Preventing and Managing Toxicities of High-Dose Methotrexate

Preventing and Managing Toxicities of High-DoseMethotrexateScott C. Howard, University of MemphisJohn McCormick, St. Jude Children’s Research HospitalChing-Hon Pui, St. Jude Children’s Research HospitalRandall K. Buddington, University of MemphisR Donald Harvey, Emory University

Journal Title: OncologistVolume: Volume 21, Number 12Publisher: AlphaMed Press: Oncologist | 2016-12-01, Pages 1471-1482Type of Work: Article | Final Publisher PDFPublisher DOI: 10.1634/theoncologist.2015-0164Permanent URL: https://pid.emory.edu/ark:/25593/st20n

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New Drug Development and Clinical Pharmacology

Preventing andManaging Toxicities of High-Dose MethotrexateSCOTT C. HOWARD,a JOHN MCCORMICK,b CHING-HON PUI,c RANDALL K. BUDDINGTON,a R. DONALD HARVEY

d

aSchoolofHealth Studies,UniversityofMemphis,Memphis,Tennessee,USA;Departments of bPharmaceutical Sciences and cOncology, St.JudeChildren’s ResearchHospital, NewYork, NewYork, USA; dDepartment ofHematology andMedical Oncology, EmoryUniversity Schoolof Medicine, Atlanta, Georgia, USADisclosures of potential conflicts of interest may be found at the end of this article.

Key Words. Methotrexate x High-dose methotrexate x Acute kidney injury x Leucovorin x Pharmacokinetics x Glucarpidase

ABSTRACT

High-dose methotrexate (HDMTX), defined as a dose higherthan500mg/m2, isused to treata rangeof adultandchildhoodcancers. Although HDMTX is safely administered to mostpatients, it cancausesignificant toxicity, includingacutekidneyinjury (AKI) in 2%–12%of patients. Nephrotoxicity results fromcrystallization of methotrexate in the renal tubular lumen,leading to tubular toxicity. AKI andother toxicitiesof high-dosemethotrexate can lead to significant morbidity, treatmentdelays, and diminished renal function. Risk factors formethotrexate-associated toxicity include a history ofrenal dysfunction, volume depletion, acidic urine, and druginteractions. Renal toxicity leads to impaired methotrexateclearance and prolonged exposure to toxic concentrations,which further worsen renal function and exacerbate nonrenal

adverse events, including myelosuppression, mucositis, der-matologic toxicity, and hepatotoxicity. Serum creatinine, urineoutput, and serummethotrexateconcentrationaremonitoredto assess renal clearance, with concurrent hydration, urinaryalkalinization, and leucovorin rescue to prevent and mitigateAKI and subsequent toxicity. When delayed methotrexateexcretion or AKI occurs despite preventive strategies, in-creased hydration, high-dose leucovorin, and glucarpidase areusually sufficient to allow renal recovery without the need fordialysis. Promptrecognitionandeffective treatmentofAKIandassociated toxicities mitigate further toxicity, facilitate renalrecovery, and permit patients to receive other chemotherapyor resume HDMTX therapy when additional courses areindicated. The Oncologist 2016;21:1471–1482

Implications for Practice: High-dose methotrexate (HDMTX), defined as a dose higher than 500 mg/m2, is used for a range ofcancers. Although HDMTX is safely administered to most patients, it can cause significant toxicity, including acute kidney injury(AKI), attributable tocrystallizationofmethotrexate in the renal tubular lumen, leading to tubular toxicity.WhenAKIoccursdespitepreventive strategies, increased hydration, high-dose leucovorin, and glucarpidase allow renal recovery without the need fordialysis. This article, based on a review of the current associated literature, provides comprehensive recommendations forprevention of toxicity and, when necessary, detailed treatment guidance to mitigate AKI and subsequent toxicity.

INTRODUCTION

Methotrexate is an antimetabolite that interferes with themetabolism of folic acid. After entry into the cell, methotrexate ispolyglutamated, binds dihydrofolate reductase (DHFR) with anaffinity 1,000-fold greater than that of folate, and competitivelyinhibits conversion of dihydrofolate to tetrahydrofolate (Fig. 1).Tetrahydrofolate is essential for biosynthesis of thymidine andpurines, which are needed for synthesis of DNA. Blockade oftetrahydrofolate synthesis by methotrexate leads to inability ofcellstodivideandtoproduceproteins.Methotrexateisanessentialcomponentof therapy for acute lymphoblastic leukemia (ALL) andis active againstmany types of cancer; this justifies its presence ontheWorld Health Organization’s list of essential medicines.

Methotrexate is administered at doses that range from12 mg intrathecally and 20 mg/m2 orally, intramuscularly, or

intravenously as weekly maintenance chemotherapy for ALLto doses as high as 33,000 mg/m2 intravenously for someother indications [1]. Doses of 500 mg/m2 or higher givenintravenouslyaredefinedashigh-dosemethotrexate(HDMTX)and are used to treat a variety of adult and pediatric cancers,including ALL, osteosarcoma, and lymphomas [2–4]. HDMTXtherapy can cause significant toxicity, which not only leads tomorbidity andoccasionalmortality butmay also interrupt cancertreatment,potentially leadingto inferioranticanceroutcomes.Toprevent unacceptable toxicity, it must be given with rigorouslystandardizedsupportivecare[1],whichdiffersacrosstumortypesand treatment protocols (Table 1). When patients experiencedelayed methotrexate excretion, without timely recognitionand treatment, the prolonged exposure to toxic methotrexate

Correspondence: ScottC.Howard,M.D.,UniversityofMemphis, SchoolofHealthStudies, 3720AlumniAvenue,Memphis,Tennessee38152,USA.Telephone: 901-500-8691; E-Mail: [email protected] Received April 22, 2016; accepted for publication June 20, 2016;published Online First on August 5, 2016. ©AlphaMed Press 1083-7159/2016/$20.00/0 http://dx.doi.org/10.1634/theoncologist.2015-0164

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concentrationscan leadtosignificantmorbidityandmortality [1].Aggressivemonitoring and prompt intervention usually promotemethotrexate excretion, prevent toxicity, and allow patients toreceive subsequent HDMTX treatment [1, 5]. Here, we describecommon toxicities, review supportive care strategies, exploreapproaches tomanage toxicity, and suggest subsequent HDMTXtherapy after treatment-related toxicity occurs.

POTENTIAL TOXICITIES OF HIGH-DOSE METHOTREXATE

Acute Kidney InjuryDespite appropriate supportive care measures during admin-istration of HDMTX, acute kidney injury (AKI) develops in

2%–12%ofpatients [6].The incidencedependsonhost factors,supportive measures used, and the dose and schedule ofHDMTX. For example, 9.1% of HDMTX cycles in patients withlymphomaarecomplicatedbyAKI, comparedwithonly1.5%ofcycles in patients with sarcomas [7, 8]. Nephrotoxicity causedby HDMTX arises through crystal nephropathy, which occurswhenmethotrexate and its metabolites precipitate within therenal tubules. Becausemethotrexate is acidic, drug crystals arenot present in urine with an alkaline pH, as alkalinizationgreatly increases methotrexate solubility and excretion.Crystal-induced nephropathy initially manifests as an asymp-tomatic elevation in serum creatinine and then progresses totubular necrosis and more severe renal injury.

Figure 1. Mechanism and site of action ofMTX and of rescue strategies for delayedMTX elimination. AfterMTX enters cells through theRFC, it is polyglutamated, then competitively and reversibly inhibits the activity of DHFR, thus preventing formation of FH4 from FH2.Thelackof FH4 inhibits DNA, RNA, and protein synthesis. LV enters cells through the RFC and allows formation of FH4 despite the presence ofMTX, which effectively rescues cells. However, whenMTX elimination is impaired and it is present at very high concentrations, very highdosesof LVarenecessary toallowentryofa sufficientamount to rescuecells fromMTXtoxicity.GlucarpidaseeliminatesextracellularMTXby converting it to nontoxic DAMPA and therefore should always be givenwith LV to provide intracellular rescue even as the glucarpidaseprevents further accumulation of intracellular MTX by removing it from the extracellular compartment.

Abbreviations: DAMPA, 4-deoxy-4-amino-N-10-methylpteroic acid; DHFR, dihydrofolate reductase; dUMP, deoxyuridine mono-phosphate; FH2, dihydrofolate; FH4, tetrahydrofolate; FH5, tetrahydrofolate; LV, leucovorin; MTX, methotrexate; RFC, reduced folatecarrier; TMP, thymidine monophosphate.

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1472 Preventing and Managing Toxicities of HDMTX

Because volume depletion and acidic urine are major riskfactors for AKI, hyperhydration and urine alkalinization aremandatory during HDMTX treatment (discussed further in thesection on supportive caremeasures) [7]. Drug-drug interactions

can also contribute to delayed methotrexate excretion andsubsequent nephrotoxicity [7]. Agents that pose the highest riskof adverse interactionare those that interferewithmethotrexateclearance by competing for renal tubular secretion (Table 2) [1].

Table 1. Selected protocols that include high-dose methotrexate for acute lymphoblastic leukemia and osteosarcoma

Study, year[reference] Methotrexate dose

Duration ofmethotrexateinfusion (hours) Leucovorin rescue dose

Time from start ofmethotrexate infusion tofirst leucovorin dose (hours)

Acute lymphoblasticleukemia

Takeuchi et al., 2002[98]

100-mg/m2 bolus, then500 mg/m2 per hour

4 15 mg every 6 hours38 doses

28

Linker et al., 2002[99]

220-mg/m2 bolus, then60 mg/m2 per hour336 hours

36 50 mg every 6 hours 36

Hill et al., 2004 [100] 6 g/m2 (age, 4 yr) 10% bolus, remainderover 23 hours

15 mg/m2 every 3 hours,then every 6 hourswhen serummethotrexate, 23 106 mM

36

8 g/m2 (age. 4 yr)

Pui et al., 2007 [58] 2 g/m2 2 10 mg/m2 every 6 hours 44

Zhang et al., 2014[101]

3–5 g/m2 24 15 mg/m2 every 6 hours,then pharmacokineticallyguided to serummethotrexate 0.1 mmol/L

36

Osteosarcoma

Souhami et al., 1997[102]

8 g/m2 (age$12 yr) Not specified 12 mg/m2 i.v. or 15 mg/m2

p.o. every 6 hours for 10doses

24

12 g/m2 (age,12 y)

Fuchs et al., 1998[103]

12 g/m2 (maximum,20 g)

Not specified 15mg/m2every6hours for12 doses

Not specified

Bacci et al., 2001[104]

12 g/m2 (escalated to14 g/m2 if the 6-hourserum methotrexatewas,1 mmol/L)

6 15 mg every 6 hours for11 doses

24

Goorin et al., 2003[105]

12 g/m2 4 15 mg every 6 hours for10 doses

24

Ferrari et al., 2005[106]

12 g/m2 4 8 mg/m2 every 6 hours for11 doses

24

Non-Hodgkinlymphoma

Koller et al., 1997[107]

200 mg/m2 over 30min, then 800 mg/m2

24 50 mg i.v. for one session,then 15 mg p.o. every 6hours or as methotrexateconcentrations define

36

Khouri et al., 1998[108]



36

Thomas et al., 2004[109]



36

Primary centralnervous systemlymphoma

Batcheloretal., 2003[110]

8 g/m2 4 Pharmacokineticallyguided until serummethotrexate, 13 106 mM

24

Wright et al., 2015[46]

2,000/5,000 mg/m2 24 15mg/m2every6hours fora total of 5 doses

24

Dalia et al., 2015[111]

8 g/m2 Not specified Not specified Not specified

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Howard, McCormick, Pui et al. 1473


Careful reviews of all medications and close monitoring ofall patients are warranted [9–15]. Whether prophylactictrimethoprim-sulfamethoxazole should be discontinuedduring HDMTX infusion is controversial. Although someprotocols suggestdeferring ituntil adequateHDMTXclearanceis documented, no strong data support this approach.

Other ToxicitiesAcute kidney injury impairs the renal clearance of methotrex-ate, resulting in the accumulation of toxic concentrations andan increased risk for additional adverse events [7]. Prolongedrenal dysfunction with increased systemic methotrexateexposure can cause myelosuppression, mucositis, hepatotox-icity, and, in severe cases, multiorgan failure [16]. Emesisoccurs in 10%–30% of patients receiving HDMTX even whenappropriate antiemetics are used; in this subgroup, anti-emetics should be escalated to completely control vomitingandadditionalhydrationprovided to replace lost fluid [17].TheAmerican Society of Clinical Oncology clinical practiceguideline for antiemetic therapy classifies HDMTX as havinglow emetic risk and recommends dexamethasone to reducethe risk for nausea and vomiting. Because 5-HT3 antagonistsare used almost universally, dexamethasone is often omitted[17]. Transient liver toxicity may include reversible chemicalhepatitis in up to 60% and hyperbilirubinemia in 25% ofcourses, respectively [18]. In up to 15% of HDMTX courses,patients report transient disturbances of the central nervoussystem (CNS), and a subset of these experience significantsymptoms, such as cortical blindness, hemiparesis, and seizure[19]. Chemical conjunctivitis occurs rarely andcanbemanagedwith local treatment; indeed, methotrexate can be safelyadministered intraocularly to control autoimmune diseasesthat affect the eye [20]. Pulmonary toxicity is observed in 0.5%of patients per year who receive weekly low-dose methotrex-ate, but rarely with HDMTX [18, 21–23]. Pulmonary functiontesting or other screening is not warranted in patients withcancer, in whom methotrexate-induced pneumonitis is rare,unless pre-existing lung compromise or other risk factorswarrant surveillance.

Animal models, particularly rats, have been used to studytoxicities of other therapeutics [24] and diseases, such asnonalcoholic steatohepatitis [25]. Rodent models have beenused for pharmacokinetic modeling of methotrexate [26],exploring leucovorin rescue [27], and preclinical studies ofinvestigational interventions to reduce toxicity (including

pentoxifylline [28], amifostine [29], melatonin [30], andactivators of peroxisome proliferator activator receptor a andg [31]). Critically, the mechanism of MTX crystal formation inthe renal tubule was elucidated in monkeys [32], and theconcept of enzymatic cleavage of MTX using glucarpidase wasfirst described in amousemodel by Chabner et al. in 1972 [33].

RISK FACTORS FOR TOXICITY DURING TREATMENT WITH

HIGH-DOSE METHOTREXATE

Several patient-related factors can increase the risk for AKI [7].Volume depletion is perhaps the most important and canresult from fluid losses due to vomiting or diarrhea,adrenal insufficiency, or renal salt wasting [7]. Reductionsin intravascular volume lead to renal hypoperfusion withsubsequent decreased urine output [7]. Precipitation ofmethotrexate crystals occurs in acidic urine (pH, 5.5) whenthe concentration of methotrexate in the renal tubulesexceeds 23 1023 molar. Intrarenal crystal formation can leadto tubule obstruction, direct toxic damage to the renal tubularepithelium (due toprolongedcontactwithmethotrexate), andhypoperfusion from afferent arteriolar vasoconstriction, eachof which independently can worsen AKI [34–36]. Polyurialeading to severe shifts in fluid balance has also been reportedwithmethotrexate infusion. Although themechanism remainsunclear, patients with methotrexate-associated polyuria re-quire especially close monitoring of fluid status and frequentadjustments in intravenous fluids tomaintain fluidbalanceandprevent renal hypoperfusion [37].

Patients with a history of toxicity with prior courses ofHDMTX have higher risk for subsequent renal toxicity [1].However, even when toxicity is severe, subsequent HDMTXcourses can generally beadministered safely after thepatientrecovers [5]. As many as 60% of adult cancer patients havesome degree of renal dysfunction, which increases their riskfor AKI [38]. Lower creatinine clearance (CrCl) before adminis-tration of HDMTX predicts renal toxicity, and both CrCl andserum creatinine concentration before HDMTX administrationcan be useful in predicting plasma methotrexate concentra-tions after infusion [8, 39]. Specific CrCl cutoff values for dosereduction or omission of subsequent HDMTX have not beenestablished, with upper cutoffs for dose reduction starting at50–60 mL/min and recommendations to omit further HDMTXwhen CrCl falls below 10–30 mL/min [40, 41]. Additional hostfactors that contribute to AKI risk include pre-existing nephrop-athy because of previous drug toxicity (e.g., from cisplatin) or

Table 2. Drugs that impair methotrexate clearance

Agents Mechanism of inhibition

Nonsteroidal anti-inflammatory drugs, penicillin andpenicillin derivatives, salicylates, probenecid, gemfibrozil,trimethoprim-sulfamethoxazole

Direct inhibition of renal excretion

Amphotericin, aminoglycosides, radiographic contrastdyes Nephrotoxicity that leads to decreased glomerularfiltration with consequent inhibition of renal excretion

Proton-pump inhibitors Unclear; potential inhibition of methotrexateBCRP-mediated renal transport

P-glycoprotein/ABCB1 inhibitors Inhibitionofmethotrexatetransport inmultipleorgans,including kidney

Levetiracetam, chloral hydrate Unclear, potential competition for tubular secretion

References [10–15].Abbreviations: ABCB1, ATP-binding cassette B1; BCRP, breast cancer resistance protein, also known as ABCG2 (ATP-binding cassette) G2.



associated disease, metabolic derangements due to the pres-ence of tumor, advanced age, and pharmacogenetic factors(such as hyperhomocysteinemia with concurrent relative orabsolute folate deficiency) (Table 2) [9, 42]. In this regard,methotrexateclearance isalsoassociatedwithpolymorphismsof SLCO1B1, which encode a hepatic solute carrier organicanion transporter that mediates disposition of many medica-tions, including methotrexate [43–45].

Delayed methotrexate excretion has been associatedwith extravascular fluid collections, including ascites, pleuraleffusions, or intracranial fluid; whether HDMTX should bedeferred to a later date in such situations depends on thebalance of risks and the benefits of deferral, but even morerigorous monitoring is warranted if one proceeds [46]. Pre-existing nephropathy is associated with substantial toxicityeven with low doses of methotrexate [47], suggesting a needfor even greater diligence during HDMTX administration in thepresence of renal dysfunction [7, 48]. Finally, delays betweenrecognition of toxicity and initiation of treatment can directlycontribute to renal and systemic toxicities [6]. In the setting ofAKI, the rise in serumcreatinine values lags behindprogressiveintrinsic renal damage, such that precise measurement offunction at a specific moment is difficult [49]. Instead, adecrease in urine output, positive fluid balance, or weightchangemay help identify patients with early AKI after HDMTXadministration, even before creatinine increases. Neverthe-less, in some cases irreversible damage to renal tubuleepithelial cells may have already occurred before the onsetof oliguria or detection of clinically significant increases inserum creatinine concentration.

Delayed methotrexate excretion has been associatedwith extravascular fluid collections, including ascites,pleural effusions, or intracranial fluid; whetherHDMTX should be deferred to a later date in suchsituations depends on the balance of risks and thebenefits of deferral, but evenmore rigorousmonitor-ing is warranted if one proceeds.

SUPPORTIVE CARE MEASURES

Inmostpatientswithnormalrenal function,HDMTXcanbegivensafely with the use of several supportive care strategies. Theseshould includeadjustingmedicationswithpotential interactions,vigorous hydration, and urinary alkalinization (target pH $ 7)before starting methotrexate infusions. The goal is to enhancethe solubility anddilutionofmethotrexate in theurineandapplyleucovorin rescue guided by serial serummethotrexate levels toprotect against potentially lethal toxicity.

Suspending Medications That Interfere WithMethotrexate ClearanceAll prescribed, over-the-counter, and nontraditional medica-tions must be reconciled and documented before HDMTXadministration begins. Because of their long half-life, somemedications (e.g., naproxen sodium) can interfere withmethotrexate elimination for many hours, with potentiallydisastrous delays in methotrexate elimination. Patients who

take such medications daily should be provided with analternative startingthedaybeforeHDMTXandcontinuinguntilmethotrexate clearance. Studies in mice documented in-hibition of renal tubule methotrexate transporters by in-domethacin and ketoprofen, with reduced methotrexateelimination and prolonged elevated levels and toxicity [24].

HydrationMore than 90% of methotrexate is eliminated by the kidneys[1]. The use of fluids to promote high urinary flow rates andalkalinize the urine protects the kidney from injury duringtreatment with HDMTX [7]. Many pediatric protocols recom-mend at least 2 hours of hyperhydration of a minimum of200 mL/m2 per hour or 100–150 mL/m2 per hour beginning12 hours before the start of methotrexate infusion andcontinuing for24–48hoursor longer if thepatienthas ahistoryof methotrexate toxicity or develops delayed methotrexateelimination [1]. In adults, rates of 150–200mL of bicarbonate-containing fluids per hour to a total of 2 L before HDMTXinfusion are often used. Strict monitoring of fluid intake andoutput is recommended during and after administration ofmethotrexate.

Urine AlkalinizationMethotrexate and its metabolites, including 7-OH-methotrexate and 4-deoxy-4-amino-N-10-methylpteroic acid(DAMPA), are poorly soluble at an acidic pH [1, 50]. Anincrease in urine pH from 6.0 to 7.0 increases the solubility ofmethotrexate and its metabolites by five- to eightfold, andalkalinization is imperative to reduce intratubular crystalformation (precipitation) [1, 50].Thus, administration of fluidswith 40 mEq/L sodium bicarbonate is recommended duringand after HDMTX administration [1, 7]. A urine pH of 7 orgreater should be required before administration of metho-trexatetoreducecrystal formation. It isalso importanttocheckurinepHvalueswitheachvoidduring the infusion toensurenoextended periods of time with acidic urine, which couldincrease the risk for precipitation, nephrotoxicity, and delayedmethotrexate elimination. The ability of the laboratory toprocess samples rapidly and notify clinicians when the pHdecreases to7or less facilitates saferHDMTXadministration. Ifa urine pH of 6.5 is identified, sodium bicarbonate at a dose of12.5mEq/m2 is administered, and for urine pH,6.5, a dose of25 mEq/m2 is given; urine pH is measured hourly throughoutHDMTX infusion because sometimes boluses of sodiumbicarbonate must be repeated to achieve alkaline urine [51].In patients with serum alkalosis and inadequate urinealkalinization, the carbonic anhydrase inhibitor acetazolamide(250–500 mg p.o. four times daily) may be added to directlyalkalinize the urine by increasing renal excretion of sodium,water, and bicarbonate, without increasing serum pH [52].

LeucovorinFor more than 30 years, leucovorin rescue has been acornerstone of HDMTX treatment (Fig. 2) [18]. Leucovorin isparticularly effective in the prevention of myelosuppression,gastrointestinal toxicity, and neurotoxicity during treatmentwith HDMTX. Chemotherapy protocols that include HDMTXalso include recommendations for the timing, dose, andduration of leucovorin administration to protect normal cells




from injury (Table 1) [1, 18]. Because leucovorin effectivelyneutralizes the effects of methotrexate, it must not be startedtoo early because it would then reduce not only toxicity butalso anticancer efficacy. In this regard, if a patient is takingleucovorinat thetimeHDMTXtreatment is scheduled tobegin,the leucovorin should be discontinued and the HDMTXdeferred until the following day.

Other Supportive Care MeasuresOther supportive care measures can be tailored according toindividual patient risk factors. For instance, HDMTX doses canbe reduced in patients with pre-existing renal dysfunction orsevere toxicity after a prior course of HDMTX, and serummethotrexate levels can bemeasured early (e.g., at hour 6 of a24-hour infusion) to make sure that there is no excessiveaccumulation [7]. During treatment with HDMTX, patientsmust also minimize exposure to other potential nephrotoxins,including those listed in Table 2 [7, 10].

MONITORING DURING TREATMENTWITH HIGH-DOSE METHOTREXATE

The pharmacokinetics of methotrexate dictate the degree ofsupportive care andmonitoring needed after treatment. Aftera HDMTX infusion with a fixed dose and duration, plasmaconcentrations can vary widely between patients andwithin apatient on different cycles. Plasma protein binding, effusions,renal function, and, to a lesser degree, hepatic function canall contribute to peak concentrations after infusion. Serialmethotrexate concentrations are obtained, with the primaryfocus on values that easily fit within the leucovorin nomo-gram timeframe (i.e., 42 hours and later) (Fig. 2). However,pharmacokinetic modeling data from Evans et al. [53] showthat values above 10 mM at 24 hours after the start of theinfusion confers a high risk for toxicity.

Methotrexate is primarily eliminated by the kidney, sorenal functionmust be assessed before, during, and after eachcourse of HDMTX. Currently usedmeasures of kidney function

include serum creatinine, urine output, urine pH, and bloodurea nitrogen [54]. A rise in serum creatinine concentrationandother parameters above normal values indicates potentialrenal dysfunction and delayed methotrexate elimination [2].Incorporation of clinical decision support in the electronichealth record can alert clinicians of acute changes in serumcreatinine or prescription of medicines that may delayelimination of methotrexate. Automated early warnings mayfacilitate prompt interventions, such as increasing the rate ofintravenous fluids, substitution of an alternative drug thatdoes not interfere with methotrexate clearance, and, inextreme cases, even stopping themethotrexate infusion earlyto prevent toxicity. Treatment protocols that include HDMTXsometimes outline strategies for dose reduction in patientswith reduced creatinine clearance [1]. The utility of alternatebiomarkers for earlier detection of renal injury is an area ofactive study [55].

Plasmamethotrexate concentrations shouldbemonitoredclosely to detect any delay in methotrexate clearance duringeach cycle of therapy [56]. Depending on the regimen, plasmamethotrexate assays may be appropriate at 24, 48, and 72hours after the start of methotrexate infusion [57]. Otherprotocols may require serum methotrexate measurements at36hours (i.e., 12hoursafter theendofa24-hour infusion)orat42 hours from the start [58]. Importantly, leucovorin doses areadjusted according to plasma methotrexate concentrations,and hydration and alkalization can be fine-tuned to optimizesafety [1]. Serum methotrexate concentrations should bemonitored with ongoing adjustments in hydration, alkaliniza-tion, and leucovorin rescue until the target of less than0.05–0.1mM is reached [1]. Plasmamethotrexate monitoringisa reliable indicator specificallyofnephrotoxicitybutmaybealimited predictor of other toxicities [59]. However, in centerswhere methotrexate levels cannot be monitored, assiduousmonitoring of urine pH and output, serum creatinine, andtwice-daily examination of mucosal membranes for evidenceof inflammation can allow safe administration of HDMTX formost patients; indeed, this practice in Recife, Brazil, wheremethotrexate levels were not available, allowed for safeadministration of hundreds of courses of HDMTX [60].

MANAGEMENT OF SPECIFIC TOXICITIES ASSOCIATEDWITH

HIGH-DOSE METHOTREXATE

NephrotoxicityAggressive supportive care measures are needed when AKIoccurs after HDMTX. Continuing to administer alkalinized i.v.fluids with addition of acetazolamide when needed to keepurinepH.7maximizesmethotrexateeliminationandreducesfurthercrystal formation innephrons. Increasing infusion ratesto the maximum tolerated amount ($3 L/m2 per day) isrecommended to maximize urine output. Attention to fluidbalance, frequent symptom assessment, pulmonary examina-tion, pulse oximetry, and chest radiography or echocardiogra-phy of patients at risk for heart failure allow aggressivehydration with minimal risks. Although pleural effusions mayoccurwith very aggressive hydration, the risk-benefit relation-ship favors continued hydration in most cases, as delayedmethotrexate elimination in most patients is primarily drivenby renal dysfunction.

Figure 2. Nomogram for the expected time-dependent decreasein serumMTX levels after completion ofMTX infusion.Nomogramfor expected serumMTX levels as a function of time from the endofa6-hour infusionofmethotrexateatadoseof7.5g/m2.Thedarkblue area represents62 SD from the mean (orange line). Valuesabove the red line indicate impending or severe toxicity. Adaptedfrom [112] with permission.

Abbreviation: MTX, methotrexate.



Extracorporeal techniques to remove excessive methotrex-ate have been used and are logical on the basis of thedistribution of methotrexate in serum and its limited proteinbinding (58%) [61]. However, results have beenmixed; becauseof ethical considerations, studies have lacked suitable controlpatientswhodidnot receivedialysis andhavebeenconfoundedby differing concomitant interventions (e.g., leucovorin doses,glucarpidase use) [1, 62]. Furthermore, retrospective analysesof differing approaches without control groups, includingplasmapheresis, charcoal hemoperfusion, high-flux hemodial-ysis, conventional hemodialysis, and peritoneal dialysis make itdifficult to identify one optimal extracorporeal strategy. High-flux hemodialysis is likely to be the most effective based ontechnique and flow rates and reduced methotrexate concen-trationsduringa6-hourperiod inoneseries,whereasperitonealdialysis is unlikely tobeeffective [63].Unfortunately, evenwhenhemodialysis iseffective,manypatientsexperienceareboundinserum methotrexate concentrations of 10%–220% of thepostprocedure values [64]. Complications of dialysis must alsobe considered, especially in critically ill patients who are athigher risk for electrolyte abnormalities, bleeding at cathetersites, and cardiac arrest. Rapid institution of extracorporealmethods is critical if they are used, but the variable results andrebound rise in methotrexate concentrations necessitatecontinuous monitoring and repeat dialysis as needed. In allcases, high doses of leucovorin should be administered untilmethotrexate has been completely eliminated; in very illpatients, continuing it for another day or two thereafter iswarranted. Leucovorin is removed by dialysis, and so it shouldbe redosed afterward [65].

HepatotoxicityHepatotoxicity after HDMTX is much less common than withthe lower, long-term oral methotrexate dosing that is used inpatients with rheumatoid arthritis, who are at risk for liverfibrosis and require regular monitoring of liver enzyme levels[66]. Indeed, almost all patients have elevations of serumalanine aminotransferase (ALT) and aspartate aminotransfer-ase (AST) values after HDMTX, but these laboratory findingshave no clinical significance and require no adjustment ofsubsequent courses of HDMTX because most cases aretransientand reversible anddonot lead tochronic liverdisease[67]. Measurement of AST, ALT, and bilirubin before eachcourse of HDMTX is advisable to assure there is no evidence ofhepatic inflammation or dysfunction that could be worsenedby methotrexate [68]. Risk factors for hepatotoxicity in long-termmethotrexate dosing include alcohol and hepatitis B andC virus infection [69–71]. Although these have not beendocumented to complicate HDMTX, avoiding alcohol andcontrolling hepatitis infection before HDMTX is warranted tominimize risk. Conversely, existing steatohepatitis may in-crease methotrexate toxicity [25].

NeurotoxicityCNS toxicity may occur after HDMTX, and concurrent in-trathecal treatment, cranial irradiation, infiltration of malig-nant cells, or concomitant CNS toxins increase the risk, as wellas confound the etiology. Depending on the populationstudied, up to 11% of patientsmay have CNS events, includingconfusion, seizures, somnolence, and headaches with or

without radiographic evidence of leukoencephalopathy [72].For example, 3% of pediatric ALL patients had acuteencephalopathy, and pharmacokinetic parameters did notpredict the onset [72]. Symptoms typically occur within24 hours, often resolve spontaneously, and rarely have long-term sequelae [19, 73]. Elevated ratios of methotrexate toleucovorinat42hoursmaypredict thoseat risk forCNStoxicity,and polymorphisms in genes associated with neurodevelop-ment (e.g., TRIO, PRKG1, ANK1, COL4A2, NTN1, and ASTN2)may also increase risk [19].

A potential mechanism of neurotoxicity is the accumula-tion of adenosine after MTX-induced reductions in purinesynthesis [74].The finding of increased adenosine in theCNS inpatientswith toxicity ledsome investigatorstoevaluate1-hour2.5-mg/kg infusions of aminophylline in pediatric ALL patientson the basis of their ability to displace adenosine from centralreceptors [75]. In the 6 patients treated, 4 had completeresolution of symptoms that had not improved after othermeasures (e.g., corticosteroids), and 2 had persistent nauseabut no other symptoms. However, no definitive studiesdemonstrate the efficacy of aminophylline in treating orpreventingmethotrexate-inducedneurotoxicity. Patientswhodevelop CNS toxicity should have all potential neurotoxinsdiscontinued and magnetic resonance imaging examinationshould be performed, particularly if symptoms do not improvewithin 24 hours of onset.

MucositisOralmucositis can become a dose-limiting toxicity, require theuse of opioids, increase infectious risk, and lead to chemo-therapy delays. The biological processes associated withmucositis ultimately result in mucosal barrier injury, basedon a systematic cascade of cellular and tissue interactionsinvolving the endothelium, extracellular matrix, metallopro-teinase, submucosal reactions, and connective tissue [76–78].Mucositis after HDMTX is caused by cellular damage to rapidlydividing epithelial cells along the entire gastrointestinal tract;inadequate or delayed leucovorin rescue can lead to impairedepithelial cell growth and regeneration.

Grade IVmucositis is anoncologicemergencyand isassociatedwith infections, the need for parenteral nutrition, increased useof health care resources, delayed chemotherapy, and even death[77]. A variety of methods have been used to prevent or treatoral mucositis, including ice chips [79, 80], glutamine andN-acetylcysteine, benzydamine hydrochloride, benzydaminehydrochloride,andprostaglandinE1andE2,butnonehasprovenbenefits in patients receiving prolonged infusions of HDMTX[78, 81]. Palifermin, a recombinant human keratinocyte growthfactor that stimulates growth of epithelial cells, reduces theincidence of mucositis after HDMTX [82, 83]. Interventions toprevent and treat mucositis have recently been reviewed, butnone is standard practice in patients receiving HDMTX [84, 85].

Small animal models, mainly rats, have been used toinvestigate the mechanisms of gastrointestinal methotrexatetoxicity [86, 87], the effect on the resident microbiome [88],and a variety of approved and experimental interventions.

MyelosuppressionAdose-limitingtoxicityofHDMTX in theabsenceof leucovorin issevere, prolonged myelosuppression; however, the frequency




and severity of cytopenias with pharmacokinetically guidedleucovorin is low. When elimination is delayed because ofthird spacing and fluid accumulations or as a result of renalinjury, neutropenia and thrombocytopenia may be severe[89].When myelosuppression occurs, standard therapies forfebrileneutropeniaand transfusionsareprovidedasclinicallyindicated. The only known strategies to prevent myelosup-pression are to prevent delayedmethotrexate elimination byavoiding interactingmedications around the time of infusionand draining effusions before treatment (or delaying HDMTXuntil effusions resolve) and to ensure optimal leucovorindosing.

GLUCARPIDASE

Enzymatic cleavage ofMTXusing glucarpidase (a recombinantbacterial carboxypeptidase G2) was first described in 1972[33]. Glucarpidase was approved by the U.S. Food and DrugAdministration in2012 forpatientswithdelayedmethotrexateelimination or AKI and plasma methotrexate concentrations.1 mmol/L [50]. Glucarpidase cleaves methotrexate intoDAMPA and glutamate, two nontoxic metabolites, and thusprovides an enzymatic method to rapidly remove methotrex-ate in patients with renal dysfunction (Fig. 1). A single dose ofglucarpidase (50 U/kg i.v. over 5 minutes) reduces plasmamethotrexate concentrations by 97% or more within 15minutes [50]. However, despite the decrease in themagnitudeand duration of systemic exposure to methotrexate afterglucarpidase, it has no effect on intracellular methotrexateconcentrations [6, 16]. Therefore, just as with dialysis, thecoadministrationofhigh-dose leucovorin is requiredtoprotectcells from toxic methotrexate concentrations until renalfunction recovers sufficiently to clear any residual methotrex-ate as it is released fromcells (Fig. 1). In fact, after glucarpidaseadministration, leucovorin should be continued until metho-trexate concentrations have been maintained at close toundetectable levels for several more days. Leucovorin shouldnot be administered within 2 hours before or after a dose ofglucarpidase because, like methotrexate, leucovorin is asubstrate for glucarpidase. Hydration and urine alkalinizationshould also be continued in patients requiring glucarpidase[50].

Within 48 hours of glucarpidase administration, only achromatographic (high-performance liquid chromatography)method can reliably measure methotrexate concentrationsbecause the DAMPA produced by enzymatic breakdown ofmethotrexate cross-reacts with methotrexate in the stan-dard immunoassay and artificially elevates the level [50].The long half-life of DAMPA (approximately 9 hours)precludes use of immunoassays for several days afterglucarpidase administration.

Correct timingof leucovorindosing relative toglucarpidaseis essential and is based on pharmacologic principles (Fig. 3)[1]. Glucarpidase is confined to the plasma; therefore,interstitial and intracellular leucovorin concentrations arenot directly affected by glucarpidase. Glucarpidase is given asa single i.v. infusion over 5 minutes and has a half-life of5.6 hours. Leucovorin should be given 2–3 hours afterglucarpidase, then every 3–6 hours at doses guided by themethotrexate concentration (pharmacokinetically guideddosing; Fig. 3) [1]. Reduction of the plasma methotrexate

concentration reduces the competition between metho-trexate and leucovorin for intracellular access. Therefore,the combination of a lower plasma methotrexate level andthe transient presence of glucarpidase probably enhances theintracellular transport of leucovorin [90]. Leucovorin iscontinued for 48 hours after glucarpidase at the doseappropriate for the preglucarpidase methotrexate concentra-tion [1]. It is important to note that in the presence of very highmethotrexate concentrations, no plasma leucovorin concen-trationmay be sufficiently high to reverse intracellular toxicitybecause of competition with the shared active transportmechanism. Urgent hemodialysis plus glucarpidase and very-high-dose leucovorin are warranted to reduce mortality [91].

Reduction of the plasmamethotrexate concentrationreduces the competition between methotrexate andleucovorin for intracellular access. Therefore, thecombination of a lower plasma methotrexate leveland the transient presence of glucarpidase probablyenhances the intracellular transport of leucovorin.

Many curable cancers require multiple courses of HDMTXtherapy. If doses are skipped or delayed, treatment outcomesmay be adversely affected [1]. For patients with delayedmethotrexate clearance, the early use of glucarpidase rescue

Figure3. Pharmacokinetically guided leucovorin rescuebasedonplasma MTX levels after high-dose MTX. Leucovorin dosing mustbe increased dramatically when plasma MTX levels are elevatedabove5mMat42hoursafter the startof theMTX infusionbecauseleucovorin must compete withMTX to enter cells via the reducedfolate carrier and the goal of leucovorin rescue is to achieve ahigh intracellular concentration of leucovorin. In color are therecommended doses of leucovorin based on the plasma MTXconcentration at each time point after the start of the MTXinfusion. For example, if at hour 60 the MTX concentration is100 mM, it falls above the red line and the recommendedleucovorin dose would be 1,000 mg/m2 every 6 hours. If at100 hours the methotrexate concentration decreases to 3 mM(above the yellow line, below the orange line), then therecommended leucovorin dose would decrease to 10 mg/m2

every 3 hours. The dotted lines indicate extrapolated valuesbased on modeling and clinical trial experience following theoriginal publication [113].

Abbreviation: MTX, methotrexate.



can facilitate a return to acceptable renal function thatallows safe administration of subsequent HDMTX courses.Christensen et al. [5] reviewed the clinical courses of 1,141pediatric oncology patients who received a total of 4,909courses of HDMTX ($1 g/m2) at St. Jude Children’s ResearchHospital from 1998 through 2010 and identified 20 (1.8% ofpatients, 0.4% of HDMTX courses) who developed AKI anddelayedmethotrexateexcretionand requiredglucarpidase. Allpatients had a return to baseline creatinine values, none diedof methotrexate toxicity, and 13 of 20 received a total of 39subsequent courses of HDMTX,whichwaswell-tolerated in allcases [5]. In a pooled analysis of efficacy data from fourmulticenter, single-arm compassionate-use clinical trials,Widemann et al. [43] showed that glucarpidase resulted in a99% or greater sustained reduction of serum methotrexateconcentrations in renally impaired patients. Of concern is thedeclining glomerular function later in life among childhoodcancer survivors who received methotrexate and othernephrotoxic chemotherapeutics; thus, prevention of AKI ispreferable to managing it [92].

Efficacy of GlucarpidaseA series of compassionate-use studies that used glucarpidasein conjunction with standard management approaches forpatients with signs of renal toxicity were published by

Widemann and colleagues about their 15-year experiencewith glucarpidase for methotrexate toxicity in 492 cancerpatients between November 1993 and June 2009 (Table 3)[6, 50, 64, 93–95].

Widemann and colleagues [6] reported additional experi-ence with glucarpidase, leucovorin, and thymidine in 100patients treated with 1–3 doses of glucarpidase and standardleucovorin rescue. An initial cohort of 35 patients receivedthymidine by continuous infusion. Thereafter, thymidine wasrestricted to patients with prolonged methotrexate exposure(.96 hours) or with substantial methotrexate toxicity. Plasmamethotrexate concentrations decreased by 99% within 15minutes after the first glucarpidase dose [6]. This analysisunderscorestheimportanceofearly leucovorindoseadjustmentand timely glucarpidase administration. Of 12 deaths, 6 wereconsidered directly related to methotrexate because patientsexperienced grade 4 myelosuppression (n 5 5), grade 3 or 4mucositis (n5 4), sepsis (n5 5), and toxic epidermal necrolysis(n 5 2). All 6 patients had received thymidine. Predictors ofgrade 4 and 5 toxicity included the presence of grade 4 toxicitybefore glucarpidase administration, inadequate initial increasein leucovorin dosing, and administration of glucarpidase morethan 96 hours after the start of the methotrexate infusion.The other patient deaths were attributed primarily to rapidcancer progression. The major risk factors for severe toxicity

Table 3. Selected studies that used glucarpidase

ParameterWidemann et al.,1995 [90]

Widemann et al., 2010 [6],Widemannet al., 1997 [64] Buchen et al., 2005 [95]

Pharmacodynamicstudy (unpublished)a

Patients (n) 43 262 65 68

Cancer types ALL, NHL, PCNSL Primarily osteosarcoma Primarily osteosarcoma Primarily osteosarcoma

Inclusion criteria: MTXconcentration (mM)

.5 or.1 at 42 hours;

.0.4 at 48 hours.2 SDs above standardelimination curve at.12 hours;$10 at42 hours

.2 SDs above standardelimination curve at.12 hours;.10 at36 hours;.5 at 42hours;.3 at 48 hours

.2 SDs above standardelimination curve at.12 hours;.50 at24 hours;.10 at 42hours;.5 at 48 hours

Inclusion criteria: renal Serum Cr$ 1.5 timesULN and urine output, 500 mL/24 hours

Serum Cr$1.5 times ULN;CrCl# 60 mL/m2

SerumCr$1.5 timesULN;decreased diuresis

SerumCr$1.5 timesULN;CrCl# 60 mL/m2

Median age (range), yr 52 (10–78) 17 (0–82) 16 (0–72) 20 (2–84)

Median pretreatmentMTX concentration(range), mM

5 (0.4–166), n5 24patientsb

35 (1–849); n5 70patients

12 (0.5–902); n5 65patients

40 (3–708);n5 23 patients

Median time from start ofMTX to first glucarpidasedose (range), d

2 (1–7) 3 (1–9) 2 (1–7) 2 (1–6)

Median glucarpidasedoses (range), g/m2

3 (0.9–12) 5.5 (0.4–19) 1–12 6.7 (1–20)

Median reduction in MTXlevels after glucarpidaseadministration

98.9% within 15 min(n5 28)c


97% within 15 min(n5 30)c


Toxicities associated withglucarpidase

Potential acute infusionreaction, type I or IIIhypersensitivity

Type I or III hypersensitivity Potential acute infusionreaction, type I or IIIhypersensitivity

Type I hypersensitivity

Toxicities associated withMTX (%)

Hematologic (70.5)Hepatic (45.5)Mucositis (43.2)Renal (29.5)

Renal (35.5)Stomatitis (32.2)Gastrointestinal (16–27)

Hepatic (45.5)Gastrointestinal (45.5)Renal (30)

Renal (38.9)Gastrointestinal (29.5)Stomatitis (27.5)

aUnpublished; data on file, BTG International Inc.,West Conshohocken, PA.bNot all patients had measurements for every parameter.cPatients whose MTX levels were measured by high-performance liquid chromatography.Abbreviations: ALL, acute lymphoblastic leukemia; Cr, creatinine; CrCl, creatinine clearance; MTX, methotrexate; NHL, non-Hodgkin lymphoma; PCNSL,primary central nervous system lymphoma; ULN, upper limits of normal.




are preexisting toxicity, inappropriate leucovorin increase, anddelayed glucarpidase administration [6].

CONCLUSIONHDMTX can be safely administered to patients with normalrenal function using hyperhydration, urine alkalization, andpharmacokinetically guided leucovorin rescue. Successfulmanagement of methotrexate toxicity requires timely recog-nition of delayedmethotrexate elimination and renal dysfunc-tion. In particular, rising serum creatinine concentration ordecreased urine output after HDMTX indicates a medicalemergency. Increased hydration, high-dose leucovorin, andglucarpidase (when necessary) effectively reduce serummethotrexate concentrations and protect cells from metho-trexate, but these measures must be administered as early aspossible to prevent further toxicity, facilitate renal recovery,and allow patients to resume HDMTX therapy after normal-ization of renal function [96, 97].

ACKNOWLEDGMENTS

We thank Thomas King, M.P.H. (BTG International Inc.) forpreparation of key figures and editorial assistance. Funding

wasprovided inpartby theNational InstitutesofHealthCancerCenter Support Core Grant (CA-21765) and the AmericanLebanese Syrian Associated Charities. Dr. Pui is an AmericanCancer Society professor.

AUTHOR CONTRIBUTIONSConception/Design: Scott C. Howard, JohnMcCormick, Randall K. Buddington,R. Donald Harvey

Provision of study material or patients: Scott C. Howard, Ching-Hon Pui, R.Donald Harvey

Collectionand/orassemblyofdata: ScottC.Howard, Ching-Hon Pui, Randall K.Buddington, R. Donald Harvey

Data analysis and interpretation: Scott C. Howard, John McCormick, Ching-Hon Pui, Randall K. Buddington, R. Donald Harvey

Manuscript writing: Scott C. Howard, John McCormick, Ching-Hon Pui, R.Donald Harvey

Final approval of manuscript: Scott C. Howard, John McCormick, Ching-HonPui, Randall K. Buddington, R. Donald Harvey

DISCLOSURES

Scott C. Howard: Sanofi, Sigma Tau (C/A), Jazz Pharmaceuticals (RF),Sanofi, Sigma Tau, Jazz Pharmaceuticals (H). The other authorsindicated no financial relationships.(C/A) Consulting/advisory relationship; (RF) Research funding; (E) Employment; (ET) Expert

testimony; (H) Honoraria received; (OI) Ownership interests; (IP) Intellectual property rights/

inventor/patent holder; (SAB) Scientific advisory board

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Preventing and Managing Toxicities of High-Dose Methotrexate