John Michael Dornish

Reduction of cis-dichlorodiammineplatinum-induced cell inactivation by benzaldehyde

The inactivating effect of a combined treatment of human cells (NHIK 3025) in culture with cis-dichlorodiammineplatinum(II) (cis-DDP) and the protein synthesis inhibitor benzaldehyde was tested. Cell inactivation was measured as loss of colony-forming ability following drug treatment. While 3.2 mM benzaldehyde had no effect on the cell survival when given alone, it reduced the effect of 10 microM cis-DDP significantly when the two drugs were added simultaneously. Scheduling experiments indicate that benzaldehyde must be present immediately before addition of, or simultaneously with, cis-DDP for optimal reduction of cell inactivation. Benzoic acid, benzyl alcohol or the protein synthesis inhibitor cycloheximide did not reduce the inactivating effect of cis-DDP. Cells synchronized by mitotic selection were used to determine the variation in the responses throughout the cell cycle. It was found that concomitant 2-hr treatment of synchronized cells with 3.2 mM benzaldehyde and 10 microM cis-DDP at various times during the cell cycle resulted in a consistently greater surviving fraction of cells than 10 microM cis-DDP alone. Benzaldehyde thus reduced the inactivating effect of cis-DDP in all phases of the cell cycle. The effect of benzaldehyde in combination with two alkylating agents, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and nitrogen mustard (HN2), was also studied. Benzaldehyde was not found to influence the effects on cell survival induced by these drugs.

Mitotic arrest and interphase inhibition induced by the pyrimidine sulfoxide NY 4138.

SummaryPhase-specific cell-cycle inhibitory effects of the sulfoxide 5-bromo-2-(2-thienylmethyl)sulfinyl-pyrimidine (NY 4138) was studied on synchronized human NHIK 3025 cells cultivated in vitro. When added to exponentially growing cells, NY 4138 induced an accumulation of metaphases. The metaphase arrest was, however, not efficient immediately after addition of the compound. While a few telophases were still present 3 h after addition of 0.1 mM, no telophases were seen 6 h after addition. Most cells exposed to metaphase arrest by NY 4138 lost their ability to form colonies. We therefore characterize the metaphase arrest induced by this drug as irreversible. For concentrations above 0.5 mM phase specific cell cycle inhibition was also observed during interphase. Cells treated in G1 only were not delayed in G1 but were delayed in S and in G2. Cells treated in S only were also delayed in S, but to a smaller extent than the cells treated in G1 only. Cells treated with 0.1 mM NY 4138 for a period of 4 h in either G1 or in S showed no significant prolongation of the subsequent mitosis (i.e. the drug was in this case not present during mitosis). However, about 20% of the cells in this case divided to form 3 daughter cells of which 1 had a normal G1 DNA-content, 1 had 1/3 and 1 had 2/3 of a normal G1 DNA-content.

In vivo and in vitro pharmacokinetics of 4,6-benzylidene-D-glucose (BG) in rats

SummaryIn vivo pharmacokinetics of 4,6-benzylidene-D-glucose (BG) was investigated in rats following an i.v. bolus injection of 85 mg BG/kg body weight. High performance liquid chromatography (HPLC) was used to characterize and quantitate BG in whole blood or serum samples. It was found that BG rapidly disappeared with a half-life (t1/2)on the order of 10 min. At the same time a metabolite appeared which eluted before the double isomer peaks of BG. It increased in concentration from 0 to 30 min after initial i.v. injection of BG. Thereafter the metabolite was slowly removed or cleared from the animals. The t1/2 of the metabolite calculated from the time of maximum concentration was found to be about 1 h. BG was also metabolized by whole rat blood at 37°C, but on a different time scale in vitro. The t1/2 of BG in the in vitro assays was now about 4 h, as compared to 10 min in vivo. BG was not metabolized in rat plasma or rat serum. In contrast to in vivo data, the metabolite of BG was not reduced upon further incubation, but remained in blood samples with no reduction for at least 24 h. In addition, we found that protein synthesis was inhibited by approximately 50% when isolated rat hepatocytes were incubated with 3.2 mM BG. BG was slowly metabolized by hepatocytes to produce a metabolite indistinguishable (by HPLC) from that found in blood samples. Analysis of the metabolite by combined gas chromatography-mass spectrometric (GC-MS) methods identified it as being 1,3-benzylidene-D-glucitol. An intracellular reduction of BG by aldose reductase is proposed to occur.

Requirement of a reactive aldehyde moiety for aldehyde-mediated protection against cis-dichlorodiammineplatinum-inducedcell inactivation

The effect of the aromatic aldehydes benzaldehyde and salicylaldehyde, the glucose-acetal derivative 4,6-benzylidene-D-glucose (BG) and the glucoside salicylaldehyde-beta-D-glucoside (helicin) on cell inactivation induced by cis-dichlorodiammineplatinum (cis-DDP) was investigated using cultured human NHIK 3025 cells. Cell inactivation was measured as loss in the ability of single cells to give rise to macroscopic colonies following drug treatment. The fraction of cells surviving a 2 hr treatment with 10 microM cis-DDP increased from 0.012 +/- 0.004 to 0.10 +/- 0.03 when treatment was combined with at least 1 mM benzaldehyde or at least 0.2 mM salicylaldehyde. Of the two sugar-aldehyde derivatives only helicin protected cells from the inactivating effect of cis-DDP, although to a much lesser extent than either benzaldehyde or salicylaldehyde. While helicin retains the aldehyde moiety of salicylaldehyde, BG does not possess any free aldehyde group. Using synchronized cells we found these effects to appear in all phases of the cell cycle. Measurements of cell-associated platinum indicated that the degree of protection from the inactivating effects of cis-DDP by these aldehydes was related to the degree of reduced platinum accumulation. We conclude that this reduced accumulation may represent an inhibition of specific cell membrane uptake sites via Schiff based formation between membrane amino groups and aldehydes.

Inactivation of human cells cultivated in vitro by the new mitotic inhibitors NY 4137 and NY 4138

The effect of the two closely related drugs, the sulfone 2-(2-thenyl)sulfonyl-5-bromopyrimidine (NY 4137), and the sulfoxide 2-(2-thenyl)sulfinyl-5-bromopyrimidine (NY 4138), a sulfoxide, on the survival of cells of the human line NHIK 3025 was investigated. Cell survival was measured as the ability of single cells to form macroscopic colonies. Two-hour treatment with 0.012 mM NY 4137 resulted in 50% inactivation. The drug concentration of NY 4138 had to be adjusted about 10 times higher than that of NY 4137 for treatment periods of 2 or 24 h to obtain similar surviving fraction after treatment of asynchronous cells. Treatment of synchronized NHIK 3025 cells with NY 4137 showed that survival varied little with cell age. Following treatment with NY 4138, however, cells were particularly sensitive in G2 and in mitosis. As the survival curves for both drugs display a plateau region, where increasing the drug dose has little or no effect on cell inactivation, the presence of resistant subpopulations of cells is considered. High-performance liquid chromatography of drug solutions in cell culture medium showed that both NY 4137 and NY 4138 bound to, or were metabolized by, medium and/or cell components. The concentration of NY 4137 in cell culture medium, however, was reduced at a higher rate than NY 4138. The half-life of NY 4137 was on the order of 5 h, while the half-life of NY 4138 was over 24 h. These observations correlate well with the relative chemical reactivities for these drugs in nucleophilic displacement reactions.

Effects of the new mitotic inhibitor pyrimidinsulfone NY 4137 on human cells in vitro and on colchicine binding to tubulin

The effect of 2-(2-thenyl)sulfonyl-5-bromopyrimidine (NY 4137) on cells of the human line NHIK 3025 was investigated. It was shown that NY 4137 induces cell cycle specific inhibition in metaphase. A dose-dependent prolongation of metaphase was found and the fraction of cells able to escape metaphase arrest declined gradually as the concentration of NY 4137 increased. A total block in metaphase was achieved with 0.016 mM and higher concentrations of NY 4137. Interphase was also prolonged in cells treated with 0.016 mM. Inhibition of valine incorporation by NY 4137 was also found to be dose-dependent. Following a 2-h exposure to 0.2 mM NY 4137 valine incorporation was inhibited by 80–85%. Inhibition of colchicine and vincristine binding to purified tubulin was also investigated. Double reciprocal plot and gel filtration chromatography showed that NY 4137 competitively inhibited colchicine binding to DEAE-cellulose purified tubulin. NY 4137 had no effect on vincristine binding to tubulin. The binding of NY 4137 to tubulin at or near the colchicine binding site may be responsible for the metaphase arrest.

The bioavailability and dose dependency of the deuterated anti-tumour agent 4,6-benzylidene-d1-d-glucose in mice and rats

The benzaldehyde derivative 4,6-benzylidene-d-glucose (BG) induces an inhibition of protein synthesis at otherwise non-toxic doses in cells grown in vitro. To increase the biological effect of BG, the hydrogen in the formyl group was exchanged with deuterium, resulting in 4,6-benzylidene-d1-d-glucose (P-1013). In this study we compared the bioavailability of BG and P-1013, since both intraperitoneal and, especially, oral administration of the drugs would be a great advantage. We also examined whether or not P-1013 displays dose-dependent pharmacokinctics. Pharmacokinetics were studied by analysing plasma samples using reversed-phase high-performance liquid chromatography (HPLC). P-1013 was given at four different doses i.v. (60, 120, 145 and 230 mg/kg) and p.o. (60, 120, 170 and 230 mg/kg) to female Bom:NMRI-numice. The bioavailability was more than 50% for all doses. The results also indicate that P-1013 shows linear pharmacokinetics, with no change being observed in the half-life (t1/2) with increasing dose and only a slightly more than proportional increase in the area under the concentration-time curve (AUC) occurring with increasing dose. A doubling in dose resulted in a 2.2-fold increase in the AUC. P-1013 and BG were also given i.v., p.o. and i.p. to female nu/nu-BALB/cABom mice and male Wistar rats. A high degree of bioavailability was found in both species, with 55–100% of the deliverered dose being absorbed. Deuteration of BG does not seem to alter its bioavailability, as we found the same bioavailability for P-1013 as for BG. We conclude that the pharmacokinetics of P-1013 does not prevent its use as a cancer treatment drug given orally.

Growth inhibition of human tumor xenografts in nude mice by treatment with the antitumor agent 4,6-benzylidene-d1-D-glucose (P-1013)

4,6-Benzylidene-d1-D-glucose, P-1013, a deuterated benzaldehyde derivative which acts as a reversible protein synthesis inhibitor in vitro, was evaluated for antitumor effects in two human tumor xenografts implanced s.c. in nude mice. The drug, dissolved in isotonic saline, was given p.o. daily for several weeks. For evaluation of drug efficacy, mean tumor volume growth curves were generated and tumor volume doubling time (TD) as well as per cent change in tumor size for the treated tumors compared to control (T/C) were calculated. P-1013 at 90 mg/kg for 49 days was effective against SK-OV-3 human ovarian carcinoma (T/C 47% at day 40 and 50% increase in TD). P-1013 at 90 mg/kg also suppressed growth of PANC-1 human pancreatic carcinoma in two experiments (T/C 44 and 50%, 20 and 40% increase in TD, respectively). There was no indication of systemic toxicity in mice receiving P-1013. Histological examinations of each tumor showed that P-1013 treatment of pancreatic xenografts reduced tumor volume without inducing greater necrosis than that comparable to respective control tumors. For the ovarian xenograft, the histological examination indicated a higher fraction of tumors with more than 50% necrotic tissue in two of the P-1013-treated groups compared with the control group (Fisher exact test, p = 0.12). It is possible that P-1013, in addition to inhibiting the rate of tumor volume growth, also induces tumor necrotization in the ovarian xenograft.

The effect of benzaldehyde on terbium fluorescence enhancement in human NHIK 3025 cells treated with cis-dichlorodiammineplatinum(II)

Abstract As a measure of platinum-DNA interaction we have examined the enhancement of terbium fluorescence in human cells treated with cis -dichlorodiammineplatinum(II) (cisplatin) in the absence and presence of benzaldehyde. While benzaldehyde alone (up to 5 mM) did not affect terbium fluorescence registered from cells, cisplatin alone caused a dose-dependent enhancement of terbium fluorescence. Simultaneous treatment of cells with cisplatin and benzaldehyde, however, suppressed terbium fluorescence enhancement, indicating a reduction in platinum-DNA interaction. Nevertheless, by use of flameless atomic absorption spectroscopy as a measure of cell-associated platinum, it was shown that a considerable amount of platinum was taken up during the simultaneous treatment with benzaldehyde and cisplatin although platinum uptake was even higher during treatment with cisplatin alone. Thus, while benzaldehyde reduces cellular uptake of platinum it also influences platinum binding to DNA.

Inhibition of protein A24 lyase by nitrosoureas

The protein A24 content of Ehrlich ascites tumor cells increased several‐fold following treatment of cell cultures with nitrosoureas, but did not increase when other alkylating agents not containing carbamoyl moieties were tested. The same nitrosoureas and, in addition, 2‐chloroethyl isocyanate inhibited an A24 lyase‐containing cytoplasmic extract in cleaving protein A24 into histone H2A and ubiquitin. It appears that carbamoylation of A24 lyase by nitrosoureas inhibits the enzyme and is responsible for the measured increases in cellular protein A24 content due to reduced turnover of this protein.