Publications by authors named "Cristina Geroni"

22 Publications

  • Page 1 of 1

Antimetastatic and Antitumor Activities of Orally Administered NAX014 Compound in a Murine Model of HER2-Positive Breast Cancer.

Int J Mol Sci 2021 Mar 6;22(5). Epub 2021 Mar 6.

Advanced Technology Center for Aging Research, Scientific Technological Area, IRCCS INRCA, 60121 Ancona, Italy.

The natural isoquinoline alkaloid Berberine (BBR) has been shown to possess several therapeutic effects, including anticancer activity. Different BBR derivatives have been designed and synthesized in order to obtain new compounds with enhanced anticancer efficacy. We previously showed that intraperitoneal (IP) administration of the BBR-derived NAX014 compound was able to counteract HER-2 overexpressing mammary tumors onset and progression in transgenic mice. However, the IP administration was found to induce organ toxicity at doses higher than 2.5 mg/Kg. In this study, we evaluated the effect of intragastric (IG) administration of 20 mg/kg of NAX014 on both safety and anticancer efficacy in HER-2/neu transgenic mice. Furthermore, cancer cell dissemination and migration, tumor cell senescence and immunological changes were examined. Our results demonstrated that IG NAX014 administration delayed the onset of mammary tumors with no negative effects on health and survival. NAX014 reduced HER-2 overexpressing BC cells migration in vitro and the frequency of lung metastasis in HER-2/neu transgenic mice. A statistically significant increase of senescence-associated expression was observed in tumors from NAX014-treated mice, and the induction of cell senescence was observed in HER-2 overexpressing BC cells after in vitro treatment with NAX014. Although NAX014 did not modulate the presence of tumor-infiltrating lymphocytes, the level of circulating TNF-α and VEGF was found to be reduced in NAX014-treated mice. The overall results address the NAX014 compound as potential tool for therapeutic strategies against HER-2 overexpressing breast cancer.
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http://dx.doi.org/10.3390/ijms22052653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961369PMC
March 2021

Characterization of water-soluble esters of nitrobenzoxadiazole-based GSTP1-1 inhibitors for cancer treatment.

Biochem Pharmacol 2020 08 29;178:114060. Epub 2020 May 29.

Department of Chemical Sciences and Technologies, University of Rome "Tor Vergata", Rome, Italy; The NAST Centre for Nanoscience and Nanotechnology and Innovative Instrumentation, University of Rome "Tor Vergata", Rome, Italy. Electronic address:

The 7-nitrobenzo[c][1,2,5]oxadiazole (NBD) derivative NBDHEX (compound 1) and its analogue MC3181 (compound 2) have been found to be potent inhibitors of tumor cell growth in vitro and therapeutically active and safe in mice bearing human melanoma xenografts. To enhance the aqueous solubility of these compounds, we synthesized the hemisuccinate of 1 (compound 3) and the phosphate monoesters of 1 and 2 (compound 4 and 5, respectively). These novel NBD derivatives displayed a solubility in the conventional phosphate-buffered saline up to 150-fold higher than that of 1, and up to 4-fold higher than that of 2. Notably, solubility of phosphates 4 and 5 in a potassium phosphate buffer at pH 7.4, was up to 500-fold higher than that of 1, and ~10-fold higher than that of 2. Compounds 3-5 retained high cytotoxicity towards cultured human melanoma and osteosarcoma cells and were cleaved in vitro by both human and murine hydrolases, thus releasing the corresponding parent compound (i.e., 1 or 2). Interestingly, esters 3-5 displayed high inhibitory activity towards the glutathione transferase (GST) isoform GSTP1-1 and showed a reactivity towards reduced glutathione comparable to that of the respective parent compound. Finally, both 4 and 5 were safe and effective when administered intravenously or orally as an aqueous solution to mice xenografted with A375 human melanoma tumors. Collectively, these results and the previously observed synergistic interaction between 1 and 2 and various approved anticancer drugs, suggest the possible utility of phosphates 4 and 5 as single agents and in combination regimens in cancers with unmet medical need, including melanoma.
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http://dx.doi.org/10.1016/j.bcp.2020.114060DOI Listing
August 2020

Enhanced Clearance of Neurotoxic Misfolded Proteins by the Natural Compound Berberine and Its Derivatives.

Int J Mol Sci 2020 May 13;21(10). Epub 2020 May 13.

Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Dipartimento di Eccellenza 2018-2022, Centro di Eccellenza sulle Malattie Neurodegenerative, Università degli Studi di Milano, 20133 Milan, Italy.

Background: Accumulation of misfolded proteins is a common hallmark of several neurodegenerative disorders (NDs) which results from a failure or an impairment of the protein quality control (PQC) system. The PQC system is composed by chaperones and the degradative systems (proteasome and autophagy). Mutant proteins that misfold are potentially neurotoxic, thus strategies aimed at preventing their aggregation or at enhancing their clearance are emerging as interesting therapeutic targets for NDs.

Methods: We tested the natural alkaloid berberine (BBR) and some derivatives for their capability to enhance misfolded protein clearance in cell models of NDs, evaluating which degradative pathway mediates their action.

Results: We found that both BBR and its semisynthetic derivatives promote degradation of mutant androgen receptor (ARpolyQ) causative of spinal and bulbar muscular atrophy, acting mainly via proteasome and preventing ARpolyQ aggregation. Overlapping effects were observed on other misfolded proteins causative of amyotrophic lateral sclerosis, frontotemporal-lobar degeneration or Huntington disease, but with selective and specific action against each different mutant protein.

Conclusions: BBR and its analogues induce the clearance of misfolded proteins responsible for NDs, representing potential therapeutic tools to counteract these fatal disorders.
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http://dx.doi.org/10.3390/ijms21103443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7279252PMC
May 2020

Antitumor activity of NAX060: A novel semisynthetic berberine derivative in breast cancer cells.

Biofactors 2018 Sep 3;44(5):443-452. Epub 2018 Sep 3.

Advanced Technology Center for Aging Research, Scientific Technological Area, IRCCS - INRCA, Ancona, Italy.

Breast cancer (BC) is the most common malignancy and the most common cause of cancer death in elderly women. We recently demonstrated that innovative compounds structurally related to and semisynthetically derived from the plant alkaloid berberine represent a promising unexplored resource for novel therapeutic tools in BC therapy. In this study, we analyzed the effectiveness of new 13-dichlorophenylalkyl berberine semisynthetic derivatives (NAX060, NAX103, NAX111, and NAX114) on the viability of BC cell lines. Our results demonstrated that the new compounds effectively inhibited the growth of a variety of human BC cell lines. In particular, the viability of HER-2 overexpressing SK-BR-3 cells was significantly reduced by the treatment with NAX060, the most active compound, in a dose and time-dependent manner. In the same tumor cell line, NAX060 induced a strong increase in sub-G1 population while G0/G1 and G2/M phase cells remarkably decreased. NAX060 withdrawal after 72 h of treatment resulted in an irreversible cell proliferation arrest and increasing cell death. Real-time PCR analyses showed that NAX060 induced the expression of some cell-cycle checkpoint molecules involved in cell senescence such as p21WAF1, p27, p16INK4a, and PAI-1. Furthermore, the HER-2 protein expression and phosphorylation, as well as the level of heparanase expression, were remarkably reduced on SK-BR-3 cells. NAX060 was effective also on HER-2 negative tumor cells, and, in particular, on human triple-negative MDA-MB-231 cells. These data suggest a potential therapeutic effect of NAX060 compound in the management of BC malignancies. Interestingly, NAX060 may represent a new useful tool also in triple-negative BC. © 2018 BioFactors, 44(5):443-452, 2018.
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http://dx.doi.org/10.1002/biof.1440DOI Listing
September 2018

Antiangiogenic and antitumor activities of berberine derivative NAX014 compound in a transgenic murine model of HER2/neu-positive mammary carcinoma.

Carcinogenesis 2015 Oct 13;36(10):1169-79. Epub 2015 Jul 13.

Advanced Technology Center for Aging Research, Scientific Technological Area, IRCCS- INRCA, Via Birarelli 8, 60121 Ancona, Italy, Experimental Animal Models for Aging Unit, Scientific Technological Area, IRCCS- INRCA, via Birarelli 8, 60121 Ancona, Italy,

Berberine (BBR) is a natural isoquinoline alkaloid with proven antiangiogenic and anticancer activities. We recently demonstrated that BBR and its synthetic derivative 13-(4-chlorophenylethyl)berberine iodide, NAX014, exert antiproliferative activity against HER2-overexpressing breast cancer cells, inducing apoptosis, modulating the expression of cell cycle checkpoint molecules involved in cell senescence, and reducing both HER2 expression and phosphorylation on tumor cells. In this study, we examined the anticancer properties of BBR and NAX014 in a transgenic mouse model which spontaneously develops HER2-positive mammary tumors. Repeated intraperitoneal injections of a safety dose (2.5mg/kg) of NAX014 delayed the development of tumors, reducing both the number and size of tumor masses. In vivo sidestream dark field videomicroscopy revealed a significant lower vessel density in mammary tumors from NAX014-treated mice in comparison with the control group. Immunohistochemical evaluation using CD34 antibody confirmed the reduced vessel density in NAX014 group. Statistically significant increase of senescence associated β-galactosidase and p16 expression, and reduced expression of heparanase were observed in tumors from NAX014-treated mice than in tumors from control animals. Finally, NAX014 treatment decreased the level of perforine and granzyme mRNA in mammary tumors. Berberine did not show any statistically significant modulation in comparison with control mice. The results of the present study indicate that NAX014 is more effective than BBR in exerting anticancer activity delaying the development of mammary tumors in mice transgenic for the HER-2/neu oncogene. The antitumor efficacy of NAX014 is mainly related to its effect on tumor vascular network and on induction of tumor cell senescence.
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http://dx.doi.org/10.1093/carcin/bgv103DOI Listing
October 2015

A novel orally active water-soluble inhibitor of human glutathione transferase exerts a potent and selective antitumor activity against human melanoma xenografts.

Oncotarget 2015 Feb;6(6):4126-43

The NAST Centre for Nanoscience & Nanotechnology & Innovative Instrumentation, University of Tor Vergata, 00133 Rome, Italy.

We designed and synthesized two novel nitrobenzoxadiazole (NBD) analogues of the anticancer agent 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX). The new compounds, namely MC3165 and MC3181, bear one and two oxygen atoms within the hydroxy-containing alkyl chain at the C4 position of the NBD scaffold, respectively. This insertion did not alter the chemical reactivity with reduced glutathione, while it conferred a remarkable increase in water solubility. MC3181 was more selective than NBDHEX towards the target protein, glutathione transferase P1-1, and highly effective in vitro against a panel of human melanoma cell lines, with IC50 in the submicromolar-low micromolar range. Interestingly, the cellular response to MC3181 was cell-type-specific; the compound triggered a JNK-dependent apoptosis in the BRAF-V600E-mutated A375 cells, while it induced morphological changes together with an increase in melanogenesis in BRAF wild-type SK23-MEL cells. MC3181 exhibited a remarkable therapeutic activity against BRAF-V600E-mutant xenografts, both after intravenous and oral administration. Outstandingly, no treatment-related signs of toxicity were observed both in healthy and tumor-bearing mice after single and repeated administrations. Taken together, these results indicate that MC3181 may represent a potential novel therapeutic opportunity for BRAF-mutated human melanoma, while being safe and water-soluble and thus overcoming all the critical aspects of NBDHEX in vivo.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4414177PMC
http://dx.doi.org/10.18632/oncotarget.2798DOI Listing
February 2015

Berberine acts as a natural inhibitor of Wnt/β-catenin signaling--identification of more active 13-arylalkyl derivatives.

Biofactors 2013 Nov-Dec;39(6):652-62. Epub 2013 Aug 24.

Institute of Biochemistry II, Jena University Hospital, Friedrich-Schiller-University Jena, Nonnenplan 2-4, 07743, Jena, Germany.

Aberrant activation of the canonical Wnt/β-catenin signaling pathway has been reported for numerous tumors of different origins. In most cases, mutations in components of the Wnt signaling pathway or in β-catenin itself were detected which ultimately induce a genetic program that promotes cell proliferation and attenuates apoptosis. Thus, targeting of Wnt/β-catenin signaling is of specific therapeutic interest. Herein, we investigated the plant-derived isoquinoline alkaloid berberine, which has been reported to have anticancer activity, and synthetic 13-arylalkyl derivatives thereof for their effects on Wnt/β-catenin signaling. Berberine did not show major effects on viability of HEK-293 embryonic kidney and HCT116 colon carcinoma cells and was not toxic in concentrations up to 20 µM. Berberine inhibited β-catenin transcriptional activity and attenuated anchorage-independent growth. As a result of berberine treatment, cellular levels of active β-catenin were reduced concomitant with an increase in the expression of E-cadherin. However, in unstimulated cells, the effects on β-catenin levels were low. A screen of synthetic 13-arylalkyl berberine derivatives identified compounds exhibiting activities superior to those of the naturally occurring parent substance with more than 100-fold lower EC50 values for Wnt-repression. Thus, berberine and its synthetic derivatives represent potential therapeutic agents to inhibit Wnt/β-catenin signaling in tumorigenesis.
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http://dx.doi.org/10.1002/biof.1133DOI Listing
July 2014

Zebularine partially reverses GST methylation in prostate cancer cells and restores sensitivity to the DNA minor groove binder brostallicin.

Epigenetics 2013 Jun 14;8(6):656-65. Epub 2013 Jun 14.

Laboratory of Molecular Pharmacology; Department of Oncology; IRCCS-Istituto di Ricerche Farmacologiche Mario Negri; Milan, Italy.

Brostallicin is a DNA minor groove binder that shows enhanced antitumor activity in cells with high glutathione S-transferase (GST)/glutathione content. Prostate cancer cells present, almost invariably, methylation of the GSTP1 gene promoter and, as a consequence, low levels of GST-pi expression and activity. In these cells, brostallicin shows very little activity. We tested whether pretreatment of heavily GST-methylated prostate cancer cells with demethylating agents could enhance the activity of brostallicin. Human prostate cancer cells LNCaP and DU145 were used for these studies both in vitro and in vivo. The demethylating agent zebularine was used in combination with brostallicin. Methylation specific PCR and pyrosequencing were used to determine the level of GST methylation. Pretreatment with demethylating agents enhanced the in vitro activity of brostallicin in LNCaP cells. Zebularine, in particular, induced an enhancement of activity in vivo comparable to that obtained by transfecting the human GSTP1 gene in LNCaP cells in vitro. Molecular analysis performed on tumor xenografts in mice pretreated with zebularine failed to detect re-expression of GST-pi and demethylation of GSTP1. However, we found demethylation in the GSTM1 gene, with consequent re-expression of GST-mu at the mRNA level. These results indicate that zebularine, both in vitro and in vivo, enhances the activity of brostallicin and that this enhancement correlates with re-expression of GST-pi and GST-mu. These findings highlight the potential therapeutic value of combining demethylating agents and brostallicin in tumors with GST methylation that poorly respond to brostallicin.
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http://dx.doi.org/10.4161/epi.24916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3857345PMC
June 2013

Down-regulation of the nucleotide excision repair gene XPG as a new mechanism of drug resistance in human and murine cancer cells.

Mol Cancer 2010 Sep 24;9:259. Epub 2010 Sep 24.

Laboratory of Molecular Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.

Background: Drug resistance is one of the major obstacles limiting the activity of anticancer agents. Activation of DNA repair mechanism often accounts for increase resistance to cancer chemotherapy.

Results: We present evidence that nemorubicin, a doxorubicin derivative currently in clinical evaluation, acts through a mechanism of action different from classical anthracyclines, requiring an intact nucleotide excision repair (NER) system to exert its activity. Cells made resistant to nemorubicin show increased sensitivity to UV damage. We have analysed the mechanism of resistance and discovered a previously unknown mechanism resulting from methylation-dependent silencing of the XPG gene. Restoration of NER activity through XPG gene transfer or treatment with demethylating agents restored sensitivity to nemorubicin. Furthermore, we found that a significant proportion of ovarian tumors present methylation of the XPG promoter.

Conclusions: Methylation of a NER gene, as described here, is a completely new mechanism of drug resistance and this is the first evidence that XPG gene expression can be influenced by an epigenetic mechanism. The reported methylation of XPG gene could be an important determinant of the response to platinum based therapy. In addition, the mechanism of resistance reported opens up the possibility of reverting the resistant phenotype using combinations with demethylating agents, molecules already employed in the clinical setting.
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http://dx.doi.org/10.1186/1476-4598-9-259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2955619PMC
September 2010

Role of glutathione transferases in the mechanism of brostallicin activation.

Biochemistry 2010 Jan;49(1):226-35

Department of Chemical Sciences and Technologies, University of Tor Vergata, Rome, Italy.

Brostallicin is a novel and unique glutathione transferase-activated pro-drug with promising anticancer activity, currently in phase I and II clinical evaluation. In this work, we show that, in comparison with the parental cell line showing low GST levels, the cytotoxic activity of brostallicin is significantly enhanced in the human breast carcinoma MCF-7 cell line, transfected with either human GST-pi or GST-mu. Moreover, we describe in detail the interaction of brostallicin with GSH in the presence of GSTP1-1 and GSTM2-2, the predominant GST isoenzymes found within tumor cells. The experiments reported here indicate that brostallicin binds reversibly to both isoenzymes with K(d) values in the micromolar range (the affinity being higher for GSTM2-2). Direct evidence that both GSTP1-1 and GSTM2-2 isoenzymes catalyze the Michael addition reaction of GSH to brostallicin has been obtained both by an HPLC-MS technique and by a new fluorometric assay. We also saw the rapid formation of an intermediate reactive species, which is slowly converted into the final products. This intermediate, identified as the alpha-chloroamido derivative of the GSH-brostallicin adduct, is able to alkylate DNA in a sequence-specific manner and appears to be the active form of the drug. The kinetic behavior of the reaction between brostallicin and GSH, catalyzed by GSTP1-1, has been studied in detail, and a minimum kinetic scheme that suitably describes the experimental data is provided. Overall, these data fully support and extend the findings that brostallicin could be indicated for the treatment of tumor overexpressing the pi or mu class GST.
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http://dx.doi.org/10.1021/bi901689sDOI Listing
January 2010

In vitro and in vivo efficacy of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) on human melanoma.

Eur J Cancer 2009 Sep 6;45(14):2606-17. Epub 2009 Aug 6.

Department of Chemical Sciences and Technologies, University of Tor Vergata, Rome, Italy.

6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) is a powerful inhibitor of the glutathione transferase P1-1 (GSTP1-1) and causes the disruption of the complex between GSTP1-1 and c-Jun N-terminal Kinase (JNK). This induces JNK activation and apoptosis in tumour cells. In the present work we assess the in vitro and in vivo effectiveness of NBDHEX on two human melanoma cell lines, Me501 and A375. NBDHEX shows IC(50) values in the low micromolar range (IC(50) of 1.2+/-0.1microM and 2.0+/-0.2 microM for Me501 and A375, respectively) and is over 100 times more cytotoxic to these cell lines than temozolomide. Apoptosis is observed in Me501 cells within 3h of the addition of NBDHEX, while in A375 cells the apoptotic event is rather late, and is preceded by a G2/M phase arrest. In both melanoma cell lines, JNK activity is required for the ability of NBDHEX to trigger apoptosis, confirming that the JNK pathway is an important therapeutic target for this tumour. NBDHEX is also both effective and well tolerated in in vivo tumour models. A tumour inhibition of 70% is observed in vivo against Me501 human melanoma and a similar result is obtained on A375 model, with 63% of tumour inhibition. These findings indicate that the activation of the JNK pathway, through a selective GSTP1-1 targeting, could prove to be a promising new strategy for treating melanoma, which responds poorly to conventional therapies.
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http://dx.doi.org/10.1016/j.ejca.2009.06.033DOI Listing
September 2009

In vitro hepatic conversion of the anticancer agent nemorubicin to its active metabolite PNU-159682 in mice, rats and dogs: a comparison with human liver microsomes.

Biochem Pharmacol 2008 Sep 11;76(6):784-95. Epub 2008 Jul 11.

Department of Pharmacology and Anaesthesiology, University of Padova, Largo Meneghetti 2, 35131 Padova, Italy.

We recently demonstrated that nemorubicin (MMDX), an investigational antitumor drug, is converted to an active metabolite, PNU-159682, by human liver cytochrome P450 (CYP) 3A4. The objectives of this study were: (1) to investigate MMDX metabolism by liver microsomes from laboratory animals (mice, rats, and dogs of both sexes) to ascertain whether PNU-159682 is also produced in these species, and to identify the CYP form(s) responsible for its formation; (2) to compare the animal metabolism of MMDX with that by human liver microsomes (HLMs), in order to determine which animal species is closest to human beings; (3) to explore whether differences in PNU-159682 formation are responsible for previously reported species- and sex-related differences in MMDX host toxicity. The animal metabolism of MMDX proved to be qualitatively similar to that observed with HLMs since, in all tested species, MMDX was mainly converted to PNU-159682 by a single CYP3A form. However, there were marked quantitative inter- and intra-species differences in kinetic parameters. The mouse and the male rat exhibited V(max) and intrinsic metabolic clearance (CL(int)) values closest to those of human beings, suggesting that these species are the most suitable animal models to investigate MMDX biotransformation. A close inverse correlation was found between MMDX CL(int) and previously reported values of MMDX LD(50) for animals of the species, sex and strain tested here, indicating that differences in the in vivo toxicity of MMDX are most probably due to sex- and species-related differences in the extent of PNU-159682 formation.
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http://dx.doi.org/10.1016/j.bcp.2008.07.003DOI Listing
September 2008

Ongoing phase I and II studies of novel anthracyclines.

Cardiovasc Toxicol 2007 ;7(2):75-9

Oncology Institute of Southern Switzerland, Ospedale San Giovanni, Bellinzona 6500, Switzerland.

Many anthracyclines are currently in clinical development with the common aim of improving selectivity. This could be achieved by improving tumor drug delivery through the identification and development of molecules with new structure, prodrugs with low molecular weight for selective release and activation, prodrugs with high molecular weight conjugated to antibody with active targeting or macromolecules with enhanced permeability and retention. There are still interfering factors to be defined, in particular chemical, with degradation steps in tumor tissues, biological, related to tumor proteases, pharmacological, with inter-individual tumor differences in the extent of accumulation. Another way to improve selectivity is to activate the drug at the tumor site, a good example of which is provided by Nemorubicin (2\"-(S)-methoxymorpholinodoxorubicin hydrochloride) in hepatocellular carcinoma. The favorable characteristics of Nemorubicin in terms of broad spectrum of significant antitumor activity in liver malignancies models, lower cardiotoxicity than Doxorubicin, make Nemorubicin a promising third-generation anthracycline, suitable for intrahepatic administration.
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http://dx.doi.org/10.1007/s12012-007-0010-8DOI Listing
October 2007

4-Demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyl-daunorubicin (PNU-159548): a promising new candidate for chemotherapeutic treatment of osteosarcoma patients.

Eur J Cancer 2005 Sep;41(14):2184-95

Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli, Via di Barbiano 1/10, 40136 Bologna, Italy.

The effectiveness of the alkycycline 4-demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyl-daunorubicin (PNU-159548, ladirubicin), a new drug with high antitumour activity against a broad range of neoplasms, was evaluated by using a panel of 32 human osteosarcoma cell lines, including cell lines resistant to doxorubicin, methotrexate, or cisplatin. PNU-159548 resulted to be highly active in all cell lines. No cross-resistance was found with conventional drugs, being PNU-159548 active also in cells resistant to doxorubicin and with a multidrug resistance phenotype (associated with MDR1 gene/P-glycoprotein overexpression), as well as in cells resistant to methotrexate or to cisplatin. Analysis of drug-drug interactions showed that PNU-159548 could be successfully used in combination with all the most important drugs currently used in OS chemotherapy. In fact, the simultaneous administration of PNU-159548 and doxorubicin, methotrexate, or cisplatin produced mostly additive or synergistic effects. Sequential exposure to PNU-159548 followed immediately by doxorubicin, methotrexate, or cisplatin was the most effective sequence of administration, invariably resulting in additive or synergistic effects in both drug-sensitive and drug-resistant osteosarcoma cell lines. In conclusion, the high in vitro effectiveness, the absence of cross-resistance with doxorubicin, methotrexate, or cisplatin and the possibility to be successfully used in combination with these drugs indicate PNU-159548 as a promising candidate to be considered for planning new therapeutic regimens for osteosarcoma patients, who show a decreased response to conventional chemotherapy.
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http://dx.doi.org/10.1016/j.ejca.2005.06.017DOI Listing
September 2005

Formation and antitumor activity of PNU-159682, a major metabolite of nemorubicin in human liver microsomes.

Clin Cancer Res 2005 Feb;11(4):1608-17

Department of Pharmacology and Anesthesiology, University of Padua, Largo Meneghetti 2, I-35131 Padua, Italy.

Purpose: Nemorubicin (3'-deamino-3'-[2''(S)-methoxy-4''-morpholinyl]doxorubicin; MMDX) is an investigational drug currently in phase II/III clinical testing in hepatocellular carcinoma. A bioactivation product of MMDX, 3'-deamino-3'',4'-anhydro-[2''(S)-methoxy-3''(R)-oxy-4''-morpholinyl]doxorubicin (PNU-159682), has been recently identified in an incubate of the drug with NADPH-supplemented rat liver microsomes. The aims of this study were to obtain information about MMDX biotransformation to PNU-159682 in humans, and to explore the antitumor activity of PNU-159682.

Experimental Design: Human liver microsomes (HLM) and microsomes from genetically engineered cell lines expressing individual human cytochrome P450s (CYP) were used to study MMDX biotransformation. We also examined the cytotoxicity and antitumor activity of PNU-159682 using a panel of in vitro-cultured human tumor cell lines and tumor-bearing mice, respectively.

Results: HLMs converted MMDX to a major metabolite, whose retention time in liquid chromatography and ion fragmentation in tandem mass spectrometry were identical to those of synthetic PNU-159682. In a bank of HLMs from 10 donors, rates of PNU-159682 formation correlated significantly with three distinct CYP3A-mediated activities. Troleandomycin and ketoconazole, both inhibitors of CYP3A, markedly reduced PNU-159682 formation by HLMs; the reaction was also concentration-dependently inhibited by a monoclonal antibody to CYP3A4/5. Of the 10 cDNA-expressed CYPs examined, only CYP3A4 formed PNU-159682. In addition, PNU-159682 was remarkably more cytotoxic than MMDX and doxorubicin in vitro, and was effective in the two in vivo tumor models tested, i.e., disseminated murine L1210 leukemia and MX-1 human mammary carcinoma xenografts.

Conclusions: CYP3A4, the major CYP in human liver, converts MMDX to a more cytotoxic metabolite, PNU-159682, which retains antitumor activity in vivo.
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http://dx.doi.org/10.1158/1078-0432.CCR-04-1845DOI Listing
February 2005

Cinnamoyl nitrogen mustard derivatives of pyrazole analogues of tallimustine modified at the amidino moiety: design, synthesis, molecular modeling and antitumor activity studies.

Bioorg Med Chem 2004 Jul;12(14):3911-21

Dipartimento di Scienze Farmaceutiche, Università di Ferrara, 44100 Ferrara, Italy.

The design, synthesis and in vitro activities of a series of cinnamoyl nitrogen mustard pyrazole analogues of tallimustine 8-13, in which the amidino moiety has been replaced by moieties of different physico-chemical features are described, and the structure-activity relationships are discussed. In spite of the relevance of these modifications on the amidino moiety, these derivatives showed significant growth inhibitory activity against mouse leukemia L1210 cells. A selected series of compounds have been evaluated for their sequence selective alkylating properties and cytotoxicity against human K562 leukemia cells. Therefore, the presence of the amidino moiety, and in general of a basic moiety, is not an absolute requirement for biological activity. Our preliminary results indicated that the compounds of this series have a pattern of alkylation similar to that of tallimustine, but they seem to be less reactive overall in alkylating naked DNA.
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http://dx.doi.org/10.1016/j.bmc.2004.04.045DOI Listing
July 2004

Cytotoxic alpha-halogenoacrylic derivatives of distamycin A and congeners.

J Med Chem 2004 May;47(10):2611-23

Pharmacia Italia S.p.A., Discovery Research Oncology, Viale Pasteur 10, 20014 Nerviano, Milan, Italy.

The mechanism of action of many antitumor agents involves DNA damage, either by direct binding of the drug to DNA or to DNA-binding proteins. However, most of the DNA-interacting agents have only a limited degree of sequence specificity, which implies that they may hit all the cellular genes. DNA minor groove binders, among which the derivatives of distamycin A play an important role, could provide significant improvement in cancer management, increasing gene specificity, due to high selectivity of interaction with thymine-adenine (TA) rich sequences. We now report and discuss the synthesis, the in vitro and in vivo activities, and some mechanistic features of alpha-halogenoacrylamido derivatives of distamycin A. The final result of this work was the selection of brostallicin 17 (PNU-166196). Brostallicin, presently in phase II clinical trials, shows a broad spectrum of antitumor activity and an apoptotic effect higher than distamycin derivative tallimustine. An important in vitro toxicological feature of brostallicin is the very good ratio between myelotoxicity on human haematopoietic progenitor cells and cytotoxicity on tumor cells, in comparison with clinically tested DNA minor groove binders. A peculiarity of brostallicin is its in vitro reactivity in the DNA alkylation assays only in the presence of glutathione. Moreover brostallicin's antitumor activity, both in in vitro and in vivo tumor models, is higher in the presence of increased levels of glutathione/glutathione-S-tranferases. These findings contribute to the definition of brostallicin as a novel anticancer agent that differs from other minor groove binders and alkylating agents for both the profile of activity and the mechanism of action and to classify the alpha-bromoacrylamido derivatives of distamycin as a new class of cytotoxics. Moreover, due to its interaction with glutathione, brostallicin may have a role for the tailored treatment of tumors characterized by constitutive or therapy-induced overexpression of glutathione/glutathione-S-tranferase levels.
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http://dx.doi.org/10.1021/jm031051kDOI Listing
May 2004

Brostallicin: a new concept in minor groove DNA binder development.

Anticancer Drugs 2004 Jan;15(1):1-6

Laboratory of Molecular Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.

Brostallicin is a bromoacryloyl derivative of distamycin A, which has shown very promising preclinical activity against a variety of human tumors both in vitro and in vivo. The drug has a limited toxicity towards bone marrow precursor cells in vitro resulting in a therapeutic index much higher than those achieved with other distamycin A derivatives. It retains activity against cancer cells resistant to alkylating agents, topoisomerase I inhibitors and cells with mismatch repair deficiency. Brostallicin has a peculiar mechanism of action involving activation upon binding to glutathione (GSH) catalyzed by glutathione-S-transferase (GST). As a consequence, cells expressing relatively high GST/GSH levels are more susceptible to treatment with brostallicin. Considering that increased levels of GST/GSH are often found in human tumors, this could represent an advantage for the drug in the clinic. Initial clinical studies indicate the tolerability of the drug and allow the determination of the optimal dose for subsequent studies. Some partial response were obtained in these initial phase I studies. Altogether, the results suggest brostallicin to be a new promising anticancer agent with a new mechanism of action. It also raises the possibility to use it in combination with other anticancer drugs currently used.
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http://dx.doi.org/10.1097/00001813-200401000-00001DOI Listing
January 2004

Enhancement of in vivo antitumor activity of classical anticancer agents by combination with the new, glutathione-interacting DNA minor groove-binder, brostallicin.

Clin Cancer Res 2003 Nov;9(14):5402-8

Laboratory of Molecular Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.

Purpose: Brostallicin (PNU-166196) is a alpha-bromoacrylic DNA minor groove binder, currently in clinical evaluation. This drug has the peculiarity of showing enhanced antitumor activity in cells with high glutathione S-transferase (GST)/glutathione content. The purpose of the study was to study multiple combinations of brostallicin with classical anticancer agents.

Experimental Design: The cis-dichloro-diammine-platinum (cDDP)/brostallicin combination was tested in the human colon carcinoma (HCT-116) model transplanted in nude mice. Two treatment schedules were tested: cDDP followed by brostallicin 48 h after or brostallicin followed by cDDP. These two schemes were selected from the observation that tumor cells in vitro show an increased activity of GST 48 h after cDDP treatment. The HCT-116 model was used also to test the irinotecan (cPT-11)/brostallicin combination. The effect of brostallicin in combination with doxorubicin (DX) was studied in the i.v. injected murine L1210 leukemia. Three administration schedules were tested. The antitumor activity of brostallicin and Taxotere was tested on the A549 lung cancer xenografts.

Results: In line with the increased GST activity observed after treatment with cDDP, the cDDP/brostallicin interaction was sequence-dependent, leading to a more than additive antitumor effect, without additional toxicity, only when cDDP was given before brostallicin. The antitumor effect of CPT-11 was enhanced significantly by brostallicin cotreatment. A more than additive antitumor effect, without additional toxicity, was observed when DX/brostallicin were sequentially administered in L1210-bearing mice. Finally, additivity was observed when brostallicin/Taxotere simultaneous combination was tested.

Conclusions: Although the precise molecular mechanism of interaction between brostallicin and the other tested cytotoxics has not yet been identified, a clear therapeutic gain is observed in preclinical models when brostallicin is combined with anticancer agents such as cDDP, DX, CPT-11, and Taxotere. These results indicate the potential therapeutic value of brostallicin in cancer combination treatment therapy.
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November 2003

Doxorubicin activity is enhanced by hyperthermia in a model of ex vivo vascular perfusion of human colon carcinoma.

World J Surg 2003 Jun 13;27(6):640-6. Epub 2003 May 13.

Department of Oncological and Surgical Sciences, Istituto di Clinica Chirurgica Generale II, University of Padova, 6th Piano, Via Giustiniani, 2 35128, Padova, Italy.

There is little information on the pharmacokinetics and pharmacodynamics of doxorubicin (DXR) administered during locoregional treatments of colon carcinoma under hyperthermic conditions. The aim of this study was to evaluate distribution and activity of DXR in healthy tissue and tumor tissues under hyperthermic conditions by using an experimental model of ex vivo isolated vascular perfusion of human colon segments bearing primary carcinoma. The influence of topoisomerase-II alpha (TPI2 alpha) expression on the anti-cancer activity of DXR combined with heat was evaluated as well. Twenty seven colon segments surgically resected for primary carcinoma were perfused ex vivo under physiological conditions (group A, n = 7), with DXR (group B, n = 6), under hyperthermic conditions (group C, n = 6), and with the combination DXR-hyperthermia (group D, n = 8). Samples of perfusate and tissues (normal and pathologic) were collected during 90 minutes of perfusion. Doxorubicin concentration in perfusate and tissues was assessed with high-performance liquid chromatography. Protein expression of TPI2 alpha, the main molecular target of DXR, was measured by image analysis in normal mucosa and tumor samples. Drug uptake was increased by heat equally in healthy and diseased tissue. Under hyperthermic conditions, DXR activity was significantly higher in pathologic mucosa than in normal mucosa. Furthermore, the activity of DXR combined with heat correlated with baseline TPI2 alpha levels in tumor tissue. From these findings, it appears that heat sensitizes tumor cells-but not normal mucosa-to DXR activity. Furthermore, protein levels of TPI2 alpha in pretreatment samples could predict tumor sensitivity to DXR.
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http://dx.doi.org/10.1007/s00268-003-6804-3DOI Listing
June 2003

Cytotoxic alpha-bromoacrylic derivatives of low molecular weight.

Bioorg Med Chem Lett 2002 Jun;12(11):1467-71

Chemistry Department, Pharmacia Discovery Research Oncology, Viale Pasteur 10, 20014 Nerviano, Milan, Italy.

In vitro and in vivo activities of a small series of alpha-bromoacrylic derivatives of low molecular weight (MW) are described and compared with those of alpha-bromoacrylic derivatives of distamycin-like frames. Low MW compounds, when lacking of a strong basic moiety, are potent cytotoxics, while analogues bearing a strong basic moiety are not. This suggests the existence of an active transport mechanism for distamycin-derived cytotoxics characterized by strong basic amidino or guanidino moieties. Low MW compounds are inactive in vivo, possibly because of the metabolic lability of alpha-bromoacrylic moiety. The same moiety is however present in a series of potent anticancer distamycin-like minor groove binders, for example, PNU-166196 (brostallicin), a fact that underlines the features of the latter.
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http://dx.doi.org/10.1016/s0960-894x(02)00177-4DOI Listing
June 2002

Brostallicin, a novel anticancer agent whose activity is enhanced upon binding to glutathione.

Cancer Res 2002 Apr;62(8):2332-6

Laboratory of Molecular Pharmacology, Department of Oncology, Mario Negri Institute of Pharmacological Research, via Eritrea 62, 20157 Milan, Italy.

Brostallicin (PNU-166196) is a synthetic alpha-bromoacrylic, second-generation DNA minor groove binder structurally related to distamycin A, presently in Phase II trials in Europe and the United States. The compound shows broad antitumor activity in preclinical models and dramatically reduced in vitro myelotoxicity in human hematopoietic progenitor cells compared with that of other minor groove binders. Brostallicin showed a 3-fold higher activity in melphalan-resistant L1210 murine leukemia cells than in the parental line (IC(50) = 0.46 and 1.45 ng/ml, respectively) under conditions in which the cytotoxicity of conventional antitumor agents was either unaffected or reduced. This melphalan-resistant cell line has increased levels of glutathione (GSH) in comparison with the parental cells. Conversely, GSH depletion by buthionine sulfoximine in a human ovarian carcinoma cell line (A2780) significantly decreased both the cytotoxic and the proapoptotic effects of brostallicin. In one experiment, human glutathione S-transferase pi (GST-pi) cDNA was transfected into A2780 cells, and four clones of A2780 with different expression levels of GST-pi were generated (i.e., two clones with high and two clones with low GST-pi expression). A 2-3-fold increase in GST-pi levels resulted in a 2-3-fold increase in cytotoxic activity of brostallicin. Similar results were obtained for GST-pi-transfected human breast carcinoma cells (MCF-7). Brostallicin showed 5.8-fold increased cytotoxicity in GST-pi-transfected versus empty vector-transfected cells with low GST-pi expression. In an in vivo experiment, A2780 clones were implanted into nude mice. The antitumor activity of brostallicin was higher in the GST-pi-overexpressing tumors without increased toxicity. Regarding the mechanism of action, brostallicin interacts reversibly with the DNA minor groove TA-rich sequences but appears unreactive in classical in vitro DNA alkylation assays. We speculated that an intracellular reactive nucleophilic species, e.g., GSH, could react with the alpha-bromoacrylamide moiety functions. Experiments on the interaction with plasmid DNA showed a change of the DNA topology from supercoiled to circular form (nicking) in the presence of GSH, whereas no change was found in its absence. In vitro incubations of brostallicin were performed with the human recombinant GST isoenzymes A1-1, M1-1, and P1-1 (alpha, mu and pi isoenzymes, respectively) in the presence of GSH. The decrease in brostallicin levels was monitored in these incubations; the rate of loss (and therefore brostallicin metabolism) was significantly higher for the M1-1 and P1-1 isoenzymes than for the A1-1 isoenzyme.
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April 2002