Nucleoside analogs. 14. The synthesis of antitumor activity in mice of molecular combinations of 5-fluorouracil and N-(2-Chloroethyl)-N-nitrosourea moieties separated by a three-carbon chain.

5-fluorouracil (5-FU) seco-nucleosdies having as the "sugar" moiety a two-carbon (C2) side chain carrying a N-(2-chloroethyl)-N-nitrosourea group were designed as molecular combinations of antimetabolite and alkylating agent, but hydrolytic release of free 5-FU was not fast enough for significant contribution to the high activity they showed against colon and breast tumors in mice. In the present study of the synthesis of the more reactive C3 seco-nucleosides, it emerged that, of various groups attached to the aldehydic center in the precursor phthalimides, only the alkoxy/uracil-1-yl type was conveniently obtained by the standard method. The methylthio/uracil-1-yl analog required relatively large amounts of reagent methanethiol, and exploration of alternatives involving alpha-chlorination of alkyl methyl sulfide or Pummerer rearrangement of its S-oxide, or successive hydrolysis and methylation of isothiouronium bromide, gave disappointing yields. For successful preparation of the alkoxy/uracil-3-yl compounds, the route used for C2 homologs required considerable experimental modification. In addition to these O,N- and S,N-acetals, some N,N-acetals bearing two 5-FU residues were prepared. The new drugs have been tested against a panel of experimental tumors in mice. Although it is evident from a parallel study that even these C3 seco-nucleosides release free 5-FU too slowly in vivo, several of them have shown impressive anticancer activity. Reviewing their performance in comparison with earlier molecular combinations, a short list of seven [B.4152 (6), B.4015 (5), B.4030 (10), B.3999 (4), B.3995 (2), B.4083 (3), and B.3996 (the N 3-substituted analog of 1)] should be investigated further. This is particularly appropriate in light of the present understanding of the mode of action of chloroethylating agents. Following a prolonged period of clinical impatience with nitrosoureas because of limited selectivity action, a new era is confidently anticipated as these powerful drugs are increasingly studied in combination with O6-benzylguanine and other more efficient inhibitors of repair enzymes like O6-alkylguanine-DNA-alkyltransferase now being developed.

Transplacental mutagenicity of N-ethyl-N-nitrosourea at the hprt locus in T-lymphocytes of exposed B6C3F1 mice.

Previous studies have compared age-related differences in total mutagenic burden in mice of differing age (preweanling, weanling, or young adult) after single intraperitoneal (i.p.) injections of ethylnitrosourea (ENU). The purpose of the present investigation was to determine the effects of time elapsed since treatment on the frequency of hprt mutant T-cells (Mf) from mice treated transplacentally with single acute vs. multiple split doses of ENU. To this end, pregnant C57BL/6 mice (n = 13-16/group), which had been bred to C3H males, were given i.p. injections of 40 mg ENU/kg bw in a single dose on day 18 of gestation, in a split dose of 6 mg ENU/kg bw on days 12 through 18 of gestation, or DMSO vehicle alone. Groups of pups were necropsied on days 10, 13, 15 (single dose only), 17, 20, 40, and 70 postpartum for T-cell isolations and hprt Mf measurements using the T-cell cloning assay. The time required to reach maximum Mfs in T-cells isolated from thymus of transplacentally treated animals was 2 weeks, the same time span as previously observed after ENU treatment of adult, weanling, and preweanling mice. Mfs in T-cells isolated from spleens of control animals averaged 2.1 +/- 0.3 (SE) x 10(-6). In spleens of mice treated transplacentally with ENU in a single dose, Mfs reached a maximum at 15 days postpartum [84.7 +/- 15.8 (SE) x 10(-6)] and decreased to lower but still elevated levels at 40 days postpartum. In spleens of mice treated transplacentally with ENU in a split dose, Mfs reached a maximum at 13 days postpartum [74.0 +/- 16.3 (SE) x 10(-6)] and decreased to background levels at 40 days postpartum. The areas under the curves describing the change in hprt Mfs over time for ENU-treated vs. control mice estimate the mutagenic potency for transplacental single- and split-dose exposures to be 1.9 and 0.8 x 10(3), respectively. Comparison of the mutagenic potency estimates for mice exposed to ENU in utero to 4-week-old mice given a similar dose of the same lot number of ENU indicates that the mouse is more susceptible to ENU-induced mutagenesis during fetal life.

Pharmacokinetic studies of chloroethylnitrosocarbamoyl-amino acid derivatives in vivo and in vitro.

The in vitro chemosensitivity of MAC 15A ascites cells to CNC-alanylalanine and CNC-glycinemethylamide was assessed using a clonogenic assay system. In vitro stability studies and in vivo pharmacokinetics were performed using a reversed-phase HPLC technique. Initial concentrations of CNC-alanylalanine and CNC-glycinemethylamide of 5.2 micrograms ml-1 and 3.2 micrograms ml-1 respectively, were required for a 70% reduction in colony formation of MAC 15A cells in vitro. The concentrations of active alkylating species generated were calculated from the drug half-lives in tissue culture medium. On this basis, a 70% cell kill was achieved by equivalent concentrations of 10.8 microM CNC-alanylalanine and 10.6 microM CNC-glycinemethylamide. Analysis of drug levels following intraperitoneal administration revealed that CNC-alanylalanine was cleared more slowly from the peritoneal cavity producing a greater drug concentration at the site of the ascitic MAC 15A tumour. These results suggested that the superior activity of CNC-alanylalanine over CNC-glycinemethylamide against MAC 15A in vivo could be attributed mainly to differences in the pharmacokinetic behaviour of the two drugs following intraperitoneal administration and that CNC-alanylalanine might have a role in the treatment of local peritoneal disease.

DNA hydroxyethylation by hydroxyethylnitrosoureas in relation to their organ specific carcinogenicity in rats.

N-Hydroxyethylnitroso-N'-ethylurea (HEENU) and N-hydroxyethylnitroso-N'-chloroethylurea (HCNU) are two of the few nitrosoureas which induce hepatocellular tumours in rats without further treatment. In the present study we have investigated whether this is due to selectively elevated levels of DNA hydroxyethylation in the target tissue. Formation of the promutagenic base O6-hydroxyethyldeoxyguanosine (O6-HEdG) in various rat tissues was determined by immuno-slot-blot assay. After a single dose by gavage (0.36 mmol/kg body wt) of HEENU, initial levels of O6-HEdG in liver and brain were close to the detection limit of 1.5 mumol/mol deoxyguanosine. In liver, steady state concentrations of 3.5 mumol/mol were reached after 6 h and maintained for at least 18 h. In brain, O6-HEdG levels were 1.7 mumol/mol after 6 h and 3.0 mumol/mol after 24 h. In a second experiment, the formation of O6-HEdG was assessed in target and non-target tissues 6 h after a single dose by gavage (0.36 mmol/kg) of HEENU, HCNU or hydroxyethylnitrosourea (HENU), which is not hepatocarcinogenic. The extent of DNA hydroxyethylation was greatest with HENU in all tissues examined. Concentrations of O6-HEdG were highest in liver (37.2 mumol/mol), followed by kidney (23.3 mumol/mol), lung (18.9 mumol/mol), brain (6.8 mumol/mol) and testes (3.8 mumol/mol). With HEENU and HCNU, levels of 1.4-3.3 mumol O6-HEdG/mol dG were observed in all tissues. In vitro, the alkylation reactions for all three compounds were nearly complete within 6 h. On a molar basis, yields of O6-HEdG in vitro were similar for HENU and HCNU and 3.7 times lower for HEENU. This suggests that the in vivo reactions of the dialkylnitrosoureas are by pathways other than or in addition to those occurring in vitro. We conclude that the hepatocarcinogenicity of HCNU and HEENU cannot be explained on the basis of their reaction with cellular DNA.

Pharmacokinetic parameters determined from the clastogenic activity of ethylnitrosourea and dimethylnitrosamine in mice in vivo.

The latency period (LP) and the time of effective activity (TEA) of ethylnitrosourea (ENU) and dimethylnitrosamine (DMN) were inferred by comparing their kinetics of micronucleated polychromatic erythrocytes (MN-PCE) formation with the kinetics induced by radiation. The results indicate that LP and TEA vary between ENU and DMN. For ENU, these parameters are very similar to radiation indicating a rapid distribution, reaction and elimination. DMN presents a very long LP which agrees with the requirement of mutagen activation. The kinetics of MN-PCE production caused by DMN showed two peaks; this could be due to the presence of two different metabolites, two types of lesions in DNA or two mechanisms of MN-PCE formation. These hypotheses do not exclude each other. The data presented here support the conclusion that the comparison of MN-PCE-formation kinetics induced by chemical agents with that caused by radiation permits one to estimate the LP and the TEA, and provide information on the possible mechanism of action of chemical mutagens.

An accurate nonlinear QSAR model for the antitumor activities of chloroethylnitrosoureas using neural networks.

The quantitative structure-activity relationship (QSAR) studies are investigated in a series of chloroethylnitrosoureas (CENUs) acting as alkylating agents of tumors by neural networks (NNs). The QSAR model is described inaccurately by the traditional multiple linear regression (MLR) model for the substitution of CENUs at N-3, whose characteristics play key roles in the biological activity. A nonlinear QSAR study is undertaken by a three-layered NN model, using molecular descriptors that are known to be responsible for the antitumor activity to optimize the input variables of the MLR model. The results demonstrate that NN models present the relationship between antitumor activity and molecular descriptors clearly, and they yield predictions in excellent agreement with the experiment's obtained values (R(2)=0.983). The R(2) value is 0.983 for the 5-8-1 NN model, compared with 0.506 for the MLR model, and the nonlinear model is able to account for 98.3% of the variance of antitumor activities.

Formation and persistence of O6-(2-hydroxyethyl)-2' -deoxyguanosine in DNA of various rat tissues following a single dose of N-nitroso-N-(2-hydroxyethyl)urea. An immuno-slot-blot study.

Rabbit antibodies against O6-(2-hydroxyethyl)-2'-deoxyguanosine (O6-HEdg) were used to develop a highly sensitive immuno-slot-blot assay for this promutagenic base which enabled the quantitation of greater than or equal to 3.6 mumol O6-HEdG/mol deoxyguanosine, corresponding to greater than or equal to 5 fmol in a 3-mug DNA sample. This assay was used to study DNA hydroxyethylation by N-nitroso-N-(2-hydroxyethyl)urea (HENU) in adult male F344 rats. Initial amounts of O6-HEdG 2 h after a single i.v. dose of 50 mg/kg were highest in kidney (81 mumol O6-HEdG/mol deoxyguanosine), followed by lung and liver (67 and 55 mumol/mol dG respectively). Formation of O6-HEdG in cerebral DNA was considerably lower (18 mumol O6-HEdG/mol deoxyguanosine), probably reflecting delayed crossing of the blood-brain barrier by HENU due to its hydrophilicity. The formation of O6-HEdG in liver and kidney was strictly proportional to dose over a range of 5-50 mg HENU/kg. Repair of O6-HEdG was very rapid in liver (apparent half-life, 12 h), and somewhat slower in kidney and lung (approximate half-life, 40 h and 48 h respectively). In contrast, 62% of the initial amount of O6-HEdG in cerebral DNA was still present after 7 days. Saturation of the hepatic O6-alkyl-guanine-DNA alkyltransferase by pretreatment with N-nitroso-dimethylamine (20 mg/kg) almost completely inhibited the removal of O6-HEdG, indicating that O6-HEdG is predominantly repaired by this repair enzyme.

Enzymatic removal of O6-ethylguanine from mitochondrial DNA in rat tissues exposed to N-ethyl-N-nitrosourea in vivo.

DNA repair is essential for maintaining the integrity of the genetic material, and a number of DNA repair mechanisms have been fairly well characterized for the nuclear DNA of eukaryotic cells as well as prokaryotes. However, little is known about DNA repair in mitochondria. Using highly sensitive immunoanalytical methods to detect specific DNA alkylation products, we found active removal of O6-ethyl-2'-deoxyguanosine (O6-EtdGuo) from rat liver mitochondrial DNA after pulse-exposure to N-ethyl-N-nitrosourea in vivo. In the kidney, O6-EtdGuo was removed from mitochondrial DNA with moderate efficiency, but nearly no removal was observed from the DNA of brain mitochondria. Among the rat tissues examined, the kinetics of O6-EtdGuo elimination from mitochondrial DNA was very similar to the kinetics of removal from nuclear DNA. O4-Ethyl-2'-deoxythymidine, another premutagenic DNA ethylation product, was stable in both mitochondrial and nuclear DNA of rat liver.

Pharmacokinetics and anti-tumour activity of B.4152, a reactive molecular combination of 5-fluorouracil and N-(2-chloroethyl)-N-nitrosourea, and its uracil analogue.

The original design of the previously described molecular combinations of 5-fluorouracil (5-FU) and a chloroethyl nitrosourea (CNU) moiety was on the basis that a single drug would be able to deliver two cytotoxic moieties to tumours following hydrolytic release in vivo of free 5-FU. Subsequently experiments have shown to date that the observed activity is due mainly to the alkylating effect of the CNU. This study investigates a molecular combination of 5-FU/CNU (B.4152) which is the most readily hydrolysed under acid conditions of all the 5-FU seco-nucleosides so far prepared, and compares it with the unsubstituted uracil analogue B.4184. In vitro cytotoxicity studies against three murine colon tumour cell lines showed large differences in IC50 values between the two analogues, those for B.4184 being > 10-fold greater than those for B.4152. These differences could not be accounted for by drug stability as half-lives in tissue culture medium were similar. In vivo, B.4152 was also more active against the tumour lines and was marrow sparing at therapeutic doses. Pharmacokinetic studies demonstrated that improved activity was not due to release of free 5-FU, but differences in activity, toxicity and plasma pharmacokinetics between B.4152 and B.4184 are quite marked, indicating that the 5-FU moiety does have an important role to play.

Tissue distribution of polybutylcyanoacrylate nanoparticles loaded with spin-labelled nitrosourea in Lewis lung carcinoma-bearing mice.

The tissue distribution of polybutylcyanoacrylate nanoparticles (PBCN) with a diameter of 127 nm, loaded with 1-(2-chloroethyl)-3-(1-oxyl-2,2,6,6-tetramethylpiperidinyl)-1- nitrosourea (spin-labelled nitrosourea, SLCNU) is described. PBCN-suspensions were intraperitoneally (i.p.) injected into Lewis lung carcinoma bearing mice. The biodistribution of PBCN in the visceral organs, blood and tumor was studied by electron spin resonance (ESR) spectroscopy. A relatively low accumulation of nanoparticles in the liver and spleen was found. The accumulation was negligible in the i.m. implanted primary tumor. SLCNU-loaded nanoparticles were mainly found in the lungs, kidneys, and heart. The highest content of the particles studied was observed in the lungs of tumor bearing experimental animals damaged by metastases. These findings suggest that PBCN offer some opportunities in the targeting of SLCNU to lung metastases.