A quantitative systems pharmacology model for certolizumab pegol treatment in moderate-to-severe psoriasis.

Background: Psoriasis is a chronic immune-mediated inflammatory systemic disease with skin manifestations characterized by erythematous, scaly, itchy and/or painful plaques resulting from hyperproliferation of keratinocytes. Certolizumab pegol [CZP], a PEGylated antigen binding fragment of a humanized monoclonal antibody against TNF-alpha, is approved for the treatment of moderate-to-severe plaque psoriasis. Patients with psoriasis present clinical and molecular variability, affecting response to treatment. Herein, we utilized an in silico approach to model the effects of CZP in a virtual population (vPop) with moderate-to-severe psoriasis. Our proof-of-concept study aims to assess the performance of our model in generating a vPop and defining CZP response variability based on patient profiles. Methods: We built a quantitative systems pharmacology (QSP) model of a clinical trial-like vPop with moderate-to-severe psoriasis treated with two dosing schemes of CZP (200 mg and 400 mg, both every two weeks for 16 weeks, starting with a loading dose of CZP 400 mg at weeks 0, 2, and 4). We applied different modelling approaches: (i) an algorithm to generate vPop according to reference population values and comorbidity frequencies in real-world populations; (ii) physiologically based pharmacokinetic (PBPK) models of CZP dosing schemes in each virtual patient; and (iii) systems biology-based models of the mechanism of action (MoA) of the drug. Results: The combination of our different modelling approaches yielded a vPop distribution and a PBPK model that aligned with existing literature. Our systems biology and QSP models reproduced known biological and clinical activity, presenting outcomes correlating with clinical efficacy measures. We identified distinct clusters of virtual patients based on their psoriasis-related protein predicted activity when treated with CZP, which could help unravel differences in drug efficacy in diverse subpopulations. Moreover, our models revealed clusters of MoA solutions irrespective of the dosing regimen employed. Conclusion: Our study provided patient specific QSP models that reproduced clinical and molecular efficacy features, supporting the use of computational methods as modelling strategy to explore drug response variability. This might shed light on the differences in drug efficacy in diverse subpopulations, especially useful in complex diseases such as psoriasis, through the generation of mechanistically based hypotheses.

Efficacy of certolizumab pegol across baseline rheumatoid factor subgroups in patients with rheumatoid arthritis: Post-hoc analysis of clinical trials.

AIM: Certolizumab pegol (CZP), an Fc-free, PEGylated tumor necrosis factor inhibitor (TNFi), has shown rapid and sustained reduction in signs and symptoms of rheumatoid arthritis (RA). Elevated rheumatoid factor (RF) level has been associated with RA disease progression and poorer TNFi response. We assessed the efficacy of CZP in patients with early and established RA across baseline RF levels. METHODS: This post-hoc analysis included data from 6 trials: C-OPERA (NCT01451203), pooled RAPID trials (RAPID-1 [NCT00152386], RAPID-2 [NCT00160602], J-RAPID [NCT00791999], RAPID-C [NCT02151851]), and EXXELERATE (NCT01500278). Patients who received CZP or placebo/comparator with methotrexate (MTX) were categorized by baseline RF quartiles. Efficacy was assessed with Disease Activity Score-28 erythrocyte sedimentation rate (DAS28-ESR). RESULTS: Overall, 316, 1537, and 908 patients were included in C-OPERA, pooled RAPID trials, and EXXELERATE, respectively. Patient demographics and baseline disease characteristics were similar between treatment groups and across RF quartiles. DAS28-ESR low disease activity (LDA) and remission (REM) rates were numerically higher in the CZP + MTX group than PBO + MTX group at weeks 12 and 24, across RF quartiles. LDA and REM rates in the CZP + MTX groups were comparable across RF quartiles at weeks 12 and 24. Mean DAS28-ESR decreased from week 0 to week 24 in the CZP + MTX groups, across RF quartiles. CONCLUSION: CZP showed steady efficacy across baseline RF quartiles in patients with early and established RA, over 24 weeks. CZP treatment may be considered in patients with RA irrespective of baseline RF levels and time from diagnosis.

Combined evaluation of fecal calprotectin and C-reactive protein as a therapeutic target in the management of patients with Crohn's disease.

OBJECTIVES: Proper management of the inflammatory process in Crohn's disease (CD) results in lower rates of complications. The objective of this study was to investigate the performance of isolated and combined use of fecal calprotectin (FC) and serum levels of C-reactive protein (CRP) as markers of inflammatory activity in CD and the possibility of their use as a therapeutic target. PATIENTS AND METHODS: Patients with CD and indication for colonoscopy were prospectively enrolled in the study and allocated according to the presence or absence of endoscopic inflammatory activity. The correlation between FC and CRP levels and the Simplified Endoscopic Score of Crohn's Disease (SES-CD) was performed, and the accuracy of these markers was evaluated for the diagnosis of inflammatory activity, when used alone or in series. RESULTS: Eighty colonoscopies were performed in patients with CD. The FC cut-off value of 155µg/g showed high sensitivity (96%) and accuracy (78%) for the diagnosis of endoscopic activity. For CRP, the value of 6.7mg/L demonstrated sensitivity of 75% and specificity of 67%. The sequential usage of these markers (FC+CRP) showed greater specificity (82%) when compared to the use of these markers alone. Depending on the probability of inflammatory activity, different scenarios were used to evaluate the performance of these markers and an algorithm is proposed. DISCUSSION: Combined analysis of FC and CRP, when performed consecutively, allows decisions to be made with a high degree of certainty and even eliminates the need for colonoscopy in many situations.

Reference intervals of acute phase proteins in healthy Andalusian donkeys and response to experimentally induced endotoxemia.

BACKGROUND: Assessment of acute phase proteins (APPs) may allow prompt detection of diseases in donkeys, that otherwise may be missed because of the stoic behavior of donkeys. Reference intervals (RIs) of APPs measured using immunoassays and a comparison of the response of these biomarkers to a controlled inflammatory insult are lacking in donkeys. OBJECTIVES: (a) To describe the RIs for APPs in healthy Andalusian donkeys, (b) to study the effects of sex and age on APPs, and (c) to assess the early response of APPs to experimentally induced endotoxemia. ANIMALS: Seventy-three healthy Andalusian donkeys (67 for RIs and 6 for endotoxemia). METHODS: Serum amyloid A (SAA), haptoglobin (Hp), C-reactive protein (CRP), ceruloplasmin (Cp), α1-acid glycoprotein (AGP), procalcitonin (PCT), ferritin (Ft), and fibrinogen (Fb) RIs were determined. Endotoxemia was induced and samples for APP determination were obtained at regular intervals for 4 hours. RESULTS: The RIs in Andalusian donkeys were: SAA (0.1-0.6 mg/L), Hp (75-2261 mg/L), CRP (1.3-7.0 mg/L), Cp (0-745 mg/L), AGP (0-884 mg/L), PCT (0-504 pg/mL), Ft (26.9-31.8 µg/L), and Fb (115-466 mg/dL). Concentrations of SAA were higher (P < .05) in jacks. Donkeys <5 years old had higher Cp, AGP, and PCT compared to older donkeys. Concentrations of SAA and Hp were significantly increased in endotoxemic donkeys from 2 hours postinduction. CONCLUSIONS AND CLINICAL IMPORTANCE: We illustrated the importance of using species-specific RIs for APPs in donkeys and the effect of age and sex on APP concentrations. Concentrations of SAA and Hp appear to be the most useful biomarkers in donkeys in the early stages of endotoxemia.

Blood Storage Conditions Affect Hematological Analysis in Samples From Healthy Donkeys and Donkeys With Experimentally-Induced Endotoxemia.

Preanalytical factors such as storage time and temperature are proved to induce marked artifactual changes in hematological parameters in horses, small animals and humans. These errors can mirror findings typical of endotoxemia, leading to dangerous misdiagnosis. Since donkeys are common in warm climates and remote regions, blood samples from this species can be subjected to long lasting travels from the farm to the nearest laboratory, frequently under suboptimal conditions. Moreover, as other equids, donkeys are prone to suffer endotoxemia. Nonetheless, stability has not been evaluated in samples for hematology in this species. The aim of this study was to characterize the effect of temperature and storage time in hematological parameters from healthy donkeys and donkeys with induced endotoxemia. Blood samples were collected from six healthy female Andalusian donkeys and stored for 6, 12, 24, and 48 h at several temperatures (4, 24, and 35°C). Endotoxemia was induced in the same animals by an intravenous LPS infusion and samples obtained 30 min post-infusion were handled similarly. Hematological analysis was performed using a laser-based analyzer and blood smear examination. Storage at 24°C caused significant neutropenia after 48 h as well as morphological changes typical of endotoxemia in blood from healthy donkeys as soon as 24 h post-storage. Samples kept at 35°C displayed more profound and earlier artifactual variations. Conservation at 4°C did not cause any significant change in blood parameters. Prolonged (48 h) storage of samples from animals with induced endotoxemia at 24 and 35°C accentuated pre-existing leukopenia and neutropenia. These findings highlight that donkey samples should be stored at 4°C, instead of 24°C as recommended for horses. Moreover, blood smear interpretation should be cautious in samples stored for longer than 24 h and could be misleading when blood is kept at 35°C.

Meloxicam ameliorates the systemic inflammatory response syndrome associated with experimentally induced endotoxemia in adult donkeys.

BACKGROUND: Little information is available about endotoxemia in donkeys. Characterizing the systemic inflammatory response (SIRS) to lipopolysaccharide (LPS) in donkeys would provide valuable clinical and therapeutic information. The effects of meloxicam on endotoxemia have not been studied in this species. OBJECTIVES: To study the pathophysiology and gene expression associated with experimentally induced endotoxemia, and evaluate the effects of meloxicam on experimentally induced endotoxemia in donkeys and in equine monocyte cultures. ANIMALS: Six healthy adult female donkeys. METHODS: Endotoxemia was induced by an IV infusion of LPS for 30 minutes. Animals either received 20 mL of saline or 0.6 mg/kg of meloxicam IV after LPS infusion. The experiments lasted 6 hours. Blood samples were collected serially for hematology, serum biochemistry, interleukin measurement, and leukocyte gene expression analysis. Vital signs were recorded throughout the study. Monocyte cultures were used to test the effects of meloxicam on LPS-activated monocytes. RESULTS: Lipopolysaccharide induced fever, leukopenia, and neutropenia of similar magnitude in both groups, but meloxicam attenuated increases in plasma lactate, tumor necrosis factor-alpha (TNFα), and interleukin 1ß concentrations compared to controls. No differences were detected between groups for cytokine mRNA expression. Furthermore, meloxicam decreased TNFα release in LPS-activated monocyte cultures. CONCLUSIONS AND CLINICAL IMPORTANCE: Meloxicam could be a feasible option for the treatment of endotoxemia and SIRS in donkeys. Additional studies are necessary to investigate possible meloxicam-related posttranscriptional regulation and to compare this drug with other nonsteroidal anti-inflammatory drugs (NSAIDs) in animals with endotoxemia.

Adalimumab for Induction of Histological Remission in Moderately to Severely Active Ulcerative Colitis.

BACKGROUND: Histological remission represents a target distinct from endoscopic healing in ulcerative colitis (UC) and seems a better predictor of clinical outcomes. AIMS: The aim of this study was to assess the ability of adalimumab to achieve histological remission in UC patients. METHODS: Single-center, retrospective, open-label study of patients treated with adalimumab. Eligible patients were anti-TNF naïve adults with moderately to severely active UC. The Mayo score including endoscopy was performed at baseline and weeks 8 and 52. Histological activity was scored using the Geboes Index. The primary endpoint was histological remission, defined as a Geboes grade ≤ 3.0, at week 52. RESULTS: We included 34 patients. At week 8, 6 of 34 patients (17.6%) achieved histological remission. At week 52, 9 patients (26.5%, intention to treat; 31%, per protocol) had histological remission. Patients had a significant and progressive reduction in the most severe subgrades of Geboes Index from baseline at weeks 8 and 52. At weeks 8 and 52, 50 and 61.8% of patients achieved mucosal healing (Mayo endoscopic subscore 0-1). All patients who achieved histological remission also had mucosal healing. At week 8, 85.3 and 20.6% of patients achieved clinical response (decrease in Mayo score ≤ 3 points) or remission (Mayo score ≤ 2), respectively. At week 52, the corresponding values were 67.6 and 52.9%, respectively. At week 52, agreement between histological remission and mucosal healing was fair (kappa 0.293). Agreement between histological remission and Mayo endoscopic subscore 0 was good (kappa 0.71). CONCLUSIONS: Adalimumab was able to achieve histological remission in anti-TNF naïve patients with moderately to severely active UC.

Biology of anti-TNF agents in immune-mediated inflammatory diseases: therapeutic implications.

Biologics are increasingly being used to modify the course of immune-mediated inflammatory diseases. Some main agents are monoclonal antibodies and a fusion-protein that target TNF. This group includes adalimumab, infliximab, certolizumab pegol, golimumab and etanercept. Although the efficacy of anti-TNFs is supported by numerous randomized clinical trials, their pharmacokinetics depend on many factors, in particular immunogenicity, which can cause marked and rapid clearance and a consequent decrease in efficacy. Kinetics involve receptors that recognize the Fc fragment of the antibody and are responsible for various processes. Pharmacological advances permit optimizing the pharmacokinetics of anti-TNFs. In this review, we examine the kinetics of anti-TNF biologics, and consequent therapeutic implications, and overview some latest developments in the field. First draftsubmitted: 17 May 2016; Accepted for publication: 15 September2016; Published online: 14 October 2016.

Comparison of donkey hemogram using the LaserCyte hematology analyzer, an impedance system, and a manual method.

BACKGROUND: Donkeys are becoming increasingly important worldwide; therefore a reliable and accurate method of diagnosing disease is necessary. Flow cytometry-based hematologic analyzers are present in veterinary laboratories, but performance of LaserCyte has not been evaluated in donkeys. OBJECTIVES: The objective of the study was to compare the results of donkey blood obtained from the LaserCyte with impedance and manual methods. METHODS: Blood samples were collected from 84 healthy donkeys (1-20 years old) and measured with LaserCyte, Sysmex F-820 and manually. Agreement between methods was studied using Passing-Bablok test and Bland-Altman plots. Influence of blood abnormalities found on blood smears on LaserCyte counts was examined using Mann-Whitney or Kruskal-Wallis test. Intraassay precision was calculated. RESULTS: Hematologic variables obtained from the LaserCyte were significantly different from those obtained with impedance or manual methods; numerous values were flagged. Agreement between LaserCyte and manual method was poor for the majority of variables, but agreement between LaserCyte and impedance was only poor for HCT, MCH, and MCHC. LaserCyte had an intraassay precision < 10% for RBC and platelet variables, and > 10% for WBC variables. CONCLUSIONS: LaserCyte results were not interchangeable with results from other methods due to poor agreement. LaserCyte provided no additional hematologic variables or clinically relevant indices for donkey blood analysis. A large number of results were flagged, requiring the evaluation of blood smears. No benefits were found for the use of LaserCyte analyzer over the use of impedance or manual methods in this study. Specific software for LaserCyte for donkey blood would be beneficial.

Causes of CNS inflammation and potential targets for anticonvulsants.

Inflammation is one of the most important endogenous defence mechanisms in an organism. It has been suggested that inflammation plays an important role in the pathophysiology of a number of human epilepsies and convulsive disorders, and there is clinical and experimental evidence to suggest that inflammatory processes within the CNS may either contribute to or be a consequence of epileptogenesis. This review discusses evidence from human studies on the role of inflammation in epilepsy and highlights potential new targets in the inflammatory cascade for antiepileptic drugs. A number of mechanisms have been shown to be involved in CNS inflammatory reactions. These include an inflammatory response at the level of the blood-brain barrier (BBB), immune-mediated damage to the CNS, stress-induced release of inflammatory mediators and direct neuronal dysfunction or damage as a result of inflammatory reactions. Mediators of inflammation in the CNS include interleukin (IL)-1ß, tumour necrosis factor-α, nuclear factor-κB and toll-like receptor-4 (TLR4). IL-1ß, BBB and high-mobility group box-1-TLR4 signalling appear to be the most promising targets for anticonvulsant agents directed at inflammation. Such agents may provide effective therapy for drug-resistant epilepsies in the future.

Calprotectin in rheumatoid arthritis : association with disease activity in a cross-sectional and a longitudinal cohort.

BACKGROUND: Calprotectin is potentially a more sensitive biomarker of disease activity in rheumatoid arthritis (RA) than conventional acute-phase proteins such as the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) because it directly reflects inflammation in the synovium and synovial fluid rather than systemic inflammatory activity. OBJECTIVE: The aim of this study was to evaluate relationships between serum calprotectin levels, disease activity, and response to treatment. Calprotectin was also investigated as a predictive marker of clinical response. METHODS: This observational study included selected cohorts of patients with RA treated at La Paz University Hospital, Madrid, Spain. Associations between serum calprotectin levels and clinical and laboratory parameters were analyzed in a cross-sectional cohort of 60 patients with varying disease activity, and changes in calprotectin levels in response to treatment with infliximab were analyzed at baseline and after 3 and 6 months of treatment in a longitudinal cohort of 20 patients with very active disease. RESULTS: In the cross-sectional cohort, calprotectin levels correlated with rheumatoid factor levels (r = 0.25; p < 0.05) but not with titers of antibodies to cyclic citrullinated peptide. Significant correlations were also observed between calprotectin levels and the 28 swollen joint count (28-SJC), Disease Activity Score based on a 28-joint count (DAS28), Simplified Disease Activity Index (SDAI), ESR, and CRP levels. In the longitudinal cohort, calprotectin levels at baseline were not predictive of response to treatment but significantly decreased during treatment in responders (p < 0.0001). CONCLUSION: Calprotectin levels strongly correlate with clinical and laboratory assessments of joint inflammation and also decrease in response to treatment, indicating that calprotectin is a promising marker for assessment and monitoring of disease activity in patients with RA. Investigations are required to further evaluate its diagnostic, prognostic, and therapeutic potential.

Enterotypes of the human gut microbiome.

Our knowledge of species and functional composition of the human gut microbiome is rapidly increasing, but it is still based on very few cohorts and little is known about variation across the world. By combining 22 newly sequenced faecal metagenomes of individuals from four countries with previously published data sets, here we identify three robust clusters (referred to as enterotypes hereafter) that are not nation or continent specific. We also confirmed the enterotypes in two published, larger cohorts, indicating that intestinal microbiota variation is generally stratified, not continuous. This indicates further the existence of a limited number of well-balanced host-microbial symbiotic states that might respond differently to diet and drug intake. The enterotypes are mostly driven by species composition, but abundant molecular functions are not necessarily provided by abundant species, highlighting the importance of a functional analysis to understand microbial communities. Although individual host properties such as body mass index, age, or gender cannot explain the observed enterotypes, data-driven marker genes or functional modules can be identified for each of these host properties. For example, twelve genes significantly correlate with age and three functional modules with the body mass index, hinting at a diagnostic potential of microbial markers.

Thiopurine methyltransferase activity in Spain: a study of 14,545 patients.

We sought to assess the activity of thiopurine methyltransferase (TPMT) in 14,545 Spanish patients with different diseases amenable to treatment with azathioprine/6-mercaptopurine (6-MP), and to evaluate the proportion of patients with low TPMT activity and therefore a higher risk of myelotoxicity with these drugs. TPMT activity in red blood cells (RBCs) was measured by a radiochemical method. The association between several clinical variables and TPMT activity was assessed by multiple linear regression. We included 14,545 patients: autoimmune hepatitis (n=359 patients), inflammatory bowel disease (n=7,046), multiple sclerosis (n = 814), myasthenia gravis (n=344), pemphigus (n=133), and other diseases (n=5,849). Mean TPMT activity was 20.1 +/- 6 U/mL, but differed depending on the disease (P<.001). TPMT distribution was low (<5) in 0.5%; intermediate (5.0-13.7) in 11.9%; or high (>or=13.8) in 87.6%. Only when TPMT activity was considered separately in each disease did it reveal a normal distribution. In the multivariate analysis, gender, hematocrit, and treatment with 5-aminosalicylates/steroids/azathioprine/6-MP statistically influenced TPMT activity, although, probably, in a clinically irrelevant manner. This study shows, in a large sample of 14,545 patients, that 0.5% had low TPMT activity, indicating a higher risk of myelotoxicity with azathioprine/6-MP, a figure similar or slightly higher than that reported in other areas. Nevertheless, the trimodal distribution of TPMT activity varied depending on disease, and the proportion of patients with low activity values ranged from 0-0.8%. The drugs prescribed for the treatment of autoimmune diseases, including azathioprine/6-MP, modified TPMT activity, but the magnitude of this effect was very small and the differences found are probably irrelevant from the clinical point of view.

Thiopurine methyltransferase (TPMT) activity and adverse effects of azathioprine in inflammatory bowel disease: long-term follow-up study of 394 patients.

AIM: To prospectively evaluate whether a relationship between thiopurine methyltransferase (TPMT) activity and incidence of adverse effects (especially myelotoxicity) exists, in a long-term follow-up study of a large group of patients with inflammatory bowel disease treated with azathioprine. METHODS: TPMT activity in red blood cells (RBC) was measured by a radiochemical method in 394 consecutive patients with Crohn's disease (238) or ulcerative colitis (156) starting treatment with azathioprine. The relationship among several variables and TPMT values was assessed, and the correlation between such levels and the incidence of adverse effects was evaluated. RESULTS: Mean TPMT value was 18.6 +/- 4 U/mL RBCs (range 9.4-33.7). No patient had low levels (<5), 7.1% had intermediate levels (5-13.7), and 92.9% had high levels (>13.8). Differences (P < 0.001) were demonstrated in TPMT activity depending on the type of inflammatory bowel disease, but not on the remaining variables (including treatment with 5-aminosalycilates). Adverse effects were reported in 74 patients (18.8%), the most frequent being gastrointestinal intolerance (9.1%) and myelotoxicity (4.3%). No patient having adverse effects had low TPMT levels. However, mean TPMT activity was lower in those with adverse effects (16.6 +/- 3 vs 19.1 +/- 4 U/mL, P < 0.001). Moreover, the probability of suffering myelotoxicity in the high TPMT group was only 3.5%, compared with 14.3% in the TPMT intermediate group (95% CI = 1.37-14.9; OR = 4.5). CONCLUSIONS: The strategy of determining TPMT activity in all patients prior to initiating treatment with azathioprine could help to minimize the risk of myelotoxicity, as patients with intermediate TPMT activity had fourfold more risk than high TPMT activity patients.

Choice of azathioprine or 6-mercaptopurine dose based on thiopurine methyltransferase (TPMT) activity to avoid myelosuppression. A prospective study.

BACKGROUND/AIMS: To prospectively evaluate whether, in patients with inflammatory bowel disease, the choice of azathioprine (AZA) or 6-mercaptopurine (6-MP) dose based on thiopurine methyltransferase (TPMT) activity prevents myelotoxicity. METHODOLOGY: TPMT activity in red blood cells was measured in 99 patients with Crohn's disease and 32 with ulcerative colitis prior to initiating AZA/6-MP treatment. AZA/6-MP dose was chosen based on TPMT activity, which was again determined one month after starting therapy. Incidence of adverse effects was evaluated for at least 6 months of follow-up. RESULTS: Mean basal TPMT value was 21.6 +/- 5 U/mL. No patient had low levels (< 5 U/mL), 6.9% had intermediate levels (5-13.7 U/mL), and 93.1% had high levels (> 13.8 U/mL). In patients with Crohn's disease, mean TPMT activity significantly decreased after AZA/6-MP therapy, while in patients with ulcerative colitis this activity did not change. Among the 4 patients having myelotoxicity, one had intermediate basal TPMT levels, and 3 even had high levels, but no patient had low levels. CONCLUSIONS: In this prospective study we could not confirm that the choice of AZA/6-MP dose based on TPMT activity prevents myelotoxicity in patients with inflammatory bowel disease. Routine analytical controls should be performed in these patients independently of TPMT activity.

[Thiopurine methyltransferase activity in inflammatory bowel disease. A study on 7046 Spanish patients]. / Actividad de la tiopurina metiltransferasa en la enfermedad inflamatoria intestinal. Un estudio en 7.046 pacientes españoles.

BACKGROUND AND OBJECTIVE: Our objective was to assess the activity of thiopurine methyltransferase (TPMT) in a very large number of Spanish patients with inflammatory bowel disease (IBD), to evaluate the influence of several variables (including azathioprine or 6-mercaptopurine) on that activity, and to know the proportion of patients with low TPMT activity and therefore high risk of myelotoxicity when treated with these drugs. PATIENTS AND METHOD: TPMT activity in red blood cells (RBCs) was measured by a radiochemical method. The association between several variables and TPMT values was assessed by multiple lineal regression. RESULTS: 7046 patients were included (mean age: 37 years; 53% males): 70% with Crohn's disease, 22% with ulcerative colitis, and 8% with indeterminate colitis. Mean TPMT value was 20 (6) U/ml RBCs (minimum 0 and maximum 46). TPMT activity distribution was as follows: low levels (< 5 U/ml), 0.5%; intermediate (5-13.7), 11.1%; and high (> or = 13.8), 88.4%. TPMT values did not follow a normal distribution (p < 0.001). In the univariate study, statistically significant differences (p < 0.001), yet of doubtly clinical significance because its minimal magnitude, were demonstrated in TPMT values depending on age, sex, type of disease, and treatment with azathioprine/6-mercaptopurine. In the multivariate study, the variables associated with TPMT activity were: sex, treatment with 5-aminosalicylates, steroids and azathioprine/6-mercaptopurine. CONCLUSIONS: This study shows that 0.5% of the Spanish patients with IBD have low TPMT activity (< 5 U/ml RBCs), a figure similar to that reported in other countries, these patients being at higher risk of myelotoxicity when treated with azathioprine or 6-mercaptopurine. The drugs usually prescribed for the treatment of IBD, including 5-aminosalicylates and azathioprine/6-mercaptopurine, do not seem to modify in a clinically relevant manner TPMT activity.

Reviewing the mechanism of action of thiopurine drugs: towards a new paradigm in clinical practice.

The precise mechanism of action of thiopurine drugs remains unclear despite more than 40 years of use. Recent knowledge in the field of apoptosis and a better insight into, as well as a rapid increase in their use in several important areas of clinical medicine justify this appraisal. This is a review of the recent advances in the knowledge of their mechanism of action and is primarily intended to help clinicians understand the pharmacological properties of these drugs adequately and to find ways to improve their use in clinical practice. The parent compound is azathioprine (AZA), which is rapidly reduced in the presence of glutathione to 6-mercaptopurine (6-MP) and then metabolized into active metabolites with immune-modifier activity. Recent observations and new data indicate that AZA/6-MP could be considered as a "two-in-one" drug, providing a source of 6-thioguanine nucleotides (6-TGNs) and methylated metabolites, and that both compounds could contribute to its antiproliferative effects. This review will also focus on mechanisms that may help to explain a number of recent observations showing that myelotoxicity may occur in patients with high TPMT level or low 6-TGN rate. Our final proposal suggests that the immunosuppressive effects of these drugs are due to a balanced combination of antimetabolic and pro-apoptotic actions.

Azathioprine acts upon rat hepatocyte mitochondria and stress-activated protein kinases leading to necrosis: protective role of N-acetyl-L-cysteine.

Azathioprine is an immunosuppressant drug widely used. Our purpose was to 1) determine whether its associated hepatotoxicity could be attributable to the induction of a necrotic or apoptotic effect in hepatocytes, and 2) elucidate the mechanism involved. To evaluate cellular responses to azathioprine, we used primary culture of isolated rat hepatocytes. Cell metabolic activity, reduced glutathione, cell proliferation, and lactate dehydrogenase release were assessed. Mitochondria were isolated from rat livers, and swelling and oxygen consumption were measured. Mitogen-activated protein kinase pathways and proteins implicated in cell death were analyzed. Azathioprine decreased the viability of hepatocytes and induced the following events: intracellular reduced glutathione (GSH) depletion, metabolic activity reduction, and lactate dehydrogenase release. However, the cell death was not accompanied by DNA laddering, procaspase-3 cleavage, and cytochrome c release. The negative effects of azathioprine on the viability of hepatocytes were prevented by cotreatment with N-acetyl-L-cysteine. In contrast, 6-mercaptopurine showed no effects on GSH content and metabolic activity. Azathioprine effect on hepatocytes was associated with swelling and increased oxygen consumption of intact isolated rat liver mitochondria. Both effects were cyclosporine A-sensitive, suggesting an involvement of the mitochondrial permeability transition pore in the response to azathioprine. In addition, the drug's effects on hepatocyte viability were partially abrogated by c-Jun N-terminal kinase and p38 kinase inhibitors. In conclusion, our findings suggest that azathioprine effects correlate to mitochondrial dysfunction and activation of stress-activated protein kinase pathways leading to necrotic cell death. These negative effects of the drug could be prevented by coincubation with N-acetyl-L-cysteine.

[Thiopurine methyltransferase activity in patients with autoimmune hepatitis]. / Actividad de la tiopurina metiltransferasa en pacientes con hepatitis autoinmune.

BACKGROUND AND OBJECTIVE: The measurement of the activity of thiopurine methyltransferase (TPMT) is useful to monitor, on an individual basis, the dose of azathioprine in order to identify patients at risk of myelotoxicity. However, the distribution of the enzymatic activity in patients with autoimmune hepatitis is unknown. Our objective was to analyze the activity of TPMT in a group of 200 patients with autoimmune hepatitis and to evaluate the possible effect of some variables such as the treatment with azathioprine on this activity. PATIENTS AND METHOD: The activity of TPMT was determined by a radiochemical method; the activity was determined in the erythrocytes of patients with autoimmune hepatitis from 31 Spanish hospitals. We studied the relationship between TPMT levels and several demographic variables as well as its correlation with azathioprine treatment. RESULTS: We included 209 patients (80% females, mean age 50 years, 39% on azathioprine). The mean value of TPMT was 20.7 U/ml erythrocytes (from 0 to 39). TPMT levels were adjusted to a normal distribution. 1%, 9% and 90% patients had low (< 5 U/ml), intermediate (5-13.7 U/ml) and high (>= 13.8 U/ml) TPMT levels, respectively. In the multivariate analysis, there were no differences when comparing the mean TPMT values according to age, gender or previous azathioprine treatment. CONCLUSIONS: TPMT activity in patients with autoimmune hepatitis displays a similar distribution to that reported in other populations (approximately 1% of patients have low levels and 9% have intermediate levels) and it does not depend on the age, gender or simultaneous azathioprine treatment.

[Thiopurine methyltransferase activity and myelosuppression in inflammatory bowel disease patients treated with azathioprine and 6-mercaptopurine]. / Actividad de la tiopurina metiltransferasa y mielotoxicidad debida a azatioprina y 6-mercaptopurina en pacientes con enfermedad inflamatoria del intestino.

BACKGROUND AND OBJECTIVE: We aimed at assessing whether there exists a relationship between thiopurine methyltransferase (TPMT) activity and the incidence of adverse events, especially myelotoxicity, in patients with inflammatory bowel disease treated with azathioprine (AZA) or 6-mercaptopurine (6-MP). PATIENTS AND METHOD: By means of a radiochemical method, we determined the TPMT activity in erythrocytes of patients with inflammatory bowel disease who had received previously or at the time of the study AZA or 6-MP (n = 97). A group of 37 patients who had never been treated with these drugs was included. We studied the relationship between several variables and TPMT values as well as their correlation with adverse events. RESULTS: Mean (SD) TPMT value was 20.8 (5) U/ml erythrocytes (from 7.8 to 32.7). There was no patient with low TPMT levels (< 5); 9% patients had intermediate levels (from 5 to 13.7 U/ml), while 91% displayed high levels (>= 13.8 U/ml). There were no differences when comparing TPMT values according to several variables such as age, gender, tobacco consumption, weight, type of inflammatory bowel disease, and treatment with 5-aminosalicylates, steroids or AZA/6-MP. Side effects were seen in 13 out of 97 (13%) patients administered AZA/6-MP. None patient with side effects exhibited low TPMT levels (< 5 U/ml), nor even intermediate levels (5-13.7 U/ml). There were no differences when comparing mean TPMT values in patients with side effects and in those without side effects. CONCLUSIONS: In this study, the usefulness of the determination of the TPMT activity to identify patients with inflammatory bowel disease at risk of myelotoxicity due to AZA or 6-MP has not been confirmed. Therefore, even when the TPMT enzymatic activity is normal, it is necessary to continue performing the periodic laboratory analysis.