DNA threading bis(9-aminoacridine-4-carboxamides): effects of piperidine sidechains on DNA binding, cytotoxicity and cell cycle arrest.

We describe the synthesis of a series of DNA-threading bis(9-aminoacridine-4-carboxamides) comprising ethylpiperidino and N-methylpiperidin-4-yl sidechains, joined via neutral flexible alkyl chains, charged flexible polyamine chains and a semi-rigid charged piperazine linker. Their cytotoxicity towards human leukaemic cells gives IC(50) values ranging from 99 to 1100 nM, with the ethylpiperidino series generally being more cytotoxic than the N-methylpiperidin-4-yl series. Measurements with supercoiled DNA indicate that they bisintercalate.

Chemotherapeutic sensitivity of normal and leukemic hematopoietic progenitor cells to N-[4-(9-acridinylamino)-3-methoxyphenyl]-methanesulfonamide, a new anticancer agent.

The sensitivities of hematopoietic colony-forming cells (CFC) to N-[4-(9-acridinylamino)-3-methoxyphenyl]-methanesulfonamide (NSC-249992) (m-AMSA) were measured with an in vitro clonogenic assay, a modification of the Robinson and Pike human marrow culture system. CFC derived from bone marrow and peripheral blood of normal subjects and patients with chronic myeloid leukemia (CML) were studied. Sensitivities to m-AMSA did not differ significantly between normal marrow and blood CFC, between normal and CML CFC, or between CML CFC obtained from patients with leukemias in chronic phase and blast transformation. Drug doses and exposure times producing in vitro hematopoietic inhibition were comparable to clinically employed drug dosages and schedules associated with hematopoietic toxicity.

Activity of 4'-(9-acridinylamino)methanesulfon-m-anisidide against Chinese hamster cells in multicellular spheroids.

The cytotoxic activity of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), a novel acridine derivative with clinical antitumor activity, has been examined in multicellular spheroids grown from Chinese hamster V79-171b cells. m-AMSA is much less effective against cells within these tumor-like structures than it is against exponential-phase V79-171b cells in monolayer cultures, the initial D0 of the survival curve for the latter being approximately 10-fold lower than for the former following a 60-min exposure to the drug. The resistance of spheroid cells to m-AMSA appears to be at least partially a result of the noncycling or slowly cycling state of the majority of these cells, although they are more sensitive than cells in plateau-phase monolayers. A further component of resistance in spheroids requires the presence of an intact spheroid structure and may be due to drug transport limitations. The use of sequential trypsinization techniques to recover cells at varying depths within spheroids demonstrates that a 60-min m-AMSA treatment preferentially kills cells nearest the spheroid surface, suggesting that tumor cells at a distance from the vasculature may limit the efficacy of m-AMSA in vivo.

Toxicity of 4'-(9-acridinylamino)methanesulfon-m-anisidide in exponential- and plateau-phase Chinese hamster cell cultures.

The antitumor acridine derivative 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) is at present being evaluated in Phase 2 clinical trials. Exposure of exponential-phase Chinese hamster V79-171b cells to physiologically attainable concentrations of m-AMSA for 60 min generates survival curves with little or no threshold region and an initial D0 of 0.245 +/- 0.019 (S.D.) microM under standard conditions of assay. A minor subpopulation of apparently drug-resistant cells is revealed at low survivals, but these cells on culturing do not display a stable drug-resistant phenotype. m-AMSA survival curves for Chinese hamster ovary cells display features similar to the above. Sensitivity of V79-171b cells to m-AMSA is maximal near pH 7.2 and is markedly reduced by the presence of fetal calf serum. Hypoxia has little effect on the toxicity of m-AMSA, and repair of potentially lethal damage has not been observed after treatment with this agent. Noncycling plateau-phase V79-171b or Chinese hamster ovary cells are markedly less sensitive to m-AMSA than are early log-phase cells. This resistance to m-AMSA appears to be related to the slowly cycling or noncycling status of plateau-phase cells, suggesting that the cytokinetic character of cell populations in vivo will be a major determinant of their sensitivity to this drug. However, the increase in resistance to m-AMSA during growth into plateau-phase appears to commence well before departure from exponential growth can be detected and may thus be a consequence of metabolic changes more subtle than the transition from a cycling to a noncycling state.

Comparison of the mutagenic and clastogenic activity of amsacrine and other DNA-intercalating drugs in cultured V79 Chinese hamster cells.

The acridine derivative amsacrine (m-AMSA) is used clinically for the treatment of acute leukemias. The mutagenic activity of this drug has been evaluated at the 6-thioguanine (6-TG) and ouabain resistance loci in cultured Chinese hamster fibroblasts (V79-171b cell line). m-AMSA was found to have weak but significant mutagenic activity at the 6-TG but not at the ouabain resistance locus, after either 1- or 45-hr exposures at concentrations causing up to 90% cell kill. Two other intercalating agents with antitumor activity, Adriamycin and actinomycin D, provided essentially identical results. All three drugs were potent inducers of micronuclei in V79-171b cells, indicating high clastogenic activity. For these intercalating agents, the yield of 6-TG-resistant mutants was approximately 100-fold lower than that for ethyl methanesulfonate after exposures causing equivalent toxicity or equivalent chromosome breakage. The acridine half-mustard ICR-191 resembled ethyl methanesulfonate rather than the other intercalating agents in providing a high yield of 6-TG-resistant mutants relative to its clastogenic activity. The tumor-inactive intercalator 9-aminoacridine demonstrated only low clastogenic activity with a lack of significant mutagenic activity at toxic concentrations. These results suggest that, for m-AMSA, Adriamycin, and actinomycin D, both cell killing and mutagenesis could be direct consequences of chromosome breakage, while 9-aminoacridine may kill cells by a different mechanism. In view of its mutagenic and clastogenic activity at clinically achievable exposures and the similarity of its genotoxic properties to Adriamycin, m-AMSA should be considered a potential carcinogen.

Topoisomerase II-mediated DNA damage produced by 4'-(9-acridinylamino)methanesulfon-m-anisidide and related acridines in L1210 cells and isolated nuclei: relation to cytotoxicity.

4'-(9-Acridinylamino)methanesulfon-m-anisidide (m-AMSA) is a DNA intercalating 9-aminoacridine with clinical activity in adult acute leukemia. m-AMSA has been shown to produce protein-linked DNA strand breaks in mammalian cells through an interaction with the nuclear enzyme DNA topoisomerase II. We have compared the effects of m-AMSA and several acridine analogues (9-aminoacridine; A, NSC 343499; B, SN 16507; C, NSC 140701; D, SN 13553) on DNA integrity and cell survival in L1210 leukemia in vitro. Cells (or isolated nuclei) were treated with drugs (0.1-50 microM) for 0.5-1.0 h and subsequently analyzed using the alkaline elution technique. All drugs, except Compound D, produced DNA-protein cross-links (DPC) in L1210 cells. At 1 microM, potency was in the order, C greater than m-AMSA greater than B greater than A much greater than 9-aminoacridine. In isolated nuclei, DPC and single-strand breaks were produced in essentially a 1:1 ratio, which is consistent with topoisomerase II-mediated protein-linked DNA breaks. Potency differences were less pronounced in nuclei than in cells. In isolated nuclei, Compound D produced extensive DPC not associated with single-strand breaks, which suggests a more complex activity for this compound. Colony formation assays demonstrated the cytotoxicity of most of these acridine analogues (C greater than B greater than A approximately equal to m-AMSA much greater than D = 9-aminoacridine). Correlation of DPC with cell kill gave similar curves for each compound. These results are evidence for a causal relationship between drug-induced topoisomerase II-mediated DNA breaks and cytotoxicity.

Phase II study of 4'-(9-acridinylamino)methanesulfon-m-anisidide (NSC 249992) in children with acute leukemia and lymphoma.

Phase I clinical studies of 4'-(9-acridinylamino)methanesulfon-m-anisidide (AMSA) using several dose schedules have shown acceptable toxicity and antitumor responses in acute leukemia and several carcinomas. Thirty-eight children with acute leukemia and non-Hodgkin's lymphoma were treated with AMSA in a total dose of 140 to 600 mg/sq m given as a daily i.v. infusion in 2 to 5 days. Maximal tolerated dose was 600 mg/sq m given in 5 days. Complete and partial remissions were seen in four of 18 patients with acute lymphocytic leukemia, zero of eight patients with acute nonlymphocytic leukemia, and one of five patients with non-Hodgkin's lymphoma. Marrow aplasia and remissions were also seen with lower doses. The major toxic effects were mucositis, fever, and sepsis which were dose related. Mild nausea and vomiting, transient elevation of serum glutamic oxaloacetic-acid-transaminase, and bilirubin were noted. All of these patients had had prior anthracycline therapy. Abnormal echocardiograms were seen in 14 of 23 patients who had echocardiograms done before and after AMSA. Seven developed congestive heart failure in association with sepsis in five and with epicardial disease in one. We conclude that AMSA possesses significant activity in childhood leukemia and lymphoma and that studies of AMSA in combination with other effective agents should be done.

Reduced formation of protein-associated DNA strand breaks in Chinese hamster cells resistant to topoisomerase II inhibitors.

DNA intercalating drugs and the epipodophyllotoxins etoposide and teniposide interfere with the action of mammalian DNA topoisomerase II by trapping an intermediate complex of the enzyme covalently linked to the 5'-termini of DNA breaks. This effect can be observed in intact cells by alkaline elution measurement of protein-associated DNA strand breaks. To assess the cytotoxic role of this effect, we have studied a subline of DC3F Chinese hamster lung cells selected for resistance to the intercalating agent 9-hydroxyellipticine. This subline (DC3F/9-OHE) was cross-resistant to other intercalators as well as to etoposide. Resistance to Adriamycin was associated with reduced uptake. However, resistance to 4'-(9-acridinylamino)methanesulfon-m-aniside and 2-methyl-9-hydroxyellipticinium was observed in the absence of changes in drug uptake, suggesting a second mode of resistance. DC3F/9-OHE cells formed fewer protein-associated DNA strand breaks in response to 4'-(9-acridinylamino)methanesulfon-m-aniside, 2-methyl-9-hydroxyellipticinium, or etoposide than did the sensitive parental cells. The same was true for isolated nuclei from these cells, which is consistent with a mode of resistance unrelated to drug uptake through the plasma membrane. These data suggest that resistance to DNA topoisomerase II inhibitors exhibited by DC3F/9-OHE cells is due in part to a modification of topoisomerase II activity.

Enhancement of the DNA breakage and cytotoxic effects of intercalating agents by treatment with sublethal doses of 1-beta-D-arabinofuranosylcytosine or hydroxyurea in L1210 cells.

4'-(9-Acridinylamino)methanesulfon-m-anisidide (m-AMSA) and other DNA intercalating agents produce protein-associated DNA strand breaks, the formation of which are mediated by topoisomerase-like chromosomal proteins. As topoisomerases would be expected to be most active during DNA replication, DNA synthesis inhibitors may alter the sensitivity of cellular DNA to intercalator-induced scission. We report that treatment of L1210 cells with 1-beta-D-arabinofuranosylcytosine (ara-C) (0.1 microM) or hydroxyurea (HU) (0.1 mM) for 18 hr resulted in a 2- to 2.4-fold enhancement of m-AMSA-induced protein-associated DNA single-strand breaks and DNA-protein cross-links as measured by alkaline elution. This enhancement was dependent on the duration of ara-C or HU treatment as well as on the concentration of ara-C or HU. Enhancement did not correlate with any alteration in cellular uptake of intercalator or with ara-C- or HU-induced alterations in the DNA synthetic rate. The DNA within nuclei isolated from ara-C- or HU-treated cells also displayed an enhanced susceptibility to m-AMSA-induced scission. There was a correlation between enhanced single-strand break formation and recruitment of cells into S-phase as well as between single-strand break formation and the production of a hypomethylated state of cellular DNA. Concurrent with the enhancement of m-AMSA-induced cellular DNA effects was a synergistic effect on m-AMSA cytotoxicity by ara-C or HU. This enhancement of intercalator effects was also found for the intercalator Adriamycin. We propose that these sublethal concentrations of ara-C and HU alter chromatin structure possibly via DNA hypomethylation and/or altered DNA-histone interactions so that intercalator-induced DNA effects are enhanced. Alternatively, the topoisomerase-like activity involved in intercalator-induced, protein-associated DNA break production may be increased in the nuclei of ara-C- or HU-treated cells.

Correlations between intercalator-induced DNA strand breaks and sister chromatid exchanges, mutations, and cytotoxicity in Chinese hamster cells.

Intercalator-induced DNA strand breaks in mammalian cells represent topoisomerase II:DNA complexes trapped by intercalators. These complexes are detected as protein-associated DNA single-strand breaks (SSB) and DNA double-strand breaks (DSB) by filter elution. Using Chinese hamster lung fibroblasts (V79 cells) that were treated for 30 min with various concentrations of 4'-(9-acridinylamino)methanesulfon-m-anisidide or 5-iminodaunorubicin, we measured DNA strand breaks (SSB and DSB), sister chromatid exchanges (SCE), mutations at the hypoxanthine:guanine phosphoribosyltransferase locus, and cell killing. Further, we correlated DNA strand breakage with the three other parameters. Both drugs induced SCE, mutations, and cell killing at concentrations which also produced reversible DNA strand breaks. While the quantity of DSB correlated with SCE, mutations, and cytotoxicity for both drugs, we found more SCE, mutations, and cytotoxicity per SSB in cells treated with 5-iminodaunorubicin than in those treated with 4'-(9-acridinylamino)methanesulfon-m-anisidide. These data show that the DSB (but not the SSB) induced by 4'-(9-acridinylamino)methanesulfon-m-anisidide and 5-iminodaunorubicin at DNA topoisomerase II binding sites correlated closely with SCE, mutations, and cell killing and could therefore be responsible for their production.

Synthesis, antitumor activity, and DNA binding properties of a new derivative of amsacrine, N-5-dimethyl-9-[(2-methoxy-4-methylsulfonylamino) phenylamino]-4-acridinecarboxamide.

The 4-(N-methylcarboxamido)-5-methyl derivative of amsacrine (NSC 249 992) has been synthesized as part of a program aimed at optimizing solid tumor activity in this series. Physicochemical studies of this analogue (N-5-dimethyl-9-[(2-methoxy-4-methylsulfonylamino) phenylamino]-4-acridinecarboxamide; NSC 343 499) indicate a slightly increased lipophilicity (estimated log p = 1.10), a decreased acridine base strength (pKa 6.40), and a 16-fold-higher association constant for double-stranded calf thymus DNA (Ka 2.1 X 10(6) M-1 at 0.01 ionic strength). Like amsacrine, the drug binds to DNA by intercalation. Inhibition of cell growth has been monitored by continuous drug exposure assays with a variety of rodent and human cell lines. The concentration for 50% inhibition varied from 6.7 nM (T-47D, a human breast carcinoma line) to 800 nM (P388/ADR, a murine cell line resistant to Adriamycin). N-5-Dimethyl-9-[(2-methoxy-4-methylsulfonylamino) phenylamino]-4-acridinecarboxamide was cytotoxic at growth-inhibitory concentrations and also induced cell cycle arrest in the G2 phase. It was active against P388 leukemia following administration by p.o., i.v., or i.p. routes, and it was superior to amsacrine, daunorubicin, and Adriamycin. It was curative towards i.v.-injected Lewis lung tumor in a proportion of animals when treatment was started on Day 1 or Day 5 after tumor inoculation. It also produced highly significant life extensions against advanced tumors (treatment starting Day 9 after i.v. inoculation or on Day 8 after s.c. inoculation) and was comparable to cyclophosphamide in its effectiveness. It is a candidate drug for clinical trial.

In vitro chemosensitivities of human tumor stem cells to the Phase II drug 4'-(9-acridinylamino)methanesulfon-m-anisidide and prospective in vivo correlations.

A potential application of the human tumor stem cell colony assay is to guide Phase II clinical investigations by identifying classes of tumors (or individual patients) which are sensitive in vitro to a new antitumor compound. We have tested human tumor stem cells from 140 tumor biopsies representing 20 different tumor types for chemosensitivity to the Phase II drug 4'-(9-acridinylamino)methanesulfon-m-anisidide. In vitro sensitivity was defined as a reduction in the number of tumor colony-forming cells to 30% of the control or less after a 1-hr exposure to one-tenth of the pharmacologically achievable plasma concentration of 4'-(9-acridinylamino)methanesulfon-m-anisidide. In vitro sensitivity was found in 29 cases: non-Hodgkin's lymphoma (2 of 2); cervical carcinoma (1 of 1); sarcoma (3 of 6); neuroblastoma (1 of 2); acute myelogenous leukemia (6 of 16); chronic myelogenous leukemia (1 of 3); melanoma (8 of 34); uterine carcinoma (1 of 5); lung carcinoma (1 of 9); ovarian carcinoma (4 of 36); and breast carcinoma (1 of 11). Prospective in vitro-in vivo correlations in eight patients with various tumor types showed that three of three patients sensitive in vitro to 4'-(9-acridinylamino)methanesulfon-m-anisidide responded in vivo, while five of five patients resistant in vitro had no clinical response. The results provide support for further evaluation of the utility of the human tumor stem cell colony assay for targeting Phase II clinical trials.

DNA damage and cytotoxicity of nitracrine in cultured HeLa cells.

The effects of nitracrine (1-nitro-9-(3,3-N,N-dimethylaminopropylamino)acridine on DNA of cultured HeLa cells were studied. DNA strand breakage and interstrand cross-linking as well as DNA-protein cross-linking were measured by means of an alkaline elution technique and were compared with the cytotoxic effect of the drug. Interstrand cross-links were not detectable in the concentration range that inhibited cell growth up to 99%. DNA single-strand breaks were found when cells were treated with highly cytotoxic doses of the drug. DNA breakage was not reparable and exhibited a tendency to increase during incubation after drug removal. The only chromatin lesion induced by sublethal doses of nitracrine were DNA-protein cross-links which persisted for 24 h after drug treatment. It is concluded that DNA breaks represent degraded DNA from dying cells, whereas DNA-protein cross-links are specific cellular lesions, which may be responsible for the cell-killing effect of nitracrine.

Comparative analysis of mutagenic potency of 1-nitro-acridine derivatives.

The anticancer effect of 1-nitro-9-hydroxyethylamino acridine (C-857), a compound belonging to the 1-nitroacridine class, has been well documented. Despite its therapeutic efficacy, the clinical development of C-857 has been impeded partly due to its high systemic toxicity. In an effort to enhance antitumor efficacy and lower toxicity, derivatives of C-857 have been synthesized with substitutions made at position C-4 and/or an esterified hydroxyl group in side chain at the C-9 position. The introduction of a methyl group at C-4 resulted in C-1748, which has a significantly higher therapeutic efficacy and is being clinically developed as an anticancer agent for solid tumors. The present study was undertaken to correlate the mutagenicity of C-857, C-1748, C-1790, C-1872 and C-1873 with their cytotoxicity and their anti-tumor efficacy. The mutagenicity of these drugs was determined using three Ames Salmonella typhimurium strains TA1537, TA98 and TA102. The bacteria were treated with different molar concentrations, ranging from 10(-3) to 10(-12) M, of the drugs and drug-induced histidine revertants were then counted after a 48 h incubation. C-1748 did not induce any revertants in both TA1537 and TA98 at a dose of 10(-6) M, whereas, C-857 at the same dose induced approximately 842 and approximately 1034 revertants respectively. In TA102, mutagenicity was lower than observed with TA98 and TA1537 with highest revertants observed at 10(-5) M with C-857 (approximately 606) and C-1748 (approximately 108). Higher mutagenicity was observed in the derivatives C-1790, C-1872 and C-1873 compared to C-1748, but lower than C-857. These studies demonstrate that C-1748 has the least mutagenic potential, with a much higher antitumor effect in prostate cancer and is a promising chemotherapeutic agent for clinical development.

Genetic toxicology of tricyclic carboxamides, a new class of DNA binding antitumour agent.

(N-[2-(Dimethylamino)ethyl]acridine-4-carboxamide (acridine carboxamide; NSC 601316) is an acridine-derived experimental antitumour agent with curative properties against Lewis lung carcinoma in mice. Although it intercalates into DNA and also appears to interact with topoisomerase II, its DNA binding properties appear distinct from other acridine derivatives such as the clinical antitumour drug, amsacrine. The mutagenic properties of acridine carboxamide, together with three related compounds containing either 9-aminoacridine or phenazine chromophores, were studied at the 6-thioguanine and ouabain loci in cultured V79 Chinese hamster fibroblasts. Each compound, when tested at concentrations causing up to 90% kill, had weak but significant activity at the 6-thioguanine but not at the ouabain locus. All drugs were potent inducers of micronuclei, indicating high clastogenic activity. There was a highly significant relationship between mutation frequency (as resistance to 6-thioguanine) and either cytotoxicity (measured as D37 in a clastogenicity assay) or clastogenicity. A broader range of compounds was also tested for microbial mutagenicity. In Salmonella typhimurium strains, none were mutagenic in TA98, TA100 or TA102 but several were mutagenic in TA1537, a frameshift tester strain. Some drugs also caused 'petite' mutagenesis in Saccharomyces cerevisiae. In general, compounds with the phenazine chromophore, which has no positive charge, were the most mutagenic in these systems. However, activity was not related to mammalian mutagenicity or antitumour effect. The results suggest that in mammalian cells, the cytotoxicity, clastogenicity and mutagenic activity of these drugs are mediated by similar mechanisms to those for amsacrine analogues, probably involving the enzyme DNA topoisomerase II.

Potential antitumor agents. 36. Quantitative relationships between experimental antitumor activity, toxicity, and structure for the general class of 9-anilinoacridine antitumor agents.

Quantitative relationships (QSAR) have been derived between antileukemic (L1210) activity and agent physicochemical properties for 509 tumor-active members of the general class of 9-anilinoacridines. One member of this class is the clinical agent m-AMSA (NSC 249992). Agent hydrophobicity proved a significant but not a dominant influence on in vivo potency. The electronic properties of substituent groups proved important, but the most significant effects on drug potency were shown by the steric influence of groups placed at various positions on the 9-anilinoacridine skeleton. The results are entirely consistent with the physiologically important step in the action of these compounds being their binding to double-stranded DNA by intercalation of the acridine chromophore between the base pairs and positioning of the anilino group in the minor groove, as previously suggested. An equation was also derived for the acute toxicities of 643 derivatives of 9-anilinoacridine. This equation took a somewhat similar form to the one modeling antileukemia potency, emphasizing the usual fairly close relationship between potency and acute toxicity for antitumor agents in general. This study demonstrated the power of QSAR techniques to structure very large amounts of biological data and to allow the extraction of useful information from them bearing on the possible site of action of the compounds concerned.

9-acridinyl and 2-methoxy-6-chloro-9-acridinyl derivatives of aliphatic di-, tri-, and tetraamines. Chemistry, cytostatic activity, and schistosomicidal activity.

9-acridinyl derivatives of 1,6-hexanediamine, 1,8-octanediamine, bis(3-aminopropyl)amine, N,N'-bis(3-amino-propyl)piperazine, and N-ethyl-1,6-hexanediamine in the form of their hydrochlorides were prepared in high yields and converted into potential hetero bis DNA intercalating diacridines. The corresponding potential homo bis DNA intercalating reagents were prepared by heating the above amines with 9-chloroacridines. The chemical stability of the acridines was examined. Their cytostatic activity against Cloudman melanoma cells, in vitro, has been determined. The strongest cytostatic activity was observed for the acridine derivatives of the tri- and tetraamines. The schistosomicidal activity of selected acridine and diacridine derivatives against Schistosoma mansoni in mice was found to be insignificant. The S. mansoni egg development was apparently suppressed by this treatment.

Absence of delayed lethality in mice treated with aclacinomycin A.

Two compounds that bind to or intercalate with DNA (DNA binders), e.g., adriamycin and 'dihydroxyanthracenedione', 9,10-anthracenedione, 1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)-amino]ethyl]amino]-, dihydrochloride salt, consistently caused delayed lethality (20-200 days after treatment) if administered intraperitoneally (IP). Both of these agents contain para-hydroxyl groups in the ring adjacent to the quinone ring. Certain analogs of these compounds (aclacinomycin A and 'anthracenedione acetate', 9,10-anthracenedione, 1,4-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-, diacetate (salt), which do not contain para-hydroxyl groups, did not cause delayed deaths when injected IP. The only difference in the molecular structure (other than the nature of their amine salts) between dihydroxyanthracenedione and anthracenedione acetate lies in the para-hydroxyl groups in the ring adjacent to the quinone ring. Another compound that binds to DNA, m-AMSA, which has neither the quinone function nor the para-hydroxyl groups, did not cause delayed deaths after IP administration.

Intraperitoneal administration of the antitumour agent N-[2-(dimethylamino)ethyl]acridine-4-carboxamide in the mouse: bioavailability, pharmacokinetics and toxicity after a single dose.

The pharmacokinetics, tissue distribution and toxicity of the antitumour agent N-[2-(dimethylamino)-ethyl]acridine-4-carboxamide (AC) were studied after i.p. administration of [3H]-AC (410 mumol/kg) to mice. The latter is the optimal single dose for the cure of advanced Lewis lung tumours. AC was rapidly absorbed into the systemic circulation after i.p. administration, with the maximal concentration (Cmax) occurring at the first time point (5 min). There was no reduction in bioavailability as compared with previous i.v. studies, but the shape of the plasma concentration-time profile was considerably different, reflecting a 3-fold lower Cmax value (20.9 +/- 3.6 mumol/l) and a longer t1/2 value (2.7 +/- 0.3 h) as compared with that observed after i.v. administration (1.6 +/- 0.6 h). Model independent pharmacokinetic parameters after i.p. administration were: clearance (C), 17.5 l h-1 kg-1; steady-state volume of distribution (Vss), 14.1 l/kg; and mean residence time (MRT), 1.46 h. High but variable tissue uptake of AC was observed, with tissue/plasma AUC ratios being 5.7 for heart, 8.4 for brain, 18.9 for kidney and 21.0 for liver but with similar elimination t1/2 values ranging from 1.3 to 2.7 h. All radioactivity profiles in plasma and tissues were greater than the respective parent AC profiles and showed prolonged elimination t1/2 values ranging from 21 h in liver to 93 h in brain. However, tissue/plasma radioactivity AUC ratios were near unity, ranging from 0.7 to 1.57, with the exception of the gallbladder (15.6), which contained greater amounts of radioactivity. By 48 h, approximately 70% of the total dose had been eliminated, with the faecal to urinary ratio being approximately 2:1. This i.p. dose was well tolerated by mice, with sedation being the only obvious side effect. No major change was observed in blood biochemistry or haematological parameters. Comparisons of Cmax, tmax and AUC values determined for AC in brain after its i.p. and i.v. administration suggest that the reduction in acute toxicity after i.p. administration is not due to reduced exposure of the brain to AC as measured by AUC but may be associated with the lower Cmax value or the slower rate of entry of AC into the brain after i.p. administration.

Influence of scheduling on therapeutic and toxic effect of AMSA in Lewis lung carcinoma.

The antitumor activity and toxic effect of AMSA were studied in Lewis lung carcinoma (3LL) at various stages of growth. The total dose of drug injected IP was 15 mg/kg, which is equivalent to the LD10. Different administration schedules were tested, these being single-injection schedules (day 1, 7, or 10 after tumor implantation) and repeated low-dose-injection regimens (days 1, 4, and 7 and days 1-7 after tumor implantation). Tumor weight inhibition, retardation of growth, reduction in the number of metastases, and median survival time of treated mice over controls were analyzed as end-points to evaluate the antitumor activity of AMSA. Early deaths and changes in white blood cell count were considered as parameters of toxicity. Our findings can be summarized as follows: (1) AMSA is only minimally effective against primary 3LL tumor at all the growth stages examined and no schedule-dependency is detected; (2) a greater reduction in metastases (70%-77%) is found when the drug is administered fractionally than when it is given in a single dose (39%-60%); (3) irreversible leukopenia is induced by the single-dose schedule of AMSA administration while after repeated low doses the white blood cell counts are in the same range of those of the control groups. Therefore, because of the schedule-dependency of toxicity and reduction in metastases, fractionated administration of AMSA at this dose level would be suitable for adjuvant chemotherapy.