Synergistic antibacterial effect of statins with the complex {[1-(4-bromophenyl)-3-phenyltriazene N 3 -oxide-κ 2 N 1,O 4 ](dimethylbenzylamine-κ 2 C 1, N 4 )palladium(II)}

Abstract The treatment of infections caused by resistant microorganisms represents a big challenge in healthcare due to limited treatment options. For this reason, the discovery of new active substances which are able to perform innovative and selective actions is of great impact nowadays. Statins and triazenes (TZC) have consolidated as a promising class of compounds, characterized by the expressive biological activity, especially antimicrobial activities. The aim of this study was to assess the in vitro synergistic antibacterial effect of the association of statins and a new TZC complex {[1-(4-bromophenyl)-3-phenyltriazene N 3-oxide-κ 2 N 1,O 4](dimethylbenzylamine-κ 2 C 1,N 4)palladium(II)} (Pd(DMBA)LBr) against American Type Culture Collection (ATCC) strains and clinical isolates. The complex and the statins showed bacterial activity of all tested strains and clinical isolates, evidencing that TZC complexion with metals can be promising. Simvastatin showed synergy when associated to the complex (FICI≤0.5), being the minimum inhibitory concentration (MIC) of 16 µg mL-1 found in 6 samples. Thus, it is possible to infer that the association between Pd(DMBA)LBr and simvastatin consists of an alternative to increase the pontential of these compounds, since statins have low toxicity.

Triazene compounds: mechanism of action and related DNA repair systems.

Triazene compounds of clinical interest (i.e. dacarbazine and temozolomide) are a group of alkylating agents with similar chemical, physical, antitumour and mutagenic properties. Their mechanism of action is mainly related to methylation of O(6)-guanine, mediated by methyldiazonium ion, a highly reactive derivative of the two compounds. The cytotoxic/mutagenic effects of these drugs are based on the presence of DNA O(6)-methylguanine adducts that generate base/base mismatches with cytosine and with thymine. These adducts lead to cell death, or if the cell survives, provoke somatic point mutations represented by C:G-->T:A transition in DNA helix. Triazene compounds have excellent pharmacokinetic properties and limited toxicity. Dacarbazine requires hepatic activation whereas temozolomide is spontaneously converted into active metabolite in aqueous solution at physiological pH. Moreover, temozolomide is fully active when administrated orally (100% bioavailability). The biological effects of triazene compounds and cell resistance to them depend on at least three DNA repair systems, (a) O(6)-alkylguanine-DNA-alkyltransferase, called also methyl-guanine methyl-transferase (MGMT); (b) mismatch repair (MMR), and (c) base excision repair (BER). MGMT is a small enzyme-like protein that removes small alkyl adducts from the O(6) position of DNA guanine through a stoichiometric and auto-inactivating reaction. This reaction consists in a covalent transfer of the alkyl group from the alkylated site in DNA to an internal cysteine residue of MGMT protein. High levels of MGMT are responsible for normal and tumour cell resistance to triazenes. Therefore, pre-treatment with MGMT inhibitors - i.e. O(6)-benzylguanine or O(6)-(4-bromotenyl)guanine (Lomeguatrib) - is followed by a great increase in the activity of triazenes against target cells expressing high MGMT levels. MMR is represented by a protein complex dedicated to the repair of biosynthetic errors generated during DNA replication. The MMR system recognizes base mismatches and insertion-deletion loops, cuts the nucleotide sequence containing the lesion, and restores the correct base sequence. Therefore, not only MGMT but also MMR is involved in target cell susceptibility to triazenes. However, the system does not suppress, but instead promotes the cytotoxic effects of triazenes. In fact, MMR is not able to repair the incorrect base pairing determined by treatment with triazenes and, according to a predominant hypothesis, it causes reiterated "futile" attempts of damage repair leading to the activation of cell cycle arrest and apoptosis. BER removes lesions due to cellular metabolism, or to physical or chemical agents. BER is able to repair N(7)-methylguanine and N(3)-methyladenine determined by treatment with triazenes. Therefore, triazene compounds can also kill tumour cells by a N(3)-methyladenine-mediated mechanism if BER activity is inhibited by chemical agents (i.e. PARP inhibitors). In conclusion, in selected cases, triazenes can represent a therapeutic alternative to treatment of neoplastic diseases including haematological malignancies. Moreover, the susceptibility of neoplastic cells to these compounds can be substantially increased through pharmacological modulation of the expression level and functional activity of DNA repair enzymes.

The clinical pharmacology of alkylating agents in high-dose chemotherapy.

Alkylating agents are widely used in high-dose chemotherapy regimens in combination with hematological support. Knowledge about the pharmacokinetics and pharmacodynamics of these agents administered in high doses is critical for the safe and efficient use of these regimens. The aim of this review is to summarize the clinical pharmacology of the alkylating agents (including the platinum compounds) in high-dose chemotherapy. Differences between conventional and high doses will be discussed.

Effects of irradiation and methyl-triazene on craniofacial development in mouse embryos: a semiautomated morphometric analysis.

OBJECTIVE: The purpose of the present study was a 2D-semiautomated morphometric analysis of craniofacial growth in nuclear magnetic resonance imaged (NMRI) mouse embryos. METHODS: The NMRI mouse embryos were exposed in utero to either a single dose of 2 Gy X-irradiation on day 9 of gestation (113 embryos) or to 1.5 mg methyl-triazene administered orally to their pregnant mothers on gestational day 10.5 (124 embryos). An additional group of 108 embryos was used as controls. Digitized pictures of embryos from gestational days 14 to 17 were taken in lateral right view using a video system. Landmarks were located and digitized for computerized analysis of growth changes in relation to developmental stages of the face. RESULTS: The results revealed that the snout of control embryos lengthens during the developmental period considered. The snout of embryos previously submitted to methyl-triazene displayed micrognathia, and all treated fetuses exhibited macroscopic signs of microcephaly with a reduced mandible. The snouts of irradiated embryos appeared shortened at the 14-day stage and continued to shorten as development proceeded. A shortening of the midface was detected macroscopically in 83% of the cases. CONCLUSION: The results of this morphometric analysis enabled us to trace the developmental progression of the induced dysmorphosis and to assess the differences compared with normal development.

Middle ear anomalies induced by hypertriazene administration in the mouse.

The middle ear is derived from various embryonic tissues. Many experiments using teratogens have been performed, employing the difference of each tissue's sensitivity to the teratogen and the tissue's critical time of development. Triazene, a foliate metabolism antagonist, produced anomalies in fetuses that resembled those associated with thalidomide in humans, the so-called the first and second branchial syndrome. In our experiment, we administrated 3,3-dimethyl-1-phenyltriazene, an inductor of triazene, to pregnant mice at 7 to 14 days of gestation resulting in unique fetal anomalies. We examined the development of the stapes, stapedial artery, facial nerve, and oval window using an optical microscopic and three-dimensional reconstruction. Middle ear and facial nerve anomalies in mice depend on the gestation day when triazene is administrated. The stapedial artery, oval window, facial nerve (horizontal segment), stapes footplate, and styloid process are affected on the 9th to 11th administration day, the annular stapedialis on the 10th to 11th day, and the malleus and incus on the 9th to 11th day. The use of the Vox View/Mac, allowed us to create three-dimensional pictures from two-dimensional slides providing an improved understanding of the relationships between anatomical structures.

Ear malformations in the mouse embryo following maternal administration of triazene, with clinical implications.

Triazene administration to 10-day pregnant mice gave rise to severe ear abnormalities, including middle ear ossicle malformations. The head of the malleus and the body of the incus were sometimes absent. In some instances, the stapes appeared dysplastic. The stapedial artery was often quite dilated, as well as other cephalic vessels, but qualitatively normal. Severe inner ear abnormalities were observed, as well as auditory nerve disruption, which was associated with central nervous system lesions. Multiple haematomas were present in the embryonic face, but ear abnormalities occurred even in the absence of local hemorrhages. We postulate a direct effect of the teratogen on the branchial mesenchyme. This model seems to be useful for the comprehension of human middle ear abnormalities, but does not constitute a phenocopy of hemicranial microscomia.

Preclinical, phase I and pharmacokinetic studies with the dimethyl phenyltriazene CB10-277.

Decarbazine is an imidazole dimethyltriazene with reproducible activity in patients with metastatic melanoma. CB10-277 is a phenyl dimethyltriazene which, like dacarbazine, requires metabolic activation to its corresponding monomethyl species for antitumour activity. In preclinical models (human melanoma xenografts and transplantable rodent tumours) CB10-277 showed a similar spectrum and level of activity when compared to dacarbazine. Pharmacokinetic studies were performed with CB10-277 in mice treated i.v. at the LD10 (750 mg m-2) and plasma analysed by HPLC. The parent drug area under the plasma concentration vs time curve (AUC) was 142 mM x minutes. Drug metabolism occurred as evidenced by the HPLC identification of the monomethyl species (AUC = 8 mM x minutes) as well as other metabolites. A Phase I trial using a short infusion with doses repeated every 21 days has been performed. Thirty-six patients received 80 courses over a dose range of 80-6,000 mg m-2. The dose limiting toxicity was nausea and vomiting which occurred in 80% of the evaluable courses > or = 900 mg m-2. The only other common side effect was a flushing or warm sensation, which occurred in over 75% of courses at > or = 1,350 mg m-2. There were no hemodynamic consequences. Responses occurred in patients with melanoma (one complete, two partial, one mixed/11), sarcoma (one mixed/6) and carcinoid (one partial/l). Pharmacokinetics were performed in 46 courses. The CB10-277 AUC increased linearly with dose (r = 0.9203, P < 0.001) up to 700 mM x minutes at 6,000 mg m-2). Evidence of CB10-277 metabolism was observed, as in mice, by detection of the monomethyl species and other metabolites. However, the plasma levels of the monomethyl species in patients (1.8 and 3.7 mM x minutes at 6,000 mg m-2) were less than those predicted from studies in mice. Despite this, antitumour activity in dacarbazine sensitive histologies was observed and additional studies with CB10-277 are recommended.

Phase I trial with pharmacokinetics of CB10-277 given by 24 hours continuous infusion.

The dose limiting toxicities of the short infusion trial of the dacarbazine analog, CB10-277, were nausea and vomiting which appeared to be related to the peak plasma level of the parent drug. In addition, based on mouse studies, these dose limiting toxicities occurred at a less than optimal level of the monomethyl metabolite, the presumed species required for antitumour activity. An alternative schedule that would avoid the parent drug peak plasma levels of short infusion, while possibly allowing an increase in the amount of monomethyl metabolite produced was considered. Thus, a 24 h continuous infusion schedule, repeated every 21 days was explored. Twenty-two patients received 42 courses with a dose range of 4,700-15,000 mg m-2. The dose limiting toxicity was myelosuppression (leucopenia and thrombocytopenia). Although nausea and vomiting also occurred, it was manageable with routine antiemetic therapy. Other toxicities included diarrhoea, hallucinations, malaise, muscle ache, headache and flushing and all were < or = WHO grade 2. Pharmacokinetic studies were performed with 13 courses which included all dose levels. The mean t1/2 of the parent drug was 178 min. Area under the concentration x time curve (AUC) at the highest dose for the parent drug and the monomethyl metabolite were 2,350 and 9 mM x minutes, respectively. This monomethyl metabolite AUC and the associated myelosuppression showed a more favourable comparison to the preclinical data determined in mice than the results from the short infusion trial of CB10-277. Therefore, the recommended Phase II dose and schedule of this drug was 12,000 mg m-2 given by 24 h continuous infusion.

In vivo depletion of O6-alkylguanine-DNA-alkyltransferase in lymphocytes and melanoma of patients treated with CB 10-277, a new DTIC analogue.

There is increasing evidence to suggest that alkylation of guanine residues in DNA at the O6 position is the critical cytotoxic event following treatment with dacarbazine (DTIC) and related drugs and that endogenous O6-alkylguanine-DNA alkyltransferase (ATase) gene expression may be a major factor in resistance to such agents. 1-p-Carboxyl-3,3-dimethylphenyltriazene (CB10-277) was recently selected for clinical evaluation as a DTIC analogue with improved solubility, stability and (possibly) metabolic activation. Serial ATase levels were measured in peripheral blood lymphocytes of nine patients and in biopsied melanoma samples of two patients undergoing treatment with 24-h continuous infusion of CB10-277 (12 g/m2). Wide individual variations in pre-treatment levels as well as in the post-treatment depletion of lymphocyte ATase were seen. Progressive depletion of lymphocyte ATase was seen during continuous infusion of CB10-277 in all patients. Complete suppression of lymphocyte ATase activity occurred in two patients whose pre-treatment ATase levels were low. Immediately following completion of the CB10-277 infusion, the median ATase activity was 17% of pre-treatment levels (range, 0-67%). At 24 h after the end of the infusion, no recovery of lymphocyte ATase activity was observed in six patients, but significant recovery to 50%, 100% and 102% of pre-treatment activity occurred in the other three. In three patients who returned for subsequent cycles of chemotherapy at 4 weeks after the first dose, pre-treatment ATase levels showed a 3- to 4-fold increase relative to the original pre-treatment values. A significant correlation was found between the extent of ATase depletion and the initial lymphocyte ATase levels (r = 0.725, P < 0.05). Haematological toxicity developed in two patients and was associated with low pre-treatment ATase activity. Depletion of tumour ATase activity was noted in these patients, with residual activity amounting to 8% and 11% of pre-treatment levels, respectively, in the biopsies melanoma tissues. These results indicate extensive metabolism of CB10-277 to a methylating agent capable of mediating alkylation of DNA and subsequent depletion of lymphocyte and tumour ATase levels and further indicate that the effects on lymphocytes may reflect effects on the target tumour.

Urogenital anomalies in fetal rats produced by the anticancer agent 4(5)-(3,3-dimethyl-1-triazeno) imidazole-4-carboxamide.

Pregnant Wistar rats injected intraperitoneally on gestational day 12 with single doses (100-1,000 mg/kg) or 600 mg/kg of 4(5)-(3,3-dimethyl-1-triazeno) imidazole-4-carboxamide (dic) were autopsied on day 21 (100-1,000 mg/kg) or at 24-hour intervals on days 13-20 (600 mg/kg). Controls received CMC on the same schedule. All fetuses were weighed and examined for urogenital system (UGS) malformations. Those given 600 mg/kg were also studied histologically. DIC produced significant growth retardation at all doses on day 21 (18-72%). UGS malformations occurred in 27-67% of the fetuses at 200-400 mg/kg and in 100% of those given 600 mg/kg or more of DIC. Abnormalities included renal growth inhibition, fusion, ectopia, and ureteropelvic dilatation. At 600 mg/kg renal and body weights were reduced 40 and 55%, respectively. Ureteropelvic dilation was common, and cortical glomeruli, nephric collecting tubules, and papillae were retarded in development. The juxtamedullary glomeruli were well developed. Proximal nephric tubular mitotic activity was 85% greater than in control animals (day 17). On the basis of pertinent morphological and physiological data, it is postulated that the dilated upper urinary tracts represent functional hydronephrosis incident to severe renal retardation and its resultant compensatory response.

[Description of a case of acute neurological toxicity after DTIC-adriamycin-vincristine therapy (author's transl)]. / Descrizione di un caso di tossicità neurologica acuta dopo trattamento con adriamicina, vincristina e dimetiltriazenoimidazolcarbossamide (NSC-45388)

A case of acute neurological toxicity was observed in a patient with a retroperitoneal fibromyxosarcoma treated with DTIC (NSC-45382), Adriamycin and Vincristine. The neurological symptoms started one hour after drug administration and rapidly declined with symptomatic therapy: no EEG and scintigraphic changes were detectable. The case is discussed in relation to central nervous system complications reported by other authors, following administration of DTIC and Adriamycin.

Combination chemotherapy for disseminated malignant melanoma.

BCNU, hydroxyurea, and imidazole carboxamide (DTIC) were administered to 89 patients with disseminated malignant melanoma. A response rate of 27% was observed. The addition of vincristine in another 89 patients did not significantly improve the response rate (30%). This includes patients who died during or after one course of therapy (less than 28 days). If the early deaths are not considered, the over-all response rate was 38%. (he best responses occurred in patients with skin, lung, and/or lymph node involvement. Liver and brain involvement heralded poor responses. This response rate appeared to be independent of age, sex or previous therapy. Moderate and severe toxicity, predominantly nausea and vomiting, was noted in most patients. The median survival for all evaluable patients was 17 months, and was independent of the regimen used.