Journal of Neuro-Oncology, Vol. 1, Nr. 1, 39-44 (1983). © Martinus Nijhoff Publishers. Printed in the Netherlands.

The inability of oral ieucovorin to elevate CSF 5-methyl-tetrahydrofolate fol lowing high dose intravenous methotrexate therapy

Jeffrey Allen, l, 2,, Gerald Rosen, J Herbert Juergens 1 & Bipin Mehta 3,4 Departments of Pediatrics I & Neurology, 2 Memorial Sloan-Kettering Cancer Center & Cornell University Medical Center and Laboratory of Drug Resistance and Cyto-Regulation/ Sloan-Kettering Institute for Cancer Research & Sloan-Kettering Division, Graduate School of Medical Sciences, Cornell University 4

Keywords: methotrexate, neurotoxicity, osteosarcoma, leucovorin rescue, leukoencephalopathy

Abstract

Osteosarcoma patients free of CNS metastases are at risk for acquiring leukoencephalopathy after receiving multiple courses of high dose intravenous methotrexate followed by oral leucovorin rescue (MTX-LV). A prospective study of the adequacy of CNS rescue of MTX biochemical toxicity by oral leucovorin was undertaken in newly diagnosed neurologically normal osteosarcoma patients. Prior to surgical resection of the primary tumor, ten patients received 4 weekly courses of MTX-LV. During the fourth weekly MTX-LV treatment, 0 and 72 hr serum and CSF determinations of MTX, 5-methyl- tetrahydrofolate (5-MTHF) and LV were made. No CSF MTX was detectable at 0 hr in any patient, but a significant elevation in CS F MTX occurred in 9/9 patients at 72 hr (mean 47.2 _+ 31.8 ng/ml or 1.04 _+ 0.7 × 10 -7 M). There was no significant change in mean CS F 5 -MTH F over 72 hr despite a rise in serum 5-MT H F. MTX exceeded 5 - M T H F in 6/9 patients in CSF, whereas only 3/8 patients had higher MTX in the serum at 72 hr. No acute systemic or neurotoxicity was seen. The failure of oral leucovorin to consistently elevate CSF 5 - M T H F levels at 72 hr in the context of significant levels of CSF MTX may result in intermittent CNS folate deficiency. The clinical and pathological syndrome of leukoencephalopathy may be related to this phenomenon and may evolve after repeated MTX-LV treatments.

Introduction

Methotrexate (MTX) has been reported to be successful in high intravenous doses in the treat- ment of osteosarcoma (1), lung cancer (2), head and neck cancer (3), brain tumors (4), and other malig- nancies (5). In order to prevent systemic toxicity, high dose methotrexate has to be combined with a 'rescue' agent, leucovorin (MTX-LV). Oral leuco- vorin (folinic acid) is converted and absorbed as 5-methyl-tetrahydrofolate (5-MTHF) which pro- vides the end-product of the inhibited enzyme, di- hydrofolate reductase. Major acute systemic side

* Recipient of American Cancer Society Junior Faculty Fellow- ship Award

effects of MTX-LV can include myelosuppression, gastrointestinal ulcerations and renal failure. These complications usually occur because of delayed ex- cretion of MTX (6).

Daily sequential serum MTX determinations may be used to predict and modify systemic toxicity by prolonging leucovorin rescue and increasing hy- dration. In our experience systemic MTX toxicity is usually averted when the serum concentration of 5-MT H F equals or exceeds that of MTX 48 hr after initiating oral leucovorin rescue (7). After a single MTX-LV treatment, cytotoxic levels of MTX may also be detected in CSF for 24-48 hr (4). However, little is known about the pharmacokinetics of CNS folate rescue by oral leucovorin or the factors pre- dicting acute or chronic CNS toxicity.

Address for reprints: Dr. Jeffrey C. Allen, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021 USA

40

Two types of MTX-LV neurologic toxicity have been described. An acute, transient type may be observed within the first or second month of ther- apy and consists of multi-focal neurological symp- toms resembling small strokes occurring over a pe- riod of two to three days. No important neuro- logical sequelae have been noted, and subsequent MTX-LV can be given without recurrence of this syndrome (8). Rarely a delayed chronic neurologic syndrome is observed which takes the form of a progressive leukoencephalopathy associated with seizures, dementia, and spasticity (9). This more se- rious syndrome appears similar to the one des- cribed in patients with ALL who have had prior cranial irradiation and parenteral and intrathecal doses of MTX (10).

The precise relationship of the chronic leuko- encephalopathy to MTX therapy, either low dose intravenous, intrathecal or high dose intravenous with leucovorin rescue, is unclear, but it is possibly related to the long term accumulation of MTX within the central nervous system producing a fo- late deficiency. In this study we examined the CNS pharmacokinetics of intravenously administered MTX and orally administered reduced folates in osteosarcoma patients. These studies were done early in their t reatment course and prior to the time neurologic or CT scan pathology has ever been observed because we suspected that subclinical neurotoxicity could be occurring at this early stage. Although we attempted to document the efficacy of folate rescue within the CNS following MTX-LV, we appreciate that CNS MTX toxicity may require rescue by other agents in addition to reduced fo- lates (11).

Patients and methods

Ten patients with pr imary osteosarcoma (three males, seven females, median age 17 years, range 9-25 years) received four weekly MTX-LV treat- ments as part of our current osteogenic sarcoma treatment protocol prior to surgical resection of their primary tumor (1). At the fourth weekly MTX-LV treatment, lumbar punctures were per- formed at 0 at 72 hr after obtaining informed con- sent. The dose of MTX was 8 g / m 2 for patients who have completed their adolescent growth spurt and 12 g / m 2 for younger patients. The rationale for

such age-dependent dosage has been described else- where (1). Methotrexate was administered in the Outpatient Department as a four hour infusion in 1 liter of D5W containing 50 meq sodium bicarbo- nate. The infusion was started shortly after the initial lumbar puncture. Antiemetics were usually given prior to the chemotherapy. Leucovorin rescue was started 24 hr after the initiation of MTX infusion. Leucovorin (10 rag) was given orally ev- ery 6 hr for a total of I 1 doses. Routine monitoring of serum MTX levels, and liver and kidney function was performed at 0, 4, 24, 48, and 72 hr after a treatment. In cases of high levels of serum MTX, the leucovorin rescue was continued. In addition to MTX, 5 - M T H F and LV were determined at 0 and 72 hr in serum and CSF. The CSF was also exam- ined for sugar, protein and cells. The CSF concen- trations of MTX, 5 -MTHF and LV were assumed to reflect the brain parenchymal concentrations as they have in previous animal experiments (12).

The quantitat ion of MTX was carried out by an enzyme-immuno assay technique available in kit form (Emit and assay, SYVA, Palo Alto, Califor- nia). Leucovorin was determined by a microbiolog- ical disc assay method using the MTX-resistant Pediococcus cerebisiae as the assay organism, and 5 - M T H F A was quantified with a differential mi- crobiological disc assay procedure (13) using MTX- resistant strains of Lactobacillus casei and Strepto- coccus faicium vat. durans.

Results

Eight of ten patients complained of headache within 24 hr after the initial lumbar puncture and the majority were lethargic. CSF protein and sugar did not change over the 72 hr period after MTX therapy. The mean CSF protein was 37.5 _+ 17 mg% at 0 hr and 40.3 _+ 22.6 rag% at 72 hr. The glucose was 60.4 _+ 6.3 rag% at 0 hr and 57.6 _+ 5.3 rag% at 72 hr. Three of ten patients had a CSF cell count above 20 cel ls /mm 3 at 72 hr, the majority of which were mononuclear cells. All initial CSF cell counts were less than 3 W B C ' s / m m 3. No evidence of bone marrow, renal or gastrointestinal toxicity was en- countered. Other than headache and lethargy, no abnormal acute neurologic symptoms were ob- served.

The distribution of MTX, 5 - M T H F and LV is

Tabel 1. Serum and CSF determinations of 5-MTHF, LV and MTX (mg/ml)* at '0' and '72' hr after MITX-LV therapy.

Serum CSF

5-MTHFA LV MTX 5-MTHFA LV MTX

41

Patient 0 hr 72 hr 0 hr 72 hr 0 hr 72 hr 0 hr 72 hr 0 hr 72 hr 0 hr 72 hr

1 34.5 197.0 <0.5 <0.5 10.5 70.0 <5.0 46 <0.5 <0.5 <5.0 65.0

2 36.5 163.5 <0.5 50.0 <5.0 46.7 22.5 14.0 <0.5 <0.5 <5.0 89.0

3 40.0 200.5 <0.5 50.0 14.0 75.0 22.5 17.7 <0.5 <0.5 <5.0 52.5 4 24.75 ND a <0.5 ND 19.5 ND 21.0 ND <0.5 ND <5,0 ND

5 27.0 207.5 <0.5 7.5 13.75 60.0 45.6 37,0 <0.5 <0,5 <5.0 11.0

6 30.0 16.5 <0.5 10.9 10.0 60.0 <5.0 3.5 <0.5 <0.5 <5.0 75.5

7 27.0 23.75 <0.5 <0.5 <5.0 4.5 <5.0 43.0 <0.5 <0.5 <5.0 5.6

8 21.0 55.0 <0.5 3.75 <5.0 86.0 21.0 16.5 <0.5 <0.5 <5.0 5.0

9 ND 2.5 ND 2.3 ND 11.5 ND 2.7 ND <0.5 ND 57.5

10 18.8 ND <0.5 19.0 14.5 17.0 <5.0 49.9 <0.5 <0.5 <5.0 65.5

Mean 30.0 ± 9.5 b 10.8 -- 5.1 15.0 --F 15.7 b -

+ S.D. 108.3 _+ 91.7 c 16.1 _+ 20.1 c 64.7 ± 45.3 c 25.5 -F 18.5 47.2 -t- 31.8 c

a ND Not determined.

b The normal values for CSF and Serum 5-MTHFA in our laboratory are 20 40 ng /m z.

c The paired t test analysis indicates a significant rise (p < 0.05) by 72 hr. * 5 n g / m l = l × 10 8M.

illustrated in the serum and CSF at 0 and 72 hr (Table 1, Figure 1 and 2). Significant rises were observed in serum 5-MTHF, LV and MTX as well as CSF MTX concentration over 72 hr.

At 0 hr (168 hr after the third MTX-LV treat- ment), the 5 - M T H F concentration was greater than MTX in 9/9 patients in serum and CSF, but 6/9 had persistent low levels of MTX in their serum (Table 1). No MTX was detectable in the CSF and

20C

-~ 16C

12c .= o

o~ 8c

E

~.4c

MTX 5-MTHF CF ~ O hr (168 hrs) 72hrs

Fig. l. Prior to the administrat ion of the fourth weekly high dose

MTX regimen, the serum 5-MTHF was in the normal range, 30.0 + 9.5 ng /ml (_+ S.D.) (N-- 9), and low levels of MTX could be detected in six of nine patients (mean = 10.8 + 5.1 ng/ml).

Seventy-two hours after MTX administrat ion and 48 hr after beginning oral LV, significant increases in serum MTX, 5- MTHF and LV were observed.

normal values of 5 - M T H F prevailed in the serum and CSF. At 72 br, an elevation in serum 5 -MTHF occurred in 5/7 patients and in serum MTX in 8/8 patients. The serum 5 - M T H F concentration ex- ceeded MTX in five of eight patients. The CSF 5 - M T H F rose in four a~ad fell in four patients at 72 hr. CSF MTX now exceeded 5 -MTHF in six of nine patients.

O ', I I MTX 5-MTHF CF MTX 5-MTHF CF

Ohr (168 hrs) 72hrs

Fig. 2, The init iaI mean CSF 5 - M T H F was 15.0 _+ ]5,7 ng/ml ( N = 9 ) and no M T X was detectable ]68 hr after the third MTX-LV therapy. Seventy-two hours after the fourth MTX- LV, there was a significant rise in CSF MTX, but the 5-MTHF

did not change. No LV was detectable in CSF and there was no significant difference between the 72-hr mean MTX and 5-

MTHF concentrations. Therefore, oral LV in the doses adminis- tered raised serum, but not CSF-MTHF concentration at 72 hr.

5C

4C

~ 3c

8 m

42

Discussion

Although the normal blood brain barrier ex- cludes a high percentage of MTX (99%) from the CNS following high dose intravenous MTX che- motherapy, cytotoxic MTX concentrations prevail in the CSF for at least 48 hr following administra- tion (4). Serum MTX levels fall rapidly during the first 12 hr with an initial tl/2 of 2-3 hr (14). The terminal serum tl/2 is 10 hr and the terminal tl/2 of CSF MTX is 8 hr (15). Eighteen hours following high dose MTX, LV is given in relatively low oral doses (10 m g / m 2) compared to the intravenous dose of MTX (8 000 mg/m2). This LV dosage ap- pears capable of restoring the intracellular pool of reduced folates in the systemic circulation, thereby, circumventing the deficiency of intracellular dihy- drofolate reductase ( D H F R ) induced by MTX. However, little is known about the pharmacokinet- ics of CNS rescue.

The serum 5 - M T H F / M T X ratio permitting cor- rection of intracellular folate deficiency is unknown and probably varies for specific organs and tumors. It is likely that 5 - M T H F rescues by not only supply- ing a source of intracellular reduced folates, but also by promoting a net egress of intracellular MTX (16). Clinically, systemic toxicity of MTX is frequently observed when the serum MTX concen- tration exceeds that of 5 - M T H F for several days and is presumably related to the inhibition of DNA synthesis in such organs as the gastrointestinal tract and bone marrow. The three patients in our study with relative excess of MTX compared to 5 - M T H F at 72 hr were spared significant systemic toxicity. Although 6/9 patients had higher CNS MTX than 5 - M T H F at 72 hr, no important neurologic symp- toms other than transient lethargy were observed.

There is evidence that 5 - M T H F more readily penetrates a normal blood brain barrier than folic acid. A group of patients with epilepsy on anticon- vulsants received 15 mg of folic acid PO per day for over 6 months. Despite a 20-fold increase in serum folate activity, no significant increment in CSF fo- late was detected. When six patients were given oral leucovorin, significant increases in CSF reduced folates (5-MTHF) were detectable by 3 months (17). In another study where leucovorin (50-100 m g / m z) was infused, intravenously, over 5 hr to prevent MTX toxicity after in t ra-Ommaya MTX administration, a significant rise in serum and CSF

5 -MTHF occurred within 1 hr and the 0/2 of serum and CSF 5 - M T H F was 7.1 and 13.1 hr, respective- ly (18). Using a radioactively labeled 5 -MTHF compound in dogs, Levitt et al. demonstrated rapid CSF equilibration within 3 hr after an IV bolus (19).

Our data indicate that the amount of oral LV (1 m g / k g / d or 20-40 rag/m2/d) administered daily for three days is not sufficient to increase CNS reduced folates in the form of 5-MTHF. Because MTX-LV therapy floods the CNS with MTX for several days, a temporary period of CNS deficiency of reduced folates presumably occurs, the duration of which has not been defined by our study. The period is greater than three days, but less than seven days.

Although a low percentage of recipients of MTX- LV acquire overt leukoencephalopathy, folate defi- ciency is most probably pernicious to the CNS and subclinical damage may be occurring. Patients with congenital folate deficiency develop not only a megaloblastic anemia and peripheral neuropathy, but also a degenerative CNS condition (20). It has been shown that the enzyme dihydrofolate reduc- tase exists in CNS tissue implicating the importance of reduced folates in CNS metabolism (21).

After each MTX-LV treatment the brain expe- riences a relatively prolonged exposure to cytotoxic levels of MTX. Most probably after multiple MTX- LV administrations the accumulation of cerebral metabolic insults can cause symptomatic and pa- thologic disease. In MTX leukoencephalopathy in- duced by either intraventricular or high dose in- travenous administration, the CT scan indicates that the earliest changes occur in periventricular white matter suggesting that transependymal ab- sorption of the drug from CSF is an important route of entry of MTX into brain parenchyma (9).

A chronic, degenerative, central nervous system disorder may occur several months after initiating therapy with MTX-LV in patients who have had no previous cranial irradiation or cerebral pathology (9). This clinical syndrome in its severest form con- sist of a progressive dementia associated with seiz- ures, spasticity and quadraparesis. A C T scan usu- ally indicates diffuse white matter hypodensity and pathologic examination of the brain reveals a mul- ti-focal coagulative necrosis of cerebral white mat- ter, as well as a necrotizing angiopathy.

In a recent study, four of six patients with severe

manifestations of this syndrome following MTX- LV therapy had cytotoxic concentrations of MTX in CSF from 4-12 days after their last MTX-LV administration (9). The neurotoxicity of MTX may be less related to its effects on DNA synthesis than it is to its antimetabolic effect on other enzymatic systems within the brain. A wide variety of reac- tions have been implicated including the synthesis of biogenic amines such as serotonin and dopam- ine, as well as vitamin B~2 dependent enzymatic processes (11). The high frequency of angionecrosis and coagulative necrosis, however, suggests that the vascular endothelium may be susceptible as well (9).

Individual susceptibility to chronic CNS toxicity following MTX-LV obviously differs since frank leukoencephalopathy is fortunately rare (9). To identify those individuals at high risk, it may be helpful to assay serum and CSF MTX and 5- M T H F 4 to 7 days after several courses of MTX- LV. Individuals with relative excess of MTX in the CSF should have their treatments stretched out or Leucovorin may be given in higher doses, orally or parenterally, for longer periods of time. It may also be important that the CSF MTN is undetectable prior to retreatment with MTX-LV. Patients may also be examined with periodic CT scans and the CSF can be assayed for myelin basic proteins. This protein is elevated in early as well as late MTX leukoencephalopathy (22).

From the therapeutic point of view for CNS neo- plasms, the prolonged elevation o fCSF MTX rela- tive to 5 -MTHF following MTX-LV therapy may be responsible for the high response rates observed in individuals with recurrent gliomas, medulloblas- tomas, and meaningeal cancer (4).

Acknowledgment

We wish to acknowledge the statistical advise of Dr David Tishler and the secretarial contributions of Karyl Plaud-Fleck in the preparation of this manuscript. This research was supported in part by The American Cancer Society, Grants No CH 173 and No CH 39U and the Elsa U. Pardee Founda- tion.

43

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