(CANCER RESEARCH 49, 6359-6364, November 15. 1989]

Dose-dependent Pharmacokinetics of Methotrexate and 7-Hydroxymethotrexate inthe Rat in Vivo1

Roy M. Bremnes,2 Lars SI0rdal,3 Erik Wist, and Jarle Aarbakke

Department of Pharmacology, Institute of Medical Biology fR. M. B., L. S., J. A.], and Department of Oncology, Institute of Clinical Medicine IE. W.j, University ofTroms«,.V-900I Troms«,Norway

ABSTRACT

The pharmacokinetics of methotrexate (MTX) and 7-hydroxymetho-trexate (7-OH-MTX) in bile, urine, and serum was studied in rats in vivoafter short-time infusions of 10, 50, 250, and 1000 mg/kg MTX. Allanimals were anesthetized and drained of bile during experiments. Thebiliary secretion rate of MTX approached saturation when serum MTXlevels surpassed 700-800 MM.causing a significant reduction in biliaryrecovery as the parent compound (49 to 32%) at MTX doses exceeding50 mg/kg. The hepatic metabolism of MTX to the 7-hydroxy metabolitewas not saturated at the doses used. Serum MTX pharmacokineticsdemonstrated dose dependency, inasmuch as doses exceeding 10 mg/kgwere accompanied by a reduced total body clearance (('/,) and biliary

clearance (f //,•).A significant finding in relation to acute hepatotoxicityreported after high-dose MTX in humans was the occurrence of choles-tasis 30-90 min after drug infusion and the observation of macroscopicprecipitations in the bile duct in five of six animals treated with 1000mg/kg MTX. In these five animals, cessation of bile secretion occurredat similar bile 7-OH-MTX levels [9800 ±1100 (SD) »IM],while thesingle rat that secreted bile throughout the experiment had a 5-fold lowerpeak 7-OH-MTX concentration in bile. Analysis of biliary precipitatesformed in vivo and in vitro found 7-OH-MTX to constitute 97% andMTX 3% of the drug content of the precipitated material.

INTRODUCTION

The antifolate agent MTX4 is widely used in cancer chemotherapy, and HD-MTX (1-34 g/m2) infusions are currently

used in several therapy regimens (1). In humans, MTX is inpart metabolized to 7-OH-MTX, which is measured in highconcentrations in the blood after HD-MTX infusions (2-12).

7-OH-MTX has been demonstrated to be cytotoxic and toaffect cellular entry, polyglutamation, and efflux of the parentcompound in vitro (3, 13-17). The limited aqueous solubilityof the metabolite, when compared to the parent drug, hasrendered it a possible mediator of renal toxicity during HD-MTX therapy (18, 19).

There are numerous reports on acute hepatotoxicity afterHD-MTX therapy (20-29) and chronic hepatotoxicity due tolong-term low-dose MTX treatment for acute leukemia andautoimmune diseases (30-44). It has been postulated that thehepatotoxicity of MTX is caused by a toxic metabolite (45, 46),but the underlying mechanisms that cause liver injury to occurafter MTX therapy remain unknown.

Compared to the frequently applied rabbit model for evaluation of MTX metabolism and 7-OH-MTX pharmacokinetics(47-52), data obtained in the rat are in better agreement with

Received 5/24/89; revised 8/16/89; accepted 8/21/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This study was supported financially by the Norwegian Cancer Society and

the Erna and Olay Aakre Foundation for Cancer Research.3 Fellow of the Norwegian Cancer Society. To whom requests for reprints

should be addressed.3 Postdoctoral fellow of the Norwegian Cancer Society. Present address: Me

morial Sloan-Kettering Cancer Center, New York, NY 10021.4 The abbreviations used are: MTX, methotrexate; 7-OH-MTX, 7-hydroxy-

methotrexate: DAMPA, 2,4-diamino-A"°-methylpteroic acid; HD-MTX, high-dose methotrexate; HPLC, high-pressure liquid chromatography.

findings in humans (53-55). Furthermore, the rat seems to bethe only animal model of human chronic MTX hepatotoxicity(56).

Previously, we studied the in vivo 7-hydroxylation in the ratafter 10 mg/kg [3H]MTX (57). Herein we report the dosedependency of MTX and 7-OH-MTX pharmacokinetics aftershort-time infusions of MTX in doses ranging from 10 to 1000mg/kg, which cover the range of HD-MTX doses used in theclinic.

MATERIALS AND METHODS

Drugs and Chemicals. L-Glutamyl-S^-pHjMTX (specific activity,37.1 Ci/mmol; purity, 99.2% by HPLC) was purchased from NewEngland Nuclear, Boston, MA. Unformulated and formulated MTXs(purity, 99% by HPLC) were gifts from Nycomed A/S, Oslo, Norway.7-OH-MTX was a gift from Dr. F. M. Sirotnak, Memorial Sloan-Kettering Cancer Center, New York, NY. Hypnorm vet. (fentanyl, 0.2mg/ml; fluanisone, 10 mg/ml) was from Janssen Pharmaceutical,Beerse, Belgium. Heparin was obtained from Nycomed A/S, andNaHCO3 was from Naflab Hospital A/S, Oslo, Norway. Insta-Gel IIscintillation liquid was from Packard Instruments Co., Groningen, TheNetherlands. Methanol and tetrahydrofuran (both HPLC grade) werefrom Rathburn Chemicals, Walkerburn, United Kingdom. All otherreagents were of analytical grade. All samples containing MTX and 7-OH-MTX were stored protected from light at -20°C for a maximum

of 4 weeks.Animals and Operations. Male Wistar rats weighing 250-370 g

(obtained from Charles River, WIGA GmbH, Sulzseld, West Germany)were used for the experiments. The rats were randomly allocated tofour groups (A, B, C, and D), each of which consisted of 6 animals. Allanimals were anesthetized and drained of bile during the experiments.Under fentanyl (0.3 mg/kg i.p.) anesthesia, all animals had their bileduct and right external jugular vein cannulated as described previously(57).

Experiments. Methotrexate solutions were prepared by dissolvingthe drug in isotonic saline with 0.1 M NaOH to concentrations of 1, 5,25, and 100 mg/ml MTX, thus allowing administration of equalvolumes to all rats. Tritiated MTX was added to an activity of 9.2 nC\/ml at each concentration applied. [3H]MTX doses of 10, 50, 250, and

1000 mg/kg were administered to group A, B, C, and D animals,respectively. The drug was administered as a continuous infusionthrough a central venous catheter for 10 min. The venous catheterswere flushed with heparinized (10 lU/ml) saline immediately afteradministration of drug and after each subsequent blood sampling.Venous samples of 200 n\ were drawn from the catheters as follows.Prior to and immediately after drug administration and subsequently2, 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 300, and 360 min aftercessation of MTX infusion. Bile samples were obtained prior to andduring the period of MTX administration (10 min), during 15-minintervals for the initial 60 min, during 30-min intervals for the next 60min, and during 60-min intervals between 2 and 6 h. Voided urine wascollected from all rats, and upon sacrificing the animals, the urinebladder was aspirated to assure complete collection. pH was measuredin voided urine and bile samples. All animals received maintenancefentanyl anesthesia, approximately 0.08 mg/kg/h, administered as single i.m. injections every hour and were hydrated with 8 ml/kg/h of 0.06M NaHCOj in isotonic saline. Venous blood gas and hematocrit samples were drawn from the venous catheters immediately prior to MTXtreatment and at the end of the experiments.

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7-OH-MTX DOSE-DEPENDENT PHARMACOKINETICS

Analytical Methods. Analysis of MTX and 7-OH-MTX concentrations in serum were performed by reverse phase HPLC as reportedpreviously (57). The assay detects both MTX and its major extracellularmetabolites 7-OH-MTX and 2,4-diamino-./Vlo-methylpteroic acid, withno interference from polyglutamates 1-3 of MTX. Urine and bilesamples were analyzed by fraction sampling and determination ofradioactivity (57). Assuming that radioactivity detection measures100% of MTX in the fractionated samples, the recoveries by HPLCanalysis of bile and urine were 94 ±7 (SD) (n = 195) and 90 ±8 (n =49), respectively, over a concentration range from 1.8 MMto 27 mM.

Calculations. Concentrations of 7-OH-MTX and MTX versus timewere plotted on semilogarithmic graphs. The serum concentrationswere analyzed according to a two-compartment open model. Pharma-cokinetic parameters were obtained by means of linear regressionanalysis in a semilogarithmic data set and refer to the biexponentialequation

C = Ae-"' + Be'*

Total clearance, CIT,was calculated by the equation

DoseCIT =

(AUCo + A/a + B/ß)

where AUC0 is the area under the curve during drug infusion (10 min),calculated by a triangular area. A and B are the zero-time intercepts ofthe extrapolated lines of the a and ßphases, respectively.

Biliary clearance was calculated by the equation

CIB =Bile flow-C«

where CB and Cs are corresponding concentrations in bile and serum,respectively. CBvalues are obtained from bile sampled for 15, 30, or 60min, whereas Cs values are calculated means of serum concentrationsat start and cessation of each bile sample interval. The central volumeof distribution, V„was obtained by dividing the dose by (A + B), andthe apparent volume of distribution in the postdistributional phase, Vß,was calculated by dividing total clearance by ß.

All results are expressed as mean ±SD. Statistical analyses wereperformed by one-way analysis of variance and estimation of leastsignificant distance (Statgraphics; STSC, Rockville, MD). Statisticalsignificance was defined as P < 0.05.

RESULTS

The results obtained after administration of 10 mg/kg MTXessentially reproduce our previous findings at this dose level(Tables 1-4; Figs. 1 and 5) (57).

The bile flow during the first hour of the experiments wassignificantly larger in group C than in groups A and B (Table1). The biliary pH, however, remained constant in all animalsthroughout the experiments. Bile MTX concentrations declinedmonophasically in a semilogarithmic plot (Fig. 1), with a significantly longer half-life in group C, 55.6 min (mean), than in

Table 1 Cumulative volumes of bile and urine excreted in bile-drained animals

The cumulative volume of bile excreted during 1 and 6 h and urine during 6 hin bile-drained animals given 10, 50, 250, and 1000 mg/kg [3H]methotrexate.

Data are given as mean ±SD.

Bile(ml)GroupABCDNoofanimals6665»Dose(mg/kg)10SO25010000-1

h1.2

±0.41.2±0.42.1

-1-0.5°*0-6

h6.2

±1.46.0±1.77.3±2.0"0-6

h3.8

±2.13.9±1.36.0+3.79.3±3.1'

*f< 0.05 between 50 and 250 mg/kg.* Volumes are omitted since bile secretion ceased within 30-90 min in 5 of 6

animals given 1000 mg/kg. One animal that died 30 min after cessation of bilesecretion has been excluded.

' P< 0.05 between 250 and 1000 mg/kg.

Ãœoo

100,000.0-,

10,000.0-

1000.0-

100.0-

10.0-

1.0-

0.1 -

100,000.0•¿�^,

î,10,000.0-

r

2 1000.0-

I; 100.0-

I LO-i

0.1 -

100.000.0-,

10,000.0-

1000.0-

100.0-

10.0-

1.0-

0.1 •¿�

1 62345

TIME(h)Fig. 1. Biliary concentrations of methotrexate (O) and 7-hydroxymethotrexate

(•)versus time following short-time infusions of 10 (A), 50 (B), and 250 (C) mg/kg [3H]methotrexate in rats. All animals were anesthetized. Data are given asmean ±SD, n = 6.

groups A and B. Peak bile MTX concentrations increased as afunction of the administered MTX doses, but in a nonlinearmanner. Whereas a dose escalation from 10 to 50 mg/kgresulted in a mean 180% increase in peak bile MTX levels,dose increments to 250 and 1000 mg/kg were accompanied bystepwise increases of 22 and 18%, respectively. As shown inFig. 2, the biliary secretion rate approached saturation whenserum levels surpassed 700 pM MTX in animals given 250 mg/kg MTX. Furthermore, a significant reduction in biliary clearance of MTX at a dose escalation from 50 to 250 mg/kg led toa correspondingly smaller biliary recovery of unaltered drug(Tables 2 and 4).

In group C animals, 7-OH-MTX concentrations ¡nbile declined monophasically with a 49.7-min (mean) half-life, whichwas significantly longer than in groups A and B (Fig. 1). Asdisposition kinetics seemed to approach an upper thresholdconcerning the ability to secrete and concentrate MTX in thebile, the biliary 7-OH-MTX concentrations increased 4- and 3-fold with dose increments from 10 to 50 and from 50 to 250mg/kg, respectively (Fig. 1). However, in group D (1000 mg/kg) cessation of bile secretion occurred within 30-90 min after

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7-OH-MTX DOSE-DEPENDENT PHARMACOKINETICS

350-1

300-

tc

-f. 250-

1

£20°-

5 150-

Iy 100-

m

50-

0-

500 1000

SERUM MEJHOTREXATE(¡M)

1500

Fig. 2. Biliary secretion rate versus serum MTX concentration in 6 anesthetized rats (each represented by a symbof) given a short-time infusion of 250 mg/kg [3H]methotrexate.

Table 2 Biliary metholrexate recovery as methotrexate and 7-hydroxy-methotrexate in bile-drained rats

Percentage of cumulative biliary recovery of administered (3H]methotrexate asmethotrexate and 7-hydroxymethotrexate in rats during 2 and 6 h after administration of 10, 50, and 250 mg/kg [3H]methotrexate. The animals were anesthe

tized and drained of bile. Data are given as mean ±SD.

GroupA

BCNo.mais6

66Dose(mg/kg)1050250Methotrexate

(%)0-250.3

±42.2 ±25.9 ±h5.6°

7.0*

6.30-652.5

±47.8 ±32.4 ±h5.9

8.1»

9.87-Hydroxymeth-

otrexate(%)0-2

h6.7

±7.3 ±8.3 ±3.0

4.12.00-67.38.49.4±

±±h3.0

4.82.4

°P < 0.05 between 10 and 50 mg/kg.*P < 0.05 between 50 and 250 mg/kg.

100.000 -,

10.000 -

10001

zo

oo

m IOD

01 23456

TIME (h)

Fig. 3. Biliary concentrations of methotrexate (O, A, V, D, O) and 7-hydroxymethotrexate (•,A, T, •¿�*) versus time following a short-time infusion of 1000mg/kg [3HJmethotrexate to 6 anesthetized rats.

MTX administration in 5 of 6 animals at biliary 7-OH-MTXlevels of 9800 ±1100 ^M (Fig. 3). In the single rat that secretedbile throughout the experiments a 5-fold lower bile concentration of the metabolite was detected. Furthermore, only thecholestatic animals demonstrated crystalline precipitates in thebile duct when laparotomized at the termination of experiments. Additionally, the bile sampled prior to cessation of bilesecretion was found to contain precipitates. Equal amounts of

MTX and 7-OH-MTX were removed during the total washingprocedure. More MTX than 7-OH-MTX was removed duringthe first washing with NaCl, while more 7-OH-MTX wasremoved during the second. Of the total MTX and 7-OH-MTXcontent in the pellets, 97.2 ±0.9% (N = 6) was 7-OH-MTX(Fig. 4). The in vivo precipitation of 7-OH-MTX in bile wasreproduced in vitro when the 7-hydroxylated metabolite and theparent compound, both at a concentration of 9000 ¿IM,wereadded to freshly collected rat bile. Of the drug content in theresulting precipitate, 97.1% was constituted by 7-OH-MTX.

The urinary recovery of unaltered drug and nonbiliary (urinary and metabolic) clearance remained unchanged at doseincrements (Tables 3 and 4). Whereas the MTX recovery as the7-hydroxy metabolite in urine (Table 3) and the volumes ofurine voided (Table 1) were significantly larger in animals ofgroup D compared to those in groups A-C. In all animals,urine pH increased slowly as a result of alkalinization withNaHCOj. Only group D animals reached pH 7 within the 6-h

experimentation period.In serum, the MTX concentrations declined rapidly in each

group of animals over the initial 10 min, followed by a slowersecond phase (Fig. 5). The pharmacokinetic variables are givenin Table 4. Doses exceeding 10 and 50 mg/kg MTX led to adose-dependent decline in total body clearance (CIT) and biliaryclearance (CIB). Animals in group D demonstrated the longest/./,0and largest Vcand Vf. The peak serum 7-OH-MTX concentrations in group D animals (300 ±210 ßM),which appearedafter onset of cholestasis in 5 of the rats, were considerablyhigher than in the other groups (means, 0.34-16.8 /¿M).Afterreaching peak levels, serum 7-OH-MTX declined monophasi-

8-

7-

6-5-4-3-2-1-

0-iiI i9LE SUPERNATANT PRECIPITATE:

Fig. 4. Five of 6 rats given 1,000 mg/kg [3H]MTX developed cholestasis, in

which precipitates were observed in the bile ducts and bile samples. Shown arethe amounts of methotrexate (D) and 7-hydroxymethotrexate (•(in supernatants,precipitates, and the in vivobile samples from which they derive. The supernatantsand precipitates were prepared in the following way. Supernatants were siphonedoff after centrifugation of bile samples at 10,000 x g for 10 min. Pellets werethen washed twice in NaCl, and supernatants and precipitates were resuspendedto the initial volume in 0.5 M NaOH. Data are given as mean ±SD, n = 5.

Table 3 Urinary methotrexate recovery as methotrexate and7-hydroxymethotrexate in bile-drained rats

Percentage of cumulative urinary recovery of administered [3H]methotrexateas methotrexate and 7-hydroxy-methotrexate in rats during 6 h after administration of 10, 50, 250, and 1000 mg/kg. The animals were anesthetized and drainedof bile. Data are given as mean ±SD.

No. of Dose Methotrexate (%),Group animals (mg/kg) 0-6 h

7-Hydroxymethotrexate(%), 0-6 h

ABCD6665°10SO250100027.4 ±4.623.1±6.030.7

-t-10.830.8±14.20.53

±0.410.07±0.010.06+0.063.7±2.3*

" One animal that died 30 min after cessation of bile secretion has been

excluded.* P < 0.05 between 250 and 1000 mg/kg.

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7-OH-MTX DOSE-DEPENDENT PHARMACOKINETICS

Table 4 Pharmacokinetic variables in rats given infusions offHJmethotrexate atfour dose levels

The rats were given 10-min infusions through a jugular vein at four differentdoses ranging from 10 to 1000 mg/kg. All animals were anesthetized and drainedof bile. Data are given as mean ±SD.

GroupNo.

ofanimalsDose(mg/kg)(Hn(min)tv,ß(min)V,(ml/kg)y,(ml/kg)Or

(ml/min-kg)Cl,(ml/min-kg)CINB

(ml/min •¿�kg)A6104.2

±0.639.5±3.028877613.69.957197r2.9'1.93.7

2.5B6504.1

±0.936.4±7.2245±43511±17110.1

±3.87.5±3.0*2.6

±1.7C62503.9

±1.041.7±7.7230±42417±

1126.9±1.13.4±0.43.5±0.6D5°10003.9

±1.2154±96*387

±59*798±138*5.0

±3.01.9±2.23.1±1.7

°One animal that died 30 min after cessation of bile secretion has been

excluded.*P < 0.05 between 250 and 1000 mg/kg.' P< 0.05 between 10 and 50 mg/kg.dP< 0.05 between 50 and 250 mg/kg.' ClNt, is nonbiliary clearance (Clm = CIT —¿�CI,).

cally with significantly longer half-lives (241 ±224 min) ingroup D animals than in groups A-C (means, 42-89 min).

At the end of the experiments, venous pH levels were 7.33-7.39 (means) in the groups, with the highest values measuredin the animals given the lowest dose of MTX. The hematocritvalues were reduced by 26.1% (mean) during the experimentsin group D animals, whereas a somewhat smaller reduction(mean, 21.2%) in hematocrit was observed in the other groups.

DISCUSSION

This is the first report concerning precipitation of 7-OH-MTX in the bile of any species. It has previously been established that the 7-hydroxy metabolite is highly insoluble inaqueous solutions and 7-OH-MTX has been found to precipitate in the renal tubules of monkeys (18, 19). Herein, we havedemonstrated that the metabolite precipitates at high concentrations in the alkaline rat bile, presumably causing the choles-tasis observed in rats treated with 1000 mg/kg MTX. This dosecorresponds to a dose of 33 g/m2 in an average 8-year-old child.Since doses up to 33.6 g/m2/24 h have been used in HD-MTX

treatment of children with acute lymphoblastic leukemia (9,58-60), these findings may be of clinical relevance. MTX dosesof this magnitude are administered as 24-h infusions in humansrather than short-time infusions as in our animal model. Evenso, peak serum concentrations of 7-OH-MTX have been detected at 337 UM (9) which exceed our measurements in ratsgiven the equivalent dose of MTX.

There have been several reports on acute hepatotoxicity withelevated serum aspartate aminotransferase and alanine aminotransferase after HD-MTX treatment in humans (20-29). Inone patient who developed serious acute hepatotoxicity, Brei-thaupt and Kuenzlen (27) found inconspicuous plasma MTXkinetics while the 7-OH-MTX kinetics deviated compared tothis patient's previous MTX courses without observed hepato

toxicity. It has further been postulated that hepatotoxicity afterMTX is caused by a toxic metabolite of MTX (45, 46), but theunderlying mechanism remains unknown. Our results may indicate that 7-OH-MTX is implicated in acute hepatotoxicityfollowing HD-MTX therapy in humans.

Saturation of biliary MTX secretion at serum levels surpassing 700-800 fiM MTX is consistent with a previous investigation by Kates and Tozer (61). The biliary transport systemdisplays Michaelis-Menten-type kinetics at high dose levels ofMTX. In rats infused with 250 mg/kg MTX the maximal rateof transport (7"max)and the transport constant analogous to the

10,000.00v

> 1000.00

100.00

10.00

LOO

0-10

0.0110,000.00

1000.00

100.00

10.00

1.00

0.10

0.0110,000.00

1000.00

100.00

10.00

1.00

0.10

0.0110,000.00-1

1000.00-

100.00-

10.00-

1.00-

0.10-

0.01-

B

0123456

TIME(h)

Fig. 5. Serum concentrations of methotrexate (O) and 7-hydroxymethotrexate(•)versus time following short-time infusions of 10 (A), 50 (B), 250 (C), and1000 (D) mg/kg [3H]methotrexate. Data are given as mean ±SD, n = 6. A single

rat of group D that ceased bile secretion within 30 min after MTX administrationdied 30 min later.

Michaelis constant (KT) were 20.7 mg/h and 411 ^M (means),respectively, somewhat higher than the findings of Kates andTozer (61), 12 mg/h and 70 ^M. Our experiments, however,were not carried out under steady state conditions which areessential for investigations of Michaelis-Menten kinetics, asdiscussed by Kates and Tozer (61). The saturation of transportexplains the disproportional increase of peak MTX concentrations in bile and the significant reduction in biliary clearance ofthe drug at MTX doses beyond 50 mg/kg, the significantlylonger biliary MTX and 7-OH-MTX half-lives following 250mg/kg MTX, and the successively smaller biliary MTX recoveries with dose increments. However, the hepatic metabolism

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of the parent compound showed no signs of saturation, as peak7-OH-MTX concentrations in bile increased almost proportionally with the dose administered with no reduction in biliaryMTX recovery as the 7-hydroxylated metabolite. In comparisonto our previous study of bile-drained rats given 10 mg/kg MTX(57), biliary recovery of 7-OH-MTX after this MTX dose was2-fold larger in the present study. This difference may be dueto variations between two different strains of Wistar rats, sincethe animals used in our previous study were obtained fromanother source. The 75% larger bile flow within the first hourof the experiments in animals treated with 250 mg/kg MTXmay be explained by osmotic choleresis, i.e., increase in bileflow, due to high concentrations of the anionic MTX and 7-OH-MTX moieties present in bile (62, 63).

Despite saturable bile secretion at high serum concentrationsof MTX, there were equal nonbiliary clearance and urinaryrecovery rates of the parent compound at all dosage levels,which corroborate with similar urinary recoveries reported intwo previous studies in rats treated with 10 and 250 mg/kgMTX, respectively (57, 64). The significantly higher recoveryof 7-OH-MTX in urine after administration of 1000 mg/kgMTX is due to significantly increased serum levels of themetabolite, which may be explained by cholestasis-related cellular injury and the resulting leakage of the hepatic metaboliteinto sinusoidal blood. The increased urine volume excretedafter 1000 mg/kg MTX may result from retention of fluidwhich would otherwise be eligible for biliary secretion.

Serum MTX elimination in rats given 10 mg/kg was tri-phasic, which is consistent with previous investigations (57,64-66). However, the pharmacokinetic variables in all animalswere analyzed according to a two-compartment model, sinceserum MTX disappearance was biphasic after MTX dosessurpassing 10 mg/kg. The apparent central volumes of distribution (Vc)and the total clearance (CIT) in animals infused with10 mg/kg MTX remained unaltered regardless of whether theywere analyzed according to a two- or three-compartment modeland were in concordance with previous studies in rats given 1or 10 mg/kg MTX (57, 67). The experiments gave evidence ofa dose-dependent decline in total body clearance (CIT) of MTX,mainly due to a reduction in biliary clearance (CIB) as animalswere given larger doses of the drug (Table 4). Central volumesof distribution in rats receiving 10 to 250 mg/kg MTX were,however, equal and consistent with previous studies (57, 67),whereas a significantly larger Vcwas found in rats given 1000mg/kg. The considerably longer terminal MTX half-life, largerKg,high peak 7-OH-MTX concentration, and long serum half-life of the metabolite after 1000 mg/kg MTX are consideredresults of cholestasis and the ensuing cessation of drug elimination by bile.

In summary, biliary excretion of MTX approaches saturationat MTX doses exceeding 50 mg/kg, whereas 7-OH-MTX formation and excretion do not. High bile 7-OH-MTX concentrations lead to in vivo cholestasis, presumably due to precipitationin bile where 7-OH-MTX constitutes 97% and MTX 3% of thedrug content in vivo and in vitro. The results indicate that 7-OH-MTX may be implicated in acute hepatotoxicity reportedafter HD-MTX therapy in humans, and they suggest that theremay be an upper limit of MTX doses regardless of leukovorinrescue.

ACKNOWLEDGMENTS

REFERENCES

The authors gratefully acknowledge the excellent technical assistanceby R. Jaeger and H. S0rensen.

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in Vivo7-Hydroxymethotrexate in the Rat Dose-dependent Pharmacokinetics of Methotrexate and

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