a radioimmunoassay for...

[CANCER RESEARCH 37, 619-624, February 1977J

MATERIALS AND METHODS

A rapid and reliable radioimmunoassay method for 1-f3-D-arabinofuranosylcytosine (ama-C)has been developed usingantibody induced in rabbits, [3Hlara-C, and a Millipore filtration technique. The sensitivity of this assay was such thatama-C,0.02 @g/ml,in plasma could be detected, and theassay was practically free from interference by deoxycytidine, cytidine, 1-/3-D-arabinofuranosyluracil, and other nucleosides, as well as from various antibiotics. Blood levelsof ama-Cin C57BL x DBA/2 F1mice were determined afterinjection of 1-(3-O-octanoyb-f3-D-arabinofuranasyl)cytosine.Relatively high ama-Clevels could be maintained for a fairlylong period. Plasmas of mouse, mat,and rabbit containedhigh esterase activity which hydrolyzed the 3'-octanoybgroup in 1-(3-O-octanayl-fJ-D-amabinofumanosyl)cytosine,whereas this activity was relatively low in dog and humanplasmas.

INTRODUCTION

ama-C,3a clinically effective antileukemic drug, exerts itscytotoxic effect only during a limited period of the cell cycle(1, 16). Furthermore, the drug, when administered to human patients or experimental animals, is degraded rapidlyto the biologically inert metabobite, ama-U(7). We have beenengaged in a study concerning the antileukemic effects of aseries of 3'-esters and 3',5'-diesters of ama-C(5, 9). Theprolonged antileukemic activity of certain of these campounds is probably related to the slow enzymatic hydrolysisof the ester groups beading to a gradual release of ama-Citself. The development of a simple and sensitive assaymethod for ama-Cwill greatly facilitate the design of dosetreatment schedules for use with leukemic patients andpharmacokinetic studies in human and animal systems (16).In this communication we describe a madiaimmunoassayprocedure for measurement of ara-C. The method is simple,reliable, and sufficiently sensitive for measurement of ama-Cin plasma, while being insensitive to interfering substancesthat affect existing microbiological and enzymatic assaysystems (6, 10).

The synthesis of S-ama-C(Ill) was achieved via the selective acylation of the 5'-hydmoxyl group of ama-Chydrochlaride in dimethylacetamide using the general procedure ofGish et a!. (4). The acylating agent used was 2,2,2-tnichbonoethylsuccinyl chloride (Ib), which was prepared by treatment of 2,2,2-trichboroethyb hemisuccinate (Ia) with thionylchloride. The crystalline 5'-ester (II), obtained in 84% yieldas the hydrochloride and readily convertible to the freebase, was then treated with zinc and acetic acid in dimethylfommamide in order to cleave the tnichboroethyb ester (17).Following ion-exchange removal of zinc ions and desaltingvia a charcoal adsorption step, analytically and chromatographically pure Ill was obtained in 59% yield (Chart 1).

2,2,2-Trichboroethyl Hemisuccinate (Ia). A solution of2,2,2-tnichloroethanol (15.0 g; 0.10 mole), succinic anhydnide (12.0 g; 0.12 mole), and tniethylammne (8.7 ml; 0.12mole) in ethyl acetate (100 ml) was heated under reflux for 1hr. The solvent was then evaporated, and a solution of theresidue in 5% aqueous sodium bicarbonate was washedtwice with ether and then acidified to pH 2 with sulfuricacid. The resulting solid was washed well with water, driedin a vacuum, and recrystallized from chloroform:hexanegiving 19.2 g (77%) of Ia m.p., 88-89Â°[reported (2) m.p.,88-90Â°]:NMA (CDCI3), 2.77 ppm (s, 4, COCÂ±12),4.86 (s, 2,O@f@2);IA (KBm),3200, 1700 cm'.

1-[5-O-(2,2,2-Trichloroethyl Succinyl)-,3-D-arabinofuranosyljcytosine (II). A mixture of Ia (2.5 g; 10 mmoles) andthianyl chloride (6.5 ml; 90 mmoles) was heated at 65Â°for 30mm and then evaporated to dryness in a vacuum and storedfor 1 hr under high vacuum. The residue, lb (IA, 1795 cmand no bands at 3200, 1710 cm), was dissolved in N,Ndimethylacetamide (15 ml) together with ama-Chydrachbaride (2.79 g; 10 mmoles) and stirred at room temperature for2 hr. The solvent was evaporated under high vatuum at 65Â°,and the residue was crystallized from 2-pnopanol giving 4.31g (84%) of lbas its hydrochloride, m.p. 158â€”161Â°.

C5H,9N3C140, (511.17)

Calculated:C 35.25, H 3.75, N 8.22, Cl 27.75Found: C 35.12, H 3.70, N 8.16, CI 27.58

1 Contribution No. 127 from The Institute of Molecular Biology.

2 To whom requests fQr reprints should be addressed.

3 The abbreviations used are: ara-C, 1-fJ-D-arabinofuranosylcytosine; ara

U, 1-$-D-arabinofuranosyluracib; S-ara-C, 1-(5.O-succinyl-/3-D-arabinofuranosyl)cytosine; NMR, nuclear magnetic resonance; O-ara-C, 1-(3-O-octanoyl-@-D-arabinofuranosyl)cytosine.

Received May 27, 1975; accepted November 11, 1976.

A sample of the hydrochloride (2.0 g; 3.9 mmoles) wasdissolved in water (25 ml), and 0.5 M sodium carbonate (4ml) was added. The resulting precipitate was collected,washed with water, dried, and crystallized from 2-prapanalgiving free base II (1.32 g, 71%), m.p. 94â€”95Â°:Amax(metha

619FEBRUARY1977

A Radioimmunoassay for 1-f@-D-ArabinofuranOSyICytOSine1

Tadashi Okabayashi,2 Shinichi Mihara, David B. Repke, and John G. Moffaft

ShionogiResearchLaboratory,Fukushima-ku,Osaka,Japan553(T.0., S.M.J,and TheInstituteof MolecularBiology,SyntexResearch,PaloAlto, California,94304(J. G. M., D. B. R.J

SUMMARY

on May 27, 2019. © 1977 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

T. Okabayashi et a!.

NH2

0 0CCi3CH,OCCH,CH2COC@@@@

0HOCCH2CH2COi@H@0@

cabCo., St. Louis, Mo. ama-Uwas a product of Calbiochem,San Diego, Calif.

Preparation of Antigen. S-ama-Cwas coupled to humanserum albumin by the method of Oliver et a!. (13). Forty mgof human serum albumin were dissolved in 4.0 ml of waterfollowed by the gradual addition of 8.01 mg of S-ama-C(23.28 @moIes)and 20 mg of 1-ethyl-3-(3-dimethylammnaprapyl)carbodiimide HCI, the pH being adjusted to 5.5 with1 M sodium bicarbonate after each addition. The reactionmixture was incubated in the dark at 25Â°far16 hr and thendialyzed against 0.15 M NaCI:0.01 M sodium phosphate, pH7.4, at 4Â°for 48 hr with 8 changes of the dialyzing solution.The conjugate gave an absorption maximum at 275 nm atpH 7.4. On the basis of the albumin content of the conjugatedetermined by the method of Lowry et al. (8) using humanalbumin as the standard, and the molecular extinction coefficients of unconjugated human albumin and S-ama-Cat 280nm, the conjugate was estimated to contain 8.2 ama-Cresidues per albumin molecule.

Immunization. Seven randomly bred rabbits were immunized with 1 mg of S-ama-Calbumin conjugate (injected 0.25mg in each foot pad) emulsified in Freund's complete adjuvant. Booster injections totaling 0.5 mg were injected atmonthly intervals, and the rabbits were bled from an earvein 1 week later. Plasma was separated by centnifugationand heated at 60Â°for 60 mm to inactivate esterase activity(see below). The heated plasma was centrifuged again, andthe supemnatant was adjusted to final concentrations of0.1% with sodium azide and 0.01% with sodium Merthiolateand then stoned at 4Â°.ama-Cantibody was demonstrable inall rabbits after 3 successive booster injections, and 3 of 7plasmas were capable of binding more than 2 nmoles of amaC pen ml of plasma atsaturation.We selecteda plasma(designated as Ab-3) far use in assay development. Thedissociation constant for ama-Cbinding to this plasma was6.6 x 10@ M as determined by a Scatchard plot (14).

Immunoassay Procedure. The standard assaymixture, ina total volume of 0.1 ml of 50 mM sodium acetate buffer (pH6.2), contained 200 j.@gof bovine serum albumin, 2 pmobesof [3H]ara-C (8.19 Ci/mmole), 10 to 50 @.dof ama-Cstandardor unknown solution, and 10 @lof antibody (diluted 1:33 inH@O).The binding reaction was initiated by adding antibody, and the reaction mixtures were incubated in an icebath for 2 hr. Bound and free ama-Cwere separated byfiltration through a Millipone filter, and the bound radioactivity was determined with a Beckman Model LS-230 liquidscintillation spectrometer (3).

Chart 2 presents a graphic representation of 6 replicateradioimmunoassays expressed as Co/Cs versus S, whereCo and Cs denote the radioactivity (cpm) of [3H]ara-C metamed on a Millipame filter (bound [3H]ara-C) in the absenceor presence of a particular amount (S pmoles/tube) ofunlabeled ama-C,respectively. The curve was linear over awide range of S. Sensitivity of this assay, defined as thelowest amount of ama-Cthat can be significantly distinguished (p < 0.05) from a control with no ama-C,was 0.7pmole. The assay precision, determined from the standardcurve, was 0.10 at 1 pmole.

Routine analyses were carried out in triplicate.Blood Level Study. An aqueous 2% suspension of 0-ama

0 0CCI,CH,OCCH,CH,CX

I (aIX OH(b)X Ci

II Ill

Chart 1.

nob) 229 nm (sh, â‚¬7,400), 273 (8,500); NMA (d6-dimethylsulfoxide) 2.71 ppm (m, 4, COCH2), 3.94 (m, 3, C4@H,C5@H2),4.27 (m, 2, C2H, C3H), 4.88 (s, 2, CCI3CH2), 5.53 (m, 2,C2OH, C3OH), 5.66 (d, I, J5.6= 7.5 Hz, C5H), 6.07 (d, I, J1'.2'= 3.5 Hz, C1H), 7.05 (bm s, 2, NH2), 7.46 (d, I, C6H).

C,5H18N3C13O8 (474.70)

Calculated: N 8.85, Cl 22.41Found: N 8.77, Cl 22.12

S-ara-C (111).Zinc dust (280 mg; 4 mmoles) and glacialacetic acid (0.40 ml) were added to a solution of II (820 mg;1.72 mmoles) in dimethylfarmamide, and the mixture wasstirred at room temperature for 6 hr. It was then filtered,and the solid was washed with water. The filtrates wereevaporated, and an aqueous solution of the residue waspassed through a column of Dowex 50 (NH4@)resin. Evaporation of the eluates and aqueous washings left a semisolid residue that was dissolved in water and passedthrough a column of charcoal (20 g of Chromato TokuseiShirasagi from Takeda Pharmaceutical Industries, Osaka,Japan). The column was thoroughly washed with water (1liter) and then eluted with 50% aqueous ethanol. Evaponation of the UV-absarbing fractions left 350 mg (59%) ofIll as a foam that was homogeneous by thin-layer chromatography (1-butanol:acetic acid:water, 5:2:3) and thinlayer electrophoresis (0.05 M Na2HPO4, pH 8.9): Amax(methanol) 229 nm (sh, a 7300), 273 (8400); [a]@3113.7Â°(c0.4, methanol); NMR (d5-pynidmne-D20) 2.33 ppm (5, 4,COCH2), 4.6 to 4.75 (m, 3, C3H, C4@H,Cs'bH),4.85 (dd, 1, Jgem= 11.5 Hz, J4',s'b 4 Hz, Cs'bH), 5.02 (dd, 1, J1',2' = 3.5 Hz,

J2,.3,= 2 Hz, C2@H),6.10 (d, 1, J56 = 7.5 Hz, C5H), 6.97 (d, 1,C,@H),8.10 (d, 1, C6H).

C13H17N3O, (343.29)

Calculated: C 45.48, H 4.99, N 12.24Found: C 45.63, H 5.19, N 11.78

0-ama-C was synthesized as described previously (5, 9).This compound had a fairly high solubility in water (335 @g/ml) and ethanol (32 mg/mb) and was thus selected as amodel for other 3'-O-acylated ama-Canalogs. In experimentsshown in Chart 3, Chart 7, and Table 1, 0-ama-Cwas used asan aqueous solution. A fine aqueous suspension of 0-ama-C(20 mg/mI) prepared by Dr. Takahashi of our laboratory wasused far a blood level study (Chart 4). [3H]ara-C (8.19 Ci/mmole) was purchased from New England Nuclear, Boston, Mass. Unlabeled ama-Cand most other nucleosidesand nucleotides , and also 1-ethyl-3-(3-dimethylaminoprapyl)cambodiimide HCI, were purchased from Sigma Chemi

620 CANCERRESEARCHVOL. 37



Radioimmunoassay for ara-C

Hydrolysis of O-ara-C by Mammalian Plasma. Esteraseactivity in mammalian plasma was measured in 20 mM phosphate buffer (pH 7.4) using 0-ama-C as the substrate. Eachtube contained 50 @M0-ama-C,0.1% bovine serum albumin,appropriate volumes of plasma, and the buffer in a finalvolume of 200 @l.The reaction was performed at 37Â°,and atdesignated time intervals proteins were precipitated by addition of 600 @Iof absolute ethanol. After centnifugation, thesupemnatant was processed as described above and the amaC content of the ethanol extract was determined. The volume of the extract subjected to madioimmunoassay wasequivalent to 0.3 @Iof the original reaction mixture andcontained a sum of 15 pmobes of ama-Cand unhydnolyzed 0-ama-C.As a check for any interference by 0-ama-C, ethanolextracts were prepared from reaction mixtures that weredevoid of 0-ama-C. Addition of 15 pmoles of 0-ama-C tomadioimmunoassay tubes containing this extract resulted inan increase of Co/Cs equivalent to the presence of 0.7pmole of ama-C.Thus, when hydrolysis mateswere small, theamount of 0-ama-C remaining was corrected using:

% O-ara-C remaining = (100% â€”% ara-C determined) x 1.047

After this correction, the error due to interference by 0-amaC should be less than 1%.

The rate of ama-Uformation during the incubation period.was neglected because it was reported that cytidine deaminase activity in mouse, mat,dog, and human plasma is weak(11). This seemed to be valid because, in hydrolysis expeniments of 0-ama-C using 1:10 dilutions of rat or mouseplasma, greater than 90% of the expected ama-Cwas found(results not shown).

RESULTS

Specificity of the Antibody. The antibody specificity wasdetermined by measuring the displacement of bound[3HJama-Cby bases, nucleosides, and nucleotides. As mdicated in Chart 3, deoxycytidmne and cytidine, which differfrom ama-Conly at the 2'-position, showed no appreciable

ioo

10 20S (ora-C pmoles/Tube)

Chart 2. Immunoassay curve for ara-C. The experiment was performedunder the standard conditions and Co/Cs was plotted against the amount ofunlabeled ara-C (5). Each point represents the mean Â±S.D. of 6 replicates.Co was3425Â±169cpm.

C (particle size, 0.8 to 1.2 @m)was injected into C57BL xDBA/2 F1(hereafter cabled BD2F1) mice i.m. in the femoralmuscle or s.c. at a dose of 200 mg/kg. (The injection andblood samplingwere performedby Dr.KenjiYamaguchiand Naomi Uchida. We thank them for their cooperation .) Atvarious times after administration, 0.3 ml of blood wasremoved from an eye vein and hepaninized. Blood from 3mice was pooled and immediately centrifuged at 3000 x gfor10 mm at4Â°.

Ta 300 @bof the resulting plasma were added 900@ ofabsolute ethanol, and , after being stirred on a Vortex mixer,themixturewas keptinan icebathfanatleast10 mm. Theprecipitate was removed by centnifugation, and 1 ml of thesupemnatant was removed from the tube and evaporated todryness under vacuum. The residue was dissolved in 1 ml ofwater, and this was designated as the ethanol extract. Thisextract was diluted further with an appropriate volume ofwater, if necessary, and aliquots, the volumes of which wereequivalent to 0.8 to 8 @Iof the original plasma, were subjected to the assay. The remainder of the extract was storedin a freezer and was used to check the validity of the assay,and also for a parallel ama-Uassay (see below and Ref. 12).When the extract was stored at â€”20Â°,its ama-Cand ama-Ucontents did not change appreciably for at least 3 months.Recovery of ama-Cin the ethanol extracts, determined byadding 3 to 15 pmales of ama-Cto 5 @lof plasma from anuntreated BD2FI mouse, was 99.4 Â±2.2% (N = 18).

Since 0-ama-C has some affinity for the antibody, it wasnecessary to ascertain whether or not the plasma containedthis nucleoside. For this purpose a 100-@laliquot of ethanolextractwas treatedwith0.1M NaOH at40Â°for10 mm and,after neutralization with 1 M HCI, the ama-Ccontent wasdetermined again. The alkali treatment hydrolyzed 0-ama-Cquantitatively to ama-C (result not shown); therefore thevalue determined after hydrolysis should be the sum of 0-ara-C and ama-C.We could not detect 0-ama-Cin any samplepresented in Chart 4.

a,

4,aa,C

.@ 50

00

0

I0@ I0@Molar Concentrations

Chart 3. Cross-reactivity of various nucleosides and the 5'-monophosphats of ara-C (ara-CMP) with ara-C antibody, Ab-3. The binding reactionwas performed under standard conditions except that compounds indicatedin the chart were added to the assay tubes. [3H]ara-C binding in the absenceof inhibitors was taken as 100%. cyd, cytidine; dcyd; deoxycytidine.

I0@ IO@I0

FEBRUARY 1977 621



T. Okabayashi et al.

cross-reactivity at concentrations 1000-fold greater thanthat of ama-C.The affinity of ama-Ufor the antibody was only0.1% of that of ama-C.On the contrary, 5'-substituted analogs, the 5'-monophosphate of ama-Cand S-ama-C,showedaffinity for the antibody equal to, ongreatenthan, that of amaC. This is not surprising because we used as an antigen forimmunization a conjugate of albumin with S-ama-C. Theaffinity of 3'-acylated ama-C,determined with 0-ama-C, differed from antibody to antibody. With the antibody used inthe present experiment, Ab-3, the affinity of 0-ama-Cwas 5%of that shown by ama-C.The following compounds showedno cross-reactivity at concentrations of 20 jIM: unidine,deoxyunidine , adenosine , guanosmne, deoxyadenosine,deoxyguanosine, uracil, cytosine, thymine, thymidine,dUMP.

We also examined the effect of antimicrobial and antineoplastic drugs, because some of these seriously interferewith a microbiological assay (6). The following drugs had noeffect on [3H]ara-Cbinding at a concentration of 0.1 mM:penicillin V, ampicillin, erythnomycin, cephabothin, tetracydine, chloramphenicol, lincomycin,nystatmn,6-mercaptapunine , 5-fluorouracil, cycbophosphamide, vinblastine, yincnistine, mitomycin C, and actinamycmnD.

Analytical Characteristics of the Immunoassay. Between pH 4.2 and 6.2, [3H]ara-C binding increased progmessively as the pH was increased, and then remained essentially unchanged to pH 9.0. The cross-reactivity of ama-Uwith the antibody became prominent, however, above pH7.0 (fan details, see Ref. 12), and accordingly we performthe assay at pH 6.2.

Under the standard assay conditions the antigen-antibodyreaction reached equilibrium within 30 mm, but for theroutine assay a 2-hr incubation was used.

The presence of most inorganic salts at a concentrationof 0.1 M did not affect [3H]ara-C binding, but considerableinhibition was noted with 0.01 M Zn2@and Cu2t

Since the lowest limit of statistical (p < 0.05) detection ofama-Cis 0.7 pmole (Chart 2) and the upper limit of plasmavolume that can be used without appreciable nonspecificeffects is 12 @l(see next paragraph and Chart 6), we mayconclude that the present assay can be used to determineama-Cin BD2F, mouse plasma at levels as low as 0.014 @g/ml (0.06 MM).

Plasma ara-C Levels after Administration of O-ara-C toBD2FI Mice. Blood bevelstudies were performed as descnibed in â€œMaterialsand Methods.â€•Typical results areshown in Chart 4.

Following i.m. injection of 0-ama-C, a maximum serumama-Clevel of 30 .tMwas achieved within 1 hr. Oventhe next24 hr, ama-Clevels decreased with an approximate half-lifeof 5 hr. With s.c. injection a larger maximum level of ama-Cwas obtained after 1 hr and the half-life was about 3 hr.

Ta check the validity of the assay, several experimentswere performed. Chart 5 shows the results obtained from arecovery experiment, which was performed after addingknown amounts of ama-Cto aliquots of an ethanol extract.As indicated in the chart, the recovery was quantitative. Tocheck the effect of possible interfering substances in theethanol extracts, increasing volumes of the extracts weresubjected to the assay. Using 2 extracts, one of which was

obtained from plasma shown to contain 11 @Mama-Candthe other from plasma containing 0.73 @Mama-C,themewasa direct, linear relationship between the determined ama-Ccontent and the volume of extract used up to a volumeequivalent to 12 @Iof original plasma (Chart 6). Na appreciable amount of 0-ama-Ccould be detected throughout theexperimental period as shown by the ama-Cvalues determined before and after hydrolysis by alkali (result notshown). Aliquots of a single ethanol extract were assayed 5times on different days, the interassaycoefficient of variation being 6.1%.The intraassaycoefficient of variation for 9ethanol extracts prepared from the same plasma was 8.3%ina singleassay.

Hydrolysis of O-ara-C by Mammalian Plasma. Studies onthe enzymatic hydrolysis of 0-ama-C were performed withplasmas diluted 1:40 in a phosphate buffer at pH 7.4. Pronounced esterase activity was found in rat, mouse, andrabbit plasmas; while dog and human plasmas showed lessactivity (Chart 7). In another experiment, the half-life of 0-

a.

00

0

0E0

U.

Chart 4. Plasma ara-C levels after administration of O-ara-C to BD2F,mice. A fine-particle water suspension of O-ara-C (200 mg/kg) was injectedi.m. ( 0) or s.c. (â€¢).

4,0Ea

â€˜Oa,CEa,a,a

0

0

0

Chart 5. Recovery test for plasma samples. To aliquots of ethanol-treatedplasmas of BD2FI mice (corresponding to 0.75 @lof the original plasma) wereadded known amounts of ara-C, and the recoveries were measured. 0, aplasma sample obtained 8 hr after i.m. injection of O-ara-C and shown (Chart3) to contain 11.0 MMara-C. The aliquot thus contained 8.2 pmoles of ara-C.., plasma of an untreated BD2F mouse.

Chart 6. Effect of increasing volumes of plasma on the radioimmunoassayfor ara-C. The same ethanol extract as indicated in Chart 4 ( 0) and anotherextract containing less ara-C [0.73 @Min original plasma (â€¢)]were used. Theamounts of the extracts used are expressed in terms of the correspondingvolumes of the original plasmas.

era-C Added (pmoles) Plasma Volume (@l)

622 CANCER RESEARCHVOL. 37



The hydrolysis experiment was performed for 4 mm at 37Â°withBD2F,mouse plasmadiluted 1:10 in 50 mM phosphatebuffer,pH7.4,

and a substrate concentration of 50 @M.The presenceofeserinesulfate at final concentrations of 0.03 to 3 j@Min radioimmu

noassaytubes did not interfere with theassay.Eserinesulfate Hydrolysis%(M)

(nmoles/tube/4 mm)inhibition0

5.42010-63.1541.910â€”[email protected]@0.21 96.1

Radioimmunoassay for ara-C

ara-C in undiluted human and dog plasma was found to beroughly 10 mm (result not shown). From the above results0-ama-Cseems to be more susceptible than 1-(5-O-adamantoyl-f3-D-amabinofuranosyl)cytosine towards the serum esterase (11). The esteraseactivity was inhibited markedly byeseninesulfate (Table 1). This might suggest the participation of choline esterasein the hydrolysis (11). However,ourattempts to hydrolyze 0-ama-C with horse and electric eelcholine esterase were unsuccessful (results not shown).

DISCUSSION

Among the published methods for determination of ama-C,microbiological (6) and enzymatic (10) methods haveproved to be useful fan routine plasma assays. Since boththese methods are affected enormously by deoxycytidineand considerably by other nucleasides such as cytidine,deoxyadenosine, and deoxyguanasine, our primary concern was to develop an immunological assay that wouldenable us quantitatively to measure ama-Cin the presence ofthesesubstances.Previouswork has indeedshown thatantibodies can be raised using conjugates of nucleaside-5'-carboxylic acids (15), but fairly high crass-reactivity between nba- and deoxynibanucleosides,and vice versa, wasobserved using this method. Since the antibody specificitywe desired was directed towards both the nature of thepynimidine base and the configuration at the 2'-position, we

Time (mm)

Chart 7. Rates of O-ara-C hydrolysis. The rates were determined with 5 @.dofthe indicated plasmas in a total volume of 200 @lof the reaction mixture.

Table 1

Inhibition by eserine sulfate of O-ara-C hydrolysis by mouse plasma

felt that it would be most desirable to conjugate ama-Cto aprotein through the relatively remote 5'-pasition. Accordingly, we have prepared an S-ara-C:human albumin conjugate and used this for immunization of rabbits. As indicatedin â€œResults,â€•deoxycytidine and other potential interferingsubstances did not have appreciable affinity fan Ab-3 atconcentrations 3 orders of magnitude higher than that ofama-C.0-ama-C showed fairly low affinity for Ab-3 when theantibody had been heated at 60Â°for 1 hr. In addition, theassay is relatively insensitive to nonspecific inhibitors asshown by its use with ethanol-treated BD2FI plasma. Furthermore, the assay is also insensitive, at least in in vitrotests, to antibiotics, the effects of which are rather seriousin a microbiological assay (6).

For determination of plasma ama-C, the plasma wastreated with ethanol and then subjected to madioimmunoassay. The recovery of ama-Cin the ethanol extracts was quantitative. Furthermore, the treatment seemed to be effectivein order to reduce nonspecific binding and also to inactivatemetabolizing enzymes such as esterase and deaminase.The validity of the radioimmunoassay was examined byseveral criteria including a recovery test and the detemmination of intra- and intenassay coefficients. Only minor interference by 0-ama-Cand by other constituents of the ethanolextracts was observed. In addition, the values determinedby the radioimmunaassay agreed well with those determined by an enzymatic assay (10), which was modified tocorrect the interference by deoxycytidmne and other substances.4 Thus the present method is simple, reliable, andsensitive enough far measurement of ama-C in biologicalmaterials.

ama-Canalogs with higher acyl groups at the 3'-O-positionhave prominent antileukemic activity against L1210 beukemia (5, 9). These compounds are relatively insoluble inwater and are inferred to be depot forms of ama-C.A preliminary blood level study using a fairly water-soluble campound, 0-ama-C (Chart 4), has indicated that, after i.m. ors.c. injection of this compound into BD2F1mice, fairly highplasma ama-Clevels can be maintained for a relatively bongtime. This is probably due to a slow rate of transbocation ofthe compound from the site of injection into the bloodstream followed by relatively rapid hydrolysis to ama-Cin theblood. We are now investigating the relationship betweenthe mateof transbocation and the blood ama-clevel using avariety of 3'-acyl-ara-C derivatives. The results will be presented elsewhere.

REFERENCES

1. Benedict, W. F., Harris, N., and Karon, M. Kinetics of 1-f3-D-Arabinofuranosylcytosine-induced Chromosome Breaks. Cancer Ass. , 30: 2477-2483, 1970.

2. Bishop, D. C., Meacock, S. C. A., and Williamson, W. A. N. Trihalogenomethyl Compounds of Potential Therapeutic Interest. VI. Some Alcoholswith Sedative Action and Their Esters. J. Chem. Soc. C, 670-673, 1966.

3. Fujimoto, M., Mihara, S., Okabayashi, T., Sugase, T., and Tarui, S.Rapid Aadioimmunoassay for Guanosine 3' ,5'-Cyclic MonophosphateUsing Tritiated Ligand. J. Biochem. Tokyo, 78: 131-137, 1975.

4. Gish, T. D., Kelly, A. C., Camiener, G. W., and Wschter, W. J. NucleicAcids. II. Synthesis of 5'-Estsrs of 1-f3-D-Arabinofuranosyl-cytosine Possessing Antileukemic and Immunosuppressive Activity. J. Med. Chem.,

4 Ide and T. Okabayashi, to be published elsewhere.

FEBRUARY1977 623



14:159â€”1162,1971.5. Hamamura, E. K., Prystasz, M., Verheyden, J. P. H., Moffatt, J. G.,

Yamaguchi, K., Uchida, N., Sato, K., Nomura, K., Shiratori, 0., Takase,S., and Katagiri, K. Reactions of 2-Acyloxyisobutyryl Halides with Nucleosides. VIII. Synthesis and Biological Evaluation of Some 3'-Acyl and3',S'-Diacyl Derivatives of 1-f3-D-Arabinofuranosylcytosine. J. Med.Chem., 19: 667-674, 1976.

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624 CANCER RESEARCH VOL. 37

T. Okabayashi et a!.



1977;37:619-624. Cancer Res Tadashi Okabayashi, Shinichi Mihara, David B. Repke, et al.

-d-ArabinofuranosylcytosineβA Radioimmunoassay for 1-

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