Jpn. J. Cancer Res. (Gann), 79, 509—516; April, 1988
`
`Pharmacokinetic Approach to Rational Therapeutic Doses for Human Tumor-bearing
`Nude Mice
`
`Makoto INABA, Tomowo I<Loaavnsnr,*l Tazuko TASHIRO and Yoshio SAKURAIH
`Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Kami—Ikebukuro, Toshima-kn,
`Tokyo 170
`
`To improve clinical predictability from therapeutic results of various antitumor agents in human
`tumor/nude mouse models it seems to be important to use a dose pharmacokinetically equivalent
`to the clinical dose. Thus, we attempted to find the dose of a given drug that can reproduce in the
`nude mouse a plasma level similar to that seen in human patients treated with an effective dose of
`the drug based on comparative pharmacokinetic studies between man and nude mouse. As a result,
`those of 3 alkylating agents, mitomycin C, 3-[(4-amino~2-methyl-5-pyrimidinyl)methyl]-l-(2-
`chloroethy1)-l-nitrosourea (ACNU) and cyclophosphamide, and those of 2 antimitotic agents,
`vincristine and vinhlastine, were estimated to be one-fourth or one-fifth of their maximum
`tolerated doses (MTD’S). 0n the other hand, in the case of adriamycin, its MTD was approxi-
`mately equivalent to its clinical dose pharmacokinetically. In contrast, clinically equivalent doses
`of 2 antimetabolites tested, 5-fluorouracil and methotrexate, were significantly greater than their
`MTD’s; i.e., their plasma levels did not reach the effective clinical ones even when their MTD’s
`were administered to the nude mice. These results suggest that the antitumor effects of most
`antitumor agents are over- or underestimated in this model when MTD’s are used as a therapeutic
`dose, and indicate that the use of clinically equivalent doses determined pharmacokinetically is
`desirable.
`
`Key words: Antitumor agents m Human tumor a Nude mouse — Plasma level —- Clinically
`equivalent dose
`
`In our previous studies,” we attempted ex-
`perimental chemotherapy of a panel of human
`gastric tumors implanted in nude mice ac-
`cording to the same regimen,
`including the
`use of the MTD”‘3 for nude mice as a thera-
`peutic dose, and evaluated the effectiveness of
`various antitumor agents in terms of response
`rate. We found that such experimental
`re—
`sponse rates showed good agreement with
`clinical ones for some drugs, but
`those of
`other drugs were much higher than their re-
`
`*' Present address: Bioscience Research Laborato-
`ries, Sankyo Co., Ltd., Hiromachi, Shinagawa—ku,
`Tokyo 140.
`*2 Present address: Kyoritsu College of Pharmacy,
`Shiba-koen, Minato—ku, Tokyo 105.
`*3 Abbreviations used: VCR, vincristine; VLB,
`vinblastine; CPM, cyclophospharnide; 5FU,
`5-
`fluorouracil; MMC, mitomycin C; ADR,
`ad-
`riamycin; MTX, methotrexate; HPLC,
`high-
`performance liquid chromatography, MTD, maxi-
`mum tolerated dose; RD, rational dose; AUC, area
`under the curve.
`
`79(4)
`
`1988
`
`spective clinical ones. This disagreement was
`often observed with drugs whose dose per
`body weight was quite different between man
`and nude mouse. These results suggested the
`importance of using appropriate dose levels in
`the nude mouse to reproduce the clinically
`equivalent effect in this model. Thus, the pur-
`pose of the present study was to find experi-
`mentally a reasonable dose of each drug for
`human tumor-bearing nude mice based on a
`comparative pharmacokinetic study between
`man and nude mouse.
`
`Theoretically, it seems most reasonable to
`use a dose that will produce a drug concentra-
`tion in the extracellular space of tumor tissue
`in mice similar to that in human patients given
`a clinical close. For most drugs which can
`easily penetrate the capillary vessel and rap—
`idly reach an equilibrium on both sides, this
`dose approximates pharmacokinetically to the
`unbound free drug concentration in the blood.
`Thus, in the present study, we administered
`various doses of selected antitumor agents to
`
`509
`
`Genentech 2099
`
`Hospira v. Genentech
`|PR2017-00737
`
`Genentech 2099
`Hospira v. Genentech
`IPR2017-00737
`
`

`

`M. INABA, ET AL.
`
`nude mice and measured their plasma levels
`by the same method as used in the clinical
`studies. Clinical data on the plasma levels of
`all antitumor agents were taken from papers
`reported by other investigators. For compari-
`son of plasma levels of both human and nude
`mouse, we attempted to determine the mouse
`dose that is needed to achieve the same plasma
`level as seen in human plasma when the pa-
`tient is treated efi‘ectively.
`
`MATERIALS AND METHODS
`
`Chemicals VCR and VLB for clinical use were
`purchased from Shionogi & Co., Osaka. 5FU,
`MMC and ADR were kindly supplied by Kyowa
`Hakko Kogyo Co., Tokyo, as pure crystals for
`experimental use. CPM, ACNU and MTX were
`provided by Shionogi & Co., Sankyo Co., Ltd.,
`Tokyo, and Lederle Japan Ltd, Tokyo, respec-
`tively.
`[3H]VCR and [3H]VLB were purchased
`from Amersham International plc, Amersham,
`England.
`Collection of Plasma Samples Each antitumor
`agent was dissolved in or diluted with sterile 0.85%
`NaCl solution just before use. Drug solution was
`injected into the tail vein of athymic Balb/c-nu/nu
`mice (Clea Japan, Inc., Tokyo). Three female mice
`weighing an average of 24 g each were used in each
`group. At specified times after injection, groups of
`mice were killed by decapitation and the trunk
`blood was
`collected from the neck into a
`heparinized tube. The plasma was obtained by
`centrifugatiou at 10,000g for 2 min in a centrifuge
`(Beckman microfuge B) and stored at 720° until
`the estimation of drug concentration could be
`performed.
`Measurement of Drug Concentration in Plasma
`Measurement of drug concentrations in plasma of
`mice was carried out by the same method as used in
`each clinical study quoted.
`assay
`MMC was analyzed by a biological
`method with E. coli B grown on agar containing
`essential medium, an assay originally developed by
`Miyamura et at.” The lower limit of sensitivity of
`this method was 0.003 Jig/ml.
`ADR in plasma was assayed by HPLC according
`to the method reported by Masuike er at.” A
`plasma sample was applied to an HPLC apparatus
`equipped with a protein-coated column and eluted
`stepwise with phosphate buffer (PB), that is, 0.005
`M KH2P04-1M H3PO4 (pH 4.5), PB-CH3CN
`(75:25), and PB. The drug was detected fluoro-
`metrically. Excitation and emission wavelengths
`were 470 and 585 nm, respectively.
`VCR concentration was measured as total radio-
`activity in accordance with the method used for
`clinical study.“ [3H]VCR solution was injected iv
`510
`
`into the nude mice, and the total radioactivity of
`the plasma (0.1 ml), without isolation of VCR, was
`measured by liquid scintillation counting.
`ACNU concentration in plasma was measured
`by HPLC according to the method of Nakamura et
`at.” Briefly, after extraction with l,2-dichloro~
`ethane, a 50% methanol solution of the extract
`was assayed. A Waters ,u-Bondapack Cl, reverse-
`phase column was used and ACNU was eluted
`from it with a solvent system consisting of PIC—B7
`plus an aqueous solution of 50% methanol at flow
`rate of 1 ml/min. The lower limit of detection by
`this method was 0.03—0.04 jug/m1.
`Regarding CPM,
`the concentrations of its ac-
`tivated metabolites, 4-hydroxy CPM and aldophos-
`phamide, were measured according to the method
`of Wagner at (11.6] In this case, a blood sample
`instead of plasma was used. In brief,
`the active
`metabolites were extracted into dichloromethane
`from the blood sample and the extract was concen-
`trated in an evaporator. A mixture of the filtered
`extract and dilute HCl solution containing 3-
`aminophenol and hydroxyammonium chloride was
`heated at 95°
`for 20 min, and the fluorescence
`emission was measured at room temperature in a
`Hitachi fluorometer, model MPFw4. Excitation and
`emission wavelengths were 350 and 515 nm, respec-
`tively. The fluorescence originating from acrolein,
`which was liberated from the active metabolites,
`was carefully measured by using blank tests. The
`minimum measurable concentration was 0.5 nmol/
`ml of blood.
`'
`
`Plasma concentrations of unbound VLB were
`determined according to Lu et of.” Plasma samples
`collected from mice injected iv with [3H]VLB were
`deproteinized by sulfosalicylic acid, neutralized
`and filtered through an Ekicrodisc 3 (0.45 am,
`Gelman Sciences Japan, Ltd.). This preparation
`was applied to an HPLC apparatus equipped with
`ju-Bondapack C13 column and eluted with a solvent
`system consisting of acetonitrile and phosphate
`buffer at a flow rate of 2 ml/min. The eluate was
`fractionated at intervals of 30 sec, and the radioac-
`tivity of VLB fractions (retention time: 11—12 min)
`was measured.
`
`The concentration of 5FU in plasma was deter-
`mined by the microbiological assay method origi—
`nally reported by Fujita,” using Staphylococcus
`aureus 209p grown on Muller-Hinton medium
`without peptone and bouillon. The detection limit
`was 0.01 ,ug/ml.
`MTX plasma concentrations were measured by
`dihydrofolate reductase inhibition assay according
`to the method of Bertino and Fischer.” Measure-
`ments were taken in a 1.5-ml quartz glass cuvette at
`30°and 340 nm wavelength, and the measurement
`time was 2 min. The lower limit of sensitivity was
`0.002 a g/ml.
`
`Jpn. J. Cancer Res. (Gann)
`
`

`

`RATIONAL DOSES FOR HUMAN TUMORS/NUDE MOUSE
`
`RESULTS
`
`to be difficult to find the
`We expected it
`dose of a given drug which would accurately
`reproduce in the nude mouse the clinically
`observed “concentration-time curve” in man,
`since the slope of the plasma clearance curve
`of most drugs is usually quite different be-
`tween man and nude mouse. As a preliminary
`experiment, three graded doses of each drug,
`for example, the MTD and one—half and one-
`fourth of it were injected into nude mice to
`measure their plasma levels at a few time
`points, and rough plasma clearance curves
`were obtained. To determine a dose for nude
`
`mouse pharmacokinetically equivalent to the
`clinically eflective dose, comparison of plasma
`levels between man and nude mouse was made
`
`with an emphasis on drug concentration in
`plasma at a relatively early time after injec-
`tion. This is very important, particularly for
`antitumor drugs with a small ditference in the
`slope of their clearance curve between the two
`species, because relatively higher drug con-
`
`centrations at early time make a greater con-
`tribution to the antitumor eiTect.
`
`The MMC, ADR, and VCR plasma clear-
`ance curves demonstrated in Fig.
`1 seem to
`belong to the above category. In the case of
`MMC, used as a reference, data for both the
`MTD (6.7 mg/kg) and 1/4 MTD (1.7 mg/
`kg) in the nude mouse are shown in compar-
`ison with those of patients given the clinical
`dosem) (Fig. l-A). The plasma level of MMC
`at its MTD was obviously higher than that in
`the case of the clinical dose. However, when
`MMC at 1.7 mg/kg was administered to nude
`mice, its plasma clearance curve was found to
`be similar to that of human patients. In the
`case of MMC, therefore, 1.7 mg/kg or there-
`abouts might be regarded as the clinically
`equivalent dose for nude mice on a pharmaco-
`kinetic basis.
`
`21 plasma level of
`to MMC,
`In contrast
`ADR similar to that in patients given a clini-
`cal therapeutic dose of 60 mg/m2 11) was ob-
`served in nude mice when the MTD was
`
`administered (Fig. l-B). With VCR, compari—
`
`A Mitomycin C
`
`B Adriamxcin
`
`10
`
`Vincrist‘ine
`
`C
`
`10
`
`
`
`
`
`
`
`Man
`
`
`
`Nude mouselOA mg/kg)
`0"""O----..__ u-—
`
`(pg/ml)
`Plasmadrugconcentration
`
`
`2
`
`4
`
`0
`
`0.5
`
`1
`
`2
`
`Time after iv administration (hr)
`Fig.1. Comparison of plasma levels of mitomycin C, adriamycin, and vincristine between man and the
`nude mouse. Plasma concentrations of antitumor agents in nude mice given a single dose of each agent (2
`doses for MMC) were measured and compared with those of human patients administered a clinical dose
`of the same agent. Determination of plasma levels of each drug was made by the same methods as used in
`clinical studies. Each value was the mean of 3 to 4 determinations with less than 20% standard deviation.
`Clinical data of each drug are quoted from the literature.” Clinical doses of MMC, ADR and VCR can
`be converted into 0.17, 1.6 and 0.033 mg/kg, respectively, assuming that mean body weight is 60 kg or 1
`m2 of body surface area corresponds to 37.5 kg of body weight.
`
`79(4)
`
`1933
`
`511
`
`

`

`M. INABA. ET AL.
`
`B
`
`Cyclophosphamide
`
`c
`
`Vinblastine
`
`10‘
`
`l
`t.
`t5
`1%
`'.
`iii.
`m
`(D
`
`(.0
`
`a
`m
`U:
`a
`"-—.
`;-<—
`
`681012
`
`03612
`
`24
`
`N l4
`
`E"M.m
`:1.
`
`E
`+4
`.2
`2
`4-1
`OJ
`C
`2
`0
`U
`
`g
`'U
`E
`m
`01
`"I
`E
`
` O
`
`1
`
`'1
`
`.01
`
`Time after iv administration (hr)
`
`Fig. 2. Comparison of plasma levels of ACNU, cyclophosphamide and vinblastine between man and the
`nude mouse. Plasma concentrations of antitumor agents in nude mice given a single dose of each agent were
`measured and compared with those of human patients administered a clinical dose of the same agent.
`Determination of plasma levels of each drug was made by the same methods as used in clinical studies. Each
`value was the mean of 3 to 4 determinations with less than 20% standard deviation. Clinical data of each
`drug are quoted from the literature.” Clinical close of VLB can be converted into 0.2 mg/kg assuming that
`l m1 of body surface area corresponds to 37.5 kg of body weight.
`
`son of the plasma level in nude mice treated
`with 0.4 mg/kg with that“ of human patients
`given an effective dose, as shown in Fig. l-C,
`reveals similar VCR levels between the two
`
`species at the early stage, i,e., up to 15 min
`after injection. On the other hand,
`in the
`second phase of clearance, the plasma level of
`VCR was significantly higher in nude mice
`than in man. If coincidence of VCR plasma
`level at the late phase between man and nude
`mouse is required, some dose less than 0.4
`mg/kg may correspond to the clinically equiv-
`alent dose.
`
`As illustrated in Fig. 2, the drugs ACNU,
`CPM, and VLB exhibited markedly different
`clearance rates between man and nude mouse.
`
`its half-lives in the
`In the case of ACNU,
`plasma of man” and nude mouse were 25 and
`4.6 min, respectively (Fig. 2-A). Thus with
`this kind of drug it is very diflicult to repro-
`duce the clinical clearance curve in nude mice
`
`by a single injection. As an approach to get a
`clearance curve of ACNU in the nude mouse
`
`512
`
`similar to the clinical one, intermittent injec-
`tion of three graded doses (8, 2, and 0.8
`mg/kg at 0, 25, and 70 min, respectively) was
`attempted, as shown in Fig. 3-A. Rather
`better tracing of the clinical clearance curve
`was thus obtained, although coincidence was
`still incomplete.
`With CPM, a steeper plasma clearance was
`also observed with the nude mouse as com-
`
`pared with the clearance in man.“ For this
`drug, plasma levels of 4-hydroxy CPM instead
`of CPM itself were compared between man
`and nude mouse, since changes in the concen-
`trations of activated CPM are thought to be
`more important as far as antitumor activity is
`concerned. However, it is hard in practice to
`mimic the clinical clearance curve in the nude
`
`mouse by intermittent injections, because the
`clearance rate of 4uhydroxy CPM in man is
`extremely slow. The AUC values of 4-
`hydroxy CPM in the plasma of both man and
`nude mouse were calculated from concentra—
`
`tion-time curves (Fig. 2—B) by computer anal-
`
`Jpn. J. Cancer Res. (Gann)
`
`

`

`RATIONAL DOSES FOR HUMAN TUMORS/NUDE MOUSE
`
`B
`
`Vinblastine
`
`
`
`
`ACNU
`
`A
`
`10
`
`\
`I
`
`\
`
`\H
`“i.
`a
`
`l
`I
`i
`‘l
`‘n
`v
`
`
`
`1
`
`.1
`
`0
`
`0.5
`
`l
`
`2
`
`E a
`
`t
`.1
`.
`v
`
`=
`O
`'5
`re
`L.
`+4
`uU
`c:
`ou
`can
`:i
`5..
`'u
`re
`
`:c
`
`5
`to
`E
`
`Time after initial administration (hr)
`Fig. 3. Comparison of clinical plasma clearance curves of ACNU and vinblastine with the
`respective curves obtained by intermittent injection of the drugs into nude mice. ACNU was
`intermittently injected into nude mice at 3 doses of 8 (0 min), 2 (25 min), and 0.8 mg/kg (70
`min). Similarly, VLB was injected at 4 doses of 1.38 (0 hr), 0.6 (6 hr), 0.3 (12 hr) and 0.1 mg/
`kg (24 hr). At appropriate intervals, the plasma concentrations were measured and compared
`with those of human patients administered a single clinical dose”) (————). Each value was the
`mean of 3 to 4 determinations with less than 20% standard deviation.
`
`ysis and found to be 16.4 and 8.37 nmol-hr/
`ml, respectively. Therefore, if the cell-killing
`activity of CPM depends on “concentration ><
`exposure time” i.e., AUC in the case of
`plasma, a doubling of the 32.5 mg/kg dose of
`CPM should correspond to the clinical dose.
`In the case of VLB, a diiference in the slope
`of the clearance curve was also seen between
`
`man and nude mouse. Its plasma level in nude
`mice administered 1.38 mg/kg of VLB was
`initially somewhat higher than that in human
`patients given 7.5 mg/m2 of VLB,” but became
`lower later due to its relatively rapid clearance
`in the nude mouse. VLB has been classified by
`cell-killing kinetic analysis as a time—depen-
`dent drug,12) suggesting that its cytotoxicity is
`not dependent on the AUC. Then, intermit-
`tent injections (1.38, 0.6, 0.3, and 0.1 mg/kg
`at O, 6,
`12 and 24 hr,
`respectively) were
`
`79(4)
`
`1933
`
`attempted, and they were found to reproduce
`the clinical clearance curve roughly, as shown
`in Fig. 3-B. However, such treatment is im-
`practical in therapeutic experiments. There-
`fore, with this drug, we tried to find a dose for
`a single injection equivalent to the above four
`intermittent dosings in terms of antitumor
`activity. Although the data are not shown, a
`single injection of 2.6 mg/kg of VLB inhibited
`in viva tumor growth to approximately the
`same extent as the intermittent administration
`
`in 2 difi'erent xenograft models.
`With the 2 antimetabolites 5FU and MTX,
`no marked difference in plasma clearance rate
`was observed between man and nude mouse,
`at
`least over the period of time examined.
`Different from the above-mentioned drugs,
`the plasma levels of 5FU and MTX in nude
`mice did not reach those in human patients
`513
`
`

`

`M. INABA. ET AL.
`
`A
`
`5-Fluorouracil
`
`B
`
`Methotrexate
`
`
`
`
`
`
`
`Plasmadrugconcentrationlug/ml)
`
`Time after iv administration (hr)
`
`Fig. 4. Comparison of plasma levels of 5-fluorouracil and methotrexate between man and the
`nude mouse. The plasma concentration of each antitumor agent in nude mice given a single dose
`of agent was measured and compared with that of human patients administered a clinical dose of
`the same agent. Determination of plasma levels of each drug were made by the same method as
`used in clinical studies. Each value was the mean of 3 to 4 determinations with less than 20%
`standard deviation. Clinical data of drugs are quoted from the literature?” The clinical dose of
`MTX can be converted into 0.42 mg/kg assuming that the mean body weight is 60 kg.
`
`treated with the therapeutic dose,”' 1‘” even
`when the MTD of MTX or greater than the
`MTD of 5FU was injected (Fig. 4).
`
`DISCUSSION
`
`In the present study, we attempted to find
`the dose of various antitumor agents for nude
`mice that could reproduce the clinical plasma
`clearance curve observed with patients treated
`effectively, because we considered from a
`pharmacokinetic point of view such a dose to
`be a reasonable therapeutic one for human
`tumor-bearing nude mice. Practically speak-
`ing, we found it diflicult to determine such a
`dose in a precise manner, since the plasma
`clearance rate of most drugs in nude mice was
`significantly greater than that found clinically
`in man. However,
`to observe the clinically
`equivalent antitumor effect
`in this human
`tumor/nude mouse model, determination of
`514
`
`such a pharmacokinetically appropriate dose
`instead of the MTD is necessary even if it is
`not so accurate. Therefore, we have desig-
`nated such a dose the “rational dose (RD)”
`and tentatively estimated such doses based on
`the present comparative study on plasma
`levels of each drug in man and nude mouse.
`With MMC and ADR, 1.7 and 12 mg/kg,
`respectively, seem to be reasonable as such a
`dose. In the case of VCR, its plasma level in
`nude mice was clearly higher than its clinical
`one in the late phase of clearance. Since clear-
`ance rates in this phase are so slow that such
`levels might predominantly represent a bound
`form of VCR, especially in the present assay
`method, we regard 0.4 mg/kg of VCR as the
`RD.
`_
`Of the 3 drugs with relatively rapid clear-
`ance in nude mice, ACNU seemed to give
`reasonable results when administered as 3 in-
`
`Jpn. J. Cancer Res. (Gann)
`
`

`

`RATIONAL DOSES FOR HUMAN TUMORS/NUDE MOUSE
`
`termittent injections to the animals. However,
`such intermittent treatment seemed impracti-
`cal in the case of CPM, since the clinically
`observed plasma clearance was too slow to be
`reproduced in nude mice by intermittent ad-
`ministration. Very recently, we found from
`experiments involving in vitro colony forming
`that
`the cell-killing activity of alkylating
`agents such as nitrogen mustard depends on
`“concentration >< exposure time” or AUC
`value. This finding was made by subjecting
`their decomposition rate in the culture
`medium during incubation to a cell-kill kinetic
`analysis”) Therefore,
`it is probably possible
`to regard the dose of CPM in nude mice
`equivalent
`to the clinically effective dose in
`terms of plasma AUC as its RD. This line of
`reasoning, however, could not be appIied in
`the case of VLB, for this drug does not dem-
`onstrate the same pharmacokinetics as CPM,
`i.e.,
`the cell-killing activity of this drug has
`been proved to be independent of the AUC.
`Accordingly, in this case, a single injection of
`2.6 mg/kg was found equivalent to intermit—
`tent administrations with respect to antitumor
`effect and was regarded as its RD.
`The estimated RD’s of 8 kinds of antitumor
`
`agents are listed with their MTD’s” in Table
`I. The RD’s of MMC, VCR, ACNU, CPM,
`and VLB corresponded to 1/5—1/4 of their
`respective MTD’s. These results can reason-
`ably explain the higher experimental response
`rates observed with these antitumor agents,
`except for VCR, as compared with the respec-
`
`Table 1. Maximum Tolerated and “Rational”
`
`Doses of Various Antitumor Agents for Nude
`Mic e
`
`MTD
`RD
`
` Dmg (mg/kg) (mg/kg) I‘D/MTD
`
`MMC
`6.7
`1.7
`ADR
`12
`12
`VCR
`1.6
`0.4
`
`
`0.25
`l
`0.25
`
`ACNU
`
`48
`
`CPM
`VLB
`
`260
`11
`
`(0 min)
`8
`(25 min)
`2
`0.8 (70 min)
`65
`2.6
`
`ca. 0.2
`
`0.25
`0.24
`
`>1
`19(><5) >19(><5)
`SFU
`
`15(><5) >15(><5) >1
`MTX
`MTD values, which were determined as maximum non-
`lethal doses, are quoted from our previous paper."
`
`79(4)
`
`1933
`
`tive clinical ones when human gastric tumors
`in nude mice were treated with the MTD.”
`
`On the other hand, the RD of ADR was very
`close to its MTD. In the case of SFU and
`MTX, their plasma levels in nude mice did not
`reach those in human patients given the effec-
`tive doses even if their MTD’s were injected
`into the nude mice. These results suggest that
`the clinical antitumor effects of these agents
`might be underestimated if treatment in nude
`mice was done even at the MTD, although the
`degree of underestimation is not clear. In this
`respect, some examples of a lack of thera-
`peutic eflicacy of 5FU‘6) and MTX”) against
`human tumor xenografts at doses that are
`effective in an in vitro clonogenic assay or in
`clinical
`treatment have been reported by a
`group in the Netherlands.
`In the succeeding paper, the results of ex-
`perimental chemotherapy using these clini-
`cally equivalent doses against a panel of
`human gastric tumors implanted in nude mice
`will be reported in comparison with those
`obtained by using the MTD’s.
`ACKNOWLEDGMENTS
`
`The present study was supported in part by a
`Grant-in-Aid for New Drug Development Re-
`search from the Ministry of Health and Welfare,
`Japan.
`(Received Oct. 22, I 987/Accepted Feb. 20, 1988)
`
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