High-dose methotrexate (above 1 g/m²) is a key component of several chemotherapy regimens used to treat children with leukemia, lymphomas, and osteosarcoma.

The availability of antidote therapy, the detailed knowledge of the pharmacokinetics of the drug, and the monitoring of the serum level in the individual patient allow for the prevention of dose-limiting toxicity upon healthy tissues and the administration of high doses with an enhanced antitumor effect, also providing an alternative for central nervous system prophylaxis in malignant hemopathies. Supportive therapy includes hyperhydration with urine alkalinization and administration of serum level-adapted antidote therapy with calcium folinate. However, nephrotoxicity remains a risk that can lead to significant morbidity and mortality. When this happens, it is suggested that the drug be replaced with another cytostatic, which affects therapeutic success.

We present the case of a child who experienced fourth-degree nephrotoxicity after the initial administration of a high dose of methotrexate, requiring renal replacement therapy.

The patient was a 13-year-old girl with an unremarkable medical history who complained of pain and a limping gait. She also had swelling in the distal third of her left thigh. The MRI revealed a 5-cm tumor mass localized to the distal metaphysis, epiphysis, and diaphysis of the left femur. There was no joint or neurovascular involvement, but there was a pronounced periosteal reaction. A biopsy was performed, and fibroblastic osteosarcoma was verified.

Neoadjuvant polychemotherapy was started according to the EURAMOS1 protocol. After applying the first course of the AP protocol (doxorubicin, cisplatin), therapy with the first course of high-dose methotrexate (12 g/m ²) was initiated, and the patient received 20 g of methotrexate with concomitant hyperhydration and urine alkalinization. The day after the infusion, there was repeated vomiting and severe oliguria, which eventually led to anuria. The laboratory tests showed steeply increasing creatinine level (from 55 µmol/L at baseline to 419 µmol/L), urea (from 4.1 mmol/L at baseline to 18.6 mmol/L), uric acid (520 µmol/L), ASAT (2356 U/l), ALAT (3534 U/l), total bilirubin (54.2 µmol/L), direct bilirubin (10.8 µmol/L), total protein (49 g/L), and albumin (31 g/L). Extremely high serum levels of methotrexate and delayed clearance were observed. At 48 hours, the level was 446.27 µmol/L; at 54 hours, it was 375 µmol/L; at 60 hours, it was 311 µmol/L; at 72 hours, it was 239 µmol/L; and at 90 hours, it was 143 µmol/L. Despite administering an antidote with high doses of calcium folinate and hyperhydration (5 l/24 hours) immediately after the medication was given, the patient’s condition did not improve. At 80 hours after infusion, the decision was made to start renal replacement therapy (hemodialysis). Four sessions were performed on 4 consecutive days. The entire clinical course is presented inFig. 1 . As a result, kidney function improved, and methotrexate levels decreased. A probable polymorphism of drug-metabolizing enzymes for the medication is discussed as the cause of the acute multi-organ toxicity—kidney and liver. At the same time, methotrexate is a key cytostatic in the treatment of osteosarcoma, and its continuation in the polychemotherapy protocol is ultimate. Following discussion, it was decided that further courses of high-dose methotrexate would be conducted, provided that carboxypeptidase G2 (glucarpidase) was available in the department and the doses were increased gradually. Three more courses followed, respectively involving 8, 12, and 18 g, then 20 g, of methotrexate. These proceeded without complications or evidence of nephrotoxicity, with the medication being eliminated in a timely manner(Table 1) . Following the operation, the patient was treated in accordance with the MAPIE arm of the postoperative phase of the protocol. The child is currently in remission and receives targeted therapy with regorafenib.

Methotrexate is an antimetabolite of an antifolate type. It competitively inhibits dihydrofolate reductase—an enzyme involved in tetrahydrofolate synthesis. Tetrahydrofolate is required for the synthesis of the nucleoside required for DNA synthesis. Therefore, methotrexate blocks the synthesis of DNA and RNA proteins. In addition, methotrexate induces inhibition of aminoimidazole carboxamide ribonucleotide (AICAR) transformylase, thereby increasing intracellular AICAR levels, which leads to the inhibition of adenosine and guanine metabolism and ultimately to the accumulation of adenosine. Adenosine induces an anti-inflammatory response by suppressing T-cell activation, downregulating B-cells, sensitizing CD95 T-cells, and inhibiting the binding of interleukin-1β to the cell surface. Administration of a high-dose regimen followed by calcium folinate antidote therapy is part of several protocols for the treatment of pediatric and adult malignancies. In patients with normal renal function, with the simultaneous administration of hyperhydration and sodium bicarbonate alkalinization of the urine, this treatment can be administered safely. Despite these measures, high-dose methotrexate-induced renal dysfunction continues to occur in about 1.8% to 36%, depending on the definition of acute kidney injury used. A polymorphism of drug-metabolizing enzymes for the drug (folylpolyglutamate synthase, gamma-glutamyl hydrolase) is discussed as the reason for its appearance. The genetic predispositions cited in the literature whose polymorphism has been proven to be associated with methotrexate-induced nephrotoxicity are AT-rich interaction domain 5B (ARID5B) and solute carrier organic anion transporter family member 1B1 (SLCO1B1).^[1–3]

The main pathogenetic mechanism of its occurrence is precipitation of methotrexate and its metabolites in the renal tubules, clinically manifesting as crystalline nephropathy, which also leads to delayed excretion of the medication. A number of studies have also been carried out to identify the risk factors for its appearance, and some of them are hypoalbuminemia, urine pH below 7 on the first day, male sex, drug interaction with other types of medication (Biseptol, nitroprusside natrium), and furosemide administration during methotrexate clearance.^[4–7] Prolonging the duration of infusion to reduce the peak concentration of methotrexate in serum and urine does not change the risk of acute kidney injury.^[8] According to the generally accepted criteria for serious side effects in patients with oncological diseases, nephrotoxicity is classified into four grades: first degree: serum creatinine increases by 1.5–2 times above baseline level; second degree: serum creatinine increases by 2–3 times above baseline level; third degree: serum creatinine increases by more than 3 times above baseline level; and fourth degree: renal replacement therapy becomes necessary.^[9] A meta-analysis of patients with osteosarcoma found an incidence rate of 1.8% for nephrotoxicity, with the highest rate being second-degree injury.^[10]

Maintenance therapy during the infusion of high-dose methotrexate includes intensive hydration—for children 100–150 ml/m²/h, starting 12 hours before and continuing 24-48 hours after the end of the infusion—along with urine alkalinization by administration of sodium bicarbonate 40 mmol/L. The purpose of alkalinization is to maintain the urine pH above 7, and when an alkaline serum pH is reached but without achieving an alkaline urine pH, the recommendation is to add acetazolamide, a carboanhydrase inhibitor, leading to an increased release of sodium, bicarbonate, and water in the urine without leading to an increase of the serum pH. In cases where there is evidence of previous nephrotoxicity, it is recommended that a lower dose of methotrexate be used in the next administration. Strict monitoring of methotrexate serum level is another mainstay in the administration of the high-dose regimen.^[11]

In the event of nephrotoxicity, characterized by an increase in serum creatinine levels, it is recommended that hyperhydration be continued to the maximum tolerated dose of 3 l/m², with alkalization and the addition of acetazolamide and leucovorin. The dose of leucovorin depends on the serum level of methotrexate, which should be monitored every 24 hours until it drops below 0.2 μmol/L.‌^[12] Regarding the application of extracorporeal purification methods, the reported results are ambivalent. High-flux hemodialysis has been shown to be the most effective method of methotrexate clearance, although a significant increase in serum methotrexate levels by 20% to 220% has been documented in several patients a few hours after the procedure.^[13] Carboxypeptidase G2 (glucarpidase, Voraxaze) is a recombinant bacterial enzyme that disintegrates circulating methotrexate to glutamate and 4-amino-4-deoxy-N10-methylpteroic acid (DAMPA), which are less water soluble and undergo hepatic metabolism and biliary excretion. The drug is highly effective in reducing methotrexate blood levels—they are falling by over 95% just 15 minutes after injection.^[14] Its use is indicated at certain methotrexate blood levels and serum creatinine levels at the 36th and 48th hours after infusion. Because of its successful use in pediatric patients with severe nephrotoxicity, the successful use of high-dose methotrexate has become possible again.^{[14, 15]} In the described case, an extracorporeal method of purification was preferred due to the impossibility of supplying Voraxaze as an unregistered medication in the country.

The presented clinical case illustrates a rare but potentially life-threatening toxicity of the high-dose methotrexate in a child with osteosarcoma. Despite supportive measures such as hyperhydration and alkalization, the extremely delayed blood clearance of the drug results in severe acute kidney injury. An extracorporeal blood purification (hemodialysis) was applied with a good response. Given the significant role of methotrexate in the child’s chemotherapy protocol, the difficult decision was made to continue administering it with a gradual increase in dosage at each subsequent cycle until the recommended dosage was reached. This case illustrates the successful re-administration of high-dose methotrexate after the occurrence of grade IV nephrotoxicity following the first cytostatic course.

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The authors have declared that no competing interests exist.