Alkylating Drugs Research Articles

Considerations in the treatment of multiple myeloma: a consensus statement from Italian experts

PURPOSE AND BASIC PROCEDURE OF THE STUDY: The availability of new targeted therapies has revolutionised the treatment of multiple myeloma (MM), for both the newly diagnosed and the relapsed and refractory settings. A panel of Italian experts provided guidelines for optimal clinical practice in the treatment of MM. The panel recommended that treatment should only be initiated in symptomatic patients. Autologous stem cell transplantation (ASCT) with melphalan is the treatment of choice in patients younger than 65 yr, and induction therapy including new drugs seems the most suitable preparatory regimen before ASCT. In patients who fail to achieve at least a very good partial response (VGPR) after transplant, a consolidation with a second transplant is of clinical benefit. Also, there is evidence that maintenance with thalidomide after ASCT in young patients failing to reach at least VGPR could prolong survival. In elderly patients, the combination of an alkylating drug with a novel agent should be considered as standard approach. Relapsed MM should be retreated after the reappearance of symptoms and signs of organ and tissue damage. Salvage regimens should include corticosteroids plus bortezomib, thalidomide or lenalidomide.

Squamous-cell carcinoma in mature cystic teratoma of the ovary: systematic review and analysis of published data

Up to a quarter of ovarian masses originate from germ cells, and many of these are mature cystic teratomas. The secondary development of malignancy is a rare but well-known phenomenon in patients with ovarian teratomas. Squamous-cell carcinoma accounts for 80% of secondary malignant transformations of ovarian teratomas. We aimed to do an up-to-date systematic review of this rare malignant transformation. 64 suitable studies provided information on 277 patients. Squamous-cell carcinoma in mature cystic teratoma was mainly found in women aged more than 50 years, with high concentrations of squamous-cell-carcinoma antigen and cancer antigen CA125, and with ovarian tumours more than 100 mm in size. Patients with FIGO stage Ia tumours had better survival than those with more advanced disease. Complete resection together with hysterectomy, bilateral salpingo-oophorectomy and lymphadenectomy for patients with advanced disease, followed by adjuvant chemotherapy with an alkylating drug was associated with higher survival, radiotherapy was not. We make proposals for investigation and treatment of this rare disorder.

BCNU-sequestration by metallothioneins may contribute to resistance in a medulloblastoma cell line

Resistance of neoplastic cells to the alkylating drug BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea] has been correlated with expression of O (6)-methylguanine-DNA methyltransferase, which repairs the O (6)-chloroethylguanine produced by the drug. Other possible mechanisms of resistance include raised levels of glutathione or increased repair of the DNA interstrand cross-links formed by BCNU. Transcriptional profiling revealed the upregulation of several metallothionein (MT) genes in a BCNU-resistant medulloblastoma cell line [D341 MED (OBR)] relative to its parental line. Previous studies have shown that MTs, through their reactive thiol groups can quench nitrogen mustard-derived alkylating drugs. In this report, we evaluate whether MTs can also quench BCNU. To demonstrate the binding of BCNU to MT, we used an assay that measured the release of the MT-bound divalent cations (Zn(2+), Cd(2+)) upon their displacement by the drug. We also measured the decomposition rates of BCNU at those reaction conditions. The rate of release of the cations was higher in pH 7.4 than at pH 7.0, which is likely a result of more rapid decomposition of BCNU (thus faster release of MT-binding intermediate) at pH 7.4 than at pH 7.0. We demonstrate that resistance to BCNU may be a result of elevated levels of MTs which act by sequestering the drug's decomposition product(s).

Synthesis and cytotoxic properties of new fluorodeoxyglucose-coupled chlorambucil derivatives

Frequently used in the treatment of malignant cells, alkylating agents, like most anticancer substances, produce adverse side effects caused by the toxicity of the agents toward normal tissues and lose efficiency through poor distribution to target sites. Our approach to developing more selective drugs with low systemic toxicity is based on the premise that the body distribution and cell uptake of a drug can be altered by attaching a neoplastic cell-specific uptake enhancer, such as 2-fluoro-2-deoxyglucose (FDG), the radiotracer most frequently used in PET for tumor imaging. Two properties of deoxyglucose, namely preferential accumulation in neoplastic cells and inhibition of glycolysis, underpin this targeting approach. Here, we report the synthesis of 19 new chlorambucil glycoconjugates in which the alkylating drug is attached to the C-1 position of FDG, directly or via different linkages. This set of compounds was evaluated for in vitro cytotoxicity against different human normal and tumor cell lines. There was a significant improvement in the in vitro cytotoxicity of peracetylated glucoconjugates compared with the free substance. Four compounds were finally selected for further in vivo studies owing to their lack of oxidative stress-inducing properties.

Immune-Potentiating Effects of the Chemotherapeutic Drug Cyclophosphamide

Cyclophosphamide (CTX) is an alkylating cytotoxic drug that primarily affects proliferating lymphocytes. CTX has been extensively used as a chemotherapeutic and disease-modifying agent against certain solid tumors, lymphomas, and some autoimmune conditions. Depending on its dose and timing of administration, CTX can also enhance immune responses. These opposing immune functions of CTX have been investigated in numerous animal models and recent clinical studies. Administration of CTX augments delayed type 1 hypersensitivity (DTH) reactions, can precipitate type 1 diabetes, and boosts antitumor responses in both vaccination and adoptive cell transfer models. Although the mechanisms by which CTX elicits these effects are not fully understood, CTX treatment has a differential effect on lymphocyte compartments, rapidly depleting B and T cells. This is followed by a recovery phase characterized by extensive proliferation and bone marrow mobilization. The proposed underlying mechanisms of augmentation of immune responses include the facilitation of homing and homeostatic proliferation by the creation of space, the skewing of Th2/Th1 responses due to the cytokine storm during the recovery phase, and the removal or inhibition of a suppressor cell population. With the identification of naturally occurring and peripheral regulatory T cells, recent studies have re-evaluated these hypotheses and suggest that CTX inhibits the function of Foxp3+ regulatory T cells. In this review, we critically evaluate relevant historical and recent data on CTX-mediated immunomodulation and discuss the resulting implications for immunotherapy.

The Sooner the Better: Rapid Transduction Enhances the Engraftment/Selection of Gene Corrected Fanconi Anemia HSC.

Fanconi anemia (FA) is amenable to genetic correction of hematopoietic stem cells (HSCs). However, as demonstrated in previous clinical gene therapy trials, successful extension of murine studies into human therapies is limited by low numbers of target HSC and poor engraftment of transduced FA HSC (Kelly et al., Mol Ther, 2007). To examine the potential biological consequences/benefits of shortened transduction we used a FA mouse model in which HSC are deficient and prone to excessive loss during in vitro manipulation. We applied a rapid transduction protocol (Mostoslavsky et al., Mol Ther, 2005) utilizing lentiviral vectors and demonstrate that this shortened transduction preserves engraftment of FA HSC to the level of C57BL/6 wt cells. Lin− Sca-1+ c-Kit+ bone marrow cells were isolated from Fanca−/− CD45.2 mice and underwent 4-hr rapid (RT) vs. 96-hr conventional (CT) transduction. An equivalent number of transduced cells were transplanted into lethally irradiated CD45.1 BoyJ mice. Analysis of engraftment chimerism three months post transplantation revealed a significantly higher level of engraftment in animals receiving RT vs. CT cells (90% +/− 14% vs. 26% +/− 31%, respectively, p=<0.01). Rapid transduction also resulted in a significant reduction of engraftment failure (0/36 animals RT vs. 20/36 animals CT). Importantly--emphasizing the FA disease-specific stem cell phenotype, RT vs. CT of C57BL/6 wt cells was associated with no significant difference in engraftment of these cells (93% +/− 1.2% RT vs. 84 +/− 19% CT, p=0.33). Analysis of peripheral blood cells expressing the proviral enhanced green fluorescent protein (eGFP) reporter gene revealed a normal distribution of B-lymphocytes (B220), T-lymphocytes (CD3 epsilon), and granulocytes (MAC-1), indicating multi-lineage engraftment of gene modified cells. In spite of this engraftment advantage, transduction efficiency was low (<30%) using RT. The 6-benzylguanine (6-BG) resistant P140K mutant of O6-methylguanine DNA methyltransferase (MGMTP140K) confers a selective advantage to tranduced HSC treated with alkylating drugs. Following RT with a MGMTP140K/ eGFP expressing lentivirus, 5/6 mice treated with 6-BG and the alkylating drug temozolomide showed a significant rise in the percentage of GFP reporter gene expression in peripheral blood. We extended this approach to the FA model by generating a tri-cistronic lentiviral vector expressing the FANCA cDNA, MGMTP140K, and eGFP. Despite modest in vivo gene marking with this vector, up to 37-fold selection (85% GFP-positive cells) was achieved following exposure of bone marrow of transplant recipients to 6-BG and the alkylating drug temozolomide in vitro. Concurrently, phenotypic correction of mitomycin C hypersensitivity of transduced Fanca−/− bone marrow cells was observed. These data suggest that RT improves stem cell engrafting capacity of FA stem cells in a relevant animal model of stem cell gene therapy. The combination of RT and in vivo selection may allow more successful reconstitution of the lympho-hematopoietic system in gene therapy applications.

Bendamustine for advanced sarcoma

A phase II trial by German researchers has shown that bendamustine, a bifunctional alkylating drug, shows encouraging results in patients with advanced soft-tissue sarcoma (STS; Cancer, published online June 28, 2007; DOI: 10.1002/cncr.22846).

MGMT: Key node in the battle against genotoxicity, carcinogenicity and apoptosis induced by alkylating agents

O 6-methylguanine-DNA methyltransferase (MGMT) plays a crucial role in the defense against alkylating agents that generate, among other lesions, O 6-alkylguanine in DNA (collectively termed O 6-alkylating agents [O 6AA]). The defense is highly important, since O 6AA are common environmental carcinogens, are formed endogenously during normal cellular metabolism and possibly inflammation, and are being used in cancer therapy. O 6AA induced DNA damage is subject to repair, which is executed by MGMT, AlkB homologous proteins (ABH) and base excision repair (BER). Although this review focuses on MGMT, the mechanism of repair by ABH and BER will also be discussed. Experimental systems, in which MGMT has been modulated, revealed that O 6-methylguanine (O 6MeG) and O 6-chloroethylguanine are major mutagenic, carcinogenic, recombinogenic, clastogenic and killing lesions. O 6MeG-induced clastogenicity and cell death require MutSα-dependent mismatch repair (MMR), whereas O 6-chloroethylguanine-induced killing occurs independently of MMR. Extensive DNA replication is required for O 6MeG to provoke cytotoxicity. In MGMT depleted cells, O 6MeG induces apoptosis almost exclusively, barely any necrosis, which is presumably due to the remarkable ability of secondarily formed DNA double-strand breaks (DSBs) to trigger apoptosis via ATM/ATR, Chk1, Chk2, p53 and p73. Depending on the cellular background, O 6MeG activates both the death receptor and the mitochondrial apoptotic pathway. The inter-individual expression of MGMT in human lymphocytes is highly variable. Given the key role of MGMT in cellular defense, determination of MGMT activity could be useful for assessing a patient's drug sensitivity. MGMT is expressed at highly variable amounts in human tumors. In gliomas, a correlation was found between MGMT activity, MGMT promoter methylation and response to O 6AA. Although the human MGMT gene is inducible by glucocorticoids and genotoxins such as radiation and alkylating agents, the role of this induction in the protection against carcinogens and the development of chemotherapeutic alkylating drug resistance are still unclear. Modulation of MGMT expression in tumors and normal tissue is currently being investigated as a possible strategy for improving cancer therapy.

DNA topoisomerase IIα (TOP2A) inhibitors up-regulate fatty acid synthase gene expression in SK-Br3 breast cancer cells: In vitro evidence for a ‘functional amplicon’ involving FAS, Her-2/neu and TOP2A genes

Fatty acid synthase (FAS), the key metabolic multi-enzyme that is responsible for the terminal catalytic step in the de novo fatty acid biosynthesis, plays an active role in the development, maintenance, and enhancement of the malignant phenotype in a subset of breast carcinomas. We recently described that a molecular bi-directional cross-talk between FAS and the Her-2/neu (erbB-2) oncogene is taking place at the level of transcription, translation, and activity in breast cancer cells. Because Her-2/neu has been linked with altered sensitivity to cytotoxic drugs, we envisioned that FAS gene expression may represent a novel predictive molecular factor for breast cancer response to chemotherapy in a Her-2/neu-related manner. We herein evaluated whether chemotherapy-induced cell damage acts in an epigenetic fashion by inducing changes in the transcriptional activation of FAS gene in breast cancer cells. To evaluate this option, FAS- and Her-2/neu-overexpressing SK-Br3 breast cancer cells were transiently transfected with a FAS promoter-reporter construct (FAS-Luciferase) harboring all the elements necessary for high level expression in cancer cells. SK-Br3 cells cultured in the presence of topoisomerase IIalpha (TOP2A) inhibitors doxorubicin and etopoxide (VP-16) demonstrated a 2- to 3-fold increase in FAS promoter activity when compared with control cells growing in drug-free culture conditions. We failed to observe any significant activation of FAS promoter following exposure to the anti-metabolite 5-fluorouracil, the alkylating drug cisplatin, or the microtubule interfering-agents paclitaxel and vincristine. Moreover, the up-regulatory effects of TOP2A inhibitors on the transcriptional activation of FAS gene expression were not significantly decreased when the FAS promoter was damaged at the sterol regulatory element binding protein (SREBP)-binding site. Considering that FAS inhibition produces profound inhibition of DNA replication and S-phase progression in cancer cells, we finally asked whether a cross-talk between TOP2A and FAS could exhibit a Her-2/neu-related bi-directional nature. TOP2A protein levels were decreased during treatment with the anti-Her-2/neu antibody trastuzumab while, concomitantly, FAS promoter activity and FAS protein expression were significantly reduced. Of note, when the expression levels of TOP2A protein were analyzed following exposure of SK-Br3 cells to increasing concentrations of the novel slow-binding FAS inhibitor C75, a dose-dependent reduction in TOP2A expression was observed. Although FAS gene is not physically located in the Her-2/neu-TOP2A amplicon, our present findings strongly suggest that a tight functional association between FAS, Her-2/neu and TOP2A genes is taking place in a subset of breast carcinoma cells.

Synthesis, Physicochemical Characterization and Preliminary Pharmacological in vitro Evaluation of Two Novel Cytotoxic Benzophenone‐Linked 3,3‐Dimethyltriazenes.

The synthesis, physicochemical characterization and preliminary pharmacological evaluation of the cytotoxic effects of two novel substances, 1-(4-benzoylphenyl)-3,3-dimethyltriazene and 1-(2-benzoylphenyl)-3,3-dimethyltriazene is presented. The cytotoxicity of the novel benzophenone-linked triazenes and of ten other 1-phenyl-3,3-dimethyl triazene derivatives as well as of the referent alkylating drug melphalan was assessed using the MTT-dye reduction assay. A panel of human tumor cell lines was used: the chronic lymphoid leukemia SKW-3, the acute promyelocyte leukemia HL-60 and its multi-drug-resistant subline HL-60/Dox. Both novel compounds showed strong cytotoxic activity, comparable to that of the referent alkylating agent melphalan, whereas the ten ring-substituted 1-phenyl-3,3-dimethyl triazenes proved to be far less active in vitro. DNA-fragmentation analysis indicated that after 24 h treatment the novel benzophenone-linked triazenes induced programmed cell death in HL-60 cells.

Alkylating drugs applied in non-cytotoxic doses as a novel compounds targeting inflammatory signal pathway

Alkylating drugs (ADs) belonging to the nitrogen mustard family are commonly used as cytostatic and immunosuppressive agents. Our previous in vitro studies demonstrated that in the case of gradual dose decrease, the number of targets for alkylation in the cell is also reduced and the drug switches from brutal cytostatic to cell growth modifier. At doses of 0.3 μg/ml and lower, the effects of ADs are no longer associated with DNA damage or stress/MAPK pathways activation. Instead, the disruption of signal transduction by the IL-2β and/or TNFα cell surface receptors is observed. As a result, ADs in the doses 100-fold lower than cytostatic ones are capable to modify lymphocyte activity including the activity of regulatory T cells. We hypothesized that ADs may have a beneficial effect in the treatment of inflammatory diseases. Indeed, the application of non-cytotoxic doses of an AD melphalan reduces the severity of murine experimental colitis. Daily administration of melphalan (25 μg/kg body weight) markedly reduced the severity of DSS-colitis as determined by clinical and histological criteria. Moreover, the beneficial effect of melphalan was also shown in asthmatic patients. In 60% of these patients histological and ultrastructural signs of bronchial epithelium regeneration were also revealed. Thus, ADs at non-cytotoxic concentrations exert beneficial effect both in acute and chronic inflammatory diseases. Such anti-inflammatory activity is thought to be due to blocking of signal transduction through various cell surface receptor including IL-2R and TNFR. Consequently different steps of inflammatory cascade turn out to be inhibited.

The influence of ATP on poly(ADP-ribose) metabolism

ATP affects poly(ADP-ribose) metabolism at two distinct sites: it inhibits poly(ADP-ribose) polymerase-1 and activates the glycohydrolase directly. The inhibitory site of ATP on poly(ADP-ribose) polymerase-1 was identified by amino acid exchange mutation to be at the arginine 34 residue in the first Zn2+ finger. Mutation of 138 arginine residue of Zn2+ finger 2 had negligible influence on the inhibitory action of ATP, pinpointing arginine 34 of the first Zn2+ finger as the specific ATP site. The glycohydrolase protein was activated by ATP when the substrate was a long-chain ADP-ribose polymer, but not with a short-chain substrate. Isolated cell nuclei also responded to both inhibition of poly(ADP-ribose) polymerase by ATP and to poly(ADP-ribose) glycohydrolase activation by ATP, demonstrating that enzymological results can be extrapolated to cellular systems. The activation of poly(ADP-ribose) polymerase in nuclei by an alkylating drug was completely suppressed by ATP, demonstrating that the bioenergetic competence of cells can regulate the cytocidal action of DNA alkylating drugs. The potential significance of bioenergetic regulation of poly(ADP-ribose) metabolism is proposed.

Therapy related Acute Myeloid Leukaemia 8 Years after Treatment for Hodgkin\'s Disease – A Case Report

Hodgkin's Disease (HD) is a curable malignancy even in Nigeria, our limitations in health care delivery notwithstanding. However, secondary malignancies especially Acute Myeloid Leukaemia (AML) may occur as late complications following alkylating cytotoxic drugs therapy, with or without radiotherapy. This is a case report of a 52-year-old man who (between 1983-1985) received 4 cycles of combination chemotherapy, namely Mustine Hydrochloride, Oncovine, Procarbazine, Prednisolone (MOPP), and then 6 cycles of combination chemotherapy consisting of Cyclophosphamide, Oncovine, Procarbazine and Prednisolone (COPP) followed by 2 doses of Adriamycin. He developed AML 8 years after complete remission. The possibility of the development of secondary malignancies as late complication of cytotoxic drug therapy should be discussed with patients receiving these drugs and long-term follow-up should be mandatory. KEY WORDS: Hodgkin's Disease, secondary malignancy, acute myeloid leukaemia, alkylating cytotoxic drugs and long-term follow up. Nigerian Journal of Health and Biomedical Sciences Vol.4(1) 2005: 14-16

Myeloablative Conditioning in Myelofibrosis using i.v. Treosulfan and Autologous Peripheral Blood Progenitor Cell Transplantation with High Doses of CD34+ Cells Results in Hematologic Responses - Follow-Up of Three Patients.

Myelofibrosis with myeloid metaplasia is a chronic myeloproliferative disorder. The only curative therapy to date is myeloablation followed by allogeneic transplantation. This approach, however, is associated with a high morbidity and mortality. Autologous transplantation after myeloablation with high-dose oral busulfan provides a palliative approach which can lead to a long-term relief of symptoms and is associated with acceptable morbidity and mortality (Anderson JE et al. 2001, Blood 98: 586). However, busulfan pharmacokinetics after oral administration can vary between patients and increased toxicity is encountered in some. On the other hand treosulfan, a bi-functional alkylating drug, can be administered safely i.v. with reliable pharmacokinetics up to the myeloablative dose range. In the study presented here, patients were stimulated with G-CSF 16 μg/kg daily for 4 days and subsequent leukapheresis of a minimum of 5 x 106 CD34+ cells/kg was performed. Myeloablation consisted of treosulfan infusions of 14 g/m2 for three consecutive days (total dose 42 g/m2) and subsequent autologous PBPCT. To date we have transplanted 3 patients, all female. Two patients (#1, #3) had symptomatic splenomegaly and severe anemia. Patient #2 had symptomatic splenomegaly and thrombocytopenia (< 100/nl). The WB nadir after treosulfan therapy was 0.06/nl (#1), 0.28/nl (#2) and 0.09/nl (#3). The time to reconstitution of leucocytes > 1/nl post transplantation was 28 days (#1, #2) and 38 days (#3) and the time for reconstitution of thrombocytes > 50/nl was 36 (#1), 22 (#2) and 33 (#3) days. The prolonged reconstitution period may have been due to the myelofibrosis and has also been observed after busulfan conditioning and PBPCT by others. There were no fever or other severe toxicity. Patients did not require total parenteral nutrition. The patients have been followed-up for 18 months post transplantation, now. The first patient (#1), who required erythrocyte transfusions twice weekly pretransplant received her last erythrocyte transfusion on day 56; her Hb-value is 11.2 g/dl at last follow-up. The second patient (#2) recovered to platelet counts higher than pre-transplantation (58/nl) at 3 months.(143/nl) and had 103/nl at last follow-up. Pat. #3 showed a marked reduction of max. spleen size and a rise in Hb from 9 g/dl to 12 g/dl after 12 months, the Hb value at 18 months is 8.7 g/dl and allogeneic transplantation is considered now. We conclude that myeloablation with treosulfan and autologous PBPCT in these three patients with myelofibrosis was safe and useful. Survival of these patients is promising up to date. The results presented warrant further exploration of this approach in a clinical study which has been initiated by our group. (cf. http://leukaemie.krebsinfo.de/kn_home/Studien/studie_101.html).

Successful Gene Therapy of Murine Paroxysmal Nocturnal Hemoglobinuria (PNH) Using Mgmt-Mediated In Vivo Selection of Genetically Corrected, Drug-Resistant Hematopoietic Stem Cells.

PNH is an acquired disorder of the hematopoietic stem cell (HSC) caused by a somatic mutation in the PIGA gene. Blood cells carrying the mutation are deficient in all glycosyl phosphatidylinositol-anchored proteins (GPI-APs). Long-term restoration of GPI-AP has been obtained in vitro using lentiviral vectors stably expressing PIGA in human HSCs. However, gene therapy for PNH might be problematic because of the lack of a growth advantage of phenotypically corrected cells. We hypothesized that providing corrected cells with a selective growth advantage might overcome this possible limitation. We chose to test this in a murine model of PNH that has GPI-AP deficient blood cells because of a somatic Piga gene mutation targeted to the HSCs (LF mice). We designed a novel bicistronic lentiviral vector (MMIP) expressing the human PIGA cDNA and the human alkylating drug resistance mutant O6-methylguanine DNA methyltransferase (MGMT G156A) under the control of the MND promoter. MGMT G156A confers protection against alkylating agents. Eight LF donor mice were injected with 5-FU. Bone marrow (BM) was harvested 5 days later for MMIP transduction performed twice at MOI 100. Transduced and mock transduced BM cells were injected into 15 lethally irradiated C57Bl/6 recipient mice. One month after BM transplantation (BMT) 8 MMIP-transduced mice were treated with the alkylating agents BCNU and O6-benzylguanine (BG) (5mg/kg and 30mg/kg). BCNU /BC treatment was repeated twice, each one month apart. Six months after BMT the percentage of donor engraftment was 75.1 ± 25% for granulocytes (G) and 88.4 ± 26% for lymphocytes (L) in the control group (n=3). Donor chimerism in the MMIP-transduced group not treated with BCNU/BG was 84.6 ± 25 and 87.8 ± 5 % (n=4). The highest donor chimerism was obtained in mice treated with BCNU/BG, 98.9 ± 0.4 and 98.6 ± 0.5 % (n=8). Next we determined the proportion of donor blood cells with a restored expression of GPI-linked proteins. Only a small proportion of GPI-AP expressing cells was found in mice receiving the mock transduced BM six months after BMT, 22±37% GPI-AP expressing RBCs; 3.1± 2% GPI-AP expressing G, and 0.5 ± 0.2% GPI-AP expressing L. In contrast, mice receiving the MMIP-transduced BM cells but not treated with BCNU/BG showed a higher level of GPI-AP+ cells: 87.8±16% for RBCs, 70.2 ±23% for G and 56.3 ± 13% for L (n=4). However, mice receiving the MMIP-transduced BM and BG/BCNU treatment, achieved an almost complete restoration of GPI-AP on peripheral blood cells: 98±3% of RBCs, 96.4 ±4.7% of G and 89.6 ± 13% of L (n=8). Findings in BM, in methylcellulose progenitor assays, and in secondary spleen colony assays confirmed that restoration of GPI-AP and BCNU/BG selection had occurred at the HSC level. This is the first report of long-term restoration of GPI-APs expression in vivo using a murine model for PNH. Our results demonstrate that BCNU/BG treatment is well tolerated (100% survival) leading to a strong in vivo selection of HSCs with restored expression GPI-AP. The selective growth advantage of converted HSCs after dual gene therapy followed by BCNU/BG treatment allows us to achieve and possibly maintain a high level of hematopoiesis with restored GPI-AP expression. The selection for the corrected HSCs might allow them to overcome a possible growth disadvantage in the competition between PNH and normal HSCs.

Electrochemical measurements confirm the preferential bonding of the antimetastatic complex 6ImH96RuCl4(DMSO)(Im)9 (NAMI-A) with proteins and the weak interaction with nucleobases*1

An electrochemical and biological study of interaction between the prototypical antimetastatic drug imidazolium trans -tetrachlorodimethylsulfoxideimidazoleruthenate (III) complex, [ImH][RuCl 4 (DMSO)(Im)] (DMSO = dimethylsulfoxide, Im = imidazole), nicknamed NAMI-A, and several biomolecules, namely DNA, bovine (BSA) and human (HSA) serum albumin, is reported. Electrochemistry offers great advantages over the existing devices based on optical techniques, since it provides rapid, simple, and low-cost information whether the interaction occurs or not. Moreover, we describe some biochemical assays to test the interaction of NAMI-A with ribonucleoprotein telomerase and protein Taq polymerase. All the data confirm the preferential interaction of NAMI-A with proteins with respect to nucleotides, especially when compared with the behaviour of the well-known alkylating drug cisplatin in the presence of the same targets.

In vivo kinetics of micronuclei induction by bifunctional alkylating antineoplastics.

The aim of the present study was to determine in vivo the kinetics of micronucleated polychromatic erythrocyte (MN-PCE) induction in mice, as an approach for studying the mechanism of micronuclei induction by mitomycin C, cis-diamine dichloroplatinum, busulfan and bis-chloroethylnitrosourea, bifuctional alkylating antineoplastic agents having different patterns of crosslink induction. The kinetics of MN-PCE induction was established by scoring the frequency of MN-PCE in 2000 PCE in peripheral blood, for periods of 8 or 10 h after acute treatment and up to 80 h, with different doses of the agent. The kinetics of MN-PCE induction and particularly the times of maximal induction by different bifunctional alkylating agents were compared with the kinetics previously obtained for ethylnitrosourea, methylnitrosourea and 6-mercaptopurine, agents that cause MN-PCE mainly in the first, second and third divisions after exposure, respectively. The results obtained in the present study allow us to conclude that: (i) bifunctional alkylating agents have very different efficiencies of genotoxic and cytotoxic action; (ii) all assayed bifunctional alkylating agents induced micronuclei during the first cell division, owing to the mistaken repair of primary lesions, e.g. excision; (iii) busulfan and bis-chloroethylnitrosourea showed an additional late mechanism of micronuclei induction, which is expressed at the third division and seems to be related to the mismatch repair process.

Electrochemical measurements confirm the preferential bonding of the antimetastatic complex [ImH][RuCl 4(DMSO)(Im)] (NAMI-A) with proteins and the weak interaction with nucleobases

An electrochemical and biological study of interaction between the prototypical antimetastatic drug imidazolium trans-tetrachlorodimethylsulfoxideimidazoleruthenate (III) complex, [ImH][RuCl 4(DMSO)(Im)] (DMSO = dimethylsulfoxide, Im = imidazole), nicknamed NAMI-A, and several biomolecules, namely DNA, bovine (BSA) and human (HSA) serum albumin, is reported. Electrochemistry offers great advantages over the existing devices based on optical techniques, since it provides rapid, simple, and low-cost information whether the interaction occurs or not. Moreover, we describe some biochemical assays to test the interaction of NAMI-A with ribonucleoprotein telomerase and protein Taq polymerase. All the data confirm the preferential interaction of NAMI-A with proteins with respect to nucleotides, especially when compared with the behaviour of the well-known alkylating drug cisplatin in the presence of the same targets.

A bicistronic SIN-lentiviral vector containing G156A MGMT allows selection and metabolic correction of hematopoietic protoporphyric cell lines.

Erythropoietic protoporphyria (EPP) is an inherited disease characterised by a ferrochelatase (FECH) deficiency, the latest enzyme of the heme biosynthetic pathway, leading to the accumulation of toxic protoporphyrin in the liver, bone marrow and spleen. We have previously shown that a successful gene therapy of a murine model of the disease was possible with lentiviral vectors even in the absence of preselection of corrected cells, but lethal irradiation of the recipient was necessary to obtain an efficient bone marrow engraftment. To overcome a preconditioning regimen, a selective growth advantage has to be conferred to the corrected cells. We have developed a novel bicistronic lentiviral vector that contains the human alkylating drug resistance mutant O(6)-methylguanine DNA methyltransferase (MGMT G156A) and FECH cDNAs. We tested their capacity to protect hematopoietic cell lines efficiently from alkylating drug toxicity and correct enzymatic deficiency. EPP lymphoblastoid (LB) cell lines, K562 and cord-blood-derived CD34(+) cells were transduced at a low multiplicity of infection (MOI) with the bicistronic constructs. Resistance to O(6)-benzylguanine (BG)/N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) was clearly shown in transduced cells, leading to the survival and expansion of provirus-containing cells. Corrected EPP LB cells were selectively amplified, leading to complete restoration of enzymatic activity and the absence of protoporphyrin accumulation. This study demonstrates that a lentiviral vector including therapeutic and G156A MGMT genes followed by BG/BCNU exposure can lead to a full metabolic correction of deficient cells. This vector might form the basis of new EPP mouse gene therapy protocols without a preconditioning regimen followed by in vivo selection of corrected hematopoietic stem cells.

Synthesis and in vivo biodisposition of [14C]-quaternary ammonium-melphalan conjugate, a potential cartilage-targeted alkylating drug.

For the purpose of developing more selective anticancer drugs that would concentrate in the malignant cartilaginous tumors (chondrosarcomas), and so improve therapeutic index through a reduction of side effects, a quaternary ammonium (QA) conjugate of melphalan was synthesized and labeled with (14)C by linking the QA moiety to nitrogen mustard via an amide bond. Comparative pharmacokinetic study of [(14)C]-melphalan and its [(14)C]-QA conjugate conducted on rats showed that the two compounds were principally excreted by the urinary way. The blood elimination of the QA conjugate was faster than that of the melphalan. In the other hand a higher rate of radioactivity derived of [(14)C]-MQA was found in feces. In the biodisposition for most organs, no striking differences were found between melphalan and its QA conjugate except for cartilages which exhibited more higher radioactivity level. Amounts of radioactivity derived from [(14)C]-QA conjugates measured in cartilaginous tissues until 1 h after injection demonstrate that the introduction of a QA moiety on melphalan allows the molecule to be carried selectively to cartilaginous tissues. As the [(14)C]-QA conjugate is radiolabeled on the chloroethyl alkylating moiety, levels of radioactivity measured in the cartilaginous tissues results from unchanged compound or metabolite having kept the active group.