Human Pharmacokinetics of 5-Formyl Tetrahydrofolate

The purpose of this study wa~ to deteraine the pharaacokinetics of calcium leucoYorin in nor•al subjects and in patients undergoing High-Dose methotrexa~e therapy for the treat•ent of neoplastic diseaseBy characterizing the disposition of leucovorin it was hoped that the the efficacy of the aethotrexate therapy might be i•proved, while at the sa•e time, the incidence of toxicity could be di•inished. An assay for the separation and quantification of leucovorin in serua was developed. The reduced folate vas extracted from seru• using re•erse-phase chromatographic •inicoluans and a paired-ion reagent. Leucovorin was eluted from this systea in methanol and e•aporated to dryness under nitrogen. The extracted samples were stable for 3 days vhen stored at -4oc. Bigh pressure liquid chroaatoqraphy vas used to separate leuco•orin from the extracted Rerua coaponents using a reverse-phase, paired-ion mode. Aamonium phosphate, dibasic, vas found to be a suitable pairing agent. The recently developed radially coapressed reverse-phase coluans vere found to provide superior resolution of serua components than vas possible with conventional stainless steel columns. The effects of flov rate, pH, column teaperature, and buffer concentration on the separation of leucoYorin vere investigated. Electrochemical detection vas used to quantify the reduced folate in the extracted serum samples. An applied potential of 0.8 volts vas used to produce a sensitive and selective aeans of detecting leucovorin. The apparent lover limit of sensitivity under the conditions employed vas 12 ng leuco•orin. The effects of pH, buffer concentration, methanol concentration, and mobile phase flow rate on detector response vere investigated. The protein binding of leucovorin and its major aetabolite 5-aethyltetrahydrofolate to hu•an serum albumin was determined. Over the concentration range of 5.0x10-78 to 1x10-•K neither folate saturated the available binding sites. Leucovorin binding was a constant 701, vhile aethyltetrahydrofolate decreased slightly over this range. When both folates vere present in the albumin solution, there was a significant decrease in the degree of binding of each. ftethotrexate did not affect the binding of leucovorin or its metabolite. The pharaacokinetics of leucovorin vas determined in 6 noraal subjects and in 5 cancer patient s. There were no siqnif icant differences in the kinetic parameters calculated fro• either group. The disposition of leucovorin is best characterized by a two coapartment open aodel. ln initial half-life of 8.79 ± 4.44 minutes and a second, slower elimination phase of 231.46 ± 31.76 minutes vas determined. The volume of the central compartment vas calculated as 5.49 ± 3.53 Liters. Leucovorin serum concentrations in 3 cancer patients receiving HighDose methotrexate vere deterained to be 3 to 11 times greater than vas predicted using methotrexate pharmacokinetic paraaeters. This study clearly deaonstrates that there are significant differences in the pharmacokinetics of leucovorin and methotrexate. The data presented in this study suggests that it aight be possible to reduce the aaount of leucovorin administered to patients being treated vith met.hotrexate. This may be expected to improve the efficacy of the antimetabolite therapy without additional risk of toxicity. ( ABSTRACT ACKNOilLEDGEftENT TABLE OF CONTENTS LIST OP TABLES LIST OP FIGURES

The protein binding of leucovorin and its major aetabolite 5-aethyltetrahydrofolate to hu•an serum albumin was determined. Over the concentration range of 5. Schwartz. 1976,. and psoriatic arthritis (Fosdick. 1968 Goldin (1978). and Djerassi (1975). have all discussed the historical background of high dose !TX therapy.
The goal of !TX research has been to identify the maximum dose and frequency of adainistration tolerated by norsal tissues that vill still be effective against tumor  (   (   page  3 adainistration of LEU or pteroylglutamic acid~ Goldin ~ alLL (1953.1954.1955.1966) and Bernbaum ~ !l·L (1965) working with leukeaic mice produced results suggestinq that proper administration of LEU subsequent to "TX could prevent toxicity in normal celis without protectinq the malignant cells. Shoenbach ~ ~1:£ (1950) reported a case in which LEU vas successfully used to reverse KTX-induced toxicity in a patient. However. it was not until the work of Djerassi ~ ~l~ (1966.1967.1968.1970) and Lefkowitz ~ !.l~L However. there is still considerable debate regarding the optiaal dose and schedule of LEU which should be employed. Penta (1975). in a review of IMD protocols discussed ten different treatment schedules while Nixon (1979) described a 100-fold range in the doses of LEU employed and labeled current regimens as arbitrary.
The applicability to pharmacokinetic analysis of each of these methods. however. has severe limitations.    513. is the formyl derivative of THF. It is one of the foras of reduced folate coenzymes found in the body. (Blakely, 1969). The term leucovorin generally refers to the chemically synthesized material containing both the -L and 1 diastereomers. while "folinic acid" or "citrovorum factor" applies to the biologically active l isomer only (Ponte!,!!!::.. .. 1979

Biochemical Role of LEU
Leucovorin was the first naturally occurinq derivative of THF to be isolated, identified, and synthesized. (Blakely, 1969  (fGAR) (Calabresi and Parks, 1975 (Patek, 1969). This phenomena has been attributed to an inability to produce reduced coenzyme foras rather than an actual lack of folic acid itself. It has been suggested in such cases that because of its ready conYersion to other reduced coenzy•e forms LEU be used to treat the anemia (Patek, 1969 ! at pH 6.9 (Bleyer, 1977). MTX has two pKa's; 4.8  synthesis is reportedly the most sensitive to THF stores (Plenderheith and Bertino. 1975 Bertino. 1975; Blakely. 1969 Goldin. 1974Goldin. .1975 (1953,1955,1956,1966) (1975) reported that the adainistration of 10 to 15 mg m-2 intrathecally, resulted in plasaa concentrations above 10-e ! , two to three times longer than the saae dose administered intravenously.

ii. Distribution
The disappearance from plas•a of intravenously administered ftTX has been described both as biphasic and triphasic.

iv. Excretion
Excretion of ftTX is primarily renal. (1973) reported that ninety-six hours intravenously adainistered MTX  The urinary excretion of LEU in tvo subjects receiving only the active isoaer of LEU was also determined. The ~vo hour cu•ulative urinary excretion in the 2 subjects vere 16.61 and 20~ of the administered dosea It was concluded f~o• these data that since the percent of dose excreted following the administration of either the racemic mixture.
or active diastereoisomer. of LEU were similar that only the ~iv~ rather than the inactive for• is rapidly excreted.

Biochemical Role of METHF
The enzyme Ns-10 methylene THF reductase, found in the liver of various vertebrates and in c~rtain bacteria (Blakely, 1969) Wagner (1966, isolated an enzyme, tentatively named triaethylsulfonium: tetrahydrofolate methyltransferase which catalyzes the methylation of TBP as shown in Figure   4.
This, however, is not a aajor mechanism for the synthesis of ~ETHF. In Another portion of the saae serum or urine sample is then acidified and exposed to sodium borohydride vhich converts The difference in METHF concentrations between the treated and untreated samples is assumed to be due to the original concentration of LEU.
Langone and Levine (1979) have published a preliminary report of a radioimmunologic assay for leucovorin using an anti-leucovorin antibody produced in rabbits. The method is reported to be sensitive to 0. 10  in which "current is measured as a with a constant potential applied at a exposed to a moving fluid" ( Shoup. 1979 irreversibly. the stable product P.   (2) transfer electrons at the surface ( Kissinger. 1977. and

This is a siaplified
The rate-determining step is considered to be diffusion through the bulk solution. which  As the potential at the working electrode drifts, an electronic feedback mechanism adjusts the potential of the auxillary electrode to compensate and return the potential difference between the working electrode and bulk solution to its operator deterained value (Kissinger, 1979).
In operation, the carrier electrolyte in the mobile phase will produce a current. This current will increase as the concentration of solute (sample) rises within the  Albumin is composed exclusively of amino acid residues; eighteen amino acids have been identified (Hughes, 1954 The association constant. Ka. can be written as: The degree of binding. r. can be expressed as the aoles of drug bound per mole of protein: Which can also be defined as: If the protein is determined to have n independent and equivalent binding sites. the quantity r can be written as follows:  Schwartz. 1979; and Pletcher and Ashbrook. 1973   In •any cases one and one-half to three hours was required.
in some instances the residual current did not decrease to acceptable levels after overnight operation. in which case the electrode vas repacked.  and methanol, allowed to air dry, and reassembled.
A potential was then applied to the working electrode for a miniaum of four hours before quantitative determinations vere attempted; in most cases the detector required ov~rnight operation to become "conditioned".
If polishing the carbon electrode with alumina did not regenerate the cell surface, an additional step was added to the polishing procedure~ A saall amount of a diamond dust ilurry vas placed on a disc of "660 vet dry" abrasive.
The cell surface was "rough" polished by this method before progressing to the aluaina paste and subsequent steps as described above.
Once a potential was impressed between working and reference electrodes, the system remained charged until the electrode surf ace required regeneration. presumably by chelating trace metals whic h may be found in the aobile phase components or in the chromatograph itself.
Ten mM Sodium EDTA was therefore added to each mobile phase as a "precautionary measure".
All mobile phases were freshly prepared on the day they were to be evaluated.

Huaan Studies · a. Human Subject Protection
A detailed report of the proposed project, its objectives, methods and potential hazards was submitted to  vas only two to three days. contrary to the veeks or aonths of stability reported for this electrode (Kissinger, 1977 The vax was evaluated, using aobile phases containing 201 to 30~ methanol in 5 mft sulfonic acid reagents, pH 3. 5.
"obile phase flow rates ranged from 0.7 to 1o0 ml. per minute. At an applied potential of 1 volt, the lover limit of detection which was achieved vith the vax, vas comparable to the Cp-0 pastes. The baseline noise, or residual current, produced vith this electrode vas lover than that vith the previous system; fever noise spikes or random peaks were generally observed.
The relatively low residual current may be attributable to the ceresin content of the electrode material which results in a stiffer paste than do aineral or silicone oil-based electrodes. The harder paste can be rigorously "polished" and clearly provides a more uniform and smooth surface than other pastes. Furthermore, because (   (   page 104 it is a stiffer material. the wax electrode material is apparently not as susceptible to distortion caused by the motion of aobile phase flowing over its surface. Such distortion is one of the major causes of excessive residual current ( Kissinger. 1977       Because the reduced folates to be chromatographed are amphoteric compounds, the use of paired-ion reagents was considered necessary. Gloor and Johnson (1977), reported that for strong and weak acids, typical counter-ions such as quaternary amines (tetrabutylaamoniua chloride for example) are generally used and, that for strong and weak bases alkyl and aryl sulfonates, such as pentane, heptane, and octane sulfonic acids are aaong the best choices. The paired-ion reagents best suited for use with amphoteric coapounds is however, less well defined. It has been suggested (Bausch, 1977) that sulfonic acids aay provide the best separation of amphoteric coapounds similar in structure to the reduced folates.
The aost commonly eaployed solvent systems in (   (   page 121 reversed-phase paired-ion chroaatography are vater/methanol or water/acetonitrile combinations. Therefore. the first mobile phases evaluated were combinations of methanol and water with SmK PIC as. (pentane sulfonic acid, Waters Assoc.). Figure 15 is the chromatogram resulting from the separation and detection of 1x10-•M LEU in a mobile phase consisting of 25~ MeOH: 75i Pie BS. p3. 5. The retention of LEU was found to be 7.25 minutes. The peak however, was broad, and eluted over a prolonged, 4 minute period. In addition the system was not suitably sensitive, as is etident froa the ~mall peak obtained for LEU.
Various combinations of paired-ion reagents, organic solvents, and pH's were prepared and evaluated. Three factors were considered in the evaluation of a mobile phase; capacity, selectivity and sensitivity for the reduced folates.

Capacity aay be calculated according to Equation xx
and aay be defined as the ability of a chromatographic system to retain a compound.   Figure 16 is the chromatogram of vhat vas considered the optimua separation obtained from the reduced folate analogs vhich could be detected. In general the capacities calculated for LEU vere relatively low, while those obtained for ~ETHF considerably larger.
The addition of aqueous buffer to some of the mobile phase systems described in Table III was    It vas not possible to devise a separation system using the c-18 column which could suitably separate the reduced folates from plasma constituents.            c. Drug Assay

Aqueous Syste•s
The electrochemical detector response to LEU or !ETHP, measured as peak height, declined rapidly from a •axiaum value observed imaediately after the cell was resurfaced.
As a result, it was necessary to generate standard curves of peak height as a function of concentration, whenever quantitative measures were to be performed.
Calcium LEU or Barium !ETBF were used as received without further purification. LEU and METBF solutions of 1x10-3! and 1x10-•ft respectively were freshly prepared in 1N KOH. 111       to analyze seraa saaples were calculated to be less than 5 percent.

b. Sample Stability
The stability of LEU in the sampling dishes was evaluated in order to deteraine the aaxiaua length of tiae that saaples could be stored in this condition.
Leucovorin was added to 20 ml. of pooled huaan serum to yield a final concentration of 1.07x10-•M~ Nine, 1 ml serum samples were then extracted and evaporated as previously de~cribed.
The samples vere randomly assigned to 3 groups of · 3 saaples each, which were then frozen until assayed. The first . qroup was assayed the day following extraction; the second, 2 days after extraction; and the third was assayed on the fifth day after extraction. The results of these assays were combined with the results of 3 assays performed immediately after the extraction process.
and appear in

Leucovorin Protein Binding
The protein binding of leucovorin vas deterained over a total drug concentration range of Sx107M to 1x10-3M. The lover liait of binding deterai~ation vas governed by assay sensitivity, the upper limit by leucovorin solubility.
Leucovorin binding to human serum albumin is essentially constant, 69.95~ ± 4.70~, over the clinically significant concentration range. Figure 25 shows the   Figure 26 prevents the conclusion that this condition has been met. Therefore, the scatchard model is not applicable for analysis of these data.
Values for affinity constants and numbers of binding sites can be estiaated by stepwise computer analysis using iterative techniques (Pletcher, 1973      The effect of ftETBF on the binding of LEU is shovn in        (.) i:: The initial estimates provided by lUTOAN were further refined using a second computer proqraa. HONLIN (Metzler.
Elfrinq. and McEvan. 1974 Normalizing the volumes of distribution by expressing these teras as either al Kg-a or al m-z rather than as al.
greatly reduces the effects of subject variability in these parameters.
The description of a biexponential decay in LEU serua concentration as a function of time is in agreement vith the report of Rothenberq ~1 !l~ (1979) in which a "rapid" decline vas followed by a second prolonged elimination phase which approaches a "plateau" in appearance.. A similar rapid distribution phase (alpha half-life 8 Leucovorin rescue has been dosed to provide an initial plasma concentration ten-fold higher than the projected MTX concentration in the patient 30 hours after the termination of the !TX infusion.               High pressure liquid chromatography vas coupled vith electrochemical detection and a sensitive, selective, and reproducible assay for LEU and !ETHF vas developed.

2.
The electroanalytical detection of reduced folates relies on the oxidation of the electroactive species at a surface electrode. The rate of oxidation, and therefore the generated current, can be affected by the electrode material, the buffer strength of the supporting electrolyte, and the strength of the applied potential.
Oil-based, vax-based, and solid electrodes were investigated; solid glassy carbon electrodes were found to provide the opt~mum balance of durability and sensitivity.        Leucovorin is similar to folic acid, an essential vitamin normally contained in the diet. It is a drug normally used with I methotrexate in cancer chemotherapy. Extemely large amounts have been administered to normal subjects with no ill or toxic effects.
The objective of this research is to study the rate leucovorin is removed from your-body . and to attempt to determine the amounts of other compounds produced by your body in your blood as a function of time. · Subjects volunteering to participate in this project will be ' required to abstain from all drugs ·and alcohol for a period of 48 hours prior to each test.
In addition, all volunteers will be · required to fast for twelve hours before starting the test and for at least four hours after the test has begun. The drug, Calcium Leucovorin, will be prepared using commercially available 10 intravenous preparations. A low dose (~ mg) of leucovorin will be administered to you directly into your arm vein by a physician using a hypodermic syri~ge.
A catheter (i . e . a small tube) will be placed in the vein of your arm for the collection of blood.
Ten milliliters (about two teaspoonfuls) will be collected before you receive the dose of leucovorin and thirteen ten milliliter blood samples will be collected at fifteen minutes, tJ;Urty minutes, forty-five minutes, one hour, one and one half hours, two hours, two and one half hours, three hours, four hours, f ive hours, six hours, seven hours and eight hours after drug administration. All blood samples will be drawn by a physician.or appropriately trained personnel. Leucovorin has been reported to be completely free of toxic .reactions except for a single report of an allergic reaction to the drug with no lasting ill effects. At the dose of leucovorin you will receive, the risk of any side effect is minimal.
Risks associated with the use of a catheter for the collection of blood samples include: infection, hematoma (i.e. soreness. and/or "black and blue" mark) or bleeding.
Medical care for problems resulting from this study will be available at Roger Williams General Hospital for emergencies ..
You will not . be compensated for injuries resulting from this study. . . . You are free to withdraw from participation in this study at any time during the investigation. You will not be identified in any publication resulting from this work.
Feel free to ask any questions you have about the study and we will be happy to answer them. If you have any additional  All those being considered for invol venent in this study will be heal thJ ma::be volunteers between the ages of twenty and fifty with no known hypersensivity to folic acid or its derivatives. Any medical abnonnality, which, in the opinion of the medical consultant or investigators,;.is likely to complicate the study or add further risk to the subject will result in the subject being excluded fran the study. All subjects will be carefully interviewed and pertinent medical information gathered.
All participants will be provided with the opp:>rtunity of reading and having carefully explained the lnfonned Consent Fonn. In addition, participants will be provided the opp:>rtunity for further questioning of the investigators. Any subject may withdraw frc:xn the study at any time during . the investigation. Medical care will be provided at Roger Williams General Hospital for medical e~ergencies resulting fran this study. Subjects will receive no canpensation for injuries received during this study.
Participants will be ·required to abstain fran all d.Iilgs and alcohol for 48 hours prior to the test period. In addition, participants will be required to fast twelve hours before d.Iilg achinistration and for at least four hours after d.Iilg administration.
All drug administration and blood sarrple collection will be under the direction of a physician at Roger Williams General Hospital in Providence, RI.
Calcium leucovorin will be administered intravenously using ccmnercially available preparations at a dose of 1(i' n:g. Blood samples will be collected inmediately before d.