Anna Kruczynski

Vinflunine, the latest Vinca alkaloid in clinical development: A review of its preclinical anticancer properties

Vinflunine is a new Vinca alkaloid uniquely fluorinated, by the use of superacid chemistry, in a little exploited region of the catharanthine moiety. In vitro investigations have confirmed the mitotic-arresting and tubulin-interacting properties of vinflunine shared by other Vinca alkaloids. However, differences in terms of the inhibitory effects of vinflunine on microtubules dynamics and its tubulin binding affinities have been identified which appear to distinguish it from the other Vinca alkaloids. Vinflunine induced smaller spirals with a shorter relaxation time, effects, which might be associated with reduced neurotoxicity. Studies investigating the in vitro cytotoxicity of vinflunine in combination therapy have revealed a high level of synergy when vinflunine was combined with either cisplatin, mitomycin C, doxorubicin or 5-fluorouracil. Furthermore, although vinflunine appears to participate in P-glycoprotein-mediated drug resistance mechanisms, it has proved only a weak substrate for this protein and a far less potent inducer of resistance than vinorelbine. Vinflunine was identified in preclinical studies as having marked antitumour activity in vivo against a large panel of experimental tumour models, with tumour regressions being recorded in human renal and small cell lung cancer tumour xenografts. Overall its level of activity was superior to that of vinorelbine in many of the experimental models used. Interestingly, an in vivo study using a well vascularised adenocarcinoma of the colon has suggested that vinflunine mediates its antitumour activity at least in part via an antivascular mechanism, even at sub-cytotoxic doses. Therefore, these data provide a favourable preclinical profile for vinflunine, supporting its promising candidacy for clinical development. Phase I evaluations of vinflunine have been completed in Europe and phase II clinical trials are now ongoing.

Antimitotic and tubulin-interacting properties of vinflunine, a novel fluorinated Vinca alkaloid

This study aimed to define the mechanism of action of vinflunine, a novel Vinca alkaloid synthesised from vinorelbine using superacidic chemistry and characterised by superior in vivo activity to vinorelbine in preclinical tumour models. In vitro vinflunine cytotoxicity proved dependent on concentration and exposure duration, with IC50 values (72-hr exposures) generally ranging from 60-300 nM. Vinflunine induced G2 + M arrest, associated with mitotic accumulation and a concentration-dependent reduction of the microtubular network of interphase cells, accompanied by paracrystal formation. These effects, while comparable to those of vincristine, vinblastine or vinorelbine, were achieved with 3- to 17-fold higher vinflunine concentrations. However, vinflunine and the other Vincas all inhibited microtubule assembly at micromolar concentrations. Vinflunine, like vinblastine, vincristine and vinorelbine, appeared to interact at the Vinca binding domain, as judged by proteolytic cleavage patterns, and induced tubulin structural changes favouring an inhibition of GTP hydrolysis. However, vinflunine did not prevent [3H]vincristine binding to unassembled tubulin at concentrations < or = 100 microM, and only weakly inhibited binding of [3H]vinblastine or [3H]vinorelbine. Indeed, specific binding of [3H]vinflunine to tubulin was undetectable by centrifugal gel filtration. Thus, the comparative capacities of these Vincas to bind to or to interfere with their binding to tubulin could be classified as: vincristine > vinblastine > vinorelbine > vinflunine. By monitoring alkylation of sulfhydryl groups, differential effects on tubulin conformation were identified with vinflunine and vinorelbine acting similarly, yet distinctively from vinblastine and vincristine. Overall, vinflunine appears to function as a definite inhibitor of tubulin assembly, while exhibiting quantitatively different tubulin binding properties to the classic Vinca alkaloids.

Novel aspects of natural and modified vinca alkaloids.

The clinical interest of Vinca alkaloids was clearly identified as early as 1965 and so this class of compounds has been used as anticancer agents for more than 30 years. Today, two natural compounds, vinblastine and vincristine and two semi-synthetic derivatives, vindesine and vinorelbine, have been registered and thus Vinca alkaloids can be considered to represent a chemical class of definite utility in cancer chemotherapy. Today, relatively few groups actively research in the chemistry of Vinca alkaloids. However, using superacidic chemistry, a new family of such compounds was synthesised and vinflunine, a difluorinated derivative, was selected for clinical testing. A consideration of the pharmacological data relating to these new derivatives appears to reveal a lack of any marked correlation between in vitro and in vivo results. Furthermore, structure/activity relationships have failed to assist the chemist in the rational design. Such rational design of new derivatives is limited by the fact that the Vinca binding site(s) on tubulin and the exact mechanism(s) of action of Vinca alkaloids remain unclear. Nevertheless, the preclinical evaluations of the new derivative vinflunine have already suggested that certain in vitro assays, in addition to in vivo experiments, could be proposed to select more rationally newer generation Vincas. Moreover, recent studies have demonstrated that certain newly identified properties, such as antiangiogenic activities, could enlarge the therapeutic usage of natural and semi-synthetic Vinca alkaloids. Thus, Vinca alkaloids remain a drug family with a continuing interest for future anticancer therapy.

Polo-like kinase 1 is overexpressed in acute myeloid leukemia and its inhibition preferentially targets the proliferation of leukemic cells

Polo-like kinase 1 (Plk1) is a major mitotic regulator overexpressed in many solid tumors. Its role in hematopoietic malignancies is still poorly characterized. In this study, we demonstrate that Plk1 is highly expressed in leukemic cell lines, and overexpressed in a majority of samples from patients with acute myeloid leukemia compared with normal progenitors. A pharmacologic inhibitor, BI2536, blocks proliferation in established cell lines, and dramatically inhibits the clonogenic potential of leukemic cells from patients. Plk1 knockdown by small interfering RNA also blocked proliferation of leukemic cell lines and the clonogenic potential of primary cells from patients. Interestingly, normal primary hematopoietic progenitors are less sensitive to Plk1 inhibition than leukemic cells, whose proliferation is dramatically decreased by the inhibitor. These results highlight Plk1 as a potentially interesting therapeutic target for the treatment of acute myeloid leukemia.

F14512, a Potent Antitumor Agent Targeting Topoisomerase II Vectored into Cancer Cells via the Polyamine Transport System

The polyamine transport system (PTS) is an energy-dependent machinery frequently overactivated in cancer cells with a high demand for polyamines. We have exploited the PTS to selectively deliver a polyamine-containing drug to cancer cells. F14512 combines an epipodophyllotoxin core-targeting topoisomerase II with a spermine moiety introduced as a cell delivery vector. The polyamine tail supports three complementary functions: (a) facilitate formulation of a water-soluble compound, (b) increase DNA binding to reinforce topoisomerase II inhibition, and (c) facilitate selective uptake by tumor cells via the PTS. F14512 is 73-fold more cytotoxic to Chinese hamster ovary cells compared with CHO-MG cells with a reduced PTS activity. A decreased sensitivity of L1210 leukemia cells to F14512 was observed in the presence of putrescine, spermidine, and spermine. In parallel, the spermine moiety considerably enhances the drug-DNA interaction, leading to a reinforced inhibition of topoisomerase II. The spermine tail of F14512 serves as a cell delivery vehicle as well as a DNA anchor, and this property translates at the cellular level into a distinct pharmacologic profile. Twenty-nine human solid or hematologic cell lines were used to characterize the high cytotoxic potential of F14512 (median IC50 of 0.18 micromol/L). Finally, the potent antitumor activity of F14512 in vivo was evidenced with a MX1 human breast tumor xenograft model, with partial and complete tumor regressions. This work supports the clinical development of F14512 as a novel targeted cytotoxic drug and sheds light on the concept of selective delivery of drugs to tumor cells expressing the PTS.

MiR-29a down-regulation in ALK-positive anaplastic large cell lymphomas contributes to apoptosis blockade through MCL-1 overexpression

Although deregulated expression of specific microRNAs (miRNAs) has been described in solid cancers and leukemias, little evidence of miRNA deregulation has been reported in ALK-positive (ALK(+)) anaplastic large cell lymphomas (ALCL). These tumors overexpress the major antiapoptotic protein myeloid cell leukemia 1 (MCL-1), a situation that could compensate for the lack of BCL-2. We report that ALK(+) ALCL cell lines and biopsy specimens (n = 20) express a low level of miR-29a and that this down-modulation requires an active NPM-ALK kinase. Murine models (transgenic mice and mouse embryonic fibroblast [MEF] cells), which allow conditional NPM-ALK fusion protein expression, showed an increase of miR-29a expression in the absence of NPM-ALK. Concordant results were observed after the abolition of NPM-ALK kinase activity (siALK or PF-2341066) in NPM-ALK(+) ALCL cell lines. In addition, we showed that low expression of miR-29a, probably through methylation repression, plays an important regulatory role in MCL-1 overexpression that could promote tumor cell survival by inhibiting apoptosis. Enforced miR-29a expression was found to modulate apoptosis through inhibition of MCL-1 expression in ALCL cell lines and in a xenografted model, with a concomitant tumor growth reduction. Thus, synthetic miR-29a represents a potential new tool to affect tumorigenesis in these lymphomas.

Vinflunine (20′,20′-difluoro- 3′,4′-dihydrovinorelbine), a novel Vinca alkaloid, which participates in P-glycoprotein (Pgp)-mediated multidrug resistance in vivo and in vitro

Vinflunine (VFL) is a novel derivative of vinorelbine (NVB, Navelbine®), which has shown markedly superior antitumor activity to NVB, in various experimental animal models. To establish whether this new Vinca alkaloid participates in P-glycoprotein (Pgp)-mediated multidrug resistance (MDR), VFL-resistant murine P388 cells (P388/VFL) were established in vivo and used in conjunction with the well established MDR P388/ADR subline, to define the in vivo resistance profile for VFL. P388/VFL cells proved cross-resistant to drugs implicated in MDR (other Vinca alkaloids, doxorubicin, etoposide), but not to campothecin or cisplatin and showed an increased expression of Pgp, without any detectable alterations in topoisomerase II or in glutathione metabolism. The P388/ADR cells proved cross-resistant to VFL both in vivo and in vitro, and this VFL resistance was efficiently modulated by verapamil in vitro. Cellular transport experiments with tritiated-VFL revealed differential uptake by P388 sensitive and P388/ADR resistant cells, comparable with data obtained using tritiated-NVB. In various in vitro models of human MDR tumor cells, whilst full sensitivity was retained in cells expressing alternative non-Pgp-mediated MDR mechanisms, cross resistance was identified in Pgp-overexpressing cells. Differences were, however, noted in terms of the drug resistance profiles relative to the other Vincas, with tumor cell lines proving generally least cross-resistant to VFL. Overall, these results suggest that VFL, like other Vinca alkaloids, participates in Pgp-mediated MDR, with tumor cells selected for resistance to VFL overexpressing Pgp, yet MDR tumor cell lines proved generally less cross resistant to VFL relative to the other Vinca alkaloids.

Preclinical activity of F14512, designed to target tumors expressing an active polyamine transport system

SummaryWe have exploited the polyamine transport system (PTS) to deliver selectively a spermine-drug conjugate, F14512 to cancer cells. This study was aimed to define F14512 anticancer efficacy against tumor models and to investigate whether fluorophor-labeled polyamine probes could be used to identify tumors expressing a highly active PTS and that might be sensitive to F14512 treatments. Eighteen tumor models were used to assess F14512 antitumor activity. Cellular uptake of spermine-based fluorescent probes was measured by flow cytometry in cells sampled from tumor xenografts by needle biopsy. The accumulation of the fluorescent probe within B16 tumors in vivo was assessed using infrared fluorescence imaging. This study has provided evidence of a major antitumor activity for F14512. Significant responses were obtained in 67% of the tumor models evaluated, with a high level of activity recorded in 33% of the responsive models. Complete tumor regressions were observed after i.v., i.p. or oral administrations of F14512 and its antitumor activity was demonstrated over a range of 2–5 dose levels, providing evidence of its good tolerance. The level of cellular fluorescence emitted by the fluorescent probes was higher in cells sampled from tumors sensitive to F14512 treatments than from F14512-refractory tumors. We suggest that these probes could be used to identify tumors expressing a highly active PTS and guide the selection of patients that might be treated with F14512. These results emphasize the preclinical interest of this novel molecule and support its further clinical development.

Inhibition of RAS-targeted prenylation: protein farnesyl transferase inhibitors revisited

The ras oncogene and its 21 kD protein product, Ras, has emerged during the last decade as a potentially exploitable target for anticancer drug development. The knowledge that Ras was readily prenylated by protein farnesyl transferase (PFTase) and that inhibition of this prenylation had functional consequences for the transformed phenotype that expressed oncogenic Ras provided the rational for the development of PFTase inhibitors. The initial enthusiasm for this approach seemed justified by the early identification of PFTase inhibitors that were able potently and specifically to block Ras processing, signalling and transformation in transformed and tumour cell lines in vitro and in certain selected animal models. More recently the recognition that geranylgeranyl transferase (GGTase) I might also be a therapeutic target is being actively researched. The last couple of years though have proved remarkable with the disclosure of a series of structurally-diverse molecules, whose major in vivo preclinical activites have been well documented against experimental animal tumours, and culminating this year in preliminary reporting of their Phase I clinical evaluations. Nevertheless, during the research and development phases of PFTase inhibitors as pharmaceutical agents for clinical use, there have been several unexpected findings which have raised intriguing and potentially crucial questions about their activities. This review aims to highlight and offer new insights into many of these issues and to bring into perspective concerns arising from basic research, as well as from clinical studies. There seems little doubt that these inhibitors of RAS-targeted prenylation represent a new generation of anticancer drugs for the preclinical researcher, whether they can be successfully exploited in clinical practice should be resolved early in the next millenium.

Physalin B, a novel inhibitor of the ubiquitin-proteasome pathway, triggers NOXA-associated apoptosis

The ubiquitin-proteasome pathway plays a critical role in the degradation of proteins involved in tumor growth and has therefore become a target for cancer therapy. In order to discover novel inhibitors of this pathway, a cellular assay reporter of proteasome activity was established. Human DLD-1 colon cancer cells were engineered to express a 4 ubiquitin-luciferase (DLD-1 4Ub-Luc) reporter protein, rapidly degraded via the ubiquitin-proteasome pathway and designed DLD-1 4Ub-Luc cells. Following treatment with reference proteasome inhibitors, the 4Ub-Luc protein accumulated in DLD-1 4Ub-Luc cells and a 80-fold increase in luciferase-produced bioluminescence signal was measured, as compared to untreated cells. The screening of over 30,000 compounds using this DLD-1 4Ub-Luc assay led to the identification of physalin B as a novel inhibitor of the ubiquitin-proteasome pathway. Indeed, physalin B induced an increase in bioluminescence from DLD-1 4Ub-Luc cells, at concentrations also producing an accumulation of ubiquitinated proteins and inhibiting TNFalpha-induced NF-kappaB activation. Physalin B did not inhibit catalytic activities of purified proteasome and interfered with cellular proteasomal catalytic activities at 4- to 8-fold higher concentrations than that required to induce significant increase in bioluminescence and accumulation of ubiquitinated proteins in DLD-1 4Ub-Luc cells. Furthermore, physalin B proved to be cytotoxic, triggered apoptosis in DLD-1 4Ub-Luc cells and induced the proapoptotic protein NOXA, characteristic of the proteasome signaling pathway. Therefore, the use of the DLD-1 4Ub-Luc assay allowed the identification of a novel inhibitor of the ubiquitin-proteasome pathway that might interfere with proteasome functions in a different way from reference proteasome inhibitors.