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Danusertib is a pan-aurora kinase inhibitor with potent activity against Abl kinase including the gatekeeper T315I mutant. A phase 1 dose escalation study of danusertib was conducted in patients with accelerated or blastic phase chronic myeloid leukemia or Philadelphia chromosome-positive acute lymphoblastic leukemia. Two dosing schedules were studied: schedule A, in which danusertib was given by 3-hour intravenous infusion daily for 7 consecutive days (days 1-7) in a 14-day cycle, and schedule B, in which the danusertib was given by 3-hour intravenous infusion daily for 14 consecutive days (days 1-14) in a 21-day cycle. A total of 37 patients were treated, 29 with schedule A and eight with schedule B. The recommended phase 2 dose for schedule A was 180 mg/m(2). Enrollment to schedule B was stopped early because of logistical problems with the frequency of infusions. Febrile neutropenia and mucositis were dose-limiting toxicities in schedule A. Four patients with T315I ABL kinase mutation, all treated with schedule A, responded. Danusertib has an acceptable toxicity profile and is active in patients with Bcr-Abl-associated advanced hematologic malignancies. This study was registered with the European Clinical Trails Data Base (EudraCT number 2007-004070-18).

PNU 145156E (formerly FCE 26644) is a noncytotoxic molecule whose antitumor activity is exerted through the formation of a reversible complex with growth/angiogenic factors, thus inhibiting their induction of angiogenesis. We studied in vitro and in vivo the activity of PNU145156E in combination with the four cytotoxic drugs doxorubicin, cyclophosphamide, methoxymorpholinyldoxorubicin (MMDX, FCE 23762, PNU152243), and 9-aminocamptothecin against M5076 murine reticulosarcoma. In vitro, PNU 145156E did not modify the cytotoxicity of the four drugs or the cell-cycle block induced by doxorubicin. In vivo, at the optimal dose of each compound, the antitumor activity was significantly increased in all combinations, with no associated increase in general toxicity being observed. In healthy mice treated with cyclophosphamide or doxorubicin the association with PNU 145156E did not enhance the myelotoxic effect induced by the two cytotoxics. These results indicate that two drugs affecting solid tumor growth through two different mechanisms – growth factor blockage and cell proliferation – can be combined, resulting in increased antitumor efficacy with no additive toxicity.

The design, synthesis, and in vitro and in vivo activities of novel benzoyl and cinnamoyl nitrogen mustard and half-mustard derivatives of distamycin A, in which the amidino moiety has been replaced by moieties of different physico-chemical features, are described and structure-activity relationships are discussed. Some amidino-modified derivatives show significant cytotoxicity and antileukemic activity.

We evaluated the myelotoxicity and the anti-tumor potential of tallimustine, three of its analogues and carzelesin, with melphalan as reference substance. Tallimustine was tested by clonogenic assays on both human bone marrow (BM) and cord blood (hCB) cells, the other compounds on hCB only. The degree of inhibition of the haemopoietic progenitors GM-CFC, CFC-E and BFU-E was evaluated after exposure to different concentrations. The same schedules were tested on five tumour cell lines. We found that the dose-response curves for tallimustine on BM and hCB cells were similar. Carzelesin was shown to be the most potent of the substances tested and to be the one with the best in vitro therapeutic index; of the distamycin analogues, the one bearing an alpha-bromoacrylic group (FCE 25450) had the best index. For melphalan, tallimustine and carzelesin, the concentration inhibiting the growth of 70% of progenitor cells in vitro (ID70) was similar to the concentrations found in the serum of patients treated at the maximum tolerated dose (MTD). We conclude that hCB cells may be used instead of BM cells for in vitro myelotoxicity tests. Therapeutic indexes can be extrapolated from this model and could help in selecting the most promising analogue for further clinical development. The in vitro-active concentrations are similar to myelotoxic concentrations in patients, suggesting a predictive value for the assay.

A new family of camptothecin derivatives is described. Their synthesis, in vitro cytotoxicity, and topoisomerase I inhibition is reported.

A series of 6-(hydroxylamino)purine and -1-deazapurine nucleosides were synthesized and tested for their antitumor and adenosine deaminase inhibitory activity. All the examined molecules displayed an in vitro activity comparable to that of the reference compounds 6-(hydroxylamino)-9-beta-D-ribofuranosylpurine (HAPR) and ara-A, their ID50 ranging from 0.9 microM to approximately 100 microM. The 6-hydroxylamino derivatives of 1-deazapurine 9, 12, and 17 and also the blocked compound 13 are inhibitors of ADA whereas the purine derivatives 4 and 6 and the nitro compounds 11 and 16 are resistant to the enzyme. 7-(Hydroxylamino)-3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3H-imi dazo[4,5- b]pyridine, the less cytotoxic but the most active ADA inhibitor in the series (Ki = 2.7 x 10(-7)), greatly potentiates the antitumor activity of ara-A in vitro.

A new family of water soluble camptothecin derivatives is described. Their synthesis, in vitro cytotoxicity, and in vivo antitumor activity is reported. Compounds 5a and 5c displayed excellent in vivo antitumor activity both ip and iv.

A new family of camptothecin derivatives is described. Their synthesis, in vitro cytotoxicity, and topoisomerase I inhibition is reported.

The design, synthesis, and in vitro and in vivo activities of novel benzoyl and cinnamoyl nitrogen mustard and half-mustard derivatives of distamycin A, in which the amidino moiety has been replaced by moieties of different physico-chemical features, are described and structure-activity relationships are discussed. Some amidino-modified derivatives show significant cytotoxicity and antileukemic activity.

A series of 6-(hydroxylamino)purine and -1-deazapurine nucleosides were synthesized and tested for their antitumor and adenosine deaminase inhibitory activity. All the examined molecules displayed an in vitro activity comparable to that of the reference compounds 6-(hydroxylamino)-9-beta-D-ribofuranosylpurine (HAPR) and ara-A, their ID50 ranging from 0.9 microM to approximately 100 microM. The 6-hydroxylamino derivatives of 1-deazapurine 9, 12, and 17 and also the blocked compound 13 are inhibitors of ADA whereas the purine derivatives 4 and 6 and the nitro compounds 11 and 16 are resistant to the enzyme. 7-(Hydroxylamino)-3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3H-imi dazo[4,5- b]pyridine, the less cytotoxic but the most active ADA inhibitor in the series (Ki = 2.7 x 10(-7)), greatly potentiates the antitumor activity of ara-A in vitro.

The design, synthesis, and cytotoxic activity of novel benzoyl and cinnamoyl sulfur mustard derivatives of distamycin A are described and structure activity relationships are discussed. These sulfur mustards are more potent cytotoxics than corresponding nitrogen mustards in spite of the lower alkylating power, while their sulfoxide analogues are substantially inactive. Cinnamoyl sulfur mustard derivative (7) proved to be one of the most active distamycin-derived cytotoxics, about 1000 times more potent than melphalan.

The design, synthesis, in vitro and in vivo activities of a series of halogenoacrylic derivatives of distamycin A are described. The structure-activity relationships indicate a key role of the reactivity of alpha-halogenoacrylic moiety. The reactivity and the putative alkylating mechanism of these compounds are different from those of the nitrogen mustards and possibly based on a Michael type reaction. This supports the hypothesis that these compounds represent a class of minor groove binders mechanistically different from tallimustine.

The design, synthesis, and cytotoxic activity of novel benzoyl and cinnamoyl sulfur mustard derivatives of distamycin A are described and structure activity relationships are discussed. These sulfur mustards are more potent cytotoxics than corresponding nitrogen mustards in spite of the lower alkylating power, while their sulfoxide analogues are substantially inactive. Cinnamoyl sulfur mustard derivative (7) proved to be one of the most active distamycin-derived cytotoxics, about 1000 times more potent than melphalan.

The design, synthesis, in vitro and in vivo activities of a series of halogenoacrylic derivatives of distamycin A are described. The structure-activity relationships indicate a key role of the reactivity of alpha-halogenoacrylic moiety. The reactivity and the putative alkylating mechanism of these compounds are different from those of the nitrogen mustards and possibly based on a Michael type reaction. This supports the hypothesis that these compounds represent a class of minor groove binders mechanistically different from tallimustine.

Oligopeptide-like derivatives structurally related to the antitumor agent tallimustine, where one or two pyrrole rings were replaced by pyrazole or thiazole rings and bearing benzoyl nitrogen mustard or bromoacryloyl moieties were synthesized and evaluated in vitro and in vivo against L1210 murine leukemia. Compounds 9 and 12 showed antitumor activity higher than or comparable with that of tallimustine.

The relationship between different chemical modifications on morpholinylanthracyclines and their ability to overcome multidrug resistance (MDR) has been evaluated testing all compounds in vitro on LoVo and LoVo/DX human colon adenocarcinoma cells and in vivo disseminated P388 and P388/DX murine leukemias. Results obtained led us to the following conclusions: 1) the insertion of the morpholinyl or the methoxymorpholinyl group on position 3' of the sugar moiety confers the ability to overcome MDR in vitro and in vivo; conversely, 4' morpholinyl compounds are effective on MDR cells only in vitro and result inactive in vivo on DX-resistant leukemia; 2) all chemical modifications performed on 3' morpholinyl or methoxymorpholinyl derivatives, that is substitutions on the aglycone or on position 2 of the morpholino ring, do not interfere with the activity of the compounds: all derivatives present in fact the same efficacy on sensitive and resistant models. It is concluded that position 3' in the sugar moiety plays a crucial role in the ability of morpholinyl-anthracyclines to overcome MDR.

The glutathione (GSH)-glutathione S-transferase (GST) detoxification system is an important element in cellular defence against injurious agents and anticancer drugs. GST isoenzymes may represent biochemical markers of neoplastic transformation, and, possibly, drug resistance is associated with altered GST-isoenzyme levels. The ability to measure GST-isoenzymes in cell populations would be useful for several biological and clinical applications. We have developed an immunofluorescence flow cytometric method for the simultaneous detection of different GST-isoenzymes and of DNA in fixed cells. Due to the impossibility of working under saturating conditions for the anti-GST antibody, a normalizing procedure was developed to permit quantitative analysis of single cells labelled with the anti-GST antibody at high dilution. A theoretical model and experimental data supported the use of this procedure. The method proposed is general and could be applied to other antibodies in order to obtain quantitative data outside saturating conditions. The method was challenged in different applications in order to compare it with other classical techniques. First, we characterized sublines resistant to different anticancer drugs with respect to variations of GST isotypes. In a second application, we studied the intercellular heterogeneity of GST content in mouse renal cells. In addition, GST was determined in aneuploid cells from solid tumor biopsies by separation from diploid cells on the basis of DNA content. Finally, GST distribution during cell-cycle progression was studied in two different cell lines by the biparametric analysis of GST/DNA. © 1995 Wiley-Liss, Inc.

The clinical use of Ca++ antagonist agents as modulators of multidrug resistance is limited by their strong vasodilator activity. This study reports data obtained by testing a series of new 1,4 dihydropyridine derivatives (DHPs) for their in vitro resistance modulating activity and their Ca++ antagonist effect. All the tested DHPs are active to increase doxorubicin activity with dose modifying factor values ranging between 2 and 47 on P388/DX cells and 12 and 36 on LoVo/DX cells. Their resistance modulating action is exerted through an increase of DX intracellular level. The Ca++ antagonist activity of DHPs, evaluated as capacity to inhibit the KCl-induced contractions in isolated Guinea pig ileum strips, is not related to their resistance modulating activity. This finding makes it possible to select, for further in vivo evaluations, compounds IX, X and XI, which have strong ability to overcome multidrug resistance and low Ca++ antagonist effect.

The objective of the experiments reported in this paper was the identification of promising anthracycline analogs on the basis of lack of cross-resistance against tumor cells presenting either P-glycoprotein multidrug resistance (Pgp-MDR) or the altered topoisomerase multidrug resistant (at-MDR) phenotype. Differently modified anthracycline analogs known to be active against MDR cells were assayed in vitro against CEM human leukemic cells, and the sublines CEM/VLB100 and CEM/VM-1 exhibiting respectively the Pgp-MDR and the at-MDR phenotype. Two classes of molecules, in which the -NH2 group in C-3' position is substituted with a morpholino, methoxymorpholino (morpholinyl-anthracycline), or an alkylating moiety, present equivalent efficacy in the drug-sensitive and the two drug-resistant sublines. These results indicate that such molecules may exert their cytotoxic effect through a mode of action different from that of "classical" anthracyclines and is not mediated through topoisomerase II inhibition. Both molecules represent novel concepts in the field of new anthracyclines derivatives.

Abstract 864 Background: Despite the availability of novel ABL tyrosine kinase inhibitors ( TKI ) in addition to imatinib mesylate, the acquisition of the T315I BCR-Abl mutation remains a major cause of resistance to registered therapeutic compounds. Some patients (Pts) may also fail therapy with ≥ 2 TKI and need additional treatment. PHA-739358 is a small ATP competitive molecule that specifically inhibits Aurora A, B and C kinases. PHA-739358 possesses high affinity binding capacity to both wild-type Abl and Abl/T315I in in vitro assays. Methods: An international, multicenter, open-label, single agent, non-comparative, phase I study is being conducted in adult Pts with advanced Chronic Myeloid Leukemia (Accelerated AP-CML/Blastic phase BP-CML) and Philadelphia-Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ ALL) resistant or intolerant to imatinib and/or 2nd generation c-Abl therapy. Primary objective of the study is to determine the Maximum Tolerated Dose (MTD) and the Dose Limiting Toxicity (DLTs) during the first cycle. Two schedules were planned. Schedule A (PHA-739358 given as daily 3-hrs iv infusion for 7 consecutive days, every 2 wks) is open. Schedule B, not yet started, foresees a more aggressive approach and is currently being amended. Results: Twenty-three Pts with CML and Ph+ ALL have been treated so far (4 with AP-CML; 8 with BP-CML and 11 with Ph+ALL). Five dose levels have been tested: 90 (N=7); 120 (N=4); 150 (N=6); 180 (N=3) and 200 (N=3) mg/m2. Only one DLT occurred at 90 mg/m2 (NCI-CTC AE Gr3 fainting). Presently the MTD has not been defined. Fifteen out of 23 Pts have confirmation of BCR-Abl T315I mutation. A response occurred in 6/14 Pts, including 3 cytogenetic (1 complete, 1 partial, 1 minimal), 5 hematologic, and 1 clinical improvement (reduction in extramedullary disease mass). One severely pretreated (chemotherapy+Imatinib, SCT, Dasatinib and Donor Lymphocyte Infusion) pt with T315I mutated Ph+ALL reached Complete Hematological Response since Cycle (Cy) 4, Complete Cytogenetic Response since Cy 8 and Molecular Response (BCR/ABL undetectable transcripts) since Cy 9. Treatment continues after 10+ months. Last cycles were given every 4 wks due to mild/moderate transaminitis. This Pt at baseline showed: Peripheral Blood Blasts=0%; Bone Marrow (BM) blasts= 33%; % of Ph+ Metaphases = 80%. Two additional Pts (AP-CML and Ph+ ALL) achieved cytogenetic responses, one minimal and one partial reached at Cy2 and Cys 1-6, respectively. Another Pt (BP-CML) who received multiple transplants (3 times) and progressed after Imatinib, Dasatinib, Bosutinib, Nilotinib and Homoharringtonine, with 4 cycles of PHA-739358 had significant improvement of a large extramedullary lesion in the left neck impeding breathing and swallowing. Significant but transient reduction in the White Blood Cells (8/13 pts) and peripheral blood blast counts (3/13 Pts) were obtained with PHA-739358. An acceptable tolerability and safety profile characterized the study therapy: max non-hematological tox (regardless of causality) was as follows (most frequent Adverse events ≥ 30 %): diarrhoea 57% (CTC Gr 3 one Pt), pyrexia 50%; headache 43% (CTC Gr 3 one Pt); dyspnoea 36% (CTC Gr 3 one Pt ) and nausea 36%. Data analysis ongoing. Conclusions: The preliminary results obtained with our study show that PHA-739358 can elicit significant response with clinical benefit in severely pretreated populations of Pts affected with advanced leukemias resistant/intolerant to Imatinib and other 2nd generation TKI. Disclosures: Schafhausen: Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi-Aventis: Speakers Bureau; Novartis: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau. Latini:Nerviano Medical Sciences: Employment. Capolongo:Nerviano Medical Sciences: Employment. Laffranchi:Nerviano Medical Sciences: Employment. Comis:Nerviano Medical Sciences: Employment.

The effects of mitoxantrone (Mx) and doxorubicin (Dx) on cytotoxicity and DNA damage as assayed by alkaline elution were studied in two human colon adenocarcinoma cell lines sensitive (LoVo) and resistant (LoVo/Dx) to doxorubicin. Mx was more cytotoxic than Dx to LoVo cells and was partially cross-resistant in LoVo/Dx. In LoVo cells, Mx produced about 5 times more DNA single-strand breaks (DNA-SSB) than Dx, but both drugs caused an equal number of DNA double-strand breaks (DNA-DSB). In LoVo/Dx cells, the number of DNA-DSB was very low for both Dx and Mx, but DNA-SSB were about 20 times higher for Mx. In LoVo cells, the number of DNA-DSB and protein-associated SSB were similar at equitoxic concentrations. For LoVo/Dx, the partial cross-resistance of Mx might be explained by the much higher number of DNA-SSB produced by this drug.

Background. The T315I BCR-ABL mutation confers high level resistance to clinically available ABL kinase inhibitors (i.e., imatinib, nilotinib, dasatinib, bosutinib, INNO-406). PHA-739358 is an aurora kinase inhibitor that selectively inhibits the ATP site of Aurora-A, Aurora-B and Aurora-C kinases, and binds with high affinity to both wild type Abl and Abl/T315I in vitro. A multicenter phase II study of PHA-739358 is being conducted in patients with chronic myeloid leukemia (CML) relapsing on imatinib or other c-ABL therapy. Methods. Seven consenting CML patients (1 in chronic phase, 1 in accelerated phase, 5 in blast phase) were initially entered in this trial and received PHA-739358 dosed at 250 or 330 mg/m2/day administered by a once-weekly 6-hour infusion for 3 consecutive weeks, every 4 weeks. Six out of seven patients had the BCR-ABL T315I mutation. Pharmacokinetic and pharmacodynamic (PD) samples were collected and analyzed during cycle 1. The PD analyses evaluated BCR-ABL inhibition (phospho-CRKL) and Aurora kinase inhibition (phospho-histone H3). Results. Two patients with T315I mutated BCR-ABL achieved a complete hematologic response (CHR) to PHA-739358. One of these patients treated in accelerated phase achieved a complete cytogenetic response (CCyR) and a complete molecular response after 3 months on the 330 mg/m2 dose level. The CCyR is ongoing after 6 months of treatment. The second patient who achieved a CHR initiated therapy in chronic phase. A minor cytogenetic response was achieved after 3 cycles of therapy at the 330 mg/m2, and a minimal cytogenetic response persists after nine months of treatment. Uncomplicated grade 4 neutropenia and an infusion-related reaction requiring acetaminophen, benadryl and hydrocortisone premedication was observed in one patient. No other grade 3/4 non-hematologic toxicity was observed. Patients with blast crisis did not have CHRs at the dose and schedule employed, although suppression of peripheral blood blasts was evident. PHA-739358 pharmacokinetics were in good agreement with those observed in other phase I - II studies. Average exposure, AUC0-t(168h), observed at 330 mg/m2/day was about 45 uM/h with Cmax values around 4 – 6 uM/h. Pharmacodynamic analyses demonstrated treatment-associated decreases of CRKL phosphorylation in 6 out of 7 patients, including both responders. Modulation of histone H3 phosphorylation was observed in 3 of 5 evaluable patients. Conclusions. Objective clinical responses to PHA-739358 have been observed in two CML patients with T315I mutations of BCR-ABL, with an acceptable tolerability and safety profile. Additional doses and schedules of PHA-739358 are being investigated in patients with advanced phase CML.

14116 Background: Nemorubicin hydrochloride (nemorubicin) is a non-conventional anthracycline in Phase II evaluation in hepatocellular carcinoma (HCC). Its mechanism of action is not fully elucidated. Although structurally related to doxorubicin, nemorubicin is a topoisomerase I inhibitor, overcomes anthracyclines resistance, is minimally cardiotoxic and is biotransformed by hepatic CYP3A4 into hundred times more cytotoxic metabolite. Phase I and II trials were conducted in Europe and China to test nemorubicin by hepatic intra-arterial (IHA) infusion in HCC patients (pts). The drug was well tolerated up to 600 mcg/m2 q4–6w; DLT was transient liver transaminase elevations. Overall, 57 HCC pts were evaluable for efficacy, with 11/57 confirmed liver CR/PRs (RR = 19.3%; 95% ci 10–31.9%) lasting 1–54+ months. Stable disease ≥ 3 months was observed in 17/57 (29.8%) pts, most with AJCC Stage III, IIIA and IVA. These data supported new trials of nemorubicin in HCC. Methods: To further characterize the mechanism of action of the drug, we generated cells (L1210) resistant to nemorubicin. Since resistant cells were more sensitive than the parental ones to UV irradiation, we reasoned that the nucleotide excision repair (NER) system might be involved in mediating the activity of nemorubicin. To test this hypothesis we used isogenic CHO cells proficient or deficient in excision repair cross-complementing (ERCC) genes, namely ERCC1 and ERCC6 genes. Results: In contrast with what is observed for most DNA damaging drugs that show resistance in the presence of high NER activity, nemorubicin is more cytotoxic in NER proficient than in deficient cells. This suggests that NER pathway plays a role in the cytotoxic effect of nemorubicin. Also, cells resistant to nemorubicin are NER-deficient and are highly sensitive to platinum derivatives and alkylating agents and synergism was found combining cisplatin with nemorubicin. Conclusions: Nemorubicin has a peculiar mechanism of action through the NER system providing the rationale for clinical combination studies with platinum derivatives. A Phase I/II trial of nemorubicin with cisplatin in HCC patients started in Italy at the end of 2005. No significant financial relationships to disclose.

The antiproliferative activity of flavone acetic acid (LM 975) was investigated on human adenocarcinoma cell lines (HCC-P2998, HCC-M1544, HCC-M1410, HT 29, LoVo), on a murine colon adenocarcinoma cell line (Colon 26), on murine pancreatic adenocarcinoma cells growing in primary culture (Pan 03) and on human normal fibroblasts (N1). No cytotoxic effects were found against human normal fibroblasts. LM 975 was active against murine adenocarcinoma Pan 03 and Colon 26, known to be sensitive in vivo too and, to variable extents, on human adenocarcinoma cell lines. LM 975 in vitro cytotoxic potency was relatively low. The high concentrations (1.0–1.4 mM ) required to obtain a cytotoxic effect are, however, pharmacologically reasonable since they are comparable with drug plasma levels in mice or in patients treated with tolerable doses. After a relatively short LM 975 treatment (2 h ) DNA, RNA and protein synthesis were inhibited in different proportions. In more sensitive cells LM 975 appeared to inhibit RNA synthesis more than DNA and protein synthesis. Inhibition of macromolecule synthesis after 2 h exposure was completely reversed in 24 h recovery. After 2 h treatment no detectable DNA breakage was found by the alkaline elution method, thus corroborating the idea that this compound does not act by causing DNA damage.

In this communication we suggest two simple ways to represent the information on the cell cycle obtained by flow cytometry, offering some advantages over the traditional plots. We show that the results of a single experiment, when reduced to the percentages of cells in G1, S, and G2M phases, can be completely expressed with a single point in a G1-G2 plan where iso-S lines are also drawn. By the use of this plot, the time course of the complete kinetics of a cell population may be shown in a single drawing. The second plot, we suggest, integrates information on cell cycle, represented by the distribution mean, with cell number.

The design, synthesis, in vitro and in vivo activities of novel benzoyl and cinnamoyl nitrogen mustard and half-mustard derivatives of distamycin A are described and structure-activity relationships are discussed. The equipotent activities of N-ethyl-N-chloroethyl half-mustards and N,N-dichloroethyl mustards and the superior activities of cinnamoyl derivatives are the most relevant features of the series.

A series of 6-hdroxylamino urine and deazapurine nucleosides were synthesized and tested for their antitumor and adenosine deaminase inhibitory activity. All the examined molecules displayed an in vitro activity comparable to that of the reference compounds HAPR and ara-A, their ID50 ranging from 0.9μM (9) to ∼100μM (5).