-500 ng/dL. Notably, this range lies considerably above the 5th percentiles for testosterone values that are listed in Tables 1 and 2. It should also be noted that all sources of the clinical thresholds listed in Table 3 also recommend the primacy of clinical signs and symptoms when interpreting total testosterone values.
Unanswered questions regarding the use of clinical threshold values are:
Should free testosterone be measured?
Testosterone circulates in the blood in a free (unbound) form and a bound form. Sex-hormone binding globulin (SHBG) and albumin are the primary sources of binding of testosterone.
Approximately 2% of total testosterone circulates in the free form. Current thinking is that the free testosterone is mostly responsible for the biological activity of the hormone, and the bound form is thought to be mostly inactive. The free and bound forms can be directly measured by a variety of methods, or a mathematical formula can be used to calculate the percentage of free testosterone, based on the values for total testosterone, SHBG and albumin.
There is substantial confusion over the best way to determine free testosterone and how to interpret the results. Unanswered questions include:
Is there a role for testing for DHT?
Testing for DHT is not commonly performed in the evaluation of men being evaluated or treated for hypo-gonadism. At this time, it is not clear how to interpret the test results. There is some evidence, however, that treatment of low-T with topical testosterone preparations can sometimes preferentially elevate the DHT levels due to the presence of 5-alpha reductase in skin and hair follicles. In theory, this phenomenon could account for so-called treatment failures of men who receive topical therapy. It is likely that as our understanding of TRT improves, there will be an increased interest in clinical testing for this metabolite of testosterone.
Is it necessary or helpful to measure estradiol in men being evaluated or treated for low testosterone?
There is now considerable evidence that estradiol levels in men play an important role in modulating the effects of testosterone on sexual function, body fat, lean muscle mass, and bone density. Nevertheless, estradiol levels are still not commonly measured in men who are being evaluated or treated for low-T. This is because the interpretive criteria for such testing are still not well understood. In some men, estradiol levels are measured in order to determine if TRT is causing an increase in estradiol due to aromatization of the testosterone. This can lead to symptoms of high estradiol such as bloating, fluid retention, and breast tenderness. Some experts now recommend calculating a ratio of testosterone to estradiol, but this approach is not yet widely accepted. Nevertheless, it is likely that clinical laboratories will experience an increased demand for estradiol testing in men as our understanding and prevalence of TRT increase.
Conclusion
From the preceding discussion, it should be apparent that our understanding of laboratory testing for low-T in men lags considerably behind the growing demand for testing and treatment of low-T. Clinical laboratories, manufacturers of in vitro diagnostic tests, and clinicians should be aware of the many unanswered questions in this field. They should also begin to prepare to educate themselves about the important changes in this field that are likely to occur in the next few years. For further details about this subject, the interested reader is referred to the following sources.
References
Bhasin S, Cunningham GR, Hayes FJ, Matsumoto AM, Snyder PJ, Swerdloff RS, Montori VM. Testosterone therapy in adult men with androgen deficiency syndromes: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2006: 91, 1995-2010.
Finkelstein JS, et al. Gonadal steroids and body composition, strength and sexual function in men. N Engl J Med 2013: 369, 1011-22.
Morgentaler A, Khera M, Maggi M, Zitzmann M. Commentary: Who is a candidate for testosterone therapy? A synthesis of international expert opinions. J Sex Med 2014: 11, 2636-45.
www.peaktestosterone.com A testosterone and men’s health blog that is an excellent source of information and peer-reviewed publications on this topic.
The author
Michael Samoszuk, M.D.
Chief Medical Officer
Beckman Coulter Diagnostics
Sekisui Diagnostics – Your global partner for over 30 years
, /in Featured Articles /by 3wmediaSekisui Diagnostics is a global diagnostics manufacturer focused on the clinical chemistry, coagulation, infectious disease, and enzyme markets. Headquartered in Lexington, MA, we have 10 facilities in 6 countries and over 500 employees worldwide. We are dedicated to delivering differentiated products, instrument systems, and services that support the improvement of patient care worldwide.
Formerly Genzyme Diagnostics, we changed our name in 2011 when we began a fresh chapter as part of the Sekisui Medical family. We remain focused on innovation and are now supported by the resources of our global parent corporation. We are a diverse company with broad product lines, a global sales and distribution network, extensive product development capabilities, state of the art manufacturing facilities, and deep diagnostics expertise.
Our core competency is to work with healthcare professionals and diagnostic manufacturers to deliver high-quality diagnostic products that help improve patient health.
Today’s healthcare professionals have a challenging job. They need to deliver quality, cost-effective test results that support patient care and facilitate effective outcomes. Accurate, reliable diagnostic tests and systems from Sekisui Diagnostics can help provide the quality results doctors, patients, and researchers expect in a fast, cost-effective, and responsible way.
Our broad product lines include:
Diagnostic manufacturers are also seeking quality products and services to best meet the needs of their customers. Manufacturers require a reliable partner with a broad portfolio developed, produced, and tested to exacting standards. We are a leading provider of quality products, supplying both off-the-shelf enzymes and reagent kits as well as customized materials and formats to an extensive number of regional and global partners. We also offer contract development and manufacturing services to meet technical specifications and branding requirements. All are backed by manufacturing and distribution operations in North America, Europe, and Asia.
By partnering with us, manufacturers benefit from our:
Today the world is becoming a smaller place, and we are more and more connected to each other. The linkage between communities around the world offers unprecedented opportunities for development and improvement of health and living standards. Globalization also brings risks, as it increases the prevalence of diseases such as diabetes, cardiovascular conditions, infectious diseases, and cancer. We are committed to taking an active role addressing these challenges by applying innovative technology to support the development of diagnostic products that can help improve global health.
Sekisui Diagnostics—Your global partner for the next 30 years, and beyond.
Carbapenemases: a major threat to healthcare
, /in Featured Articles /by 3wmediaBacterial resistance to antibiotics is a major health and economic problem recognized today by national and international policy makers. The Enterobacteriaceae belong to the commensal human gut flora and are frequently the cause of community- and healthcare-associated infections (HAI). Infections with Klebsiella pneumoniae are usually hospital-acquired and occur primarily in patients with impaired host defences while Escherichia coli are mostly involved in urinary tract infections. Acinetobacter and Pseudomonas species are opportunistic pathogens frequently isolated from healthcare settings where they cause life-threatening infections particularly in immunocompromised patients.
For several years, in Europe and worldwide, Enterobacteriaceae, mainly K. pneumoniae and E. coli but also non-fermenting bacteria (Acinetobacter baumannii and ) have become resistant to the main antibiotics, i.e. ß-lactam antibiotics, fluoroquinolones and aminoglycosides [1]. In particular, the US “Center for Disease Control and Prevention” (CDC) pointed the carbapenemase-producing Enterobacteriaceae (CPE) (or Carbapenem Resistant Enterobacteriaceae, CRE) among the three microorganisms exhibiting the most urgent health risk. Carbapenemases are indeed enzymes that inactivate ß-lactam antibiotics like carbapenems that currently constitute the last resort for treating multi-drugs resistant Gram-negative bacteria. Moreover, production of carbapenemases in these bacteria is most often associated with the expression of additional resistance mechanisms to other classes of antibiotics such as aminoglycosides, fluoroquinolones and cotrimoxazole, leading to bacteria resistant to all available antibiotics, so-called pan-resistant bacteria [2].
Carbapenemases resistance
Carbapenems are a class of broad-spectrum ß-lactam antibiotics with very broad activity and have therefore become the empirical treatment of choice in countries where infections due to Extended-Spectrum ß-Lactamases-producing bacteria are common. Resistance to carbapenems in Enterobacteriaceae is linked to either decreased permeability because of porine alteration or loss combined with production of a ß-lactamase with poor carbapenemase activity or, more worryingly, to the enzymatic breakdown of the antibiotic by a carbapenem-hydrolyzing ß-lactamase [3].These so called carbapenemases can hydrolyse and hence deactivate several kinds of antibiotics ranging from penicillins to cephalosporins, monobactams and carbapenems.
The most frequent carbapenemases in Enterobacteriaceae reported in Europe belong to three molecular classes according to the Ambler classification:
– class A carbapenemases hydrolyse almost all ß-lactams including carbapenems. Klebsiella pneumoniae carbapenemase (KPC) are the most frequent enzymes of this group that show a very high spreading capability [4] . KPC, contrary to other class A ß-lactamase, is inhibited by boronic acid and its derivatives.
– class B carbapenemases, belonging to metallo-ß-lactamases, including Verona integron-encoded metallo-ß-lactamase (VIM), IMP and the New Delhi metallo-ß-lactamase-1 (NDM-1) can hydrolyse all ß-lactams but monobactams. These enzymes are inhibited in vitro by EDTA and dipicolinic acid that are used in confirmatory tests for the presence of metallo ß-lactamases (MBL). NDM is the most frequent MBL present in Enterobacteriaceae. Originally detected in the Asian subcontinent, NDM is now spreading worldwide and causing outbreaks in Western countries.
– class D carbapenemases including the OXA carbapenem-hydrolysing oxacillinases hydrolyse penicillins but show lower activity against carbapenems, and no activity against extended-spectrum cephalosporins [5)]. OXA-48 is the main enzyme of this family and is now spreading worldwide even in the community although only a few cases are reported in the USA. OXA-48 hydrolyses all penicillins including temocillin. The resistance to temocillin primarly evidenced by a Belgian group [6] is now recommended as a marker of the possible presence of OXA-48. Resistance to carbapenems in OXA-48-producing CPE is variable with minimal inhibitory concentration againt carbapenems varying between less than 0.5 µg/mL to more than 256 µg/mL. This mechanism is very difficult to detect and no confirmatory test currently exists as OXA-48 is not specifically inhibited by clavulanic acid, boronic acid or EDTA. To date, only expensive molecular tests are able to confirm the presence of OXA-48. The rapid and global expansion of CPE is a threat to healthcare and patient safety worldwide, as it seriously curtails the ability to cure infections. Infections due to CPE are associated with higher in-hospital morbidity and mortality [7].
Carbapenemases epidemiology
According to the report summarizing the results from 39 European countries [8], six levels of occurrence of CPE have been defined i.e. endemic situation (level 1), inter-regional spread (level 2), regional spread (level 3), sporadic hospital outbreaks (level 4), single hospital outbreaks (level 5) and sporadic occurrence (level 6).
Nevertheless, specific occurrence may also vary depending on the type of CPE.
Discussion
Carbapenemase-producing Enterobacteriaceae (CPE) are an emerging threat to healthcare and are frequently resistant to many other antibiotics besides carbapenems leaving few treatment options [9, 10]. Rapid diagnostic tests that can be performed directly on clinical specimens or blood cultures are urgently needed in order to save an average of 24 hours compared to the results obtained by culture. Some commercial tests of this type targeting carbapenemases from CPE currently exist and are based either on molecular amplification of specific genes associated with resistance or on molecular hybridization.
Some tests are able to target all carbapenemases of clinical interest, or other resistance mechanisms alongside with accurate species identification.
However, these tests require specific equipment and are extremely expensive (80 Euros being an average price).
Multiplex real-time PCR tests allow the detection of the genes encoding for the main carbapenemases directly from samples or feces but do not detect all variants of the genes of interest in a single operation.
Other molecular biology tests can be performed on isolated colonies from culture. These tests are costly (average cost of 40 €), quite labour-intensive and often do not deliver results before the next morning, when the susceptibility testing is already available. Molecular biology tests only partially meet the needs in carbapenemase identification. Either these tests do not cover the complete range of targets or cannot distinguish between different carbapenemases and, in any case, do not give information on the level of gene expression and thus the level of bacterial resistance. The high price of these techniques and/or the need for expensive equipment, dedicated areas and specially trained personnel restrict their use to a limited number of specialized laboratories.
Rapid phenotypic tests directly performed on bacterial colonies and based on the hydrolysis of a carbapenem with colorimetric shift are now available at a reasonable price. MALDI-TOF mass spectrometry is also proposed, however, this technology requires the use of expensive equipment together with specific software analysis. All the above phenotypic tests only partially meet the needs of clinical laboratories. On the one hand, most of them require the use of antibiotics with stability problems, and secondly, the time for obtaining a result with these tests is not totally satisfactory in terms of integration into the laboratory workflow that would ensure results in a short time and allow quick decision for optimal impact. On the other hand these phenotypic tests do not identify the exact type of carbapenemase and ideally require subsequent procedures using a molecular method to achieve identification. Among CPE, OXA-48 represents the most challenging resistance mechanism to be identified that would need a rapid and easy to use test to be performed in routine labs.
References
1. Carbapenemase-producing bacteria in Europe. Interim results from the European Survey on carbapenemase-producing Enterobacteriaceae (EuSCAPE) project 2013.
2. Souli M, Galani I, and Giamarellou H. Emergence of extensively drug-resistant and pandrug-resistant Gram-negative bacilli in Europe. Euro Surveillance 2008; 13(47).
3. Nordmann P, Naas T, and Poirel L. Global spread of carbapenemase producing Enterobacteriaceae. Emerging Infectious Diseases 2011; 17(10): 1791–1798.
4. Naas T, Cuzon G, Villegas M-V, Lartigue M-F, Quinn JP, and Nordmann P. Genetic structures at the origin of acquisition of the ß-lactamase blaKPC gene Antimicrobial Agents and Chemotherapy 2008; 52(4): 1257–1263.
5. Nordmann P, Naas T, and Poirel P. Global spread of carbapenemase producing Enterobacteriaceae. Emerging Infectious Diseases 2011; 17(10): 1791–1798.
6. Glupczynski Y, Huang TD, Bouchahrouf W, Rezende de Castro R, Bauraing C, Gérard M, Verbruggen AM, Deplano A, Denis O, Bogaerts P. Rapid emergence and spread of OXA-48-producing carbapenem-resistant Enterobacteriaceae isolates in Belgian hospitals. Int J Antimicrob Agents 2012; 39(2):168-72.
7. Borer A, Saidel-Odes L, Riesenberg K, Eskira S, Peled N, Nativ R, et al. Attributable mortality rate for carbapenem-resistant Klebsiella pneumoniae bacteremia. Infect Control Hosp Epidemiol 2009, 30:972–6.
8. Carbapenemase-producing Enterobacteriaceae in Europe: a survey among national experts from 39 countries, February 2013, Euro Surveill. 2013;18(28):pii=20525.
9. Cantón R, Akóva M, Carmeli Y, Giske CG, Glupczynski Y, Gniadkowski M, et al. Rapid evolution and spread of carbapenemases among in Europe, Clin Microbiol Infect. 2012;18(5):413–31.
10. Hawkey PM. The growing burden of antimicrobial resistance, J Antimicrob Chemother. 2008;62(Suppl 1):i1–i9.
The authors
Isabelle OTE, Laetitia AVRAIN, Pascal MERTENS, Thierry LECLIPTEUX
R&D Department, Coris BioConcept,
Parc Scientifique Crealys,
29A, rue Jean Sonet,
B-5032 Gembloux, Belgium
Rapid and simultaneous analysis of multiple drugs in hair samples using dynamic multiple reaction monitoring
, /in Featured Articles /by 3wmediaHair analysis for forensic diagnostics is gaining popularity in both research and applied settings. Commercially available dynamic multiple reaction monitoring (Dyn-MRM) software applied to hair samples can provide drug-use history for several months. The cost-effective drug test using Dyn-MRM software facilitates analysis of over 200 analytes in a 10-minute chromatographic run.
by Professor D. P. Naughton and Professor A. Petróczi
Background
Considerable global efforts are expended to address substance abuse which has major effects on public health and quality of life, as well as on economic and societal prosperity. This grand challenge impacts on a wide range of healthcare, regulatory and research endeavours. Key examples include destruction of lives through abuse of class A drugs, efforts to reduce doping in sport, attempts to address alcohol abuse and the lethal dangers of ‘legal highs’ (novel psychoactive substances).
Healthcare and regulatory officials engage in a wide range of activities to combat substance abuse. These include criminalization, banning substances in sport, education programs to prevent and efforts to understand and alter drug-related behaviour. However, in many cases there is an unmitigated failure to address the issues around substance misuse or abuse.
For example, for doping in sports, where vast efforts and resources are expended, indirect assessment of doping produces prevalence figures some 10-fold higher than positive doping test rates [1]. These frequent prevalence reports, at odds with figures from analytical tests, corroborate the belief that current anti-doping testing regimes are far from adequate [1]. Using doping in sport as an exemplar, major improvements in approaches to test for prohibited substances are needed. The advent of more advanced instrumentation aids testing in a number of ways. Increased sensitivity and affordability are very important but so are software developments that provide capability to monitor several hundred substances in one liquid chromatography-tandem mass spectrometry (LC-MS/MS) cycle of less than 10 minutes. These advances bring opportunities that require both instrumentation updates and frequent training updates for staff.
Hair analysis
Despite major advances in instrumentation and software, there are still obstacles to performing successful drug tests that benefit the drug taker. In sport, doping practices are frequently highly advanced, with some athletes taking heed of in-depth knowledge about most key parameters including generic testing methods, masking drugs and advances in detection for specific substances. The burden of proof has shifted to acquiring samples both in and out of competition as well as ensuring that appropriate tests are performed on each sample [2]. Owing to varied pharmacokinetics, analyte distribution and analytical procedures used, testing for a wide range of drugs in all samples is prohibitive [3]. Current testing approaches using biofluids impart considerable practical and financial consequences for the testing regime. Furthermore, cases of microdosing, masking and using novel substances make life challenging for the anti-doping officials.
Drug testing based on biofluids presents a number of issues that add considerably to cost but also are restrictive in terms of the number of tests required to cover a suitable duration owing to the pharmacokinetic profiles of many drugs. Where drugs or their metabolites are washed out efficiently after cessation of use, detection is less viable. The relatively short half-lives of many substances means the window for detection can be limited, which affects the success of occasional testing. The cost of supervised sampling along with the requirement for biofluid storage and handling to avoid sample corruption or infection is prohibitive for major levels of testing. Focusing on doping in sport, further complexities arise through variations in the lists of prohibited substances for testing in and out of competition [4]. The advantage of a longer window of detection via hair-analysis is suited to out of competition testing where a cumbersome system currently exists for sampling which is intrusive and controversial [5]. Thus, new approaches that allow a single test to be conducted simultaneously for (i) a wide range of substances and (ii) covering a prolonged period such as a 3-month window, would be valuable in sport for out of competition testing but also, beyond sport, for social drugs and new psychoactive substances.
In contrast to drug tests based on biofluids, hair analysis provides a range of advantages including: ease of sampling, ability to conduct multiple tests on one cut-hair sample to cover a prolonged duration (a typical 3-cm hair sample is equivalent to approximately 3 months’ growth), lack of issues with infection risk, facile storage at room temperature, lack of requirement to process tissue containing genetic data, and good stability of many drugs and metabolites in the hair matrix.
Instrumentation advances
We recently reported a hair-based method, using liquid chromatography–tandem mass spectrometry (LC-MS/MS), for the analysis of substances of forensic nature [6]. The multi-drug/metabolite assay employs a dynamic multiple reaction monitoring (Dyn-MRM) method using proprietary software [7, 8]. It allows both screening and validated confirmatory analysis depending on the focus of the investigation. This approach has several benefits: (a) the Dyn-MRM software is suited to screen over 200 compounds on a single chromatographic run of under 10 minutes, (b) full validated methods for compounds can be incorporated into the software, (c) hair samples provide the opportunity to cover longer windows for detection in one test (e.g. approximately 3-month history covered in a 3-cm sample), and (d) the software is designed to allow ready adoption of new compounds of interest. The advantage of Dyn-MRM is that multiple reaction monitoring is employed with a focus on scanning for specific peaks at their selected elution times. This efficient method allows the analysis of large numbers of analytes simultaneously in a short run (Fig. 1). In our report, the proprietary software has been extended and applied to cover a range of drugs and metabolites of interest to forensic investigations including cognitive enhancers, amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine, opioids, steroids and sedatives. The chromatographic run is calibrated by a test mixture containing approximately 20 substances and further tailoring would be required to match a specific remit such as the WADA (World Anti-Doping Agency) prohibited list more closely [4].
Conclusion and future perspectives
The application of Dyn-MRM software to screen for a large range of drugs brings considerable advantages to laboratories involved in drug testing. The ease of use and ability to add new compounds to the screening database are noteworthy. Coupling this commercially available software to hair analysis adds the extra dimension of being able to screen for drug use over several months in one hair sample. This advance will add considerably to the efficiency of drug testing but will remain as an adjunct to other testing methods for out of competition testing in sport as it will not cover all analytes of interest to anti-doping officials [4]. Some substances are unlikely to be found in hair (e.g. performance enhancing peptides) and for other substances there will be issues with establishing a threshold – either for endogenous substances (e.g. testosterone) or for substances consumed through diet (e.g. drugs used in farming). Further limitations are that (i) a single use of a drug may be undetectable owing to the low levels deposited in hair, and (ii) more research is warranted to ascertain the effects of hair type and colour on analyte uptake and stability. In spite of these limitations, hair analysis coupled to modern advances in instrumentation sensitivity and software capabilities is promising in many scenarios especially to obtain a prolonged history of abuse and where ‘zero tolerance’ is applied (e.g. for synthetic steroids). In addition, hair analysis may have a role in support of the Athlete Biological Passport through analysis of indirect biomarkers of doping [9].
References
1. de Hon O, Kuipers H, van Bottenburg M. Prevalence of doping use in elite sports: A review of numbers and methods. Sports Med. 2015; 45(1): 47–69.
2. World Anti-Doping Agency (WADA). International Standards. 2015; https://www.wada-ama.org/en/what-we-do/international-standards.
3. Maennig W. Inefficiency of the anti-doping system: Cost reduction proposals. Subst Use Misuse 2014; 49(9): 1201–1205.
4. WADA. List of prohibited substances and methods. 2015; http://list.wada-ama.org/.
5. Hanstad DV, Loland S. Elite athletes’ duty to provide information on their whereabouts: Justifiable anti-doping work or an indefensible surveillance regime? Eur J Sport Sci. 2009; 9(1): 3–10.
6. Shah I, Petroczi A, Uvacsek M, Ranky M, Naughton DP. Hair-based rapid analyses for multiple drugs in forensics and doping: application of dynamic multiple reaction monitoring with LC-MS/MS. Chem Cent J. 2014; 8(1): 73.
7. Agilent Technical Overview. Ion optics innovations for increased sensitivity in hybrid MS systems. Agilent Technologies USA 5989-7408EN. 2007; http://www.chem.agilent.com/Library/technicaloverviews/Public/5989-7408EN_HI.pdf.
8. Stone P, Glauner T, Kuhlmann F, Schlabach Tim, Miller K. New dynamic MRM mode improves data quality and triple quad quantification in complex analyses. Agilent Technologies USA 5990-3595EN. 2009; http://www.chem.agilent.com/Library/technicaloverviews/Public/5990-3595en_lo%20CMS.pdf.
9. Vernec AR. The athlete biological passport: an integral element of innovative strategies in antidoping. Br J Sports Med. 2014; 48(10):817–819.
The authors
Declan P. Naughton* PhD, Andrea Petróczi PhD
School of Life Sciences, Kingston University, London, UK
*Corresponding author
E-mail: D.Naughton@kingston.ac.uk
Therapeutic drug monitoring of mycophenolic acid and its glucuronide by HPLC/UV
, /in Featured Articles /by 3wmediaA simple and rapid method for simultaneous determination of mycophenolic acid (MPA) and its glucuronide (MPAG) in plasma using high-performance liquid chromatography (HPLC) with UV detection is described. MPA is an immunosuppressant used in kidney, liver and heart transplantation to prevent organ rejection. Owing to MPA’s narrow therapeutic window and considerable variability within and between patients, the routine monitoring of MPA concentrations is suggested.
by C. Misch and Prof. P. Tang PhD
Background
Mycophenolate mofetil (MMF) and enteric-coated mycophenolate sodium (EC-MPS) are widely used to prevent organ rejection after organ transplantation. Following administration, both prodrugs are rapidly hydrolysed to mycophenolic acid (MPA), the active immunosuppressant. MPA is able to suppress the synthesis of guanosine nucleotides in T and B lymphocytes, principally via noncompetitive, selective and reversible inhibition of inosine monophosphate dehydrogenase. MPA is primarily metabolized by the uridine diphosphate glucuronyl transferase to an inactive glucuronide (MPAG), which is transported from liver into bile. Biliary MPAG then enters the gastrointestinal (GI) tract, where it is converted back to MPA, which is then recycled into the bloodstream via the enterohepatic circulation pathway. Several studies have documented that variation in MPA plasma concentrations are unpredictable and variability in plasma concentrations of MPA within and between individuals are high [1–4]. The highly variable set of patient situations on MPA therapies can cause variable risk for adverse effects such as hematologic and GI toxicity. Therapeutic drug monitoring (TDM) of MPA and MPAG can aid clinicians develop personalized therapy strategies to avoid toxicity and maintain efficacy.
For measuring MPA and MPAG concentrations in biological samples, high-performance liquid chromatography (HPLC) has been the primary technique. Scrutinizing all reported technologies, mass spectrometry is generally superior in sensitivity, selectivity and specificity to other detectors. However, the purchase, maintenance and running costs of mass spectrometry are high. From an economic standpoint, HPLC/UV methods [5–10] allow cost-effective assay while provide adequate sensitivity, selectivity and specificity for measuring clinically relevant concentrations of MPA (0.5–5 μg/mL) and MPAG (5–100 µg/mL). The intent of this application was to develop a simple and rapid HPLC/UV method for the determination of MPA and MPAG concentrations in plasma.
Experimental details
Apparatus and materials
The instrument and analytical conditions are listed in Table 1. MPA and internal standard clonazepam were obtained from Sigma (St. Louis, MO). MPAG was from TRC (Toronto Research Chemicals). All other chemicals used were analytical grade or HPLC grade. Separate stock solutions of clonazepam, MPA and MPAG were prepared by accurately weighing and dissolving it in an appropriate amount of methanol.
Calibration/sample preparation
For constructing calibration curves, the concentration ranges of MPA and MPAG were set to 0.1–20 and 1–200 µg/mL, respectively. To 0.1 mL of blank plasma, 0.1 mL each of clonazepam, MPA, MPAG and methanol were added; the mixture was vortex-mixed for 1 min. After centrifugation for 10 min at 10 000 rpm, the supernatant was transferred to an autosampler vial. To 0.1 mL of patient plasma, 0.1 mL clonazepam and 0.3 mL methanol were added and processed as stated above
Results and discussion
Chromatographic separation
A typical chromatogram is presented in Figure 1. These compounds resolved without any overlapping of their peaks or ambiguity in identification. All compounds were eluted within 14 min. No interference was observed in patient samples containing endogenous matrix components, metabolites, xenobiotics and concomitant medication (see Table 2).
Linearity
Good linearities (1/x weighted) were obtained for MPA and MPAG with coefficient of determination (r2) values >0.990 from 0.1 to 20 µg/mL (for MPA) or 1 to 200 µg/mL (for MPAG). The percentage deviation was <15%. Method validation
Method accuracy and precision data are presented in Table 3. Overall the percentage recovery of MPA and MPAG ranged from 93 to 105%, indicating the consistent, precise, and reproducible extraction efficiency of the method. Both within-run (n=6) and between-run (n=30) precisions were <9%. Comparison between two HPLC-UV methods
Figure 2a and 2b illustrate comparisons between the current method and reference method. The reference method was also based on a HPLC-UV procedure. The correlation between the two methods was good; the linear regression statistics indicated both r2 values >0.990 (P<0.0001). The linear regression equation for MPA correlation was y = 1.018 x + 0.031 with a standard error value of 0.24; where y, the current method and x, the reference method. The linear regression equation for MPAG correlation was y = 0.984 x − 0.292 with a standard error value of 5.08.
MPA and MPAG concentrations in plasma
Figure 3 illustrates considerable variability of MPA and MPAG concentrations in patient plasma. MPA concentrations ranged from 0.3 to143 µg/mL; MPAG concentrations ranged from 1.2 to 457 µg/mL; MPAG : MPA mole ratio ranged from 0.5 to 186. The mean values for MPA, MPAG and MPAG : MPA were 9.5 µg/mL, 62.3 µg/mL and 13.5, respectively. Clearly, this assay can aid clinicians develop personalized therapy strategies to avoid toxicity and maintain efficacy.
Conclusion
This method includes single dilution step, protein precipitation, ultracentrifugation and gradient chromatography. Sample preparation is rapid and efficient. This method avoids the use of more complex liquid–liquid extraction or solid-phase extraction procedure, which substantially decreases set-up time. This method has been applied to measure MPA and MPAG concentrations in plasma for pharmacokinetic studies and for monitoring clinical use of MPA prodrugs.
References
1. Bullingham RE, Nicholls AJ, Kamm BR. Clinical pharmacokinetics of mycophenolate mofetil. Clin Pharmacokin. 1998; 34: 429–455.
2. Shaw LM, Korecka M, et al. Mycophenolic acid pharmacodynamics and pharmacokinetics provide a basis for rational monitoring strategies. Am J Transplant. 2003; 3: 534–542.
3. Staatz CE, Tett SE. Clinical pharmacokinetics and pharmacodynamics of mycophenolate in solid organ transplant recipients. Clin Pharmacokinet. 2007; 46:13–58.
4. Cattaneo D, Baldelli S, Perico N. Pharmacogenetics of immunosuppressants: progress, pitfalls and promises. Am J Transplant. 2008; 8: 1374–1383.
5. Indjova D, Kassabova L, Svinarov D. Simultaneous determination of mycophenolic acid and its phenolic glucuronide in human plasma using an isocratic high-performance liquid chromatography procedure. J Chromatogr B Analyt Technol Biomed Life Sci. 2005; 817: 327–330.
6. Patel CG, Akhlaghi F. High-performance liquid chromatography method for the determination of mycophenolic acid and its acyl and phenol glucuronide metabolites in human plasma. Ther Drug Monit. 2006; 28: 116–122.
7. Bahrami G, Mohammadi B. An isocratic high performance liquid chromatographic method for quantification of mycophenolic acid and its glucuronide metabolite in human serum using liquid-liquid extraction: application to human pharmacokinetic studies. Clini Chim Acta. 2006; 370: 185–190.
8. Mino Y, Naito T, et al. Simultaneous determination of mycophenolic acid and its glucuronides in human plasma using isocratic ion pair high-performance liquid chromatography. J Pharm Biomed Anal. 2008; 46: 603–608.
9. Watson DG, Araya FG, et al. Development of a high pressure liquid chromatography method for the determination of mycophenolic acid and its glucuronide metabolite in small volumes of plasma from paediatric patients. J Pharm Biomed Anal. 2004; 35: 87–92.
10. Westley IS, Sallustio BC, Morris RG. Validation of a high-performance liquid chromatography method for the measurement of mycophenolic acid and its glucuronide metabolites in plasma. Clin Biochem. 2005; 38: 824–829.
The authors
Catherine Misch MLT and Peter Tang* PhD
Department of Pathology and Laboratory Medicine, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA
*Corresponding author
E-mail: peter.tang@cchmc.org
New psychoactive substances pose a challenge for drug testing laboratories
, /in Featured Articles /by 3wmediaNew psychoactive substances (NPS) reach the recreational drugs market at a fast pace and are of concern because of potential health risks. In addition to not being legally regulated, NPS escape detection in standard drug tests. Drug testing laboratories, therefore, must adapt their analytical methods to also cover these new substances. For screening and confirmation of NPS, mass-spectrometric multicomponent methods are useful.
by Prof. Olof Beck and Prof. Anders Helander
New psychoactive substances
The emergence of new drugs of abuse that are designed to circumvent narcotics legislation by slight chemical structural modifications of already classified drugs represents an ever increasing problem [1, 2]. Nowadays, this phenomenon is commonly termed ‘new psychoactive substances’ or ‘NPS’, but also other names such as designer drugs, legal highs, research chemicals, smart drugs, bath salts, and spice have been and are used. The NPS problem is of global concern but may vary in extent between countries, partly due to national differences in legislation and drug culture. Statistics from the EU Early Warning System operated by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) and Europol on the number of NPS reported for the first time in Europe on a yearly basis gives a good insight on the progress of this phenomenon (Fig. 1) [2]. Over the past 6 years particularly, it has escalated to the level of more than 100 new substances in 2014 (i.e. about two new substances each week on average). The NPS market was long dominated by stimulants and synthetic cannabinoids but currently comprises all classes of abused substances [2].
Problems related to NPS
NPS are of particular concern because they can be sold openly in web-based shops and elsewhere and thereby reach new drug users that are attracted by their ‘legal’ status. Of public concern are the unforeseen toxic effects of NPS, as using these uncontrolled and unsafe substances and products may lead to severe intoxication and even death [1, 3]. In Sweden, the progress of the NPS phenomenon and associated harmful effects has been followed in a collaborative project between the Department of Laboratory Medicine at the Karolinska University Hospital and the Karolinska Institutet, and the Swedish Poisons Information Center [3, 4]. This project, named STRIDA, enrolls patients with suspected NPS intoxication presenting in emergency departments all over the country. By combining the results from laboratory investigations of serum and urine samples with clinical information, new knowledge about NPS prevalence and toxicity is compiled. Since the start in 2010, the STRIDA project has documented over 2000 non-fatal but often severe acute intoxication cases involving a large number of different NPS. Polydrug use is commonly seen in these cases [3].
NPS in drug screening
One reason for using NPS instead of conventional drugs of abuse may be that NPS often remain undetected in standard drug testing procedures. Accordingly they are especially attractive alternatives for individuals who want to minimize the risk of being detected, such as in workplace drug testing and drug rehabilitation programmes.
The established procedure for drug testing is to use initial screening by immunoassays and then to confirm positive samples using methods based on the more sensitive and selective mass spectrometry (MS) technique. On one hand, the NPS present a challenge for the immunoassay screening, as available methods are typically directed only towards the conventional substances, e.g. amphetamines (amphetamine and methamphetamine), tetrahydrocannabinolcarboxylic acid (THC, cannabis), morphine (heroin), and benzoyl ecgonine (cocaine). On the other hand, as NPS are often designed to mimic and are chemical derivatives of conventional drugs, there is a possibility that certain NPS will also bind to (i.e. cross-react with) the antibodies used in immunoassay screening methods. And this is indeed the case. However, when these ‘false-positive’ screening results are subjected to confirmatory analysis by methods based on MS detection, they will turn out negative (i.e. ‘false-negative’ for drug use), if the MS method is only directed toward the standard set of abused drugs.
Cross-reactivity of NPS in immunoassays
When ecstasy (3,4-methylenedioxymethamphetamine, MDMA) became established as a street drug, interest emerged to detect it in immunoassay screening. MDMA and its metabolite 3,4-methylenedioxyamphetamine (MDA) were found to be detectable in existing assays for amphetamine and methamphetamine, due to a high degree of cross-reactivity for these compounds [5]. Likewise, also other new amphetamine-like substances were detectable [6].
However, although many NPS showed low cross-reactivity in commercial immunoassays [7, 8], the stimulant methylenedioxypyrovalerone (MDPV) was reported to cross-react in the CEDIA phencyclidine test [9]. A study from the authors’ laboratory comprising 45 NPS confirmed that several possessed chemical similarities leading to high cross-reactivity in the immunochemical screening tests commonly employed in routine urine drug testing [10]. The detectability of NPS observed to possess cross-reactivity was further confirmed by analysis of urine specimens from authentic intoxication cases included in the STRIDA project (Table 1). Given a more widespread use of new drugs among individuals subjected to drug testing, an increased number of unconfirmed positive screening results may occur.
The cross-reactivity for NPS in current screening assays may be seen as a problem or as a possibility to detect more substances. One possibility for improved drug testing is to include the most common new substances in the confirmation methods. As ecstasy became established as an illicit drug, new immunochemical screening tests for amphetamine/methamphetamine were developed that also included MDMA and MDA. Authentic case samples were used to demonstrate the capability of several commercial amphetamine class screening tests to detect MDMA/MDA. At that time, cross-reactivity towards the new ‘amphetamine’ analytes was wanted [5]. With the advent of the large number of NPS, both legal and illegal, the strategy to also cover new substances in the screening assays for classical narcotic drug substances may not be feasible. For example, the multitude of new synthetic cannabinoids (‘spice’) have not been incorporated in screening tests for THC, but resulted in the development of new independent tests [11].
One approach put forward to understand the potential of immunoassays to detect NPS is to use molecular similarity models [12]. Interestingly, the work of Petrie and co-workers [13] included such a molecular modelling method to predict the cross-reactivity of 261 amphetamine-like compounds. However, when comparing the theoretical data with our experimental data for one compound, the predicted reactivity for butylone was 10 times lower than that observed. In a more recent publication, it was proposed that molecular similarity models could be used to design new immunoassays with sensitivity for a larger number of target compounds [14].
NPS analysis by mass spectrometry
Another analytical strategy to cover NPS in drug testing is to employ MS-based ‘screening’ methods. As part of the STRIDA project, a multicomponent analytical MS method for NPS analysis in urine and serum specimens has been developed [15]. The method uses MS in combination with liquid chromatography (LC-MS/MS in selected-reaction monitoring mode) and is continuously updated as new NPS appear. There are also other methods for multicomponent screening of drugs in urine and plasma/serum, which proves that this technology can be employed in routine drug testing [16].
The LC-MS/MS technique has great potential for drug testing and for clinical laboratories in general. There are examples of laboratories that have already successfully replaced immunoassay screening by MS methods, also for the conventional drugs of abuse [17]. One way to make this possible and cost-effective is to use simple sample preparation procedures, e.g. a simple dilution of urine with internal standards [16]. When studying the cross-reactivity of 30 NPS in commercial ELISA tests for serum and blood, only a few were found to display cross-reactivity, and it was therefore proposed that MS methods should be used in future drug screening [18]. One attraction of MS-based screening is that accurate results are already obtained from the initial analytical step, which may be especially important in cases of acute intoxication (Fig. 2).
Potential of high-resolution MS
One promising technique for drug screening is high-resolution MS (HRMS) [19]. In the HRMS technique, the acquisition of data can be made with an untargeted design. Thousands of substances can be monitored at the same time without the need for optimizing MS parameters for each compound. In addition, new compounds can be searched for retrospectively.
Conclusion
The NPS present a challenge for drug testing laboratories and calls for novel drug screening strategies. It is likely that the current broader spectrum of abused psychoactive drugs will persist in at least in the foreseeable future. This new drug situation has put the performance of drug testing into focus and indicates that drug testing laboratories will play a more important role, as on-site drug screening using dipsticks is likely to lose significance.
References
1. Lewin AH, Seltzman HH, Carroll FI, Mascarella SW, Reddy PA. Emergence and properties of spice and bath salts: A medicinal chemistry perspective. Life Sci. 2014; 97: 9–19.
2. EMCDDA. New psychoactive substances in Europe. An update from the EU Early Warning System (March 2015). 2015. Available at: http://www.emcdda.europa.eu/attachements.cfm/att_235958_EN_TD0415135ENN.pdf.
3. Helander A, Bäckberg M, Hultén P, Al-Saffar Y, Beck O. Detection of new psychoactive substance use among emergency room patients: results from the Swedish STRIDA project. Forensic Sci Int. 2014; 243: 23–29.
4. Helander A, Bäckberg M, Beck O. MT-45, a new psychoactive substance associated with hearing loss and unconsciousness. Clin Toxicol. 2014; 52(8): 901–904.
5. Hsu J, Liu C, Hsu CP, Tsay WI, Li JH, Lin DL, Liu RH. Performance characteristics of selected immunoassays for preliminary test of 3,4-methylenedioxymethamphetamine, methamphetamine, and related drugs in urine specimens. J Anal Toxicol. 2003; 27: 471–478.
6. Apollonio LG, Whittall IR, Pianca DJ, Kyd JM, Haher WA. Matrix effect and cross-reactivity of select amphetamine-type substances, designer analogues, and putrefactive amines using Bio-Quant direct Elisa presumptive assays for amphetamine and methamphetamine. J Anal Toxicol. 2007; 31: 208–213.
7. Kerrigan S, Mellon MB, Banuelos S, Arndt C. Evaluation of commercial enzyme-linked immuno assays to identify psychedelic phenethylamines. J Anal Toxicol. 2011; 35: 444–451.
8. Bell C, George C, Kicman AT, Traynor A. Development of a rapid LC-MS/MS method for direct urinalysis of designer drugs. Drug Test Anal. 2011; 3: 496–504.
9. Macher AM, Penders TM. False-positive phencyclidine immunoassay results caused by 3,4-methylenedioxypyrovalerone (MDPV). Drug Test Anal. 2012; 5: 130–132.
10. Beck O, Rausberg L, Al-Saffar Y, Villen T, Karlsson L, Hansson T, Helander A. Detectability of new psychoactive substances, ‘legal highs’, in CEDIA, EMIT, and KIMS immunochemical screening assays for drugs of abuse. Drug Test Anal. 2014; 6: 492–499.
11. Arntson A, Ofsa B, Lancaster D, Simon JR, McMullin M, Logan B. Validation of a novel immunoassay for the detection of synthetic cannabinoids and metabolites in urine specimens. J Anal Toxicol. 2013; 37: 284–290.
12. Krasowski MD, Pizon AF, Siam MG, Giannoutsos S, Iyer M, Ekins S. Using molecular similarity to highlight the challenges of routine immunoassay-based drug of abuse/toxicology screening in emergency medicine. BMC Emerg Med. 2009; 9: 5.
13. Petrie M, Lynch KL, Ekins S, Chang JS, Goetz RJ, Wu AHB, Krasowski MD. Cross-reactivity studies and predictive modeling of “Bath Salts” and other amphetamine-type stimulants with amphetamine screening immunoassays. Clin Toxicol. 2013; 51: 83–91.
14. Krasowski MD, Ekins S. Using cheminformatics to predict cross reactivity of “designer drugs” to their currently available immunoassays. J. Cheminform. 2014; 6: 22.
15. Al-Saffar Y, Stephanson NN, Beck O. Multicomponent LC-MS/MS screening method for detection of new psychoactive drugs, legal highs, in urine – experience from the Swedish population. J Chromatogr B 2013; 930: 112–120.
16. Beck O, Ericsson M. Methods for urine drug testing using one-step dilution and direct injection in combination with LC-MS/MS and LC-HRMS. Bioanalysis 2014; 6 : 2229–2244.
17. Eichhorst JC, Etter ML, Rousseaux N, Lehotay DC. Drugs of abuse testing by tandem mass spectrometry: A rapid, simple method to replace immunoassays. Clin Biochem. 2009; 42: 1531–1542.
18. Swortwood MJ, Hearn WL, DeCaprio AP. Cross-reactivity of designer drugs, including cathinone derivatives, in commercial enzyme-linked immunosorbent assays. 2014; 6: 716–727.
19. Maurer HH. What is the future of (ultra) high performance liquid chromatography coupled to low and high resolution mass spectrometry for toxicological drug screening? J Chromatogr A 2013; 1292: 19–24.
The authors
Olof Beck*1,3 PhD and Anders Helander2,3 PhD
1Department of Clinical Pharmacology, Karolinska University Laboratory Huddinge, Sweden
2Department of Clinical Chemistry, Karolinska University Laboratory Huddinge, Sweden
3Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden
*Corresponding author
E-mail: olof.beck@karolinska.se
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.
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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.
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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.
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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