[CANCER RESEARCH 52, 3604-3609. July I, 1992]

L-Histidinol: Preclinical Therapeutic Studies in Combination with AntitumorAgents and Pharmacokinetic Studies in Mice1

Daniel Zaharko,2 Jacqueline Plowman, William VVaud,Donald Dykes, and Louis Malspeis

Pharmacology Branch [D. Z., J. P.] and Laboratory of Pharmaceutical Chemistry [L. M.], Developmental Therapeutics Program, Division of Cancer Treatment, NationalCancer Institute, Bethesda, Maryland 20892, and Southern Research Institute, Birmingham, Alabama 35205 [W. W., D. D.]

ABSTRACT

Therapeutic studies were conducted with L-histidinol, in combinationwith cyclophosphamide, bischloroethylnitrosourea, 5-fluorouracil, phen-ylalanine mustard, or m-platinum(H)diammine dichloride, in severaltransplantable tumors in mice. These tumor types included murine LI 210P388 leukemias, M5076 sarcoma, mammary 16/C adenocarcinoma, human LOX melanoma, and colon III-29 adenocarcinoma. Therapeuticbenefits of adding i.-histidinol to a regimen, compared to the regimenalone, were marginal. Pharmacokinetic studies indicated a rapid clearanceof L-histidinol following a bolus dose (250 mg/kg ¡.p.),peak plasmaconcentration of 200 Mg/ml (1.4 HIM), and ft phase f., of 12.6 min.Maximum tolerable plasma steady state concentrations with a 24-hinfusion (2000 mg/kg/24 h) were no greater than 25 Mg/ml(0.18 mivi).

INTRODUCTION

HIST3 [3-(4-imidazolyl)-2-amino-l -propanol, NSC 74518]

has been proposed as a modulator for anticancer agents (1).Publications of preclinical results to date have been somewhatcontroversial. It has been suggested that HIST protects the hostagainst toxicity of some anticancer agents and enhances theantitumor activity of some agents (2-8). Others have reportedthat HIST reduces the incorporation of 5-FU into RNA anddoes not enhance the antitumor effects of 5-FU (9,10). A recentstudy reports that in vitro exposure to HIST, at concentrationsof 1.5-4.5 mivi, reverses the multidrug resistance attribute of aChinese hamster ovary cell line resistant to colchicine, dauno-rubicin, and vinblastine (11).

The unpublished database files of the National Cancer Institute, Division of Cancer Treatment, Developmental Therapeutics Program, indicate that HIST was screened in 1963 for invivo antitumor activity in the murine models L1210, Lewis lungcarcinoma, and sarcoma 180, in a single-agent treatment modality, with negative results. A more recently published reportindicates that HIST, with a special schedule of administration,yielded significant activity against spontaneous, autochthonous,CDSFi breast tumors, at tolerable doses (12).

We report here our results of studies in which we focused ontwo aspects of HIST use. We wished to determine whether theuse of HIST in combination with currently effective chemo-therapeutic agents reduced their toxicity and increased theiranticancer activity. We also studied the pharmacokinetics ofHIST in mice, in order to determine plasma concentrations andexposure times of HIST that can be attained in vivo at thetolerable doses used in therapeutic studies.

Received 1/27/92; accepted 4/20/92.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.

1National Cancer Institute, Division of Cancer Treatment, Contracts N01-CM-73726 and N01-CM-67903 were used to obtain data for this manuscript.

2To whom requests for reprints should be addressed, at Pharmacology Branch,Developmental Therapeutics Program, Division of Cancer Treatment, NationalCancer Institute, 9000 Rockville Pike, Executive Plaza North, Room 841, Bethesda, MD 20892.

'The abbreviations used are: HIST, L-histidinol; CPA, cyclophosphamide;BCNU, bischloroethylnitrosourea; 5-FU, 5-fluorouracil; L-PAM, phenylalaninemustard; Cis-Pt, cu-platinum(II)diammine dichloride; ILS, increase in life span;CD2F,, BALB/c X DBA/2 F,.

MATERIALS AND METHODS

Animal Therapeutic Studies

Drugs. HIST and the antitumor drugs were provided by the DrugSynthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute (Bethesda, MD). The agents were prepared as solutions in the followingvehicles: HIST, distilled water, pH 7.1-7.4; BCNU, saline containing2% ethanol; CPA, 5-FU, L-PAM, and Cis-Pt, saline.

Mice and Tumors. Mice, obtained from various commercial suppliers,were housed either in open-top, stainless steel cages (immunocompetentmice) or in Microisolator cages (Lab Products, Inc., Maywood, NJ)(athymic mice). Immunocompetent and athymic mice were housed inseparate barrier facilities. Mice were allowed commercial mouse foodand water ad libitum. Tumor lines (murine P388 and LI 210 leukemias,M5076 sarcoma, mammary 16/C adenocarcinoma, human HT-29 colon tumors, and LOX amelanotic melanoma) were obtained from theDevelopmental Therapeutics Program Tumor Repository (Frederick,MD).

Evaluation of Antitumor Activity. For studies using the murine leukemias, CD2F] mice were implanted either i.p. with IO6 P388 cells ors.c. with IO5 LI210 cells. For studies using the murine solid tumors,C57BL/6 x C3HF, mice were inoculated with M5076 cells i.p. (IO6)or s.c. (5 x IO6)or with fragments (30-60 mg) of mammary 16/C s.c.For studies using the human tumor xenografts, NCr-n«athymic micewere implanted either i.p. with IO6 LOX melanoma cells or s.c. withtumor fragments (30-60 mg) of CX-1 colon carcinoma xenograft. Thetumor implantation day was designated as day 0. HIST and the anti-tumor drugs were administered according to the schedules and routeslisted in the tables. In each experiment, HIST was evaluated alone andin combination with several dosage levels of an antitumor drug, witheach dosage being administered to both tumor-bearing (7-10/testgroup) and normal (6-8/test group) mice. Tumor-bearing control mice(20-40/experiment) were not treated with agent vehicle. Mice wereobserved for life span and/or tumor growth.

Quantitation of Antitumor Activity. For LI210, P388, M5076 (i.p.),and LOX tumors, antitumor activity was assessed on the basis ofpercentage of median ILS and net log,0 cell kill. Calculations of netlogio cell kill were made from the tumor-doubling time that wasdetermined from an internal tumor titration consisting of implantsfrom serial 10-fold dilutions (13). Each experiment contained a titration. Long term (30-60 day) survivors were excluded from calculationsof percentage of ILS and tumor cell kill. To assess tumor cell kill atthe end of treatment, the survival time difference between treated andcontrol groups was adjusted to account for regrowth of tumor cellpopulations that may occur between individual treatments (14).

For M5076 (s.c.) and mammary 16/C murine tumors, antitumoractivity was assessed on the basis of percentage of ILS and delay intumor growth (treated compared with control). The delay in tumorgrowth is the unweighted average of the differences of the median times(days) after implant for the treated and control groups to attain thetumor sizes of 500 and 1000 mg. Drug-induced deaths, tumor-freesurvivors, and any other animal whose tumor failed to reach the sizesof 500 and 1000 mg were excluded.

For HT-29 colon tumor xenograft, antitumor activity was assessedon the basis of delay in tumor growth (treated compared with control)and the maximum change in tumor weight (group mean tumor weighton the day producing the optimum percentage of tumor/control valueminus group mean initial tumor weight).

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1.-HISTIDINOL THERAPEUTICS AND PHARMACOKINET1CS

Liquid Chromatograph}'

HIST dihydrochloride (M, 214; lot MS-11-285-1) was obtained

from the Developmental Therapeutics Program, Division of CancerTreatment, National Cancer Institute. The purity of the compound was>99%, based upon the spectral and liquid Chromatographie data provided. Sodium pentanesulfonate was purchased from the Regis Chemical Co. (Morton Grove, IL). Fluoraldehyde (o-phthalaldehyde/2-mer-captoethano!) derivatizing solution was obtained from Pierce ChemicalCo. (Rockford, IL). The methanol was glass-distilled OmniSolv Grade(E. M. Science, Cherry Hill, NJ). The glacial acetic acid was analyticalreagent grade (Mallinckrodt, Paris, KY), and the inorganic chemicalswere analyzed reagent grade (J. T. Baker Chemical Co., Phillipsburg,NJ). The water, double distilled and deionized, was filtered through a0.2-^m Nylon 66 filter (Rainin Instrument Co., Woburn, MA) beforeuse. Phenylboronate extraction columns of 1-ml capacity were purchased from Analytichem International (Harbor City, CA). The hepa-

rinized microhematocrit capillary tubes were purchased from the AlzaCorporation (Palo Alto, CA).

Chromatography was performed using a model 114 pump (BeckmanInstruments, Berkeley, CA), WISP 712 automatic injector (WatersAssociates, Milford, MA), Spectroflow 980 programmable fluorescencedetector (ABI Analytical, Kratos Division, Ramsey, NJ) containing a5-n\ flow cell (path length, 8 mm), and model 3390A recording integrator (Hewlett-Packard, Avondale, PA). The system was equipped with a4.6-mm x 25-cm stainless steel column, packed with 5-^m Ultrasphere-ODS, and a Kratos model URS/051 post-column reactor. A Cahn 25

electrobalance (Cahn Instruments, Inc., Ceritos, CA) was used forweighing milligram quantities of material during the preparation ofstock solutions. An Eppendorf model 5412 microcentrifuge (Brink-

mann Instruments, Westbury, NY) was used for centrifuging assaysamples contained in 1.5-ml polypropylene microcentrifuge tubes(VWR Scientific, San Francisco, CA).

HIST was isolated from plasma using a phenylboronate solid-phase

extraction column. Prior to the addition of plasma, the column wasconditioned with methanol (1 ml), followed by water (1 ml) and saturated sodium borate solution (5 ml). Plasma (350 /<!)was mixed withsaturated sodium borate solution ( 1 ml) and passed through the phenylboronate extraction column. The column was then successivelywashed with saturated sodium borate solution (2 ml) and water (1.5ml). The eluant was centrifuged at 12,000 x g for 4 min prior to liquidchromatography. Liquid Chromatography of an aliquot (50 /¿I)of theeluant from the phenylboronate column was performed using a 5-¿tmUltrasphere-ODS column (Beckman Instruments, Berkeley, CA), with0.1 M phosphate buffer, pH 5, containing 5 HIMsodium pentanesulfonate as the mobile phase. The mobile phase solution was degassed byplacing the flask in a ultrasonic bath for 15 min prior to chromatography. Chromatography was performed at ambient temperature, with aflow rate of 1.0 ml/min. The effluent from the analytical column wasmixed with o-phthaladehyde/2-mercaptoethanol reagent in the post-column reaction chamber before being passed to the detector (15).Fluorescence detection was monitored using 300-nm excitation and a470-nm cutoff filter. The flow rate of the derivatizing solution was 0.24

ml/min. The integrator was configured to report peak areas. Calibrationcurves were prepared daily from freshly prepared stock solutions ofHIST, and all samples were assayed in duplicate. The peak area ofHIST was plotted against concentration. The best-fit straight line wasdetermined by least squares regression analysis. The concentrationrange of the standard curves was 0.12-3.0 jig/ml. The concentrationsof HIST in the unknown samples were calculated using the result ofthe regression analysis. The relative recovery of HIST from standardsassayed during a 6-week period ranged from 98.1 to 102.5% of theadded amount, and the coefficient of variation of the averaged slopesof the linear regression curves was 1.17% (n = 6). Similar assay resultswere obtained when fresh mouse plasma samples that were spiked withHIST were analyzed immediately before and after storage at -20°Cfor

24 h.

Animal Pharmacokinetic Studies

Studies on the plasma pharmacokinetics of HIST following i.p.administration were performed with unfasted male HaríanCD2F, mice.Dosing solutions were prepared immediately prior to treatment bydissolving the compound in normal saline. Administration of the dosingsolution (100 //I) was performed without anesthesia, using a 27-gauge,0.5-inch hypodermic needle, by a 1-min injection into the i.p. cavity.After drug administration, mice were bled through the retro-orbitalplexus, under ether anesthesia, using heparinized microhematocrit capillary tubes. Each mouse provided approximately 0.75-1 ml of wholeblood, which was centrifuged for 5 min at 12,000 x g. Two 350-j¿laliquots of plasma were transferred to individual microcentrifuge tubesfor immediate assay. Samples were reassayed in cases where the analyzed concentrations for the individual replicates deviated by more than10% from their average. Geometric mean plasma concentrations werecalculated from the average determinations of at least three mice ateach time point. Dosing solutions for s.c. infusion were preparedimmediately prior to treatment, by dissolving the compound in sterilewater for injection. The osmotic minipumps containing the dosingsolution were equilibrated at 37°Cin isotonic saline prior to implanta

tion. The rate of delivery from the minipump was 6.44 ^1/h.

Pharmacokinetic Analysis

The geometric mean plasma concentrations of HIST following i.p.administration were pharmacokinetically analyzed using the nonlinearleast squares regression program NONLIN (16). The three-compartment open model was selected, using Ir weighting of data.

RESULTS

The role of HIST in improving the therapeutic antitumoractivity of five antitumor agents (CPA, BCNU, Cis-Pt, L-PAM,and 5-FU) on leukemia and solid tumors, in a number of studies,is described below.

Mouse Leukemia P388 and 1,121(1. Table 1 illustrates theresults from a P388 i.p. model in mice in which the agentsHIST and BCNU were injected i.p. The dose range of BCNUused alone ranged from nonlethal to lethal doses. The optimaldose was 40 mg/kg (ILS of 139% and cell kill of 7.9 log); thiswas also the highest nonlethal dose for tumor-bearing mice.The earliest death in this group of 10 treated tumor-bearingmice was 20 days and the latest death was 31 days, with amedian of 23.5 days. Since the median day of death of controlmice was 9.8 days and all mice dying in the treated group hadevidence of splenomegaly and ascites, no lethal toxicity wasconsidered to have occurred. However, for non-tumor-bearingmice only 3 of 8 mice survived for 45 days; therefore, somedelayed drug toxicity was suggested. Toxicity appeared to bereduced in non-tumor-bearing mice receiving the same dose ofBCNU in combination with HIST, inasmuch as 6 of 7 micesurvived 45 days. HIST at either 250 or 125 mg/kg gave similartherapeutic results, and we report in Table 1 only the data atthe 250 mg/kg dose level, which gave the better modulatoryresponse in combination with BCNU at its optimal dose of 40mg/kg. This beneficial modulatory effect of HIST at 40 mg/kgBCNU is indicated by an increase in median survival time oftumor-bearing mice from 23.5 to 38.5 days (ILS, 139% to292%) and an increase in 60-day survivors from 1 of 10 to 5 of10. A similar experiment repeated with 250 mg/kg doses ofHIST but with a more closely spaced dose range of BCNU(25.6, 32, 40, and 50 mg/kg) resulted in a modulatory effect ofHIST at 40 mg/kg BCNU. This dose of BCNU alone gave twodrug-related deaths in this experiment with tumor-bearing mice.The ILS for BCNU at 40 mg/kg increased from 111% to 179%

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L-mSTIDINOL THERAPEUTICS AND PHARMACOKINETICS

Table 1 Effect of HIST on BCNU activity in P3S8 leukemiaCD2F, mice were inoculated i.p. on day 0 with 10' P388 leukemia cells. Treatment was i.p. bolus on day 5 with five doses of HIST given 2 h apart and with one

dose of BCNU given at the same time as the second HIST dose.

BCNU(mg/kg)906040270906040270HIST(mg/kg/

injection)250250250250250Msr(days)11.714.323.520.29.811.112.838.521.39.9ILS(%)194513910613302921171login

change intumor burden at end

of treatment*ToxiccToxic-7.9-5.80"ToxicToxic-8.3-6.5060-daysurvivors

(tumorous)0/101/101/100/100/200/100/105/100/100/1045-daysurvivors

(normal)0/80/83/88/87/80/83/86/78/88/8

°MST, median survival time.'' login cell kill calculations based on dying mice only (13).1Toxic designation assessed on the basis of day of death, absence of ascites, and spleen size at necropsy.'' Approximate number of tumor cells on day 5 was 2 x 10".

Table 2 Effect of HIST on 5-FU activity in P388 leukemiaCD2F, mice were inoculated i.p. on day 0 with 10" P388 leukemia cells. Treatment was i.p. bolus on day 1 with five doses of HIST given 2 h apart and with one

dose of 5-FU given at the same time as the second HIST dose.

5-FU(mg/kg)240012006003001500240012006003001500HIST(mg/kg/

injection)250250250250250250MST*(days)10.215.016.016.316.710.512.115.316.815.815.311.9ILS(%)-342525559154560504513login

change intumor burden

at end oftreatment*ToxiccToxic-3.5-3.7-4.0Toxic-3.1-4.0-3.4-3.1-0.930-day

survivors(tumorous)0/80/80/80/80/80/200/80/80/80/80/80/830-day

survivors(normal)0/62/62/66/66/66/60/61/64/66/66/66/6

" MST, median survival time.*'See Table 1.

with HIST. There was no increase in 60-day survivors (2 of 10with BCNU alone and 0 of 7 with HIST plus BCNU). Therewere 8 of 8 and 6 of 6 45-day survivors in the respective groupsof non-tumor-bearing mice. These data indicate that, at theoptimal dose of BCNU, there is a slightly enhanced ILS effectresulting from HIST; however, this effect is not very apparentat either the next higher or the next lower dose. The increasein 60-day survivors can be quite variable from experiment toexperiment.

A third experiment with advanced P388 leukemia was conducted with higher HIST doses (400, 500, and 600 mg/kg) andBCNU doses of 38.4, 48.0, 60, and 75 mg/kg. At the optimaldose of BCNU (38.4 mg/kg), estimated tumor cell kill was 8.2log (180% ILS), with 2 of 8 mice being 60-day survivors;however, only 2 of 6 non-tumor-bearing mice survived 60 daysat this dose. The non-tumor-bearing mice died between days 9and 15, whereas the tumor-bearing mice died between days 20and 37 at a dose of 38.4 mg/kg. With 400, 500, and 600 mg/kg HIST in combination with BCNU (38.4 mg/kg), estimatedlog cell kill of tumor and ILS were 7.3 and 130%, 6.2 and112%, and 8.2 and 170%, with 0 of 6, 1 of 8, and 2 of 8 60-daysurvivors, respectively; however, 6 of 6, 4 of 6, and 4 of 6 non-tumor-bearing mice survived 60 days with this combination.These results, in contrast to the results obtained with HISTdoses of 250 and 125 mg/kg, indicate no improvement intherapeutic benefit but, similarly to the lower doses of HIST,indicate some improvement in the reduction of toxicity.

Table 2 shows the results of the modulation of 5-FU activity

on P388 leukemia by HIST. Although HIST did reduce thetoxicity of 5-FU slightly, as indicated in both tumor-bearingand normal mice, it did not increase the tumor cell kill or lifespan in a consistent manner or to a significant degree. Anexperiment with a dose of 500 mg/kg HIST gave similar results.

Two experiments were conducted with the s.c. LI210 leukemia, in CD2F] mice, with CPA in combination with HIST(data not shown). The agents were administered, in the samemanner as the 5-FU and HIST combination, on day 5 followingtumor implant on day 0. The dosage range of CPA was 150-600 mg/kg, with the optimal dose of CPA alone, 400 mg/kg,resulting in 133% ILS and 8.0 log of cell kill, with no 30-daysurvivors. This dose was lethal to 1 of 8 non-tumor-bearingmice, whereas the next two higher doses, 500 and 600 mg/kg,were lethal to 2 of 8 and 8 of 8 non-tumor-bearing mice,respectively. Although HIST at 250 mg/kg/injection decreasedthe lethality slightly, to 0 of 8 and 7 of 8 for 500 and 600 mg/kg CPA, respectively, it had little effect on the therapeuticresults, as evidenced by 150% ILS and 8.1 log of cell kill, withno 30-day survivors (500 mg/kg CPA).

Mouse Solid Tumors. The modulation of several antitumoragents (BCNU, Cis-Pt, 5-FU, and L-PAM) by HIST was evaluated in several solid tumors (s.c. and i.p. M5076 sarcoma, s.c.16/C mammary adenocarcinoma, s.c. HT-29 colon, and i.p.human LOX melanoma). These results are summarized inTables 3-5. With BCNU treatment of murine s.c. M5076 sarcoma, the range of median life spans over a dose range of 16-48 mg/kg was 46-52 days. HIST did not modulate this BCNU

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L-HIST1DINOL THERAPEUTICS AND PHARMACOKINETICS

Table 3 Combination chemotherapy with HIST in mouse solid tumorsHIST was administered as an i.p. bolus at 0, 2, 4, 6, and 8 h or as a 24-h s.c. infusion with an Alzet minipump. The chemotherapeutic agent was given coincidental!)1

at 2 h with the second bolus dose of HIST or when the s.c. infusion was started. Mice were C57BL/6 x C3H F¡.

Tumor

i.p. M5076 sarcoma

s.c. mammary 16/Cadenocarcinoma

Treatment dose range (mg/kg) and schedule

Range ofmedian lifespans (days)

Optimal dose(mg/kg) ILS (%)

Range of delays intumor growth

(days)"

Range of log,0changes in

tumor burdenat end of

treatment*

s.c. M5076 sarcoma i.p. BCNU (16-48) ondays2and9i.p.HIST (250) every 2 hx5,on days 2 and9BCNU

+ HIST (seeabove)Controls46-522647-5126483910009616-30-119-38

i.p. Cis-Pt (6-16) on days 5and 9

s.c. HIST (1440-1800) 24-hinfusion on days 5 and 9

i.p. HIST (250) every 2 h x5, on days 5 and 9

Cis-Pt (9-12) + S.C.HISTinfusion (as above)

Cis-Pt (9-12)-(-i.p. HIST(as above)

Controls

i.p. 5-FU (67-505) on days 2and 9

i.p. HIST (250) every 2 h x5, on days 5 and 9

5-FU + HIST (as above)Controls

11-42

19-20

20

10-14 (toxic)

10-12 (toxic)

21

17-40

20

18-3721

Toxic

Toxic

ISO

150

103

-8

-3

-52

-51

88

-8

73

-4.7 to -5.7

+0.6

+0.6

10-16

7-20

' The average of difference in median days (tumor-treated - tumor-untreated) to reach the tumor sizes of 500 and 1000 mg.'' log,o cell kill calculations based on dying mice only (13).

effect, inasmuch as the range for the combination was 47-51days. The ILS for the optimal dose also was not improved,being 100% with BCNU and 96% with HIST plus BCNU.There was a hint of increased activity with HIST plus BCNU(19-38 days), compared with BCNU alone (16-30 days), in themaximum delay of tumor growth. With Cis-Pt treatment of i.p.M5076 sarcoma over a dose range of 6-16 mg/kg, the range ofmedian life spans was 11-42 days. For Cis-Pt in combinationwith HIST given as either infusions or i.p. injections, the rangeof median life spans was 10-14 days, which indicates a toxiccombination, as do the negative values for ILS at the optimalCis-Pt doses. With 5-FU treatment of s.c. mammary 16/C,there was no indication of benefit with HIST, either by medianlife spans or by ILS at optimal 5-FU doses. The 20-day maximum delay in tumor growth with HIST plus 5-FU gave a hintof increased activity, compared with 5-FU alone.

Human Tumor Xenografts. The human solid tumors growingas xenografts in athymic mice were treated with the agents Cis-Pt and L-PAM in combination with HIST, to determinewhether HIST could improve the marginal antitumor activityof these agents (see Tables 4 and 5). In the s.c. human HT-29colon, the addition of HIST to either Cis-Pt or L-PAM treatment had little effect on the optimum percentage of treated/control value or the maximal delay in tumor growth. LOXmelanoma, implanted i.p., was treated with Cis-Pt in combination with HIST given either as multiple bolus injections oras constant infusions. In neither case, even though treatmentwith Cis-Pt alone increased the life span of the mice, did HISTconfer any beneficial effects. The infusion dose of HIST usedsuccessfully alone in earlier studies with C57BL/6 x C3HF,mice was toxic when used either alone or in combination withCis-Pt in these athymic mice.

Pharmacokinetics. Plasma concentration-time profiles following bolus i.p. administrations of HIST were fitted to a three-compartment open model (Fig. 1). The plasma profiles of HIST

were monitored to 480 min after dosing, at which time plasmaHIST concentration had declined approximately 1000-fold.The pharmacokinetic parameters, determined by computer fits,for the two i.p. studies are shown in Table 6. The data in Fig.1 suggest that repetitive i.p. bolus doses of 250 mg/kg givenevery 2 h in the therapeutic studies would maintain peak andtrough plasma HIST concentrations of 200 to 2 ¿ig/ml.

Plasma concentrations of HIST were determined during thes.c. infusion of HIST at a rate of 2000 mg/kg/24 h, using theALZA Osmet-24 minipump (Fig. 2). The observed geometricmean plasma concentrations at 3, 6, and 16 h were 16.82,14.54, and 18.31 /ig/ml, respectively. These values were belowprojected steady state plasma concentrations of 23 i<n/ml calculated by using the i.p. bolus dose of 250 mg/kg and theplasma clearance value. At 24 h after implantation of the

Table 4 Combination chemotherapy with HIST inhuman HT-29 colon xenografts

HIST and the chemotherapeutic agents were administered as described inTable 3. Male athymic NLR-nu mice were implanted s.c. on day 0 with HT-29tumor fragments.

Range of delays in Range inTreatment dose range (mg/kg) Earliest day tumor growth optimal T/C

and schedule of death" (days)* (%)c

i.p. Cis-Pt (4-32) on days 17and24i.p.

HIST (125, 250) every 2 hx5,on days 17 and24Cis-Pt

+ HIST (asabove)i.p.L-PAM (5-40) on day18i.p.HIST (125, 250) every 2 hx5,on day 18L-PAM

+ HIST (asabove)Controls28>542724>4723>47-8

to13-6

toO-7

to 11-Oto5-1

to3-2

to 560-12469-15346-17267-9489-9350-94

" Mice were sacrificed starting on day 47 for humanitarian reasons, because of

tumor size.* The average of difference in median days (tumor-treated —¿�tumor-untreated) to

reach the tumor sizes of 500 and 1000 mg.' T/C is the maximum change in tumor weight after treatment, relative to

untreated controls.

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L-HISTIDINOL THERAPEUTICS AND PHARMACOKINET1CS

Table 5 Combination chemotherapy with HIST in human LOX melanoma i.p. xenograftsHIST and Cis-Pt were administered as described in Table 3. Female athymic NCr-nu mice were implanted i.p. on day 0 with 10* LOX cells.

Treatment dose range (mg/kg)andschedulei.p.

Cis-Pt (6-28) ondaysSand9i.p.HIST (500, 375)every2h x 5, on days 5 and9Cis-Pt

+ HIST (asabove)Controlsi.p.

Cis-Pt (6-21) ondays5and9s.c.HIST (2000) 24-hr in

fusion on days 5 and9Cis-Pt+ HIST (asabove)ControlsRange

of medianlife span(days)10-3822-2310-372333-4576-720Rangeof ILS(%)-56

to69-2toO-55

to6364

to123-66-67

to -70Optimal

dose(mg/kg)121621Range

of loglo changesin tumor burden atend oftreatment"-0.3

to-1.3+0.4

to+0.5+0.2

to-1.2-1.610-2.5Toxic*Toxic*

"See Table 1.* Toxic in tumor-bearing mice but not in normal mice.

10°

-j 102

S

ico

10'

10"100 200 300 400 500

WINS

Fig. 1. Geometric mean plasma concentrations of HIST after administrationof a single i.p. dose of 250 mg/kg to CD2F, mice. Each point is the mean of three

Table 6 Pharmacokinetic parameters for HIST in miceGroups of three mice per time point were injected i.p. with HIST and bled at

the times indicated in Fig. 2.Parameter"y,yty,v«.IM4»UCLpiitm,MRT*AUC%

AUC (y phase)Unitsliter/kgliter/kgliter/kgliter/kgminminhml/min/kg

hng/mlxmin%250

mg/kgdose0.440.511.342.291.7912.641.9160.93

0.63410316500

mg/kgdose0.800.301.963.065.2016.552.9365.17

0.78767213.5

°Pharmacokinetic analysis was by NONLIN (15).* MRT, mean residence time.c AUC, area under the concentration-time curve.

minipumps, the geometric mean plasma level was 24.71 Mg/ml.These data indicate that results obtained using the s.c. route ofinfusion are reasonably close to the results expected from ani.v. infusion.

DISCUSSION

The results of these therapeutic studies indicate that, forsome anticancer agents, HIST can improve the antitumor effectand protect against host toxicity, but only in a marginal manner.Table 1 illustrates that a very small tumor burden does notcorrelate well with long term (60-day) survivors in experiments

10'

_J1B

U

Oo<co

1013Eo§10°(-

ftffff=T==~

**:

--]

i t t i i\)200 400 600 800 1000 1200 140016(M\NS

Fig. 2. Geometric mean plasma concentrations of HIST during s.c. infusionat a rate of 2000 mg/kg/24 h in CD2F, mice. Each point is the mean of threemice.

with P388 leukemia. Other investigators concluded that HISTaugments the effectiveness of BCNU in a very dramatic manner,when they considered only long term survivors and did notconsider the estimated degree of cell kill resulting from treatment (3). Our interpretation is that the number of long termsurvivors may increase dramatically with only a marginal decrease in tumor burden when the tumor burden is extremelysmall (13).

Recent studies showing reversal by HIST of a multidrug-resistant phenotype of Chinese hamster ovary cells indicatedthat 3 HIM medium concentrations, with 48-h exposure, wererequired for this effect (11). Other studies have shown that fivebolus i.p. doses of HIST (200-250 mg/kg/dose) at 2-h intervals

are required for therapeutic synergism with antitumor agents(1-5, 8) and that an i.v. infusion of 2000 mg/kg/24 h is themaximal tolerable dose in mice (6, 7). Our pharmacokineticexperiments in mice indicate that 250 mg/kg bolus i.p. dosesof HIST result in peak plasma concentrations of 200 Mg/ml(1.4 HIM)that decrease to 2 ¿ig/ml(0.01 mivi) within 2 h. Theplasma steady state concentration following a 24-h infusion of2000 mg/kg/24 h was no greater than 25 Mg/ml (0.18 mivi). It,therefore, appears that mice cannot be exposed, without lethaleffects, to the 1.5-5 mivi plasma concentrations for the 48 hreported to be necessary to reverse multidrug resistance.

Although these studies do not support the use of HIST in3608

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I-HISTIDINOL THERAPEUTICS AND PHARMACOK1NETICS

combination with antitumor agents, there are aspects of thisagent which are interesting. A study reported that growthinhibition resulting from HIST treatment of B-16 melanomain vitro is accompanied by increases in activities of NADPHcytochrome c reducíase and y glutamyl transpeptidase, lipidaccumulation, and cell enlargement (17). This finding mayindicate histidinol selectivity for certain tumors. There is arecent report of antitumor activity of HIST alone in a mammarytumor model in mice with an intensive schedule of HISTadministration (5 times each day at 2-h intervals on four successive days each week, for a total of 3 weeks) (12). Our data,with HIST alone, do not support this concept in the tumorstested with a variety of HIST schedules. However, none of ourschedules reached the dose intensity with the multiple coursesof treatment used in the report cited above.

ACKNOWLEDGMENTS

We thank Caroline Bazikian and Nicole Harris for help in typingthe manuscript.

REFERENCES

1. Warrington, R. C. A novel approach for improving the efficacy of experimental cancer chemotherapy using combinations of anticancer drugs and ihistidinol. Anticancer Res., 6: 451-464, 1986.

2. Warrington, R. C., Muzyka, T. G., and Fang, W. D. Histidinol mediatedimprovement in the specificity of 1-0-n-arabinosylcytosine and 5-fluorouracilin LI 210 leukemia-bearing mice. Cancer Res., 44: 2929-2935, 1984.

3. Warrington, R. C., and Fang, W. D. i.-Histidinol improves the selectivityand efficacy of alkylating agents and daunomycin in mice with P388 leukemia. Br. J. Cancer, 60: 652-656, 1989.

4. Warrington, R. C., Cheng, I., and Fang, W. D. Effects of L-histidinol on the

susceptibility of P815 mastocytoma cells to selected anticancer drugs in vitroand in DBA/2J mice. J. Nati. Cancer Inst., 78: 1177-1193, 1987.

5. Warrington, R. C., and Fang, W. D. Histidinol mediated enhancement ofthe specificity of two anticancer drugs in mice bearing leukemic bone marrowdisease. J. Nati. Cancer Inst., 74: 1071-1077, 1985.

6. Edelstein, M. B., and Heilbrunn. L. K. Infused i.-histidinol and cisplatin:schedule, specificity, and proliferation dependence. J. Nati. Cancer Inst., HI:298-301, 1989.

7. Edelstein, M. B., and Heilbrunn, L. K. Specificity, schedule, and proliferationdependence of infused i -histidinol after 5-fluorouracil in mice. Cancer Res.,48: 1470-1475, 1988.

8. Ridgway, H. J., and Stewart, D. P. L-Histidinol improves the therapeuticefficacy of some conventional antitumor agents against the advanced B16melanoma in mice. Proc. Am. Assoc. Cancer Res., 29:480, 1988.

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10. Sawyer, R. C., Stolfi, R. L., and Martin, D. S. Effect of L-histidinol on themetabolism of 5-fluorouracil in the BALB/c x DBA/8F, murine tumorsystem. Cancer Res., 48: 6664-6668, 1988.

11. Warrington, R. C., and Fang, W. D. Reversal of multidrug-resistant pheno-type of Chinese hamster ovary cells by i.-histidinol. J. Nati. Cancer Inst., 81:798-803, 1989.

12. Stolfi, R. L., and Martin. D. S. Chemotherapeutic activity of i.-histidinolagainst spontaneous, autochthonous murine breast tumors. Chemotherapy,36:435-440, 1990.

13. Schabel, F. M., Jr., Griswold, D. P., Jr., Laster, W. R., Jr., Corbett, T. H.,and Lloyd, H. H. Quantitative evaluation of anticancer agent activity inexperimental animals. Pharmacol. Ther., Ì:411-435, 1977.

14. Lloyd, H. H. Application of tumor models toward the design of treatmentschedules for cancer chemotherapy. In: B. Drewinko and R. M. Humphrey(eds.), Growth Kinetics and Biochemical Regulation of Normal and Malignant Cells, pp. 455-469. Baltimore: Williams & Wilkins Co., 1977.

15. Allenmark, S., Bergstrom, S.. and Enerback, L. A selective postcolumn o-phthalaldehyde-derivatization system for the determination of histamine inbiological material by high-performance liquid chromatography. Anal.Biochem., ¡44:98-103, 1985.

16. Metzler, C. M., Elfring, G. L., and McEwen, A. J. A package of computerprograms for pharmacokinetics modeling. Biometrics, 30: 562-563, 1974.

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1992;52:3604-3609. Cancer Res   Daniel Zaharko, Jacqueline Plowman, William Waud, et al.   Antitumor Agents and Pharmacokinetic Studies in Micel-Histidinol: Preclinical Therapeutic Studies in Combination with

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