Mustard Group Research Articles

Aneugenic potential of the anticancer drugs melphalan and chlorambucil. The involvement of apoptosis and chromosome segregation regulating proteins

Previous findings showed that the anticancer drugs p-N,N-bis(2-chloroethyl) amino-l-phenylalanine (melphalan, MEL) and p-N,N-bis(2-chloroethyl)aminophenylbutyric acid (chlorambucil, CAB) belonging to the nitrogen mustard group, in addition to their clastogenic activity, also exert aneugenic potential, nondisjunction and chromosome delay. Their aneugenic potential is mainly mediated through centrosome defects. To further investigate their aneugenicity we (a) studied whether apoptosis is a mechanism responsible for the elimination of damaged cells generated by MEL and CAB and (b) investigated if proteins that regulate chromosome segregation are involved in the modulation of their aneugenic potential. Apoptosis was studied by Annexin-V/Propidium Iodide staining and fluorescence microscopy. The involvement of apoptosis on the exclusion of cells with genetic damage and centrosome disturbances was analyzed by DAPI staining and immunofluorescence of β- and γ-tubulin in the presence of pan-caspase inhibitor. The expressions of Aurora-A, Aurora-B, survivin and γ-tubulin were studied by western blot. We found that (a) apoptosis is not the mechanism of choice for selectively eliminating cells with supernumerary centrosomes, and (b) the proteins Aurora-A, Aurora-B and survivin are involved in the modulation of MEL and CAB aneugenicity. These findings are important for the understanding of the mechanism responsible for the aneugenic activity of the anticancer drugs melphalan and chlorambucil.

Interaction between staining and degradation of a composite resin in contact with colored foods

Composite resins might be susceptible to degradation and staining when in contact with some foods and drinks. This study evaluated color alteration and changes in microhardness of a microhybrid composite after immersion in different colored foods and determined whether there was a correlation between these two variables. Eighty composite disks were randomly divided into 8 experimental groups (n = 10): kept dry; deionized water; orange juice; passion fruit juice; grape juice; ketchup; mustard and soy sauce. The disks were individually immersed in their respective test substance at 37 ºC, for a period of 28 days. Superficial analysis of the disk specimens was performed by taking microhardness measurements (Vickers, 50 g load for 45 seconds) and color alterations were determined with a spectrophotometer (CINTRA 10- using a CIEL*a*b* system, 400-700 nm wavelength, illuminant d65 and standard observer of 2º) at the following times: baseline (before immersion), 1, 7, 14, 21 and 28 days. Results were analyzed by ANOVA and Tukey's test (p < 0.05). Both variables were also submitted to Pearson's correlation test (p < 0.05). The passion fruit group underwent the greatest microhardness change, while the mustard group suffered the greatest color alteration. Significant positive correlation was found between the two variables for the groups deionized water, grape juice, soy sauce and ketchup. Not all color alteration could be associated with surface degradation.

A phase II study of second-line bendamustine in relapsed or refractory small cell lung cancer (SCLC).

e17505 Background: Bendamustine is an alkylating agent with a nitrogen mustard group and purine-like benzimidazole group. Bendamustine in combination with carboplatin has shown efficacy as first line therapy in extensive stage SCLC with RR 73%, TTP 5.2 months and OS 8.3 months [Köster W et al. J Tho Onc. 2007;2(4):312-6]. This study aims to examine the safety and efficacy of single agent bendamustine as 2nd or 3rd line therapy in patients (pts) with SCLC. Methods: This is an open-label, multicenter phase II trial. Eligible pts had previously treated SCLC, up to 2 prior regimens, PS 0-2, evaluable/measurable disease, adequate marrow, renal and hepatic function. Pts with stable treated brain metastases were allowed. Pts were treated with bendamustine (120mg/m2 IV days 1 and 2 every 3 weeks) up to 6 cycles and evaluated every 2 cycles. Primary endpoint was TTP; secondary endpoints include RR, PFS, OS, and toxicity. Results: 28 pts have enrolled to date (target accrual 60); 19 pts were evaluable for response; there were 7 PR, 7 SD and 5 PD. Median TTP was 2.5 months. At the time of analysis, 12 pts were alive. Median number of cycles given was 2. 5 pts required dose reductions due to AEs. Grade 3/4 AEs included neutropenia 14.3%, fatigue 10.7%, anemia and diarrhea 7.1% and less commonly, thrombocytopenia, nausea, infection, hyponatremia, pulmonary at 3.6%. Conclusions: These preliminary data indicate that single agent bendamustine appears to be well tolerated and effective in the second or third line setting for patients with SCLC. Accrual is ongoing.

Mechanism of Action: The Unique Pattern of Bendamustine-Induced Cytotoxicity

Bendamustine has demonstrated substantial efficacy in the treatment of hematologic malignancies and continues to distinguish itself from other alkylating agents with regard to its activity in tumor cells. The mechanistic and clinical differences associated with bendamustine may be directly related to its unique structural features. Although the precise mechanisms of action are still poorly understood, bendamustine is associated with extensive and durable DNA damage. The increased potency of bendamustine may be due to secondary mechanisms such as inhibition of mitotic checkpoints, inefficient DNA repair, and initiation of p53-dependent DNA-damage stress response, all of which lead to mitotic catastrophe and apoptosis. It has also been hypothesized that the presence of a benzimidazole ring in addition to the nitrogen mustard group may influence the way bendamustine interacts with DNA and/or confer antimetabolite properties. Further elucidation of the mechanisms of action for bendamustine and the signaling pathways involved in the response to bendamustine-induced DNA damage is essential to maximize its therapeutic potential, identify biomarkers for response, and understand the potential for synergy with other agents involved in DNA damage and inhibition of DNA repair. This review will discuss the current understanding and hypotheses regarding bendamustine mechanisms of action and suggest future investigations that would shed light on the many unanswered questions.

Bendamustine in the Management of Non-Hodgkins Lymphoma

Bendamustine is a unique bifunctional cytotoxic agent that includes a nitrogen mustard group with alkylating properties and a benzimidazole ring with potential antimetabolite properties. It shows only partial cross-resistance with other alkylators in vitro and remains highly active in heavily pretreated patients. The history of bendamustine began in the former East German Democratic Republic, where it was developed in the early 1960s. Re-discovered in the 1990s, it demonstrated excellent response rates in several clinical trials with a favorable side-effect profile leading to its approval in 2008 by the U.S. Food and Drug Administration for the treatment of patients with chronic lymphocytic leukemia and for the treatment of patients with rituximab-refractory, indolent B-cell non-Hodgkin lymphoma. Further studies showed efficacy in the first-line treatment of B-cell NHL and in the treatment of patients with multiple myeloma. We review the data on bendamustine published so far and discuss the future role of this promising agent in the treatment of patients with lymphoproliferative malignancies.

Abstract 3664: Bendamustine induces a dose-dependent cell cycle arrest in Hela cells that results in different mitotic outcomes

Abstract Bendamustine (BDM) was originally synthesized in the 1960s but has only recently been approved by the FDA for the treatment of chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. BDM consists of a bifunctional alkylating nitrogen mustard group and a benzimidazole ring and has been suggested to have both alkylator and purine analogue activities. Insights into the molecular mechanisms by which efficacy is achieved are scarce. Furthermore, the effects of BDM on solid tumors also remains under-investigated. Here we evaluate the effects of BDM on Hela cells and attempt to discern its effects on the cell cycle. Hela cells were treated with different concentrations of BDM, with the antimetabolite gemcitabine, or with the alkylator MMS. Interestingly, we found dose-dependent effects of BDM on cell cycle progression. Using synchronized cells, we observed that a low dose of BDM blocked cells in G2, similar to MMS, while a 4-fold higher dose of BDM arrested cells in S phase, as seen with gemcitabine. We next investigated the effect of abrogating the BDM-induced S and G2 cell cycle arrests with the Chk1 inhibitor UCN-01. To track the fates of cells time-lapse microscopy was conducted on cells expressing H2B: GFP. Cells were first arrested in G2 with low BDM or MMS before addition of UCN-01. Cells were observed to enter mitosis, align their chromosomes but then were delayed from entering anaphase. The cells eventually exited mitosis, with a small number of cells displaying lagging chromosomes. In contrast, cells arrested in S phase with high dose BDM or with gemcitabine, followed by UCN-01 showed highly aberrant mitotic figures. Although these cells were also delayed in mitosis, many of them died in mitosis rather than exit. We stained the mitotic cells generated by G2 or S phase inhibitors and found that cells arrested in G2 and forced into mitosis displayed normal looking metaphases, with aligned centromere/kinetochores on the spindle. However, the cells arrested in S phase followed by UCN-01 led to the generation of ‘mitosis with unreplicated genomes’ (MUGs). This phenomenon is characterized by fragmented mitotic chromatin and detached centromeres and kinetochores away from bulk DNA, as confirmed by electron microscopy (see accompanying poster Beeharry et al.). Furthermore, UCN-01 only induces formation of MUGs in cells arrested in with high BDM or gemcitabine. Although cells arrested in G2 by low BDM or MMS can also enter mitosis after UCN-01 treatment, they do not exhibit fragmented centromeres (MUGs). These observations are consistent with the notion that BDM can act as a nucleoside analogue or as an alkylator, depending on the dose. BDM must therefore inhibit different processes in a dose-dependent manner. Furthermore, tumor cell response may differ depending on the sensitivity of these two cellular processes to BDM. These findings maybe critical for predicting sensitization by inhibitors of Chk1 kinase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3664.

Bendamustine in B-Cell Malignancies: The New 46-Year-Old Kid on the Block

On March 20, 2008, bendamustine HCl (Treanda, Cephalon, Inc.) was approved by the U.S. Food and Drug Administration (FDA) for treatment of chronic lymphocytic leukemia (CLL). This approval was based on a randomized, multicenter trial comparing the test drug ( n = 162) to chlorambucil ( n = 157) as a

Safety and Efficacy of Bendamustine with or without Rituximab for the Treatment of Heavily Pretreated CLL and Lymphoma Patients. A Multicenter Retrospective Study.

Abstract 1662Poster Board I-688 IntroductionBendamustine is an alkylating agent with a nitrogen mustard group and a purine like benzimidazol group. Recently this drug was introduced in Italy. We analized all pts treated in sixteen haematological Italian centers with Bendamustine alone or in combination with anti-CD20 antibody. Patients and methodsOn an intention to treat basis pts who have received at least one complete cycle were evaluable for response and toxicity. The treatments consisted of: Bendamustine 60-90 mg/m2 days 1,2 alone or in combination with Rituximab 375 mg/m2 day 0, every 21 or 28 days. 173 pts were analized, median age was 67 (range 31-87), 114 were male, 63 chronic lymphatic leukaemia 41 indolent non-follicular lymphoma, 26 diffuse large B cell lymphoma, 26 follicular lymphoma, 15 mantle cell lymphoma, 2 Peripheral T cell lymphoma. Pts were heavily pretreated, the median number of previous treatments was 3,5 (range 1-8), 121 pts have experienced more than three chemotherapy schemes. One hundred and twenty-seven pts were previously treated with Rituximab and 24 performed an autologous peripheral blood stem cell transplantation. The Bendamustine pre-treatment condition was: 70 relapsed pts, 40 with refractory disease and 63 with a progressive disease after partial response. The median number of Bendamustine cycles was 4.3 (range 1-11). ResultsAll patients were evaluable for response: 48 pts (28%) obtained a complete remission, 78 (45%) a partial response or stable disease with an overall response rate of 73% and 47 were non responders. According to histotype we observed that 10/15 pts (67%) with mantle cell lymphoma obtained a response (6 CR;4 PR), 37/41 (9 CR; 28 PR) indolent non follicular lymphoma and 25/26 (96%) follicular lymphoma obtained a response (12 CR;13 PR), 46/63 CLL obtained a response and 8/26 (31%) DLBCL obtained a response to therapy (4 CR;4 PR), none of the two T lymphoma pts responded to therapy. With a median period of observation of 12 months (1-46) 121 (70%) pts are alive and 83 pts are in complete remission or with stable disease without any other treatment. The overall survival was 82%, 72%, 68% and 27% respectively for indolent, CLL, mantle cell and diffuse large B cell lymphoma. The progression free survival was 31%, 27%, 15% and 10% respectively for indolent, CLL, mantle cell and diffuse large B cell lymphoma. Fifthy-two pts died, 47 for progressive disease, 11 due to infection and sepsis (6%) and 2 due to other causes not related with therapy or disease. In this group of heavily pretreated pts 760 cycles were performed. The extrahematological toxicity was mild the most important problem were infections (Herpes Zooster, pneumonia, enteritis) reported in 16 pts, hepatic in 4 and cardiologic in 2 pts. The hematological toxicity was trombocytopenia grade 3-4 in 20 pts (12%), neutropenia grade 3-4 in 40 pts (23%) and anemia grade 3-4 in 19 pts (11%). DiscussionIn conclusion this retrospective study shows that treatment with Bendamustine alone or in combination with Rituximab is a safe and efficacy regimen in a subset of pluriresistent patients. This data shows also that the best results could be obtained in indolent lymphoma and CLL incouraging data in mantle cell lymphoma are reported. DisclosuresNo relevant conflicts of interest to declare.

Novel Cytotoxic Agents

Picoplatin Picoplatin is a new cisplatin analog designed to avoid the development of platinum resistance. Preclinical studies in human ovarian xenografts showed complete and durable tumor regression with picoplatin, with continued remission even after the cessation of treatment on day 28 at maximum tolerated dose. In comparison, maximum tolerated doses of cisplatin induced partial tumor regression during treatment and rapid regrowth after cessation of treatment. In the same model, picoplatin caused marked volume reduction in cisplatin-resistant tumor xenografts. In a randomized multicenter study, 77 patients with previously treated small cell lung cancer (SCLC) were treated with picoplatin 150 mg/m intravenously every 3 weeks until disease progression. The study population was subdivided into chemotherapy refractory (failure to achieve response or progressive disease during the first-line cisplatin-based chemotherapy), resistant (initial response with relapse within 3 months from the last cycle of therapy), and sensitive (relapse between 3 and 6 months after last cycle of therapy). Most patients had refractory (44 patients) or resistant (27 patients) disease, whereas only six patients had sensitive disease. Response rates were observed in 9% of patients, and median survival was nearly identical for refractory and sensitive patients at 27 and 26 weeks, respectively. The most common toxicity was myelosuppression and most notably, there were no grade 3 to 4 nephrotoxicity, neurotoxicity, or ototoxicity. The Study of Picoplatin Efficacy After Relapse is an ongoing randomized phase III trial comparing overall survival for picoplatin and best supportive care with best supportive care alone in previously treated patients with SCLC. Bendamustine Bendamustine is a cytotoxic agent combining a purinelike benzimidazol and a bifunctional alkylating nitrogen mustard group. This agent, synthesized in the early 1960s, has significant activity against hematological malignancies including chronic lymphocytic leukemia, indolent non-Hodgkin lymphoma, and multiple myeloma, being approved in the United States for the treatment of chronic lymphocytic leukemia and indolent non-Hodgkin lymphoma in 2008. Several ongoing trials are evaluating the efficacy of bendamustine in solid tumors.1 Bendamustine was initially evaluated in two trials for previously untreated patients with lung cancer. In the first trial, 43 patients with lung cancer were treated with single agent bendamustin 70 mg/m for 4 days every 4 weeks to a maximum of six cycles. There were no responses among the 23 patients with non-small cell lung cancer (NSCLC), whereas nine of 22 patients (40%) with SCLC achieved partial response (PR). The treatment was very well tolerated, with the main grade 3 toxicities consisting of nausea and vomiting (7%), diarrhea (4.6%), leukopenia (4.6%), and thrombocytopenia (4.6%). In the second study, 26 patients with extensive disease SCLC received bendamustine 120 mg/m on days 1 and 2 every 3 weeks for a maximum of six cycles. Among the 22 evaluable patients, complete response (CR) was observed in two patients (9%), PR in eight patients (36%), and stable disease (SD) in four patients (19%). Survival time was not reported, and there were no grade 3 or 4 toxicities. The same schedule of bendamustine (120 mg/m on days 1 and 2 every 3 weeks) was tested in 21 patients with extensive disease-SCLC relapsing more than 2 months after completion of first-line treatment. Response rate was 29% with additional 29% achieving SD. Median time to progression, overall survival, and 1-year overall survival were 4 months, 7 months, and 16%, respectively. The main toxicity was neutropenia, including three patients with grade 3, one patient with grade 4, one patient with neutropenic fever, and one treatment-related death due to sepsis. In a multicenter German trial, 56 patients with SCLC were treated with a combination of carboplatin area under the curve of 5 on day 1 and bendamustin 50 mg/m on days 1 and 2 every 3 weeks. Among the 55 evaluable patients, the response rate was 73% with 1 CR (1.8%) and 39 PR (71%). Main toxicities included 46% grade 3 to 4 leukopenia, 26% grade 3 to 4 thrombocytopenia, and 3.6% treatment-related deaths. In summary, bendamustine seems to be a promising agent worthy of further investigation in the treatment of patients with SCLC. However, the role of bendamustine in SCLC may be further defined in randomized phase III stud*Washington University School of Medicine, St. Louis, Missouri; and †Penn State Hershey Cancer Institute, Pennsylvania. Disclosure: The authors declare no conflicts of interest. Address for correspondence: Chandra P. Belani, MD, Penn State Hershey Cancer Institute, PA. E-mail: cbelani@hmc.psu.edu Copyright © 2009 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/09/0411-1082 Santa Monica Supplement Journal of Thoracic Oncology • Volume 4, Number 11, Supplement 3, November 2009

The role of GSTA2 polymorphisms and haplotypes in breast cancer susceptibility: A case-control study in the Portuguese population

Glutathione-S-transferases (GSTs) are a super-family of phase II metabolizing enzymes that catalyse the detoxification of a large range of endogenous and exogenous toxic compounds, playing an important role in protecting cells against damage, through glutathione conjugation with electrophilic substances. Polymorphic variation in these enzymes that affect its activity seems to be related to individual susceptibility to various human diseases, including cancer. Of the GST super-family, the alpha class GSTs have commonly been described as one of the most versatile class, since it is responsible for detoxification of compounds such as bilirubin, bile acids and penicillin, thyroid and steroid hormones, allowing its solubilization and storage in the liver. Among the alpha class, GSTA1 and GSTA2 isoforms are the most widely expressed in human tissues. Additionally, these enzymes can catalyse conjugation of the nitrogen mustard group of alkylating anticancer drugs, some heterocyclic amines and alpha,beta-unsaturated aldehydes. Since some risk factors for increased breast cancer risk could be related to high production of reactive oxygen species during the metabolism of estrogens by catechol estrogens, or to the exposure to genotoxic compounds, and some of these toxic compounds are usually metabolized by GSTA2, we carried out a hospital based case-control study in a Caucasian Portuguese population (291 breast cancer patients without familiar history of breast cancer and 547 controls matched for age, sex and ethnicity) in order to evaluate the potential modifying role of three non-synonymous polymorphisms in the GSTA2 gene (P110S Ex 5+56C>T;, rs2234951; S112T Ex5+63G>C, rs2180314 and E210A Ex7+83A>C, rs6577) on the individual susceptibility to breast cancer. Our data show that the studied polymorphisms are in strong linkage disequilibrium, but no association was observed between individual GSTA2 polymorphisms and haplotypes and individual susceptibility to breast cancer.

Chlorambucil-Adducts in DNA Analyzed at the Oligonucleotide Level Using HPLC-ESI MS

Chlorambucil (N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid) is a bifunctional alkylating drug belonging to the nitrogen mustard group and is widely used as an anticancer agent. As the antitumor activity of the nitrogen mustards is based on the formation of adducts with genomic DNA, calf thymus DNA-Chlorambucil adducts were the major target in this study. Calf thymus DNA was incubated with Chlorambucil to induce the formation of a wide variety of adducts. Subsequently, enzymatic digestion of the DNA was performed using Benzonase and Nuclease S1 aiming at the production of oligonucleotides. Separation and structure elucidation of the individual DNA-Chlorambucil adducts was achieved using HPLC interfaced to electrospray ionization ion trap mass spectrometry. Both trinucleotide and tetranucleotide Chlorambucil adducts were detected. The majority of the detected trinucleotide adducts involved monofunctional alkylation with guanine being the hotspot for alkylation. Only a few bifunctional trinucleotide adducts both intra- and interstrand cross-links were found. On the contrary, cross-linked adducts were the major detected tetranucleotides in which the intrastrand cross-links predominated over the interstrand cross-links. To a lesser extent, monofunctional guanine alkylated tetranucleotides were detected as well. With MS(n) experiments, the detailed structures of Chlorambucil adducts of the tri- and tetranucleotides were determined.

Somatic Growth after Fontan and Mustard Palliation

Patients with complex congenital heart disease frequently develop early growth failure; however, the long-term outcome for growth after surgery for single ventricle or anatomic right ventricle as systemic ventricle is not clear. This study was designed to determine long-term growth in patients following the Fontan and Mustard operations. We retrospectively reviewed the growth parameters of children who had previously undergone the Fontan (n = 80) or Mustard (n = 66) palliation at the Riley Hospital for Children, Indiana. Both the Fontan and Mustard groups had normal height and weight at birth. At the time of their Fontan or Mustard palliation, there was a significant retardation in weight (Z-score: -0.98 and -1.79, respectively) and height (Z-score: -0.96 and -1.03, respectively). Both cohorts postoperatively demonstrated significant catch-up in their weights. Although the Mustard group normalized their heights, the Fontan patients continued to demonstrate short statures in long-term follow-up. Children with single ventricles and those with palliated d-loop transposition of the great arteries suffer somatic growth delay prior to definitive surgery, despite being of normal size at birth. Catch-up growth in weight occurs after the Fontan and Mustard operations. In the Mustard population, height also normalizes, whereas in patients with univentricular circulation, height remains abnormally low.

Synthesis and Cytotoxicity of Pyrrole-Amino Acid Dipeptides Containing Phosphonyl Group

A series of pyrrole-amino acid dipeptide were synthesised by chloroform reaction and coupling reaction using DCC/HOBT as promoting additives. The structure of compound was confirmed by 1H NMR, 31P NMR, MS and IR. The biological activity data showed that the dipeptide containing nitrogen mustard group inhibited all four cells that were examined.

Bendamustine and rituximab (BR) are effective in the treatment of relapsed or refractory indolent and mantle-cell lymphomas: Long-term follow-up of a phase II study

8034 Bendamustine combines a purine-like benzimidazol and bifunctionally alkylating nitrogen mustard group. The aim of this multicenter- study was to evaluate the progression-free survival (PFS), response rate and toxicity of BR in patients with mantle-cell or low-grade lymphomas in 1st to 3rd relapse or refractory to previous treatment. A median of 4 courses per patient were administered to 63 pts. Bendamustine was given at a dose of 90 mg/m2 on day 1 and 2 combined with 375 mg/m2 Rituximab on day 1 for a max. of 4 cycles every 4 weeks. Histologies: 24 follicular, 16 mantle cell, 17 lymphoplasmacytoid, 6 marginal zone lymphoma. 57 of 63 pts responded to BR corresponding to an overall response rate of 90% with a CR-rate of 60%. In mantle cell lymphomas BR showed a considerable activity achieving a response rate of 75% with a CR-rate of 50%. In an updated analysis the median time of PFS was 30 months and the median duration of overall survival has not yet been reached. With a median follow-up period of 3 years two secondary neoplasms have been observed (bronchial carcinoma). No MDS was observed to date. These results demonstrate that the combination of Bendamustine and Rituximab is a highly active regimen in the treatment of low-grade lymphomas and mantle cell lymphomas. Based upon these encouraging results the Studiengruppe indolente Lymphome (StiL) initiated two Phase III trials that compare BR to established chemo-immunotherapy regimens: BR vs R-CHOP in untreated pts, and BR vs Fludarabine- R in relapsed pts. These studies should provide considerable guidance for the appropriate role of Bendamustine in the treatment of low-grade and mantle cell lymphomas. No significant financial relationships to disclose.

Cyclophosphamide as a potent inhibitor of tumor thioredoxin reductase in vivo

Cyclophosphamide (CTX) is in the nitrogen mustard group of alkylating antineoplastic chemotherapeutic agents. It is one of the most frequently used antitumor agents for the treatment of a broad spectrum of human cancers. Thioredoxin reductase (TrxR) catalyze the NADPH-dependent reduction of thioredoxin and play an important role in multiple cellular events related to carcinogenesis including cell proliferation, apoptosis, and cell signaling. This enzyme represents a promising target for the development of cytostatic agents. The purpose of this study is to determine whether CTX could target TrxR in vivo. Lewis lung carcinoma and solid H22 hepatoma treated with 50-250 mg/kg CTX for 3 h lost TrxR activity in a dose-dependent fashion. Over 75% and 95% of TrxR activity was lost at the dose of 250 mg/kg. There was, however, a recovery of TrxR activity such that it attained normal levels by 120 h after a dose of 250 mg/kg. In addition, we found that CTX caused a preferential TrxR inhibition over other antioxidant enzymes, such as glutathione peroxidase, catalase, and superoxide dismutase. We also used ascites H22 cells to investigate cancer cells response after TrxR was inhibited by CTX in vivo since CTX is needed to be activated by liver cytochrome P450 enzymes. The time course and dose-dependent changes of cellular TrxR activity were similar with those in tumor tissue. CTX caused a dose-dependent cellular proliferation inhibition which was positively correlated with TrxR inhibition at 3 h. Furthermore, when 3 h CTX-treated cells with various TrxR backgrounds, harvested from ascites-bearing mice, were implanted into mice, the proliferations of these cells were again proportionally dependent on TrxR activity. The TrxR inhibition could thereby be considered as a crucial mechanism contributing to anticancer effect seen upon clinical use of CTX.

Bendamustine and rituximab act synergistically in vitro and are effective in the treatment of relapsed or refractory indolent and mantle cell lymphomas

6565 Bendamustine combines a purine-like benzimidazol and bifunctionally alkylating nitrogen mustard group. The drug is active in a variety of lymphoproliferative disorders without crossresistance to cyclophosphamide. We investigated simultaneous combination of rituximab with bendamustine and found synergistic effects of apoptosis in vitro on lymphoma cell lines (DOHH-2, WSU-NHL) as well as on ex vivo cells of pat. with CLL. Apoptosis was determined by Annexin V and JC-1. The synergistic effects of rituximab in combination with bendamustine were evenly strong when rituximab was applied at a dose, at which half of the CD20 molecules per cell were saturated. Based on our in vitro results we initiated a phase-II study to examine efficacy and toxicity of bendamustine combined with rituximab (BR) as therapy of 1st, 2nd or 3rd relapse. Treatment schedule: 6 x Rituximab 375 mg/qm day 1 combined with 4 x Bendamustine 90 mg/qm day 2+3, every 4 weeks. Included entities: 17 immunocytomas, 16 mantle cell (MCL), 24 follicular, 6 marginal zone lymphomas. 63 pat. entered the study. Results: 57 pat. responded to therapy yielding to an ORR of 90% with a CR-rate of 60%. In MCL an ORR of 75% was achieved with an CR-rate of 50%. All pat. with immunocytome achieved a remission with a CR-rate of 53%. The median duration of remission was 25 months. BR induced considerable low hematological toxicity with only 16% granulocytopenias grade 3 and 4. We conclude that BR seems to be very effective in the treatment of relapsed indolent and mantle cell lymphomas reaching an overall response rate of 90% and a CR rate of 60%. Based upon the encouraging results obtained with BR in this study we initiated two Phase III trials that compare BR to established chemo-immunotherapy regimens. One trial compares BR versus R-CHOP in pat. with previously indolent lymphomas to demonstrate a non-inferiority of BR in comparison to R-CHOP with respect to efficacy but to show a more favorable toxicity profile. The second trial compares BR to fludarabine/rituximab in pat. with relapsed indolent lymphomas. These studies should provide considerable guidance for the appropriate role of bendamustine in the treatment of indolent lymphomas. No significant financial relationships to disclose.

Utilizing a D-Amino Acid as a Drug Carrier for Antineoplastic Nitrogen Mustard Groups

The evaluation of an alkylating nitrogen mustard agent that utilizes D-alanine as a drug carrier for three chloroethyl substituents (ClCH2CH2-) is shown. Various important pharmacological properties were determined including polar surface area, partition coefficient, molar volume, polarizability, numbers of -OH and -NH2 groups, and aqueous solubility. The synthetic approach utilizes 1,2-dichloroethane reaction with the primary amine of D-alanine resulting in chloroethyl (ClCH2CH2-) substituents. A nitrogen mus-tard group results, and this agent showed alkylation activity in aqueous solution directed toward a nucleophilic primary amine group. Kinetics of alkylation activity is determined by placing p-chloroaniline and the nitrogen mustard agent in sodium bicarbonate buffered aqueous solution at physiological pH 7.4 and 37°C. Samples taken from the test solution are injected with fluorescamine that reacts specifically with primary amine functional groups. Absorbance measurements obtained at 400 nm by UV-Vis spectrometer indicates the relative amounts of nonalkylated p-chloroaniline in the test solution. The D-alanine nitrogen mustard agent effectively alkylated the nucleophilic primary amine of p-chloroaniline with zero-order kinetics. The rate constant was determined to be 7.445E-04 mol/l/min. Formula weight, polar surface area, Log P, molar volume, violations of Rule of 5 for D-alanine mustard agent are 276.59, 29.543 angstroms2, 1.605, 222.3 cm3, and zero violations, respectively. Cluster analysis of molecular properties showed D-alanine mustard agent to be quite similar to cyclophosphamide. This agent showed good druglikeness and zero violations of the Rule of 5, indicating good bioavailability. Molecular properties calculated for the mustard agent are numerically comparable to some clinical anticancer drugs.

Bendamustine in Combination with Thalidomide and Prednisolone (BPT) in Patients with Refractory or Relapsed Multiple Myeloma (MM): Preliminary Results of a Phase I Clinical Trial.

Thalidomide is an active single agent in advanced relapsed or refractory MM. Treatment, however, is associated with significant dose-dependent toxicity limiting his application. Combination of low dose thalidomide with bendamustine and prednisolone might be a way to maintain efficacy of the drug without dose limiting toxicity. The cytotoxic agent bendamustine combines a purine-like benzimidazol with a bifunctionally alkylating nitrogen mustard group. The drug is active in a variety of lymphoproliferative disorders with modest non-hematological and hematological toxicities. Previous clinical studies confirmed the efficacy of bendamustine monotherapy and in combination with prednisolone in patients with newly diagnosed and relapsed MM. In a phase III study of newly diagnosed patients, overall response rate for bendamustine and prednisone was 75% and shown to be superior to melphalan and prednisone in regard to progression free survival and quality of life. The purpose of this phase I study was to test the feasibility of the combination of BPT in advanced MM patients.The treatment consists of a fixed dose of bendamustine (60 mg/qm) i.v. day 1, 8, and 15 and prednisolone (100 mg) p.o. day 1, 8, 15, and 22. At the same time, thalidomide will be given orally in 4 patients cohorts with escalating doses, starting at 50 mg to a maximum of 200 mg daily. Cycles will be repeated every 28 days for a minimum of 2 and a maximum of 10 cycles until appearance of significant treatment-related toxicity or disease progression. Till now, four patients with stage IIIa MM were enrolled in the first dose level with 50 mg thalidomide daily. All patients had two prior treatment lines and three were refractory. Median age was 67 years (range 64 – 69 years).Results: All four patients completed at least 2 cycles of BPT-treatment (2 completed 6 cycles, 1 completed 3 cycles and 1 completed 2 cycles) and were evaluable for toxicity and efficacy. All patients responded after the first two cycles of therapy in three of them with decrease of the myeloma protein of more than 90%. One showed a reduction of the myeloma protein of 30%. Although median follow up is still short, no progression of disease was observed yet.Thalidomide in a dose of 50 mg daily given during 4.5 months median (range 2–6 months) was well tolerated. Most common site effects were constipation (three patients WHO grade 2) and somnolence (two patients WHO grade 1). None of the four patients developed dose-limiting hematotoxicity as defined by an ANC < 1,0 x 109/l for >7 days or an ANC < 0,5 x 109/l for > 3 days or platelet count < 25 x 109/l. Neutropenia was reported in 1 patient (WHO grade 2) but no thrombocytopenia was observed. No grade 3 or 4 non-hematological toxicity was encountered and no dose modification was required.BPT with a dose of 50 mg thalidomide daily is well tolerated in patients with relapsed or refractory MM. The next cohort of patients is currently treated with Thalidomide 100 mg daily to evaluate the maximal tolerable doses of the BPT regimen.

1,2-benzisoxazole phosphorodiamidates as novel anticancer prodrugs requiring bioreductive activation.

Several 1,2-benzisoxazole phosphorodiamidates have been designed as prodrugs of phosphoramide mustard requiring bioreductive activation. Enzymatic reduction of 1,2-benziosoxazole moiety is expected to result in the formation of imine intermediate due to the cleavage of the N-O bond. The imine should then be spontaneously hydrolyzed to a ketone metabolite, thereby facilitating base-catalyzed beta-elimination of cytotoxic phosphoramide mustard. As expected, the proposed prodrugs 4, 9, and 12 were at least 3-5-fold more potent cytotoxins than control compounds 5 and 15, which lack in the phosphoramide mustard group. Upon incubation with phenobarb-induced rat liver S-9 fraction, compounds 4, 9, and 12 underwent extensive NADPH-dependent metabolism with concomitant generation of alkylating activity under both hypoxic and oxic conditions. Corresponding ketone metabolites were detected for 9 and 15. NADPH-dependent bioreduction of 15 to its ketone metabolite 16 was located in the microsomal fraction and inhibited by SKF-525A and pCMBA. Compared with phenobarb-induced rat liver microsomal fraction, incubation of 15 with rat or human p450 reductase microsomes showed moderate generation of 16. Microsomal cytochrome p450 and/or p450 reductase appear to be involved in the reductive metabolism of 1,2-benzisoxazole moiety under hypoxic as well as oxic conditions.

The discovery of the antiplatelet effect of aspirin: a personal reminiscence

The discovery of the antiplatelet effect of aspirin: a personal reminiscence