- Email: [email protected]
A sensitive and specific radioimmunoassay for 1-β-d -arabino-furanosylcytosine
Cancer Letters, Elsevier Scientific
24 (1984)
173
173-178
Publishers
Ireland
Ltd.
A SENSITIVE AND SPECIFIC RADIOIMMUNOASSAY l-p-D-ARARINO-FURANOSYLCYTOSINE
NORLAKI OH-ISHI’
SHIMADA, TAKAO UEDA, and TADAAKI OH-OKAa
TETSUYOSHI
YOKOSHIMA,
FOR
JUN-ICHI
Tokai Laboratory, DaiichiPure Chemicals Co. Ltd., 2,117 Tokai-mura, Ibaraki 319-l 1 and aPharmaceuficals Division, Asahi Chemical Industry Co. Ltd., Asahi-machi, Nobeoka, Miyazaki 882 (Japan) (Received 8 May 1984) (Revised version received 1 August (Accepted 4 August 1984)
1984)
SUMMARY
In order to determine the blood level of l-b-D-arabinofuranosylcytosine (Ara-C), an antileukemic agent, a sensitive and specific radioimmunoassay (RIA) system using anti-AraC serum, [ 5-3H] Ara-C and a dextrancoated charcoal method has been developed. The anti-Ara-C serum obtained from a guinea pig was hardly cross-reactive with 1-fl-D-arabinofuranosyluracil (Ara-U), tetrahydrouridine (THU) and other Ara-C analogues. The RIA system for AraC could detect concentrations as low as 60 pg/ml in plasma. Average of the intra- and inter-assay variancies at 5,10, 20 ng/ml were 4.3% and 5.6%, respectively. AraC in blood samples obtained from human patients who orally received iV4 -palmitoyl-l-13 -D-arabinofuranosylcytosine (PL-AC) was determined by the present RIA system.
INTRODUCTION
New treatment methods for acute non-lymphocytic leukemia, such as treatment with Ara-C in low dosage [5] and with N4-acyl-l-O-D-arabinofuranosylcytosine (Acyl-Arab) [ 1,2,7] , were recently developed. Acyl-Ara-C administered to human or animals is converted slowly to Ara-C, and then shows long-lasting anti-tumor activity. Although these treatment methods were proved to be clinically effective, the blood level of Ara-C in these patients was lower than that in patients treated with Ara-C in conventional dosage. For pharmacokinetic study of these treatment methods, therefore, a more sensitive and specific determination method of Ara-C is required. We have developed a sensitive and specific RIA system for Ara-C and applied it to determine the blood level of Ara-C in patients administered PL-AC, an Acyl-Ara-C derivative. 0304-3835/84/$03.00 Published and Printed
0 1984 in Ireland
Elsevier
Scientific
Publishers
Ireland
Ltd.
174 MATERIALS
AND METHODS
Preparation of antigen l-( 5’-O-Succinyl-P-D-arabinofuranosyl)cytosine(S-Ara-C) and its conjugate with human serum albumin (Sigma Chemical Co.) were prepared by the method described by Okabayashi et al, [lo] . Immunization The conjugate of S-Ara-C with albumin in 0.9% NaCl was emulsified in an equal volume of Freund’s complete adjuvant (Calbiochem-Behring Co.) by a homogenizer. Twenty Hartley guinea pigs (male, 250 g) were immunized. Each guinea pig was subcutaneously injected with 0.5 ml of the emulsion containing 0.5 mg of the conjugate at multiple sites. Immunization was repeated 9 times every 2 weeks in the same manner as the initial injection, except that the dosage of the conjugate was reduced to 0.25 mg. The guinea pigs were bled 10 days after the last immunization. Antibodies directed against Ara-C were produced in 2 of the guinea pigs. Antiserum which showed the higher specific binding to [ 5-3H] Ara-C (37% of the radioactivity bound at a final dilution of 1: 2800) was used in the assay. RIA for Ara-C The diluent for reagents was 0.01 M phosphate buffer containing 0.5% bovine serum albumin (Sigma Chemical Co.) and 0.9% NaCl (pH 7.4). To each tube was added 0.4 ml of the above buffer, 0.1 ml of Ara-C (Sigma Chemical Co.) standard or unknown solution, 0.1 ml of antiserum diluted to 1: 400 and 0.1 ml (approximately 10,000 dpm) of [5-3H]Ara-C (RCC Amersham, 26 Ci/mmol). The tubes were incubated for 16 h at 4°C. The bound and free Ara-C were separated by the dextran-coated charcoal method. To each tube was added 1 ml of dextran-coated charcoal (100 mg of dextran T-70 (Pharmacia Fine Chemicals) and 1000 mg of charcoal (Norit) per 100 ml of 0.01 M phosphate buffered saline (pH 7.4)). After incubating for 30 min at 4”C, the tubes were centrifuged at 3000 rev./min for 15 min at 4°C. The radioactivity of the supernatant fluid was determined in a liquid scintillation counter (Aloka Model LSC-903) using RIAFLUOR (New England Nuclear) as the scintillator. Blood level study Two patients were orally administered PL-AC at a dosage of 6.8 and 11.7 mg/kg, respectively. The blood samples obtained in heparinized syringes, which contained THU (50 fig of THU per 1 ml of the blood) to inhibit the activity of cytidine deaminase, were immediately chilled to 4°C followed by centrifugation, and the plasma were stored at -20°C until assayed. RESULTS Specificity To test the specificity
of the antiserum,
Ara-C analogues
such as Ara-U,
175
5’-monophosphate of AraC, cytidine, uridine, thymidine, 2’-deoxycytidine, 2’deoxyuridine, cytosine, uracil, thymine (Sigma Chemical Co.) and THU, PL-AC (Asahi Chemical Industry Co,), were offered to cross-reactivity studies. It has been reported that the cross-reactivity of antiserum prcduced against Ara-C was affected by pH of the assay medium, i.e. Ara-C is determined at pH 6.2, while Ara-U is determined at pH 8.6 [9,10]. The crossreactivity of anti-AraC serum which was used in this study was practically constant between pH 6.2 and 8.6, so it was tested at pH 7.4. Cross-reactivity was expressed as the relative potency of each Ara-C analogue compared to Ara-C, for the 50% displacement of [ 5-3H] Ara-C. As shown in Table 1, the antiserum was hardly cross-reactive with Ara-U, THU and other Ara-C analogues except for 5’-monophosphate of Ara-C (7.9%). Recovery Recovery experiments were performed by adding 3 different amounts of Ara-C along with THU to normal human plasma (5, 10 and 20 ng/ml), and assaying them by the RIA. As shown in Table 2, the averages of 10 replicates at 3 concentrations were 107.2%, 102.9% and 97.4X, respectively, and the mean recovery was 102.5%. Limit of detection The limit of detection of the RIA for Ara-C was estimated as 60 pg/ml by extrapolating 95% confidence limit of B0 samples to the standard curve. Ara-C in a biological fluid can be routinely detectable in the concentration range between 100 pg/ml and 100 ng/ml using 100 ~1 of sample in this assay system.
TABLE 1 CROSS-REACTIVITY
OF ARA-C ANALOGUES
IN THE RIA FOR ARA-C
Compound
Cross-reactivity ( W)”
Ara-C Ara-U THU Ara-C-5’-monophosphate Cytidine Uridine Thymidine X’-Deoxycytidine 2’-Deoxyuridine Cytosine Uracil Thymine PL-AC
100 0.1 0 7.9 0.03> 0.03> 0 0.3 0 0 0 0 0
aAs determined by the relative amounts needed for 50% displacement of [5-‘H]AraG.
176
TABLE 2 RECOVERY
AND PRECISION OF THE ARAC
Sample concentration (nglml)
Recovery (W) (n = 10) Mean + SD.
5 10 20
107.2 + 6.5 102.9 +_2.8 97.4 + 4.2
Average
102.5
RIA SYSTEM
Intraassay variance (n = 10) Found ( ng/ml)a
Inter-assay variance (n = 10) Found ( ng/ml)a
Means f S.D.
cv (%)
Mean + S.D.
cv (%)
5.36 f 0.32 10.29 f 0.28 19.50 + 0.84
6.0 2.7 4.3
5.25 -_b0.35 10.60 + 0.45 19.80 + 1.17
6.7 4.2 5.9 5.6
4.3
aCalculated by the first degree formula of logit-log conversion of standard curve.
Precision
The precision in the RIA for Ara-C was estimated from the coefficient of variation (CV) in intra- and inter-assay system. To determine intra-assay CV, 10 replicates of Ara-C-containing plasma were measured in the same assay at 3 different concentrations, that is, 5, 10 and 20 ng/ml. As shown in Table 2, the intraassay CV value at each concentration was 6.0%, 2.7% and 4.3%, respectively, and the mean value was 4.3%. These samples containing above amounts of Ara-C were also measured 10 times in the different assay to determine inter-assay CV. The inter-assay CV value at each concentration was 6.7%, 4.2% and 5.9% respectively, and the mean value was 5.6% Plasma Ara-C level after oral administration
of PL-AC
to human patients
Figure 1 shows the plasma Ara-C levels in patients at various intervals after oral administration of PL-AC. The peak concentrations of Ara-C appeared at 2 h (3.8 ng/ml) and 6 h (11.5 ng/ml) after administration at a dosage of 6.8 and 11.7 mg/kg, respectively. Further results will be discussed elsewhere.
0.1 ’ ’ ’ 123
1
6
,
12
I
24
Time ( hr) Fig. 1. Plasma AraC levels after oral administration of PL-AC to human patients. Dosage: A, 6.8 mg/kg; .,11.7 mg/kg.
177 DISCUSSION
RIA systems for Ara-C have been reported by Okabayashi et al. [lo] and Piall et al. [ll].Limits of detection of these RIAs were 20 ng/ml and 1 ng/ml in the determination of plasma Ara-C. These RIAs are suitable to monitor whether therapeutically effective concentration of Ara-C is maintained after intravenous administration of Ara-C in conventional dosage to patients. Blood level of Ara-C in patients treated with Ara-C in low dosage [ 121 or with Acyl-Ara-C [ 131 is, however, lower than that in patients treated with Ara-C in conventional dosage. A more sensitive and specific determination method of Ara-C is, therefore, required to facilitate the design of the treatment schedules and perform pharmacokinetic studies. The RIA for Ara-C described in the present paper is sufficiently sensitive for the determination of plasma Ara-C after oral administration of PL-AC, an Acyl-Ara-C derivatives (Fig. 1). Furthermore, both RIA systems previously reported were sensitive to 5’-phosphorylated Ara-C. The cross-reactivity with 5’-monophosphate of Ara-C was almost equal to [lo] or greater (125%) than [ 111 that of Ara-C, while in the present RIA, the cross-reactivity was lower (7.9%). These results are reasonable, because 5’-hemisuccinate of AraC (in Ref. 10 and the present study) or 5’-monophosphate of Ara-C (in Ref. 11) were used as a hapten to conjugate with carrier protein. However, the interference of phosphorylated Ara-C in determining plasma AraC seems to be negligible, since phosphorylated AraC is found only in cells [ 31. In animal or human bodies, Ara-C is converted to Ara-U by an omnipresent deaminase. It is known that the activity of this enzyme is high in human spleen, liver and mouse kidney, but is weak in rats, dogs and in plasma of humans and mice [6,8]. It has also been reported that THU was a potent and specific inhibitor of the enzymatic deamination of Ara-C when the concentration of THU was more than 1.7 X lo-’ M [4]. In this assay system, therefore, sufficient amount of THU, which did not interfere with the assay, was added to samples to inhibit the activity of deaminase. Under these conditions, the rate of Ara-U formation during the period of assay was negligible. The RIA system described here appeared to be useful for the measurement of Ara-C in biological materials. Pharmacokinetic studies of plasma Ara-C after administration of Acyl-Ara-C to humans are now being undertaken using the present RIA, and will be discussed elsewhere. .ACKNOWLEDGEMENTS
The blood level study was performed as a part of the research of a cooperative study group on AAC (chairmen: Drs. K. Kimura and Y. Sakurai). We would like to thank Drs. J. Hattori, S. Nakamura, T. Yoshida and S. Ohtake, Faculty of Medicine, Kanazawa University, Japan, for offering plasma samples of patients.
178 REFERENCES 1 Akiyama, M., Oh-&hi, J., Shirai, T., Akashi, K., Yoshida, K., Nishikido, J., Hayashi, H., Usubuchi, Y., Nishimura, D., Itoh, H., Shibuya, C. and Ishida, T. (1978) The synthesis of new derivatives of 1-p-D-arabinofuranosyl-cytosine. Chem. Pharm. Bull., 26,981-984. 2 Aoshima, M., Tsukagoshi, S., Sakurai, Y., Oh-ishi, J., Ishida T. and Kobayashi, H (1976) Antitumor activities of newly synthesized N’-acyl-l-p-D-arabinofuranosylcytosine. Cancer Res., 36,2726-2732. 3 Bender, R.A., Zwelling, L.A. and Doroshow, J.H. (1978) Antineoplastic drugs: clinical pharmacology and therapeutic use. Drugs, 16,46-87. 4 Camiener, G.W. (1968) Studies of the enzymatic deamination of aracytidine. V. Inhibition in vitro and in vivo by tetrahydrouridine and other reduced pyrimidine nucleosides. Biochem. Pharmacol., 17, 1981-1991. 5 Castaigne, S., Daniel, M.T., Tilly, H., Herait, P. and Degos, L. (1983) Does treatment with ARAC in low dosage cause differentiation of leukemic cells? Blood, 62,85-86. 6 Ho, D.H.W. (1973) Distribution of kinase and deaminase of l-p-o-arabinofuranosylcytosine in tissues of man and mouse. Cancer Res., 33, 2816-2820. 7 Kimura, K., Yamada, K., Uzuka, Y., Maekawa, T., Takaku, F., Shimoyama, M., Ogawa, M., Amaki, I., Osamura, S., Ito, M., Sakai, Y,, Oguro, M., Hattori, K., Hoshino, A., Hirota, Y., Ohta, K., Nakamura, T., Masaoka, T., Kimura, I. and Ichimaru, M. (1982) Phase I study of N4 -behenoyl-l-P-D-arabinofuranosylcytosine and its phase II study in adult acute leukemia. Cm-r. Chemother. Immunother., Proc. 12th Int. Congr. Chemother., 1306-1308. 8 Neil, G.L., Buskirk, H.H., Moxley, T.E., Manak, R.C., Kuentzel, S.L. and Bhuyan, B.K. (1971) Biochemical and pharmacologic studies with l-p-D-arabinofuranosylcytosine 5;adamantoate (NSC-117614), a depot form of cytarabine. Biochem. Pharmacol., 20,3295-3308. 9 Okabaysshi, T., Mihara, S. and Moffatt, J.G. (1977) A radioimmunoassay method for l-p-D-arabinofuranosyluracil using antibodies directed against 1-p-D-arabinofuranosylcytosine. Cancer Res., 37,625-628. 10 Okabayashi, T.,Mihara, S., Repke, D.B. and Moffatt, J.G. (1977) A radioimmunoassay for l-p-D-arabinofuranosylcytosine. Cancer Res., 37, 619- 624. 11 Piall, E.M., Aherne, G.W. and Marks, V.M. (1979) A radioimmunoassay for cytosine arabinoside. Br. J. Cancer, 40, 548-556. 12 Sawada, H., Yamagishi, M., Mochizuki, T., Ishikura, H., Izumi, Y., Park, Y.H., Wano, M., Usui, T. and Uchino, H. (1983) Low Dose of AraC for remission induction in aged patients with atypical nonlymphocytic leukemia (ANLL) and refractory aremia with excess of blasts (RAEB). Jpn. J. Clin. Hematol., 24,1464-1472. 13 Ueda, T., Nakamura, T., Ando, S., Kagawa, D., Sasada, M., Uchino, H., Johno, I. and Akiyama, Y. (1983) Pharmacokinetics of N4-behenoyl-1-P-~arabinofuranosylcytosine in patients with acute leukemia. Cancer Res., 43,3412-3416.
24 (1984)
173
173-178
Publishers
Ireland
Ltd.
A SENSITIVE AND SPECIFIC RADIOIMMUNOASSAY l-p-D-ARARINO-FURANOSYLCYTOSINE
NORLAKI OH-ISHI’
SHIMADA, TAKAO UEDA, and TADAAKI OH-OKAa
TETSUYOSHI
YOKOSHIMA,
FOR
JUN-ICHI
Tokai Laboratory, DaiichiPure Chemicals Co. Ltd., 2,117 Tokai-mura, Ibaraki 319-l 1 and aPharmaceuficals Division, Asahi Chemical Industry Co. Ltd., Asahi-machi, Nobeoka, Miyazaki 882 (Japan) (Received 8 May 1984) (Revised version received 1 August (Accepted 4 August 1984)
1984)
SUMMARY
In order to determine the blood level of l-b-D-arabinofuranosylcytosine (Ara-C), an antileukemic agent, a sensitive and specific radioimmunoassay (RIA) system using anti-AraC serum, [ 5-3H] Ara-C and a dextrancoated charcoal method has been developed. The anti-Ara-C serum obtained from a guinea pig was hardly cross-reactive with 1-fl-D-arabinofuranosyluracil (Ara-U), tetrahydrouridine (THU) and other Ara-C analogues. The RIA system for AraC could detect concentrations as low as 60 pg/ml in plasma. Average of the intra- and inter-assay variancies at 5,10, 20 ng/ml were 4.3% and 5.6%, respectively. AraC in blood samples obtained from human patients who orally received iV4 -palmitoyl-l-13 -D-arabinofuranosylcytosine (PL-AC) was determined by the present RIA system.
INTRODUCTION
New treatment methods for acute non-lymphocytic leukemia, such as treatment with Ara-C in low dosage [5] and with N4-acyl-l-O-D-arabinofuranosylcytosine (Acyl-Arab) [ 1,2,7] , were recently developed. Acyl-Ara-C administered to human or animals is converted slowly to Ara-C, and then shows long-lasting anti-tumor activity. Although these treatment methods were proved to be clinically effective, the blood level of Ara-C in these patients was lower than that in patients treated with Ara-C in conventional dosage. For pharmacokinetic study of these treatment methods, therefore, a more sensitive and specific determination method of Ara-C is required. We have developed a sensitive and specific RIA system for Ara-C and applied it to determine the blood level of Ara-C in patients administered PL-AC, an Acyl-Ara-C derivative. 0304-3835/84/$03.00 Published and Printed
0 1984 in Ireland
Elsevier
Scientific
Publishers
Ireland
Ltd.
174 MATERIALS
AND METHODS
Preparation of antigen l-( 5’-O-Succinyl-P-D-arabinofuranosyl)cytosine(S-Ara-C) and its conjugate with human serum albumin (Sigma Chemical Co.) were prepared by the method described by Okabayashi et al, [lo] . Immunization The conjugate of S-Ara-C with albumin in 0.9% NaCl was emulsified in an equal volume of Freund’s complete adjuvant (Calbiochem-Behring Co.) by a homogenizer. Twenty Hartley guinea pigs (male, 250 g) were immunized. Each guinea pig was subcutaneously injected with 0.5 ml of the emulsion containing 0.5 mg of the conjugate at multiple sites. Immunization was repeated 9 times every 2 weeks in the same manner as the initial injection, except that the dosage of the conjugate was reduced to 0.25 mg. The guinea pigs were bled 10 days after the last immunization. Antibodies directed against Ara-C were produced in 2 of the guinea pigs. Antiserum which showed the higher specific binding to [ 5-3H] Ara-C (37% of the radioactivity bound at a final dilution of 1: 2800) was used in the assay. RIA for Ara-C The diluent for reagents was 0.01 M phosphate buffer containing 0.5% bovine serum albumin (Sigma Chemical Co.) and 0.9% NaCl (pH 7.4). To each tube was added 0.4 ml of the above buffer, 0.1 ml of Ara-C (Sigma Chemical Co.) standard or unknown solution, 0.1 ml of antiserum diluted to 1: 400 and 0.1 ml (approximately 10,000 dpm) of [5-3H]Ara-C (RCC Amersham, 26 Ci/mmol). The tubes were incubated for 16 h at 4°C. The bound and free Ara-C were separated by the dextran-coated charcoal method. To each tube was added 1 ml of dextran-coated charcoal (100 mg of dextran T-70 (Pharmacia Fine Chemicals) and 1000 mg of charcoal (Norit) per 100 ml of 0.01 M phosphate buffered saline (pH 7.4)). After incubating for 30 min at 4”C, the tubes were centrifuged at 3000 rev./min for 15 min at 4°C. The radioactivity of the supernatant fluid was determined in a liquid scintillation counter (Aloka Model LSC-903) using RIAFLUOR (New England Nuclear) as the scintillator. Blood level study Two patients were orally administered PL-AC at a dosage of 6.8 and 11.7 mg/kg, respectively. The blood samples obtained in heparinized syringes, which contained THU (50 fig of THU per 1 ml of the blood) to inhibit the activity of cytidine deaminase, were immediately chilled to 4°C followed by centrifugation, and the plasma were stored at -20°C until assayed. RESULTS Specificity To test the specificity
of the antiserum,
Ara-C analogues
such as Ara-U,
175
5’-monophosphate of AraC, cytidine, uridine, thymidine, 2’-deoxycytidine, 2’deoxyuridine, cytosine, uracil, thymine (Sigma Chemical Co.) and THU, PL-AC (Asahi Chemical Industry Co,), were offered to cross-reactivity studies. It has been reported that the cross-reactivity of antiserum prcduced against Ara-C was affected by pH of the assay medium, i.e. Ara-C is determined at pH 6.2, while Ara-U is determined at pH 8.6 [9,10]. The crossreactivity of anti-AraC serum which was used in this study was practically constant between pH 6.2 and 8.6, so it was tested at pH 7.4. Cross-reactivity was expressed as the relative potency of each Ara-C analogue compared to Ara-C, for the 50% displacement of [ 5-3H] Ara-C. As shown in Table 1, the antiserum was hardly cross-reactive with Ara-U, THU and other Ara-C analogues except for 5’-monophosphate of Ara-C (7.9%). Recovery Recovery experiments were performed by adding 3 different amounts of Ara-C along with THU to normal human plasma (5, 10 and 20 ng/ml), and assaying them by the RIA. As shown in Table 2, the averages of 10 replicates at 3 concentrations were 107.2%, 102.9% and 97.4X, respectively, and the mean recovery was 102.5%. Limit of detection The limit of detection of the RIA for Ara-C was estimated as 60 pg/ml by extrapolating 95% confidence limit of B0 samples to the standard curve. Ara-C in a biological fluid can be routinely detectable in the concentration range between 100 pg/ml and 100 ng/ml using 100 ~1 of sample in this assay system.
TABLE 1 CROSS-REACTIVITY
OF ARA-C ANALOGUES
IN THE RIA FOR ARA-C
Compound
Cross-reactivity ( W)”
Ara-C Ara-U THU Ara-C-5’-monophosphate Cytidine Uridine Thymidine X’-Deoxycytidine 2’-Deoxyuridine Cytosine Uracil Thymine PL-AC
100 0.1 0 7.9 0.03> 0.03> 0 0.3 0 0 0 0 0
aAs determined by the relative amounts needed for 50% displacement of [5-‘H]AraG.
176
TABLE 2 RECOVERY
AND PRECISION OF THE ARAC
Sample concentration (nglml)
Recovery (W) (n = 10) Mean + SD.
5 10 20
107.2 + 6.5 102.9 +_2.8 97.4 + 4.2
Average
102.5
RIA SYSTEM
Intraassay variance (n = 10) Found ( ng/ml)a
Inter-assay variance (n = 10) Found ( ng/ml)a
Means f S.D.
cv (%)
Mean + S.D.
cv (%)
5.36 f 0.32 10.29 f 0.28 19.50 + 0.84
6.0 2.7 4.3
5.25 -_b0.35 10.60 + 0.45 19.80 + 1.17
6.7 4.2 5.9 5.6
4.3
aCalculated by the first degree formula of logit-log conversion of standard curve.
Precision
The precision in the RIA for Ara-C was estimated from the coefficient of variation (CV) in intra- and inter-assay system. To determine intra-assay CV, 10 replicates of Ara-C-containing plasma were measured in the same assay at 3 different concentrations, that is, 5, 10 and 20 ng/ml. As shown in Table 2, the intraassay CV value at each concentration was 6.0%, 2.7% and 4.3%, respectively, and the mean value was 4.3%. These samples containing above amounts of Ara-C were also measured 10 times in the different assay to determine inter-assay CV. The inter-assay CV value at each concentration was 6.7%, 4.2% and 5.9% respectively, and the mean value was 5.6% Plasma Ara-C level after oral administration
of PL-AC
to human patients
Figure 1 shows the plasma Ara-C levels in patients at various intervals after oral administration of PL-AC. The peak concentrations of Ara-C appeared at 2 h (3.8 ng/ml) and 6 h (11.5 ng/ml) after administration at a dosage of 6.8 and 11.7 mg/kg, respectively. Further results will be discussed elsewhere.
0.1 ’ ’ ’ 123
1
6
,
12
I
24
Time ( hr) Fig. 1. Plasma AraC levels after oral administration of PL-AC to human patients. Dosage: A, 6.8 mg/kg; .,11.7 mg/kg.
177 DISCUSSION
RIA systems for Ara-C have been reported by Okabayashi et al. [lo] and Piall et al. [ll].Limits of detection of these RIAs were 20 ng/ml and 1 ng/ml in the determination of plasma Ara-C. These RIAs are suitable to monitor whether therapeutically effective concentration of Ara-C is maintained after intravenous administration of Ara-C in conventional dosage to patients. Blood level of Ara-C in patients treated with Ara-C in low dosage [ 121 or with Acyl-Ara-C [ 131 is, however, lower than that in patients treated with Ara-C in conventional dosage. A more sensitive and specific determination method of Ara-C is, therefore, required to facilitate the design of the treatment schedules and perform pharmacokinetic studies. The RIA for Ara-C described in the present paper is sufficiently sensitive for the determination of plasma Ara-C after oral administration of PL-AC, an Acyl-Ara-C derivatives (Fig. 1). Furthermore, both RIA systems previously reported were sensitive to 5’-phosphorylated Ara-C. The cross-reactivity with 5’-monophosphate of Ara-C was almost equal to [lo] or greater (125%) than [ 111 that of Ara-C, while in the present RIA, the cross-reactivity was lower (7.9%). These results are reasonable, because 5’-hemisuccinate of AraC (in Ref. 10 and the present study) or 5’-monophosphate of Ara-C (in Ref. 11) were used as a hapten to conjugate with carrier protein. However, the interference of phosphorylated Ara-C in determining plasma AraC seems to be negligible, since phosphorylated AraC is found only in cells [ 31. In animal or human bodies, Ara-C is converted to Ara-U by an omnipresent deaminase. It is known that the activity of this enzyme is high in human spleen, liver and mouse kidney, but is weak in rats, dogs and in plasma of humans and mice [6,8]. It has also been reported that THU was a potent and specific inhibitor of the enzymatic deamination of Ara-C when the concentration of THU was more than 1.7 X lo-’ M [4]. In this assay system, therefore, sufficient amount of THU, which did not interfere with the assay, was added to samples to inhibit the activity of deaminase. Under these conditions, the rate of Ara-U formation during the period of assay was negligible. The RIA system described here appeared to be useful for the measurement of Ara-C in biological materials. Pharmacokinetic studies of plasma Ara-C after administration of Acyl-Ara-C to humans are now being undertaken using the present RIA, and will be discussed elsewhere. .ACKNOWLEDGEMENTS
The blood level study was performed as a part of the research of a cooperative study group on AAC (chairmen: Drs. K. Kimura and Y. Sakurai). We would like to thank Drs. J. Hattori, S. Nakamura, T. Yoshida and S. Ohtake, Faculty of Medicine, Kanazawa University, Japan, for offering plasma samples of patients.
178 REFERENCES 1 Akiyama, M., Oh-&hi, J., Shirai, T., Akashi, K., Yoshida, K., Nishikido, J., Hayashi, H., Usubuchi, Y., Nishimura, D., Itoh, H., Shibuya, C. and Ishida, T. (1978) The synthesis of new derivatives of 1-p-D-arabinofuranosyl-cytosine. Chem. Pharm. Bull., 26,981-984. 2 Aoshima, M., Tsukagoshi, S., Sakurai, Y., Oh-ishi, J., Ishida T. and Kobayashi, H (1976) Antitumor activities of newly synthesized N’-acyl-l-p-D-arabinofuranosylcytosine. Cancer Res., 36,2726-2732. 3 Bender, R.A., Zwelling, L.A. and Doroshow, J.H. (1978) Antineoplastic drugs: clinical pharmacology and therapeutic use. Drugs, 16,46-87. 4 Camiener, G.W. (1968) Studies of the enzymatic deamination of aracytidine. V. Inhibition in vitro and in vivo by tetrahydrouridine and other reduced pyrimidine nucleosides. Biochem. Pharmacol., 17, 1981-1991. 5 Castaigne, S., Daniel, M.T., Tilly, H., Herait, P. and Degos, L. (1983) Does treatment with ARAC in low dosage cause differentiation of leukemic cells? Blood, 62,85-86. 6 Ho, D.H.W. (1973) Distribution of kinase and deaminase of l-p-o-arabinofuranosylcytosine in tissues of man and mouse. Cancer Res., 33, 2816-2820. 7 Kimura, K., Yamada, K., Uzuka, Y., Maekawa, T., Takaku, F., Shimoyama, M., Ogawa, M., Amaki, I., Osamura, S., Ito, M., Sakai, Y,, Oguro, M., Hattori, K., Hoshino, A., Hirota, Y., Ohta, K., Nakamura, T., Masaoka, T., Kimura, I. and Ichimaru, M. (1982) Phase I study of N4 -behenoyl-l-P-D-arabinofuranosylcytosine and its phase II study in adult acute leukemia. Cm-r. Chemother. Immunother., Proc. 12th Int. Congr. Chemother., 1306-1308. 8 Neil, G.L., Buskirk, H.H., Moxley, T.E., Manak, R.C., Kuentzel, S.L. and Bhuyan, B.K. (1971) Biochemical and pharmacologic studies with l-p-D-arabinofuranosylcytosine 5;adamantoate (NSC-117614), a depot form of cytarabine. Biochem. Pharmacol., 20,3295-3308. 9 Okabaysshi, T., Mihara, S. and Moffatt, J.G. (1977) A radioimmunoassay method for l-p-D-arabinofuranosyluracil using antibodies directed against 1-p-D-arabinofuranosylcytosine. Cancer Res., 37,625-628. 10 Okabayashi, T.,Mihara, S., Repke, D.B. and Moffatt, J.G. (1977) A radioimmunoassay for l-p-D-arabinofuranosylcytosine. Cancer Res., 37, 619- 624. 11 Piall, E.M., Aherne, G.W. and Marks, V.M. (1979) A radioimmunoassay for cytosine arabinoside. Br. J. Cancer, 40, 548-556. 12 Sawada, H., Yamagishi, M., Mochizuki, T., Ishikura, H., Izumi, Y., Park, Y.H., Wano, M., Usui, T. and Uchino, H. (1983) Low Dose of AraC for remission induction in aged patients with atypical nonlymphocytic leukemia (ANLL) and refractory aremia with excess of blasts (RAEB). Jpn. J. Clin. Hematol., 24,1464-1472. 13 Ueda, T., Nakamura, T., Ando, S., Kagawa, D., Sasada, M., Uchino, H., Johno, I. and Akiyama, Y. (1983) Pharmacokinetics of N4-behenoyl-1-P-~arabinofuranosylcytosine in patients with acute leukemia. Cancer Res., 43,3412-3416.