Nemorubicin

Nemorubicin is an anticancer drug with IC50 value of 0.08μM [1].

Nemorubicin is a morpholinyl analog of doxorubicin and is developed to be an anticancer drug. It is less cardiotoxic and more cytotoxic against multidrug-resistant tumor cells. The SRB in vitro assay shows nemorubicin can inhibit the proliferation of HCC cell lines with average IC50 value of 0.08μM. It is much more potent(from 10 to 380 times) than other anticancer drugs. The treatments of nemorubicin at doses of both 25μg/kg and 50μg/kg cause growth inhibition of human HCC xenografts BEL-7402 and Zip-177 in nude mice. When the dose is up to 100μg/kg, the high toxicity causes the death of mice. In phase I study, nemorubicin shows a terminal half-life in eight patients of 11.1±10.8 h. And the hematotoxicity tests show a <70% colony growth inhibition without correlation between the growth inhibition effect and the degree of myelotoxicity in the same patient. Besides that, nausea and vomiting is a limit of the application of nemorubicin [1, 2].

References:
[1] Shengtao Yuan, Xiongwen Zhang, Lijuan Lu, Chenghui Xu, Weiyi Yang and Jian Ding. Anticancer activity of methoxymorpholinyl doxorubicin (PNU 152243) on human hepatocellular carcinoma. Anti-Cancer Drugs. 2004, 15(6): 641-646.
[2] Cristiana Sessa, Massimo Zucchetti Michele Ghielmini, Jean Bauer, Maurizio D'Incalci Jolanda de Jong, Huguette Naegele, Simona Rossi Maria Adele Pacciarini, Letizia Domenigoni Franco Cavalli. Cancer Chemother Pharmacol. 1999, 44:403-410.

The interaction of nemorubicin metabolite PNU-159682 with DNA fragments d(CGTACG)(2), d(CGATCG)(2) and d(CGCGCG)(2) shows a strong but reversible binding to G:C base pairs.[Pubmed:23154079]

Bioorg Med Chem. 2012 Dec 15;20(24):6979-88.

The antitumor anthracycline Nemorubicin is converted by human liver microsomes to a major metabolite, PNU-159682 (PNU), which was found to be much more potent than its parent drug toward cultured tumor cells and in vivo tumor models. The mechanism of action of Nemorubicin appears different from other anthracyclines and until now is the object of studies. In fact PNU is deemed to play a dominant, but still unclear, role in the in vivo antitumor activity of Nemorubicin. The interaction of PNU with the oligonucleotides d(CGTACG)(2), d(CGATCG)(2) and d(CGCGCG)(2) was studied with a combined use of (1)H and (31)P NMR spectroscopy and by ESI-mass experiments. The NMR studies allowed to establish that the intercalation between the base pairs of the duplex leads to very stable complexes and at the same time to exclude the formation of covalent bonds. Melting experiments monitored by NMR, allowed to observe with high accuracy the behaviour of the imine protons with temperature, and the results showed that the re-annealing occurs after melting. The formation of reversible complexes was confirmed by HPLC-tandem mass spectra, also combined with endonuclease P1digestion. The MS/MS spectra showed the loss of neutral PNU before breaking the double helix, a behaviour typical of intercalators. After digestion with the enzyme, the spectra did not show any compound with PNU bound to the bases. The evidence of a reversible process appears from both proton and phosphorus NOESY spectra of PNU bound to d(CGTACG)(2) and to d(CGATCG)(2). The dissociation rate constants (k(off)) of the slow step of the intercalation process, measured by (31)P NMR NOE-exchange experiments, showed that the kinetics of the process is slower for PNU than for doxorubicin and Nemorubicin, leading to a 10- to 20-fold increase of the residence time of PNU into the intercalation sites, with respect to doxorubicin. A relevant number of NOE interactions allowed to derive a model of the complexes in solution from restrained MD calculations. The conformation of PNU bound to the oligonucleotides was also derived from the coupling constant values.

Virtual Cross-Linking of the Active Nemorubicin Metabolite PNU-159682 to Double-Stranded DNA.[Pubmed:28068470]

Chem Res Toxicol. 2017 Feb 20;30(2):614-624.

The DNA alkylating mechanism of PNU-159682 (PNU), a highly potent metabolite of the anthracycline Nemorubicin, was investigated by gel-electrophoretic, HPLC-UV, and micro-HPLC/mass spectrometry (MS) measurements. PNU quickly reacted with double-stranded oligonucleotides, but not with single-stranded sequences, to form covalent adducts which were detectable by denaturing polyacrylamide gel electrophoresis (DPAGE). Ion-pair reverse-phase HPLC-UV analysis on CG rich duplex sequences having a 5'-CCCGGG-3' central core showed the formation of two types of adducts with PNU, which were stable and could be characterized by micro-HPLC/MS. The first type contained one alkylated species (and possibly one reversibly bound species), and the second contained two alkylated species per duplex DNA. The covalent adducts were found to produce effective bridging of DNA complementary strands through the formation of virtual cross-links reminiscent of those produced by classical anthracyclines in the presence of formaldehyde. Furthermore, the absence of reactivity of PNU with CG-rich sequence containing a TA core (CGTACG), and the minor reactivity between PNU and CGC sequences (TACGCG.CGCGTA) pointed out the importance of guanine sequence context in modulating DNA alkylation.

Nemorubicin and doxorubicin bind the G-quadruplex sequences of the human telomeres and of the c-MYC promoter element Pu22.[Pubmed:26922833]

Biochim Biophys Acta. 2016 Jun;1860(6):1129-38.

BACKGROUND: Intra-molecular G-quadruplex structures are present in the guanine rich regions of human telomeres and were found to be prevalent in gene promoters. More recently, the targeting of c-MYC transcriptional control has been suggested, because the over expression of the c-MYC oncogene is one of the most common aberration found in a wide range of human tumors. METHODS: The interaction of Nemorubicin and doxorubicin with DNA G-quadruplex structures has been studied by NMR, ESI-MS and molecular modelling, in order to obtain further information about the complex and the multiple mechanisms of action of these drugs. RESULTS AND CONCLUSIONS: Nemorubicin intercalates between A3 and G4 of d(TTAGGGT)4 and form cap-complex at the G6pT7 site. The presence of the adenine in this sequence is important for the stabilization of the complex, as was shown by the interaction with d(TTGGGTT)4 and d(TTTGGGT)4, which form only a 1:1 complex. The interaction of doxorubicin with d(TTAGGGT)4 is similar, but the complex appears less stable. Nemorubicin also binds with high efficiency the c-MYC G-quadruplex sequence Pu22, to form a very well defined complex. Two Nemorubicin molecules bind to the 3'-end and to the 5'-end, forming an additional plane of stacking over each external G-tetrad. The wild type c-MYCPu22 sequence forms with Nemorubicin the same complex. GENERAL SIGNIFICANCE: Nemorubicin and doxorubicin, not only intercalate into the duplex DNA, but also result in significant ligands for G-quadruplex DNA segments, stabilizing their structure; this may in part explain the multiple mechanisms of action of their antitumor activity.

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.[Pubmed:18671948]

Biochem Pharmacol. 2008 Sep 15;76(6):784-95.

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.