[Chinese Journal of Cancer 27:10, 363-367; October 2008]; 2008 Sun Yat-Sen University Cancer Center Clinical Research Paper Effects of infusion duration of high-dose methotrexate on cerebrospinal fluid drug levels in lymphoma patients Xu-Bin Lin, 1, 2 Ning-Ning Zhou, 1, 2 Su Li,1, 2 Qing-Qing Cai, 1, 2 Zhong-Jun Xia, 1, 2 Hai Liao, 1, 2 Yan Gao 1, 2 and Hui-Qiang Huang 1, 2, * 1 State Key Laboratory of Oncology in South China; Guangzhou, Guangdong P.R. China; 2 Department of Medical Oncology; Cancer Center; Sun Yat-sen University; Guangzhou, Guangdong P.R. China Key words: methotrexate, lymphoma, cerebrospinal fluid, serum, drug concentration Background and Objective: Methotrexate (MTX) levels of higher than minimal therapeutic level in cerebrospinal fluid (CSF) is essential for the therapeutic effects on central nervous system (CNS) lymphoma. The effect of infusion schedules on MTX penetrating into CSF has not been clearly understood. This study was to evaluate the effect of duration of venous infusion of high-dose MTX (HD-MTX) on drug levels in CSF, and to define the optimal schedule of HD-MTX infusion with high efficiency and low toxicity in CNS lymphomas. Methods: Thirty-four non-hodgkin's lymphoma (NHL) patients received 6-hour or 24-hour continuous venous infusion of MTX (1 3 g/m 2 ). CSF samples were obtained at 0 h after the end of HD-MTX infusion, and serum samples were obtained at 0 h, 24 h, and 48 h after the end of HD-MTX infusion. MTX concentrations were measured by high-pressure liquid chromatography and compared. Results: The serum concentration of MTX at the end of infusion was higher in 6-hour group than in 24-hour group. The CSF concentration of MTX was significantly higher in 6-hour group than in 24-hour group (0.70 μmol/l vs. 0.49 μmol/l, p = 0.044). A weak positive correlation between CSF and serum levels of MTX was observed (r = 0.295, p = 0.002). CSF levels of MTX were much higher in the patients with CNS involvement than in those without CNS involvement. The occurrence rates of grade II IV mucositis were 15.4% in 6-hour group and 37.8% in 24-hour group; those of grade III IV myelosuppression were 46.2% in 6-hour group and 67.6% in 24-hour group Conclusion: The shorter duration (6 h) of MTX administration is thought to be more beneficial on the aspects of reducing toxicity and enhancing CNS pharmacokinetics. *Correspondence to: Hui-Qiang Huang; State Key Laboratory of Oncology in South China and Department of Medical Oncology; Cancer Center; Sun Yat-sen University; Guangzhou, Guangdong 510060 P.R. China; Tel.: 86.20.87343350; Fax: 86.20.87343350; Email: huang_sysu@163.com Submitted: 04/08/08; Revised: 05/23/08; Accepted: 07/09/08 This paper was translated into English from its original publication in Chinese. Translated by: Wei Liu on 08/25/08. The original Chinese version of this paper is published in: Ai Zheng (Chinese Journal of Cancer), 27(10); http://www.cjcsysu.cn/cn/article.asp?id=14742 Previously published online as a Chinese Journal of Cancer E-publication: http://www.landesbioscience.com/journals/cjc/article/7979 Methotrexate (MTX), an antifolate, is widely used in the treatment of leukemia, chorionic cell carcinoma, tumors of the head and neck, osteosarcoma, lymphoma, and so on. High-dose methotrexate (HD-MTX) can penetrate the blood-brain barrier, therefore, it is an important drug for the prevention and treatment of metastatic and primary tumors in the central nervous system (CNS). 1 Effective therapeutic concentration and adequate action time of MTX in CNS is essential for the prevention and treatment of CNS tumors. Although high concentration of MTX in cerebrospinal fluid can be obtained by intrathecal injection, the concentration in the brain parenchyma is low, therefore, it only has good therapeutic effect on tumor metastasis of the meninges, and intrathecal injection of MTX can not substitute intravenous administration of HD-MTX for the whole CNS. 2 In order to obtain better preventive and therapeutic effect, and avoid toxicity of HD-MTX as far as possible, we used different dose schedules of HD-MTX to detect serum and cerebrospinal fluid concentration of MTX, which would provide experimental evidence for clinical medication. Subjects and Methods Subjects data. Thirty-four patients with pathologically confirmed non-hodgkin s lymphoma (NHL), treated in the Department of Medical Oncology of Sun Yat-sen University Cancer Center from December, 2004 to July, 2007, were selected. Of the 34 cases of NHL, eight were primary central nervous system lymphoma, while 26 were systematic NHL; 17 were retrospective cases, while another 17 were self-control cases. Three patients received single HD-MTX treatment, ten received HD-MTX combined CHOP regimen, 14 received HD-MTX combined high-dose cytarabine, and seven HD-MTX combined temozolomide. None of the patients received central radiotherapy. The 34 patients received a total of 92 times of HD-MTX chemotherapy: 6-hour group received 29 times of HD-MTX, while 24-hour group received 63 times of HD-MTX. Seventeen patients were treated by both HD-MTX dose schedules alternatively. The patients received 1-6 cycles of chemotherapy. were treated for 1 6 courses. The clinical features of patients in 6-hour group were comparable with those in 24-hour group, as indicated by Chi-Square test and Mann-Whitney U test (Table 1). Medication of MTX. The dose of MTX was 1.0 3.0 g/m 2. For 6-hour schedule, 10% of the total dose was administered by www.landesbioscience.com Chinese Journal of Cancer 363
Table 1 Clinical characteristics of patients in 6-hour and 24-hour MTX infusion groups MTX,methotrexate; Burkitt, Burkitt-like lymphoma; DLBCL, diffuse large B-cell lymphoma; Mantle, mantle cell lymphoma; PTL, peripheral T-cell lymphoma; LBL, lymphoblastic lymphoma; CNS, central nervous system; PS, performance status; IPI, international prognostic index; Ara-C, arabinoside cytarabine; TMZ, temozolomide. intravenous infusion for 30 min, and the remaining dose was administered in the same manner for 5.5 h. Leucovorin (LV) rescue began at 12 h after MTX withdrawal, which was administered by intravenous injection once every 6 h in a dose of 30 mg for 6 times or more than 6 times according to serum MTX concentration. For 24-hour schedule, 10% of the total dose was administered by intravenous infusion for 30 min, and the remaining dose was administered in the same manner for 23.5 h. LV rescue began at 12 h after MTX withdrawal, which was performed in the same way as above. Urine hydration and alkalinization was performed with routine methods. Sample collection. Venous blood was collected at 0 h, 24 h, and 48 h after MTX withdrawal for serum separation. Lumbar puncture was performed at 0 h after MTX withdrawal and 1 ml of cerebrospinal fluid was collected. MTX concentration detected by high performance liquid chromatography. The concentration of MTX in serum and cerebrospinal fluid samples was detected by AGILENT1100 high performance liquid chromatograph, with C18 column (Dalian Elite, 250 mm), 0.1 mol/l KH 2 PO 4 -K 2 HPO 4 and methanol (87:13, v/v) as mobile phase, a flow rate of 1.0 ml/min, and detected at UV 313 nm. Toxicity assessment. Toxicity was assessed according to WHO toxicity evaluation criteria. Statistical methods. MTX concentration was presented as mean ± SD, median and full range. Mann-Whitney U test, Wilcoxon self-control test, and non-parametric Spearman correlation analysis were adopted for statistical analysis. Multiplicity was analyzed by multiple linear regression and variables were selected by Stepwise method (criteria: Probability-of-F-to-enter 0.05, Probability-of- F-to-remove 0.10). Measurement data were tested by Pearson Chi-Square test. p < 0.05 was considered of significance. The actual dose of MTX was 1.0 3.7 g/m 2. In order to eliminate changes in MTX concentration due to dose differences, MTX concentration was adjusted to 1.0 g/m 2 for correlation test, which was realized via dividing MTX concentration by MTX dose (g/m 2 ). Statistical analysis was performed by SPSS12.0 software. Results Serum and cerebrospinal fluid concentration of MTX. Serum concentration of MTX was significantly higher in HD-MTX 6-hour group than in 24-hour group. It was rapidly decreased at 24 h after drug administration in both groups, which was slightly higher in 6-hour group than in 24-hour group. There was no significant difference between the two groups at 48 h after drug administration. Cerebrospinal fluid concentration of MTX at 0 h after drug administration in 6-hour group was higher than that in 24-hour group [0.73 μmol/l vs. 0.52 μmol/l (corrected value), p = 0.068] (Table 2 and Fig. 1). Cerebrospinal fluid concentration of MTX exceeded 0.5 μmol/l in all patients and exceeded 1 μmol/l in 24 (83%) patients in 6-hour group, while exceeded 1 μmol/l in only 41 (65%) patients in 24 h group. Coefficient variations (CV) of corrected serum and cerebrospinal fluid concentration of MTX 364 Chinese Journal of Cancer 2008; Vol. 27 Issue 10
Table 2 Serum and CSF concentration of MTX after the 6-hour and 24-hour infusion of HD-MTX Mann-Whitney U test. Table 3 CSF concentration of MTX in self-compared patients All values are presented as mean ± SD of relevant groups. Figure 1. Serum and CSF concentration of methotrexate (MTX) at 0 h after 6-hour and 24-hour infusion of high-dose MTX. were 65% and 121% respectively in 6-hour group, and 57% and 110% respectively in 24-hour group. There was also great variability in serum and cerebrospinal fluid concentration of MTX in the same individual between different courses of treatment. To decrease the effects of interindividual variations and other factors on cerebrospinal fluid concentration of MTX, 17 patients received two MTX dose schedules alternatively. The dose of MTX was the same as those in other chemotherapeutic regimens. A total of 63 case-times of HD-MTX treatment were performed, with 26 case-times of 6-hour administration and 37 case-times of 24-hour administration. Cerebrospinal fluid concentrations of MTX for 6-hour and 24-hour administration of each patient were averaged as self-control of pairing data. MTX concentration was also significantly higher in 6-hour group than in 24-hour group (p = 0.044) (Table 3). Cerebrospinal fluid concentration of MTX after 6-hour administration was decreased to (2.94 ± 5.65)% of the 0-hour level, and that after 24-hour administration was decreased to (5.03 ± 6.62)% of the 0-hour level. Correlation analysis on factors that influencing MTX concentration. Considering the great variability in serum and cerebrospinal fluid concentrations of MTX, we performed univariate and multivariate analyses on factors that might influence corrected MTX concentration. The factors involved sex and age, pathologic type, PS score, IPI score, CNS invasion, serum MTX concentration and du ration of HD-MTX administration. Univariate analysis indicated that serum MTX concentration at 0 h was correlated to duration of HD-MTX administration (p < 0.001), while cerebrospinal fluid concentration of MTX was positively correlated with serum MTX concentration at 0 h, CNS invasion and age. Multivariate analysis, with Y = -0.156 + 0.009X1 + 0.517 X2 as the regression equation, showed that cerebrospinal fluid concentration of MTX was correlated to serum MTX concentration at 0 h (r = 0.295, p = 0.002) and CNS invasion (r = 0.253, p = 0.008) (Fig. 2). Corrected cerebrospinal fluid concentration of MTX was significantly higher in the patients with CNS invasion than in those without CNS invasion [(1.18 ± 1.37) μmol/l vs. (0.69 ± 0.63) μmol/l, p = 0.038]. Toxic reaction. In order to eliminate differences in toxicity due to chemotherapeutics other than HD-MTX and dose difference, toxicity evaluation was performed only in 17 self-controlled patients. Grade III-IV hematologic toxicity occurred in 46.2% (12/26) and 67.6 % (25/37) (p = 0.089) courses of treatment in 6-hour and 24-hour www.landesbioscience.com Chinese Journal of Cancer 365
Figure 2. Correlations of CSF MTX concentration to serum MTX concentration and CNS involvement. groups, respectively. The occurrence rates of grade II IV mucositis (manifested as oral mucositis and diarrhea) were 15.4% (4/26) and 37.8% (14/37) in 6-hour and 24-hour groups (p = 0.05). Discussion MTX forms MTX poly glutamate (MTXPG) under the function of intracellular glutamate synthase. Competitive inhibition of dihydrofolate reductase by MTX and MTXPG, together with inhibition of thymidylic acid synthase and transferase required by initiating purine synthesis by MTXPG will influence synthesis of nucleic acid and protein. MTXPG can stay in cells longer than MTX and can not be rescued easily by LV, which contribute to its cytotoxic effect. Relatively low concentration of LV only has rescuing effect on normal cells since more MTXPG with longer chains are formed in tumor cells as compared with normal cells. Certain extracellular concentration and maintenance time of MTX is necessary for MTX to kill tumor cells. For intravenous administration of HD-MTX, the concentration of MTX in cerebrospinal fluid is usually used to reflect that in brain parenchyma. Animal experiment showed that MTX concentration in mouse brain tumor tissue was higher than that in cerebrospinal fluid, and the latter was able to reflect MTX concentration in brain tumor tissue effectively. 3 When cerebrospinal fluid concentration exceeds 1 μmol/l, MTX will have therapeutic effect on CNS lymphoma. Half-time of MTX in cerebrospinal fluid is about 7 16 h, which is influenced by permeability of the bloodbrain barrier. 2,4 Previous studies showed that intravenous administration of low-dose MTX can not penetrate the blood-brain barrier effectively: cerebrospinal fluid concentration of 0.1 μmol/l could not be obtained by administration of 500 mg/m 2 MTX, while minimal effective therapeutic concentration of 0.5 μmol/l in cerebrospinal fluid could be obtained when the dose of MTX reached 3 g/m 2. 5 Currently, there are several administration schedules of MTX for the prevention and treatment of lymphoma CNS invasion. The duration of administration varies from 3 h to 24 h for different schedules, while the effect of duration of intravenous administration of HD-MTX on its penetration through the blood-brain barrier is still unclear. When treat pediatric leukemia with continuous infusion of 5 g/m 2 MTX for 24 h, cerebrospinal fluid concentration of MTX in 20 40% patients can not reach 1 μmol/l; when treated with infusion of 3 g/m 2 MTX for 24 h, cerebrospinal fluid concentration of MTX is only 0.35 0.98 μmol/l. Vassal et al. 5 reported that median cerebrospinal fluid concentration of MTX was 2.3 μmol/l when treated pediatric NHL by continuous infusion of 3 g/m 2 MTX for 3 h, while it was 17.1 μmol/l (3.7 55 μmol/l) when treated adult patients with tumorous meningitis by continuous infusion of 8 g/m 2 MTX for 4 h. These results suggest that different durations of administration might influence cerebrospinal fluid concentration of MTX. 5-10 The present study compared the influence of 6-hour and 24-hour administration of HD-MTX on its CNS concentration. Our results showed that in the schedule of short delivery time, MTX can penetrate the blood-brain barrier more effectively, so that effective therapeutic concentration in CNS can be obtained rapidly; while in 24-hour schedule, more than 1/3 of treatment courses can not reach effective therapeutic concentration in CNS. On account of the great individual differences of serum and cerebrospinal fluid concentration of MTX, 6,8 self-control can be adopted to eliminate the influences of individual differences and other factors, such as CNS invasion and drug interaction, on cerebrospinal fluid concentration of MTX, so that the effect of different infusion durations on cerebrospinal fluid concentration of MTX can be reflected accurately. We detected cerebrospinal fluid concentration of MTX in 17 self-controlled patients, and found that cerebrospinal fluid concentration of MTX was significantly higher in 6-hour group than in 24-hour group, validating the results of previous retrospective studies. 5-10 Higher cerebrospinal fluid concentration of MTX can achieve better tumor control rate and survival benefit. Hiraga et al. 11 used 100 mg/kg MTX to treat PCNSL, and found that peak concentration of MTX in cerebrospinal fluid in 3-hour schedule group was significantly higher than that in 6-hour schedule group, tumors diminished more rapidly, response rate was increased, and survival time was prolonged. Previous studies indicated that when the blood-brain barrier was integral, the ratio of MTX concentration in cerebrospinal fluid to that in serum was about 2%, which would be significantly increased when the blood-brain barrier was injured. 2,4 Even the same schedules might result in great variation of MTX concentration, while predictive factors for serum or cerebrospinal fluid concentration of MTX haven t been found yet. 6-8 The present study showed that the ratio of cerebrospinal fluid MTX concentration to serum MTX concentration was 5.03% in 24-hour group and 2.94% in 6-hour group, suggesting that due to slow penetration of MTX into the blood-brain barrier, prolonging the maintenance of serum MTX steady state concentration would improve the penetration of MTX into CNS. In spite of this, serum peak concentration of MTX in 6-hour group was 5 times of that in 24-hour group, making the amount of MTX entered into CNS in 6-hour group still higher than that in 24-hour group, and resulting in therapeutic concentration of 1 μmol/l in CNS more effectively in 6-hour group. Previous studies verified correlation of various intensities between cerebrospinal fluid and serum concentration of MTX. 6,8,12 Multivariate analysis in our study showed that cerebrospinal fluid concentration of MTX had correlation with CNS lymphoma invasion in addition to weak positive correlation with serum concentration. Cerebrospinal fluid concentration of MTX in lymphoma patients with CNS invasion was significantly higher than that in patients without CNS invasion, indicating that lymphoma CNS invasion would lead to damage of the blood-brain barrier and thereby increase the permeability for MTX. 366 Chinese Journal of Cancer 2008; Vol. 27 Issue 10
Mucositis and bone marrow depression are main toxicity of HD-MTX, and the major risk factor of toxicity is exposure time to MTX rather than its peak concentration. 13 The threshold concentrations of MTX for producing intestinal tract mucous membrane and bone marrow toxicity are only 5 μmol/l and 10 μmol/l, which are far lower than serum steady state concentration of MTX, therefore, the maintenance time of its steady state concentration becomes the determinative factor of toxicity. Observation on 17 self-controlled cases in our study showed that the occurrence of mucositis and bone marrow suppression was significantly less in 6-hour group than in 24-hour group. Serum concentration of MTX in 6-hour group was rapidly decreased to below toxic threshold concentration, which significantly shortened the exposure time of mucous membrane and myeloid tissue to MTX as compared with that in 24-hour group. In addition, LV rescue in 6-hour group was performed relatively earlier, which significantly decreased the toxicity of short time administration of HD-MTX. In summary, 6-hour intravenous administration of 1 3 g/m 2 HD-MTX can obtain higher MTX concentration in CNS, reach therapeutic concentration of 1 μmol/l in CNS more effectively with lower peripheral toxicity and better tolerance as compared with 24-hour administration. There is weak positive correlation between cerebrospinal fluid and serum concentration of MTX. Lymphoma CNS invasion would lead to damage of the blood-brain barrier, resulting in enhanced permeability for MTX and increased cerebrospinal fluid concentration of MTX. Whether higher cerebrospinal fluid concentration of MTX can increase therapeutic effect on CNS lymphoma needs to be validated by randomized controlled study. References: [1] Borsi JD, Schuler D, Moe PJ. Methotrexate administered by 6-h and 24-h infusion: a pharmacokinetic comparison [J]. Cancer Chemother Pharmacol, 1988,22(1):33-35. [2] Tetef ML, Margolin KA, Doroshow JH, et al. Pharmacokinetics and toxicity of high-dose intravenous methotrexate in the treatment of leptomeningeal carcinomatosis [J]. Cancer Chemother Pharmacol, 2000,46(1):19-26. [3] Guo P, Wang X, Liu L, et al. Determination of methotrexate and its major metabolite 7-hydroxymethotrexate in mouse plasma and brain tissue by liquid chromatography-tandem mass spectrometry [J]. J Pharm Biomed Anal, 2007,43(5):1789-95. [4] Green MR, Chowdhary S, Lombardi KM, et al. Clinical utility and pharmacology of highdose methotrexate in the treatment of primary CNS lymphoma [J]. Expert Rev Neurother, 2006,6(5):635-652. [5] Vassal G, Valteau D, Bonnay M, et al. Cerebrospinal fluid and plasma methotrexate levels following high-dose regimen given as a 3-hour intravenous infusion in children with non- Hodgkin s lymphoma [J]. Pediatr Hematol Oncol, 1990,7(1):71-77. [6] Millot F, Rubie H, Mazingue F, et al. Cerebrospinal fluid drug levels of leukemic children receiving intravenous 5 g/m2 methotrexate [J]. Leuk Lymphoma, 1994,14(1-2):141-144. [7] Milano G, Thyss A, Serre Debeauvais F, et al. CSF drug levels for children with acute lymphoblastic leukemia treated by 5 g/m2 methotrexate. A study from the EORTC Children's Leukemia Cooperative Group [J]. Eur J Cancer, 1990,26(4):492-495. [8] Seidel H, Andersen A, Kvaløy JT, et al. Variability in methotrexate serum and cerebrospinal fluid pharmacokinetics in children with acute lymphocytic leukemia: relation to assay methodology and physiological variables [J]. Leuk Res, 2000,24(3):193-199. [9] Tang W, Wang YX, Zhang DL, et al. Monitoring of concentration in serunl and cerebrospinal fluid of high-dose methotrexate an d it s adverse drug reaction in acute lymphocyte leukemia children [J]. Chin J Clin Pharmacy, 2002,11(3):150-152. [in Chinese] [10] Zhan JY, Ye TZ, Wu ZL, et al. Clinical study on high-dose methotrexate for treating acute lymphocytic leukemia in children [J]. J Appl Clin Pediatr, 2005,20(7):652-654. [in Chinese] [11] Hiraga S, Arita N, Ohnishi T, et al. Rapid infusion of high-dose methotrexate resulting in enhanced penetration into cerebrospinal fluid and intensified tumor response in primary central nervous system lymphomas [J]. J Neurosurg, 1999,91(2):221-230. [12] Lippens RJ, Winograd B. Methotrexate concentration levels in the cerebrospinal fluid during high-dose methotrexate infusions: an unreliable prediction [J]. Pediatr Hematol Oncol, 1988,5(2):115-124. [13] Rask C, Albertioni F, Bentzen SM, et al. Clinical and pharmacokinetic risk factors for highdose methotrexate-induced toxicity in children with acute lymphoblastic leukemia-a logistic regression analysis [J]. Acta Oncol, 1998,37(3):277-284. www.landesbioscience.com Chinese Journal of Cancer 367