Mitochondrial Cumulative Damage Induced by Mitoxantrone: Late Onset Cardiac Energetic Impairment

Mitoxantrone (MTX) is a chemotherapeutic agent, which presents late irreversible cardiotoxicity. This work aims to highlight the mechanisms involved in the MTX-induced cardiotoxicity, namely the effects toward mitochondria using in vivo and in vitro studies. Male Wistar rats were treated with 3 cycles of 2.5 mg/kg MTX at day 0, 10, and 20. One treated group was euthanized on day 22 (MTX22) to evaluate the early MTX cardiac toxic effects, while the other was euthanized on day 48 (MTX48), to allow the evaluation of MTX late cardiac effects. Cardiac mitochondria isolated from 4 adult untreated rats were also used to evaluate in vitro the MTX (10 nM, 100 nM, and 1 μM) direct effects upon mitochondria functionality. Two rats of MTX48 died on day 35, and MTX treatment caused a reduction in relative body weight gain in both treated groups with no significant changes in water and food intake. Decreased levels of plasma total creatine kinase and CK-MB were detected in the MTX22 group, and increased plasma levels of lactate were seen in MTX48. Increased cardiac relative mass and microscopic changes were evident in both treated groups. Considering mitochondrial effects, for the first time, it was evidenced that MTX induced an increase in the complex IV and complex V activities in MTX22 group, while a decrease in the complex V activity was accompanied by the reduction in ATP content in the MTX48 rats. No alterations in mitochondria transmembrane potential were found in isolated mitochondria from MTX48 rats or in isolated mitochondria directly incubated with MTX. This study highlights the relevance of the cumulative MTX-induced in vivo mitochondriopathy to the MTX cardiotoxicity.     

The time course of gastric methotrexate intolerance in patients with rheumatoid arthritis and psoriatic arthritis

Objectives

This study aimed to evaluate the incidence and the time course of methotrexate (MTX)-associated gastric intolerance in patients with rheumatoid arthritis and psoriatic arthritis.

Methods

Four hundred twenty subjects undergoing MTX treatment for rheumatoid arthritis (n = 346) and psoriatic arthritis (n = 74) were retrospectively assessed. The incidence and time course of gastric MTX intolerance resulting in treatment discontinuation were investigated. In addition, the relations between gastric intolerance and patient characteristics, including gender, age, diagnosis, and rheumatoid factor (RF) positivity, were examined.

Results

Overall, oral MTX discontinuation rate due to gastric intolerance was 28.6 %. The time to discontinuation for oral MTX was 8.1 ± 11.5 months on average, with more than half of the discontinuations occurring within the first three months of treatment. Discontinuation was not associated with gender, age, diagnosis, or RF positivity. More than half of the patients that switched to a parenteral treatment regimen (52.6 %, 20/38) could tolerate the agent.

Conclusions

Gastric MTX intolerance usually develops within the first year of treatment and presents a major obstacle to long-term treatment retention in patients with rheumatologic disease. However, parenteral MTX appears to be a good alternative for patients intolerant of oral MTX.     

Reduction of plasma IL-6 but not TNF-α by methotrexate in patients with early rheumatoid arthritis: a potential biomarker for radiographic progression

Objective

To determine the effect of methotrexate (MTX) on plasma levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α and to investigate their associations with clinical and radiographic responses in patients with early rheumatoid arthritis (RA).

Methods

Sixty-two untreated RA patients with the disease duration of ≤36 months in whom MTX was initiated were consecutively identified in our prospective RA cohort and included in this study. Concomitant use of prednisolone and synthetic disease-modifying anti-rheumatic drugs with MTX was allowed, but patients who used biological agents were excluded. Plasma IL-6 and TNF-α levels were measured at the time of diagnosis (baseline) and 1 year later. The relationships of the clinical and radiographic data with plasma levels of IL-6 and TNF-α were analyzed.

Results

The median age of the patients was 57 years, 49 patients were female, and the median disease duration was 3 months. Forty-six (74.2 %) patients were anti-cyclic citrullinated protein antibody-positive. Serum C-reactive protein (CRP), plasma IL-6, and DAS28 decreased significantly (p <0.001) after MTX treatment, but plasma TNF-α did not. Radiographic progression was significantly correlated with disease activity score and plasma IL-6 levels but not with CRP or TNF-α after MTX treatment. Patients with plasma IL-6 level above 4.03 pg/ml showed clinically relevant radiographic progression with a sensitivity of 91.7 % and a specificity of 88.0 %.

Conclusion

In this early RA cohort, we demonstrated a significant (p <0.001) reduction of plasma IL-6, but not TNF-α, during MTX treatment. The post-treatment IL-6 level was a strong indicator of radiographic progression.     

Apoptotic Cell Death in Cultured Cardiomyocytes Following Exposure to Low Concentrations of 4-Hydroxy-2-nonenal

Lipid peroxidation (LP), induced by oxidative stress, is associated with degenerative processes. 4-Hydroxy-2-nonenal (HNE), a highly reactive diffusible product of LP, is considered by-product and mediator of oxidative stress. Its level increases under pathological conditions such as cardiovascular diseases. In this study, we partially characterized the mechanisms of HNE-mediated cytotoxicity in cardiomyocytes. After establishing that pathophysiological doses of HNE trigger cell death dependent on the incubation time and dose of HNE (LD₅₀ = 4.4 μM), we tackled the mechanisms that underlie the cell death induced by HNE. Our results indicate that HNE rapidly increases intracellular Ca²⁺; it also increases the rate of reactive oxygen species generation and causes a loss of mitochondrial membrane potential (ΔΨm) as well as a decrease in the ATP and GSH levels. Such alterations result in the activation of caspase-3 and DNA breakdown, both characteristic features of apoptotic cell death, as well as disruption of the cytoskeleton. Moreover, the nucleophilic compounds N-acetyl-cysteine and β-mercapto-propionyl-glycine, and the synthetic antioxidant Trolox exert a potent antioxidant action against HNE damage; this suggests its use as effective compounds in order to reduce the damage occurred as consequence of cardiovascular disorders in which oxidative stress and hence LP take place.     

Diosmin Prevents Isoproterenol-Induced Heart Mitochondrial Oxidative Stress in Rats

Cardiac mitochondrial oxidative stress causes mitochondrial damage that plays an important role in the pathology of myocardial infarction. The preventive effects of diosmin on cardiac mitochondrial oxidative stress in isoproterenol-induced myocardial infarcted rats were evaluated. Rats were pretreated with diosmin (10 mg/kg body weight) daily for 10 days. Myocardial infarction was induced in rats by isoproterenol (100 mg/kg body weight) injection twice at an interval of 24 h (on 11th and 12th day). Isoproterenol-induced myocardial infarcted rats showed a significant increase in the levels of cardiac diagnostic markers, heart mitochondrial lipid peroxidation, calcium ion, and a significant decrease in the levels of heart mitochondrial glutathione peroxidase, reduced glutathione, glutathione-S-transferase, isocitrate, malate, α-ketoglutarate, and succinate dehydrogenases. Transmission electron microscopic findings revealed damaged mitochondria with loss of cristae, swelling, and vacuolation in isoproterenol-induced rats’ heart. Diosmin pretreatment showed significant preventive effects on all the biochemical parameters, and the structure of mitochondria was evaluated. Furthermore, the transmission electron microscopic study confirms the biochemical findings. The antioxidant and negative inotropic effects of diosmin inhibited cardiac mitochondrial oxidative stress and prevented mitochondrial damage in myocardial infarcted rats.     

Factors associated with methotrexate dosing and therapeutic decisions in veterans with rheumatoid arthritis

The purpose of this study is to explore patient factors associated with differences in methotrexate (MTX) dosing and to compare patient factors and MTX-dosing patterns between those who remained on MTX monotherapy and those who were switched or had additional therapy. A retrospective cohort of 7,017 patients with newly diagnosed rheumatoid arthritis (RA) was identified in the United States Department of Veterans Affairs administrative databases between 1 October 1999 and 30 September 2009. Regression analyses were used to study the association of MTX start and maximum dose attained with various patient characteristics and compare differences between groups who had therapeutic change (having switched to or added another anti-rheumatic agent or having steroids increased by 2.5 mg of prednisone or equivalent) with those remaining on MTX monotherapy. Abnormal serum creatinine (>1.5 mg/dL) was associated lower start and peak MTX doses (p?<?0.01). Older RA patients were less likely to attain peak MTX dose of 15 mg or more (p?<?0.01). Males and patients 75 and older (compared with <45) had lower risk of therapeutic change (hazard ratio, [HR] 0.80, 95 % confidence interval [CI] 0.72–0.90, and HR 0.42, 95% CI 0.42–0.36–0.50, respectively). Patients who attained higher peak MTX dose had lower risk of therapeutic change compared with those dosed at less than 15 mg/week (HR 0.85, 95% CI 0.77–0.92 for 15 to <20 and HR 0.79, 95% CI 0.72–0.86 for 20 or more). Injectable MTX use conferred lower risk of therapeutic change (HR 0.64, 95% CI 0.52–0.78). Two thirds did not attain a maximum MTX dose of 20 mg/week or more before therapeutic change occurred. Older age and renal insufficiency were barriers to the use of higher MTX maximum dosages. Use of injectable MTX and higher maximum MTX dose were independently associated with higher likelihood to remain on MTX monotherapy. Further studies are needed to explore targeted interventions that may optimize MTX dosing to improve success rates of MTX monotherapy.     

Feeding S-adenosyl-l-methionine attenuates both ethanol-induced depletion of mitochondrial glutathione and mitochondrial dysfunction in periportal and perivenous rat hepatocytes

Mitochondrial glutathione plays an important role in maintaining a functionally competent organelle. Previous studies have shown that ethanol feeding selectively depletes the mitochondrial glutathione pool, more predominantly in mitochondria from perivenous hepatocytes. Because S-adenosyl-l-methionine (SAM) is a glutathione precursor and maintains the structure and function of biological membranes, the purpose of the present study was to determine the effects of SAM on glutathione and function of perivenous (PV) and peri-portal (PP) mitochondria from chronic ethanol-fed rats. SAM administration resulted in a significant increase in the basal cytosol and mitochondrial glutathione in both PP and PV cells from both pair-fed or ethanol-fed groups. When hepatocytes from ethanol-fed rats supplemented with SAM were incubated with methionine plus serine or N-acetylcysteine, mitochondrial glutathione increased in parallel with cytosol, an effect not observed in cells from ethanol-fed rats without SAM. Feeding equimolar N-acetylcysteine raised cytosol glutathione but did not prevent the mitochondrial glutathione defect. In addition, SAM feeding resulted in significant preservation of cellular adenosine triphosphate (ATP) levels (23% to 43%), mitochondrial membrane potential (17% to 25%), and the uncoupler control ratio (UCR) of respiration (from 5.1 ± 0.7 to 7.3 ± 0.6 and 2.1 ± 0.3 to 6.1 ± 0.7) for PP and PV mitochondria, respectively. Thus, these effects of SAM suggest that it may be a useful agent to preserve the disturbed mitochondrial integrity in liver disease caused by alcoholism through maintenance of mitochondrial glutathione transport.     

Concomitant iguratimod therapy in patients with active rheumatoid arthritis despite stable doses of methotrexate: a randomized, double-blind, placebo-controlled trial

Objectives

To investigate the efficacy and safety of iguratimod (T-614) in Japanese patients with active rheumatoid arthritis who had inadequate response to stable background methotrexate (MTX) alone.

Methods

In this multicenter, double-blind, controlled trial, a total of 253 patients were randomized at 2:1 ratio to either the iguratimod group or the placebo group. Iguratimod was orally administered at dosages of 25 mg/day for the first 4 weeks (25 mg once daily) and 50 mg/day for the subsequent 20 weeks (25 mg twice daily). MTX at dosage of 6 or 8 mg/week was administered to patients in both groups.

Results

The rate of 20 % improvement in American College of Rheumatology criteria (ACR20) at week 24 was 69.5 % in the iguratimod group compared with 30.7 % in the placebo group (P < 0.001). Significant improvements in the ACR50, ACR70, Health Assessment Questionnaire Disability Index, Disease Activity Score 28 <3.2, and rheumatoid factor were also observed. The most commonly reported adverse events (AEs) were blood iron decrease, nasopharyngitis, and lymphocyte decrease. These AEs were mild or moderate in severity. No deaths occurred.

Conclusion

The study results suggest that iguratimod in combination with MTX was efficacious and had a manageable safety profile.     

High-dose methotrexate in Egyptian pediatric acute lymphoblastic leukemia: the impact of ABCG2 C421A genetic polymorphism on plasma levels,what is next?

Purpose

High-dose methotrexate (HD-MTX) is a cornerstone antineoplastic drug in most treatment protocols of pediatric acute lymphoblastic leukemia (ALL). Among the membrane efflux transporters of MTX, the human breast cancer resistant protein is the second member of the G subfamily of ATP-binding cassette (ABC) efflux pump (ABCG2). A single-nucleotide polymorphism (SNP) in ABCG2, the exchange of C to A at position 421, represents 13 % in the Middle Eastern population. We studied the effect of this SNP on the plasma levels of HD-MTX in Egyptian pediatric ALL.

Methods

Two hundred ALL patients were recruited from Children’s Cancer Hospital Egypt-57357, and all were treated according to the St Jude Total XV protocol. Determination of plasma MTX levels was done at 23, 42 and 68 h. Genotyping of C421A of ABCG2 was done by polymerase chain reaction-restriction fragment length polymorphism.

Results

We found 14.5 % of the variant allele of the ABCG2 C421A SNP. The statistical association between ABCG2 421C>A SNP and the cutoff toxic plasma level of 24 h HD-MTX infusion at different time points tested was not statistically significant. There was no statistical significance between steady-state plasma concentration in patients with and without with this SNP.

Conclusion

To date, this is the largest study on Egyptian ALL patients for this SNP. This study shows that there is no effect of ABCG2 421C>A on plasma concentrations of HD-MTX. Replacing candidate gene association studies with genome-wide studies of HD-MTX is now mandatory and is part of our research blueprint.     

Effects of methotrexate on the oxidative metabolism of cultured rabbit articular chondrocytes

OBJECTIVE: The mechanism of action of methotrexate (MTX) in inflammatory joint disease is still unclear. We examined the possible interactions of MTX with the oxidative metabolism of rabbit articular chondrocytes. METHODS: Cell cultures of articular chondrocytes enzymatically isolated from juvenile New Zealand white rabbits were incubated 24 h with either MTX (0.22 or 1.1 microM), bacterial lipopolysaccharide (LPS, 50 microg/ml), or both. Cytofluorometry was then performed using 2',7'-dichlorofluorescein diacetate (DCFH-DA), rhodamine 123 (Rh123), or propidium iodide (PI). These fluorochromes allow evaluation of cellular production of H2O2, mitochondrial membrane potential, and cell viability, respectively. In a separate experiment, we used the Griess colorimetric technique to evaluate cellular nitric oxide (NO) production. RESULTS: Addition of MTX alone (0.22 or 1.1 microM) inhibited spontaneous production by chondrocytes of H2O2 (p < 0.01 and p < 0.001, respectively) and NO (p < 0.01 both concentrations). The LPS induced increase in H2O2, production was inhibited by MTX at 0.22 and 1.1 microM (p < 0.01 both concentrations), whereas the LPS induced increase in NO synthesis was not influenced by MTX, even at 1.1 microM. MTX did not significantly modify mitochondrial activity or cell viability. CONCLUSION: MTX at therapeutic concentrations in vitro inhibits the production of H2O2 and NO by unstimulated chondrocytes, and only the H2O2 overproduction by LPS stimulated chondrocytes. These properties may contribute to the therapeutic effect of MTX in RA.     

Phase II dose–response study of abatacept in Japanese patients with active rheumatoid arthritis with an inadequate response to methotrexate

Objective

The objective of this study was to assess the response to abatacept at doses of 2 mg/kg and 10 mg/kg compared to placebo in patients with active rheumatoid arthritis (RA) with an inadequate clinical response to methotrexate (MTX).

Methods

In this multicenter, placebo-controlled, double-blind, parallel-group, dose–response study, 195 Japanese patients with active RA with an inadequate response to MTX were randomized 1:1:1 to receive 10 mg/kg or 2 mg/kg abatacept plus MTX, or placebo plus MTX, for 24 weeks.

Results

Abatacept demonstrated a dose–response relationship when given at 2 and 10 mg/kg. Based on the American College of Rheumatology criteria (20, 50, and 70 %), the responses to 10 mg/kg abatacept were significantly greater than those to placebo at week 24 (p < 0.001). Smaller yet statistically significant responses were also seen in the 2 mg/kg abatacept group. Overall rates of adverse events, serious adverse events, and treatment discontinuations because of adverse events were comparable in all three groups.

Conclusions

Abatacept (2 mg/kg and 10 mg/kg) showed a dose–response relationship in Japanese patients with active RA with an inadequate clinical response to MTX. Administration of abatacept in combination with MTX for 24 weeks was well tolerated.     

Mitochondrial glutathione content determines the rate of liver regeneration after partial hepatectomy in eu- and hypothyroid rats

BACKGROUND/AIMS: Mitochondrial glutathione has been postulated to affect mitochondrial function and liver regeneration. METHODS: Mitochondrial respiration, total and oxidized glutathione, and liver regeneration were assessed after partial hepatectomy in glutathione-depleted and in hypothyroid rats with/without supplementation of glutathione ester. RESULTS: Mitochondrial, cytosolic and circulating glutathione levels were lower in glutathione-depleted rats. Hepatectomy was followed by significant changes of intra- and extracellular glutathione and of mitochondrial respiration. In glutathione-deficient rats, the recovery of mitochondrial function and the liver regeneration were delayed. Administration of glutathione ester partially corrected the fall of cytosolic and mitochondrial glutathione following hepatectomy, reduced mitochondrial oxidative damage, and accelerated the restoration of mitochondrial respiration and the rate of liver regeneration. In hypothyroid rats, intracellular glutathione homeostasis and mitochondrial respiration were impaired already at baseline; slower regeneration and mitochondrial oxidative alterations were observed after hepatectomy. Glutathione ester ameliorated the regenerative response in hypothyroid rats by providing higher concentrations of cytosolic and mitochondrial glutathione. CONCLUSIONS: Glutathione depletion and hypothyroidism affect the mitochondrial function during liver regeneration. Liver regenerates more slowly in glutathione-depleted and in hypothyroid rats. The earlier restoration of mitochondrial function and the higher rate of proliferation in glutathione ester treated rats suggest that the maintenance of intracellular glutathione facilitates liver regeneration.     

A systemic review and meta-analysis of the clinical efficacy and safety of total glucosides of peony combined with methotrexate in rheumatoid arthritis

To assess the efficacy and safety of the combination of total glucoside of peony (TGP) and methotrexate (MTX) for the treatment of rheumatoid arthritis (RA). Randomized controlled trial (RCT) data on the traditional Chinese active component TGP combined with MTX vs. MTX alone for the treatment of RA was collected by searching the Pubmed, Embase, Cochrane Library, CNKI, VIP Journals database, and Wanfang database up to February 2017. Study selection, data extraction, data synthesis, and data analyses were performed according to the Cochrane standards. A total of eight RCTs involving 522 participants were included in this meta-analysis. Compared with MTX alone, the use of TGP combined with MTX exhibited better therapeutic effects for the treatment of RA (P = 0.004). In addition, TGP combined with MTX caused a more significant decrease in erythrocyte sedimentation rate (ESR) (P < 0.0001) and swollen joint count (SJC) (P < 0.00001). However, no significant differences were found in C-reactive protein (CRP) (P = 0.19), duration of morning stiffness (DMS) (P = 0.32), or tender joint count (TJC) (P = 0.23) between the two groups. In addition, adverse events were more frequently reported in the MTX monotherapy group than in the TGP and MTX combination group (P = 0.0007). Our study demonstrates that TGP combined with MTX is more effective than MTX alone for the treatment of RA. Nevertheless, the adverse effects of the combination of TGP and MTX need to be further assessed. Due to the poor methodological quality of included trials, well-designed, multi-center, and large-scale RCTs are necessary to draw a more definitive conclusion.     

Combination of Cl-IB-MECA with paclitaxel is a highly effective cytotoxic therapy causing mTOR-dependent autophagy and mitotic catastrophe on human melanoma cells

Purpose

Metastatic melanoma is the deadliest form of skin cancer. It is highly resistant to conventional therapies, particularly to drugs that cause apoptosis as the main anticancer mechanism. Recently, induction of autophagic cell death is emerging as a novel therapeutic target for apoptotic-resistant cancers. We aimed to investigate the underlying mechanisms elicited by the cytotoxic combination of 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5′-N-methyl-uronamide (Cl-IB-MECA, a selective A₃ adenosine receptor agonist; 10 μM) and paclitaxel (10 ng/mL) on human C32 and A375 melanoma cell lines.

Methods

Cytotoxicity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction, neutral red uptake, and lactate dehydrogenase leakage assays, after 48-h incubation. Autophagosome and autolysosome formation was detected by fluorescence through monodansylcadaverine-staining and CellLight^® Lysosomes-RFP-labelling, respectively. Cell nuclei were visualized by Hoechst staining, while levels of p62 were determined by an ELISA kit. Levels of mammalian target of rapamycin (mTOR) and the alterations of microtubule networks were evaluated by immunofluorescence.

Results

We demonstrated, for the first time, that the combination of Cl-IB-MECA with paclitaxel significantly increases cytotoxicity, with apoptosis and autophagy the major mechanisms involved in cell death. Induction of autophagy, using clinically relevant doses, was confirmed by visualization of autophagosome and autolysosome formation, and downregulation of mTOR and p62 levels. Caspase-dependent and caspase-independent mitotic catastrophe evidencing micro- and multinucleation was also observed in cells exposed to our combination.

Conclusions

The combination of Cl-IB-MECA and paclitaxel causes significant cytotoxicity on two melanoma cell lines through multiple mechanisms of cell death. This multifactorial hit makes this therapy very promising as it will help to avoid melanoma multiresistance to chemotherapy and therefore potentially improve its treatment.     

Increased human dermal microvascular endothelial cell survival induced by cysteamine

Background

Cystinosis is an autosomal recessive disease caused by intralysosomal cystine accumulation, treated with cysteamine. Recently, new adverse effects of cysteamine were reported. Skin biopsies showed microvascular proliferation (angioendotheliomatosis). To examine the mechanism of angioendotheliomatosis associated with cysteamine toxicity, we examined the effect of cysteamine on human dermal microvascular endothelial cells (HDMVEC).

Methods

After cysteamine exposure (range 0–3.0 mM) during 24 h, cell viability was measured using water soluble tetrazolium salt-1 (WST-1) in both control HDMVEC and fibroblasts. Cell proliferation and apoptosis rate were measured in HDMVEC by bromodeoxyuridine (BrdU) incorporation and caspase 3 and caspase 7 activity, respectively. Intracellular glutathione (GSH) was measured in HDMVEC after cysteamine exposure of 0, 0.1 or 1.0 mM. Medium and cysteamine were refreshed every 6 h to mimic the in vivo situation. Next, cell viability in HDMVEC was measured after 24 h of GSH exposure (range 0–10.0 mM).

Results

HDMVEC viability and proliferation increased after cysteamine exposure 0.03–3.0 mM (p?<?0.01) and 0.03–1.0 mM (p?=?0.01) respectively; cell viability in fibroblasts was not affected by incubation with cysteamine. Apoptosis remained unaffected by incubation with 0–1.0 mM cysteamine, 3.0 mM caused increased apoptosis. Intracellular GSH was significantly increased after incubation with cysteamine 0.1 mM (p?=?0.02) and 1.0 mM (p?<?0.01). HDMVEC viability increased after exposure to GSH 1.0–5.0 mM (p?<?0.01).

Conclusion

Cysteamine concentrations, similar to those described in plasma of cystinosis patients, stimulate HDMVEC viability and proliferation and increase intracellular GSH content. We postulate that this mechanism might underlie angioendotheliomatosis induced by cysteamine.     

Folic acid alters methotrexate availability in patients with rheumatoid arthritis

OBJECTIVE: To evaluate the effects of repeated doses of folic acid on the pharmacokinetics of methotrexate (MTX) in patients with rheumatoid arthritis. METHODS: We studied 20 patients (ages 30-78 years) who received MTX intramuscularly (10 mm/week). MTX was administered alone or after treatment with folic acid (5 mg tablet once daily) for 13 days. Plasma samples were collected 2 and 8 h after dose intake. MTX concentrations in plasma and ultrafiltrate samples were measured by fluorescence polarization immunoassay. A Bayesian approach was used to determine individual MTX pharmacokinetic variables to minimize the number of samples collected. RESULTS: Folic acid supplementation led to reduced plasma MTX levels 2 and 8 h after MTX administration and reduced area under the plasma MTX concentration versus time curve (AUC) (about 20%; p < 0.02). Total clearance of MTX and Vd were higher when patients were also receiving folic acid than when they were taking MTX alone (p < 0.02). The plasma protein binding of MTX remains unchanged. CONCLUSION: The lower plasma MTX concentrations in patients taking folic acid supplements could be interpreted as increased cellular uptake of MTX; the folic acid supplements would promote the sequestering of MTX intracellularly. The decrease of MTX concentrations leads to reduced AUC; it is also possible that there are reduced AUC combined with increased intracellular folate levels. These results reopen the question of whether folic acid should be used immediately in all patients when MTX is begun.     

Analysis of intracellular methotrexate polyglutamates in patients with juvenile idiopathic arthritis: Effect of route of administration on variability in intracellular methotrexate polyglutamate concentrations

Objective

Intracellular methotrexate (MTX) polyglutamates (MTXGlu) have been shown to be potentially useful biomarkers of clinical response in adult patients with rheumatoid arthritis. The present study was undertaken to measure intracellular MTXGlu concentrations in a cohort of patients with juvenile idiopathic arthritis (JIA) to determine the predictors of MTXGlu variability in these patients.

Methods

Blood samples were obtained from patients with JIA who were being treated with a stable dose of MTX for ≥3 months. Clinical data were collected by chart review. Concentrations of MTXGlu_1–7 in red blood cell lysates were quantitated using an innovative ion‐pairing chromatography procedure, with detection by mass spectrometry.

Results

Patients with JIA from a single center (n = 99; mean ± SD age 117.8 ± 56.5 months, 69 female) were included in the analysis. The mean ± SD dose of MTX was 0.51 ± 0.25 mg/kg per week, with a median treatment duration of 18 months (interquartile range 3–156 months). MTX was administered subcutaneously in 66 patients (67%). Fifty‐six patients (57%) had active arthritis at the time of the clinic visit. Total intracellular MTXGlu (MTXGlu_TOT) concentrations varied 40‐fold, with a mean ± SD total concentration of 85.8 ± 48.4 nmoles/liter. Concentrations of each MTXGlu subtype (MTXGlu_1–7) were measured individually and as a percentage of MTXGlu_TOT in each patient. MTXGlu₃ was the most prominent subtype identified, comprising 42% of MTXGlu_TOT, and the interindividual variability in the concentration of MTXGlu₃ was the most highly correlated with that of MTXGlu_TOT (r = 0.96). The route of MTX administration was significantly associated with MTXGlu_1–5 subtypes; higher concentrations of MTXGlu_{1 + 2} were observed in patients receiving oral doses of MTX, whereas higher concentrations of MTXGlu_3–5 were observed in patients receiving subcutaneous doses of MTX (P < 0.0001).

Conclusion

In this cohort of patients with JIA, the MTXGlu_TOT concentration varied 40‐fold. Individual MTXGlu metabolites (MTXGlu_1–7), which have, until now, not been previously reported in patients with JIA, were detected. The route of MTX administration contributed to the variability in concentrations of MTXGlu_1–5.

     

Measurement of piperacillin plasma concentrations in cancer patients with suspected infection

Background

Piperacillin (PIP) in combination with tazobactam is commonly used for anti-infective treatment in cancer patients. PIP exerts a time-dependent killing. Thus, the maintenance of plasma concentrations above a pre-defined target concentration for a pre-defined time may be relevant for optimal efficacy. It is assumed that PIP-plasma concentrations above the clinical breakpoint of the target pathogen [Pseudomonas aeruginosa, clinical breakpoint at minimal inhibitory concentration (MIC) 16 mg/L] should be reached for 100% of the dosing interval or >4xMIC (64 mg/L) for 50% of the dosing interval. Whereas studies in the intensive-care setting have shown underdosing in patients with sepsis, little is known about PIP-plasma concentrations in cancer patients.

Methods

Data of 56 cancer patients who received piperacillin/tazobactam (PIP/TAZ, 4.5 g three times daily) as empiric therapy for suspected infection were analysed at baseline and 4 h after the infusion.

Results

Median trough concentrations in steady state [median 3 days (IQR 3–5) after start of PIP/TAZ] were 4.6 mg/L (95% CI 0.3–136.3) and median PIP-plasma concentrations 4 h after infusion were 46.2 mg/L (95% CI 10.1–285.6). A second evaluation 5 days (IQR 4–7) after start of PIP/TAZ confirmed these results: trough concentrations were 2.7 mg/L (95% CI 0.5–6.3), concentrations after 4 h 28.0 mg/L (95% CI 1.7–47.3). A good renal function was associated with lower plasma concentrations (r = ?0.388, p < 0.003). Detailed pharmacokinetic measurements in six patients showed low maximum plasma concentration (median 165 mg/L) and a rapid decline of plasma concentrations (median plasma half time 1.38 h).

Conclusion

In conclusion, piperacillin plasma concentrations in cancer patients are below target levels warranting prospective trials to investigate therapeutic drug monitoring.

     

Cell-free DNA as a potential marker to predict carbon tetrachloride-induced acute liver injury in rats

Purpose

Finding an optimal biomarker for the noninvasive evaluation of acute liver injury (ALI) may be of great value in predicting clinical outcomes and investigating potential treatments. We investigated cell-free DNA (CFD) as a potential biomarker to predict carbon tetrachloride-induced ALI in rats.

Methods

Forty-five Sprague–Dawley rats were randomly assigned to three groups. ALI was induced by carbon tetrachloride via a nasogastric tube at 1, 2.5, or 5 ml/kg of a 50 % solution. Fifteen additional rats underwent a sham procedure. Blood samples were drawn at time t which was 0 (baseline), 3, 6, 12, 24, 48, 72, 96, and 120 h for the measurements of CFD, glutamate–pyruvate transaminase (GPT), glutamate–oxaloacetate transaminase (GOT), and total bilirubin. Prothrombin time and histology were examined at 24 and 120 h following injection of 5 ml/kg carbon tetrachloride in 18 additional rats and in 10 control rats.

Results

CFD levels in rats subjected to carbon tetrachloride-induced ALI were significantly increased in all blood samples starting at 12 h after the induction of ALI (p < 0.001), reaching peak levels at 24 h. Blood GOT, GPT, and total bilirubin were elevated in all blood samples starting at 3 h after the induction of ALI (p < 0.0001), reaching peak levels by 48 h. A positive correlation was demonstrated between CFD levels and GOT (R ² = 0.92), GPT (R ² = 0.92), and total bilirubin (R ² = 0.76). CFD levels correlated with liver damage seen on histological examination, as well as predicted liver damage, at 24 h after ALI.

Conclusions

CFD may be a useful biomarker for the prediction and measurement of ALI. There is no evidence to suggest that CFD is superior to other available noninvasive biomarkers.