[CANCER RESEARCH 44, 4172-4176, September 1984]

Development and Application of a Sensitive Enzyme Immunoassay for7-A/-(p-Hydroxyphenyl)mitomycin C1

Kunio Fujiwara,2 Hideki Nakamura, Tsunehiro Kitagawa, Noriaki Nakamura, Atsushi Saito, and Kohei Hará

Faculty of Pharmaceutical Sciences, Nagasaki University, Bunkyo-machi 1-14 [K.F.. H.H., T.K.] and Second Department of Internal Medicine, School of Medicine,Nagasaki University, Sakamoto-machi 1-14 [N.N., A.S., K.H], Nagasaki 852, Japan

ABSTRACT

An antibody specific for 7-A/-<p-hydroxyphenyl)mitomycin C (M-

83) was developed and used in a simple and sensitive enzymeimmunoassay for detection of this anticancer drug in serum. Theimino group of M-83 was covalently coupled to mercaptosuccinylgroups introduced into bovine serum albumin with a cross-linker,/V-maleoylaminobutyric acid. The resulting conjugate was thenused for the production of a specific antibody to M-83 in rabbits.Enzyme labeling of M-83 was performed using 0-D-galactosidase(EC 3.2.1.23) via m-maleoylbenzoic acid. Antibody production

was of sufficiently high titer in rabbits to allow the developmentof a highly sensitive enzyme immunoassay for the free drugwhich accurately measures 15 pg of M-83 per assay tube. Thisassay was monospecific to M-83 and showed almost no cross-

reactivity with a variety of other mitomycin analogues (mitomycinA, B, and C; porfiromycin; and acetylmitomycin C). Using thisassay, preliminary pharmacokinetic study was undertaken usingserial serum samples obtained from a patient who received thedrug i.v., revealing a biphasic fashion of the kinetics, with an a-serum half-life of 9.7 min and a /3-serum half life of 80 min. Ani.v. injection of M-83 into rats and assay of serum concentrationrevealed a similar biphasic response with an a-serum half-life of8.3 min and a /3-serum half-life of 62 min.

INTRODUCTION

M-833 is a derivative of MMC with higher antitumor activity

against Hirosaki ascite sarcoma (18) and P388 ascite leukemia(8, 9,15) and lower bone marrow toxicity (12) as compared withMMC. These advantages may allow M-83 to have an antitumor

effect superior to that of the present MMC, and the drug hasbeen on clinical trial in Japan.

It is considered that dosing of anticancer drug for clinicalefficacy is aimed at achieving those concentrations of the drugwithin body fluids which selectively inhibit tumor cell growthwithout producing side effects. Thus, pharmacokinetic data willbe useful for its optimal clinical usage. Previous pharmacokineticstudies of M-83 were undertaken by a laborious microbiological

assay technique that lacks sensitivity and specificity or by theuse of the 14C-labeled drug (16), thus using radioactivity as an

indirect measure of the drug itself.Quite recently, we developed an EIA for MMC, parent com-

1This work was partially supported by Cancer Research Grant 57015079 from

the Ministry of Education of Japan.2To whom requests for reprints should be addressed.3The abbreviations used are: M-83, 7-AI-(p-hydroxyphenyl)mitomycin C; MMA,

MMB, and MMC, mitomycin A, B, and C, respectively; Ac-MMC, acetylmitomycinC; EIA, enzyme immunoassay; MABA, N-mateoylaminobutyric acid; MBA, m-maleoylbenzoic acid; MSBSA, mercaptosuccinyl bovine serum albumin; BSA, bovine serum albumin; M-83-0-Gal, 7-N-<p-hydroxyphenyl)mitomycin C-0-D-galacto-

sidase conjugate.Received February 13, 1984; accepted June 6, 1984.

pound of M-83, using the bifunctional cross-linking agents MABAand MBA for preparing the immunogen and for the /3-o-galacto-

sidase labeling of the drug, respectively (5, 6). The assay washighly sensitive and specific to MMC, and it hardly detected itschemical derivative M-83.4

This paper describes the methodology for the antibody production, the labeling of M-83 with /3-o-galactosidase to act as a

tracer, the initial characterization of antibody specificity, and thetechnique developed for the measurement of M-83 by EIA. Initialapplication of the assay to measure M-83 levels in a patient and

rats demonstrates its utility in the assessment of basic pharmacokinetic distribution.

MATERIALS AND METHODS

Materials. M-83, MMA, MMB, and MMC were obtained from KyowaHakko Kogyo Co., Ltd., Tokyo, Japan. Ac-MMC and porfiromycin wereprepared according to the method of Matsui ef al. (13). /3-o-Galactosidase

(EC 3.2.1.23) from Escherichia coli was obtained from Boehringer/Mannheim, Mannheim, Germany. MABA and MBA were synthesized bythe methods of Rich ef a/. (17) and Kitagawa and Aikawa (11), respec

tively.Introduction of Maleimide Group into M-83. M-83 was acylated with

MABA through an imide bond by the mixed carbonic anhydride methodof Anderson ef a/. (1). A solution containing 1 mg (5.46 Mmol) of MABAand 0.7 n\ of N-methylmorpholine in 500 n\ of tetrahydrofuran (freshlydistilled off calcium hydride) was cooled at -5° and mixed with 1 u\ of

isobutyl chloroformate under vigorous stirring. Ten min later, 2.33 mg(5.46 ^mol) of M-83 dissolved in 1.0 ml of tetrahydrofuran were furtheradded, and the reaction mixture was stirred in the dark for 15 hr at 4°.

After the solvent was removed under vacuum, 5 ml of ethyl acetate and5 ml of 0.33 M citric acid were added to the residue, and the mixturewas shaken vigorously. The water layer was discarded, and the ethylacetate layer was further washed twice with the citric acid, subsequently3 times with 1 N sodium bicarbonate, and finally with saturated sodiumchloride. The ethyl acetate solution was dried over anhydrous sodiumsulfate and was concentrated under vacuum. The resulting MABA-

acylated M-83 gave a single spot when chromatographed on a Silica Gel

60F2M plate.Coupling Reaction for Hapten. The preparative process was similar

to our previous method of synthesizing immunogen for MMC (6). Acetyl-

MSBSA (10 mg), containing 17 acetylmercaptosuccinyl groups per BSAmolecule, was incubated in 0.2 ml of freshly prepared 0.1 M hydroxyl-amine, pH 7.2, at 25°for 30 min to remove the protecting acetyl group.

After dilution with 2.0 ml of 20 mw phosphate buffer, pH 7.0, containing3 M urea, the resulting MSBSA was added immediately to the MABA-

acylated M-83 in 50 n\ of tetrahydrofuran and then incubated for 30 min

with vigorous stirring. The mixture was chromatographed on a columnof Sephadex G-100 (2.8 x 42 cm) with 0.1 M phosphate buffer, pH 7.0,containing 3 M urea. Then, the purified conjugate was examined spectrc-photometrically and was estimated to contain about 3.4 molecules of M-83 per BSA molecule assuming the molar extinction coefficients of M-83

' Unpublisheddata.

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EIAofM-83

to be 6,000 at 280 nm and 11,800 at 380 nm and those of BSA to be43,600 at 280 nm.

Antibody Production in Rabbits. An aliquot, containing about 1.0 mgof the M-83-BSA complex, was emulsified with an equal volume ofFreund's complete adjuvant. Three white female rabbits received the

complex by i.m. and s.c. injections. This was followed 3 times by biweeklybooster injections of 0.5 mg of the complex. The rabbits were bled froman ear vein 2 weeks after each injection. The samples of serum wereseparated by centrifugation, heated at 55° for 30 min, and stored at-30°.

These serum samples were used to examine the levels of anti-M-83antibody produced by the following procedure. The M-83-0-Gal conjugate

prepared in the next section was incubated with solutions of antiserumfor 14 hr. This was followed by incubation for 4 hr with the secondantibody (goat anti-rabbit IgG and normal rabbit serum). The uptake ofM-83-/3-Gal in the precipitate was measured as a quantitative index of

bound antibody (6). As a result, the antibody titer in the serum increasedgradually with the booster injections and peaked 2 weeks after the finalinjection (data not shown).

Preparation of M-83-/9-Gal. Labeling of M-83 with .(-o-galactosidasewas performed by the help of MBA as a bifunctional cross-linker whichmight avoid a cross-reaction with the antibody produced against theimmunogen linkage group of MABA (14). MBA-acylated M-83 was synthesized from MBA (1 mg; 4.61 ¿imol)and M-83 (2.13 mg; 5 /»mol)andpurified by what is essentially a repetition of the procedure for MABA-acylated M-83. The MBA-acylated M-83 (approximately 5 Mg; 8.0 nmol)in 50 n\ of tetrahydrofuran was added directly to a solution of ß-o-

galactosidase (78 ^g; 0.14 nmol) (3) in 1.0 ml of 0.1 M phosphate buffer(pH 7.0), and incubated for 30 min with stirring. The mixture was thenchromatographed on a column of Sepharose 6B (2 x 38 cm) with 20 mMphosphate buffer, pH 7.0, containing 0.1 M NaCI, 1 HIM MgCI2, 0.1%BSA, and 0.1% NaN3 (Buffer A). The elution profiles of the enzyme andimmunoreactive enzyme activities are shown in Chart 1, revealing thatthe immunoreactive enzyme activity of the conjugate was eluted inparallel with the pure enzyme activity; however, the immune specificityof the conjugate, defined as the immunoreactive enzyme activity in thepresence of free M-83 (36.8 ng), is better differentiated from M-83 in the

latter peak fractions than in the early peak fractions. Fractions 16 to 18of the main peak were thus chosen for the EIA. The conjugate wasstable for within 3 months in Buffer A at 4°without any loss of immu

noreactive enzyme activity. The Km of the substrate (7-/3-o-galactopyra-nosyloxy-4-methylcoumarin) against the M-83-|8-Gal conjugate was

found to be 0.50 HIM, which was slightly larger than that of the pureenzyme (0.33 mM).

Measurement of /3-o-Galactosidase Activity. Fifty n\ of diluted enzyme solution were incubated with 0.15 ml of 0.1 mM 7-/3-o-galactopy-ranosyloxy-4-methylcoumarin in Buffer A at 30°for 1 hr. The reaction

was stopped by the addition of 2.5 ml of 0.2 M glycine-NaOH buffer (pH10.3), and the 7-hydroxy-4-methylcoumarin liberated was measured

spectrofluorometrically at wavelengths of 365 nm for excitation and 448nm for emission. The amount of the conjugate was expressed in unitsof /3-D-galactosidase activity, defining 1 unit of the enzyme activity as the

amount that hydrolyzes 1 ^mol of the substrate per min.EIA Method. The assay was performed by the double antibody

method (19). An antiserum, M-83-(i-Gal, and unlabeled M-83 were diluted

in Buffer B (0.06 M sodium phosphate, pH 7.4, containing 0.01 M EDTA,0.1% BSA, and 0.1% NaN3). Fifty Mlof M-83-/3-Gal conjugate (60 fiunits),50 fi\ of M-83 or sample, as appropriate, and 50 ¿ilof a 1/620,000

solution of the antiserum were mixed, giving a final reaction volume of150 ¿il,and the mixture was incubated at 25°for 14 hr. Then, 50 n\ of a

10% solution of goat anti-rabbit IgG and 50 ¿dof a 0.33% solution of

normal rabbit serum were added. After further incubation for 4 hr, theimmune precipitate was washed twice by the addition of 1.0 ml of BufferA and centrifuged at 2500 rpm for 15 min in a refrigerated centrifuge.The supernatant was decanted, and the enzyme activity in the immuneprecipitate was measured.

Pharmacokinetic Analysis of M-83. The human subject studied was

800

8 IO IS 20 28 30

FRACTION NUMBER (3.0 ml/tub«)Chart 1. Chromatogram of M-83-.i-Gal conjugate on a column of Sepharose

68. •,enzyme activity of the conjugate, measured by the use of 5 n\ from eachfraction at 30°for 5 min; •,immunoreactive enzyme activity, measured by EIA asdescribed in •Materialsand Methods" except that 5 ^l of the conjugate and a 1/

1000 solution of anti-M-83 serum were used in the absence of M-83; O, immunespecificity, determined by the same manner in the presence of 36.8 ng of M-83.

a 65-year-old male patient receiving treatment for cholangiocarcinoma atthe Medical College of Nagasaki University. The patient received M-83by i.v. bolus at 20 mg M-83 per body (57.5 kg). Serial samples of serum

were obtained via an i.v. catheter from the opposite arm from which thedrug was administered. Seven ml of blood drawn prior to dosing (Time0) and 2 ml each at 1,3, 5,15, 30, 60,120,180, 240, and 300 min werecentrifuged at 2500 rpm for 15 min, and the serum was separated andstored at -30°. Analysis of blood levels of M-83 was performed using

50 /¿Ieach of those sera diluted 2.5, 10, or 100 times with Buffer B, asdescribed above. The unknown amount of M-83 present in the serum

was quantitated by interpolation on the linearized standard curve performed with serum obtained prior to drug administration.

Two male Wistar rats, each weighing 250 g, received M-83 at a dose

of 1.8 mg/kg via the femoral vein. Serial blood samples were collectedat 3, 10, 30, 60, 120, 180, 240, and 300 min from the same femoralvein. Serum analysis was the same as described previously for thehuman subject.

RESULTS

M-83 was coupled to BSA with the intention of producing aspecific anti-M-83 antibody. The coupling procedure is based onthe reaction of a maleimide group coupled to a hapten and asulfhydryl group introduced into a BSA by thiolation accordingto our previous method (6). However, the acylation reaction ofM-83 with MABA was slightly modified and done by a mixedcarbonic anhydride method, and the resulting MABA-acylatedM-83 was extracted in acidic pH and basic pH with ethyl acetateseparating unreacted MABA and M-83. This process of purification of MABA-acylated M-83 was important for the subsequentconjugation reaction with the thiol groups of MSBSA to preparea homogeneous conjugate of M-83-MABA-BSA, not giving unwanted MABA-BSA conjugate. After gel filtration of the M-83-MABA-BSA conjugate on Sepahdex G-100, it was determined

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K. Fujiwara et al.

that 3.4 mol of M-83 bound each mol of BSA.The M-83-BSA conjugate elicited the production of anti-M-83

antibody in each of 3 rabbits, the highest titer of which was seen8 weeks after the initial inoculation. The dilution curves of thisantiserum were prepared to decide its concentrations suitablefor EIA. As was shown in Chart 2, the binding potency ofantiserum to M-83-/3-Gal conjugate was relatively high in theabsence of M-83 and decreased gradually with increasing dilu

tions, and it was better inhibited by a fixed amount of nonlabeledM-83 (36.8 ng), especially in rather higher diluted antiserum. The

antiserum was thus decided to use an initial dilution of approximately 1/620,000 with a specific binding value in the absence offree drug of 32%. Using this concentration of antiserum, anoptimal incubation time for the immune reaction of anti-M-83antibody with the labeled M-83 (in the absence of unlabeled M-

83) and subsequently with a second antibody was examined(Chart 3). The first-step and the second-step immune reactions

almost reached the equilibrium in 14 and 4 hr, respectively, at25°.Thus, we chose these incubation times for EIA.

IO5 K> O5 (O6

DILUTION (fold)Chart 2. Dilution curves of anti-M-83 antiserum. Several dilutions of the anti-

serum collected 8 weeks after the initial inoculation were prepared with Buffer B,and 50 nl each were incubated with 50 p\ of the M-83-fi-Gal (60 nunits) (7")for 14hr at 25°.This was followed by further incubation for 4 hr with the secondantibody(goat anti-rabbit IgG and normal rabbit serum). The uptake of M-83-/3-Galin theprecipitate was measured (B) and expressed as B/T (•).Inhibitionexperiments forbinding on the antibody were carried out in the presence of 36.8 ng of free M-83by the same manner (O).

Chart 3. Time dependence of the immune reaction of anti-M-83 serum with M-83-iJ-Galand with the second antibody. ».diluted anti-M-83 serum (1/620,000)incubated with M-83-/J-Gal (60 Munits) at different time intervals (the first-stepreaction), followed by 4-hr incubation with the second antibody (the second-stepreaction);O, first-step immune reaction fixed at 14 hr, with the periods of second-step reaction varied. For definition of B/T, see legend to Chart 2.

A dose-response standard curve of M-83 obtained in a buffer

system is shown in Chart 4. The curve was essentially linear ona logit-log plot between 6 pg and 3 ng. The lowest detectableamounts of M-83 which were significantly different from zero

varied 1 to 10 pg. For practical purposes, the lower and upperlimits of sensitivity were arbitrarily set at 15 pg and 2 ng perassay tube, respectively. Intraassay precision, defined as theratio between the standard deviation of Y and the slope of theregression line between mean values of logit Y and the corresponding log X, was X = 0.12, 0.040, and 0.082 at 15.5 pg, 140pg, and 1.26 ng, respectively, whereas interassay precision (X =0.131, 0.052, and 0.088 at each of the same doses as above in6 different assays) was also indicative of a reproducible EIAtechnique.

Quantification of the drug in samples of normal human serum(50 p\) was also performed by adding known concentrations. Thestandard curve yielded was essentially linear and parallel withthat in the buffer system (Chart 4). For practical purposes, thelower and upper limits of the sensitivity were arbitrarily set at 30pg and 7 ng per assay tube, respectively. Intraassay precision(X = 0.096, 0.042, and 0.073) and interassay precision (X =0.139, 0.055, and 0.086; n = 6) were obtained at 28.7 pg, 259pg, and 2.33 ng, respectively. The presence of 50 /J serumslightly reduced the inhibiton of M-83. Thus, instead of obtaining50% inhibition with 150 pg of M-83, 400 pg were required. Thespecific binding (B°)was the same as that in a buffer system.

A standard curve was also constructed with 5 and 20 n\ ofnormal human pooled urine added. Urine had no effect on theshape of the standard curve or the percentage of binding.

The change in immunoreactivity of M-83 following incubationin normal human serum or urine was examined. Known concentrations of M-83 were incubated in serum or urine for up to 24hr at 37°, and aliquots of the mixture were periodically with

drawn, diluted 100 times with Buffer B, and measured by EIAusing the buffer system. By both incubations, no appreciable

3.0

2.0

1.0

à 0.0

-1.0

-2.0

-3.0' ' '""' ••I

9 10 100 1000 10000

M-83/tub«iS.D. (pg)

Chart 4. Dose-response standard curves for EIA for M-83 in the buffer system(A)or the serum system (B). The response, Y, to the dose, X, can be defined as alogit function of Y so that logit Y = In (V/1 - Y). For experimental purposes, Y isfurther defined as the ratio between antibody-bound enzyme activity (B) and theenzyme activity bound at zero concentration of unlabeled M-83 (B°).Thus, theresponse logit B/B°to dose log M-83 concentration is plotted. Each point represents the mean for 6 replicates; oars, S.D.

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EIA of M-83

Charts. Standard curve for M-83 and cross-reactivity ofsome mitomycin analogues with anti-M-83 serum. The curvesshow the amount (percentage) of bound enzyme activity forvarious doses of M-83 or the mitomycin analogues (8) as aratio of that bound using the M-83-o'-Gal alone (B°).Antiserum

used had been collected 8 weeks after starting immunization.»,M-83; O, Ac-MMC; D, porfiromycin; A, MMC; •,MMA; A,

MMB.

10° tor r io3 io4 io8

UNLABELED DRU6 (pg/lubt)

K)6

decrease in M-83 immunoreactivity was caused with an almost

complete recovery.Since several mitomycin analogues have been identified (7,

10,13), we sought to characterize a specific part of the moleculewhich contains the antigenic determinant. Calibration curvesshowing the relationship between concentrations of unlabeledagents and the percentage of bound M-83-ß-Galwere plotted

(Chart 5). When MMA, MMB, MMC, or porfiromycin was substituted for unlabeled M-83 in the EIA, it can be seen that very littlecompetition with M-83 was observed. Ac-MMC, which pos

sesses the acetyl group on the aziridine moiety of MMC, produced only very slight cross-reaction of 0.017% at the amount

of the drug for a 50% inhibition.Initial application of the EIA was made to measure M-83 levels

in serum of a patient with cholangiocarcinoma following administration of M-83 in a single dose of 0.35 mg/kg i.v. The disappearance of M-83 immunoreactivity from the sera is given inChart 6. Results were analyzed according to a 2-compartment

model following an i.v. bolus injection. Estimated from the curve,the «-phaseserum half-life was 9.7 min, whereas the /3-phasehalf-life was 80 min.

Animal experiments were also designed to follow the serumlevels of M-83 by the EIA. Two rats were given 1.8 mg of M-83/

kg by rapid injection into the femoral vein. The results in Chart 7show a similar biphasic fashion of the kinetics with a rapid initialclearance (distribution) followed by a slower elimination phase.The rat a-phase serum half-life was 8.3 min, whereas the ß-phase half-life was 62 min as an average of 2 rats. Because the

sensitivity of the assay was known to be at least 1.4 pmol/ml, itwould appear that additional values beyond the 5-hr time point

are possible to obtain.

DISCUSSION

Numerous analogues of MMC have been prepared in the hopeof obtaining compounds with improved therapeutic properties(2), since the toxicity of MMC, particularly its myelosupressiveeffect, has prevented it from gaining wider and more rapidacceptance in cancer chemotherpay. M-83 has recently emerged

as a clinical agent with such a hopeful possibility (8, 9, 12, 15,18) and has been undergoing clinical trial in Japan.

Nonisotopic EIA for the quantification of M-83 will contribute

extensively to an understanding of the pharmacology.A heterobifunctional cross-linker, MABA, was used to prepare

the immunogen as could be done in the EIA for MMC (6). In

O I234S6TIME AFTER INJECTION (hr)

Chart 6. Concentrations of M-83 in serum of a patient with time after the initialdose. Samples of serum obtained at various times after M-83 injection wereassayed using EIA as described in "Materials and Methods.* Data represent the

average of triplicate samples with the mean coefficient of variation for all humanpoints of 5.2%.

100

Õ—»•

S

m 0.01 -

0.001I 23456

TIME AFTER INJECTION (hr)Chart 7. Concentrations of M-83 in serum of rats with time after the initial dose.

Two rats, each weighing about 250 g, were given injections of 1.8 mg of M-83 perkg. The assay was done by the same manner as that for samples of human serum.The mean coefficient of variation for all points on the rat curves was 6.2%.

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order to obtain a homogeneous preparation of M-83-BSA conjugate which would be expected to produce a sensitive andspecific antibody directed toward the drug, we modified theprocedure slightly so that MABA-acylated M-83 prepared by amixed carbonic anhydride method was separated from the non-reacted MABA and the hapten drug by extracting with ethylacetate, and this was followed by reaction with a carrier protein,MSBSA. The M-83-BSA conjugate thus synthesized elicited inrabbits the production of a considerably high titer of anti-M-83antibody, such that the 620,000-fold diluted antiserum still boundabout 30% of the added M-83-/3-Galin control tubes containingno labeled M-83 and made possible a sensitive assay for M-83.

The present extraction technique in synthesizing M-83-/3-Galusing MBA as a cross-linker led also to a preparation of theconjugate which rather better competed with a small amount offree M-83 for binding on the antibody than did that preparedwithout the extraction processes, giving a steeper dose-response curve in the EIA (data not included). Besides, once MBA-acylated M-83 is prepared, the conjugation with /3-o-galactosid-ase and the subsequent Chromatographiepurification are easy,rapid, and safe to perform within 1 day, and the resulting enzymeconjugate was found to be stable within 3 months in Buffer A.

Using both anti-M-83 antiserum and M-83-ß-Ga\conjugate, itis noted that a highly sensitive EIA for M-83 was developedwhich allows the reproducible measurement of as little as 15 pgof the drug per assay tube. The sensitivity is about 21 times thatof the previously reported EIA for MMC at the amount of thedrug required to inhibit immune binding by 50% [150 pg of M-83; 3.2 ng of MMC (6)]. This assay was quite specific to M-83and does not detect MMC analogues (MMA, MMB, MMC, Ac-MMC, and porfiromycin). Among them, Ac-MMC was only minimally reactive with the anti-M-83 antibody with a cross-reactivityvalue of 0.017%. This cross-reactivity might suggest the antibody recognition for the sites substituted by an acetyl groupthrough an imide bond on the aziridine ring of MMC. This mightbe reasonable since anti-M-83 antibody was produced againstM-83-BSA conjugate in which M-83 is bound by peptide bondsvia the imino group of M-83.

It was also found that with this EIA no cross-reactivity existedwith other anticancer drugs (Adriamycin, daunomycin, 5-fluoro-uracil, and actinomycin D), indicating that the EIA for M-83 isappropriate for use in clinical evaluation of combination chemotherapy since anticancer drugs are often used with other chem-otherapeutic agents.

The utility of the EIA was demonstrated with its application tomeasure serum levels of M-83 in a patient and rats. Despitespecies differences between human and rat, a similar biphasicpharmacokinetic pattern was observed with a rapid initial clearance followed by a slower elimination phase. Fujita (4) hasrecently reported using microbiological assay that the clearanceof M-83 from the sera of patients obeys a 2-compartment modelwith a /3-phasehalf-life of about 18 min, which was, however,calculated only from the serum concentrations of M-83 within 90min after the dosing. By the present EIA following the serumlevels of M-83 in a patient during 5 hr after i.v. administration, it

was revealed that the /3-phase serum half-life was 80 min.Obviously, these results must be confirmed in more patientsbefore a firm conclusion can be made about actual half-lives andspecies variation.

ACKNOWLEDGMENTS

The authors thank Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan, for providingM-83 and mitomycm analogues; Dr. J. Shibasaki, Nagasaki University, for thevaluable suggestions for pharmacokinetic study; and Or. M Ishiguro of this facultyfor excellent advice in the preparation of this manuscript.

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4176 CANCER RESEARCH VOL. 44

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1984;44:4172-4176. Cancer Res   Kunio Fujiwara, Hideki Nakamura, Tsunehiro Kitagawa, et al.  

-Hydroxyphenyl)mitomycin Cp-(NImmunoassay for 7-Development and Application of a Sensitive Enzyme

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