[Quantitative analysis of corneal subbasal nerve plexus with in vivo confocal laser scanning microscopy]. / Quantitative Analyse des subbasalen Nervenplexus der Kornea mittels in vivo konfokaler Laser-Scanning-Mikroskopie.

BACKGROUND: An analysis of the corneal subbasal nerve plexus (SNP) allows an evaluation of the peripheral neuropathy in cases of degenerative diseases. In order to study the SNP structures quantitatively the automatically calculated morphological and topological parameters are required. METHODS: In vivo confocal laser scanning microscopy (Heidelberg Retina Tomograph II/Rostock Cornea Module) was performed in healthy volunteers as well as patients with severe diabetic neuropathy. An adapted image processing algorithm was used to preprocess, segment and evaluate quantitatively the nerve fibers of the SNP. Data sets were analysed statistically. RESULTS: The developed algorithm allows an automated detection of SNP structures. Furthermore, it allows the collection of data based on morphological and topological parameters. The main parameters that show significant differences between healthy cornea and cases of diabetic neuropathy are nerve fibre density and length, number of branching, tortuosity and number of terminal and crossing points. All parameters of the measurements can be used isolated, combined or weighted for quantification of the SNP networks. CONCLUSION: The presented fully automated preprocessing eliminates a large number of motion-induced artefacts. The quality of the resulting pictures allows an automated quantification using characteristic measurements. This represents an in vivo, non-invasive technology analysing degenerative changes of SNP especially in the course of diabetes mellitus.

Efficacy and safety of a new ready-to-use recombinant human growth hormone solution.

We report 24-month interim results of two multicenter phase III studies in previously untreated children with growth failure secondary to GH deficiency (GHD) that were paramount to the development of a new recombinant human GH (rh- GH, somatropin), approved as the first 'biosimilar' in Europe. Study 1 consisted of 3 parts performed in 89 children. The objective was to compare efficacy and safety of the lyophilized formulation of the new somatropin [Somatropin Powder (Sandoz)] with a licensed reference rhGH preparation and the liquid formulation of the new somatropin [Somatropin Solution (Sandoz)] and to assess long-term efficacy and safety of this ready-to-use Somatropin Solution. Study 2 was performed in 51 children and designed to demonstrate efficacy and safety of Somatropin Powder and to confirm its low immunogenic potential; rhGH was given sc at a daily dose of 0.03 mg/kg. Primary [body height, height SD score (HSDS), height velocity, and height velocity (HV) SD score (HVSDS)] and secondary [IGF-I and IGF binding protein 3 (IGFBP-3)] efficacy endpoints and safety parameters were assessed regularly. In study 1, all treatments showed comparable increases in growth. The baseline-adjusted difference between Somatropin Powder and the reference rhGH product in mean HV was -0.20 cm/yr (95% confidence interval (CI) [-1.34;0.94]) and in mean HVSDS was 0.76 (95% CI [-0.57;2.10]) after 9 months. These very small differences demonstrate comparable therapeutic efficacy between the two treatments. The results of study 2 were consistent with those seen in study 1. Equivalent therapeutic efficacy and clinical comparability in terms of safety and immunogenicity between Somatropin Powder and the reference rhGH product and between Somatropin Powder and Somatropin Solution was demonstrated. The safety and immunogenicity profiles were similar and as expected from experience with rhGH preparations.

Apoptosis induced by proteasome inhibition in cancer cells: predominant role of the p53/PUMA pathway.

The proteasome has emerged as a novel target for antineoplastic treatment of hematological malignancies and solid tumors, including those of the central nervous system. To identify cell death pathways activated in response to inhibition of the proteasome system in cancer cells, we treated human SH-SY5Y neuroblastoma cells with the selective proteasome inhibitor (PI) epoxomicin (Epoxo). Prolonged exposure to Epoxo was associated with increased levels of poly-ubiquitinylated proteins and p53, release of cytochrome c from the mitochondria, and activation of caspases. Analysis of global gene expression using high-density oligonucleotide microarrays revealed that Epoxo triggered transcriptional activation of the two Bcl-2-homology domain-3-only (BH3-only) genes p53 upregulated modulator of apoptosis (PUMA) and Bim. Subsequent studies in PUMA- and Bim-deficient cells indicated that Epoxo-induced caspase activation and apoptosis was predominantly PUMA-dependent. Further characterization of the transcriptional response to Epoxo in HCT116 human colon cancer cells demonstrated that PUMA induction was p53-dependent; with deficiency in either p53 or PUMA significantly protected HCT116 cells against Epoxo-induced apoptosis. Our data suggest that p53 activation and the transcriptional induction of its target gene PUMA play an important role in the sensitivity of cancer cells to apoptosis induced by proteasome inhibition, and imply that antineoplastic therapies with PIs might be especially useful in cancers with functional p53.

[Knowledge and use of Pap-smear among HIV-positive women]. / Importanza della conoscenza del Pap-test e suo utilizzo tra le donne HIV-positive.

AIM: HIV-positive women are at increased risk for preneoplastic lesions and invasive cervical cancer (ICC). The occurrence of these lesions can be substantially reduced by appropriate cervico-vaginal screening protocols (i.e., Pap-test). The aim of study was to assess: 1) awareness of Pap-smear and 2) the association between awareness of Pap-smear and screening attitudes of HIV-positive women. METHODS: Three-hundred and ninety HIV-positive women who attended the HIV outpatient gynecological unit of the National Institute for Infectious Diseases, Rome, from January 2003 to April 2005 were included in this investigation. These 390 women were interviewed to assess whether they were aware that Pap-test was a preventive tool against cervical cancer. In addition, past history of Pap-test, socioeconomic condition, history of HIV infection, and sexual habits were investigated. Odds ratios (OR) and 95% confidence intervals (CI) were used to assess the association between knowledge of Pap-test and covariates. RESULTS: Of these 390 HIV-positive women, 54.6% were not aware that Pap-test could prevent ICC. Women with a low educational level (OR = 6.6) or women who originated from Africa (OR = 6.5) were more likely to be unaware of Pap-test. Lack of Pap-test awareness was strongly associated with negative history for lifetime Pap-test (OR = 4.7). CONCLUSIONS: We showed that a large proportion of HIV-infected women are not aware that ICC could be prevented through Pap-test screening, and that lack of Pap-test screening is strongly associated with lack of awareness. The need for Pap-test counseling targeted to HIV-infected women clearly emerges from our findings.

Valsartan versus ACE inhibition after bare metal stent implantation--results of the VALVACE trial.

The use of ACE inhibitors (ACE-i) represents an Ia recommendation in the treatment of patients with STEMI and NSTEMI. However, results of smaller studies suggest an increase of in-stent-restenosis under ACE-i administration. The effects of ACE-i and valsartan after bare metal stent implantation of the culprit type B2/C lesion should be compared. Seven hundred patients were treated either by ACE-i in cases of LVEF<50% or 80 mg valsartan in cases of LVEF> or =50%. Restenosis rates after 6 months were analysed in 399 patients under valsartan and 224 patients under ACE-i with control angiography and major adverse cardiac events (death, infarction, reintervention) in a follow-up of up to 4 (mean 2.6) years in all patients. In-stent-restenosis was found in 19.5% under valsartan and in 34% under ACE-i (p<0.005). In diabetic patients, restenosis occurred in 24% under valsartan and in 43% under ACE-i (p<0.01). In initial acute coronary syndrome (ACS), restenosis rate was 14% under valsartan and 43% under ACE-i (p<0.0001). In stable angina, restenosis rates were 26.5% and 27.5%, respectively. Total MACE rates revealed significant differences in ACS due to reintervention rates of 22% and 7% under ACE-i and valsartan (p<0.0001). The administration of 80 mg valsartan after bare metal stent implantation leads to a reduction of in-stent-restenosis compared to ACE-i. This effect is mainly due to beneficial effects of valsartan in cases with initial ACS. Major differences between ACE-i and valsartan are discussed including inflammation, activation of neutrophils, mode of bradykinin activation, AT2 receptor stimulation and apoptosis of smooth muscle cells.

Drug-resistant tuberculosis in HIV-infected persons: Italy 1999-2000.

BACKGROUND: Patients with HIV infection may be a valuable target for assessing the impact of drug-resistant tuberculosis (TB). PATIENTS AND METHODS: An observational, prospective study was conducted in 96 infectious disease hospital units in Italy during 1999-2000. A total of 140 HIV-infected patients with diagnosis of TB and with an isolate tested for drug susceptibility entered the analysis. Drug resistance (DR) was defined as resistance to either isoniazid (INH) or rifampin (RIF), while multidrug resistance (MDR) was defined as resistance to INH and RIF. RESULTS: A total of 117 (83.6%) episodes of TB were classified as new cases and 23 (16.4%) as previously treated cases. Prevalence of resistance to INH or RIF was 12.8% and 4.3% among new cases, and 17.4% and 26.1% among previously treated cases, respectively. Prevalence rates of DR and MDR were 14.5% and 2.6% among new cases and 30.4% and 12.5% among previously treated cases, respectively. No statistically significant risk factors associated with DR or MDR TB emerged in this analysis. CONCLUSION: High prevalence rates of DR and MDR are present among HIV-infected TB patients in Italy, in particular among previously treated cases.

Successful recanalization of an occluded coronary artery by percutaneous coronary intervention, systemic administration of tirofiban, a glycoprotein IIb/IIIa inhibitor, and intracoronary thrombolysis with alteplase.

A 51 year-old male was admitted to our institution with subacute inferior myocardial infarction. Coronary angiography showed thrombotic occlusion of the right coronary artery. Percutaneous coronary intervention including the delivery of 3 stents was unsuccessful (TIMI grade 0 flow). In addition to an ongoing systemic administration of tirofiban, a glycoprotein IIb/IIIa inhibitor, the patient received intracoronary thrombolysis (ICT) with alteplase (recombinant tissue type plasminogen activator, rt-PA). There was complete reperfusion on control angiography the following day (TIMI grade 3 flow); 7 months later, there was still TIMI grade 3 flow. To our knowledge, this is the first report on systemic administration of tirofiban combined with ICT.

Effects of recombinant growth hormone therapy on thyroid hormone concentrations.

BACKGROUND AND OBJECTIVE: There are numerous, often contradictory reports on the effects of growth hormone (GH) therapy on thyroid function. The aim of this study was to assess the effect of such therapy on serum concentrations of thyroid hormones in GH-deficient children euthyroid prior to the treatment, and to determine the necessity of thyroid hormone administration in these patients. MATERIAL AND METHODS: The study included 32 GH-deficient patients in the first stage of sexual development, in whom disorders of thyroid function could be excluded. The inclusion criteria were based on clinical examination and levels of thyroxine (T4), triiodothyronine (T3), free thyroxine (fT4), free triiodothyronine (fT3), reverse triiodothyronine (rT3), thyrotropin (TSH) before and after stimulation with thyrotropin-releasing hormone (TRH). Recombinant growth hormone (rGH) (Genotropin 16U, Pharmacia) was administered at a dose of 0.7 U/kg/week. Fasting blood samples were drawn before treatment and after 3, 6, 9 and 12 months of therapy. Thyroid hormones were measured using RIA and IRMA methods. RESULTS: There were no physical signs of hypothyroidism in the patients examined during 12 months of rGH administration, and the satisfactory growth rate was achieved. T4 levels decreased in the first 3 months but remained within the normal range, and then returned to the values prior to the treatment. A similar trend was observed for fF4, with 28.5% of patients exhibiting fF4 levels below the normal in the 3rd month. An increase during the first 3 months of therapy was observed in the cases of T3 (statistically non-significant) and fT3, and these values then fell to levels within the normal range of patients' age. During treatment, TSH levels decreased but remained within the normal range. CONCLUSIONS: A transient decrease in T4 concentrations in the 3rd month with unchanged T3 and an increase in fT3 concentrations probably result from the effect of rGH on the peripheral metabolism of thyroid hormones. The results obtained do not support the use of thyroid hormone therapy with levothyroxine during the first year of rGH therapy in patients who are initially euthyroid.

Contralateral suppression of TEOAE in diabetic children. Effects of 1.0 kHz and 2.0 kHz pure tone stimulation--preliminary study.

The medial efferent system and its regulating outer hair cell function have not been previously studied in diabetic children. In this study, the group comprised 32 diabetic children, aged 6.0-16.0 years, with diabetes lasting 2.0-9.0 years, with normal tonal and impedance audiometry. A control group consisted of 30 healthy children with similar age and sex distribution. Contralateral stimulation (CS) was performed using 1.0 and 2.0 kHz pure tones on the level of 30 and 50 dB SL. Effects of CS on transient evoked otoacoustic emissions (TEOAE) elicited by click of a level equal to 70 and 60 dB SPL were investigated. Analysis included assessment of TEOAE amplitude and 0.8 kHz frequency bandwidth (0.8-FBW) amplitudes (signal/noise) centred at 1.0; 2.0; 3.0; 4.0; 5.0 kHz. TEOAE-RA recorded for stimulus 80, 70 and 60 dB SPL without CS were decreasing: average values respectively 7.3, 4.7 and 3.9 dB SPL. In the group of diabetic children TEOAE amplitudes, recorded for different click levels without CS, were similar to these recorded in healthy children. It suggested that normal function of the cochlea was preserved, mostly outer hair cells. However, the obtained effects of CS, in comparison with healthy children, were weaker and not so regular. Statistical analysis revealed that the reduction of TEOAE amplitudes for adequate 0.8-FBW in the control group was significantly higher, for both 1.0 kHz and 2.0 kHz CPTs of 30 dB SL and 50dB SL, in comparison with diabetic children. It is concluded that the suppressive effect on OAE in diabetic children is rather weak and seems to be associated with pathological changes in medial olivo-cochlear myelinated fibres.

Spectroscopic studies of the tungsten-containing formaldehyde ferredoxin oxidoreductase from the hyperthermophilic archaeon Thermococcus litoralis.

The electronic and redox properties of the iron-sulfur cluster and tungsten center in the as-isolated and sulfide-activated forms of formaldehyde ferredoxin oxidoreductase (FOR) from Thermococcus litoralis (Tl) have been investigated by using the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD) spectroscopies. The results reveal a [Fe4S4]2+,+ cluster (Em=-368mV) that undergoes redox cycling between an oxidized form with an S=0 ground state and a reduced form that exists as a pH- and medium-dependent mixture of S=3/2 (g=5.4; E/D=0.33) and S=1/2 (g=2.03, 1.93, 1.86) ground states, with the former dominating in the presence of 50% (v/v) glycerol. Three distinct types of W(V) EPR signals have been observed during dye-mediated redox titration of as-isolated Tl FOR. The initial resonance observed upon oxidation, termed the "low-potential" W(V) species (g=1.977, 1.898, 1.843), corresponds to approximately 25-30% of the total W and undergoes redox cycling between W(IV)/ W(V) and W(V)/W(VI) states at physiologically relevant potentials (Em= -335 and -280 mV, respectively). At higher potentials a minor "mid-potential" W(V) species, g= 1.983, 1.956, 1.932, accounting for less than 5 % of the total W, appears with a midpoint potential of -34 mV and persists up to at least + 300 mV. At potentials above 0 mV, a major "high-potential" W(V) signal, g= 1.981, 1.956, 1.883, accounting for 30-40% of the total W, appears at a midpoint potential of +184 mV. As-isolated samples of Tl FOR were found to undergo an approximately 8-fold enhancement in activity on incubation with excess Na2S under reducing conditions and the sulfide-activated Tl FOR was partially inactivated by cyanide. The spectroscopic and redox properties of the sulfide-activated Tl FOR are quite distinct from those of the as-isolated enzyme, with loss of the low-potential species and changes in both the mid-potential W(V) species (g= 1.981, 1.950, 1.931; Em = -265 mV) and high-potential W(V) species (g=1.981, 1.952, 1.895; Em = +65 mV). Taken together, the W(V) species in sulfide-activated samples of Tl FOR maximally account for only 15% of the total W. Both types of high-potential W(V) species were lost upon incubation with cyanide and the sulfide-activated high-potential species is converted into the as-isolated high-potential species upon exposure to air. Structural models are proposed for each of the observed W(V) species and both types of mid-potential and high-potential species are proposed to be artifacts of ligand-based oxidation of W(VI) species. A W(VI) species with terminal sulfido or thiol ligands is proposed to be responsible for the catalytic activity in sulfide-activated samples of Tl FOR.

Self-care of young diabetics in practice.

UNLABELLED: To determine whether young diabetics follow the most important rules of diabetes educational programs as remembering to carry 'soluble sugars' and insulin shots in their daily life, the analysis using 263 simple and anonymous questionnaires was performed. The study involved 183 IDDM children from Kansas-USA (76 boys, 107 girls) and 80 IDDM children from Silesia, Poland (36 boys, 44 girls) of mean age 12.95 +/- 2.65, mean IDDM duration 4.77 +/- 3.15 years, mean HbA1c 8.53 +/- 1.93%. We found: (1) 79.85% of all IDDM children carry something to treat or prevent hypoglycemia, more girls than boys, and more American than Polish ones (p < 0.01). (2) Only 59.92% of IDDM children carry 'soluble sugars' (no statistically significant differences between sexes and nationalities). (3) No correlation between carrying 'sugars' and age, metabolic control, number of glycemia measurements, forgetting insulin injections and kind of insulin therapy. (4) Duration of diabetes was not correlated with carrying 'sugars' whereas it was correlated with carrying 'other food' (p < 0.05). (5) 53.4% of children never forget insulin injections (p < 0.01). (6) Forgetting insulin shots was correlated with IDDM duration in boys group, with increasing level of HbA1c and with smaller number of glycemia measurements in all children groups, with the intensive insulin therapy in girls and Polish groups (p < 0.01 in all cases). CONCLUSIONS: 1). Diabetic children quite often carry nothing or 'non-soluble sugars' with themselves to treat hypoglycemia. 2). Diabetic children more often forget to shoot insulin injections than to 'carry sugars'. 3). Children with a longer duration of diabetes more often forget their insulin injections. 4). Older girls with longer duration of IDDM demonstrating worse metabolic control check their glycemia less frequently and more often forget the insulin injections.

Increased trend of type 1 diabetes mellitus in children's population (0-14 years) in Upper Silesia region (Poland).

UNLABELLED: THE PURPOSE OF THE STUDY was to provide current and reliable information about trends in the incidence rates of Type 1 Diabetes in the Upper Silesia region in children aged 0-15 years in the period of 9 years. RESULTS: Incidence ratio in the examined population increased from 4.71 in 1989 up to 10.16/100,000/year in 1997. In the analysed group no significant statistical differences dependent on sex were found. During the examined period, the incidence ratio has increased significantly. It was observed that the extent of change varied in different age groups. The youngest children (0-4 years old) showed the greatest increase of the incidence ratio from 1.09/10(5) in 1989 up to 6.75/10(5) in 1997. The highest incidence ratio was observed in the group of 10-14 year-olds. CONCLUSIONS: In the examined period dramatic increase (more than 200%) of the incidence ratio of type 1 diabetes was reported in the children's population (0-14 year-olds) in Upper Silesia. The fastest increase in this ratio was established in the group of youngest children (0-4 years old).

[Congenital adrenal hyperplasia conditioned by 21beta-hydroxylase deficiency - clinical considerations]. / Wrodzony przerost kory nadnerczy uwarunkowany niedoborem 21beta-hydroksylazy - rozwazania kliniczne.

The authors of the paper present three cases of nonclassical forms of congenital adrenal hyperplasia conditioned by 21 beta-hydroxylase deficiency occuring within the same family. They point out significant difficulties related to the detection of such anomalies, and they analyse in detail clinical symptoms occuring in this disease entity.

Hypoglycemia induced by hydroxychloroquine in a type II diabetic treated for polyarthritis.

A 77-year-old man with type II diabetes taking a stable dose of subcutaneous, twice daily human insulin developed symmetrical, inflammatory, rheumatoid factor positive polyarthritis. Within 2 weeks of starting therapy with prednisone 5 mg daily and hydroxychloroquine 400 mg daily he had 2 episodes of severe hypoglycemic coma requiring emergency care. His blood glucose became controlled again when his insulin was decreased by 37%. There are no reported cases of hypoglycemia in diabetic or nondiabetic patients treated with hydroxychloroquine. Hydroxychloroquine has been reported to reduce insulin requirements in refractory type II diabetes by an average of 30%. When hydroxychloroquine is initiated for the treatment of polyarthritis in a type II diabetic requiring insulin or sulfonylurea treatment, blood glucose levels should be monitored closely and the insulin dose may need to be reduced.

The Met243 sulfonium ion linkage is responsible for the anomalous magnetic circular dichroism and optical spectral properties of myeloperoxidase.

The heme group of myeloperoxidase shows anomalous optical properties, and the enzyme possesses the unique ability to catalyze the oxidation of chloride. However, the nature of the covalently bound heme macrocycle has been difficult to identify. In this work, the electronic and magnetic properties of the heme groups in oxidized and reduced forms of wild-type and Met243Thr mutant myeloperoxidase and wild-type lactoperoxidase have been investigated using variable-temperature (1.6-273 K) magnetic circular dichroism (MCD) spectroscopy along with parallel optical absorption and electron paramagnetic resonance studies. The results provide assessment of the spin state mixtures of the oxidized and reduced samples at ambient and liquid helium temperatures and show that the anomalous MCD properties of myeloperoxidase, e.g. red-shifted and inverted signs for bands in the high-spin ferric and low-spin ferrous forms compared to other heme peroxidases and heme proteins in general, are a direct consequence of a novel electron-withdrawing sulfonium ion heme linkage involving Met243.

Energy requirement for eating in cattle.

We determined the energy requirement for the activity associated with eating in an experiment with four adult Hinterwälder steers weighing 640+/-25 kg BW (mean+/-SD), using indirect calorimetry in respiration chambers. Heat production was measured during 3 h with and 3 h without ad libitum access to wheat straw, and the energy cost of eating was calculated as the difference, which was on average 27+/-13 J/(min x kg BW) (mean+/-SD). Straw intake, the time spent for eating, and the number of jaw movements were recorded. Values for the energy cost of eating reported in the literature are similar to those in this study, and values for ruminating amount to approximately 27% of those for eating. The energy cost of eating (literature values) varies greatly between feeds and feed treatments, when related to the amount of feed ingested, but it was relatively constant when related to time spent eating and was similar for cattle, sheep, and horses, when related to BW or metabolic BW (35 J/[min x kg BW] or 124 J/ [min x kg BW.75]). Calculations indicated that in high-quality roughage 10% of the ME and in untreated straw approximately 30% of the ME provided by the feed are used for eating and ruminating. This might be the main reason for the lower efficiency of ME utilization in roughages. The energy requirement for eating and ruminating should therefore be considered as a distinct proportion of the total ME requirement.

Characterization of the mononickel metallocenter in H134A mutant urease.

A mutant form of Klebsiella aerogenes urease possessing Ala instead of His at position 134 (H134A) is inactive and binds approximately half the normal complement of nickel (Park, I.-S., and Hausinger, R. P.(1993) Protein Sci. 2, 1034-1041). The crystal structure of the H134A protein was obtained at 2.0-A resolution, and it confirms that only Ni-1 of the two nickel ions found in the native enzyme is present. In contrast to the pseudotetrahedral geometry observed for Ni-1 in native urease (where it is liganded by His-246, His-272, one oxygen atom of carbamylated Lys-217, and a water molecule at partial occupancy), the mononickel metallocenter in the H134A protein was found to possess octahedral geometry and was coordinated by the above protein ligands plus three water molecules. The nickel site of H134A urease was probed by UV-visible, variable temperature magnetic circular dichroism, and x-ray absorption spectroscopies. The spectroscopic data are consistent with the presence of Ni(II) in octahedral geometry coordinated by two histidylimidazoles and additional oxygen and/or nitrogen donors. These data underscore the requirement of Ni-2 for formation of active urease and demonstrate the important role of Ni-2 in establishing the proper Ni-1 coordination geometry.

Cyclopent[a]anthraquinones as DNA intercalating agents with covalent bond formation potential: synthesis and biological activity.

A series of mitomycin C (MMC) analogues, namely cyclopentanthraquinone derivatives, were synthesized via Diels-Alder cyclization of naphthoquinone with 1-vinylcyclopent-1-enes. These new compounds are planar structures, like MMC, and bear an aziridine ring and a methyl carbamate side chain. After bioreduction, they are anticipated to be capable of intercalating into double-stranded DNA and bind covalently. Structure-activity relationships were studied. Of these compounds, 2,3-aziridino-4-[[(methylamino)carbonyl]methyl] cyclopent[alpha]anthracene-6,11-dione (4) was shown to have inhibitory activity against several leukemic and solid tumor cell lines. Mice (BDF1) bearing Lewis lung adenocarcinoma were treated with 4 and MMC (i.p., QD x 5). At a dose of 30.0 mg/kg, compound 4 was as effective as MMC (0.8 mg/kg). Compound 4 appears to be less toxic than MMC. DNA unwinding assay indicated that 4 is able to intercalate into DNA double strands and is also a topoisomerase II inhibitor.