A model system for studying covalent binding of food carcinogens MeIQx, MeIQ and IQ to DNA and protein

We have studied the covalent binding of carcinogenic aminoimidazoazaarene compounds to macromolecules in a microsomal model system. The 14C-labelled compounds were incubated with rat-liver microsomes, and binding to macromolecules was measured after their precipitation on glass filters, which were washed several times in organic solvents. The amount of radioactivity was determined by liquid scintillation counting. Covalent binding was dependent on the addition of NADPH, with an optimal concentration of about 1 mM. The binding appeared to follow saturation kinetics when carcinogen concentrations were lower than 200 microM, with Km values of less than 20 microM. At 50 microM, 2-amino-3,4-dimethylimidazo[4,5-f]-quinoline (MeIQ) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) bound more effectively than 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). When DNA was included in the incubations, binding to this macromolecule was ten-fold less per milligram than binding to proteins. In comparison with microsomes from untreated animals, those from rats treated with beta-naphthoflavone caused up to nine-fold more binding of MeIQx, six-fold more of IQ and three times as much of MeIQ. Induction by Aroclor 1254 caused up to 17-fold more binding, whereas induction by phenobarbital caused up to three-fold more binding. The effects of the inducers were greatest for MeIQx and IQ, while smaller effects were seen for MeIQ. The results are most consistent with cytochrome P450-dependent metabolic activation of the carcinogens to hydroxylamine metabolites, for which an isoenzyme(s) inducible by polyaromatic and polychlorinated hydrocarbons is most effective. To our knowledge, this is the first report that MeIQx is metabolized to reactive species capable of covalent binding to macromolecules.     

Dose-response studies of MeIQx in rat liver and liver DNA at low doses

2-Amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx) is aheterocyclic amine mutagen found in cooked meats and is carcinogenicin mice and rats at high doses (mg/kg body wt). Humans, however,are exposed to low amounts (p.p.b.) in the diet, and the effectscaused by exposure to human equivalent doses of MeIQx have beendifficult to determine accurately. We report on the effect ofMeIQx exposure on liver bioavailability, hepatic DNA bindingand MeIQx persistence in both liver tissue and liver DNA afteracute (24 h), and subchronic (7 day and 42 day) exposures inmale Sprague-Dawley rats. Male Sprague-Dawley rats were administered[2-¹⁴C] MeIQx either by gavage or in the diet for 1, 7 or 42days (1x10^-6mg/kg day up to 3.4x10^-2 mg/kg day dose) and the[2-¹⁴C]MeIQx was measured by accelerator mass spectrometry (AMS).Assessment of the kinetics of hepatic MeIQx DNA adduct formationover 42 days (1.1x10^-4 mg [2-¹⁴C]MeIQx kg daily dose) showsthat steady-state [2-¹⁴C]MeIQx tissue concentrations of 138± 15 pg/g liver and DNA adduct levels of 113 ±10 ag adduct/µg DNA were reached at 14–28 days and28 days respectively. The relationship between administereddose and either hepatic MeIQx DNA adduct levels or MeIQx tissuelevels are linear for the 24 h, 7 day and 42 day exposures.Furthermore, MeIQx adducts persist for at least 14 days afterexposure ceases. These data suggest that bloavailability andDNA adduction by MeIQx increase linearly with increasing dosefor both acute and subchronic exposures. These data also showthat MeIQx DNA adducts are useful in predicting daily exposureand support a linear extrapolation in the risk assessment ofMeIQx. However, the quantitative relationship between DNA adductsand tumor formation will also depend on the specific tissueand the subsequent steps needed for tumor progression.     

Prostaglandin H synthase-catalyzed metabolism and DNA binding of 2-naphthylamine

The oxidation of the bladder carcinogen 2-naphthylamine (2-NA) by prostaglandin H synthase (PHS) in vitro was examined. Oxygen uptake studies of 2-NA oxidation in the presence of glutathione, as well as extensive product analysis data, are consistent with a one-electron mechanism of 2-NA oxidation by PHS. The formation of 2-nitrosonaphthalene is not observed under any condition. Metabolism studies with a purified PHS preparation confirm that 2-NA oxidation is dependent upon the peroxidase activity of the enzyme complex, and that a variety of organic hydroperoxides may support the reaction. Horseradish peroxidase oxidizes 2-NA to the same products but, depending on pH, in very different proportions from those obtained with PHS. Oxidation of 2-NA by a one-electron chemical oxidant results in a product profile similar to that obtained in the enzymatic systems. The above data are consistent with a one-electron mechanism of 2-NA oxidation by PHS. The metabolism data provide evidence for the formation of two types of potentially reactive electrophiles: 2-imino-1-naphthoquinone and a free radical species. We further examine the time course of covalent binding of [3H]2-NA products to DNA in vitro, and compare this with the reaction of authentic [3H]2-amino-1-naphthol (2-A-1-N) product(s) with DNA and protein. A significant amount of the PHS-catalyzed binding of 2-NA to DNA is derived from a short-lived intermediate; furthermore, the time course of binding is very rapid. Conversely, the binding to DNA of 2-A-1-N (presumably in the form of 2-imino-1-naphthoquinone) occurs to a lower extent and is not time dependent under the conditions studied. 2-A-1-N binds to protein, however, at a rapid rate and to three orders of magnitude greater extent than to DNA. The PHS-catalyzed binding of 2-NA to DNA was studied under several conditions; binding was shown conclusively to result from the peroxidase activity of the PHS complex. In addition, greater levels of binding were observed at pH 5.0 than at pH 7.6, and when catalyzed by horseradish peroxidase/H2O2 rather than PHS. These are conditions under which 2-A-1-N formation is negligible or nonexistent. These results demonstrate that in the PHS system, a reactive product(s) in addition to 2-A-1-N is generated which binds to DNA, and that this product is probably a free radical.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ellagic acid metabolism and binding to DNA in organ explant cultures of the rat

Ellagic acid (EA) is a plant phenolic compound with postulated antimutagenic and anticarcinogenic activity. In this study, explants of esophagus, forestomach, colon, bladder, trachea, lung and liver from male Sprague-Dawley rats (130-140 g) were incubated in culture medium containing [3H]EA (20 microM, 4.5 microCi/ml) for 24 h at 37 degrees C. After extraction, purification and quantitation of explant DNA significant differences in the binding of EA to the DNA was observed. The most binding occurred in esophagus and the least in lung. Analysis of the organsoluble fraction of the culture medium by high performance liquid chromatography yielded 3 metabolites of EA. None of the metabolites were identified. Elution of water-soluble metabolites from an alumina column showed that there were sulfate ester, glucuronide and glutathione conjugates of EA in the explant culture medium from all the organs. The profile of water-soluble conjugates was very similar between colon and forestomach and between trachea and lung. These results indicate that EA binds to DNA in different tissues and that tissues metabolize EA to both organosoluble and water-soluble products.     

The consequences of fruit and vegetable fibre fermentation on their binding capacity for MeIQx and the effects of soluble fibre sources on the binding affinity of wheat bran preparations

Fruits and vegetables provide dietary fibre some of which ispartly soluble in the upper gut; in the colon it is highly fermentable.Using alcohol-insoluble residues prepared from a range of fruitsand vegetables the effects of fermentation on the changes incomposition and binding capacity have been assessed for thehydrophobic mutagen 2-amino-3,8-dimethylimidazo[4,5-f]lquinoxaline(MeIQx). Fermentation was extensive and resulted in destructionof most of the pectic polysaccharides. Of the unfermented vegetablefibre only cabbage had a measurable hydrophobic binding capacity.The binding capacities of unfermented apple, carrot and sugarbeet were negligible. After fermentation, binding capacities,(per mg of fermented residue), increased. Although fermentedcabbage was found to have the highest capacity of the fruitand vegetable fibres this remained less than the least effectiveof unfermented wheat bran samples which had a relatively highaffinity for MeIQx. Mucin inhibited the binding of MeIQx towheat bran fibre but apple fibre did not The results show thatthe contribution of fruit and vegetable fibre to a hydrophobicbinding matrix in the colon is insignificant and the suggestedharmful effect of fruit and vegetable fibre, maintaining hydrophobicmutagens in solution, can be prevented by the presence of wheatbran fibre.     

Distribution,metabolism, and irreversible binding of hexamethylmelamine in mice bearing ovarian carcinoma

The covalent binding of hexamethylmelamine (HMM) and its metabolites was studied in liver, tumor, blood, kidney, spleen, lung, brain, heart, and small intestine after a single IP injection of 2,4,6-14C-hexamethylmelamine (50 mg/kg) to C57Bl/6J female mice bearing 20-day-old M5076/73A ovarian cancer. Covalent binding to tissue macromolecules was measured 2, 10, and 40 h after injection of the drug. At 2 h liver and small intestine showed the highest levels of irreversibly bound metabolites, the lowest being found in brain and heart. Except in the small intestine, where a decrease was observed between 2 and 10 h, the level of covalent binding was constant up to 40 h. HMM metabolism was also studied. Tissue distribution of pentamethylmelamine (PMM), 2,2,4,6-tetramethylmelamine (TMM), and 2,4,6-trimethylmelamine (TriMM) was determined at the three times considered. At 2 h the drug was already extensively metabolized, TriMM being the major metabolite among those determined.     

Comparative metabolism and DNA binding of 6-nitro-5-methylchrysene and 5-methylchrysene

The metabolic activation in mouse skin of the strong carcinogen,5-methylchrysene (5-MeC) was compared to that of the inactivecompound, 6-nitro-5-methylchrysene (6-NO₂-5-MeC). Metabolitesof 6-NO₂-5-MeC, formed using rat liver homogenates, were identifiedbased on their spectral properties and were used as markersfor studies performed in vivo. In mouse epidermis in vivo, theidentified metabolites of 6-NO₂-5-MeC were trans-1,2-dihydro-1,2-dihydroxy-6-nitro-5-methylchrysene(6-NO₂-5-MeC-1,2-diol), the precursor to a bay region dihydrodiolepoxide, trans-9,10-dihydro-9,10-dihydroxy-6-nitro-5-methylchrysene,and 6-nitro-5-hydroxymethylchrysene. The levels of 6-NO₂-5-MeC-1,2-diolformed in mouse epidermis from 6-NO₂-5-MeC were greater thanthose of the proximate carcinogen trans-1,2-dihydro-1,2-dihydroxy-5-methylchrysene(5-MeC-1,2-diol) formed from 5-MeC. The further metabolism of6-NO₂5-MeC-1,2-diol was examined in mouse epidermis under conditionssimilar to those described previously for 5-MeC-1,2-diol. Theextents of formation of 1,2,3,4-tetraols from both dihydrodiolswere similar. The chromatographic patterns of DNA adducts formedin mouse epidermis from 6-NO₂-5-MeC and 5-MeC were qualitativelysimilar; however, the extent of formation of DNA adducts from5-MeC was 15-fold greater than from 6-NO₂-5-MeC. The reactionsbetween calf thymus DNA and the bay region 1,2-diol-3,4-epoxidesof 5-MeC and 6-NO₂-5-MeC were compared; the levels of adductsformed from the bay region diol epoxide of 5-MeC were aboutfour times greater than those formed from the bay region diolepoxide of 6-NO₂5-MeC. The results indicate that the relativelylow DNA binding in vivo of 6-NO₂-5-MeC may be responsible forits apparent lack of tumorigenicity compared to 5-MeC. It islikely that nitro substitution at the 6-position of 5-MeC interfereswith the structural requirements of the 1,2-diol-3,4-epoxidewhich are necessary for specific DNA interactions.     

Dose-dependent skin ulcers in mice treated with DNA binding antitumor antibiotics

Summary The DNA-binding agents daunomycin (DAUNO), mithramycin (MITH), dactinomycin (ACT-D), amsacrine (mAMSA) and esorubicin (ESO) were tested for local vesicant potential in a quantitative intradermal mouse skin model. Only MITH, which adlineates but doses not intercalate DNA, did not produce dose-dependent skin ulcerations in the mouse. The anthracycline antibiotics DAUNO and ESO produced the largest skin ulcers when administered intradermally at clinically relevant doses (adjusted on the basis of comparable body surface areas). Numerous local pharmacologic adjuvants were tested for activity to decrease skin ulceration patterns in mice given one of the DNA intercalators. Inactive local adjuvants included heat, cold, saline, hyaluronidase, glucorticosteroids and isoproternol. Only one adjuvant, topical dimethylsulfoxide (DMSO), was found to reduce DAUNO skin lesions. A single topical DMSO application significantly decreased ulceration size to almost half of control levels. However, it was ineffective for the other intercalating agents. These results show that the DNA intercalators DAUNO, ESO and ACT-D are potent vesicants in a mammalian skin model. These vesicant agents must be administered cautiously to prevent extravasation. No single local adjuvant treatment can be recommended for extravasation of these drugs in the clinic. One significant exception is DAUNO, where topical DMSO may reduce clinical toxicities.Supported in part by the Public Health Service grants CA-23074, CA-31078 and CA-17094 from the National Cancer Institute, Department of Health and Human Services, Bethesda, MD, USA     

DNA metabolism in the immunocompetent organs of C57BL mice in chemical carcinogenesis]

DNA synthesis was determined in small lymphocytes nuclei in mice C57BL during the process of methylcholanthrene induced carcinogenesis. Both in the pretumor period and in animals with a tumor the DNA synthesis in lymph node lymphocytes nuclei was found to be decreased. Inhibition of the DNA synthesis is greatly pronounced in lymphocytes adjacent to the site of lymph node tumor development.     

Disposition and metabolism of the food mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in rats

The disposition and metabolism of a common food mutagen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), was studied in rats. Five rats of both sexes were given a single oral dose of 14C-labeled MeIQx (3-4 mg/kg body wt). The male rats excreted 36% of the radioactivity and 15% of the mutagenic activity of the dose given in the urine collected during the first 24 h. In the females the corresponding urine contained 41% of the radioactivity and 12% of the mutagenicity. During the next 48 h only 1-3% of the radioactive dose was excreted in urine. The remaining dose was excreted in the feces except of less than 1% that was retained by the tissues after 72 h. The liver and kidney retained more radioactivity than other organs. In a separate study the metabolites of bile, urine and feces of both sexes were investigated. After a single oral dose of 20 mg 14C-labeled MeIQx/kg body wt, three major non-mutagenic metabolites were identified. These were 2-amino-4(or 5)-(beta-D-glucuronopyranosyloxy)-3,8-dimethylimidazo[4,5-f] quinoxaline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxalin-4(or 5)-yl sulfate and N-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl) sulfamate. Another two metabolites present in bile, urine and feces were 2-(beta-D-glucuronopyranosylamino)-3,8-dimethylimidazo[4,5-f ] quinoxaline and 2-amino-8-hydroxymethyl-3-methylimidazo[4,5-f]quinoxalin-4 (or 5)yl sulfate. All metabolites were essentially non-mutagenic. Most of the mutagenicity still present in bile, urine and feces could be explained by unchanged MeIQx. Unchanged MeIQx was the most abundant form excreted in urine.     

Covalent binding of [2-14C]2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx) to mouse DNA in vivo

Female BALB/c mice were administered intragastrically with equimolaramounts of either [2-¹⁴C]2-amino-3,8-dimethyl-[4,5-f]quinoxaline(MeIQx) or 2-acetylamino[9-¹⁴C]fluorene (2AAF). DNA was isolatedfrom tissues of mice killed either 6 or 24 h after administration.Analysis of liver DNA nucleotide digests by HPLC analysis revealedthat all of the radioactivity was attributable to adduct formation.The specific activities of DNA samples were converted to covalentbinding indices (CBI, µmol adduct per mol DNA nucleotides/mmolchemical applied per kg animal body weight). CBI values of 25and 9 were determined for 2AAF and MeIQx in the livers of micekilled 6 h after dosing. The values were in general agreementwith the moderate carcinogenic potency of these compounds. Thespecific activities of DNA preparations obtained from the kidneys,spleens, stomachs, small intestines and large intestines ofmice treated with MeIQx and killed 6 h after dosing were 5-to 35-times less than those obtained with the liver. DNA isolatedfrom the lungs (a target organ for MeIQx tumorigenicity) ofMeIQx-treated mice was not radiolabelled at the limit of detection(CBI <0.3). With the exception of the gastrointestinal tract,the specific activities of DNA samples isolated from mice killed6 h after administration were higher than those from mice killedafter 24 h.     

Effect of butylated hydroxyanisole on the metabolism of benzo[a]-pyrene and the binding of metabolites to DNA, in vitro and in vivo, in the forestomach, lung, and liver of mice

The effects of butyldted hydroxyanisole (BHA) administrationon the amounts of benzo[a]pyrene (BP) metabolite-DNA adductsformed in vivo in the forestomach of A/HeJ mice were investigated48 h after oral administration of BP. BP was administered tomice in amounts known to result in BPInduced neoplasia in certaintissues. Analysis of deoxyribonudeosides by h.p.l.c. showedthat several BP metabolite-DNA adducts were formed in this tissue.The major identified adduct was the (±)-7ß,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDEI) deoxyguanosine adduct. Addition of BHA to the diet inhibitedBPDE I-DNA adduct formation in the forestomach. The inhibitionof BPDE I-DNA adduct formation by BHA occurred under the sameexperimental conditions as does inhibition of tumor formationby this compound. These results in forestomach and previousresults in lung and forestomach demonstrated that inhibitionof the formation of the BPDE I-DNA adduct in the target tissueis a possible mechanism by which BHA inhibits BP-induced neoplasia.BP metabolism and DNA binding were also studied under in vitroconditions using microsomes prepared from forestomach, lung,and liver of A/HeJ mice. The amount of BPDE-DNA adduct formedin vitro is either equal to or lower than the amount of BP phenol-oxide-DNAadduct formed. BPDE I-DNA adduct formation was not significantlydifferent in incubations containing microsomes prepared fromBHA-treated or untreated mice. These results suggest that alterationsof the microsomal monooxygenases induced by BHA feeding arenot sufficient to account for the observed decreases in BPDE-DNAadduct formation in vivo. The monooxygenases were apparentlyaltered by BHA feeding as indicated by the substantial changesin the metabolic profile of BP and the decrease in the formationof the BP phenol-oxide-DNA adducts in the forestomach. The exclusionof glutathione transferases from the in vitro incubations couldaccount for the lack of effect of BHA treatment on BPDE-DNAadduct formation. BHA enhancement of ghitathione transferaseactivity has been postulated to play a role in the anticarcinogenicaction of BHA.     

N-acetylcysteine and S-methylcysteine inhibit MeIQx rat hepatocarcinogenesis in the post-initiation stage

N-acetylcysteine (NAC) and S-methylcysteine (SMC), water soluble organosulfur compounds contained in garlic, were evaluated for chemoprevention of hepatocarcinogenesis after 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) initiation in rats. Intergastric treatment with NAC or SMC five times a week resulted in decreased numbers and areas of preneoplastic, glutathione S-transferase placental form (GST-P) positive foci of the liver in a dose-dependent manner. Moreover, cell proliferation was reduced in GST-P positive foci by NAC and SMC. Insulin-like growth factor I (IGF-I) and inducible nitric oxide synthase (iNOS) mRNA expressions were found downregulated in the liver by NAC. The studies indicate that NAC can serve as a chemopreventive agent for rat hepatocarcinogenesis induced by MeIQx by reducing cell proliferation, which may involve IGF-I and iNOS downregulation.     

Role of metabolism and DNA adduct formation in the induction of sister chromatid exchanges in human lymphocytes by diethylstilbestrol

Recent studies have shown that diethylstilbestrol (DES) induces sister chromatid exchanges (SCEs) in lymphocytes from pregnant and premenopausal women but has only a slight effect on lymphocytes from post-menopausal women or men. In this study blood specimens from premenopausal women were used to define the role of different metabolic pathways on DES-induced formation of SCEs and to determine whether conditions resulting in induction of SCEs also induced detectable levels of DNA adducts. Exposure of lymphocytes in vitro to 0-40 microM DES induced a concentration-dependent increase in SCEs. Addition of indomethacin to the cultures partially abolished DES-induced SCEs, suggesting involvement of prostaglandin synthetases in the formation of specific DES metabolites that cause SCEs. alpha-Naphthoflavone, an inhibitor of cytochrome P-450 monooxygenases, had no effect on DES-induced SCEs. Cells exposed to DES at doses sufficient to cause large increases in SCE induction did not have adducts detectable by a 32P-postlabeling assay capable of revealing adducts at a level of 1 adduct/10(9) normal nucleotides.     

Role of reactive oxygen intermediates in the interferon-mediated depression of hepatic drug metabolism and protective effect of N-acetylcysteine in mice

Interferon (IFN) and IFN inducers are known to depress hepatic microsomal cytochrome P-450 levels, and the liver toxicity of IFN was reported to be lethal in newborn mice. We have observed that administration to mice of IFN and IFN inducers caused a marked increase in liver xanthine oxidase activity. Because this enzyme is well known to produce reactive oxygen intermediates and cytochrome P-450 was reported to be sensitive to the oxidative damage, we have tested the hypothesis that a free radical mechanism could mediate the depression of cytochrome P-450 levels by IFN. Administration to mice of the IFN inducer polyinosinic-polycytidylic acid (2 mg/kg i.p.) caused a 29 to 52% decrease in liver cytochrome P-450. Concomitant p.o. administration of the free radical scavenger, N-acetylcysteine (as a 2.5% solution in drinking water), or the xanthine oxidase inhibitor, allopurinol (100 mg/kg), protected against the IFN-mediated depression of P-450 kg), protected against the IFN-mediated depression of P-450 levels. The results suggest that an increased endogenous generation of free radicals, possibly due to the induction of xanthine oxidase, is implicated in the IFN-mediated depression of liver drug metabolism. The relevance of these data also extends to cases in which this side effect is observed in pathological situations (e.g., viral diseases and administration of vaccines) associated with an induction of IFN.     

The covalent binding of polycyclic hydrocarbons to DNA in the skin of mice of different strains.

The binding of three tritium-labelled carcinogenic polycyclic hydrocarbons, 7,12-dimethylbenz (a) anthracene (DMBA), benzo (a) pyrene (BP) and 3-methylcholanthrene (MCA) to DNA in mouse skin has been studied in C57BL, DBA/2 and Swiss mice following topical application of the hydrocarbons. DNA isolated from the treated areas was hydrolysed to deoxyribonucleosides and chromatographed on Sephadex LH20 columns. The levels of binding of hydrocarbon to DNA were determined from the amount of radioactivity eluted from Sephadex LH20 columns in those fractions containing hydrocarbon-DNA adducts and the radioactivity shown, by rechromatography on AG5OWX4 columns, to be due to tritium incorporation into normal deoxyribonucleosides was not included. C57BL mice were treated with doses of DMBA ranging from 0.025 μmol to 1 μmol/mouse and the levels of hydrocarbon bound to DNA 19 h after treatment were determined; there was no evidence for a threshold dose below which no binding to DNA occurs, and the same hydrocarbon-DNA product peaks were obtained at all doses. The levels of binding of DMBA (1 μmol/mouse) to DNA in skin were compared in C57BL, DBA/2 and Swiss mice at times varying from 6 h to 8 days after treatment. DMBA became bound to similar extents in Swiss and C57BL mice and to a slightly greater extent in DBA/2 mice; the rate of disappearance of bound DMBA from DNA was similar in all three strains. DMBA (0.1 μmol/mouse) was bound to DNA in C57BL and DBA/2 mice to similar extents 19 h after treatment and to a slightly lesser extent in Swiss mice. The ratios of the sizes of the hydrocarbon-DNA product peaks varied with the time after treatment, but were similar at any given time for the three strains. Both BP (1 μmol/mouse) and MCA (1 μmol/mouse) were bound to DNA to similar extents 19 h and 48 h after treatment in all three strains. BP (0.1 μmol/mouse) was bound to DNA in the order DBA/2>C57BL> Swiss 19 h after treatment. The levels of binding for all three hydrocarbons in the different strains do not show a correlation with the reported susceptibilities of the three strains to polycylic hydrocarbon carcinogenesis.     

Pharmacokinetics and distribution of SN 28049, a novel DNA binding anticancer agent, in mice

Purpose

N-[2-(Dimethylamino)ethyl]-2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b]-1,6-naphthyridine-4-carboxamide (SN 28049) is a potent DNA binding topoisomerase II poison that shows excellent antitumour activity in a colon-38 murine tumour model in comparison to standard topoisomerase II poisons. We report here the preclinical pharmacokinetics of SN 28049.

Methods

C57 Bl/6 mice (n = 3 per time point) were treated with a single i.v., i.p. or p.o. administration (8.9 mg/kg). Plasma and tissue samples were analysed using a validated LC/MS method utilizing a homologue as an internal standard.

Results

The assay range was 0.062–2.5 μM with a quantitation limit of 0.062 μM and a detection limit of 0.025 μM. Acceptable intra- and inter-assay accuracy (95–105%) and precision (<6.5% RSD) were achieved. Following i.v. administration, SN 28049 demonstrated 2-compartment model kinetics with a volume of distribution of 42.3 ± 4.1 l/kg, a plasma clearance of 12.1 ± 0.5 l/h per kg and distribution and elimination half-lives of 0.15 ± 0.02 and 2.8 ± 0.2 h (mean ± SE), respectively. For all administration routes, SN 28049 concentrations in normal tissues (brain, heart, liver, lung, and kidney) were 12- to 120-fold higher than those in plasma, but half-lives and mean residence times were similar. The i.p. and p.o. bioavailabilities were 83.1 ± 1.5 and 54.5 ± 1.1%, respectively. In the tumour tissue, elimination half-life (9.1 ± 0.7 h) and the mean residence time (18.2 ± 0.7 h) were significantly (P < 0.001) longer than those of plasma and normal tissues. The tumour area under the concentration–time curve (AUC) (1,316 ± 66 μM h) was also 693-fold greater than the plasma AUC, and considerably higher (~5-fold) than any other tissue examined, indicating selective uptake and retention of SN 28049 in the tumour.

Conclusion

We conclude that SN 28049’s high tumour exposure and long tumour retention time is likely to contribute to its high antitumour activity in vivo.