A10: Antidotes in Depth

Folates refer to the metabolically active reduced forms of folic acid, including dihydrofolate and tetrahydrofolate. These folates are vital to cellular biochemistry, including the synthesis of purines and DNA. Folic acid must be reduced in vivo by dihydrofolate reductase to tetrahydrofolate. Dihydrofolate reductase inhibitors such as methotrexate prevent this reduction. Leucovorin (folinic acid) and levoleucovorin, and compared with folic acid, they do not require dihydrofolate reductase for activation. Therefore, either leucovorin or levoleucovorin is the primary antidote for a patient who receives an overdose of methotrexate or another dihydrofolate reductase inhibitor.

Methanol is metabolized to the active and toxic formic acid. Folates, including folic acid and leucovorin, speed up the conversion of formic acid to nontoxic metabolites. Because methanol does not interfere with the synthesis of tetrahydrofolate, either folic acid or leucovorin is acceptable for a patient poisoned by methanol who necessitates enhanced formate metabolism. Preliminary evidence also suggests a role for folic acid to enhance ­arsenic elimination.

In the early 1930s when Lucy Wills went to Bombay to study pregnant textile workers with macrocytic anemia, she found that a yeast extract given to these poor people with a nutritionally deficient diet corrected and prevented their anemia.¹¹ Mitchell isolated the active ingredient from spinach in 1941 and named it folic acid from the Latin folium meaning leaf.¹¹ Subsequently, folic acid was synthesized and its chemical structure identified. Since then, the many roles of folate and natural or induced folate deficiency continue to be studied.

Folic acid (pteroylglutamic acid), an essential water-soluble vitamin, consists of a pteridine ring joined to PABA (para-aminobenzoic acid) and glutamic acid.¹⁰ Folic acid is the most common of the many folate congeners that exist in nature and perform essential cellular metabolic functions. Folic acid is often called vitamin B₉. After absorption, folic acid is reduced by dihydrofolic acid reductase (DHFR) to dihydrofolic acid and then tetrahydrofolic acid (THF), which accepts one carbon groups. Tetrahydrofolic acid serves as the precursor for several biologically active forms of folic acid, including 5-formyltetrahydrofolic acid (5-formyl THF), which is best known as folinic acid, leucovorin, and citrovorum factor. These biologically active forms of folate are enzymatically interconvertible and function as cofactors, providing the one carbon groups necessary for many intracellular metabolic reactions, including the synthesis of thymidylate and purine nucleotides, which are essential precursors of DNA.^{29,32,36,38,43} The minimum daily requirement of folic acid is normally 50 µg, but pregnant women and nutritionally deprived, acutely ill patients may require 100 to 200 µg.^10,12

Leucovorin is a mixture of the active and inactive diastereoisomers of 5-formyl THF of which the levo form is active and available as ­levoleucovorin.⁶ Both are available as the calcium salt, with the same chemical formula, C₂₀H₂₁CaN₇O₇, and a molecular weight (MW) of 511 Da. Both are rapidly metabolized to several active folates, including 5-methyltetrahydrofolate (5-CH₃-THF). The dose of levoleucovorin is half of leucovorin.

After a DHFR inhibitor such as methotrexate inhibits the formation of tetrahydrofolic acid, the intracellular machinery for the synthesis of indispensible thymidylate and purine nucleotides comes to a halt, and DNA production ceases. Leucovorin and levoleucovorin are biologically active forms of folic acid and bypass this inhibition of DHFR caused by methotrexate.

Folate catalyzes the formation of carbon dioxide and water from formic acid, the final metabolic step in methanol elimination. Because there is no inhibition of the formation or recycling of active folate, either folic acid or leucovorin is beneficial.

Early investigations suggest that folic acid may aid in the methylation and subsequent elimination of arsenic. Folate supplementation in folate-deficient subjects was done to enhance the elimination of arsenic and potentially decreased chronic arsenic toxicity.^5,7,8,30 Studies remain to be performed to determine whether folate supplementation in folate-replete individuals is beneficial.

Leucovorin Pharmacokinetics

Whereas leucovorin is naturally formed in the body as the active (l) isomer, the commercial preparation is the racemic mixture, which means it consists of equal amounts of the inactive (d) and active (l) isomers. The pharmacokinetics of the racemic mixture of leucovorin and its active metabolite were studied after a single intravenous (IV) infusion and as a constant infusion in normal human volunteers.^39,40 During constant infusion of 500 mg/m²/d, the steady-state concentration for the active isomer was 2.33 µmol, the half-life was 35 minutes, and the volume of distribution (Vd) was 13.6 L. The active isomer is metabolized to an active metabolite (l-5-CH₃-THF) that achieved a steady-state concentration of 4.85 µmol and a half-life of 227 minutes. Similar values were achieved for half-life and Vd after single IV doses ranging from 25 to 100 mg. The inactive d-isomer achieved higher concentrations and had a much longer half-life with oral administration, which is saturable and stereoselective, resulting in absorption of the active isomer that is four to five times greater than that of the inactive isomer. Studies of stereospecific oral absorption demonstrate that 100% of the l-leucovorin is absorbed, but only 20% of the d-leucovorin is absorbed at this dose.¹⁹ One study detected no adverse effects of the inactive isomer on the intracellular uptake of the active isomer and concluded that giving the active isomer provided no pharmacokinetic advantage over the racemic mixture.³⁵

Two hundred milligrams of levoleucovorin was compared to 400 mg of leucovorin, each administered as a 2 hour IV infusion as a crossover study in 40 healthy volunteers. The area under the curve and the maximum serum concentrations of l-5-CH₃-THF were similar for both.

Preliminary evidence suggests that l-5-CH₃-THF enters the CSF after systemic administration of both leucovorin and levoleucovorin. However, the concentration achieved is one to three orders of magnitude less than is normally obtained after intrathecal methotrexate. Intrathecal administration of leucovorin and levoleucovorin is contraindicated.

The pharmacokinetics of IV leucovorin was compared with intramuscular (IM) and oral administration in male volunteers given 25 mg. The mean peak of the active l-5-CH₃-THF concentration was 258 ng/mL (5.5 × 10^–1µmol/L) at 1.3 hours after IV administration compared with 226 ng/mL at 2.8 hours for IM and 367 ng/mL at 2.4 hours for oral administration.

The pharmacokinetics of orally administered leucovorin was studied in healthy, fasted male volunteers in single doses ranging from 20 to 100 mg and 200 mg IV over 5 minutes compared with 200 mg orally.^23,31 Bioavailability decreased from 100% for the 20-mg dose to 78% for the 40 mg dose and ultimately to 31% for the 200 mg dose. A microbiologic assay was used to measure total tetrahydrofolates (reduced and active folates). Normal serum folate concentrations are approximately 0.05 μmol/L.¹⁴ The 200 mg oral dose produced a peak serum concentration of 1.82 µmol/L compared with 0.66 µmol/L for the 20-mg oral dose and 27.1 µmol/L for the 200 mg IV dose.^23,31

Methotrexate, an antimetabolite, is a structural analog of folic acid, differing only in the substitution of an amino group for a hydroxyl group at the number 4 position of the pteridine ring (Chap. 51). Methotrexate binds to the active site of DHFR, rendering it incapable of reducing folic acid to its biologically active forms and incapable of regenerating the necessary active forms required for the synthesis of purine nucleotides and thymidylate.³⁷ At physiologic pH, the binding between methotrexate and DHFR is competitive, with an inhibition constant of about 1 µmol/L.³⁴ Leucovorin is a reduced, active form of folate. As such, it does not require DHFR for enzymatic interconversion to the form required for purine nucleotide and thymidylate formation. Folic acid is unable to counteract methotrexate toxicity because after methotrexate therapy, DHFR is unavailable to convert folic acid to an active reduced form. Leucovorin rescue is the term used to describe the practice of limiting the toxic effects of high-dose methotrexate therapy.

Monkeys experimentally made folate deficient develop methanol toxicity at lower methanol concentrations.²⁴ Administering folic acid to normal monkeys accelerates formate metabolism.²⁴ Pretreatment with folic acid or leucovorin decreased both formate concentrations and the accompanying metabolic acidosis without affecting the rate of methanol elimination.²⁴ Leucovorin remained effective in hastening the metabolism of formate when given 10 hours after methanol administration.²⁷

The hepatic concentrations of total folate, leucovorin, and folate dehydrogenase (which increases leucovorin concentrations) are all diminished in methanol poisoned humans.¹⁶ In an analysis of a single methanol-poisoned patient who was given folic acid, ethanol, and hemodialyzed, the half-life of formate was 1.1 hours.²⁸ In another methanol poisoned patient treated without folic acid, the formate half-life was 2.8 hours.¹³ These comparative data are inadequate to draw definitive conclusions but may support the therapeutic role of folate in addition to that of fomepizole (Antidotes in Depth: A30) and hemodialysis.

Arsenic contamination of drinking water has plagued millions, causing increased manifestations of chronic arsenic toxicity, including cancer and cardiovascular, dermatologic, and neurologic problems. Arsenic is methylated to monomethylarsonic acid (MMA) and then to dimethylarsinic acid (DMA). Early evidence suggests that nutritional deficiencies facilitate arsenic methylation, encouraging further study of folate supplementation along with other nutrients with the hope of diminishing the chronic health effects of arsenic toxicity.^5,7,8,30 The absolute methylation amount and product probably also depends on the degree of upregulation.^9a

Reports of adverse reactions to parenteral injections of folic acid, leucovorin, or levoleucovorin are uncommon. However, adverse reactions may include allergic or anaphylactoid reactions.¹⁰ Seizures are rarely associated with leucovorin or levoleucovorin administration.^6,25 The calcium content of leucovorin and levoleucovorin warrants a slow IV infusion at a rate not faster than 160 mg/min in adults. There is 0.004 mEq of calcium per milligram of leucovorin calcium injection. Extremely large doses of leucovorin on the order of 1000 mg every 3 hours might lead to hypercalcemia.⁴⁴ Neither leucovorin nor levoleucovorin should be administered intrathecally.^15,18,33,42

Leucovorin and levoleucovorin are not antidotes for 5-fluorouracil (5-FU), and both can enhance both the therapeutic and toxic effects of fluoropyrimidines such as 5-FU.^19,22 Both leucovorin and levoleucovorin are readily converted to 5,10-methylenetetrahydrofolate, which enhances the binding of the active metabolite of fluorouracil to thymidylate synthase, thereby potentiating the inhibition.

Many protocols recommended separating leucovorin and levoleucovorin from glucarpidase by 2 hours (Antidotes in Depth: A11); otherwise, leucovorin acts as a substrate for glucarpidase.

There is a potential for dosing errors when interchanging ­leucovorin and levoleucovorin. The dose of levoleucovorin is half the dose of leucovorin.

Leucovorin and levoleucovorin are Food and Drug Administration (FDA) pregnancy category C drugs. Human studies are lacking, and animal reproductive studies have not been conducted. Breast milk excretion is unstudied.

Folic acid is an FDA category A drug and is safe during pregnancy and compatible with breastfeeding.

After overdose of methotrexate, a dose of leucovorin estimated to produce the same plasma concentration as the methotrexate dose should be given as soon as possible, preferably, within one hour. One mole of methotrexate weighs 455 Da, and 1 mol of leucovorin calcium weighs 511 Da, with the MW of the leucovorin portion equal to 471 Da. Because of the safety of leucovorin and because of the toxicity of methotrexate, underdosing leucovorin should be avoided. Although serum methotrexate concentrations are often closely followed in patients on diverse oncologic regimens,^2,3 in the overdose setting, or in methotrexate toxicity related to treatment for tubal pregnancies, it is inappropriate to wait for a serum concentration before initiating treatment with leucovorin.¹ The toxic threshold for methotrexate is reported to be 1 × 10^–8 mol/L (0.01 µmol/L or 10 nmol/L).⁴ Normal serum folic acid concentrations are in the range of 13 to 43 nmol/L. If the patient’s exposure to methotrexate is not for therapeutic purposes there is no need to permit any methotrexate to remain unantagonized by leucovorin.

As an example, if a child unintentionally ingests 100 (2.5 mg) methotrexate tablets for a total dose of 250 mg, only part of this dose is absorbed because methotrexate absorption is saturable.⁹ The bioavailability of methotrexate decreases from 100% with doses less than 30 mg/m² to approximately 10% to 20% with doses greater than 80 mg/m². In this case, it is safe to assume that a bioavailability of 50% would result in an absorbed dose of methotrexate of less than 125 mg. For this exposure, an IV dose of 125 mg of leucovorin should be given over 15 to 30 minutes. This dose of IV leucovorin would be expected to produce serum concentrations in excess of that of the methotrexate, given that the Vd of leucovorin is about 25% less than methotrexate and the MWs are similar. This dose of IV leucovorin should be repeated every 3 to 6 hours until the serum methotrexate concentration is less than 0.05 µmol/L, preferably zero. This differs from recommendations in patients receiving methotrexate therapeutically (see later discussion). The methotrexate half-life may vary from 5 to 45 hours, depending on the dose and the patient’s kidney function. For this reason, leucovorin therapy should be continued for 12 to 24 doses (3 days) or longer if methotrexate concentrations are unavailable. Patients who may develop third-space storage in ascites or pleural effusions may also require leucovorin dosing for an extended period of time. Patients with bone marrow toxicity require more prolonged dosing because plasma half-lives of methotrexate do not reflect persistent intracellular concentrations.

Systemic Methotrexate Toxicity

The routine dose of leucovorin for “leucovorin rescue” after high-dose methotrexate therapy with doses of 12 to 15 g/m² ranges from 10 to 25 mg/m² IM or IV every 6 hours for 72 hours to 150 mg/m² every 3 hours in patients with renal compromise and delayed elimination. If a neonate must be treated, a benzyl alcohol–free preparation must be used because of the toxicity of benzyl alcohol in neonates (Chap. 55).⁴¹ For methotrexate overdoses, equimolar serum leucovorin concentrations should provide adequate protection, but because precise determinations are invariably delayed, leucovorin administration should be initiated without delay.

As a rough guide, a single dose of 25 mg of IV leucovorin in an adult produces a peak concentration of the active l-5-CH₃-THF metabolite of approximately 258 ng/mL, which is 0.55 µmol/L.¹⁹ A dosage of about 150 mg every 4 hours in an adult achieves a steady-state concentration of about 4.85 µmol/L.³⁹ And although the dose of leucovorin can be as high as 1000 mg/m² every 6 hours, this is rarely warranted and cannot adequately compete with serum concentrations of methotrexate above 100 µmol/L under these circumstances, glucarpidase should be strongly considered. An IV leucovorin dose of 150 mg/m² every 3 to 6 hours should be effective in all but the most severe overdoses and should be administered IV as soon as possible over 15 to 30 minutes but not faster than 160 mg/min in adults because of the calcium content. This dose should be continued a minimum of several days or until the serum methotrexate concentration falls below 0.05 µmol/L in the absence of bone marrow toxicity. One case series of 11 patients receiving methotrexate over 4 hours at 10 to 12 g/m² for osteosarcoma or 3.5 g/m² for central nervous system lymphoma required high-dose leucovorin rescue for methotrexate concentrations at high risk for toxicity at 24, 48, or 72 hours, usually because of acute kidney injury. The dosage of leucovorin ranged from 0.24 to 10 g/day and was titrated downward as the methotrexate concentration fell. It took an average of 11 ± 3 days for the methotrexate concentration to drop below 0.1 µmol/L (Table A10–1).

The package insert for leucovorin rescue after high-dose methotrexate as well as the methotrexate package insert suggest the following guidelines when methotrexate concentrations are drawn 24 hours or more after the start of the methotrexate infusion (Table A10–1):^19,26

An IV leucovorin dose of 100 mg/m² every 3 to 6 hours should be effective in all but the most severe overdoses. A constant IV infusion of 21 mg/m²/h has been safely administered for 5 days. Figure A10–1 offers a nomogram for pharmacokinetically guided rescue after high-dose methotrexate.² A transition to oral administration of leucovorin depends on the serum concentration of methotrexate and whether adequate serum concentrations of leucovorin can be achieved orally. In adults, a 200 mg oral dose of leucovorin produces a peak serum concentration of 1.82 μmol/L compared with 27.1 μmol/L with a 200 mg IV dose.

Levoleucovorin, the active l isomer of folinic acid, is available and should be dosed at half the dose of the racemate leucovorin.⁶

Administration of activated charcoal (AC) precludes the subsequent administration of oral leucovorin. In addition to leucovorin, other modalities to treat patients with methotrexate overdoses include AC and urinary alkalinization as well as glucarpidase (carboxypeptidase G) and extracorporeal removal (Chap. 51).

Intrathecal Methotrexate Overdose

Unintentional overdose with intrathecal methotrexate is potentially quite serious and is dose dependent.^15,20 In these cases, IV leucovorin and not intrathecal leucovorin should be administered. Intrathecal leucovorin was considered a major factor in the death of a child given a slightly higher dose of intrathecal methotrexate than was prescribed.¹⁸ Not all intrathecal methotrexate overdoses require aggressive intervention, but consultation with experienced hematologists/oncologists and medical toxicologists is warranted (Special Considerations: SC3).¹⁷ Consideration should be given to providing intrathecal glucarpidase.

Either folic acid or leucovorin (folinic acid) should be administered parenterally at the first suspicion of methanol poisoning. Folic acid is most commonly used. No complications are reported with the use of 50 to 70 mg of IV folic acid every 4 hours for the first 24 hours in the treatment of methanol-poisoned patients.²⁸ The precise dosage necessary is unknown, but 1 to 2 mg/kg every 4 to 6 hours is probably sufficient. Folic acid should be continued until the methanol and formate are eliminated. Because the first dose is usually administered before hemodialysis, a second dose should be administered at the completion of hemodialysis because this highly water-soluble vitamin will have been eliminated.

Leucovorin (folinic acid) powder for injection is available in 50, 100, 200, and 350-mg vials. Each milligram of leucovorin contains 0.004 mEq of calcium. Reconstitution with sterile water for injection—5 mL to the 50 mg vial, 10 mL to the 100-mg vial, or 20 mL to the 200 mg vial—results in a final concentration of 10 mg/mL. Adding 17.5 mL of sterile water for injection to the 350-mg vial results in a final concentration of 20 mg/mL. These lyophilized products contain no preservatives. The only inactive ingredient is sodium chloride added to adjust tonicity. Reconstitute with Sterile Water for Injection, USP, when doses greater than 10 mg/m² are used and use immediately. Further dilute in 100 to 1000 mL of 0.9% sodium chloride or D₅W for infusion.²¹ Leucovorin is also available in a single-use vial as a solution for injection at a concentration of 10 mg/mL in a 50-mL vial. Because of the calcium content, the rate of IV administration should not be faster than 160 mg/min in adults.¹⁹ Leucovorin is also available orally in a variety of strengths, including 5, 10, 15, and 25 mg tablets.

Levoleucovorin⁶ lyophilized powder for injection is available in a single-use 50 mg vial containing the equivalent of 50 mg of levoleucovorin as the calcium pentahydrate salt and 50 mg of mannitol. Reconstitution with 5.3 mL of 0.9% sodium chloride injection, yields a concentration of 10 mg/mL.⁵ Levoleucovorin is also available as a sterile solution in a single-use 175 mg vial that contains 17.5 mL of sterile solution in which each milliliter contains levoleucovorin calcium pentahydrate equivalent to 10 mg of levoleucovorin and 8.3 mg of sodium chloride. Because of the calcium content, the rate of IV administration should not be faster than 160 mg/min (16 mL of reconstituted solution/min).⁶ Further dilution to concentrations of 0.5 mg/mL in 0.9% sodium chloride injection or 5% dextrose injection is acceptable but should be used within 4 hours when stored at room temperature.⁶

Folic acid is available parenterally in 10 mL multidose vials with 1.5% benzyl alcohol in concentrations of 5 or 10 mg/mL from a variety of manufacturers. After it has been opened, the vial must be kept refrigerated.

If administration to neonates is necessary, a benzyl alcohol–free preparation must be used because of the toxicity of benzyl alcohol in neonates (Chap. 55).

• Leucovorin (folinic acid) is the primary antidote for a patient who receives an overdose of methotrexate.

• Leucovorin is the biologically active, reduced form of folic acid, the synthesis of which is prevented by methotrexate.

• Only leucovorin (folinic acid) is an acceptable antidote for a patient with methotrexate toxicity, but either folic acid or leucovorin is acceptable for a patient poisoned by methanol.

• After a methanol overdose, folic acid enhances the elimination of formate (Table A10–2).

References