1987;47:6142-6146.Cancer Res Merrill J. Egorin, Eleanor G. Zuhowski, Adam S. Cohen, et al. Hexamethylene Bisacetamide MetabolitesPlasma Pharmacokinetics and Urinary Excretion of Updated version http://cancerres.aacrjournals.org/content/47/22/6142Access the most recent version of this article at: E-mail alerts related to this article or journal. Sign up to receive free email-alerts SubscriptionsReprints and . pubs@aacr.org Department atTo order reprints of this article or to subscribe to the journal, contact the AACR Publications Permissions . permissions@aacr.org Department atTo request permission to re-use all or part of this article, contact the AACR PublicationsResearch. on February 22, 2014. 1987 American Association for Cancer cancerres.aacrjournals.orgDownloaded from Research. on February 22, 2014. 1987 American Association for Cancer cancerres.aacrjournals.orgDownloaded from [CANCER RESEARCH 47, 6142-6146, November 15. 1987]Plasma Pharmacokinetics andUrinary Excretion of Hexamethylene BisacetamideMetabolites1Merrill J. Egorin2, Eleanor G. Zuhowski, AdamS. Cohen, Linda A. Geelhaar, Patrick S. Callery, andDavidA. VanEchoDivisions ofDevelopmental Therapeutics andMedical Oncology, Universityof Maryland Cancer Center [M. J. E., E. G. Z., A. S. C., D. A. V. E.J, Divisionof MedicalOncology, Department of Medicine, University of Maryland School of Medicine [M. J. E., D. A. K E.], and Department of Medicinal Chemistry/Pharmacognosy,UniversityofMaryland School ofPharmacy [L. A. G., P.S. C.I, Baltimore, Maryland 21201ABSTRACTIn order to further understand theclinical toxicities of hexamethylenebisacetamide (HMBA) and to allowappropriate in vitro studies, wedeveloped asuitable gas Chromatographie assay andquantified plasmaconcentrations andurinary excretion offour metabolites whichwehadpreviouslyidentifiedin urine of patients receiving 5-dayHMBAinfusionsat 4.8-43.2 g/m2/day. 6-Acetamidohexanoic acid(AcHA) wasthe majorplasma metabolite andreached steady state concentration (C) by24h.AcHAC,, increased from0.120.02(SD) mM at 4.8g/m2/day to 0.72mM at 43.2 g/m2/day. The CAcHA:CHMBA ratio decreased withincreasing HMBAdosage. At dosages below24g/m2/dayplasma C of/V-acetyl-1,6-diaminohexane (NADAH), theinitial metaboliteof HMBA,were below the limitof detectionof ourassay. With HMBAinfusions of24, 33.6, and43.2 g/m2/day, C,, of NADAH were0.16 0.05, 0.14 0.06, and0.190.04mM, respectively. C NADAH:CHMBA ratiosat 24, 33.6, and43.2g/m2/daywere0.18 0.06, 0.080.02, and0.310.05, respectively. Plasma C,, of 1,6-diaminohexane and 6-aminohex-anoicacidwerebelow the limit of detection of our assay. Eachpatient'surinary excretion ofNADAH, AcHA, and 1,6-diaminohexane wasconsistent fromdayto day. Thefraction of doseexcreted in urineasAcHAwas not affectedby HMBAdosageandaccountedfor12.7 3.9%of thedaily dose. The percentage of daily HMBA dose accounted for byexcretionof NADAHdecreased withincreasing HMBAdosage(10.86.0%at4.8 g/m2/day to 4.21.2% at33.6g/m2/day). Urinary excretionof 1.6-diaminohexane always accounted for less than 3%of the dailydose. Our results indicatethat: (a) plasmaconcentrations of AcHAalonecannot explainthe degreeof acidosisobservedwith toxic doses of HMBA;(b) NADAH ispresent inplasma at concentrations that wehavefoundtocause differentiation invitro-, and(c) the probable rate-limiting stepin HMBAmetabolismis the initial deacetylation.INTRODUCTIONHMBA3 (NSC 95580) induces in vitro morphological andfunctional differentiation of murine and human leukemic andsolid tumor cell lines (1-13). Among the class of agents thathavethepotential forinducing thedifferentiation of tumor cellsand that represent an exciting and novel approach to thechemotherapy of neoplasia (14-19), HMBA has a number ofcharacteristics which render it of greatest potential clinical use(20). HMBA was selected for introduction into clinical trialsbecause, of a series of bisacetamides tested, it approachedmaximum differentiation potency (1-5). Inaddition, carefullyconducted clinical and pharmacokinetic studies have documented the ability to achieve concentrations of HMBA inReceived3/5/87; revised8/17/87; accepted 8/24/87.The costs of publication of this article weredefrayed inpart bythe paymentof pagecharges. This article must therefore behereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solelytoindicate this fact.1Supported inpart by American Cancer Society Institutional Research GrantIN-174D, Maryland Cancer Program/University of Maryland, and ContractNO1-CM47734 awarded by theNational Cancer Institute. Department of HealthandHuman Services.2Towhomrequests for reprints should be addressed, at Division of Developmental Therapeutics, University ofMaryland Cancer Center, 655 W. BaltimoreSt., Baltimore. MD21201.3The abbreviations usedare: HMBA, hexamethylene bisacetamide: NADAH,A'-acetyl- 1,6-diaminohexane; DAH, 1,6-diaminohexane; AcHA, 6-acetamido-hexanoic acid; AmHA, 6-aminohexanoic acid; C, steady state concentration.patient plasma equal tothe concentrations required for induction of differentiation invitro(29, 30). Inthis regard, HMBAdiffers fromdifferentiating agents, such as dimethyl sulfoxideandyV-methylformamide, that haveundergone previous clinicalevaluation (21-28). However, adverse effects have been notedwhen HMBA has been administered to humans (29, 30). AtHMBA dosages greater than or equal to33.6 g/m2/day, metabolic acidosis and neurotoxicity occur as dose-limiting toxicities. Platelet count suppression, although not always dose limiting, alsooccurs with HMBA therapy.We have recently utilized gas chromatography/mass spec-trometry toidentify fivemetabolites of HMBA inthe urine ofpatients treated with HMBA (31). These metabolites includedthe major metabolite, AcHA; the monodeacetylated product,NADAH; the bis-deacetylated diamine, DAH; and the aminoacid, AmHA and its lactam, caprolactam (Fig. 1). The importance of quantifying the amounts of each of these metabolitesin bodyfluids of patients treated withHMBA ledustodevelopagasChromatographie analysis which would allowthe routineandsensitive assayofHMBA metabolites in biological samples(32). We have nowused this assay to quantify the concentrations of various HMBA metabolites in plasma and urine ofpatients treated inour phase I trial of HMBA administered by5-daycontinuous infusion (29).MATERIALS AND METHODSPatient Selection andEvaluation. All patients entered into this studyhad histolgica! proof of malignant disease for which conventionalchemotherapy had proven ineffective and for which no other investi-gational therapy with established efficacywas available. Before entryinto this study, each patient had the investigational nature of thetreatment explained, and aninformed consent, approved bythe Institutional Review Board, was signed. Additional details of eligibilitycriteria and the patient population treated have been published previously(29).Reagents. DAH, AcHA, AmHA, acetic anhydride, trifluoroaceticanhydride, 2,2,2-trifluoroethanol, 1,2-diphenylethylamine, and cadaverine wereobtained fromthe Aldrich Chemical Co., Inc. (Milwaukee,WI). NADAH was synthesized from DAH and acetic anhydride asdescribed previously (31).Drug Schedule and Administration. HMBA was supplied by theInvestigational Drug Branch of the National Cancer Institute (Be-thesda, MD). Each500-ml bottlecontained 15 g of HMBAas a solutionin 0.154 MNaCl. HMBA was administered bycontinuous infusionthrough afree-flowing peripheral or central venous catheter. The rateofHMBA infusion wascontrolled byaTravenol Flo-Gard 8000volumetric infusion pump (Travenol Laboratories, Inc., Deerfield, IL). Thestarting dose was 4.8 g/m2/day for 5 consecutive days andapproximatedone-half of thecanine toxicdoselow (20). Doses wereescalated to43.2g/m2/day for 5days according toamodified Fibonacci schedule (29).Doses werenot escalated within individual patients.Sample Acquisition. Heparinized bloodsamples were obtained beforeandatmultiple times duringandafter theHMBAinfusion. Specifically,samples wereobtained at 0, 1, 3, 5, 8, 12, 16, 23, 47, 71, 95, and 120hduring the 5-day infusion and at 5, 15, 30, 60, 120, 240, 360, and6142Research. on February 22, 2014. 1987 American Association for Cancer cancerres.aacrjournals.orgDownloaded from PHARMACOKINETICS AND EXCRETION OF HMBA METABOLITESAcNHCH2(CH2)4 Che xome thyle ne bis oce tomideAcNHCH2(CH2)4CH2NH2/V - oce lyl -1,6 -diominohe xone[AcNHCH2(CH2)4CHO]6- oce tomidohe xonolH2NCH2 (CH2)4CH2NH21,6 - diominohe xoneAcNHCHj(CH2)4COOH6-oce tomtdohe xonoic acid(major me tabolite )H2NCH2 ( CH2>4COOH6"ominohe xonoic ocidFig. 1. Proposed metabolic pathways leading tothe metabolites of HMBA inhumans, a. deacetylase: b, monoamine oxidase;