The Absorption, Distribution, Excretion, and Biotransformation of the Carcinostatic L-(2-Chloroethy!)-3-Cyclohexyl-L-Nitrosourea in Animals1

Home , Cyclohexylamine

[CANCER RESEARCH 30, 1330-1337, May 1970] The Absorption, Distribution, Excretion, and Biotransformation of the Carcinostatic l-(2-Chloroethy!)-3-cyclohexyl-l-nitrosourea in Animals1

Vincent T. Oliverio, Wesley M. Vietzke, Mary K. Williams, and Richard H. Adamson

Laboratory of Chemical Pharmacology, National Cancer Institute, NIH, Bethexda, Maryland 20014

SUMMARY tions have recently produced a family of antitumor agents, the nitrosoureas, with great efficacy in rodent The physiological disposition of l-(2-chloroethyl)-3- tumor screens (6, 8, 16, 18) and man (1, 13). Only one of cyclohexyl-1-nitrosourea, a highly active agent against these agents, BCNUL (NSC 409962), has received inten intraperitoneal and intracranial mouse leukemia L1210, sive clinical trial and was found to be effective against a was studied in mice, rats, dogs, and monkeys with the wide spectrum of advanced solid tumors, including 14C label in each of 3 positions of the molecule, the eth- Hodgkin's disease (1, 9, 11), acute lymphocytic leukemia, ylene, carbonyl, and cyclohexyl moieties. l-(2-Chloro- and its meningeal spread in children (7, 15). ethyl)-3-cyclohexyl-l-nitrosourea is lipid soluble and, Although intensive investigations have been made of after parenteral dosage, rapidly degraded in mouse and the biochemistry (5, 20, 21) and pharmacological disposi dog plasma with 2 exponential phases. The half-life of tion of BCNU in animals and man (2, 10), the active the initial phase is about 5 min, while the 2nd phase ex moiety of this agent is still unknown. This is attributable tends over 1 hr. During the 6 hr after i.v. injection of to the pronounced instability of BCNU in biological sys ethylene-labeled 1-(2-chloroethyl)-3-cyclohexyl-1 -nitro- tems and to the high reactivity of its daughter products sourea in dogs, radioactivity in the cerebrospinal fluid (12). exceeded that of plasma 3-fold. With the label in the cy Another related compound, CCNU (NSC 79037, Chart clohexyl moiety, plasma radioactivity was about 50% of 1), is at least as or more active than BCNU against cerebrospinal fluid UC levels. The drug following biotrans mouse leukemia L1210 and is more lipid soluble, which formation is primarily excreted by the kidneys with ex might enhance passage across the blood-brain barrier. cretion being essentially complete during the first 24 hr Also, in contrast to BCNU, CCNU has only a single ÃŸ- in rodents and monkeys, but more protracted in dogs. chloroethyl group and a cyclohexyl group which can serve as a "handle" in following the metabolic fate of the Biliary secretion and reabsorption from the gastrointesti nal tract have been demonstrated. The cyclohexyl por drug, since it might be expected to be less reactive than tion of the molecule was bound extensively (40 to 60%), the chloroethyl moiety and not as susceptible to degrada while the ethylene moiety was not bound to plasma pro tion. Therefore, we have studied the physiological dispo teins of dogs. In mice, 10 to 20% of the carbonyl and 4 to sition of CCNU in animals in hopes of better elucidating 6% of ethylene carbon atoms were recovered in expired its own metabolic fate and mode of action and that of CO< 1 day after parenteral or oral dosage. The detection the class of nitrosoureas. of cyclohexylamine, N, A"-dicyclohexylurea, among other predominant unidentified molecular species, sup ports the hypothesis of intermediate hydroxydiazoalkane MATERIALS AND MBTHODS and isocyanate formation during the degradation of nitro- Radioactive CCNU was obtained from the Cancer soureas in vivo. However, the identified catabolites and Chemotherapy National Service Center with the 14C la cyclohexyl isocyanate were found to be inactive against bel in 3 separate positions of the molecule: the carbon the mouse leukemia L1210. atoms of the 2-chloroethyl moiety (ethylene-14C-labeled CCNU, 1.37 mCi/mmole), the carbonyl moiety (car- INTRODUCTION bonyl-14C-labeled CCNU, 1.14 mCi/mmole) and uni formly throughout the carbon atoms of the 3-cyclohexyl ring (cyclohexyl-"C-labeled CCNU, 1.13 mCi/mmole). In the search for new and more effective antitumor agents, the combined efforts of several research institu The radioactively labeled drugs were of greater than 95% purity as determined by thin-layer chromatography on ' A portion of this work was presented at the 59th Annual Meeting of the American Association for Cancer Research, Atlantic City, N. J., 2The abbreviations used are: BCNU, 1,3-bis(2-chloroethyl)-l-nitro- April 11to 15, 1968. sourea; CCNU, l-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea; DMSO, Received August 28, 1969;accepted December 4, 1969. dimethyl sulfoxide; CSF, cerebrospinal fluid.

1330 CANCER RESEARCH VOL. 30

to recover from anesthesia, and given the drug; bile was ii r~\ collected for 24 hr without the use of anesthesia. Clâ€”CH2â€”CH2 â€”Nâ€”Câ€”NH-( > Dogs. Twelve female mongrel dogs, weighing approxi mately 15 kg, were given a single i.v. injection of 15 mg/ kg CCNU-UC while under pentobarbital anesthesia. NO Blood samples were withdrawn by an indwelling fem Chart 1. Molecular structure of CCNU, NSC 79037. oral vein catheter and CSF was sampled from an in dwelling spinal needle at the basal cistern. Urine was Silica Gel G plates as described for BCNU (2), except collected continuously for 6 hr by an indwelling Foley that chloroform:95% ethanol (98:2, v/v) was used as catheter. The catheter was then removed and urine sam the developing solvent. ples were collected up to 48 hr from dogs individually All labeled compounds were stored in the dark at 0Â° housed in large metabolism cages. Urine, CSF, and dose until use. For p.o. administration or i.p. injections in solutions were counted directly for total radioactivity and mice and rats, the dose was prepared immediately prior plasma samples were counted following oxygen flask to administration by dissolving CCNU in 95% ethanol combustion as described for BCNU (2). Urine, collected and diluting with propylene glycol until the final volume through a catheter during the first 6 hr, plasma, and CSF contained a glycol:alcohol ratio of 5:0.6 (v/v). For i.v. samples were also assayed for intact CCNU and metabo administration in dogs and monkeys, the compound was lites as described below. A modified Toribara method (3) dissolved in either a mixture of 99% ethanol:0.9% NaCl was used to determine plasma protein binding of radio solution:DMSO (7:5:5,v/v) or 99% ethanol:propylene activity. glycol (7:3, v/v). Monkeys. Four female rhesus monkeys (Macaca mu Radioactive determinations on all dose solutions and latta) were lightly anesthetized with phencyclidine and biological samples were carried out as described pre placed in a metabolism chair. The animals were given a viously for BCNU (2). single i.v. injection of 10 mg/kg labeled CCNU, and blood and CSF samples were obtained by venipunctures Distribution and Excretion of Radioactivity and lumbar punctures at designated time periods. Urine was collected continuously to the end of the experiment by an indwelling Foley catheter. The monkeys were fed Mice. CDFi male mice were given 50 mg/kg (approxi apples and banana pellets and were hydrated by constant mate LDi,,, effective antitumor dose) of each of the la i.v. infusion of dextrose (5%) in 0.9% NaCl solution dur beled CCNU preparations p.o. or i.p. The animals were ing the experiment. One monkey was sacrified 5 days kept in glass metabolism cages, and urine and stool were after drug injection, and the residual radioactivity was collected separately at various time intervals by the anal determined in the organs and tissues as described for cup technique (14). When assays were to be made for parent compound mice. and biotransformation products, the urine was collected in flasks containing 0.2 ml 0.1 N hydrochloric acid im Recovery and Identification of CCNU and Metabolites mersed in a Dry Ice bath. Organs and tissues were ex in Biological Fluids cised from sacrificed animals and homogenized in dis tilled water, and dried aliquots were combusted by the The modified Greiss reaction (2) was used to identify oxygen flask technique to determine radioactivity content intact CCNU in solution and on thin-layer chromato- as described earlier for BCNU (2). Expired >4CO>was grams (Silica Gel G). The thin-layer plates and radio- collected from each of 6 animals over a 24-hr period fol autograms of the plates were prepared as previously de lowing drug administration, and the radioactivity was scribed in the BCNU studies (2). Urine samples were assayed by Steinberg's (17) anthracene system. Urine spotted on plates directly, with a standard reference solu and plasma samples were processed and chromato- tion of CCNU. For assay of intact CCNU, 1.0-ml sam graphed on thin-layer Silica Gel G plates to determine ples of plasma and CSF were extracted twice with 2.0 the presence of parent compound and metabolites as de ml ether in a stoppered tube. The combined ether layers scribed below. were evaporated under a gentle stream of nitrogen at 45Â° Rats. Male Sprague-Dawley rats, weighing approxi and 0.13 ml ice-cold ethanol (95%) was added to the tube mately 250 g, were given 5 mg/kg of each of the labeled which was then agitated on a Vortex mixer for several CCNU compounds by the i.p., i.m., or intragastric sec. Duplicate 50-//1 samples were then spotted on a thin- routes. Urine and feces were assayed for radioactivity as layer chromatogram, one at the origin, and the other described for mice. Biliary excretion studies were carried ahead of the solvent front which served as a control out in 12 animals over a 24-hr period. Animals were ini to indicate the amount of radioactivity at the origin. tially placed under pentobarbital anesthesia, and plastic The plates were then developed in a system of chloro catheters were sutured into hepatic segments of the form: 95% ethanol (98:2, v/v) and the radioactive spots common bile ducts for collection of the total bile. The were either located by using a Baird-Atomic model RSC- animals were then placed in a restraining cage, allowed 363 radiochromatogram scanner or by preparing a radio-

MAY 1970 1331

Table 1 treated with appropriate solvent) consistently had median Excretion of radioactivity in mice after a single parenteral or survivals of 8 to 9 days. Drugs used for antitumor testing oral dose of 50 mg/kg CCNU-" C were CCNU, yv,/V'-dicyclohexylurea (K and K Labora Cumulative % of dose tories, Inc., Plainview, N. Y.), cyclohexyl isocyanate Position of (Aldrich Chemical Company, Cedar Knolls, N. }.), and label Ethylene-"C Carbonyl-"C Cyclohexyl-"C cyclohexylamine (J. T. Baker Chemical Company, Phil- lipsburg, N. J.). CCNU was suspended in 1% methyl i.p. p.o. i.p. p.o. I.p. p.o. cellulose, cyclohexyl isocyanate was suspended in 0.5% (jrÂ¡ne methyl cellulose, cyclohexylamine was diluted with wa 6 hr 46.4 16.0 28.8 32.6 57.7 58.7 ter, and N, jV'-dicyclohexylurea was dissolved in DMS( 24 hr 86.2 79.9 57.3 57.9 78.4 88.7 and then diluted to a final concentration of 10 or 20C Co2(24hr) 3-5 5-8 24-4 10-6 !Ã¬ Â° DMSO. ;V,/V'-Dicyclohexylurea was given to mice a 04 4'5 doses up to 100 mg/kg; all other drugs were injected i Total 89.7 86.9 83.0 69.9 78.8 93.2100

80 Table 2 &Q Chromatographie properties, color reactions, and relative distribution of urinaryinjection biotransformation of 50 mg/kg'a, products cyclohexyl- from '' mice C-labeled 24 hr after CCNU a single i.p.' . _

, R Kvalue in Â§ Compound ÃŽAâ„¢dose S0lvem" CÂ°'orâ€ž o

=CCNU ABC c

SO.l 0.90 0.89 0.76 Purple * JV.yv'-Dicyclohexyl- 35 0.13 0.71 0.73 Yellow" urea CyclohexylaminePurple"The 14 18 0.07 0.52 0.56 solvent systems were: A. chloroform :95% ethanol (98:2): B. n-butyl alcohol: acetic acid:water (4:1:2): and C, isopropyl alcohol: ammonium hydroxide: water (9: 1: 1). 'Color reaction given by: 'Griess reagent (2): ^-dimethylaminoazo- IO 20 3060MINUTES 40 50 benzene (Ehrlich's reagent): and ninhydrin reagent.?v_lililÃ) Chart 2. Plasma disappearance of CCNU in mice during 1st hr fol lowing a single i.p. injection of 50 mg/kg cyclohexyl-'4C parent drug. Each point represents the average value of pooled samples from 4 mice. autogram. The radioactivity in the spots were then deter mined quantitatively by cutting out the appropriate areas in strips and placing them in scintillation counting bottles Table 3 Distribution of radioactivity in mice 24 hr after a single i.p. injection of containing 18 ml toluene-phosphor solution (3 g/liter 50 mg/kg CC/VÃ•AÃ•4C PRO and 100 mg/liter POPOP). Urine samples from mice and dogs were chromato- "C(% Position graphed on thin-layer plates with 3 solvent mixtures as oflabelLiverKidneyLungBrainSpleenSmall developing systems: A, chloroform:95% ethanol (98:2, dose)0.70.3SO.lSO. (cpm/mg)0.3 dose)0.30.2Ã¡O.ISO.mg)12291441165123 v/v); B, n-butyl alcohol: acetic acid: water (4:1:2): 33n.d. and C, isopropyl alcohol:ammonium hydroxide:water 0.2n.d.n.d.n.d.2.00.8n.d.n.d.n.d.1.01.410.657.964.0Cyclohexyl- (9:1:1). System A was suitable for migration of intact CCNU only while Systems B and C were used to chro- 1SO.l2.8) 1SO.l0.2 matograph biotransformation products as well as parent intestine CCNU. In all procedures, known reference compounds LargeintestineStomachGonadsHeartMuscleCarcassFecesCO,UrineTotalElh>lene-(%0.3Ã•SO. 3.8SO. were chromatographed with biotransformation products as indicated in "Results." 1so.lgo.iSO. 1SO.lSO.ln.d.1.40.4078.484.7(cpm/

i2.4<0.13.586.296.6"C:cmT/6368331323161420Carbonyl-"C(%dose) Antitumor Studies in Mice

Male and female CDF, mice were inoculated i.p. with 0.1 ml 1:1,000 dilution of L1210 ascitic fluid obtained from a donor mouse on Day 7. The inoculum contained about 8 X 10' cells. Control mice (nondrug-treated, but

1332 CANCER RESEARCH VOL. 30

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1970 American Association for Cancer Research. l-(2-Chloroethy[)-3-cyclohexyl-l-nitrosoiirea doses that ranged from nontoxic to lethal. Mice were time intervals up to 4 hr did not yield radioactive com treated by i.p. injections on Day 1 only (24 hr after inocu ponents which might have been present in the urine as lation) or daily from Day 1 until death. Ten mice were these conjugates. Thus, approximately 75% of the UC used for each dose level of each drug and the same num label in the cyclohexyl moiety of CCNU represent other ber was used for solvent or untreated controls. Results biotransformation products which have not been identi are expressed as percentage of increase in median sur fied. Urine samples of mice treated with the carbonyl- vival over controls. labeled compounds were only examined for the presence of unchanged CCNU. Chart 2 depicts the rapid decline of intact CCNU in RESULTS the plasma of mice (pooled samples from 4 mice at each designated time) after a single i.p. injection. The half-life Mice. Despite the route of administration and the posi during the first 30 min of exponential fall appears to be tion of the label in the parent drug, excretion was rapid in the order of 5 to 6 min. Thereafter, the drug disap and predominantly by the urinary route (Table 1). More pears more slowly with an estimated half-life of approxi than 75% of the dose was recoverable in the urine 24 hr following administration of the ethylene-uC- or cyclo- mately 100 min. Although the chemical half-life of hexyl-14C-labeled CCNU. Following dosage with the CCNU in plasma appears to be of shorter duration than carbonyl-MC-labeled CCNU, more than 50% of the radio BCNU (2, 10), the plasma disappearance patterns of each drug are similar. activity appeared in the urine during the ensuing 24 hr. The latter reduction in urinary excretion of UC is ac In mice given a single p.o. dose of 50 mg/kg cyclo hexyl-labeled CCNU, radioactivity was detectable in countable by the greater proportion of the radioactivity which appeared in the expired UCO:>. The cyclohexyl plasma as early as 3 min and peaked at 10 min following drug administration. Despite this rapid absorption, less moiety of the parent molecule apparently remains intact since no radioactive I4CO2 was expired by mice during than 0.5% of the plasma radioactivity was associated with intact parent compound. the 24-hr period following administration of the cyclo- The 24-hr tissue distribution studies (Table 3), regard hexyl-labeled CCNU. With each of the labeled CCNU less of the position of the label in the administered preparations, fecal excretion of radioactivity was mini CCNU, showed a high percentage of isotope in the intes mal after parenteral or oral dosage. tines, liver, and carcass. When the results were calcu In urine samples examined by thin-layer chromatog- lated in terms of tissue specific activity, the differences raphy on Silica Gel G followed by spraying with Griess reagent, less than 0.1% of the radioactivity could be asso ciated with intact CCNU used as a standard reference. Table 4 Biliary and urinary excretion of radioactivity in rats 24 hr after a single Radioactivity from urine samples of mice treated with i.m. or intragastric injection of 5 mg/kg CCNU-nC ethylene-labeled CCNU did not migrate from the origin of thin-layer plates in either polar or nonpolar solvent Each value represents the average obtained from 2 animals. systems with the exception of intact drug (^0.1%). Position Conversely, urine samples of mice treated with cyclo oflabelEthylene-"CCarbonyl-"CCyclohexyl- (%dose)21.818.325.024.053.825.2Bile(%dose)39.146.941.234.925.232.0Total60.965.266.258.979.057.2 hexyl-labeled CCNU yielded a number of distinct radio active spots which migrated from the origin to give various RF values in several polar solvent systems. Incu bation of untreated mouse urine with cyclohexyl-labeled CCNU at 37Â°for periods up to 1 hr did not yield the bio- NCRoutei.m.Â¡â€¢g-Â°i.m."â€¢g-i.m.'â€¢g-Urine transformation products obtained from in vivo experi ments. In these control incubations, only unchanged " Intragastric. CCNU was recovered. From urine samples of treated animals, in addition to a trace of unchanged CCNU, 2 of the radioactive spots were identified in 2 Chromato graphie systems as cyclohexylamine (14 to 18% of radio Table 5 active dose) and N, /V'-dicyclohexylurea (3 to 5%). The Urinary excretion of radioactivity in dogs after a single i.v. injection of 15 mg/kg CCNU-^C R F values for both compounds in the various solvent sys tems using the known reference compounds are shown doseEthylene-NCÂ£0.11.48.117.536.767.9Cyclohexyl-'4C1.62.018.638.764.484.3percentage of in Table 2. Cyclohexylamine spots were located on thin- Timeinterval(hr)0.51362448Cumulative layer plates sprayed with ninhydrin reagent and then heated for 10 min in an oven at 110Â°.TV,/V'-dicyclohex ylurea spots were located on the chromatograms sprayed with 4-dimethylaminoazobenzene and exposed to con centrated hydrochloric acid vapors. Incubation of the urine samples of mice treated with the cyclohexyl-labeled CCNU with sulfatase or glucuronidase at 37Â°for various

MAY 1970 1333

3500 out by biliary excretion studies in rats summarized be low. Rats. Recovery of radioactivity in the urine (~75%) 3000 and feces (~4%) of 2 rats given i.p. doses of the ethyl- W ene- or cyclohexyl-labeled CCNU was similar to recover II / \ CIâ€”CH2â€”CH2â€”N-C-NH-Â¿ * ÃŒ ies obtained in mice. Biliary and urinary excretion of NO the labeled CCNU compounds was studied in 12 male Sprague-Dawley rats. In the first 6 hr following intragastric or i.m. injection of 5 mg/kg CCNU-14C, 15 to 2000 30% of the dose was excreted, and by 24 hr (Table 4) 25 S CL to 47% of the dose was recovered in the bile. Minimal Ã¼ differences were found when the position of the label in the molecule varied. Urinary excretion of radioac tivity in most of the animals was somewhat decreased, 1000 averaging about 23% (with the exception of 2 animals) of the 14Cdose, but the total recovery via bile and urine approximated the recovery obtained with noncannu- 500 lated rats. Dogs. The results of urinary excretion of radioactivity in female mongrel dogs given a single i.v. injection of 2345 either ethylene-uC-labeled CCNU or cyclohexyl-14C- HOURS labeled CCNU are summarized in Table 5. Following Chart 3. Plasma and CSF levels of CCNU and biotransformation injection of ethylene-uC-labeled CCNU, urinary excre products in a female mongrel dog following a single i.v. injection of 15 tion of radioactivity is somewhat more protracted during mg/kg cyclohexyl-"C parent drug. the first 24 hr as compared to excretion observed follow ing administration of the cyclohexyl-14C-labeled drug. In the animal treated with cyclohexyl-14C CCNU, urine col 3500 lected through a catheter during the first 6 hr contained approximately 10% of the dose as cyclohexylamine and less than 1% as /V,/V'-dicyclohexylurea. The remaining 3000 radioactivity (about 28% of the dose) could not be identi-

/o_l \Z \l 1 CIâ€”CH2-CH2â€”* * Nâ€”Câ€”NH-( II / ) ^'_ 2000 T/ ' N 14500 NOÃŒo ,/ \ â€”\ |U3500 .o-0"1*V,*-â€ž PLASMA-1* C hmn/r ^ v'"'^* Â£(J12500 *'*^ ,'â€” â€”URINE -"**-' 70ff*> ^M1500 PLASMALO\kÂ«IOOOP-V /^-|L ^'KAâ€” J*c .-'' â€”â€¢"''-â€¢' \ --. xÂ«^ Â¿5001 A800 \ "â€¢â€¢ 60i_2 *~~~~-~t_ ~^_*>/*xÂ« _^1^ csr- I4c -j \CCHU-I4C\ 50z3U40HI1 ^f ^lv^^**^? ,' j i5500 0â€” _i tf'/'- 110-t-T-A--,-.!. jb^JL 1 1 1 1 7MINUTES30 50 2 3 4 5 6 HOURSChart 302010C1\â€”/II \CI /â€” 4. Plasma and CSF levels of CCNU and biotransformation â€”CH2â€”CH2-N-C-NH-^ Â«) products15mg/kg in a female mongrel dog following a single i.v. injection of drug.fromethylene-"C parent NOâ€” /'' ,1.1,1.1.1.1n1,1,2 relativelyhighorgan to organ were not apparent. The 45HOURS 6 6 10 12 " 1 3 concentration of isotope in the intestines, and not in1009080rf DAYS the feces, suggested that radioactivity derived from the Chart 5. Urinary excretion and plasma and CSF levels of radioac drug was rapidly excreted in the bile and had undergone tivity in a female rhesus monkey following a single i.v. injection of 10 enterohepatic recirculation. This possibility was borne mg/kg cyclohexyl-14CCCNU.

1334 CANCER RESEARCH VOL. 30

6000 plasma radioactivity with a subsequent second peak as was seen with BCNU (2) probably reflects the change in lipid solubility of the molecular species associated with 100 1600 the UC. It appears that the ethylene portion of the CCNU molecule has no difficulty in crossing the blood- 1400 90 jL.O URINE-*C .--â€¢'"' brain barrier while the cyclohexyl portion, perhaps by virtue of its substantial plasma protein-binding and lipid- 1200 80 insoluble characteristics, is concentrated in the CSF to a 1000' much less extent. Of particular pertinence is the finding So that, in dogs given the ethylene-14C-labeled CCNU par- lu > enterally, no plasma protein binding of radioactivity was 60 800 observed, which suggests that the binding occurs mainly with the cyclohexyl moiety. It is also apparent from the l 50 600 :s observation of the extremely short half-life of both the o cyclohexyl- and ethylene-14C-labeled CCNU in dog u 40 400 plasma that only a minute portion of the radioactivity in the CSF represented entry of intact parent compound. 30 200 Indeed, this was the case when CSF radioactivity was analyzed for intact CCNU. 20 Monkeys. The urinary excretion and concurrent ** II / \ CI-CH2-CH2-N-C-NH-Â¿ ) plasma and CSF levels of radioactivity following injection IO of cyclohexyl-14C-labeled CCNU in the monkey were NO similar to the pattern obtained in the dog. In 1 of 2 mon keys studied (Chart 5), the plasma half-life of radioactiv 4 6 B IO 12 I 3 5 ity after the initial fall-off was about 24 hr. The CSF HOURS DAYS levels of 14C reached about 24% of the plasma radioac Chart 6. Urinary excretion and plasma and CSF levels of radioactiv ity in a female rhesus monkey following a single i.v. injection of 10 mg/ tivity level at 2 hr and about 8% at 24 hr. Almost 90% of kg ethylene-MC CCNU. the radioactive dose had been excreted in the urine dur ing the 5 days following drug injection. In each of the 2 monkeys given a single i.v. injection fied with a particular molecular species. In animals of ethylene-14C-labeled CCNU, there was a rapid rising treated with CCNU labeled in either position, less than appearance of radioactivity in the CSF, but there was 0.1% of the radioactive dose was excreted as intact par also a more rapid disappearance of label when compared ent drug. to a similarly treated dog. This is illustrated for 1 of the Charts 3 and 4 depict the plasma and concurrent CSF treated animals in Chart 6. As observed with the dogs levels of intact CCNU and total radioactivity in 2 female and monkeys given the cyclohexyl-14C-labeled CCNU, mongrel dogs given a single i.v. dose of cyclohexyl-'4C- labeled CCNU and ethylene-'4C-labeled CCNU, respec following the rapid initial falloff of radioactivity in the plasma, which probably represents mostly intact lipid- tively. With the cyclohexyl-labeled CCNU (Chart 3), soluble parent compound, the drug undergoes biotrans- radioactivity enters the CSF rapidly, but is only about formation, resulting in release of a lipid-insoluble fraction 55% of the plasma level between 30 and 60 min after in jection of the drug. Most of this radioactivity represents ether-nonextractable products of the parent compound since the initial rate of decline of the ether-extractable Table 6 Distribution of residual radioactivity in afemale rhesus monkey 5 days intact CCNU in both plasma and CSF is very rapid, with after a single i.v. injection ofethvlene-'4C CCNU (10 mg/kg, a half-life of about 5 min. ÃŒ8.3X10scpm) These results might be explained on the basis of activity plasma protein binding of the cyclohexyl portion of the OrganLiverBileKidneySpleenLungHeartMuscleSmall CCNU molecule which would partially exclude its entry (cpm/g)9,4101,18057039031030024019018000 into the CSF. In dogs given the cyclohexyl-14C-labeled CCNU, plasma protein binding of radioactivity of about 45% initially rose to about 60% by the end of 6 hr. Approximately 0.5 hr following i.v. administration of ethylene-'4C-labeled CCNU in dogs, the level of radio activity in the CSF was at least 3-fold greater than that of plasma. This radioactivity was almost entirely ether intestineLarge nonextractable since the ether-extractable parent CCNU intestineOvaryBrainSpecific in plasma and CSF was rapidly degraded during the first 10 min following injection of drug. The initial dip in

MAY 1970 1335

Table 7 The data from mice given CCNU p.o. suggest that the Antitumor effect of CCNU and catabolites against drug is probably degraded prior to and/or simultaneously mouse leukemia LI2IO with the absorption process since the radioactivity in plasma samples at early time intervals following drug of median dosage was almost entirely associated with nonparent CompoundCCNUCyclohexylamineCyclohexyldoses(mg/kg)1.25-1015-506.25-5050-150'7.5-1515-75'50-100TreatmentscheduleDailyDaydose(mg/kg)5-1025-356.25-25507.51550-100Increaseinsurvivalover controls265â€” molecular entities. The primary excretory route of the parent compound or its biotransformation products (or both) is through the kidneys, with biliary excretion 1onlyDailyDay â€¢00000 and reabsorption from the gastrointestinal tract playing a predominant role. The cyclohexyl portion of the mole 1onlyDailyDay cule is bound to plasma proteins, whereas the chloroethyl iso segment is not. The carbonyl and chloroethyl carbons are cyanateN,N'-D\cy\o-hexylureaRange 1onlyDailyOptimal partially recoverable in expired CO^ while the cyclohexyl carbon atoms are not. Thus, the cyclohexyl ring is prob ' Eighty to 90% survivors at 8 months following inoculation. ably not extensively degraded in vivo. Finally, in rodents, ' Range from a nonlethal to a lethal dose. the parent compound and/or its biotransformation prod ucts do not appear to be localized selectively in any of the tissues or organs examined. In the monkey, how into the blood stream which is characterized by the sec ever, retention of some fragment(s) of CCNU was ob ond peak. Here too, after the initial falloff of radioactiv served 5 days following parenteral drug administration. ity, the plasma-14C half-life was about 24 hr. Future studies will be pursued to determine whether re One of the monkeys treated with ethylene-14C-labeled tention of the bound radioactivity in the monkey liver is CCNU was sacrificed after 5 days, and the radioactivity associated with a particular cell fraction or other tissue was determined in the organs and tissues designated in sites. Table 6. As was similarly found in studies with MC- The above observations parallel those made in studies labeled BCNU (2), in contrast to the homogeneous spe of the fate of BCNU in animals (2) and indicate that cific activity of organs in the mouse at 24 hr following there is probably little difference between the physiologi administration of ethylene-14C-labeled CCNU, the mon cal disposition of these 2 drugs which have similar anti- key showed appreciable concentration of radioactivity in tumor potencies in experimental animal tumors. Re the liver 5 days following treatment. Bile taken from the cently, however, CCNU has shown a marked superiority gallbladder also contained significantly high radioactive over BCNU in the treatment of an experimental mouse counts. Despite this, the intestinal radioactivity was con ependymoblastoma (percutaneous). The availability of siderably less. CCNU isotopically labeled in various positions of the Antitumor Studies in Mice. Table 7 presents results of molecule has afforded a clearer elucidation of in vivo fate the effect of CCNU and 3 of the catabolites of the parent of the different segments of the nitrosourea molecule. compound which have been identified: namely, cyclo- Therefore, on the basis of the results obtained here and hexyl isocyanate, cyclohexylamine, and W, /V'-dicyclo- from previous studies with BCNU in this laboratory (2, hexylurea against leukemia L1210. Only CCNU had 10), the in vivo biotransformation of CCNU is envisioned antitumor activity; the other compounds were inactive as proceeding according to the in vitro decomposition when tested in doses up to 100 mg/kg or in doses that scheme for BCNU proposed by Montgomery et al. (12). produced lethality. CCNU showed greater antitumor ef Following parenteral administration of a single dose of fect when given as a single large dose on Day 1 than CCNU in animals, a small but measurable quantity of when given daily. When given on Day 1, CCNU at the lipid-soluble parent compound enters the CSF before optimal doses (25, 30, 35 mg/kg) consistently gave 80 to degradation occurs. The parent compound could then de 90% long-term survivors (8 months). grade similarly in blood, brain, and CSF, according to the suggested scheme (1) which involves initial rear rangement of the CCNU molecule to an oxazolidine in DISCUSSION termediate which almost instantaneously cleaves to form ethylenediazohydroxide and cyclohexyl isocyanate. Hy These studies have established several important facts drolysis of the isocyanate would result in the amine. In concerning the physiological disposition of CCNU in ani direct evidence for isocyanate formation was obtained mals. in the present studies by identification of radioactive The parent compound is lipid soluble, but the major cyclohexylamine and N, /V'-dicyclohexylurea in the urine portion of the drug entering the CSF following paren- of mice given cyclohexyl-labeled CCNU and 14CO2 in teral administration is associated with its lipid-soluble expired air following administration of carbonyl-labeled degradation products. The chemical and radioactive CCNU. Radioactive cyclohexylamine and N, /V'-dicyclo- half-lives of the parent compound are extremely short, hexylurea were also detected in the urine of a dog treated being approximately 5 min in both plasma and CSF of with cyclohexyl-14C CCNU. Cyclohexylamine and CO2 dogs and plasma of mice during the initial disappearance would be expected to arise from hydrolysis of the isocy phase. anate. The amine would then be excreted largely un-

1336 CANCER RESEARCH VOL. 30

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1970 American Association for Cancer Research. I-(2-ChloroethyÃ)-3-cyclohexyl-l-nitmsoiirea changed in the urine which is in accordance with results 2. DeVita, V. T., Denham, C., Davidson, J. D., and Oliverio, V. T. of studies of the metabolic fate of cyclohexylamine in The Physiological Disposition of the Carcinostatic 1,3-Bis-(2-chlo- rabbits reported by Elliott et al. (4). The small amount of roethyl)-l-nitrosourea (BCNU) in Man and Animals. Clin. Phar- urea derivative isolated probably resulted from the inter macol. Therap., 8: 566 577, 1967. action of the amine with free isocyanate. Access of the 3. Dixon, R. L., and Adamson, R. H. Antitumor Activity and Phar macologie Disposition of Cytosine Arabinoside (NSC 63878). Can isocyanate and the amine into the CSF might be limited cer Chemotherapy Rept., 48: 11-16, 1965. by the demonstrated plasma protein binding of the cyclo- 4. Elliott, T. H., Lee-Yoong, N. Y., and Tao, R. C. C. The Metabo hexyl moiety of CCNU. The results of the present exper lism of Cyclohexylamine in Rabbits. Biochem. J., 109: IIP 12P, iments shed little or no light on the fate of the chloroethyl 1968. moiety. The recovery of a small portion of the chloroethyl 5. Gale, G. R. Effect of 1,3-Bis-(2-chloroethyl)-l-nitrosourea on Ehr group of CCNU in expired CO2 does demonstrate 2-car- lich Ascites Tumor Cells. Biochem. Pharmacol., 14: 1707 1712, bon metabolism. Furthermore, since it was demonstrated 1965. that the chloroethyl carbon atoms were not bound to 6. Green, S. The Effect of 1,3-Bis-(2-chloroethyl)-l-nitrosourea on plasma protein, it might be questioned whether the left Nicotinamide Adenine Dinucleotide Glycohydrolase of Mouse and side of the molecule is bound at all. However, the hy- Rat Neoplastic and Normal Tissue. Cancer Res., 26: 2481 2484, 1966. droxy diazoalkane or subsequent metabolic products may 7. Iriarte, P. V., Hananian, J., and Cortner. J. A. Central Nervous well be bound to some site other than protein. Indeed, System Leukemia and Solid Tumors of Childhood: Treatment with this has been suggested as the mechanism of action of l,3-Bis-(2-chloroethyl)-l-nitrosourea. Cancer, 19: 1187 1194, 1966? drugs with alkylating-like activity (19). 8. Johnston, T. P., McCaleb, G. S., Opliger, P. S., and Montgomery, We still have not explained the carcinostatic activity or J. A. The Synthesis of Potential Anticancer Agents. XXXVI. N- the delayed toxicity of these compounds for liver, kidney, Nitrosoureas. II. Haloalkyl Derivatives. J. Med. Chem., 9: 892-911, or marrow. Certainly, the postulated intermediate bio- 1966. 9. Lessner, H. E. BCNU (1,3-Bis(/3-chloroethyl)-l-nitrosourea. Ef transformation products cyclohexyl isocyanate, cyclohex fects on Advanced Hodgkin's Disease and Other Neoplasia. Can ylamine, and dicyclohexylurea, both the latter being ex cer, 22; 451-456, 1968. creted in the urine, were all inactive against the L1210 10. Loo, T. L., Dion, R. L., Dixon, R. L., and Rail, D. P. The Antitu- mouse leukemia under our experimental conditions. mor Agent, 1,3-Bis-(2-chloroethyl)-l-nitrosourea. J. Pharm. Sci., Also, on the basis of the infinitesimal detection of in 55: 492 497, 1966. tact parent compound in the plasma of mice given 11. Moertel, C. G., Reitemeier, R. J., Hahn, R. G. Therapy of Ad CCNU P.O., it is questionable whether the parent com vanced Gastrointestinal Cancer with 1,3-Bis-(2-chloroethyl)-l- pound is, indeed, the active carcinostatic agent. Since nitrosourea (BCNU). Clin. Pharmacol. Therap., 9: 652-656, 1968. CCNU is equally active by the oral and parenteral routes 12. Montgomery, J. A., James, R., McCaleb, G. S., and Johnston, T. P. against the murine leukemia L1210 (8), the activity most The Modes of Decomposition of 1,3-Bis-(2-chloroethyl)-l-nitro- likely emanates from biotransformation product(s). The sourea and Related Compounds. J. Med. Chem., 10: 668 674, 1967. 13. Nies, B. A., Thomas, L. B., and Freireich. E. J. Meningeal Leuke above biotransformation products represent only several mia: A Follow-up Study. Cancer, 18: 546-553, 1965. of the numerous possible metabolic products of CCNU 14. Oliverio, V. T., and Davidson, J. D. The Physiological Disposition and further efforts will be made to isolate, identify, and of Dichloromethotrexate-CI"1 in Animals. J. Pharmacol. Exptl. determine the role of other chemical moieties in the Therap., Â¡37:76-83, 1962. antitumor effectiveness of CCNU and other nitrosoureas. 15. Rail, D. P., Ben, M., and McCarthy, D. M. l ,3-Â¿/.v-fÃ-Chloroethyl- Finally, the observed high levels of both urinary and bil l-nitrosourea (BCNU): Toxicity and Initial Clinical Trial. Proc. iary excretion of both sides of the CCNU molecule indi Am. Assoc. Cancer Res., 4: 55, 1963. cate that both liver and kidney are exposed to relatively 16. Schabel, F. M., Jr.. Johnston. T. P., McCaleb, G. S.. Montgomery, high concentrations of metabolic degradation products J. A., Lester, W. R., and Skipper, H. E. Experimental Evaluation which might be bound and adversely affect cellular func of Potential Anticancer Agents. VIII. Effects of Certain Nitro soureas on Intracerebral L1210 Leukemia. Cancer Res., 23: 725- tion at a later period. 733, 1963. 17. Steinberg, D. A New Approach to Radioassay of Aqueous Solu ACKNOWLEDGMENTS tions in the Liuqid Scintillation Spectrometer. Anal. Biochem., 1: 23-39, 1960. We are grateful for the able technical assistance of Miss Noreen 18. Sugiura, K. Effect of BCNU on a Spectrum of Tumors. Proc. Am. Considine, Mrs. Margie Beaudet, Mr. Ernie Owens, and Mr. Sidney T. Assoc. Cancer Res., 6: 62, 1965. Yancey. Special thanks are due Mrs. Connie Caddington for prepara 19. Wheeler, G. P. Studies Related to the Mechanism of Action of tion of the manuscript. Cytotoxic Alkylating Agents: A Review. Cancer Res.. 22: 651 688, 1962. REFERENCES 20. Wheeler, G. P., and Bowdon, B. J. Effects of 1,3-Bis-(2-chloroethyl)-l-nitrosourea upon the Synthesis of Protein and Nucleic l. DeVita, V. T., Carbone, P. P., Owens, A. H., Jr., Gold, G. L.. Acids in Vivo and in Vilro. Cancer Res., 25; 1770-1778, 1965. Krant, J. J., and Edmonson, J. Clinical Trials with 1,3-Bis-(2-chlo- 21. Wheeler, G. P., and Bowdon, B. J. Effects of 1,3-Bis-(2-chloro- roethyl)-l-nitrosourea, NSC 409962. Cancer Res., 25: 1876-1881, ethyl)-l-nitrosourea and Related Compounds upon the Synthesis of 1965. DNA by Cell-free Systems. Cancer Res., 28: 52-59, 1968.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1970 American Association for Cancer Research. The Absorption, Distribution, Excretion, and Biotransformation of the Carcinostatic 1-(2-Chloroethyl)-3-cyclohexyl-1-nitrosourea in Animals

Vincent T. Oliverio, Wesley M. Vietzke, Mary K. Williams, et al.

Cancer Res 1970;30:1330-1337.

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