Topical effects of cidofovir on cutaneous rabbit warts: treatment regimen and inoculum dependence.

The present study examined topical effects of cidofovir on cutaneous rabbit warts. Based on an inoculum-dependency study, each New Zealand White rabbit was inoculated with a high and low titer of cottontail rabbit papillomavirus (CRPV) at four sites on each dorsolateral area. Inoculation with 50 ID(50) induced papillomas at 100% of the inoculation sites within 16+/-1 days, and the wart growth curve plateaued within approximately 7 weeks. With an inoculum of 5 ID(50), 80% of the inoculated sites developed papillomas within 21+/-1 days and their size plateaued at a later time. Cidofovir was applied topically twice daily on the inoculated sites at a concentration of 1% for 18 days, starting at three different time points. In the first experiment, treatment was initiated 7 days post-inoculation. One of the inoculated sides received cidofovir or the vehicle, PBS, while the other side was left untreated. With this treatment regimen, cidofovir significantly delayed the time of onset and the growth rate of papillomas induced with the high titer of inoculum. It completely prevented papilloma-induction on the sites inoculated with the low titer of CRPV. Reversible side-effects of cidofovir were observed on the directly treated area including erythema, necrosis, and flaking. Both therapeutic and side-effects were limited to the sites of direct exposure. In the second experiment, one of the two sides in each group of rabbits received cidofovir or vehicle starting on day 29 post-inoculation. With this treatment regimen, cidofovir significantly reduced wart growth against the low titer only. Topical treatment initiated on day 49 post-inoculation was not effective on warts initiated with either viral titer. These results demonstrated that topical cidofovir could be very effective against papillomavirus-induced wart growth if it is initiated early during the infection, especially against low titers of inoculum.

Cytokinetics of a novel 1,2,3-triazene-containing heterocycle, 8-nitro-3-methyl-benzo-1,2,3,5-tetrazepin-4(3H)-one (NIME), in the human epithelial ovarian cancer cell line OVCAR-3.

The mechanism of action of the novel tetrazepinone 8-nitro-3-methyl-benzo-1,2,3,5-tetrazepin-4(3H)-one (NIME), structurally related to the antitumour drug temozolomide, was studied in the human ovarian tumour cell line OVCAR-3. NIME preferentially inhibited DNA synthesis over protein and RNA syntheses at 3 and 24 hr post-treatment. A Maxam-Gilbert sequencing assay showed that NIME induced barely detectable levels of guanine N7 alkylation in an isolated DNA strand, in contrast to temozolomide, a strong alkylating agent containing, like NIME, a cyclic 3-methyl-1,2,3-triazene moiety. Alkaline sucrose density-gradient sedimentation, at concentrations 2- to 10-fold lower than the ones used in the DNA sequencing assay, showed significant DNA damage in OVCAR-3 cells 24 hr after treatment with NIME. This was accompanied by a significant accumulation of cells in late S and G2M. Cell cycle arrest was transient and was reversed after 2-3 days following drug treatment. This was in agreement with bivariate bromodeoxyuridine/propidium iodide analysis, which showed that at 100 microM, a concentration at which the majority of the cells arrested in late S and G2M, a significant fraction of bromodeoxyuridine positive (S-phase) cells escaped the block. In an attempt to elucidate the mechanism underlying these effects, the degradation of NIME in cell culture medium was analyzed by GC-MS (gas chromatography coupled with mass spectrometry). The results showed that, in contrast to temozolomide, NIME did not convert to an open-chain alkyltriazene in cell culture medium, but to a major benzimidazole product, which exerted a minor effect on the cell cycle. This suggests that NIME, despite containing a 3-(alkyl)-1,2,3-triazene moiety, does not act by DNA alkylation but probably by generating a short-lived genotoxic species during its degradation to 6,5-benzofused derivatives.

Comparative studies between the effects of mitozolomide and two novel tetrazepinones PYRCL and QUINCL on NIH:OVCAR-3 cells.

UNLABELLED: Cytotoxicity, reduction of macromolecule synthesis and cell cycle perturbations by two novel 3-(2-chloroethyl)-tetrazepinones, PYRCL and QUINCL were compared with those produced by the structurally related 3-(2-chloroethyl)-tetrazinone, mitozolomide, in the OVCAR-3 cell line. METHODS: Macromolecule synthesis was determined by incorporation of 3H-thymidine, 3H-uridine and 3H-leucine into acid-precipitable fractions of OVCAR-3 cell extracts. Maxam-Gilbert sequencing was used to compare the DNA alkylating sites induced by the tetrazepinones, with those created by mitozolomide. Alkaline sucrose-density sedimentation was employed to detect genomic DNA damage. Also, the effects of the tetrazepinones on the cell cycle were determined by univariate flow cytometry. RESULTS: At 3 h post-treatment, mitozolomide appeared as a selective inhibitor of DNA synthesis, while both tetrazepinones inhibited the synthesis of all three macromolecules. At 24 h post-treatment, the inhibition of DNA synthesis was observed to increase in cells treated with mitozolomide, while it decreased in those previously exposed to the tetrazepinones. Also at 24 h post-treatment, mitozolomide induced accumulation of cells in S(late)/G2M at low concentrations and in S-middle at high concentrations. In contrast, at the same recovery time, cells treated with the tetrazepinones accumulated specifically in G2M, the strength of the block being dose-dependent. At an equimolar concentration, the tetrazepinones induced weaker guanine N-7 alkylation than mitozolomide. By 24 h after treatment, cells exposed to the tetrazepinones showed significantly greater DNA fragmentation than those previously treated with mitozolomide. CONCLUSION: In summary, based on (a) their effects on DNA, RNA, protein synthesis and on the cell cycle, (b) their alkylating power and (c) their interactions with DNA, the 3-(2-chloroethyl)tetrazepinones appeared to kill tumor cells by a novel mechanism which may significantly differ from that of their 3-(2-chloroethyl)-tetrazinone counterpart, mitozolomide.

Design and mechanism of action of a novel cytotoxic 1,2,3-triazene-containing heterocycle, 3,5-dimethyl-pyrido-1,2,3,5-tetrazepin-4-one (PYRZ), in the human epithelial ovarian cancer cell line NIH:OVCAR-3 in vitro.

The mechanism of action of the novel heterocycle 3,5-dimethyl-pyrido-1,2,3,5-tetrazepin-4-one (PYRZ), structurally related to temozolomide, was studied in the human ovarian tumour cell line OVCAR-3. Our results showed that, despite its marked structural similarities to temozolomide, PYRZ presents properties that are atypical of 1,2,3-triazene-containing alkylating agents. In a Maxam-Gilbert DNA sequencing assay, PYRZ showed background levels of DNA alkylation, in contrast to temozolomide which strongly alkylated DNA preferentially at guanine residues. At high concentrations, PYRZ inhibited the synthesis of DNA, RNA and protein 3 h after treatment, in contrast to temozolomide which, in previous work, was found to preferentially inhibit DNA synthesis in OVCAR-3 cells. In cells exposed to PYRZ, alkaline sucrose density-gradient centrifugation showed a dose-dependent increase in DNA fragmentation only 12 and 24 h after treatment. PYRZ induced increasing accumulation of cells in late S and G2+M 6-24 h after treatment. This also contrasts with previous work that showed delayed cell cycle arrest induced by temozolomide in OVCAR-3 cells and in the murine leukaemia L1210 cells. Cell-killing kinetics by PYRZ showed a series of sigmoidal dose-response curves with 50-90% cell killing attained as early as 24 h after treatment in the 25-100 microM dose range. (IC50 clonogenic assay 18 microM). The results suggest that the mechanism of cell killing by PYRZ may be different from that of its parent drug temozolomide, and other alkyl-triazene-containing molecules of the same class.

Ontogeny of Big endothelin-1 effects in newborn piglet pulmonary vasculature.

Endothelin-1 (ET-1), a 21-amino acid peptide produced by endothelial cells, results from the cleavage of preproendothelin, generating Big ET-1, which is then cleaved by the ET-converting enzyme (ECE) to form ET-1. Big ET-1, like ET-1, is released by endothelial cells. Big ET-1 is equipotent to ET-1 in vivo, whereas its vasoactive effects are less in vitro. It has been suggested that the effects of Big ET-1 depend on its conversion to ET-1. ET-1 has potent vasoactive effects in the newborn pig pulmonary circulation, however, the effects of Big ET-1 remain unknown. Therefore, we studied the effects of Big ET-1 in isolated perfused lungs from 1- and 7-day-old piglets using the ECE inhibitor, phosphoramidon, and the ETA receptor antagonist, BQ-123Na. The rate of conversion of Big ET-1 to ET-1 was measured using radioimmunoassay. ET-1 (10(-13) to 10(-8) M) produced an initial vasodilation, followed by a dose-dependent potent vasoconstriction (P < 0.001), which was equal at both ages. Big ET-1 (10(-11) to 10(-8) M) also produced a dose-dependent vasoconstriction (P < 0.001). The constrictor effects of Big ET-1 and ET-1 were similar in the 1-day-old, whereas in the 7-day-old, the constrictor effect of Big ET-1 was less than that of ET-1 (P < 0.017).(ABSTRACT TRUNCATED AT 250 WORDS)

Newborn piglet lungs release endothelin-1: effect of alpha-thrombin and hypoxia.

Endothelin-1 (ET-1) is a 21 amino acid vasoconstrictor peptide produced by endothelial cells, the expression of which is modulated by a variety of vasoconstrictors, vasodilators, and inflammatory mediators. Hypoxia has been shown to increase ET-1 expression and release in cultured endothelial cells from the systemic circulation, but reports are contradictory regarding the pulmonary circulation. In this study, the release of ET-1 and its cellular localization in the isolated perfused newborn piglet lung were examined under control conditions and after stimulation with hypoxia or alpha-thrombin (positive control). In the control condition, perfusion pressure remained stable during the study period, and a progressive increase in levels of immunoreactive ET-1 (irET-1) was noted. When alpha-thrombin was added to the perfusion fluid, a slow gradual increase in perfusion pressure was produced and the levels of irET-1 were significantly greater than those measured in the control preparations. Finally, hypoxia produced a significant increase in the perfusion pressure; however, the release of irET-1 did not differ significantly from the control, if anything, the net release across the lung was diminished. In all conditions, immunocytochemistry using antiserum to human-porcine ET-1 revealed the presence of high ET-1-like immunoreactivity in epithelial cells of bronchi, bronchioles, and terminal bronchioles. In addition, endothelial cells of large and medium-size pulmonary arteries were only moderately immunoreactive for ET-1. These findings indicate that the neonatal pig lung can produce and release ET-1, and that its release can be increased by certain stimuli like alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Maturational changes in endothelium-derived relaxations in newborn piglet pulmonary circulation.

It is accepted knowledge that the endothelium can profoundly affect vascular tone through the release of vasoactive substances. The maturational changes in the role of the endothelium-derived relaxing factor (EDRF) and ATP-dependent K+ channels in the neonatal pulmonary circulation were investigated in isolated perfused lungs from 1- and 7-day-old piglets. The EDRF inhibitor, N omega-nitro-L-arginine (L-NNA), had potent dose-dependent constrictor effects on the pulmonary vasculature with normal and raised tone. The constrictor effect of L-NNA was greater (P < 0.05) in the 1-day-old than in the 7-day-old lungs and was significantly (P < 0.005) attenuated by pretreatment with the EDRF precursor, L-arginine. Furthermore, we studied the possibility of developmental changes in the sensitivity of smooth muscle cells to EDRF by testing sodium nitroprusside, nitric oxide, and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). All caused a decrease in perfusion pressure, but only sodium nitroprusside elicited a greater (P < 0.01) effect in the 1-day-old. Endothelin-1 (ET-1) and bradykinin (BK) elicited dilator responses that were significantly (P < 0.05) reduced in the presence of L-NNA. Interestingly, the dilator response to ET-1 was more marked (P < 0.001) in the younger group, whereas no age difference was noted with BK. Finally, lemakalim, a K+ channel activator, caused a vasodilation of equal magnitude at both ages. In summary, EDRF and ATP-dependent K+ channels appear to play a role in the control of the newborn piglet pulmonary vasculature.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence against the involvement of a cytochrome P-450 mechanism in pulmonary hemodynamics in the newborn pig.

Control mechanisms operating through a cytochrome P-450 system have emerged lately as a possible important determinant of pulmonary hemodynamics. Their action may be expressed in the adjustment of vascular tone under both physiologic and pathophysiologic conditions. One such condition is the pulmonary constrictor response to hypoxia. The identity of the effector agent, or agents, is not known, though there are data implicating monooxygenase products of arachidonic acid. From this premise, we wanted to evaluate the effect of cytochrome P-450 inhibitors on basal pulmonary vascular tone during normoxia, and their effect upon hypoxic pulmonary vasoconstriction response. Experiments were performed in an isolated, perfused lung preparation from 1- and 7-day-old piglets, and the effects of two cytochrome P-450 inhibitors (metyrapone and ketoconazole) were tested on the perfusion pressure. At 10(-5) and 10(-4) M, metyrapone caused a modest, but significant, increase in pulmonary pressure (p less than 0.05) in 7-day-old preparations, while it was without effect in the 1-day-old preparation. Similarly, ketoconazole at concentrations from 10(-6) M upwards increased the perfusion pressure in the older animal (p less than 0.01). Responses to the inhibitors were not seen in preparations that had been pretreated with a cyclooxygenase inhibitor (indomethacin, 2.8 x 10(-6) M) or a dual cyclooxygenase-lipoxygenase inhibitor (BW755C, 10(-5) M). Hypoxic vasoconstriction was marginally enhanced by 10(-4) M metyrapone, while it was affected inconsistently by 10(-5) M ketoconazole. We conclude that vasoactive agents formed through cytochrome P-450 reactions have a minor role, or no role at all, in the control of pulmonary hemodynamics in the newborn pig.

Endothelin-1 has a dilator effect on neonatal pig pulmonary vasculature.

Endothelin-1 (ET-1), a 21-residue potent vasoconstrictor peptide produced by endothelial cells, was reported to cause vasodilation in the systemic and pulmonary vascular beds. Therefore, in isolated perfused lungs from 7-day-old piglets, we studied the effects and the mechanisms responsible for the dilator effect of ET-1. ET-1 produced a mild transient decrease in perfusion pressure at low doses (less than 10(-7) M/g dry lung); at higher doses, a potent long-lasting vasoconstriction was noted. Indeed, the constrictor effect of ET-1 was at least equal to or greater than that of U-44069 and prostaglandin D2 (PGD2). When the vascular tone of the preparation was increased with U-46619, another stable endoperoxide analogue, the dilator response to low doses of ET-1 was increased, while the constrictor response remained unchanged. Indomethacin (2.8 x 10(-6) M) and glybenclamide (an ATP-sensitive potassium channel inhibitor) (10(-5) M) did not alter the responses to ET-1. The endothelium-derived relaxing factor (EDRF) inhibitor Nw-nitro-L-arginine (2 x 10(-4) M) not only inhibited the dilator response to ET-1 almost completely, but also potentiated the constrictor response. Finally, Nw-nitro-L-arginine alone had a mild vasoconstrictor effect in newborn pig lung. The results of these studies indicate that ET-1 has both vasodilator and vasoconstrictor activity in neonatal pig pulmonary vascular bed. This vasodilator activity may be mediated by EDRF.(ABSTRACT TRUNCATED AT 250 WORDS)