Francesca Mattioli

1,8-Naphthyridines IV. 9-Substituted N,N-dialkyl-5-(alkylamino or cycloalkylamino) [1,2,4]triazolo[4,3-a][1,8]naphthyridine-6-carboxamides, new compounds with anti-aggressive and potent anti-inflammatory activities

The title compounds (8) were synthesized through the cyclocondensation of the corresponding N-substituted 4-amino-2-chloro-1,8-naphthyridine-3-carboxamides (4) with the proper hydrazides, in order to evaluate their anti-inflammatory and anti-aggressive properties. Several compounds 8 exhibited high anti-inflammatory activity (carrageenin-induced paw edema assay in the rat) along with appreciable anti-aggressive properties (isolation-induced aggressiveness test in mice). With respect to anti-inflammatory activity, the most active compounds (8n and 8c) produced a 61% edema inhibition at the 25 mg/kg dose, and 50 or 35% inhibition, respectively, at the 12.5 mg/kg dose. The structure-activity relationships are discussed.

Genotoxic and carcinogenic effects of antipsychotics and antidepressants.

This review provides a compendium of the results of genotoxicity and carcinogenicity assays performed on marketed antipsychotics and antidepressants. Of the 104 drugs examined, 47 (45.2%) do not have retrievable data. The remaining 57 (54.8%) have at least one and often more than one genotoxicity and/or carcinogenicity test result. Of these 57 drugs, 33 (57.9%) have at least one positive finding: 24 tested positive in at least one genotoxicity assay, 14 in at least one carcinogenicity assay, and 5 gave a positive response in both. Concerning the predictivity of genetic toxicology findings for the result(s) of long-term carcinogenesis assays, 25 drugs have both genotoxicity and carcinogenicity data: 8 of them (32.0%) were neither genotoxic nor carcinogenic, 8 (32.0%) were carcinogenic in at least one sex of mice or rats but tested negative in genotoxicity assays, 4 (16.0%) tested positive in at least one genotoxicity assay but were non-carcinogenic, and 5 (20.0%) gave a positive response in at least one genotoxicity assay and in at least one carcinogenicity assay. Only 16 (15.4%) of the 104 drugs examined have all data required by present guidelines for testing of pharmaceuticals, but it should be considered that a large fraction of them were developed and marketed prior to the present regulatory climate.

Species, sex and inter-individual differences in DNA repair induced by nine sex steroids in primary cultures of rat and human hepatocytes.

Sex steroids, due to the generally negative responses observed in routinely employed standard genotoxicity assays, are considered epigenetic carcinogens. Some doubts on this conviction are raised by the results of recent studies providing evidence that cyproterone acetate and two structural analogues, chlormadinone acetate and megestrol acetate, are genotoxic in female rats but only for the liver, and in primary human hepatocytes from donors of both genders. The experimental evidence suggests that the metabolic activation of these molecules to reactive species and the consequent formation of DNA adducts occur only in the intact hepatocyte. Since the possibility that other sex steroids cause a liver-specific genotoxic effect cannot be ruled out a priori, we investigated nine drugs of this family for their ability to induce DNA repair synthesis in primary cultures of rat and human hepatocytes. Each steroid was tested in cultures from at least two male and two female donors of each species. Hepatocytes were exposed for 20h to sub-toxic concentrations ranging from 1 to 50 micro M, and DNA repair induction was measured by quantitative autoradiography. In primary rat hepatocytes, induction of DNA repair indicative of a frankly positive response was detected in cultures from: 2/2 males and 3/3 females with drospirenone, 2/2 males and 1/2 females with ethinylestradiol, 1/2 males and 1/2 females with oxymetholone, 1/2 males with norethisterone, 1/4 females with progesterone, and 1/4 males with methyltestosterone. Consistent negative responses were obtained with testosterone and stanozolol. A few inconclusive responses were observed in rat hepatocytes exposed to progesterone, medroxyprogesterone, norethisterone, methyltestosterone and oxymetholone. In contrast, under the same experimental conditions the nine sex steroids provided frankly negative responses in the large majority of cultures of primary hepatocytes from both male and female human donors; the only exceptions being the inconclusive responses obtained in cultures from two of the donors exposed to norethisterone and to ethinylestradiol, and from one of the donors exposed to testosterone, methyltestosterone, and stanozolol. These results and previous findings concerning cyproterone and its structural analogues suggest that sex steroids differ for their ability to induce DNA repair, and that their genotoxicity may be: (i) different in rat and human hepatocytes, (ii) dependent on the sex of the donor, and (iii) affected by inter-individual variability.

Evaluation of omeprazole genotoxicity in a battery of in vitro and in vivo assays

Omeprazole, a proton pump inhibitor of wide use in the treatment of gastric acid-related disorders, was evaluated for its genotoxic effects in both rat and human cultured cells and in the intact rat. DNA repair synthesis, as revealed by autoradiography, was detected in primary cultures of metabolically competent rat hepatocytes exposed to concentrations ranging from 10 to 100 mg/l, but the responses cannot be considered as clearly positive. Under the same experimental conditions any significant evidence of DNA repair was absent in primary hepatocytes from two human donors. At the same concentrations a modest but dose-related increase of micronucleated cells, that reached the level of statistical significance at 33 mg/l, was present in primary rat hepatocytes and in one of two human donors. In human lymphocytes exposed to subtoxic concentrations ranging from 0.78 to 12.5 mg/l a reproducible concentration dependent clastogenic effect was absent. In partially hepatectomized female rats treated with a single p.o. dose of 1000 mg/kg, the frequency of micronucleated cells was 5.2-fold higher than in controls in the liver, but only 2.0-fold higher in polychromatic erythrocytes of the bone marrow. In rats of the same sex given azoxymethane as initiator of colon carcinogenesis the oral administration for 8 successive weeks of 10 mg/kg omeprazole on alternate days increased the response to azoxymethane, as indicated by the occurrence in colon mucosa of a modest but statistically significant increase in both the average number and size of aberrant crypt foci. Taken as a whole, our results suggest that omeprazole behaves as a weak genotoxic agent for the rat liver. Reliable information about the potential genotoxic risk to humans requires further studies on primary cells from a wide number of donors.

1,5-Benzodiazepines IX. A new route to substituted 4H-[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5-amines with analgesic and/or anti-inflammatory activities

Abstract A new two-step synthetic pathway to substituted 4 H -[1,2,4]triazolo[4,3- a ][1,5]benzodiazepin-5-amines 2a–p is described. The cyclocondensation of (dimethylamino)benzodiazepinone 1a with hydrazides afforded triazolobenzodiazepinones 5 which in turn reacted with suitable primary or secondary amines, in the presence of titanium tetrachloride, to give the desired 5-amino-derivatives 2a–p . When compounds 5 were treated with the Lawessons reagent thiolactams 6 were obtained, which then reacted with sodium hydride and proper alkyl halides to yield 5-(alkylthio)derivatives 7a–d . Compounds 2a–h, j–p , and 7a–d were tested for their analgesic and anti-inflammatory activities, as well as for their acute toxicity and gross behavioral effects. The analgesic activity appeared noteworthy in the writhing test, where fifteen compounds were more effective than both the reference drugs acetylsalicyclic acid and dipyrone, but was less evident in the hot plate test. An anti-inflammatory activity, lower than that of indomethacin but reaching the level of statistical significance, was displayed in the carrageenin-induced edema assay by five of the nineteen test compounds.

Genotoxicity testing of chloramphenicol in rodent and human cells

The results of this work, carried out to extend the limited information at present available on the genotoxic potential of chloramphenicol (CAP), indicate that in millimolar concentrations this antibacterial agent produced a minimal amount of DNA fragmentation in both V79 cells and metabolically competent rat hepatocytes. Moreover, a level of DNA-repair synthesis indicative of a weak but positive response was detected in primary cultures of liver cells obtained from 2 of 3 human donors, and a borderline degree of repair was present in those prepared from rats. The promutagenic character of CAP-induced DNA lesions was confirmed by a low but significant increase in the frequency of 6-thioguanine-resistant clones of V79 cells, which, however, was absent when the exposure was done in the presence of co-cultured rat hepatocytes. Finally, oral administration to rats of 1/2 LD50 CAP did not increase the incidence of either micronucleated polychromatic erythrocytes or micronucleated hepatocytes. Taken as a whole these findings suggest that CAP should be considered a compound intrinsically capable of producing a very weak genotoxic effect, but only at concentrations about 25 times higher than those occurring in patients treated with maximal therapeutic dosages.

Evaluation of auramine genotoxicity in primary rat and human hepatocytes and in the intact rat

Auramine, a dye previously found to be a liver carcinogen in both mice and rats, was evaluated for its DNA-damaging and clastogenic activities in primary cultures of rats and human hepatocytes and for the induction of DNA single-strand breaks in the liver and urinary bladder mucosa of intact rats. A similar dose-dependent frequency of DNA fragmentation was revealed by the alkaline elution technique in metabolically competent primary cultures of both rat and human hepatocytes exposed for 20 h to subtoxic concentrations ranging from 10 to 32 microM. In contrast, neither rat nor human hepatocytes displayed an increased frequency of micronuclei after a 48-h exposure to the same auramine concentrations. In rats given a single oral dose of 125, 250 or 500 mg kg-1 auramine, the Comet assay revealed a significant increase in the frequency of DNA lesions in the liver and in the urinary bladder mucosa, the effect being slightly more marked in the liver. Taken as a whole and compared with previous findings, these results suggest that auramine is biotransformed into reactive species in target organs of both rats and humans, and that this dye might play by itself the main role in the increased incidence of bladder cancer which has been judged as causally related to its manufacture.

Dna damage in peripheral blood lymphocytes of patients with cirrhosis related to alcohol abuse or to hepatitis B and C viruses

Background Both alcohol abuse and hepatitis B or C virus infections are implicated in the development of hepatocellular carcinoma, but it is still controversial whether the pathogenetic mechanism is epigenetic or genotoxic. Aim Considering that alcohol promotes the generation of reactive oxygen species and both viruses infect peripheral lymphocytes, in this study we investigated the occurrence of DNA fragmentation in peripheral blood lymphocytes from patients with alcoholic cirrhosis and from patients with cirrhosis related to B and C viruses, and analyzed the correlation between the degree of DNA fragmentation and the Child–Pugh score used to assess the degree of hepatic insufficiency. Methods The study population consisted of two groups: group I involved 12 patients with alcoholic cirrhosis; group II involved 25 patients with hepatic B virus or hepatic C virus cirrhosis. The control group involved 20 healthy individuals. The degree of DNA fragmentation in peripheral blood lymphocytes was determined with the alkaline Comet assay that provides two indexes of the frequency of DNA single-strand breaks and alkali-labile sites, the tail length and the tail moment. Results Mean values of both tail length and tail moment were significantly increased (P<0.001) in lymphocytes from 12 patients with alcoholic cirrhosis and in lymphocytes from 25 patients with HBV or HCV cirrhosis, as compared with average tail length and tail moment values of lymphocytes from 20 healthy individuals. A significant positive correlation was found to exist between the degree of DNA fragmentation present in lymphocytes of each of the 37 patients with alcoholic or viral cirrhosis and the corresponding value of the Child–Pugh score. Conclusion The occurrence of DNA fragmentation in peripheral blood lymphocytes reflects a direct genotoxic effect of either alcohol or HBV and HCV and suggests that the same genotoxic effect may operate in the liver and contribute to hepatocarcinogenesis.

Update of carcinogenicity studies in animals and humans of 535 marketed pharmaceuticals.

This survey is a compendium of information retrieved on carcinogenicity in animals and humans of 535 marketed pharmaceuticals whose expected clinical use is continuous for at least 6 months or intermittent over an extended period of time. Of the 535 drugs, 530 have the result of at least one carcinogenicity assay in animals, and 279 (52.1%) of them gave a positive response in at least one assay. Only 186 drugs (34.8%) have retrievable information on carcinogenicity in humans, and 104 of them gave to a variable extent evidence of a potential carcinogenic activity. Concerning the correlation between results obtained in animals and epidemiological findings, 58 drugs gave at least one positive result in carcinogenicity assays performed in animals and to a variable extent displayed evidence of carcinogenicity in humans, but 97 drugs tested positive in animals and were noncarcinogenic in humans or vice versa. Our findings, which are in agreement with previous studies, indicate that the evaluation of the benefit/carcinogenic risk ratio should be always made in prescribing a drug.

In vitro and in vivo antiproliferative activity of metformin on stem-like cells isolated from spontaneous canine mammary carcinomas: translational implications for human tumors

BackgroundCancer stem cells (CSCs) are considered the cell subpopulation responsible for breast cancer (BC) initiation, growth, and relapse. CSCs are identified as self-renewing and tumor-initiating cells, conferring resistance to chemo- and radio-therapy to several neoplasias. Nowadays, th (about 10mM)e pharmacological targeting of CSCs is considered an ineludible therapeutic goal. The antidiabetic drug metformin was reported to suppress in vitro and in vivo CSC survival in different tumors and, in particular, in BC preclinical models. However, few studies are available on primary CSC cultures derived from human postsurgical BC samples, likely because of the limited amount of tissue available after surgery. In this context, comparative oncology is acquiring a relevant role in cancer research, allowing the analysis of larger samples from spontaneous pet tumors that represent optimal models for human cancer.MethodsIsolation of primary canine mammary carcinoma (CMC) cells and enrichment in stem-like cell was carried out from fresh tumor specimens by culturing cells in stem-permissive conditions. Phenotypic and functional characterization of CMC-derived stem cells was performed in vitro, by assessment of self-renewal, long-lasting proliferation, marker expression, and drug sensitivity, and in vivo, by tumorigenicity experiments. Corresponding cultures of differentiated CMC cells were used as internal reference. Metformin efficacy on CMC stem cell viability was analyzed both in vitro and in vivo.ResultsWe identified a subpopulation of CMC cells showing human breast CSC features, including expression of specific markers (i.e. CD44, CXCR4), growth as mammospheres, and tumor-initiation in mice. These cells show resistance to doxorubicin but were highly sensitive to metformin in vitro. Finally, in vivo metformin administration significantly impaired CMC growth in NOD-SCID mice, associated with a significant depletion of CSCs.ConclusionsSimilarly to the human counterpart, CMCs contain stem-like subpopulations representing, in a comparative oncology context, a valuable translational model for human BC, and, in particular, to predict the efficacy of antitumor drugs. Moreover, metformin represents a potential CSC-selective drug for BC, as effective (neo-)adjuvant therapy to eradicate CSC in mammary carcinomas of humans and animals.