Akihiko Nakagawa

Antitumor activity and novel DNA-self-strand-breaking mechanism of CNDAC (1-(2-C-cyano-2-deoxy-?-d-ARABINO-Pentofuranosyl) cytosine) and itsN4-palmitoyl derivative (CS-682)

We have studied the antitumor activity and the novel DNA‐self‐strand‐breaking mechanism of CNDAC (1‐(2‐C‐cyano‐2‐deoxy‐β‐d‐arabino‐pentofuranosyl)cytosine) and its N4‐palmitoyl derivative (CS‐682). In vitro, CS‐682 showed strong cytotoxicity against human tumor cells comparable with that of CNDAC; both compounds displayed a similar broad spectrum. In vivo, however, orally administered CS‐682 showed a more potent activity against human tumor xenografts than CNDAC, 5′‐deoxy‐5‐fluorouridine, 5‐fluorouracil and 2′,2′‐difluorodeoxycytidine. Moreover, CS‐682 was effective against various human organ tumor xenografts at a wide dose range and with low toxicity, and was effective against P388 leukemic cells resistant to mitomycin‐C, vincristine, 5‐fluorouracil or cisplatin in syngeneic mice. CNDAC, an active metabolite of CS‐682, had a prolonged plasma half‐life after repeated oral administrations of CS‐682 but not after oral administrations of CNDAC itself. This difference may partially explain the higher antitumor activity of CS‐682 relative to CNDAC. In both CNDAC‐ and CS‐682‐treated carcinoma cells, CNDAC 5′‐triphosphate (CNDACTP) was generated and incorporated into a DNA strand. High performance liquid chromatography (HPLC) and mass spectrometric analysis of the nucleosides prepared by digestion of the DNA from the CNDAC‐treated cells detected ddCNC (2′‐C‐cyano‐2′,3′‐didehydro‐2′,3′‐dideoxycytidine), which was shown to be generated only when the self‐strand‐breakage of CNDACTP‐incorporated DNA occurred. The cytotoxicity of CNDAC was completely abrogated by the addition of 2′‐deoxycytidine and was low against cells with decreased deoxycytidine kinase. Our results suggest that CNDAC is converted to CNDACMP by deoxycytidine kinase and that the resulting CNDACTP incorporated into a DNA strand as CNDACMP may induce DNA‐self‐strand‐breakage. This novel DNA‐self‐strand‐breaking mechanism may contribute to the potent antitumor activity of CS‐682. Int. J. Cancer 82:226–236, 1999.

Automated high-performance liquid chromatographic method for the determination of antipyrine and its metabolites in urine : Some preliminary results obtained from smokers and non-smokers

Abstract A simple, accurate and fully automated high-performance liquid-chromatographic method was developed for the simultaneous determination of antipyrine (AP), 3-hydroxymethylantipyrine (3HMA), 4-hydroxyantipyrine (4OHA) and norantipyrine (NORA) in urine. This method requires no extraction step and only one chromatographic run with the use of a reversed-phase system. The coefficient of variation (%) (n = 8 each) was: 4.14 for AP, 2.31 for 3HMA, 3.48 for 4OHA, and 2.71 for NORA. The method was applied to studies on AP metabolism in three smokers and three non-smokers who received an oral 10 mg/kg dose of AP. These preliminary results suggest that smokers appear to excrete more 4OHA and NORA in the urine than non-smokers.

Application of molecular-secondary-ion mass spectrometry for drug metabolism studies. I. Direct analysis of conjugates by thin-layer chromatography-secondary-ion mass spectrometry.

Molecular-secondary-ion mass spectrometry (SIMS) is a suitable method for the analysis of nonvolatile substances such as conjugated metabolites of drugs. We have developed a simple method for the direct SIMS measurement of conjugates following thin-layer chromatography without any extraction procedure. After separation with a butanol-acetic acid-ethanol-water (3:1:1:1, v/v) system, the spot was cut out and attached to a SIMS probe. The conjugates of p-nitrophenol and 4-hydroxyantipyrine were measured. The quantitative application of the method is also discussed, using deuterium-labelled internal standards for p-nitrophenol conjugates.

Enzyme immunoassay of human plasma 11-dehydrothromboxane B2

11-Dehydrothromboxane B2 (11-dhTXB2) is a proposed marker compound for thromboxane A2 formed in vivo. An enzyme immunoassay was established for determination of the plasma concentration of this compound. The assay was based on a horseradish peroxidase-linked immunoassay utilizing polyclonal anti-11-dhTXB2 antibody obtained from a rabbit, and enabled determination of 11-dhTXB2 in the range of 2 to 500 pg/tube with an IC50 of 36 pg. The cross-reactivities with TXB2, 2,3-dinor-TXB2 and other prostanoids were less than 0.05%. Validity of the enzyme immunoassay was confirmed by a radioimmunoassay utilizing a monoclonal antibody. The plasma 11-dhTXB2 was immunoaffinity-purified by one step using an immobilized monoclonal antibody. The mean plasma level of 11-dhTXB2 in six male volunteers was 4.0 +/- 0.3 pg/ml by this enzyme immunoassay.

Superiority of plasma 11-dehydro-TXB2 to TXB2 as an index of in vivo TX formation in rabbits after dosing of CS-518, A TX synthase inhibitor

We compared plasma 11-dhTXB2 and TXB2 for their ability to reflect in vivo TX formation in rabbits treated with AA and CS-518, a TX synthase inhibitor. The average plasma level of TXB2 in rabbits was much higher than that of 11-dhTXB2, probably because of artificial formation of TXB2 during blood sampling. CS-518 (1 mg/kg, p.o.) caused a long-lasting suppression of the 11-dhTXB2 level, and its inhibitory effect on 11-dhTXB2 was much more extensive than that on TXB2. AA-infusion for 5 min resulted in transient and remarkable increases of both TXs, and prevention of such increases by CS-518 pretreatment (1 mg/kg, i.v.) was shown: inhibitions of 11-dhTXB2 and TXB2 were 85% and 40%, respectively. The inhibitory effect caused by CS-518, which was more clearly observed on plasma 11-dhTXB2 than on TXB2, was due to not only the completely inhibited levels without artificial formation but also the durable high levels based on the long half-life of 11-dhTXB2 in AA-infused rabbits. CS-518 injection during sustained AA-infusion also resulted in a 2-fold faster disappearance of plasma 11-dhTXB2 than was seen without CS-518, despite its long half-life. Considering the absence of artificial formation, the long half-life, and the good response to change of TX formation, plasma 11-dhTXB2 is superior to TXB2 as an index for monitoring in vivo TX synthase activity and its pharmacological modification with AA and CS-518.

Determination of Furosine in Hair by Liquid Chromatography–Tandem Mass Spectrometry

A sensitive and reliable analytical method for determining furosine in hair has been developed using liquid chromatography-tandem mass spectrometry. Fructose-N(omega)-formyl-d4-DL-lysine was synthesized and used as an internal standard. By the present method, glycated lysine levels in hair could be determined as fructose-lysine in only 0.1 mg of hair sample, ranging from 35.1-72.6 ng/mg hair among five healthy volunteers, which corresponded to 0.0635-0.153% of the total lysine contents in hair determined by amino acid analysis.

Enzyme-Linked Immunosorbent Assay of CS-518, a Thromboxane Synthetase Inhibitor, in Rabbit Plasma and Platelets

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for determination of CS-518, a novel thromboxane synthetase inhibitor. Antisera against CS-518 were obtained from rabbits immunized with bovine serum albumin linked to CS-518 via carboxylic acid introduced into the imidazolyl ring (for ELISA-1) or via 6-carboxylic acid directly (for ELISA-2). Each of two CS-518 derivatives was conjugated to horseradish peroxidase by a N-succinimidyl ester method, and it was used as a labeled-antigen in homogeneous combination with antisera. In ELISA-1, CS-518 was detectable in a range of 5pg-1ng, and all cross-reactivities with main metabolites were less than 5%, in contrast to high affinity to the taurine and glucuronic acid conjugates of CS-518 in ELISA-2. Validity of ELISA-1 was confirmed by a high-performance liquid chromatography and ELISA-1 enabled specific determination of CS-518 in plasma samples deproteinized by methanol. When ELISA-1 was applied to determine CS-518 in platelets after oral administration to rabbits, CS-518 uptake up to maximum capacity in platelets (4.2-5.4 x 10(6) M) and slow elimination of CS-518 from platelets (T1/2 = 36-41 hr) were observed independent of CS-518 doses. These results confirm that CS-518 binds to thromboxane synthetase in platelets with high affinity.