clinical pharmacokinetics of the antipurine antifolate (6r...

Vol. 1. /479-1486. December /995 Clinical Cancer Research 1479

Clinical Pharmacokinetics of the Antipurine Antifolate (6R)-5,1O-

Dideaza-5,6,7,8-tetrahydrofolic Acid (Lometrexol) Administered

with an Oral Folic Acid Supplement1

Stephen R. Wedge, Sudsawat Laohavinij,

Gordon A. Taylor, Alan Boddy,

A. Hilary Calvert, and David R. Newell2Cancer Research Unit. The Medical School, University of Newcastle-

upon-Tyne. Framlington Place. Newcastle-upon-Tyne. NE2 4HH,

United Kingdom

ABSTRACT

(6R)-5,1O-Dideaza-5,6,7,8-tetrahydrofolic acid (lome-

trexol) is an antipurine antifolate which selectively inhibitsglycinamide ribonucleotide formyltransferase. Lometrexol

pharmacokinetics were evaluated in 17 patients (32 courses)

as part of a Phase I study in which folic acid supplementa-

tion was used to improve tolerance to the drug, its clinicalutility being previously limited by severe cumulative toxic-

ity. Lometrexol was administered as an i.v. bolus every 4

weeks at a starting dose of 12 mg/m2, with subsequent

interpatient dose escalation to 16, 30, and 45 mg/m2. p.o.

folic acid (5 mg/day) was given for 7 days before and 7 days

after lometrexol administration. The disposition of total

lometrexol in plasma was best described by a biexponential

model for data acquired up to 12 h after drug administra-tion, although triexponential plasma pharmacokinetics wereoften found to give a more adequate description when datawere available at later time intervals (24 h and greater).

Mean plasma half-lives (± SD) for model-dependent analy-sis were t012a 19 ± 7 mm, t112 �3 256 ± 96 mm, and t112y

(where measurable) 1 170 ± 435 mm. Lometrexol area under

plasma concentration versus time curve was proportional to

the dose administered. Moderate plasma protein binding of

lometrexol was evident (78 ± 3%) with an inverse linear

relationship between fraction of unbound lometrexol and

the concentration of serum albumin. The volume of distri-

bution oflometrexol at steady state was between 4.7 and 158

l/m2. Renal elimination of lometrexol, studied in 19 patients(21 courses), was considerable, accounting for 56 ± 17% of

the total dose administered within 6 h of treatment, and 85

± 16% within 24 h of treatment. These recoveries of Un-changed lometrexol indicate that the drug does not appear

to undergo appreciable systemic metabolism at the range of

concentrations studied.

Received 2/24/95: revised 8/15/95: accepted 8/I 7/95.

I � R. W. and S. L. were supported by Eli Lilly and Company (India-napolis, IN). Financial support was also provided by the North of

England Cancer Research Campaign.

2 To whom requests for reprints should be addressed. Phone: 44-19 1-

222-8233: Fax: 44-191-222-7556.

Lometrexol pharmacokinetics were also examined inseven patients who received 45 or 60 mg/m2 lometrexol as

part of a separate study of the drug given with folinic acid

rescue 5-7 days after treatment. No marked differences

were evident in lometrexol plasma half-lives, plasma clear-

ance, or the extent of plasma protein binding, indicating that

there is not a pronounced pharmacokinetic interaction be-

tween lometrexol and folic acid.

INTRODUCTION

Lometrexol is a folate analogue which selectively inhibits

GAR3 formyltransferase. an enzyme essential for de izos’a purine

biosynthesis (1, 2). This antipurine antifolate exhibits a broad

spectrum of antitumor activity in murine and human xenograft

tumor models. in which the established antifolate methotrexate

demonstrates little or no effect (3).

In early Phase I clinical studies with lometrexol. significant

clinical toxicity was evident, characterized by severe mucositis

and myelotoxicity (thrombocytopenia and leucopenia). which

limited drug administration to only one or two courses (4-7).

These toxicities were unexpected. occurring at drug concentra-

tions which were approximately one hundredth of the 10%

lethal dose in mice (8). A number of clinical responses were

documented, including activity against malignant fibrous histi-

ocytoma (5). non-small cell lung cancer. breast cancer. and

colonic adenocarcinoma (7), which stimulated studies aimed at

the pharmacological amelioration of lometrexol toxicity. Exper-

imentation in mice revealed that the therapeutic index of lome-

trexol was highly dependent on dietary folic acid intake (9. 10),

and suggested that folic acid administration could reduce tox-

icity. without ablating antitumor activity. To enable the devel-

opment of a tolerable and effective schedule for the routine

clinical use of lometrexol, a Phase I study was initiated in which

S mg folic acid/day were given for 7 days before and after

lometrexol administration. After 7 days. this dose of folic acid

resulted in an increase in plasma folate levels from 3 to 64 ng/ml

to 6 to 180 ng/ml in the patients studied.

The Phase I study of lometrexol with ftlate supplementa-

tion provided an opportunity. for the first time. to conduct

detailed clinical pharmacokinetic studies with lometrexol: a

comprehensive pharmacological examination in humans being

previously prohibited by the lack of clinical utility and avail-

ability of a suitable assay. The principal objectives of this

I The abbreviations used are: GAR, glycinamide ribonucleotide: CV.coefficient of variation: AUC. area under the lometrexol plasma con-

centration versus time curve: ClinT. total plasnia clearance: GFR. gb-

merular filtration rate: Vd�, volume of distribution at steady state: FBP,

folate-binding protein.

on July 2, 2018. © 1995 American Association for Cancer Research.clincancerres.aacrjournals.org Downloaded from

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1480 Lometrexol with Folic Acid Supplementation

clinical pharmacokinetic study were: (a) to determine the

plasma pharmacokinetics in patients receiving multiple courses

of lometrexol, and thereby define the relationship between

lometrexol AUC and dose, and intra- and interpatient variability

in AUC; (b) to investigate the extent of lometrexol plasma

protein binding; (c) to measure urinary excretion of lometrexol;

and (d) to evaluate the effect of folic acid supplementation on

lometrexol pharmacokinetics to determine whether the im-

proved tolerance of lometrexol produced by folic acid adminis-

tration is a consequence of a pharmacokinetic interaction.

MATERIALS AND METHODS

Patient Eligibility. Patients eligible for this study had a

histiologically documented malignant solid tumor, which was

either refractory to established therapies, or for which no stan-

dard therapy existed. All patients had a predicted life expectancy

of at least 12 weeks, and had recovered from the toxic effects of

previous treatment before entering into the study. i.e., they had

not received any major therapy or investigational drug for at

least 4 weeks (6 weeks if prior therapy included chemotherapy

with a compound known to have delayed toxicity, e.g., a nitro-

sourea). Exclusion criteria included factors which could have

interfered with lometrexol disposition/toxicity or folic acid absorp-

tion and comprised: (a) concomitant medication with probenecid,

trimethoprim, co-trimoxazole, pyrimethamine, prednisolone, anti-

epileptics, or allopurinol; (b) extensive radiotherapy; and (c) in-

flammatory, ulcerative bowel disease or malabsorption syndrome.

All patients were required to have adequate organ function

prior to treatment, with hepatic function characterized by bil-

irubin levels of <25 �i.molIliter, and renal function by a creat-

mine measurement of < 120 �i.molIIiter and a 51Cr-EDTA clear-

ance of >50 ml/min. Informed written consent was given

according to local regulatory requirements.

Study Design. Folic acid (Approved Prescription Ser-

vices Ltd., Leeds, United Kingdom) was given daily as a single

5-mg tablet for 7 days before and 7 days after lometrexol

administration at 4-week intervals. Lometrexol (Lilly Research

Centre, Surrey, United Kingdom) was reconstituted in 0.9%

(v/v) saline and administered as a rapid i.v. bolus over 0.5-1.0

mm at a concentration of 1-10 mg/ml. Patients were admitted to

the Department of Medical Oncology, Newcastle General Hos-

pital, to receive lometrexol and were observed for an additional

24 h after drug administration to ensure that acute toxicity was

not apparent. The performance status of patients was assessed at

least once a week, for a period of 4 weeks, following bometrexol

therapy.

The trial design required three patients, previously un-

treated with lometrexol, to be treated at each dose level. The

first patient entered at each dose level was followed up for 3

weeks before the next patient was entered. At least two patients

per dose level received two courses before dose escalation.

Toxicities were evaluated according to the WHO criteria. If

repeated courses at a given dose level were tolerated without

toxicity greater than WHO grade II, doses were escalated ac-

cording to the clinical judgment of the investigator with ap-

proval of the Medicines Control Agency (London, United King-

dom) and the Local Ethics Committee. The starting dose of

lometrexol was 12 mg/m2, with subsequent escalation to 16, 30,

and 45 mg/m2. Dose escalation increments were determined by

clinical experience at the previous dose level and by data from

a parallel study of lometrexol given with folinic acid ( 1 1 ). No

intrapatient dose escalation occurred.

Pharmacokinetic Studies. Lometrexol pharmacokinet-

ics was determined in 17 patients (32 courses) receiving folic

acid supplementation and in an additional 7 patients (7 courses)

who did not receive folic acid. Plasma samples from patients

receiving bometrexol without folate supplementation were

kindly provided by Drs. C. Sessa and F. Cavalli (Ospedale San

Giovanni, Bellinzona, Switzerland), who were responsible for

an alternative Phase I study that involved folinic acid adminis-

tration ( I 5 mg every 6 h for 1 2 doses), starting 5-7 days after

treatment with lometrexol (I 1).

Blood samples were collected by venipuncture into vacu-

tamer tubes placed on ice and containing the sodium salt of

EDTA as an anticoagulant, and were taken before treatment and

at 5, 15, 30, and 45 mm and at 1, 1.5, 2, 4, 6, 8, 12, and 24 h,

and in some patients at 48. 72, and 96 h, after bometrexol

administration. Samples were immediately centrifuged (1000 X

g, 8 mm, 4#{176}C),and plasma was removed by aspiration with a

Pasteur pipette. Plasma was stored at -20#{176}Cprior to analysis.

The plasma lometrexol concentration was measured by the

HPLC method of Wedge et a!. ( 12), which uses derivitization

and fluorescence detection. Briefly. patient samples were

thawed at room temperature and diluted to I ml with control

human plasma (Red Cross Transfusion Service, Newcastle-

upon-Tyne, United Kingdom) to contain 10-250 ng/ml lome-

trexol. Samples were further diluted ( 1 : 1) with aqueous formic

acid [1% (v/v): pH 3.7] containing 100 ng C’tt-desmethylene

lometrexol (Lilly Research Centre) as an internal standard.

Following rotary mixing and centrifugation, each sample was

subjected to solid-phase extraction using a CS ( I cm) Bondelut

cartridge (Analytichem International, Harbour City. CA). Eluted

samples were evaporated to dryness using a Speedvac concen-

trator (Savant Ltd., Farmingdale, New York) and reconstituted

in 13% (v/v) aqueous formic acid. Oxidation of bometrexol and

the internal standard was achieved by incubation (37#{176}C.90 mm)

with a suspension of manganese dioxide (0.2 mg/mI) in water

and terminated by the addition of a I : 1 mixture of S M NaOH

and 1% (w/v) ammonium carbonate (pH 5) to samples on ice.

Samples were centrifuged (13,000 X g, 10 mm), and 100 p.1 of

the supernatant were analyzed chromatographically. Chromato-

graphic analysis was achieved using an Apex II (Cl 8. 3 p.m; 150

x 4.6 mm) analytical column (Jones Chromatography, Hen-

goed, Glamorgan, South Wales, United Kingdom) and a mobile

phase of I 2% (w/v) acetonitrile in I % (v/v) aqueous acetic acid

(pH 5) containing tetramethylammonium hydrogen sulfate

(0. 1 7 1 g/liter) as an ion pair reagent. Elution was isocratic, at a

flow rate of 1 ml/min, and analyte measurement was by fluo-

rescence detection (E5, 325 nm; EN,, 450 nm).

To assess intraassay variation, each assay was calibrated

using a five-point standard curve of duplicate lometrexol stan-

dards in the range 10-250 ng/ml, prepared in control human

plasma, and extracted/analyzed at the same time as patient

samples. Quantitation was achieved using internal standardiza-

tion by a comparison of peak height ratios. with peak heights

being quantified using Minichrom Software (VG Data Systems

Ltd., Altrincham, Cheshire, United Kingdom). All calibration



Male 6Female I 1

Median age, yr (range) 49 (30-67)WHO performance status

0 4

I 5

2 8

Primary tumor

Breast 4

Ovarian 3

Malignant melanoma 3

Others 7Prior treatment

Chemotherapy 14

Radiotherapy 9

Clinical Cancer Research 1481

curves were linear (r2 > 0.997), the lower limit of determination

of the assay was 10 ng/ml, and the intraassay CV at nominal

concentrations of 10, 50, and 250 ng/ml was always <8.5%. In

addition, three lometrexol quality assurance samples (10, 50,

and 250 ng/ml), prepared every 2-3 months in bulk, were

assayed in triplicate to assess the interassay CV, which was

always <6%.

Pharmacokinetic Parameters. Plasma lometrexol data

were analyzed using both model-independent and model-depen-

dent analyses. In model-independent analyses. the AUC was

calculated using the log trapezoidal rule (13) with extrapolation

to infinity, using the terminal phase rate constant calculated by

the compartmental analysis. For model-dependent analyses. a

biexponential or triexponential equation was fitted to the con-

centration versus time data using a nonlinear-weighted least-

squares parameter estimation program (ADAPT II, kindly pro-

vided by Dr. S. D. Z. D’Argenio and A. Schumitzky.

Biomedical Simulations Resource, Los Angeles. CA). Data

were weighted as the reciprocal of the estimated variance, where

the SD of the output was assumed to be proportional to the

estimated concentration (constant CV). The model providing the

best fit to each data set was determined using the precision of

the parameter estimates and consideration of the Akaike infor-

mation criterion ( 14). The parameters derived were used to

calculate model-independent (AUC, CITOT, and Vd.,.) or model-

dependent (AUC, CITOT. t112Ot� t112�3, and t111”)’) pharmacokinetic

parameters (13, 15).

Plasma Protein Binding. Lometrexol plasma proteinbinding was determined using [‘4Cllometrexol (specific activ-

ity, 13 �i.Ci/mg). which was kindly provided by Dr. M. D’Incalci

(Istituto Mario Negri, Milano, Italy). This compound was radio-

labeled at the carbonyl group of the benzyl moiety, and had a

radiochemical purity of >88%, as determined by HPLC. Each

patient plasma sample ( 1 ml) was spiked with 3.5 �ig [ ‘4C]lome-

trexol, rotary mixed, and an aliquot (100 p.1) was removed. The

remainder was then subjected to ultrafiltration using an Amicon

Centrifree Micropartition Unit (Amicon Corp., Upper Mill,

Stonehouse, Gloucestershire, United Kingdom) and centrifuga-

tion (1000 X g, 10 mm, 4#{176}C),after which an aliquot (100 p.1) of

ultrafiltrate was removed. [ ‘4C}Lometrexol in the ultrafiltrate

and prefiltered plasma was determined by liquid scintillation

counting, and the ratio was used to calculate the percentage of

unbound lometrexol. Nonspecific binding of [ ‘4Cllometrexol to

the filter was determined to be <4% by ultrafiltering solutions

of f’4Cllometrexol in PBS (0.1 M, pH 7.4; 1 ml) and was

therefore ignored. Samples of plasma from each patient. taken at

< I h and at 12-1 20 h after lometrexol administration were

examined to assess any potential concentration dependency of

lometrexol plasma protein binding, but no significant differ-

ences in binding were found (P = 0.1 1, paired t-test). The mean

of each pair of analyses was therefore used to: (a) relate the

unbound fraction of lometrexol with serum albumin and plasma

protein concentrations and (b) calculate the unbound Vd�� (15).

Urinary Excretion. Urinary excretion was studied in 13

patients (14 courses) for which there was evaluable plasma

pharmacokinetics available, and in an additional 6 patients (7

courses) for which plasma pharmacokinetics was not measured.

Urine samples for lometrexol analysis were collected for 24 h

after drug administration at 6-hour intervals, and 20 ml aliquots

Table I Patient characteristics

Evaluab be patients (courses) 17 (32)

Sex

were stored at -20#{176}Cprior to analysis. Samples were thawed at

room temperature and diluted in control urine to ensure that the

bometrexol concentration would be between 2 and 25 p.g/ml

(i.e. , the dilution factor used was estimated according to the dose

of lometrexol administered and the volume of urine produced in

a 6-h period). The method for sample preparation was as de-

scribed for plasma samples, except C’#{176}-desmethylene bome-

trexol was added at a concentration of I 5 jig/mI, and evaporated

samples were not oxidized, but resuspended in 150 �i.1 of the

mobile phase and 50 p.1 were analyzed chromatographically.

Chromatographic analysis (12) involved a Spherisorb C6 (5 �.am;

150 X 4.6 mm) analytical column (Jones Chromatography) and

isocratic elution with a mobile phase of 13% (w/v) acetonitrile

in aqueous phosphoric acid [1% (v/v), pH 3.2] at a flow rate of

I .5 mI/mm. Each assay included a five-point standard curve

(duplicate samples within the range 0.2-10 pg/ml). and quality

assurance standards at 0.2, 2, and 10 �ig/ml were assayed in

triplicate. The intraassay and interassay CVs for these assays

were found to be <2% and <3%, respectively. Lometrexol

excretion in urine was expressed as a percentage of the admin-

istered dose, and renal clearance was calculated as the ratio of

the total amount excreted and the AUC.

Statistical Methods. All values expressed with a margin

of error represent the mean ± SD. Levels of significance were

calculated using Student’s t test, where P < 0.05 was considered

indicative of a significant difference between groups. The rela-

tionship between lometrexol dose and pharmacokinetic param-

eters was assessed by linear regression analysis and/or a Spear-

man rank correlation.

RESULTSPharmacokinetics of Lometrexol Administered with a

Folic Acid Supplement. The characteristics of the patients

studied are shown in Table I , and plasma lometrexol concen-

tration-time profiles from representative patients receiving 12,

16, 30, or 45 mg/m2 lometrexol are shown in Fig. 1 . Data

collected within the first 12 h of lometrexol administration were

found to be best described by a biexponential equation when

evaluated by compartmental analysis. However. at doses of 30

and 45 mg/m2 lometrexol and where data were available at later



A‘�. 10E

.�)

C

0

0

C00C00

05<0

0E0

-j

0 150 300 450 600 750

Time (mm)

B=. 100E

.�)

�100

0

� 10C.,C0C., 0.105<0

� 0.01E0

�10.001

Fig. 1 Representative plasma disposition curves for total bometrexol in

patients receiving folic acid supplementation: A, 12 mg/m2 (Li), 16mg/m2 (A); B, 30 mg/m2 (0), 45 mg/m2 (#{149})bometrexol. The lines are

those generated by compartmental analysis.

0 1000 2000 3000

Time (mm)


0.1

time points (�24 h), plasma elimination could be described

more accurately by a triexponential equation. The most appro-

pnate equations (biexponential or triexponential) fitted to each

data set all had coefficients of determination (ri) of >0.92, i.e.,

0.99 median (range, 0.92-0.99).

Pharmacokinetic parameters are shown in Table 2 with

parameters for patients receiving more than two courses of

lometrexol being represented as the mean and SD of all courses

studied in that patient. Model-dependent analysis of lometrexol

pharmacokinetics resulted in a t’/2a and t’/213of 19 ± 7 mm and

256 ± 94 mm, respectively, and a twy (where measurable) of

1170 ± 435 mm.

Calculation of both lometrexol AUC and C1TOT by either a

model-independent or model-dependent analysis was not found

to result in any significant difference (P 0.14 and P 0.33,

respectively, paired t test), and there were strong linear relation-

ships for both parameters for both analyses (r > 0.98).

Model-independent analysis indicated that lometrexol

AUC was linearly related to dose (r = 0.88, P < 0.001 ; Fig. 2),

i.e. , plasma clearance of lometrexol was not dose dependent.

However, two patients had a lometrexol AUC and C1TOT which

differed markedly from that observed in other patients treated at

the same dose level. One patient (patient 12) receiving 30

mg/m2 lometrexol had a consistently greater AUC than for

others receiving the same dose, i.e., 2.08-2.28 mg/ml . mm

compared with 1.38 and 1.50 mg/ml . mm (Table 2 and Fig. 2).

Reduced lometrexol clearance in this patient may have been due

to an underlying early left ventricular failure, which could have

decreased cardiac output and thereby reduced tissue perfusion,

combined with a relatively low pretreatment GFR of 75 ml/min,

which may have influenced the renal excretion of lometrexol. In

contrast, at a dose of 45 mg/m2 lometrexol. one patient (patient

19) had a much lower AUC than was measured in others (1.27

mg/mi . mm compared with values of I .60-2.20 mg/mI . mm),with a correspondingly higher C1TOT (35.8 mI/min/m2 compared

to values of 23. 1-27.7 ml/min/m2). This may be attributable to

an unusually large lometrexol Vd�� in this patient of I 5.8 liters!

m2, which in turn could have been caused by the presence of a

bilateral pleural effusion, combined with a high pretreatment

GFR of 165 mI/mm which would have promoted extensive renal

excretion of lometrexol.

No consistent change in the model-independent AUC was

evident following more than one course of lometrexol, with the

possible exception of three patients receiving 45 mg!m2 lome-

trexol every 4 weeks (patients 13, 16, and 17), in whom small

increases in AUC after the second and third courses of bome-

trexol were observed. In patients receiving more than three

courses of bometrexol, no consistent change in clearance could

be found, i.e. , the intrapatient CV of plasma clearance was 14%

(median; range, 3-21%; ii = 5).

4000 5000 The bometrexol Vd�� varied between 4.7 and 15.8 l!m2, and

the unbound lometrexol � calculated from measurements of

lometrexol plasma protein binding (see below), varied between

18.1 and 67. 1 l/m2. The larger Vd.�. of two patients receiving 45

mg/m2 lometrexol may be attributed to the fact that both patients

had pleural effusions. Even if data from these two patients are

excluded, rank correlations are observed between dose level

(mg!m2) and both � (r� = 0.84. P < 0.001) and unbound Vd��

(r� 0.66, P < 0.01).

Plasma Protein Binding of Lometrexol. The percent-

age of unbound lometrexol in plasma was 22 ± 3%. indicating

plasma protein binding of 78 ± 3%. Protein binding determined

at two concentrations of lometrexol (3.5-4.7 and 7.7-2 1 . I p.g!

ml) revealed that binding was not concentration dependent (P

0. 1 1 , paired t test). An inverse linear relationship was apparent

between the lometrexol binding and serum albumin concentra-

tion (r = 0.88; P < 0.001; Fig. 3). The relationship between

unbound lometrexol and total serum protein was not found to be

significant (r 0.38; P > 0.05), when one patient with partic-

ularly low total serum protein (55 g/liter) was removed from the

analysis.

Urinary Excretion of Lometrexol. The 0-24-h cumu-

lative urinary excretion data (Fig. 4) for I 9 patients (2 1 courses)

indicated that the major elimination route for lometrexol was

renal excretion, with 85 ± 16% of the administered dose being

excreted within 24 h of drug administration and 56 ± 17%

within the first 6 h. Urinary excretion studied in two patients

who received two consecutive courses of lometrexol did not

reveal any consistent change in excretion following the second

course of treatment. Two patients had 0-24-h lometrexol un-

nary recoveries of > I 00% of the administered dose, which was

likely to be due to inaccuracies in the measurement of urine



Patient/course

1/1-3

2/2

3/1, 2

4/2-45/I. 2

6/I

8/2

9/1, 3,4

I 2/1-4

13/1-3

14/1

15/I

16/I , 2

17/1. 2

18/I

19/I

20/I

Mean ± SD(ii = 17)

Renal

Dose level

(mg/m)

12

t1,2a

(mm)

12 ± 2

(mm)

224 ± 31

tiifY(mm)

AUC

(mg/mI . mm)

Total

clearance

(mI/minim2)

clearance

(mllminl

m2)

16.2

Vd�

(liters/ni)

6.1 ± 0.5

Unbound

Vd�

(liters/m)

28.70.57 ± 0.12 21.6 ± 4.1

12 19 243 0.73 16.8 4.7 18.0

12 18, 24 276. 289 0.57, 0.56 20.8. 21.4 12.1 6.7, 6.8 35.3

16 11 ± 2 222 ± 38 0.77 ± 0.09 20.8 ± 2.1 6.0 ± 0.4 31.8

16 12. 13 226. 235 0.82, 0.83 19.1, 19.6 14.5 5.5, 5.8 32.9

16 18 205 0.62 26.0 18.5 6.1 26.8

30 19 282 1.52 20.2 7.1 34.9

30 12 ± 3 248 ± 23 1.38 ± 0.16 22.0 � 3.0 22.5 7.2 ± 0.2 39.2

30 24± 19 295±246 1394±573 2.17±0.11 13.9±0.5 9.8± 1.0 53.5

45 27 ± 6 525 ± 223 2.05 ± 0.42 22.5 ± 4.7 22.0 10.8 ± 3.2 39.4

45 37 392 2.20 23.1 19.7 12.4 53.645 9 123 795 1.74 27.2 24.3 12.0 51.0

45 14, 24 160. 512 1.60, 2.00 27.7. 22.5 27.7 8.7, 10.8 31.4

45 28, 19 265,209 1.77,2.18 25.4,20.8 23.1 7.4,8.8 37.0

45 14 147 791 2.13 22.0 15.6 9.2 36.5

45 22 175 1811 1,27 35.8 39.3 15.8 67.2

45 23 195 1062 1.79 25.4 29.5 10.9 48.2

19±7 256±96 1170±435 22.6 ± 4.7 21.2 ± 7.5

C

E

E

EC)

3.

2

1

0

.

.r = 0.87

p <0.001

.

.

40

05<0

� 30�E0

�0C

0

f 20�

10

r = 0.88p < 0.001

.

..

.

, t I � I 4 4

0 10 20 30 40 50 20 30 40 50

Dose level (mg/m2) Serum albumin (g/l)

Fig. 2 Relationship between bometrexol dose and

from noncompartmental analysis. Data are given in Table

AUC calculated

2. and the line

Fig. 3 Relationship between serum albumin concentration

percentage of lometrexol which was unbound to total plasma

and the

protein.

is that generated by linear regression analysis. The line is that given by linear regression analysis.


Table 2 Pharmacokinetic parameters for lometrexol administered with folic acid supplementation

volume. The 0-24-h urinary excretion of bometrexol was found

to correlate linearly with the dose administered (r = 0.69; P <

0.0 1 ) even when these patients were omitted from the analysis,

and a significant rank correlation was also evident (r.� = 0.72;

P < 0.001).

Pharmacokinetics of Lometrexol Administered without

Folic Acid Supplementation. Lometrexol pharmacokinetics

were studied in seven patients who received 45 or 60 mg!m2

lometrexol without folic acid supplementation. A triexponential

compartmental model was found to best describe plasma elim-

ination of lometrexol in four of seven patients (Table 3) and a

biexponential curve in the remainder. For most data sets a good

curve fit was observed, the coefficient of determination (r�)

being greater than 0.89 in every case (median, 0.99; range,

0.89-0.99). The t’ 2a and (‘/43 determined from these analyses

were 17 ± 8 mm and 169 ± 51 mm, respectively. with a t’ .‘y

(where measurable) of 2593 ± 1671 mm. As was found with

patients receiving folic acid, AUC and C1TOT calculated using

model-dependent analysis were not significantly different from

those calculated using model-independent analysis (P = 0.30

and P = 1 .00). The lometrexol Vd�.. in these patients was highly

variable, i.e. , 5.7-28.0 liters!m2 and 8.5-3 1.8 liters!m2 at 45 and

60 mg!m2 bometrexol, respectively.

Plasma protein binding of lometrexol in patients treated

without folate supplementation was virtually identical to that in

patients receiving folic acid, with a value for the unbound

bometrexol fraction of 22 ± 4% and 78 ± 4% for the protein

bound fraction.



�. 1200

I �I0 60’

I�‘ 20’0C

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III

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/I

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12 16 30 45

Dose level (mg/m2)

Fig. 4 Relationship between lometrexol dose and the percentage of the

administered dose excreted in urine within 24 h of drug treatment. Each

column represents an individual patient treated with either I 2, 16. 30, or

45 mg/m2 lometrexol.


0’

DISCUSSION

This study describes the first detailed examination of the

plasma pharmacokinetics of lometrexol, a prototype antipurine

antifolate, administered as an iv. bolus to patients receiving

folate supplementation. The plasma elimination of lometrexol

was adequately described by a biexponential equation when data

are collected up to 12 h after drug administration, although the

inclusion of samples taken at later time points (i.e. , 24 h and

greater) would indicate that a tniphasic model was a more

appropriate description. The a, �3, and “s’ phase half-lives deter-

mined for lometrexol were comparable to those measured for

the classical dihydrofolate reductase inhibitors methotrexate

( 16) and edatrexate ( 10-ethyl- lO-deaza-aminopterin; Ref. I 7),

but not with the thymidylate synthase inhibitor Tomudex, which

differs in having a much longer t#{189}’y of 50-100 h (18). The data

reported here agree with those of Young et a!. (5) who per-

formed a preliminary pharmacokinetic study of lometrexol in

patients not receiving folic acid, using a particle concentration

immunofluorescence assay, and identified biphasic and triphasic

plasma pharmacokinetics. A tertiary elimination phase could

potentially result from release of lometrexol from hepatic stores

or enterohepatic cycling of the drug; two possibilities which

have also been proposed to describe the t�,2’y of methotrexate

(19, 20). The involvement of enterohepatic cycling has also

being implicated in the disposition of dichloromethotrexate in

humans (21) and CB3988 (C2-desamino-C2-methyl-N’#{176}-propar-

gyl-2’-trifluoromethyl-5,8-dideazafolic acid) in the rat (22). The

involvement of such mechanisms in the disposition of lome-

trexol are supported by whole-body autoradiographic studies

using ‘4C-labeled lometrexol in mice maintained on a folate-

deficient diet, which indicate that the liver is the primary organ

for drug accumulation (23). This terminal elimination phase

indicates persistence of lometrexol, which could be related to

the cumulative toxicity encountered in early Phase I studies.

The AUC in individual patients studied after multiple

courses of lometrexol was found to be minimally cumulative in

three patients and noncumulative in five patients. Minimal ac-

cumulation of lometrexol on repeated treatment was also de-

scribed in the earlier Phase I study of Young et a!. (5). Similarly.

in patients receiving repeated Tomudex treatment, no accumu-

lation is evident ( 1 8). Lometrexol AUC was found to increase

linearly with dose, an observation which is in agreement with

data for other classical antifolates such as CB3717 (N’#{176}-propar-

gyl-5,8-dideazafolic acid; Ref. 24), Tomudex (18), and edatrex-

ate (17), but not for methotrexate for which it has been sug-

gested that there is a nonlinear relationship between dose and

AUC following bolus administration (25).

That there was a tendency for the lometrexol Vd�� (and

unbound Vd��) to be larger in patients receiving higher doses of

the drug may be indicative of concentration-dependent protein

binding. Thus, protein binding of lometrexol may be a saturable

phenomenon, with greater drug concentrations resulting in a

larger unbound fraction. Unbound lometrexol would be subject

to rapid cellular uptake and hence an apparently larger �

This possibility is supported by the observation that the fraction

of lometrexol unbound is inversely related to serum albumin

concentration (Fig. 4). A relationship between Vd�� and dose has

also been observed in pharmacokinetic studies with Tomudex in

rats (26).

The Vd�� range (4.7-15.8 liters!m2) from patients treated

with 12-45 mg/m2 lometrexol was, on average, smaller than

that measured in patients who did not receive folate supplemen-

tation, but were treated with 45 and 60 mg/rn2 lometrexol

(5.7-3 1 .8 liters/rn2), and that reported for patients treated in a

previous study (14-32 liters!m2) with doses of 15-60 mg/rn2

(27). This observation may again reflect the possible concentra-

tion-dependent plasma protein binding of lometrexol and the

effect that higher lometrexol concentrations would therefore

have on drug disposition.

Despite these findings, the in vitro protein-binding data did

not indicate that concentration-dependent protein binding of

lometrexol was statistically significant (P > 0.05) between the

concentration ranges of 3.5 and 4.7 and 7.7 and 21 .0 p.g/ml,

although a comparison of binding by a paired Student’s t test

resulted in a P value of 0.1 1, which would indicate a trend

toward reduced lometrexol binding at higher concentrations.

The magnitude of lometrexol protein binding (78%) was

comparable to that of methotrexate (28, 29), but not with the

thymidylate synthase inhibitors CB3717 and Tomudex which

bind more extensively (97% and >90%; Refs. 24 and 26). Since

lometrexol has a high affinity for membrane-bound FBP (30,

31), it should be noted that a component of the protein binding

measured in this study may have involved binding to soluble

FBP. Soluble FBP present in plasma is thought to function as a

folate transport protein (32) and has a Mr 35,000l00,000 (33),

which is above the threshold (30,000) used to determine protein

binding.

Any condition influencing lometrexol protein binding, e.g.,

changes in protein conformation, hyperbilirubinemia, or dis-

placement by concomitant drugs, could increase the cellular

uptake and renal clearance of the drug. The inverse linear

relationship between unbound lometrexol and the concentration

of albumin, which comprises approximately 50% of plasma



�-�-- Table 3 Pharmacokinetic parameters for bometrexol administered without folic acid supplementation

Patient/course

1/1

2/I

3/I

4/I

5/I

6/I

Dose level t,12a ‘I/2� tI/2� AUC

(mg/m2) (mm) (mm) (mm) (mg/mI ‘ mm)

45 12 136 4020 1.48

45 27 217 1.73

45 22 164 1.18

60 14 128 3885 1.53

60 11 198 1935 3.56

60 6 103 533 2.62

60 27 240 1.70

17 ± 8 169 ± 51 2593 ± 1671

7/I

Mean ± SD(F’ = 7)

Totalclearance

(ml/min/m2)

Vd�

(liters/m2)

28.0

UnboundVd��

(liters/m2)

125.731.2

26.0 5.7 29.5

38.2 6.8 28.5

37.6 31.8 09.0

16.8 13.1 68.2

23.7 9.0 37.9

35.8

30.1 ± 8.1

8.5 46.1

3. Shih. C.. Grindey, G. B.. Houghton, P. J., and Houghton, J. A. in vito

antitumour activity of 5. lO-dideazatetrahydrofolic acid (DDATHF) and


proteins. also suggests that albumin concentration may be such

a factor. As a determinant of lometrexol toxicity. however,

plasma protein binding may be of lesser importance if drug

dissociation is induced by high-affinity binding to membrane-

bound FBP or the reduced folate carrier, proteins which facili-

tate the cellular uptake of lometrexol. This could also hold true

for renal excretion, if renal tubular cells contain proteins that are

involved in active secretion of bometrexol.

Renal excretion was found to be the major route of lome-

trexol elimination. with approximately 85% of the administered

dose excreted as unchanged drug within 24 h after bolus iv.

injection. the majority of which (56%) occurred within the first

6 h. The percentage of the lometrexol dose excreted in urine was

found to be comparable to that reported for methotrexate (70-

94%; Ref. 34), but much greater than that for other antifolates in

humans, i.e., edatrexate (13-55%), CB3717 (27%) and tomudex

(12%; Refs. 17, 18, 24), and CB3988 (< 10%) in the rat (22),

which are predominantly eliminated by biliary clearance. Meth-

otrexate is reabsorbed by renal tubules at low concentrations

( 16), but actively secreted when at high concentrations (35),

with reabsorption being attributed to the pteridine ring and

secretion to the p-aminobenzoylglutamate moiety (36). Thus,

bometnexol also has the structural requirements (i.e. , a 5-deaza

pteridine ring and a benzoylglutamate moiety) for bidirectional

transport by renal tubular cells. Indeed, active secretion of

lometrexol was suggested in this study by the finding that the

ratio of unbound renal clearance:GFR (data not shown) was

significantly > I (range, 1 .2 1-2. 15). That a weak linear relation-

ship was observed between the percentage of the lometrexol

dose recovered in the urine and the dose administered could

reflect saturability of lometrexol renal tubular reabsorption. In

comparison. dose-dependent urinary excretion of methotrexatehas also been suggested within a comparatively low dose range

of 25-100 rug (25), but not in high-dose methotrexate treatment

where doses of 50-300 mg/kg are administered (37).

Although systemic metabolism of lometrexol was not stud-

ied, the finding that the majority of the dose was recovered in

urine unchanged and that no metabolite(s) of lometrexol was

detected by HPLC would indicate that the drug does not un-

dergo extensive systemic metabolism at the range of doses

studied. Quinazoline antifolates are also poorly metabolized

(22), while in comparison, the lipophilic nonpolyglutamatable

dihydrofolate reductase inhibitor trimetrexate is found to un-

dergo 95% biotransformation (38).

Folic acid supplementation reduces the toxicity of bome-

trexol, enabling the administration of up to four repeated courses

of treatment (39). The mechanism(s) which underlies modula-

tion of lometrexol toxicity remains to be identified, but the

possibility exists that folic acid may increase the plasma clear-

ance of bometrexol, such that the AUC is lower for a given dose.

However, this would seem unlikely since comparison of plasma

pharmacokinetic parameters for patients receiving 45 mg/m2

lometrexol every 4 weeks, with or without folate supplementa-

tion, showed no marked differences. Unfortunately, samples

were not available to study the urinary excretion and renal

clearance of lometrexol in patients not receiving folic acid.

The disposition of lometrexol administered with a folate

supplement is similar to that of methotrexate in terms of the

kinetics of plasma elimination, extent of protein binding. and

urinary excretion. Plasma elimination of lometrexol was not

markedly different in patients who did not receive a folate

supplement before and during lometrexol administration, and

the improved tolerance of the drug when given with folate

supplementation is therefore unlikely to be due to a pharmaco-

kinetic interaction.

ACKNOWLEDGMENTSWe thank Drs. H. Schmidt (Lilly Research. Brussels, Belgium) and

J. Walling (Lilly Research, Basingstoke, Surrey, United Kingdom) for

supplying bometrexol and C’#{176}-desmethylene bometrexol, and for their

support and encouragement. Thanks are also due to the research nurses,

data managers. and medical staff in the Department of Medical Oncol-

ogy. Newcastle General Hospital. who cared for the patients treated with

bometrexol and obtained samples for the pharmacokinetic study. Finally.the help of Drs. C. Sessa, 0. Pagani, and F. Cavalli in supplying plasma

samples from patients treated without folate supplements is greatly

appreciated.

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1995;1:1479-1486. Clin Cancer Res S R Wedge, S Laohavinij, G A Taylor, et al. with an oral folic acid supplement.dideaza-5,6,7,8-tetrahydrofolic acid (Lometrexol) administered Clinical pharmacokinetics of the antipurine antifolate (6R)-5,10-

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