Accumulation of Plasma Reduced Folates
`After Folic Add Administration
`
`David G. Priest, John C. Schmitz, and Marlene A. Bunni
`
`The pharmacol<inetics of folic acid, and resultant me(cid:173)
`tabolites thereof, have been determined after admin(cid:173)
`istration orally and int~avenously at 25 mg/m 2 and 125
`mg/m 2
`• Saturation behavior was observed for uptake of
`folic acid into plasma and with regard to metabolism to
`methylenetetrahydrofolate and tetrahydrofolate as
`. well as methyltetrahydrofolate. Repetitive oral admin(cid:173)
`istration every 6 hours resulted in consistently elevated
`levels of each metabolite pool with the same general
`saturation behavior as observed with single dose ad(cid:173)
`ministration. This repetitive oral administration is con(cid:173)
`cluded to be a suitable means to provide uniform ele(cid:173)
`vation of metabolites that could offer protection from
`undesirable toxic effects of drugs such as MT A.
`Semin Oneol 26 (suppl 6):38-41. Copyright © 1999 by
`W.B. Saunders Company.
`
`W HILE folic acid (FA) has been used as a
`
`vitamin supplement for many years, it has
`more recently received interest as a means to mod(cid:173)
`ulate the activity of antifolates. l -4 This interest has
`centered on toxicity reversal, permitting higher
`dosages of cytotoxic drugs to be administered
`safely. Much of the basis for use of FA as a phar(cid:173)
`macologic modulating agent is associated with
`studies conducted in animal models. l ,s-7 However,
`human trials have been recently undertaken to
`determine the feasibility of this approach in the
`clinical setting.3,4
`Folic acid itself is unlikely to be pharmacologi(cid:173)
`cally active. It is far more likely that the reduced
`metabolites of FA are the active species. The pre(cid:173)
`dominant reduced folate found in human plasma is
`5-methyltetrahydrofolate (5-CH3FH4 ). 8 However,
`following the administration of FA, other metab(cid:173)
`olites also arise, albeit to a lesser extent than the
`major metabolite. The degree to which each of
`these metabolites participates in the modulatory
`activity of FA is not clear, but their presence in
`
`From the Dej)artment of Biochemistry and Molecular Biology,
`Medical University of South Carolina, Charleston, Sc.
`Sponsored by Eli Lilly and Company. The )'esearch was supported
`by Grant No. CH-461 from the American Cancer Society and by
`Gmnt No. CA 22754 from the National Institutes of Health.
`Dr Priest is a consultant for and has l'eceived honoral'ia from Eli
`Lilly and Comj)any.
`Address l'eprint request l'equests to David G. Pl'iest, PhD, De(cid:173)
`partment of Biochemistl'Y and Molecular Biology, Medical Univer(cid:173)
`sity of South Carolina, 171 Ashley Ave, Charleston, SC 29425.
`CoPYlight © 1999 by \V.B. Saunders Comj)any
`0093-7754/99/2602-0606$10.00/0
`
`plasma makes each a candidate. Hence,. the de(cid:173)
`pendence of plasma accumulation of each metab(cid:173)
`olite pool on dose, schedule, and administration
`route can provide useful information to guide the
`appropriate administration of FA in conjunction
`with antifolates .
`
`MATERIALS AND METHODS
`Materials
`Folic acid was obtained from Lyphomed (Deerfield, IL). Radio(cid:173)
`labeled fluorodeoxyuridine monophosphate ([3H]FdUMP) was
`purchased from Moravek Biochemicals (Brea, CAl. Nicotinamide
`adenine dinucleotide phosphate and all other reagents were pur(cid:173)
`chased from Sigma Chemical Co (St Louis, MO). ThymidyIate
`synthase (3.7 U/mg protein) was purified from an Eschelichia coli
`strain that overproduces Lactobacillus casei thymidylate synthase.9
`The E coli strain was a gift from D. Santi (University of Califomia,
`San Francisco). 5,l0-Methylenetetrahydrofolate reductase (0.62
`U/mg protein), 10-formyltetrahydrofolate deacylase (1.1 mU/mg
`protein), and dihydrofolate reductase (1.25 U/mg protein) were
`purified from pig liver, beef liver, and methotrexate-resistant L
`casei, respectively.1O-12
`
`Folic Acid Administration
`
`Ten volunteers were randomly separated into two groups of
`five each. One group was administered FA at a single dose of 25
`mg/m2; the other group received 125 mg/m2. Each group was
`administered FA both orally and intravenously (IV). A 2-week
`washout period lapsed before the same volunteer was given the
`same dose by the altemative administration route. In addition,
`four more volunteers were recruited and administered FA at
`total daily doses of 100 mg/m2 in doses of 25 mg/m2 every 6
`hours for 3 days. After a 2-week washout period, the same
`volunteers received 500 mg/m2 FA daily in doses of 125 mg/m2
`every 6 hours for 3 days.
`Sample Collection and Preparation for Analysis
`Blood samples were collected and immediately centrifuged at
`400g for 5 minutes at 4°C. The resultant plasma was diluted
`into an equal volume of cold 50 mmol/L Tris-HCI buffer (pH
`7.4) that contained 100 mmol/L sodium ascorbate and was
`stored at _20°C. For routine analysis of folates, plasma samples
`were placed in a boiling water bath for 5 minutes and centri(cid:173)
`fuged to remove precipitated protein. The resultant superna(cid:173)
`tant used for estimation of reduced folates ranged from 0.1 }.LL
`(lO}.LL diluted 100-fold) to 100 }.LL, depending on FA dose and
`sampling time. 8
`
`Estimation of Reduced Folates
`The temary complex assay described previously was used to
`quantitate reduced folates.s,13 This assay is based on enzymatic
`. cycling of reduced folates
`to methylenetetrahydrofolate
`(CH2FH4 ) followed by entrapment into a stable temary com(cid:173)
`plex with excess L casei thymidylate synthase and [3H]F_
`
`38
`
`Seminars in Oncology. Vol 26. No 2, Suppl 6 (April), 19·99: pp 38-41
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1103-0001
`
`

`
`FOLIC ACID ADMINISTRATION
`
`39
`
`25 mg/m2
`
`A
`
`- -0 - IV
`-0 - - Oral
`
`125 mg/m2
`
`B
`
`been evaluated by the ternary complex assay in
`plasma over a 24-hour period.s It can be seen in
`Fig 1A that at the lower dose, essentially identical
`'levels of FA are achieved after approximately 3
`hours, whether FA is adtninistered orally or IV.
`This is consistent with relatively efficient absorp(cid:173)
`tion of FA into the blood stream at this dose.
`Furthermore, when area under the curve (AUC)
`values for oral versus IV administration were com(cid:173)
`pared (6,977 ± 747 nmol/L e hr and 11 ,632 ± 817
`nmol/Le hr, respectively), nearly 60% of an oral
`dose is absorbed into the circulatory system at the
`25 mg/m2 dose. On the other hand, administration
`of 125 mg/m2 FA results in much less efficient
`absorption behavior. Figure 1B shows that at no
`point is the same plasma level of FA achieved.
`Likewise,
`the oral versus
`IV AUC values
`
`25 mg/m2
`
`A
`
`~
`~
`cd
`~
`0::
`
`4000
`
`3000
`
`2000
`
`1000
`
`0
`
`30000
`
`25000
`
`20000
`
`15000
`
`10000
`
`5000
`
`15
`
`20
`
`Time (hrl
`
`Fig I. Plasma pharmacokinetics of FA. Each group of five
`volunteers was given FA IV and orally at doses of 25 mg/m 2 (A)
`or 125 mg/m 2 (8). Folic acid was estimated by the ternary
`complex-based assay. Points represent the average from dupli(cid:173)
`cate analyses of samples from five volunteers in each dosage
`group. Error bars represent SEM.
`
`dUMP.14 Additional enzymes and cofactors were added as
`necessary to cycle each reduced folate to the CHzFH4 form.
`Bound PH]FdUMP, which is equivalent to CHzFH4, was de(cid:173)
`termined by scintillation counting following separation on
`Sephadex 0-25 minicolumns. Since CHzFH4 can potentially
`be converted to tetrahydrofolate (FH4l under these conditions,
`the sum of these folates is reported. ls
`
`RESULTS AND DISCUSSION
`
`Folic acid typically has been administered orally
`at very low doses, consistent with its role as a
`vitamin.3 Thus, little information is available re(cid:173)
`garding its pharmacokinetic properties when ad(cid:173)
`ministered as a pharmacologic agent. To examine
`the behavior of FA and its metabolites at pharma(cid:173)
`cologically relevant doses, human volunteers have
`been administered FA doses of 25 mg/m2 and 125
`mg/m2 both orally and IV. Parent compound and·
`corresponding reduced folate metabolites have
`
`~
`E
`'<I'
`::c:
`f;r..
`CI')
`::c:
`()
`I
`L!'.l
`
`~
`~
`
`100
`
`50
`
`0
`
`400
`
`300
`
`200
`
`100
`
`125 mg/m2
`
`B
`
`O~----r----'-----r-----.--~
`o
`20
`15
`5
`10
`Time (hrl
`
`Fig 2. Plasma accumulation of 5-CH3FH4 after administra(cid:173)
`tion of FA IV and orally at doses of 25 mg/m 2 (A) or 125 mg/m 2
`(8). 5-CH3FH4 was estimated by the ternary complex-based
`assay. Points represent the average from duplicate analyses of
`samples from five volunteers in each dosage group. Error bars
`represent SEM.
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1103-0002
`
`

`
`40
`
`PRIEST, SCHMITZ, AND BljNNI
`
`(14,127 ± 1,790 v 83,066 ± 6,663) yield an esti(cid:173)
`mate of absorption of only 17%. Hence, there is
`clear saturation of FA absorption into the blood
`stream as the dose is increased from 25 mg/m2 t~
`125 mg/m2
`•
`5-CH3FH4, the predominant circulating re(cid:173)
`duced folate, became elevated substantially follow(cid:173)
`ing FA administration (Fig 2). At the lower dose
`(Fig 2A), the accumulation of this metabolite is
`'essentially identical whether FA is given IV or
`orally. This is true with regard to both peak accu(cid:173)
`mulation (Cmax ) and AUC. At the higher dose,
`oral administration results in both C max and AUC
`values that are only approximately half those for
`IV administration. When comparison is made for
`C max or AUC between FA oral doses of 25 mg/m2
`versus 125 mg/m2
`, it can be seen that the fivefold
`increase in dose results only in approximately a
`twofold increase in this metabolite pool. These
`results are consistent with saturation of FA absorp(cid:173)
`tion as a major component of the limited increase
`in 5-CH3FH4 elevation as the dose is increased
`from 25 to 125 mg/m2
`• However, there is also some
`saturation of metabolism at the higher dose be(cid:173)
`cause when administered IV, wherein the entire
`dose is available for metabolism, 5-CH3FH4 C max
`is only increased approximately threefold and
`AUC approximately fourfold, as a result of the
`fivefold increase in dose (25 mg/m2 v 125 mg/m2
`).
`Hence, there is a dose-dependent limitation of
`5-CH3FH4 accumulation after FA administration
`that is largely due to uptake saturation with a
`smaller contribution from saturation of metabolic
`capacity.
`In addition to 5-CH3FH4, the methodology
`used permits evaluation of the more labile, but
`potentially more important, reduced folate pool,
`CH2FH+ + FH4 (Fig 3). This pool behaves phar(cid:173)
`macokinetically much like the 5-CH3FH4 pool
`(Fig 2) and achieves maximal accumulation that is
`approximately half that of the more stable metab(cid:173)
`olite pool. As such, this pool can have a significant
`quantitative importance to FA modulation. The
`CH2FH4 + FH4 pool, like the 5-CH3FH4 pool,
`shows a strong dose-dependence for the time re(cid:173)
`quired to achieve maximal accumulation. This is
`true for both IV and oral dosing and suggests that
`metabolic capacity is the origin of this delay in
`peak achievement.
`While the prolonged elevation associated with
`the relatively long time to peak for all metabolite
`pools after a single dose results in an expanded
`
`25 mg/m2
`
`A
`
`- -0 - IV
`- -0 - Oral
`
`20
`
`10
`
`04L--------------------~~
`125 mg/m2
`
`B
`
`75
`
`50
`
`25
`
`O~---,----,---~----.---~
`o
`5
`15
`20
`10
`Time (hr)
`
`Fig 3. Plasma accumulation of CH 2FH. + FH. after admin(cid:173)
`istration of FA IV and orally at doses of 25 mg/m 2 (A) or 125
`mg/m2 (8). CH 2 FH. + FH. was estimated by the ternary
`complex-based assay. Points represent the average from dupli(cid:173)
`cate analyses of samples from five volunteers in each dosage
`group. Error bars represent SEM.
`
`opportunity for administration of other drugs
`whose modulation is sought, single daily doses do
`not result in consistent elevation over the entire
`period. Hence, multiple doses were evaluated to
`further extend this modulatory window. Folic acid
`was administered to volunteers orally every 6
`hours for 3 days at total daily doses of 100 mg/m2
`and 500 mg/m2
`• Metabolite pools, as well as parent
`compound, ,were monitored (Fig 4). This admin(cid:173)
`istration schedule resulted in achievement of rel(cid:173)
`atively constant metabolite elevation at approxi(cid:173)
`mately twofold higher levels than were observed at
`C max for equivalent single doses. Parent compound
`behavior was less uniform than metabolite pools
`and could suggest some circadian dependence of
`uptake or elimination. Generally, the same satu(cid:173)
`ration of uptake is observed as when single doses
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1103-0003
`
`

`
`7500
`
`~
`E
`~ 5000
`co
`
`()
`;!:1
`
`r2
`
`2500
`
`~
`3
`~'"'
`"' £ C)
`
`J,
`
`0
`
`600
`
`400
`
`200
`
`0
`
`~ 250
`'"'
`~ "'
`+ '"' ~ 100
`
`200
`
`150
`
`0-1
`~
`C)
`
`50
`
`0
`
`0
`
`12 24 36 48 60 72 84 96 108 120
`
`Time (hr)
`
`Fig 4. Plasma accumulation of FA (A), 5-CH3FH. (B), and
`CH2FH. + FH. (C) following administration of oral FA to four
`volunteers. Folic acid was administered at a total daily dose of
`100 mg/m2 (open diamonds) given four times daily (25 mg/m2/
`dose) for 3 days. After a 2-weel< washout period, the same
`volunteers received a total dose of 500 mg/m2 FA (open boxes)
`given four times daily (125 mg/m2/dose) for 3 days. Plasma
`folates were estimated by the ternary complex assay. Points
`represent the average from duplicate analyses of samples from
`four volunteers in each dosage group. Error bars represent
`SEM.
`
`to 125 mg/m2
`are increased from 25 mg/m2
`•
`Hence, this schedule permits a substantially higher
`sustained metabolite level, which may, in turn,
`provide a more consistent means for modulating
`the antitumor activity of drugs that target reduced
`folate-metabolizing enzymes.
`In summary, FA administered orally exhibits
`dose-dependent saturation of absorption and, to a
`lesser extent, metabolism. The relatively pro(cid:173)
`longed elevation of plasma-reduced folate metab(cid:173)
`olites can be extended further with multiple dos(cid:173)
`ing, which also leads to achievement of twofold
`higher levels than can be achieved maximally with
`a single dose. Hence, FA administered in conve(cid:173)
`nient oral doses can be used to provide sustained
`
`FOLIC ACID ADMINISTRATION
`
`41
`
`Folic acid
`
`A
`
`elevation of reduced folate metabolites which
`likely are the agents that modulate the toxicity of
`antifolates such as MT A.
`
`REFERENCES
`
`1. Smith GK, Amyx H, Boytos CM, et al: Enhanced anti(cid:173)
`tumor activity for the thymidylate synthase inhibitor 1843U89
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`2. Alati T, Worzalla ]F, Chin C, et al: Augmentation of the
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`the anti purine antifolate
`lometrexol
`(DDATHF) given with oral folic acid. Invest New Drugs 14:
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`5. Schmitz ]C, Grindey GB, Schultz RM, et al: Impact of
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`7. Rees C, Kimbell R, Valenti M, et al: Effects ofleucovorin
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`9. Pinter K, Davisson V], Santi DV: Cloning, sequencing,
`and expression of the Lactobacillus case; thymidylate synthase
`gene. DNA 7:235-241, 1988
`10. Matthews RG: Methylenetetrahydrofolate reductase
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`11. Huennekens FM, Scrimgeor KG: NIO-Formyltetrahydro(cid:173)
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`12. Dunlap RB, Harding NG, Huennekens FM: Thymidy(cid:173)
`late synthetase from amethopterin-resistant Lactobacillus case;'
`Biochemistry 10:88-97, 1971
`13. Priest DG, Doig MT: Tissue folate polyglutamate chain
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`thase-fluorodeoxyuridylate ternary complexes. Methods Enzy(cid:173)
`mol 122:313-319, 1986
`14. 'Santi DV, McHenry CS, Perriard ER: A filter assay for
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`15. Priest DG, Schmitz ]C, Bunni MA, et al: Phannacoki(cid:173)
`netics of leucovorin metabolites in human plasma as a function
`of dose administered orally and intravenously. ] Nat! Cancer
`Inst 83:1806-1812, 1991
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1103-0004