Plasma Methotrexate Concentrations Research Articles

Ultrafast Selective Quantification of Methotrexate in Human Plasma by High-Throughput MALDI-Isotope Dilution Mass Spectrometry

A new analytical MS method using isotope dilution combined with MALDI-triple quadrupole MS/MS has been developed and validated for the determination of methotrexate and 7-hydroxymethotrexate in plasma. Methotrexate, methotrexate-d3, 7-hydroxymethotrexate and 7-hydroxymethotrexate-d3 were monitored by selected reaction monitoring using the transitions m/z 455.2→308.2, 458.2→311.2, 471.2→324.2 and 474.2→327.2 for methotrexate, methotrexate-d3, 7-hydroxymethotrexate and 7-hydroxymethotrexate-d3, respectively. The LLOQ was 1 nmol/l for methotrexate and 7-hydroxymethotrexate while the limit of detection was 0.3 nmol/l for both analytes. The new developed method was cross-validated by a fluorescence polarization immunoassay and tested for its clinical feasibility by measuring plasma samples from patients suffering from acute lymphoblastic leukemia. Plasma methotrexate concentrations ranged between 66.0 and 954 nmol/l and observed 7-hydroxymethotrexate/methotrexate ratios ranged between 0.1 and 32.4, respectively. The new method showed comparable analytical performances as the fluorescence polarization immunoassay, but analyte specificity and sensitivity of the newly developed method were significantly better.

Impact of Abcc2 [Multidrug Resistance-Associated Protein (Mrp) 2], Abcc3 (Mrp3), and Abcg2 (Breast Cancer Resistance Protein) on the Oral Pharmacokinetics of Methotrexate and Its Main Metabolite 7-Hydroxymethotrexate

The ATP-binding cassette (ABC) transporters ABCC2 [multidrug resistance-associated protein (MRP) 2], ABCC3 (MRP3), and ABCG2 (breast cancer resistance protein) are involved in the efflux of potentially toxic compounds from the body. We have shown before that ABCC2, ABCC3, and ABCG2 together influence the pharmacokinetics of the anticancer and antirheumatic drug methotrexate (MTX) and its toxic metabolite 7-hydroxymethotrexate (7OH-MTX) after intravenous MTX administration. We now have used Abcc2;Abcc3;Abcg2(-/-) and corresponding single and double knockout mice to investigate the relative impact of these transporters on MTX and 7OH-MTX pharmacokinetics after oral MTX administration (50 mg/kg). The plasma areas under the curve (AUC(plasma)) in Abcg2(-/-) and Abcc2;Abcg2(-/-) mice were 1.7- and 3.0-fold higher than those in wild-type mice, respectively, suggesting additive effects of Abcc2 and Abcg2 on oral MTX pharmacokinetics. However, the AUC(plasma) in Abcc2;Abcc3;Abcg2(-/-) mice was not different from that in wild-type mice, indicating that Abcc3 protein is necessary for increased MTX plasma concentrations in the absence of Abcc2 and/or Abcg2. Furthermore, 2 h after administration, MTX liver levels were increased in Abcg2-deficient strains and MTX kidney levels were 2.2-fold increased in Abcc2;Abcg2(-/-) mice compared with those in wild-type mice. The absence of Abcc2 and/or Abcg2 also led to significantly increased liver and kidney levels of 7OH-MTX. Our results suggest that inhibition of ABCG2 and/or ABCC2, genetic polymorphisms or mutations reducing expression or activity of these proteins may increase the oral availability of MTX. Such conditions may also present risk factors for increased MTX-related toxicity in patients treated with oral MTX.

Polymorphisms of the SLCO1B1 gene predict methotrexate‐related toxicity in childhood acute lymphoblastic leukemia

Methotrexate (MTX) is an important component of the therapy for childhood acute lymphoblastic leukemia. Treatment with high-dose MTX often causes toxicity, recommending a dose reduction and/or cessation of treatment. Polymorphisms in genes involved in the MTX metabolism have been associated with toxicity with controversial results. The discrepancies could be due to differences in treatment protocols among studies, small, or non-homogeneous populations or the use of different toxicity criteria. The aim of the present study was to analyze the possible correlation of polymorphisms of genes involved in the MTX metabolism with the toxicity during therapy with the well-established LAL/SHOP protocol. We analyzed 10 polymorphisms in seven genes (MTHFR, TS, SHMT1, RFC1, ABCB1, ABCG2, and SLCO1B1) from the MTX metabolism in 115 Spanish pediatric B-ALL patients, using MTX plasma concentration as an objective and quantifiable marker of toxicity. We confirmed the suitability of MTX plasma levels as a toxicity marker. We found a statistically significant association between MTX plasma concentration and the SLCO1B1 rs11045879 CC genotype (P = 0.030). The rs4149081 AA genotype, in the same gene, could also be an indicator for high-MTX plasma concentrations. We did not find any significant association in the other genetic polymorphisms analyzed. Identification of the rs4149081 and rs11045879 SLCO1B1 polymorphisms in children with ALL could be a useful tool for monitoring patients at risk of low-MTX clearance in order to avoid MTX-related toxicity.

High-Dose Methotrexate-Induced Renal Dysfunction: Is Glucarpidase Necessary for Rescue?

TO THEEDITOR: Widemann et al 1 report the use of glucarpidase, leucovorin, and thymidine for high-dose methotrexate-induced renal dysfunction. When patients develop delayed excretion of methotrexate and impaired renal function after the administration of high-dose methotrexate there are two equally important imperatives. First, we must protect the patients from the potential toxicity of prolonged exposure to the antifolate drug methotrexate. Potential toxicities include myelosuppression, mucositis, and hepatoxicity, and can be lethal. The second imperative is to have the patient’s renal function return to normal to allow continuation of the chemotherapy needed to treat the underlying malignancy. The authors performed an analysis to determine which factors predicted greater than or equal to grade 4 toxicity. They noted that inappropriate increase in leucovorin predicted perfectly for development of greater than or equal to grade 4 toxicity and therefore could not be included in their multiple factor logistic regression analysis. We have reported our experience treating patients who have delayed methotrexate excretion and renal failure after high-dose methotrexate administration using high-dose leucovorin as the sole form of rescue. 2 All patients in our experience were salvaged. None of our patients died. The authors report that the median time to recovery of renal function for their cohort was 22 days. Our experience, albeit considerably smaller, achieved at least as good a time to recovery of normal renal function. One possible interpretation of the results that the authors report is that glucarpidase was not an essential component of rescue for patients experiencing high-dose methotrexate-related renal dysfunction and that similar results could have been achieved with the use of high-doses of leucovorin alone. Glucarpidase is unquestionably an excellent way to lower the plasma methotrexate concentration. This effect, however, has no impact on intracellular concentrations of methotrexate or on renal function. Protection of cells from intracellular methotrexate still requires the administration of high-doses of leucovorin and renal recovery appears to take place independently of glucarpidase administration. The authors’ conclusion that early intervention with the combination of leucovorin and glucarpidase is highly effective in patients who develop high-dose methotrexate-related renal dysfunction may be true but the authors should consider the possibility that similar results could be achieved with leucovorin alone.

Suivi thérapeutique pharmacologique du méthotrexate à faible dose dans les maladies inflammatoires

Methotrexate is prescribed to low-dose, ranging from 7.5 mg to 15 mg and until 25 mg if necessary, pulse once a week, in inflammatory pathologies, in particular in rheumatoid arthritis and psoriasis. The therapeutic answer and the frequency of adverse reactions are very variable from a patient to the other one, consequences of a large interindividual variability of the pharmacokinetic parameters of methotrexate, in particular bioavailability, suggesting a genetic support. Numerous polymorphisms being involved (carriers of influx and efflux, enzymes of the metabolism and of the mechanism of action of methotrexate), their determination with the aim of an individualized prescription does not seem realistic at the moment. On the other hand, an exposure-effect relationship, not so much by considering the plasma concentrations of methotrexate, but those of its polyglutamate derivatives in red blood cells, was described. Their determination should be able to contribute to a faster adaptation of dosages, or to a well-argued change of molecule in case of non clinical response. Although other studies are necessary to specify which markers would be the most relevant, which would be the best moment for their determination and to refine the therapeutic range, this approach seems promising. But currently, the level of proof of the therapeutic drug monitoring of low dose methotrexate in inflammatory disease was classified "remaining to evaluate".

Use of calcium folinate in the management of accidental methotrexate ingestion in two dogs

2 English Pointers were suspected of having consumed toxic doses of methotrexate, a dihydrofolate reductase inhibitor frequently used in human and veterinary chemotherapeutic protocols. Potentially toxic plasma concentrations of methotrexate were detected in both dogs. Results of physical examination, a CBC, blood gas analysis, and serum biochemical analysis were predominantly unremarkable, although 1 dog had mild hyponatremia (1372 mmol/L; reference range, 140 to 153 mmol/L) and mild hypocalcemia (1.03 mmol of ionized calcium/L; reference range, 1.13 to 1.33 mmol of ionized calcium/L). Point-of-care determination of plasma methotrexate concentrations was not available; thus, palliative care was provided. Emesis was induced in both dogs by SC administration of apomorphine, and 3 doses of a suspension of activated charcoal with sorbitol were administered orally over a 6-hour period. Fluid diuresis was initiated in both dogs by administration of a compound sodium lactate solution, and N-acetylcysteine was administered IV to both dogs as a hepatoprotectant. A solution of calcium folinate (also known as leucovorin) was administered IV to both dogs to mitigate the effects of ingested methotrexate. No adverse effects associated with calcium folinate administration were identified, and no clinical or pathological evidence of methotrexate intoxication was detected. IV administration of calcium folinate appeared to prevent the pathological sequelae of methotrexate intoxication without adverse effects. Administration of calcium folinate is recommended for the treatment of dogs with suspected or confirmed methotrexate overdose.

Difficulty Measuring Methotrexate in a Patient with High-Dose Methotrexate-Induced Nephrotoxicity

An 18-year-old male presented with pain and swelling in his left leg that he noted after playing football. An x-ray of the affected leg showed a destructive lesion that prompted a concern for malignancy. Subsequent tests, including magnetic resonance imaging, a bone scan, and a needle biopsy of the lesion, confirmed nonmetastatic osteosarcoma in the left proximal tibia. The patient was started on a standard regimen of chemotherapy. He received 4 cycles of high-dose methotrexate (HDMTX)3 with leucovorin rescue and 2 cycles of cisplatin and doxorubicin, which he tolerated well. Each HDMTX course involved the intravenous administration of 20 g methotrexate (MTX) over 4 h. The patient experienced delayed MTX clearance after the first cycle but showed typical clearance after the subsequent 3 cycles. He then underwent a planned radical resection of the tumor with allograft placement. After the patient recovered from surgery, chemotherapy resumed, and the patient received 2 additional cycles of cisplatin and doxorubicin and 1 additional cycle of HDMTX. His treatment was interrupted when he had to undergo surgery for a wound infection in the affected leg. After recovery from the second surgery, the patient received a sixth HDMTX cycle. After this cycle, the patient developed acute nephrotoxicity, which was manifested by marked renal dysfunction and delayed MTX clearance. His plasma creatinine concentration increased from 0.8 mg/dL (8 mg/L) at the start of the cycle to 6.8 mg/dL (68 mg/L) after he received HDMTX. Plasma MTX concentrations were 1700 μmol/L at 24 h after infusion, 450 μmol/L at 48 h, and 350 μmol/L at 72 h. The patient was treated with aggressive hydration, diuresis, and 1500 mg leucovorin intravenously every 6 h for several days. These measures did not reduce MTX below the toxic concentration, however, and the decision was made to give the patient glucarpidase …

Pediatric-Type Therapy Including Lineage-Targeted Methotrexate to Improve Early Minimal Residual Disease Response and Survival In Adult Acute Lymphoblastic Leukemia (ALL): Interim Analysis of Northern Italy Leukemia Group Study 10/07

AbstractAbstract 2131 Background.Some studies have shown how the use of pediatric-type therapy (PDT) and minimal residual disease (MRD)-oriented programs could improve outcome of adult ALL. Study Design.We included PDT elements in a pilot trial started January, 2008. Protocol 10/07 consisted of standard induction and late reinduction courses plus 3 PDT blocks (modified after BFM therapy) alternating with 3 lineage-targeted Methotrexate (LTM) blocks (B-precursor: 2.5 g/m2; T-precursor: 5 g/m2; Ph+ or age >55 years: 1.5 g/m2). LTM targeted a Methotrexate plasma concentration of ∼35 and ∼65 micromol/L in B- and T-ALL, respectively, in line with a therapeutic concept developed at St. Jude's Hospital (Memphis, TN, USA) in childhood ALL. The program was integrated by (a) a randomised, radiation-free central nervous system (CNS) prophylaxis study comparing intrathecal Methotrexate, Ara-C, and Prednisone (×12) vs. liposomal Ara-C (DepoCyte 50 mg, ×6 [B-ALL]-8 [T-ALL]); (b) the molecular evaluation of MRD at pre-fixed treatment weeks (w), to optimise risk stratification and indications for allogeneic stem cell transplantation (allo-SCT); and (c) by concurrent imatinib in patients with Ph+ ALL. The whole chemotherapy plus MRD study sequence was as follows: prephase (Prednisone, Cyclophosphamide) → induction (Idarubicin, Vincristine, Asparaginase, Dexamethasone) + w4 MRD → PDT1 (Cyclophosphamide, Vincristine, Idarubicin, Dexamethasone, Ara-C, 6-mercaptopurine) → LTM1 (plus high-dose Ara-C) + w10 MRD → PDT2 → LTM2 (plus Asparaginase) + w16 MRD → PDT3 → LTM3 (plus HD Ara-C) + w22 MRD → reinduction (Idarubicin, Vincristine, Cyclophosphamide, Dexamethasone). CNS prophylaxis was administered only during PDT blocks, with a minimum interval >15 days from/to LTM cycles. Risk classes were standard (SR: pre-B and WBC<30; cortical T and WBC<100; CR at cycle 1) and high (HR: other subsets). Allo-SCT was prescribed as soon as possible to all patients with Ph/t(4;11)+ ALL or other highly adverse cytogenetics; with WBC >100, or CD1a-negative early or mature T phenotype; and to SR/HR patients with MRD >10-4 after LTM1 (w10) or detectable at any level after LTM3 (w22). Autologous SCT followed by maintenance was a suitable alternative when allo-SCT was not feasible. All patients not eligible to frontline allo-SCT and with low positive (<10-4) or negative MRD results were submitted to standard maintenance. Results.Seventy-five of 81 evaluable patients (median age 40 years [range 18–65], 54% male, 23% T-ALL, 60% HR, 20% Ph+) achieved CR (92.5%); 55 of them (73.5%) were alive in CR1 at time of interim analysis (April, 2010). Sixteen patients relapsed (21.5%) and four died in CR of therapy-related complications (one after allo-SCT and 3 aged >55 years after a PDT cycle). With a maximum follow-up slightly >2 years (27.5 mos.), projected overall survival (OS) at 1.5 years is 73% (95% CI 57%-83%), and disease-free survival (DFS) 66% (95% CI 52%-77%). The best results were observed in patients aged 55 years and less (n=65, OS 79%) and those with T-ALL (n=20, OS 86%). These findings correlated well with a favourable early MRD response in Ph-negative subsets: 49% of evaluable cases achieved a major MRD response (<10-4) at end of induction cycle (w4: MRD negative 6/13 T [46%] vs. 7/24 B [29%]; <10-4 2 T and 3 B; overall 61.5% T and 42% B), as did 64% of the patients after LTM1 (w10: MRD negative 11/15 T [73%] and 16/27 B [59%]; <10-4 2 T and 1 B; overall 87% T and 63% B). MRD results were predictive of CR durability. Pooled MRD data from all time-points (w4-22) indicated a probability of CR of 92% in 18 patients with all results <10-4 vs. 60% in 19 patients with at least one MRD value >10-4 (P=0.039). The flexible risk- and MRD-adapted SCT policy resulted in high early transplantability rate in patients with this indication (70% in T-ALL). Finally, the randomised CNS prophylaxis trial preliminarily confirmed the feasibility of intrathecal DepoCyte as planned (total randomised patients = 57). Conclusions.This regimen was highly active, yielding a >90% CR rate in unselected adults aged up to 65 years, with a major early MRD response of 63% in Ph-negative B-ALL and 87% in T-ALL. The toxicity observed after PDT blocks in a first group of patients aged >55 years required a reduction of PDT dose intensity in this age group. LTM therapy proved feasible, up to 5 g/m2 in T-ALL, and remarkable early OS/DFS rates were obtained in T-ALL and in unselected patients aged <55 years. Disclosures:Bassan: Mundipharma: Consultancy, Research Funding. Off Label Use: Study is devoted to assess feasibility/toxicity/value of intrathecal liposome-encapsulated cytarabine (DepoCyte) vs standard intrathecal therapy as prophylaxis of CNS recurrence in adult ALL.

Glucarpidase, Leucovorin, and Thymidine for High-Dose Methotrexate-Induced Renal Dysfunction: Clinical and Pharmacologic Factors Affecting Outcome

To assess the role of the recombinant bacterial enzyme, glucarpidase (carboxypeptidase-G(2)), leucovorin, and thymidine in the management and outcome of patients with high-dose methotrexate (HDMTX) -induced nephrotoxicity. Patients with HDMTX-induced nephrotoxicity received one to three doses of intravenous (IV) glucarpidase and leucovorin rescue. The initial cohort (n = 35) also received thymidine by continuous IV infusion. Subsequently, thymidine was restricted to patients with prolonged exposure (> 96 hours) to methotrexate (MTX) or with substantial MTX toxicity at study entry. Plasma MTX, leucovorin, and 5-methyltetrahydrofolate (5-mTHF) concentrations were measured pre- and postglucarpidase. Toxicities were monitored, and logistic regression analysis was used to assess the relationship of baseline characteristics to the development of severe toxicity and death. Glucarpidase was administered at a median of 96 hours (receiving thymidine, n = 44) and 66 hours (not receiving thymidine, n = 56) after the start of the MTX infusion. Plasma MTX concentrations decreased within 15 minutes of glucarpidase by 98.7%. Plasma 5-mTHF concentrations also decreased more than 98% after administration of glucarpidase. Of 12 deaths, six were directly attributed to irreversible MTX toxicity. Presence of grade 4 toxicity before administration of glucarpidase, inadequate initial increase in leucovorin dosing, and administration of glucarpidase more than 96 hours after the start of the MTX infusion were associated with development of grade 4 and 5 toxicity. Early intervention with the combination of leucovorin and glucarpidase is highly effective in patients who develop HDMTX-induced renal dysfunction. Severe toxicity and mortality occurred in patients in whom glucarpidase rescue was delayed and occurred despite thymidine administration.

Association of a genetic variant in the ABCC2 gene with high methotrexate plasma concentrations in pediatric malignancies.

9522 Background: High-dose Methotrexate (HD-MTX) forms a cornerstone in the treatment of several pediatric malignancies including acute lymphoblastic leukemia (ALL) and osteosarcoma. Delayed MTX el...

Treatment of Methotrexate Intoxication with Various Modalities of Continuous Extracorporeal Therapy and Glucarpidase

Methotrexate, administered for treatment of pediatric and adult malignancies, is a direct renal toxin, which can lead to renal dysfunction, decreased methotrexate clearance, elevated methotrexate concentrations, and systemic toxicity. Although plasma methotrexate concentrations have been shown to decline precipitously after a single dose of glucarpidase, this drug is investigational and available only through compassionate use. Therefore, alternative treatments for methotrexate removal may be required. We describe a 13-year-old girl (body surface area 1.2 m(2)) with osteosarcoma who was treated with high-dose methotrexate 12 g/m(2) infused over 4 hours. Forty-eight hours after the infusion, her plasma methotrexate concentrations were elevated at 446 micromol/L. She exhibited severe signs of methotrexate toxicity, including encephalopathy, liver failure, and acute kidney injury, and could not tolerate conventional hemodialysis. Over the next 12 days, the patient was treated with continuous venovenous hemodialysis (CVVHD), single-pass albumin dialysis (SPAD), continuous venovenous hemodiafiltration (CVVHDF), and glucarpidase to enhance methotrexate elimination. Compared with standard CVVHD, SPAD did not significantly increase methotrexate removal as measured by elimination half-life and methotrexate saturation coefficient. The highest clearance rate among extracorporeal therapies was achieved by CVVHDF, with an effluent rate of 4950 ml/hour. The patient's clinical condition steadily improved, and all extracorporeal therapies were stopped 168 hours after methotrexate administration. The patient was discharged home and continued with chemotherapy, including methotrexate, which was dosed based on iothalamate glomerular filtration rates on the day before infusion. Although extracorporeal treatments appeared to enhance methotrexate clearance, the administration of glucarpidase resulted in the most rapid percentage decline (86%) in methotrexate concentration. Until glucarpidase is readily available, intermittent hemodialysis should be used to enhance methotrexate clearance. If the patient is unable to tolerate hemodialysis, use of CVVHDF with maximum effluent rates will enhance methotrexate clearance.

Abcc2 (Mrp2), Abcc3 (Mrp3), and Abcg2 (Bcrp1) are the main determinants for rapid elimination of methotrexate and its toxic metabolite 7-hydroxymethotrexate in vivo

The multidrug transporters ABCC2, ABCC3, and ABCG2 can eliminate potentially toxic compounds from the body and have overlapping substrate specificities. To investigate the overlapping functions of Abcc2, Abcc3, and Abcg2 in vivo, we generated and characterized Abcc3;Abcg2-/- and Abcc2;Abcc3;Abcg2-/- mice. We subsequently analyzed the relative impact of these transport proteins on the pharmacokinetics of the anticancer drug methotrexate (MTX) and its main, toxic, metabolite 7-hydroxymethotrexate (7OH-MTX) after i.v. administration of MTX (50 mg/kg). Whereas in single and double knockout mice, the plasma and liver concentrations of MTX and 7OH-MTX decreased rapidly after MTX administration, in the Abcc2;Abcc3;Abcg2-/- mice, they remained very high. One hour after administration, 67% of the MTX dose was still present in livers of Abcc2;Abcc3;Abcg2-/- mice as MTX or 7OH-MTX versus 7% in wild-type, showing dramatic liver accumulation of these toxic compounds when Abcc2, Abcc3, and Abcg2 were all absent. Furthermore, the urinary and fecal excretion of the nephrotoxic metabolite 7OH-MTX were increased 27- and 7-fold, respectively, in Abcc2;Abcc3;Abcg2-/- mice. Thus, Abcc2, Abcc3, and Abcg2 together mediate the rapid elimination of MTX and 7OH-MTX after i.v. administration and can to a large extent compensate for each other's absence. This may explain why it is still comparatively safe to use a toxic drug such as MTX in the clinic, as the risk of highly increased toxicity due to dysfunctioning of ABCC2, ABCC3, or ABCG2 alone is limited. Nevertheless, cotreatment with possible inhibitors of ABCC2, ABCC3, and ABCG2 should be done with utmost caution when treating patients with methotrexate.

Germline Genetic Variation in an Organic Anion Transporter Polypeptide Associated With Methotrexate Pharmacokinetics and Clinical Effects

Methotrexate plasma concentration is related to its clinical effects. Our aim was to identify the genetic basis of interindividual variability in methotrexate pharmacokinetics in children with newly diagnosed acute lymphoblastic leukemia (ALL). We performed a genome-wide analysis of 500,568 germline single-nucleotide polymorphisms (SNPs) to identify how inheritance affects methotrexate plasma disposition among 434 children with ALL who received 3,014 courses of methotrexate at 2 to 5 g/m(2). SNPs were validated in an independent cohort of 206 patients. Adjusting for age, race, sex, and methotrexate regimen, the most significant associations were with SNPs in the organic anion transporter polypeptide, SLCO1B1. Two SNPs in SLCO1B1, rs11045879 (P = 1.7 x 10(-10)) and rs4149081 (P = 1.7 x 10(-9)), were in linkage disequilibrium (LD) with each other (r(2) = 1) and with a functional polymorphism in SLCO1B1, T521C (rs4149056; r(2) > 0.84). rs11045879 and rs4149081 were validated in an independent cohort of 206 patients (P = .018 and P = .017), as were other SLCO1B1 SNPs residing in different LD blocks. SNPs in SLCO1B1 were also associated with GI toxicity (odds ratio, 15.3 to 16.4; P = .03 to .004). A genome-wide interrogation identified inherited variations in a plausible, yet heretofore low-priority candidate gene, SLCO1B1, as important determinants of methotrexate's pharmacokinetics and clinical effects.

Effect of allopurinol versus urate oxidase on methotrexate pharmacokinetics in children with newly diagnosed acute lymphoblastic leukemia

Allopurinol and urate oxidase are both effective in preventing or treating hyperuricemia during remission induction therapy for lymphoid malignancies, but to the authors' knowledge, their effects on concomitant anticancer drug therapy have not been compared. The authors compared plasma methotrexate pharmacokinetics in pediatric patients with newly diagnosed acute lymphoblastic leukemia who received concomitant allopurinol (n = 20) versus those treated with nonrecombinant or recombinant urate oxidase (n = 96) during high-dose methotrexate administration before conventional remission induction therapy. The minimum plasma concentration of uric acid was significantly (P < .0001) lower after urate oxidase treatment than the concentration after allopurinol treatment. Methotrexate clearance was significantly higher (median, 117.1 mL/minute/m(2) vs 91.1 mL/minute/m(2); P = .019) in patients who received urate oxidase. A higher proportion of patients in the allopurinol group had elevated methotrexate plasma concentrations (36% vs 7%; P = .003) and experienced mucositis (45% vs 16%; P = .003) after methotrexate treatment compared with the urate oxidase group. The lower rate of methotrexate clearance in patients who received allopurinol likely reflected a less potent hypouricemic effect of allopurinol, leading to precipitation of uric acid in renal tubules. Hence, during remission induction therapy for lymphoid malignancies, the authors concluded that renally excreted drugs should be monitored closely, especially in patients who are receiving allopurinol.

Examination of the Effect of Increasing Doses of Etoricoxib on Oral Methotrexate Pharmacokinetics in Patients With Rheumatoid Arthritis

The authors designed 2 randomized controlled studies to examine the effects of etoricoxib 60 to 120 mg daily on methotrexate pharmacokinetics in 50 rheumatoid arthritis (RA) patients on stable doses of methotrexate (7.5-20 mg). Patients received oral methotrexate at baseline and on days 7 and 14. In study 1, patients received etoricoxib 60 mg (days 1-7) and then 120 mg (days 8-14); in study 2, patients received etoricoxib 90 mg (days 1-7) and then 120 mg (days 8-14). For study 1, the AUC(0-infinity) geometric mean ratio (GMR) (90% confidence interval [CI]) for day 7 versus baseline was 1.01 (0.91, 1.12) for etoricoxib 60 mg; the area under the plasma concentration-time curve from zero to infinity (AUC(0-infinity)) GMR (90% CI) for day 14 was 1.28 (1.15, 1.42) for etoricoxib 120 mg. For study 2, the AUC(0-infinity) GMR (90% CI) for day 7 versus baseline was 1.07 (1.01, 1.13) for etoricoxib 90 mg; the AUC(0-infinity) GMR (90% CI) for day 14 was 1.05 (0.99, 1.11) for etoricoxib 120 mg. In summary, etoricoxib 60 and 90 mg had no effect on methotrexate plasma concentrations. Although no effect on methotrexate pharmacokinetics was observed with etoricoxib 120 mg in study 2, GMR AUC(0-infinity) fell outside the prespecified bounds in study 1. Standard monitoring of methotrexate-related toxicity should be continued when etoricoxib and methotrexate are administered concurrently, especially with doses >90 mg etoricoxib.

Involvement of Multidrug Resistance-Associated Protein 1 in Intestinal Toxicity of Methotrexate

Methotrexate (MTX) causes dose-limiting gastrointestinal toxicity due to exposure of intestinal tissues, and is a substrate of the multidrug resistance-associated protein (MRP) 1. Here we examine the involvement of MRP1, which is reported to be highly expressed in the proliferative crypt compartment of the small intestine, in the gastrointestinal toxicity of MTX. MTX was intraperitoneally administered to mrp1 gene knockout (mrp1 ((-/-))) and wild-type (mrp1 ((+/+))) mice. Body weight, food and water intake were monitored, intestinal histological studies and pharmacokinetics of MTX were examined. mrp1 ((-/-)) mice more severely decreased body weight, food and water intake than mrp1 ((+/+)) mice. Almost complete loss of villi throughout the small intestine in mrp1 ((-/-)) mice was observed, whereas the damage was only partial in mrp1 ((+/+)) mice. Plasma concentration and biliary excretion profiles of MTX were similar in mrp1 ((-/-)) and mrp1 ((+/+)) mice, though accumulation of MTX in immature proliferative cells isolated from mrp1 ((-/-)) mice was much higher compared to mrp1 ((+/+)) mice. Immunostaining revealed localization of Mrp1 in plasma membrane of the intestinal crypt compartment in mrp1 ((+/+)) mice, but not in mrp1 ((-/-)) mice. Mrp1 determines the exposure of proliferative cells in the small intestine to MTX, followed by gastrointestinal toxicity.

Intra-articular treatment of inflammatory arthritis with microsphere formulations of methotrexate: pharmacokinetics and efficacy determination in antigen-induced arthritic rabbits

Methotrexate (MTX) encapsulated microspheres release MTX in the joint in a slow, controlled manner following intra-articular injection in healthy rabbits. The objective of this study was to determine the pharmacokinetics of MTX and to evaluate the efficacy following intra-articular treatment of MTX-loaded microspheres in an antigen-induced inflammatory arthritis rabbit model. Arthritis was induced in both knee joints of rabbits using ovalbumin. Rabbits were intra-articularly treated with MTX solution or MTX microspheres and plasma concentrations of MTX were determined in the first 8 h following intra-articular treatment. Rabbits were killed 14 days following treatment and histological analysis of rabbit joints was conducted to determine efficacy. Arthritis was successfully induced in the joints of rabbits with the observation of histopathological features resembling rheumatoid arthritis. No significant differences were detected between MTX solution and MTX microspheres treated groups compared to phosphate buffered saline (control) animals. MTX microspheres effectively delivered the drug to the intra-articular space. However, a high degree of inter-animal variability, the severity of the disease induced and insufficient length of the observation period were suggested to be possible causes for the lack of therapeutic responses to MTX-loaded microspheres treatment.

Co‐administration of proton pump inhibitors delays elimination of plasma methotrexate in high‐dose methotrexate therapy

To assess whether or not co-administration of proton pump inhibitors (PPIs) is a risk factor for delayed elimination of plasma methotrexate (MTX) in high-dose MTX (HDMTX) therapy for malignant diseases. To assess the effects of PPI co-administration on elimination of plasma MTX, we examined plasma MTX concentration data on 171 cycles of HDMTX therapy performed in 74 patients. We performed multiple logistic regression analysis to evaluate PPI co-administration as a risk factor. Inhibitory potencies of omeprazole, lansoprazole, rabeprazole and pantoprazole on MTX transport via breast cancer resistance protein (BCRP, ABCG2) were also investigated in an in vitro study using membrane vesicles expressing human BCRP. We identified co-administration of PPIs as a risk factor for delayed elimination (odds ratio 2.65, 95% confidence interval 1.03, 6.82) as well as renal and liver dysfunction. All four PPIs inhibited BCRP-mediated transport of MTX, with half-maximal inhibitory concentrations of 5.5-17.6 microM--considerably higher than the unbound plasma concentrations of the PPIs. Our results support previous findings suggesting that PPI co-administration is associated with delayed elimination of plasma MTX in patients with HDMTX therapy. This drug interaction, however, cannot be explained solely by the inhibitory effects of PPIs on BCRP-mediated MTX transport.

Increasing Systemic Exposure of Methotrexate by Active Efflux Mediated by Multidrug Resistance-Associated Protein 3 (Mrp3/Abcc3)

The aim of this study was to investigate the functional importance of multidrug resistance-associated protein (Mrp)3/Abcc3 and Mrp4/Abcc4 in the pharmacokinetics of methotrexate. Compared with the corresponding wild-type mice, the plasma concentrations of methotrexate given orally were similar in Abcc4(-/-) mice and were significantly lower in Abcc3(-/-) mice. Pharmacokinetic parameters related to hepatobiliary transport were determined under steady-state conditions in wild-type and Abcc3(-/-) mice that were given a constant intravenous infusion of methotrexate. The biliary clearance, based on the plasma concentration, was 1.6-fold greater in Abcc3(-/-) mice than in wild-type mice (23 and 15 ml/min/kg, respectively, P < 0.05). Because the basolateral uptake and canalicular efflux clearances of methotrexate were similar in wild-type and Abcc3(-/-) mice, this result suggests that the basolateral efflux clearance of methotrexate is decreased in the liver of Abcc3(-/-) mice. Furthermore, a lower fraction of absorption of methotrexate (F(a) F(g)) was suggested in Abcc3(-/-) mice (0.49 and 0.29 in wild-type and Abcc3(-/-) mice, respectively). The mucosal-to-serosal transport rate of methotrexate, determined in vitro using everted sacs, was highest in the duodenum and was significantly decreased in Abcc3(-/-) mice compared with wild-type mice. This is ascribed to the reduced intrinsic efflux clearance of methotrexate across the serosal membrane (22 and 5.3 mul/min/sac in wild-type and Abcc3(-/-) mice, respectively, P < 0.05). These results suggest that Mrp3 mediates basolateral efflux of methotrexate in the liver and duodenum, thereby serving to increase systemic exposure, whereas Mrp4 is likely to play only a limited role in the systemic methotrexate exposure.

Reduced folate carrier-1 80G&gt;A polymorphism affects methotrexate treatment outcome in rheumatoid arthritis

The folate antagonist methotrexate (MTX) is a drug currently used in the treatment of rheumatoid arthritis (RA). MTX enters the cells through the reduced folate carrier (RFC-1) and is activated to polyglutamates. Previous studies have shown that RFC-1 expression may influence the efficacy of therapy with MTX. The studies suggest that G80A polymorphism in RFC-1 is associated with altered folate/antifolate levels and the subjects carrying homozygous mutant 80AA genotype tend to have higher plasma folate and MTX concentrations and higher erythrocyte polyglutamate levels compared with those with the wild type or heterozygous genotype. It is possible that this polymorphism might influence MTX treatment outcome in patients with RA. In the present study, we examined the association between RFC-1 G80A polymorphism and treatment outcome in patients with RA administered MTX. The study was carried out on 174 patients diagnosed with RA treated with MTX (7.5-15.0 mg weekly) plus low doses of methylprednisone. The RFC-1 80G>A polymorphism (resulting in a histidine-to-arginine substitution at codon 27 of RFC-1) was detected using a polymerase chain reaction-restriction fragment length polymorphism method. The probability of remission of RA symptoms was 3.32-fold higher in carriers of 80AA genotype as compared with patients with 80GG genotype (P=0.021, OR=3.32, 95% CI: 1.26-8.79). The frequency of A allele among MTX responders was 62.1, compared to 47.8% in a group of poor MTX responders (P=0.013, OR=1.78, 95% CI: 1.13-2.81). Moreover, the increase of aminotransferase activity was noted more frequently in carriers of 80AA genotype. The present data suggest that evaluation of RFC-1 gene 80G>A polymorphism may be a useful tool to optimize MTX therapy in patients with RA.