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The Oncologist, Vol. 12, No. 11, 1299-1308, November 2007; doi:10.1634/theoncologist.12-11-1299
© 2007 AlphaMed Press

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Clinical Pharmacology

Glucarpidase (Carboxypeptidase G2) Intervention in Adult and Elderly Cancer Patients with Renal Dysfunction and Delayed Methotrexate Elimination After High-Dose Methotrexate Therapy

Stefan Schwartza, Klaus Bornerb, Krystina Müllera, Peter Martusc, Lars Fischera, Agnieszka Korfela, Timothy Autond, Eckhard Thiela

aMedizinische Klinik III, bInstitut für Klinische Chemie und Pathobiochemie, and cInstitut für Biometrie und Klinische Epidemiologie, Charité, Campus Benjamin Franklin, Berlin, Germany; dProtherics PLC, Runcorn, United Kingdom

Key Words. Carboxypeptidase G2 • Methotrexate • Toxicity • Kidney failure • Aged

Correspondence: Stefan Schwartz, M.D., Medizinische Klinik III, Hämatologie, Onkologie und Transfusionsmedizin, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany. Telephone: 49-30-8445-2337; Fax: 49-30-8445-4468; e-mail: stefan.schwartz{at}charite.de

Received December 11, 2006; accepted for publication September 12, 2007.

Disclosure: S.S. has acted as a consultant for and received support from Protherics PLC. P.M. has acted as a consultant for Protherics PLC. T.A. is an employee of, owns stock in, and has received support from Protherics PLC. This study was supported in part by a research grant from Protherics PLC, Runcorn, UK.


   Learning Objectives
Top
 Learning Objectives
Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 Conclusion
 Acknowledgments
 References
 
After completing this course, the reader will be able to:

  1. Identify risk factors for kidney failure associated with high-dose MTX therapy.
  2. Discuss the therapeutic options in patients with delayed MTX elimination.
  3. Discuss the potential risks and benefits of glucarpidase intervention.

Access and take the CME test online and receive 1 AMA PRA Category 1 CreditTM at CME.TheOncologist.com


   ABSTRACT
Top
 Learning Objectives
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 Conclusion
 Acknowledgments
 References
 
Objective. Leucovorin and extracorporeal removal of methotrexate (MTX) have limited efficacy in delayed MTX elimination after high-dose methotrexate (HD-MTX) therapy. Glucarpidase (carboxypeptidase G2) cleaves MTX into nontoxic metabolites, but experience with this enzyme is limited in adult patients. We evaluated the effects of glucarpidase intervention in adult and elderly patients with delayed MTX elimination.

Patients and Methods. Forty-three patients (age, 18–78 years) with MTX serum concentrations (sMTX) of 1–1,187 µmol/l received glucarpidase, leucovorin rescue guided by MTX immunoassay, and standard supportive care. MTX and MTX metabolites were quantified in serum (24 patients) and urine (8 patients) by high-performance liquid chromatography. Contributory risk factors, toxicities, and survival were recorded in all patients.

Results. Glucarpidase was well tolerated and resulted in an immediate >97% reduction in sMTX, with a 0.2%–35% urinary recovery of the total MTX dose as inactive MTX metabolites. Forty (93%) of 43 patients had normalization (n = 25) or improvement (n = 15) of their serum creatinine. Frequent grade III–IV MTX toxicities were hematological (60%) and mucositis (35%); only eight (19%) patients developed grade III–IV nephrotoxicity. Ten (23%) of 43 patients experienced fatal complications associated with HD-MTX therapy. Patients with three or more contributory risk factors for delayed MTX elimination had a significantly poorer survival than patients with fewer than three risk factors (hazard ratio, 3.64; confidence interval, 1.14–17.54).

Conclusions. Glucarpidase is well tolerated and produces a rapid inactivation of substantial amounts of MTX. However, overall results are still unsatisfactory in adult and elderly patients, suggesting that earlier recognition of delayed MTX elimination and more rapid intervention are needed.


   INTRODUCTION
Top
 Learning Objectives
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 Conclusion
 Acknowledgments
 References
 
High-dose methotrexate (HD-MTX) is frequently used in various malignancies, including acute lymphoblastic leukemia, osteosarcoma, central nervous system (CNS) lymphoma, and leptomeningeal cancer [1–4]. With leucovorin rescue and standard supportive care, HD-MTX carries a low risk for severe treatment-related toxicity. However, around 2%–10% of patients experience grade ≥2 HD-MTX–related nephrotoxicity, which could result in delayed further treatment or may occasionally be life threatening. Frequencies of grade 3 or 4 HD-MTX–related nephrotoxicity vary from only 0.6% in osteosarcoma patients to 4%–5% in mostly elderly patients with primary CNS lymphoma [5–7]. Precipitation of methotrexate (MTX) in renal tubules is thought to be the main mechanism causing HD-MTX–induced renal failure, and consequently, prolonged exposure to toxic blood MTX concentrations. Various types of renal replacement therapies have been used to enhance MTX clearance, but these require invasive access, have a limited effect in lowering blood MTX levels with rebound increase in MTX levels on termination, and are not always readily available [8].

Soon after the introduction of MTX into anticancer therapy, a loss of its biological activity was observed after contact with several bacteria. In 1967, a bacterial enzyme that cleaves glutamate from folates was identified and named carboxypeptidase G [9, 10]. Carboxypeptidase G1, isolated from Pseudomonas stutzeri, was successfully used to lower MTX blood levels in animal models and man, but its bacterial source was lost [11, 12]. In 1983, the gene for carboxypeptidase G2 (now termed glucarpidase), derived from Pseudomonas sp. strain RS-16, was cloned into Escherichia coli, allowing the production of sufficient amounts of this enzyme for therapeutic purposes [13, 14]. Enzymatic cleavage of glutamate from MTX by glucarpidase results in formation of the nontoxic metabolite 2,4-diamino-N10-methylpteroic acid (DAMPA) [15]. In an early study, glucarpidase, together with thymidine and leucovorin, was given to 20 patients, mainly children and young adults, who experienced HD-MTX–induced renal failure. Glucarpidase caused a pronounced and rapid reduction in circulating MTX, and only mild-to-moderate MTX toxicity occurred [16]. A subsequent study evaluated glucarpidase treatment in 65, mostly pediatric, patients with HD-MTX–induced renal failure. Following glucarpidase intervention, grade 3 or 4 MTX-related toxicities occurred in up to 40% of patients, but only four patients died as a result of HD-MTX–related myelosuppression or infectious complications [17].

To extend these observations in a larger cohort of adult patients with HD-MTX–induced renal failure, including patients of advanced age, we initiated a nationwide study in Germany. In particular, we were interested to assess the amount of enzyme-inactivated MTX by quantification of MTX metabolites in urine samples. Furthermore, we evaluated risk factors contributing to renal injury during HD-MTX therapy [18–21].


   PATIENTS AND METHODS
Top
 Learning Objectives
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 Conclusion
 Acknowledgments
 References
 
Eligibility and Enrolment
Patients receiving MTX at a dose of >1 g/m2 body surface area (BSA) for a solid tumor or hematopoietic malignancy were eligible if their serum MTX concentration (sMTX) remained at >5 µmol/l at ≥42 hours after the start of MTX therapy. Patients with renal failure (serum creatinine [sCrea] >1.5x the upper limit of normal and/or oliguria) were also eligible if this occurred <42 hours after the start of MTX therapy or their sMTX remained at >1 µmol/l at ≥42 hours or >0.4 µmol/l at ≥48 hours after the start of MTX therapy. Following patient registration (24-hour service), the study drug was immediately dispatched by a courier service.

The study was approved by the ethical committee of the Charité and registered with the national regulatory authority (Bundesamt für Arzneimittel und Medizinprodukte, Bonn, Germany). Informed and written consent was always obtained according to the Declaration of Helsinki. The study protocol was offered to 110 centers collaborating within the German multicenter study group for treatment of adult acute lymphoblastic leukemia.

Glucarpidase Treatment and Supportive Care
Glucarpidase, manufactured by the Centre for Applied Microbiology and Research (Salisbury, UK), was provided as a lyophilisate (1,000 units per vial) [13, 14]. Following reconstitution in isotonic saline, glucarpidase was given i.v. over 5 minutes (scheduled dosage, 50 units/kg). A second glucarpidase treatment was optional in patients with sMTX >0.1 µmol/l ≥24 hours after the first intervention. Continuation of supportive care measures (i.v. fluid intake ≥3 l/m2 BSA within 24 hours, urine pH ≥7, leucovorin) was recommended until sMTX was reduced to <0.1 µmol/l twice within 24 hours. Patients with sMTX ≤5 µmol/l received 15–75 mg/m2 BSA of leucovorin i.v. every 6 hours, according to a previously published dosing schedule [22]. Patients with sMTX >5 µmol/l received leucovorin doses as follows: leucovorin (mg) = sMTX (µmol/l) x body weight (kg). Decisions on secondary glucarpidase treatment and leucovorin rescue were based on a readily available MTX immunoassay.

Patient Monitoring
Adverse events and toxicity were assessed daily. Serum creatinine, electrolytes, and blood cell counts were monitored daily and differential blood count, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, bilirubin, prothrombin time, and activated partial thromboplastin time were measured thrice weekly. Information on the following risk factors, possibly contributing to HD-MTX–induced renal failure, were requested: body mass index (BMI), any comedication potentially interfering with MTX excretion or with known nephrotoxicity, whether the urine pH was measured as <7 at any time or the i.v. fluid intake was <3 l/m2 BSA within 24 hours, any hepatic dysfunction at baseline, presence of a third space, urinary tract obstruction, or baseline renal dysfunction. Clinical, routine laboratory, and toxicity data [23] were recorded in standardized case report forms. sMTX was determined locally by a commercially available fluorescence polarization immunoassay (Abbott, Wiesbaden, Germany) [24].

Pharmacokinetic Sampling
Serum samples for high-performance liquid chromatography (HPLC) analyses were requested immediately before and 15, 60, and 240 minutes after glucarpidase treatment (either primary or secondary), and thereafter once daily. To inactivate glucarpidase, tubes prefilled with 1 N hydrochloric acid were provided. Blood samples were immediately put on ice and, after centrifugation, serum was transferred into the prepared tubes to give a final concentration of 0.1 N hydrochloric acid.

In a subset of patients, whole urine sampling was started after registration until glucarpidase treatment and continued thereafter until sMTX declined to <0.1 µmol/l. The total urine volume of each container and time intervals of urine sampling were recorded. Specimens were drawn from each urine container after gentle mixing. All samples were stored at least at –20°C until HPLC analysis.

HPLC Assay of MTX and MTX Metabolites in Serum and Urine
Concentrations of MTX, 7-OH-methotrexate (OH-MTX), DAMPA, and OH-DAMPA were determined by isocratic reversed-phase HPLC with UV detection at 315 nm. Serum samples were deproteinized with perchloric acid. Urine samples were diluted with buffer. Separation was performed on a C18 column filled with spherical beads (NucleosilTM; Macherey & Nagel, Düren, Germany) or a monolithic rod (Chromolith Speed RodTM; E. Merck, Darmstadt, Germany). Validation of the method yielded a limit of quantitation of 0.08 µmol/l for serum, a precision between series of 4.3%–6.9% (coefficient of variation), and recovery rates of 96%–105%. Commercially available standards for MTX, OH-MTX, and DAMPA were used (Sigma-Aldrich, Deisenhofen, Germany), whereas OH-DAMPA was produced by enzymatic cleavage of OH-MTX with glucarpidase and identified in chromatograms. Approximate concentrations of OH-DAMPA were estimated by reference to the calibration of OH-MTX.

The amounts of recovered MTX and MTX metabolites within urine sampling periods were calculated as follows: urine concentration (mol/l) x urine volume (l) = amount of excreted substance (mol). The percentages of molar quantities of the total MTX dose administered, recovered from urine samples as MTX and MTX metabolites, were calculated.

Immunogenicity Testing
Antiglucarpidase IgG antibody responses were assessed using an enzyme-linked immunosorbent assay. Briefly, microtiter plate wells were coated with glucarpidase prior to incubation with serum samples. Biotinylated protein G (Biogenesis, Poole, UK) and streptavidin horseradish peroxidase (DAKO, Ely, UK) were then added, followed by tetramethylbenzidine as a chromogenic reagent (Europa Bioproducts, Wicken, UK). A positive signal was indicated by an increase in optical absorbance, measured at 450 nm, above the limit of detection (LOD). The LOD was defined as the mean plus two standard deviations of absorbance values from control samples. Serum from subjects who had not been exposed to glucarpidase served as negative controls, whereas a rabbit antiglucarpidase antibody (CytImmune Sciences Inc., College Park, MD) was used as a positive control.

Serum samples that tested positive for antiglucarpidase antibodies were subsequently assayed to determine the effects on glucarpidase activity. The activity of glucarpidase, added to test samples, was determined using an enzyme-substrate spectrophotometric method, where cleavage of the substrate, MTX (Sigma-Aldrich, Gillingham, UK), was recorded by UV absorbance at 320 nm against time (Ultrospec 4000; Amersham Biosciences, Amersham, UK). By comparing the gradients of reactions, the enzyme activity in test versus control samples was calculated. A significant reduction in enzyme activity was considered when the glucarpidase activity was reduced to <80%.

Statistical Methods
Because of skewed distributions of continuous variables, medians and ranges are given for descriptive analyses. Survival data were analyzed using the Cox proportional hazard model. Patients whose death was unrelated to HD-MTX were censored at their last follow-up. The level of significance was 0.05 (two-sided). SPSS® for WindowsTM, release 11.5 (SPSS Inc., Chicago, IL), was used for statistical analyses.


   RESULTS
Top
 Learning Objectives
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 Conclusion
 Acknowledgments
 References
 
Baseline and Treatment Characteristics
Forty-three patients with acute lymphoblastic leukemia (n = 13), lymphoma with (n = 16) or without (n = 12) CNS involvement, germ cell tumor (n = 1), or osteosarcoma (n = 1) were registered (Table 1). Treatment with HD-MTX was given within (n = 26) or outside (n = 17) clinical trials. Treatment centers were academic medical centers (n = 29), large community hospitals with an inpatient capacity ≥700 (n = 11), or small community hospitals (n = 3). HD-MTX infusion times were 3–6 hours (n = 20) and 23–26 hours (n = 23). At registration, sMTX was in the range of 1–1,187 µmol/l (median, 10.5 µmol/l). The majority of patients (40/43) already had elevated sCrea and three patients developed oliguria (Table 1). The median time from the start of HD-MTX infusion to first glucarpidase treatment was 56 hours. At first treatment, the majority of patients (n = 32) received a glucarpidase dose close to the protocol recommendation of 50 units/kg (47–58 units/kg). The remaining 11 patients received lower doses (10–31 units/kg) because of a transient shortage of supply during the early part of the study. Except for an allergic skin reaction with pruritus (grade III), which first occurred 3 days after treatment in one patient, and fever (grade II) in another patient, glucarpidase was tolerated without side effects. Both events resolved without sequelae. A second dose of glucarpidase was administered to three patients 24–41 hours after the first dose; no side effects were reported after the second dose (Table 1). In all patients, total doses of leucovorin were 180–35,820 mg (median, 3,380 mg). At 42 hours after the start of the HD-MTX infusion, ratios of the leucovorin dose administered to the scheduled leucovorin dose varied in the range of 1%–215% (median, 27%).


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  		Table 1. Patient and treatment characteristics

 
Risk Factors for HD-MTX–Induced Renal Failure
Twenty-six (60%) patients who were overweight (BMI, 25–43 kg/m2) received 1.5–8 g/m2 (median, 2.9 g/m2) MTX (Table 2). Twenty-one (49%) patients had comedications with nephrotoxic potential (n = 7) or known interference with renal MTX elimination: salicylates (n = 5), nonsteroidal anti-inflammatory drugs (n = 6), beta-lactam antibiotics (n = 8), sulfonamides (n = 7), and aminoglycosides (n = 1). A urine pH <7 was recorded in 15 (35%) patients. Other risk factors were reported in 5%–23% of patients (Table 2). Two patients (5%) had diarrhea and none of the patients had urinary tract obstruction. Only four (9%) patients had no identifiable risk factor.


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  		Table 2. Risk factors contributing to delayed methotrexate (MTX) clearance in 43 patients treated with glucarpidase

 
Effects of Glucarpidase on Serum Levels of MTX and MTX Metabolites
In this study of a rare medical emergency, serum samples from 24 (56%) of 43 patients were available for HPLC analyses. In the remaining 19 patients, serum samples were not provided for a variety of reasons (e.g., glucarpidase treatment outside usual working hours with lack of appropriate laboratory facilities). In 24 patients with available samples, sMTX decreased markedly within 7–50 minutes (median, 15 minutes) after intervention with glucarpidase from a median of 5.1 µmol/l (range, 0.4–165.9 µmol/l) to a median of <0.1 µmol/l (range, <0.1–1 µmol/l), which corresponds to a >97% reduction (Fig. 1). Simultaneously, DAMPA serum levels increased, with a median DAMPA increase to MTX decrease ratio of 70% (data not shown).


Figure 1
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  		Figure 1. Immediate reduction in serum methotrexate (MTX) levels in 24 patients treated with glucarpidase.

 
Serial serum samples were assayed to detect rebound increases in sMTX during follow-up (median, 105 hours). In all 24 evaluable patients, the median sMTX increased from a nadir of <0.1 µmol/l (range, <0.1–1 µmol/l) to 0.24 µmol/l (range, <0.1–14.8 µmol/l) during follow-up, which corresponds to a median sustained reduction in sMTX of 97% (range, 18%–99%). In 23 patients, sMTX rebound increases did not exceed 1.2 µmol/l. The remaining patient had a sMTX of 165.9 µmol/l, and showed a rapid decline to 1 µmol/l within 15 minutes after glucarpidase treatment with a subsequent maximum increase to 14.8 µmol/l at 85 hours after intervention (Fig. 2A). Nine of 11 patients with early (<56 hours) glucarpidase intervention, compared with 10 of 13 patients with late (≥56 hours) intervention, had sustained ≥90% reductions in sMTX (not significant).


Figure 2
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  		Figure 2. Serum MTX and DAMPA levels following glucarpidase treatment. (A): Patient with baseline serum MTX level of 165.9 µmol/l and low urine output (0.1 l/hour). (B): Patient with baseline serum MTX level of 43.5 µmol/l and high urine output (0.31 l/hour).

Abbreviations: DAMPA, 2,4-diamino-N10-methylpteroic acid; MTX, methotrexate.

 
Repeat glucarpidase treatment during the same cycle of HD-MTX was given to three patients. In two of these patients, sMTX prior to the second intervention was greatly overestimated because of interference of DAMPA in the immunoassay (sMTX immunoassay, 4.1 and 4.1 µmol/l; sMTX HPLC, 0.13 and 0.08 µmol/l). In these two patients, sMTX further decreased slightly to 0.11 and <0.08 µmol/l, respectively, after repeat intervention (Fig. 2B). The remaining patient had a sMTX of 0.5 µmol/l (HPLC) and a low concentration of DAMPA (0.01 µmol/l) prior to repeat intervention. In this patient, sMTX declined from 0.5 µmol/l to undetectable after repeat intervention.

The median reduction in serum concentration of OH-MTX in 21 patients was 43% (range, 18%–99%), whereas three patients had 101%–138% increases in OH-MTX concentration within 7–15 minutes after glucarpidase administration (data not shown).

Urine Recovery of MTX Metabolites After Glucarpidase Treatment
Urinary excretion of DAMPA and OH-DAMPA was quantified in eight patients during an 85- to 217-hour follow-up. Proportions of the total MTX dose, recovered as DAMPA and OH-DAMPA, varied from 0.2%–35% (Fig. 3). In two patients, the urine recovery rates of DAMPA and OH-DAMPA were 35% and 8.9%, compared with only ≤3.7% in the other six patients. These two patients had higher pretreatment sMTX than all other patients (≥28.3 µmol/l versus ≤5.8 µmol/l), with the exception of a single patient, and received their first glucarpidase intervention early (48 and 50 hours, versus ≥50 hours in all other patients after the start of HD-MTX). One patient received glucarpidase at 50 hours after the start of HD-MTX when his sMTX was 165.9 µmol/l, but his DAMPA and OH-DAMPA urine recovery was only 3.7%. This patient had an average urine output of only 0.1 l/hour and a sCrea of 310 µmol/l prior to glucarpidase, compared with a median urine output of 0.3 l/hour and a sCrea of ≤238 µmol/l in all other patients. Furthermore, this particular patient was treated with a single dose of only 11 units/kg glucarpidase, compared with a median glucarpidase dose of 50 units/kg in all other patients. Finally, the serum DAMPA concentrations in this patient remained >40 µmol/l (Fig. 2A), whereas all other patients had steadily declining serum DAMPA concentrations (Fig. 2B).


Figure 3
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  		Figure 3. Recovery of the inactive MTX metabolites DAMPA and OH-DAMPA from urine samples in eight patients following glucarpidase treatment.

Abbreviations: DAMPA, 2,4-diamino-N10-methylpteroic acid; MTX, methotrexate.

 
Immunogenicity of Glucarpidase
Serum samples for immunogenicity testing, obtained 7–41 days after glucarpidase treatment, were available from seven patients. In three of these seven patients, antiglucarpidase antibodies were first detected 7–17 days after glucarpidase administration, whereas the remaining four patients did not show antibody responses during 7–41 days of follow-up. All positive samples were additionally tested for their effect on glucarpidase activity. Only one of four antibody-positive samples showed a slight (23%) reduction in glucarpidase activity.

Recovery from Renal Failure, MTX Toxicity, and Outcome
The median peak sCrea in all 43 patients was 232 µmol/l. In 40 (93%) of 43 patients, the sCrea remained normal (n = 1), returned to normal (n = 24), or improved (n = 15) after glucarpidase intervention. Three (7%) of 43 patients showed an increasing sCrea during follow-up. In nine and 16 patients, the sCrea returned to baseline values and <1.5x the baseline level within a median of 28 days and 19 days after the start of HD-MTX, respectively. In 26 and 19 patients, the sCrea remained above these thresholds within median observation periods after the start of HD-MTX of 36 days and 12 days (eight patients with missing data), respectively. Five patients received hemodialysis (n = 3), hemofiltration (n = 3), or hemoperfusion (n = 1) for 2–13 days, although no absolute necessity for this was reported for four of them. The remaining patient had a decline in sCrea after glucarpidase administration, but subsequently required hemofiltration as a result of severe infection with renal failure.

Renal toxicity of any grade was the most frequently reported MTX-related toxicity (98%), but only eight (19%) patients developed grade III–IV renal toxicity (Table 3). Among other grade III–IV toxicities, hematotoxicity (60%) and mucositis (35%) were the most frequently reported. Grade IV neurotoxicity occurred in two patients with acute lymphoblastic leukemia who concurrently received a single intrathecal MTX administration (15 mg): severe depression with hallucinations (n = 1) and fatal progressive cerebral coma (n = 1). There was no evidence of an accidental intrathecal MTX overdose in either patient.


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  		Table 3. Toxicities occurring after high-dose methotrexate in 43 patients treated with glucarpidase for delayed methotrexate elimination

 
Ten (23%) of 43 patients died as a result of complications associated with HD-MTX: infection (n = 7), uremia and seizures (n = 1), MTX neurotoxicity (n = 1), and peritonitis with multiorgan failure (n = 1). All seven patients with fatal infectious complications had grade IV neutropenia, three of these seven patients received other chemotherapy agents in addition to HD-MTX, and death occurred in these patients 4–38 days (median, 18 days) after the start of HD-MTX. Nine (21%) patients died as a result of causes unrelated to HD-MTX: subsequent chemotherapy (n = 5), hematopoietic stem cell transplantation (n = 1), disease progression (n = 2), and cardiovascular event (n = 1).

Statistical analyses revealed no significant impact of the time of glucarpidase intervention relative to the time of MTX administration, the sMTX prior to glucarpidase administration, whether there was a sustained reduction in sMTX, the glucarpidase dose, or age on toxicities and survival. Similar analyses in subsets of patients treated with HD-MTX alone or in combination with other cytostatic drugs did not show any significant impact on toxicities and survival. In addition, the total leucovorin dose or the ratio of the leucovorin dose administered to the leucovorin dose scheduled according to sMTX at 42 hours did not correlate with toxicities and survival, except for a trend toward less severe mucositis in patients with high total leucovorin doses. However, survival in patients with three or more contributory risk factors for delayed elimination of MTX was significantly less than that of patients with fewer than three risk factors (hazard ratio, 3.64; confidence interval, 1.14–17.54; p = .03).


   DISCUSSION
Top
 Learning Objectives
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
Conclusion
 Acknowledgments
 References
 
HD-MTX–induced renal failure with persistence of toxic blood MTX levels is a rare but life-threatening complication that occurs more frequently in adults, particularly those with advanced age and CNS lymphoma [5–7]. Hemodialysis, hemoperfusion, or a combination of the two in this condition have been repeatedly investigated, but these invasive procedures require prolonged application, might add treatment-related morbidity, and result in a limited decline in blood MTX levels with frequent rebound increases [8].

In contrast, glucarpidase is well tolerated, easy to administer, and causes rapid, pronounced, and, in the majority of patients, sustained reductions in circulating MTX, as demonstrated by this study and previous trials [16, 17]. MTX is modified intracellularly by sequential addition of glutamate molecules. These MTX polyglutamates are retained within cells and are the main mediators of MTX cytotoxicity through inhibition of various folate-dependent enzymes, including dihydrofolate reductase [25]. Thus, leucovorin rescue is still required after glucarpidase intervention as glucarpidase does not hydrolyze intracellular MTX. However, removal of MTX from the circulation is expected to affect tissue levels indirectly, by preventing further MTX uptake. Leucovorin is also a substrate for glucarpidase, but with lower affinity for the enzyme than MTX, and there is a risk that interaction of glucarpidase with leucovorin may decrease the efficacy of leucovorin rescue [14].

In two patients with repeat intervention, glucarpidase showed diminished efficacy, which was likely caused by the presence of the metabolite DAMPA. Also, an immune response to glucarpidase could potentially cause a reduction in enzyme activity or adverse effects upon repeat administration. We found antiglucarpidase antibodies in three of seven study patients, but only one of four antibody-positive samples showed a slight (23%) reduction in glucarpidase activity in vitro. More data on the immunogenicity of glucarpidase are needed before considering repeat use of this enzyme.

According to a previously used definition of renal recovery (sCrea <1.5x baseline), 16 patients recovered within a median of 19 days, and 19 patients still had a sCrea above this threshold within a median observation period of 12 days [6]. This suggests that the renal recovery in this study was somewhat slower than in patients treated with supportive care measures, including extracorporeal removal of MTX but not glucarpidase, whose reported median time to renal recovery was 16 days [6]. The severity of renal damage in this study, as indicated by the median peak creatinine level, was, however, higher (232 µmol/l = 3 mg/dl) than that of previously evaluated patients treated with supportive care not including glucarpidase or dialysis-based interventions (2.1 mg/dl), but was less than in patients who received extracorporeal removal of MTX (3.9 mg/dl) [6]. Remarkably, some patients in this study received nephrotoxic agents in addition to HD-MTX, which may have contributed in part to delays in renal recovery. It remains unclear whether DAMPA, which is regarded as virtually inert but around 10-fold less soluble than MTX, might have caused delays in renal recovery. DAMPA is cleared by the kidneys and metabolized into OH-DAMPA and DAMPA glucuronides [15, 16]. It is unknown whether variations in DAMPA metabolism have a clinical impact, requiring more refined studies with superior analytical techniques. In patients with normal renal function, around 80% of the MTX dose is excreted renally within 24 hours [26]. It is noteworthy that, in this study, up to 35% of the total MTX dose was recovered as inactive metabolites from the urine later than 48 hours after the start of the MTX infusion.

We observed a high frequency of grade III–IV MTX-related toxicities, which is not unexpected for patients with prolonged MTX exposure. In the two major previous trials evaluating glucarpidase, frequencies of fatal HD-MTX–associated complications were <7%. In contrast, we observed a strikingly high (23%) MTX-related fatality rate, which in part could be explained by the higher median age of our study patients compared with those of both previous trials (54 versus ≤16 years) [16, 17]. The use of HD-MTX in adult patients is associated with significant treatment-related mortality, and fatality rates of 9%–10% have been seen in trials evaluating HD-MTX in previously untreated adult patients, who had renal toxicity rates of only ≤14% [27–29]. In contrast, the patients in this study all had evidence of delayed elimination of MTX, which likely increased the risk for fatal MTX-related events. Except for scarce data from case reports and small case series, toxicity data and fatality rates in adult/elderly patients with HD-MTX–associated renal failure are largely unknown, which limits an estimate of the benefits of glucarpidase intervention in this particular patient subgroup. The survival and HD-MTX–related toxicities in this study did not correlate with age, baseline sMTX, time of intervention with glucarpidase, glucarpidase dose, degree of sustained reduction in sMTX, additional use of chemotherapy agents other than MTX, or inappropriately low leucovorin doses. Risk factors contributing to delayed MTX elimination were identified in the majority (91%) of patients, and the presence of multiple (three or more) risk factors correlated with fatal HD-MTX–associated complications. This emphasizes that patients should be carefully evaluated before HD-MTX is considered. Overweight is not an established risk factor associated with HD-MTX–induced renal failure, but there was a remarkably high number of overweight study patients. Pharmacokinetic data from an obese patient indicate increases in the volume of distribution and systemic clearance of MTX, which may compensate each other [30]. However, nephrotoxicity with delayed MTX elimination was reported in another obese patient treated with an intermediate dose of MTX [31]. Thus, dose adaptation guided by ideal body weight or a proposed score system might reduce the risk for HD-MTX–related toxicity in overweight patients [32].


   CONCLUSION
Top
 Learning Objectives
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 Conclusion
Acknowledgments
 References
 
In this study, glucarpidase intervention was safe and effectively reduced plasma MTX exposure in patients with delayed MTX elimination. Glucarpidase may occasionally result in antibody formation that has the potential to reduce enzyme activity upon repeat administration. Patients in this study still had a high frequency of severe toxicities, which reflects the high risks associated with delayed MTX elimination in adult patients. No conclusion can be drawn about the effects of glucarpidase on toxicity from this single-arm study, and further studies evaluating the potential of glucarpidase to reduce the risk for severe or fatal HD-MTX–induced toxicities in patients at risk are warranted. Currently, a more careful selection of patients considered suitable for HD-MTX and the use of optimal supportive care strategies should always be attempted.


   ACKNOWLEDGMENTS
Top
 Learning Objectives
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 Conclusion
 Acknowledgments
References
 
The authors are grateful to E. Borner and H. Hartwig for skillful technical assistance. We are also indebted to N. Gökbuget and D. Hoelzer from the GMALL study center for their continuous support and to R. Melton for his efforts in the early phase of this study. Results from this study were presented in part at the 40th Annual Meeting of the American Society of Clinical Oncology, New Orleans, Louisiana, June 5–8, 2004.

Participating investigators and centers: L. Basovski and H. Döhner (Medizinische Universitsklinik III, Ulm); J. Beyer and A. Neubauer (Medizinische Universitätsklinik, Marburg); A. Gerhardt and R. Pasold (Klinikum Ernst von Bergmann, Potsdam); A. Giagounidis and C. Aul (St. Johannes-Hospital, Duisburg); B. Halle and H. Bodenstein (Klinikum, Minden); P. Hernaiz-Driever and G. Henze (Charité, Campus Virchow-Klinikum, Berlin); D. Kämpfe (Klinikum, Lüdenscheidt); J. Kern and K. Wilms (Medizinische Universitätsklinik II, Würzburg); W. Kern and W. Hiddemann (Universitätsklinikum Grosshadern, München); A. Klink and M. Königsmann (Medizinische Universitätsklinik, Magdeburg); B. Lehmann and L. Trümper (Universitätsklinikum, Göttingen); L. Leimer and W.E. Aulitzky (Robert Bosch Krankenhaus, Stuttgart); M. Leithäuser and M. Freund (Universitätsklinikum, Rostock); A. Matzdorff and H. Pralle (Universitätsklinikum, Giessen); C. Müller and M. Lübbert (Medizinische Universitätsklinik I, Freiburg); K. Oevermann and A. Ganser (Medizinische Hochschule, Hannover); N. Peter and H.B. Steinhauer (Carl-Thiem-Klinikum, Cottbus); R. Plettig and G. Ehninger (Universitätsklinikum Carl Gustav Carus, Dresden); R. Schabath and W.D. Ludwig (Charité, Campus Buch, Berlin); C. Schicht and R. Andreesen (Universitätsklinikum, Regensburg); H. Schieder and R. Kuse (Allgemeines Krankenhaus St. Georg, Hamburg); B. Schmid and H.A. Dürk (St. Marien-Hospital, Hamm); S. Schwartz and E. Thiel (Charité, Campus Benjamin Franklin, Berlin); T. Spulak and L. Balleisen (Evangelisches Krankenhaus, Hamm); H. Steiniger and S. Seeber (Universitätsklinikum, Essen); U. Wedding and K. Höffken (Medizinische Universitätsklinik II, Jena); K. Wolf and M. Hänel (Klinikum, Chemnitz); A. Yaman and R. Schwerdtfeger (Deutsche Klinik für Diagnostik, Wiesbaden); I. Zirpel and H.J. Illiger (Klinikum, Oldenburg).


   REFERENCES
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 Learning Objectives
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 Conclusion
 Acknowledgments
 References
 

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