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Background: Nitrosoureas are chemotherapeutic alkylating agents, widely used in the treatment of malignant diseases. One of them (streptozotocin) is the standard for the therapy of neuroendocrine tumours. We synthesised a nitrosourea compound, aranoza, which has been effective in preclinical studies on experimental tumour models. Objectives: The current research deals with the development of the pharmaceutical dosage form of aranoza and the evaluation of the quality of the finished product. Methods: Sterilizing filtration followed by freeze drying was used to prepare the finished product. The acidity of the solutions was determined potentiometrically. Aranoza content in solution and lyophilisate was determined by spectrophotometry method. Results: We suggested the composition of the drug for parenteral use. The created product includes polyvinylpyrolidone and sorbic acid for solution stabilization and regulation of acidity, respectively. We also selected the method for aranozasolution preparation and showed that filters of mixed cellulose esters have optimal characteristics for filtration sterilisation. We chose lyophilisation to obtain a stable product: the freezing temperature was determined as -40°C to -45°C and the duration of freezing as 3–4 hr. The finished product has characteristics corresponding to requirements of the European Pharmacopoeia and State Pharmacopoeia of the Russian Federation and maintained its quality for the time of observation of 36 months. Conclusion: We created the algorithm for the development of a lyophilised antitumor drug, which allowed to obtain an appropriate pharmaceutical dosage form of a nitrosourea compound, aranoza, purposed for the treatment of neuroendocrine tumours.

Implementation of the seventh amendment to the EU Cosmetics Directive has driven much research into suitable in vitro alternative assays to support satisfactory risk assessments. One such assay is the reconstructed skin micronucleus (RSMN) assay. First reported in 2006, further development occurred and a standard protocol was published in 2011. To evaluate and optimise the assay at Covance Laboratories, we tested nine chemicals [4-nitrophenol (4-NP), cyclohexanone (CH), 2-ethyl-1,3-hexanediol (2-EHD), methyl methansulfonate (MMS), mitomycin C (MMC), ethyl nitrosourea (ENU), benzo[a]pyrene (BaP), cyclophosphamide (CPA) and vinblastine (VIN)] using the EpiDerm™ 3D skin model (MatTek Corporation®, IVLSL, Bratislava, Slovakia) and compared the data using the standard 48-h treatment regimen and also an emerging 72-h treatment protocol. The EpiDerm™ tissue has reportedly some metabolic capacity but data using 48-h treatments has provided mixed results. Our investigations demonstrate that the two chemicals requiring metabolic activation (BaP and CPA) were negative following the 48-h protocol but were clearly positive following 72-h treatment. Furthermore, Replication Index (RI) data showed higher RI values in vehicle control treatments (indicating increased cell division) across the treatment set following 72-h treatments. A general greater magnitude of micronucleus (MN) induction was also observed following test chemical treatment. These data suggest that the 72-h treatment protocol is more suitable as a standard approach for the detection of clastogenic, aneugenic and metabolically activated chemicals in the RSMN assay. For further assay optimisation, we compare the statistical power of scoring cells from duplicate or triplicate cultures per treatment concentration and provide recommendations.

Background: Glioblastoma multiforme (GBM) is a rare and deadly disease, with a reported average incidence rate of 3.19 cases per 100,000 inhabitants. Fotemustine, a third-generation nitrosourea with an alanine phosphor carrier that facilitates cellular penetration, has been extensively investigated in the setting of recurrent/progressive disease after initial treatment. Fotemustine is usually administered following a schedule consisting of 3 doses every week, followed by maintenance doses administered every 3 weeks. Methods: In this phase I/II trial, we aimed to assess whether the use of a biweekly regimen allowed administration of higher dose than the standard 100mg/m2 dose approved per label indication in a population of patients with recurrent GBM. In this phase I/II trial, fotemustine was administered intravenously over 1hour every 2 weeks at either 120 or 140mg/m2 doses for up to 1 year, until disease progression, unacceptable toxicity, or patient's request to withdraw from the study. The phase I part of the trial was conducted following the classic 3+3 study design. The phase II part of the trial was a single-arm study. The primary efficacy endpoint was the percentage of patients who had not progressed after 24 weeks (PFS-24). Results: Thirty-seven patients were enrolled in this phase I/II trial from August 2006 to November 2011. Treatment was well tolerated in the overall population. Main severe toxicity was grades 3 and 4 thrombocytopenia, which occurred in 4 of 6 patients treated at the 140mg/m2 dose level and in 3 of 31 patients treated at 120mg/m2. Median PFS and overall survival were 12.1 (1-40.2) weeks and 19.7 (1-102) weeks, respectively. Conclusion: We conclude that fotemustine can be safely administered at 120mg/m2 biweekly. The efficacy of such modified schedule and doses should be compared to the biweekly schedule at 80mg 2 and the standard weekly schedule at 80 to 100mg/m2.

BACKGROUND Despite decades of research, nearly all patients with glioblastoma (GBM) face recurrent disease. Temozolomide (TMZ), the drug of choice for GBM, methylates guanine bases at the O6 position (O6meG). These lesions are normally repaired by O6meG-methyltransferase (MGMT). When MGMT is deficient (e.g. in the case of promoter hypermethylation), O6meG lesions generate mispairs with thymine and activate the mismatch repair system (MMR), which is thought to be critical for the cytotoxicity of TMZ. Numerous studies have established that acquired MMR deficiency is a common mechanism of TMZ resistance that emerges in recurrent GBM. However, reversion of MGMT-methylation status is not common in recurrent GBM. Recent work from our group demonstrated that TMZ synergizes with ATR inhibitors (ATRi’s) in MGMT-methylated GBM cells. The goal of this project was to identify the specific types of alkylating agents that synergize with ATRi’s in an MGMT-methylated, MMR-deficient molecular context. RESULTS We used GBM cell lines U251 and LN229, which are both MGMT-methylated and exquisitely sensitive to TMZ in combination with ATRi at baseline, and created MMR-deficient versions through stable knockdown of MSH2. We determined that MSH2 knockdown conferred remarkable resistance in U251 and LN229 to TMZ treatment in comparison to their respective MMR-proficient counterparts. In addition, we were unable to detect synergy between ATRi’s and TMZ in the setting of MSH2 knockdown. In contrast to TMZ, we found that treatment with chloroethyl nitrosourea (CNU) alkylating agents--including lomustine--with ATRi’s was extremely effective in killing MGMT-methylated, MMR-deficient GBM cells. CONCLUSION Here, we report an exquisite synergistic interaction between ATRi’s and CNU agents in MMR-deficient, MGMT-methylated GBM cells. Future work in the lab will examine the interaction of CNU and ATRi’s with radiation therapy (RT). Because ATRi’s are known radiosensitizers, we believe that novel combinations of ATRi’s, lomustine, and re-RT may have promise in recurrent GBM.

The drug resistance of CENUs induced by O(6)-alkylguanine-DNA alkyltransferase (AGT), which repairs the O(6)-alkylated guanine and subsequently inhibits the formation of dG-dC cross-links, hinders the application of CENU chemotherapies. Therefore, the discovery of CENU analogs with AGT inhibiting activity is a promising approach leading to novel CENU chemotherapies with high therapeutic index. In this study, a new combi-nitrosourea prodrug 3-(3-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)-1-(2-chloroethyl)-1-nitrosourea (6), designed to release a DNA cross-linking agent and an inhibitor of AGT, was synthesized and evaluated for its antitumor activity and ability to induce DNA interstrand cross-links (ICLs). The results indicated that 6 exhibited higher cytotoxicity against mer(+) glioma cells compared with ACNU, BCNU, and their respective combinations with O(6)-benzylguanine (O(6)-BG). Quantifications of dG-dC cross-links induced by 6 were performed using HPLC-ESI-MS/MS. Higher levels of dG-dC cross-link were observed in 6-treated human glioma SF763 cells (mer(+)), whereas lower levels of dG-dC cross-link were observed in 6-treated calf thymus DNA, when compared with the groups treated with BCNU and ACNU. The results suggested that the superiority of 6 might result from the AGT inhibitory moiety, which specifically functions in cells with AGT activity. Molecular docking studies indicated that five hydrogen bonds were formed between the O(6)-BG analogs released from 6 and the five residues in the active pocket of AGT, which provided a reasonable explanation for the higher AGT-inhibitory activity of 6 than O(6)-BG.

Nucleosides can be considered as specific carriers for the nitrosourea moiety with altered pharmacological properties such as improved water solubility, lower myelotoxicity with enhanced cell membrane transport. Several seconucleoside nitrosourea compounds have been reported to have anticancer activity against solid tumors such as, murine adenocarcinomas of the colon 13 (MAC 13), MAC 15A, colon 38 and mammary carcinoma. In an effort to have a good understanding of the role of the structural features on the anticancer activity of these seconucleosides, we have constructed the quantitative structure activity relationship (QSAR) involving 26 seconucleoside nitrosourea compounds having anticancer activity against murine adenocarcinomas of the colon 15A (MAC 15). Traditional QSAR studies require knowledge of several physicochemical properties to obtain a good QSAR. To circumvent this problem, we have made use of quantum chemical methods to calculate several electronic and molecular properties of these compounds and used these properties to obtain the best QSAR using statistical procedures. Semi-empirical quantum chemical RM1 methods were used to optimize the molecular structures. From the optimized structure, hundreds of molecular and electronic properties were calculated and correlated with the anticancer activities of the compounds. The best correlation was obtained using heuristic and multi-linear regression methods. We were able to obtain a best QSAR with correlation coefficient, R2=0.8993. We find that the average structural information contents of the second order, average bond order of oxygen atom and maximum columbic interaction for H-N bond play significant roles in influencing the anticancer activity of the selected compounds. The QSAR obtained in our study can be used to predict the anticancer activity of new seconucleoside nitrosourea analogs, prior to resorting to any experimental studies.

A theoretical study has been carried out for internal rotation of nitrosoureas at the B3LYP/6-311G* level of theory. For each nitrosourea compound, two ground state structures have been found and the E isomer is predicted to be more stable than the Z isomer. Two transition state conformations for the isomerization have also been obtained and the calculated results show that the isomerization through TS1 is easier than that through TS2. The relationship between energy barrier and toxicity has also been investigated. It is concluded that the carcinogenic potency increases along with the decrease of rotational energy barrier.

Background: A meta-analysis of chemotherapy for glioblastoma multiforme (GBM) was performed. We sought to update prior analyses by focusing exclusively on GBM, including new trials of novel treatments, assessing effectiveness of individual treatment categories and presenting data in a clinically useful format. Methods: A search of MEDLINE and EMBASE was conducted for randomised controlled trials of chemotherapy in GBM. Results: Relative risks (RRs) for survival in 16 trials comparing chemotherapy with no chemotherapy were 1.18 (95% CI 1.08, 1.30) at 6 months, 1.53 (95% CI 1.26, 1.86) at 12 months and 2.12 (95% CI 1.60, 2.80) at 24 months. Nitrosourea compounds, local therapy (e.g. carmustine [1,3-bis [2-chloroethyl]-1-nitrosourea] wafers) and temozolomide were all more effective than no chemotherapy. Absolute increases in survival at 6, 12 and 24 months were 11%, 8% and 1%, respectively, for nitrosourea compounds; 8%, 24% and 5%, respectively, for local therapy; and 4%, 15% and 17%, respectively, for temozolomide. Efficacy of local therapy and temozolomide peaked at 12 and 18 months, respectively. After 2 years, nitrosourea compounds no longer provided clinically relevant benefit (number needed-to-treat [NNT] = 100; effect size [ES] = 0.17 SD), local therapy had diminishing returns (NNT = 20) that remained clinically relevant (ES = 0.71 SD) and temozolomide continued to show good efficacy (NNT = 5.9; ES = 0.74 SD). Survival was not significantly improved with multi-agent versus single-agent nitrosourea-based therapy in five trials: 6-month RR 0.91 (95% CI 0.71, 1.16); 24-month RR 1.33 (95% CI 0.72, 2.46). Conclusion: Although nitrosourea compounds, local therapy and temozolomide are all effective in the treatment of GBM, local therapy and temozolomide may be associated with greater response, with clinically significant benefits extending to 24 months. The timing of peak benefits of local and temozolomide therapy suggests this combination may be more effective than single-agent chemotherapy and warrants further study.

Zinc has been shown to be accumulated in N-methyl-N-nitrosourea (MNU)-induced rat mammary tumors. Zinc is required for cell proliferation and tumorigenesis is characterized by dysregulation of cell proliferation. An accumulation of zinc in mammary tumors perhaps indicates a reliance on zinc to sustain tumor growth. Limiting zinc supply by means such as reduced zinc intake should negatively modulate mammary tumorigenesis. Our objective was to determine the effects of zinc status on MNU-induced mammary tumorigenesis in sexually mature female rats. Twenty-one-day-old Sprague-Dawley rats were assigned to low-zinc (3 mg zinc/kg diet) or adequate-zinc (12 mg zinc/kg diet) ad libitum or pair-fed control group (n = 25-33 rats/ group). On day 50 of age, all rats were intraperitoneally injected with MNU (50 mg/kg body wt) to induce mammary tumorigenesis. Rats were further maintained on their assigned diet until 14 weeks post-MNU injection. Total food intake and overall body weight gain were lower in low-zinc rats than in adequate-zinc ad libitum control rats, but were similar to adequate-zinc pair-fed control rats. Plasma zinc concentration was lower in low-zinc rats than in adequate-zinc ad libitum and pair-fed control rats, confirming moderately low-zinc status in low-zinc rats. Tumor incidence (46 versus 84 and 80%; P < 0.05) and tumor multiplicity (0.8 versus 5.0 and 2.6 tumors/rat; P < 0.05) and tumor number (28 versus 123 and 66 tumors) were reduced in low-zinc rats compared with that in adequate-zinc ad libitum and pair-fed control rats, respectively. Tumor latency in low-zinc and adequate-zinc pair-fed control rats was not significantly different, but was longer than in adequate-zinc ad libitum control rats (P < 0.05), suggesting that reduced food intake associated with low-zinc intake prolonged tumor latency. Tumor burden and size were not affected by zinc intake. Overall, our observations showed that moderately low-zinc status suppressed MNU-induced rat mammary tumorigenesis.

Phospholipid metabolism is tightly involved in tumor growth regulation and tumor cell survival. The response of phospholipid metabolism to chloroethyle nitrosourea treatment is investigated in a murine B16 melanoma model. Measurements of phospholipid derivatives are performed on intact tumor tissue samples using one- and two-dimensional proton NMR spectroscopy. During the tumor growth inhibition phase under treatment, tumors overexpress phosphocholine, phosphoethanolamine, glycerophosphocholine and glycerophosphoethanolamine, whereas phosphatidylcholine and phosphatidylethanolamine levels are maintained to control levels. During re-growth, which remained quantitatively much below control growth, chloroethyle nitrosourea-treated melanoma tumors overexpress phosphocholine and phosphoethanolamine only. In treated melanoma, phosphatidylcholine levels show an inverse relationship with tumor growth rates. In conclusion, chloroethyle nitrosourea-treated melanoma tumors maintain their phosphatidylcholine levels and exhibit transformed phospholipid metabolism phenotype, by mechanisms that could participate in tumor cell survival.

Tumor-treating fields (TTF) increase patient survival in glioblastoma, and are approved for recurrent (2011) and de novotumors (2016). Since 2012, 52 patients with gliomas at Columbia University were treated with TTF. Given preclinical data suggesting synergy between TTF and PD1-targeted therapy, the rates and outcomes of combination TTF and immunotherapy were analyzed. Of 52 patients, 40% were women. Median age at diagnosis was 58 (23-84) years. WHO glioma grade was II (6%), III (10%), or IV (84% of patients). TTF was used at recurrence in 42 (81%), with 0-4 prior recurrences. Prior therapy included bevacizumab (20%), resection (gross-total in 54%), radiation (60Gy in 85%), and temozolomide (100%). 11 (75%) patients met a compliance goal of 75%. Only 2 (4%) experienced skin toxicity requiring interruption. Concurrent systemic therapy included temozolomide (31%), nitrosourea (8%), bevacizumab (37%), or immunotherapy (54% of patients). At interim follow-up, progression free (PFS) and overall (OS) survival were 3.6 and 21.5 months, respectively. Objective response rate (ORR) based on RANO was increased in patients treated with concurrent immunotherapy (26% vs. 13%). Among GBM patients treated with concurrent immunotherapy, median PFS was significantly increased (4.6 vs. 3.5 mo, p=0.04), and survival trended upward (8.3 vs. 6.5 mo, p=0.32). After long-term follow-up, PFS, OS, and impact of compliance rates will be re-analysed. TTF improves survival for glioblastoma patients and immunotherapy shows promise in other cancers. The possible synergy between these modalities in preclinical studies and this retrospective series warrants further study in a randomized controlled trial. We propose a single-arm phase II trial of concurrent PD1 inhibitor and TTF for patients with bevacizumab-naïve glioblastoma, at diagnosis or recurrence. OS is the primary endpoint; secondary endpoints include PFS, ORR, and predictive value of PDL1 expression, and impact of compliance. Results from the EF-14 trial will serve as a historical control.

Adding lomustine to the standard combination of idarubicin and cytarabine for the treatment of old patients with acute myeloid leukemia improves complete response rates and overall survival. These findings emphasize the importance of dose intensity and raise the question of a unique role for nitrosoureas in the treatment of this disease.

Glioblastomas are highly aggressive adult brain tumors with poor clinical outcome. In the central nervous system (CNS) the blood-brain barrier (BBB) is the most important limiting factor for both development of new drugs and drug delivery. Here, we propose a new strategy to treat glioblastoma based on transferrin (Tf)-targeted self-assembled nanoparticles (NPs) incorporating zoledronic acid (ZOL) (NPs-ZOL-Tf). NPs-ZOL-Tf have been assessed on the glioblastoma cell line U373MG-LUC that showed a refractoriness in vitro to temozolomide (TMZ) and fotemustine (FTM). NPs-ZOL-Tf treatment resulted in higher in vitro cytotoxic activity than free ZOL. However, the potentiation of anti-proliferative activity of NPs-ZOL-Tf was superimposable to that one induced by NPs-ZOL (not armed with Tf). On the other hand, NPs-ZOL-Tf showed a higher antitumor efficacy if compared with that one caused by NPs-ZOL in immunosuppressed mice intramuscularly bearing U373MG-LUC xenografts, inducing a significant tumor weight inhibition (TWI). The experiments performed on mice with intracranial U373MG-LUC xenografts confirmed the efficacy of NPs-ZOL-Tf. These effects were paralleled by a higher intratumour localization of fluorescently-labeled-NPs-Tf both in intramuscular and intracranial xenografts. In conclusion, our results demonstrate that the encapsulation of ZOL increases the antitumor efficacy of this drug in glioblastoma through the acquisition of ability to cross the BBB.

The exposure of cells to O6-benzyl-N2-acetylguanosine (BNAG) and several guanine derivatives is known to reduce the activity of O6-alkylguanine-DNA alkyltransferase (MGMT) and to enhance the sensitivity of Mer+ (methyl enzyme repair positive) tumour cells to chloroethylnitrosoureas (CENUs) in vitro and in vivo. High water solubility and the pharmacokinetic properties of BNAG make it a candidate for simultaneous administration with CENUs by the i.v. route in human clinical use. In vivo we have shown previously that BNAG significantly increases the efficiency of N'-[2-chloroethyl]-N-[2-(methylsulphonyl)ethyl]-N'-nitrosourea (cystemustine) against M4Beu melanoma cells (Mer+) through its cytostatic activity by the i.p. route, but also increases its toxicity. To investigate the toxicity of BNAG and cystemustine when administered simultaneously in mice, we compared the maximum tolerated dose and LD50 doses of cystemustine alone or in combination with 40 mg kg(-1) BNAG by the i.p. route. The toxicity of cystemustine was enhanced by a factor of almost 1.44 when combined with BNAG. To compare the therapeutic index of cystemustine alone and the cystemustine/BNAG combination, pharmacological tests were carried out in nude mice bearing Mer+ M4Beu human melanoma cells. Isotoxic doses were calculated using the 1.44 ratio. The treatments were administered three times by the i.v. route on days 1, 5 and 9 after s.c. inoculation of tumour cells. Although the toxicities of the treatments were equal, BNAG strongly enhanced tumour growth inhibition. These results demonstrate the increase of the therapeutic index of cystemustine by BNAG and justify the use of BNAG to enhance nitrosourea efficiency in vivo by i.v. co-injection.

The antiproliferative activity of two nitrosourea-methionine-steroid conjugates, N-[N'-(2-chloroethyl)-N'-nitrosocarbamoyl] (CNC)-L-methionine-testosterone-17-ester and CNC-L-methionine-dihydrotestosterone-17-ester, was investigated in two meningioma low-passage primary cultures that had been derived from human meningiomas and characterized with regard to proliferation pattern and progesterone receptor content. Treatment was given over 4 days. Cytotoxicity (concentrations investigated: 50, 100, and 200 microM) was compared with the effects of the equimolar mixtures of CNC-Met plus steroid. The steroid-linked nitrosoureas were superior to their unlinked components. CNC-Met itself displayed no or marginal activity, whereas the steroid component displayed significant activity.

In the present study we investigated the susceptibility of high passages of the rat glial transplantation tumour G-XIII to chemotherapy using nitrosourea compounds. We observed a significant increase in lifespan (ILS) of animals treated with BCNU (37%, p0.01) and CCNU (27%, p0.01). There were no difference in the efficiency between these two substances. Using a semi-quantitative score system no histopathological changes were observed which were associated with the response to therapy. The only predicative parameter in the present study was the quantitative DNA distribution pattern. There was a close correlation between treatment and the occurrence of unimodal DNA distribution patterns indicating clonal regrowth of recurrent tumours. Moreover, we also observed a correlation of the DNA distribution pattern of recurrent tumours with the result of experimental chemotherapy. Survival times of animals suffering from tumours containing unimodal DNA histogram was significantly longer than survival times of rats with multimodal DNA distribution, i.e. bimodal or broad DNA histograms. A unimodal, near-diploid stem line was only present in treated animals suggesting that these clones are more resistant against therapy using nitrosourea compounds. Our data indicate DNA cytophotometry as comprehensive tool for the monitoring of therapy response and the design of experimental chemotherapy using rat glial tumours.

Alkylating agents, such as temozolomide (TMZ) and fotemustine (FTM) are widely used in recurrent glioblastoma (GBM) regimes. Several strategies have been proposed to prevent resistance to these agents, by combining or sequencing them. We report the results of a pilot study of patients with refractory GBM receiving a regime of twice-daily dosing of temozolomide administered on day 1, (with an initial oral dose of 200 mg/m(2) and a second oral dose of 75 mg/m(2) 12 h later), followed by fotemustine in a single i.v. infusion at 75 mg/m(2) on day 2, repeated every four weeks. Enrolment was stopped at 15 patients due to lack of effectiveness of this schedule for patients with GBM. Toxicity was mild, with no grade 4 side effects reported. Results indicate that our temozolomide -FTM combined schedule is not effective, although well tolerated, in non responsive patients with GBM. Further strategies are required to improve the outcome of these patients.

N-Nitroso-compound DNA adduct formation in vivo and occurrence of aberrant crypt foci (ACF) were studied in the rat colon mucosa after a single, local treatment with a carcinogen, N-methyl-N-nitrosourea (MNU), using a simple surgical approach. A segment of F344 rat colon was ligated to make a pouch and injected with MNU solution. For the study of DNA adduct formation, the solution contained 50 microCi of [3H]MNU. The results demonstrated that similar ranges of carcinogen dose, i.e. 0.15 x 10(-2) - 1.5 x 10(-2) M MNU, could induce both DNA adduct formation and appearance of ACF in the rat colon with both parameters showing a nearly linear dose dependence. HPLC analysis revealed the DNA adducts to include both 7-methylguanine (7-mGua) and O6-methylguanine (O6-mGua) with the 7-mGua/O6-mGua ratio being 8.2-11.3:1 in the system used. Assessment of ACF development from 4 to 16 weeks after MNU treatment at a dose of 7.5 x 10(-2) M showed the numbers to increase up to the 8th week, followed by a decrease at weeks 12 and 16, when 40% of the ACF counted at the peak time point were still present. The percentage of large ACF (> or = 4 crypts/ACF) significantly increased with time. These results indicate a clear relation between DNA adducts and preneoplastic lesions, i.e. ACF. In conclusion, DNA adduct formation and ACF can be efficiently and simply detected in vivo by using the method described in the present paper.

A combination of two new drugs — ifosfamide and nimustine (ACNU), a water-soluble nitrosourea compound — was studied as salvage therapy for small cell lung cancer (SCLC). A phase I trial determined that nimustine 75 mg/m2 and ifosfamide 4 g/m2 given intravenously on day 1 was associated with moderate leucopenia as the main toxicity. A phase II trial was then conducted in 25 patients. As a second-line treatment, after initial therapy with a multiple drug regimen, 2 of 19 (10.5%) patients had an objective response; no responses were observed in 6 patients who received IMA as third-line treatment. The responses were observed in patients who had good response to first-line chemotherapy with a long (> 5 months) treatment-free interval. IMA was shown to have limited activity as salvage therapy for SCLC. Further investigations are necessary to determine the exact role of IMA as first-line treatment for SCLC.

Fecapentaenes are a group of fecal mutagens produced by anaerobic microflora of the colon. The potential of fecapentaene-12 (FP-12) to promote tumor development was tested in a rat colon carcinogenesis model using N-methyl-N-nitrosourea (MNU) as the initiating agent. Two groups of female F-344 rats were initiated by intrarectal instillations of MNU (2 mg in 0.5 ml H2O, 3 times a week, for 3 weeks; MNU and MNU + FP-12 groups). Two additional groups (FP-12 and Control) were given H2O without carcinogen. In the post-initiation phase, rats of the MNU + FP-12 and FP-12 groups were intrarectally administered 400 ng of FP-12 in 0.5 ml T-E buffer, twice a week, for 24 weeks, whereas the MNU and Control groups received the vehicle only. Tumors were found only in the MNU and MNU + FP-12 groups, their number being higher in the latter. The number of carcinoma bearing rats as well as the average number of carcinomas per rat were significantly higher (P0.05) in the MNU + FP-12 group as compared to the MNU-alone values. Aberrant crypt foci (ACF) were found in all carcinogen-treated rats, including those that did not contain tumors, whereas none were observed in the FP-12 and Control groups. The average number of ACF/cm2 was also significantly higher in the MNU+FP-12 group, as was the case for the average number of ACF containing10 aberrant crypts per focus. These findings suggest that FP-12 can express promoting activity in chemical induced colon carcinogenesis.

We observed the tissue delivery of a novel water-soluble nitrosourea, 1-(2-chloroethyl)-3-(methyl-alpha-D-glucopyranos-6-yl)-1-nitros our ea (MCNU) in rats bearing experimental brain tumors by conducting autoradiography on all. Prior to this study, the development of a streaming phenomenon was ascertained (and thus finding the optimum velocity for intra-arterial infusion) by 14C-iodoantipyrine (IAP) autoradiography. Furthermore, a single pass extraction value of MCNU was measured. At an arterial infusion rate of 0.2 ml/min., the streaming phenomenon was recognized but the tracer was fairly evenly distributed at a rate of 1.0 ml/min. On the other hand, the single pass extraction value for MCNU was 0.18 +/- 0.036 (mean +/- S.D., n = 3, under pentobarbital anesthesia). It was suggested that MCNU is very unlikely to be transported into the normal rat brain. We conducted 14C-MCNU autoradiography to observe tissue distribution of MCNU following its intra-arterial and intravenous infusions in a brain tumor model using rats. The normal side (the side where no infusions were given) and the cerebral cortex at the side affected by the tumor (the side where the infusion was given) showed hardly any uptake of 14C-MCNU in both the intra-arterial and intravenous infusion groups. The tumorous section was divided into the periphery and the center to measure tissue concentration of the tracer in each section. Compared against the cortical section, the periphery and the center showed significant increases in the concentration (approximately 11 to 15 times and 3 to 7 times, respectively, the figure for the cortical region) for both the intra-arterial and intravenous groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The denitrosation of a novel nitrosourea, 1-(2-chloroethyl)-3-[(2-dimethylaminosulfonyl)-ethyl]-1-nitrosoure a, tauromustine (TCNU), has been investigated in liver cytosol from various species and with a pool of cytosolic rat liver glutathione transferases (GSTs). In addition, the denitrosation of related nitrosoureas, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) have been investigated. By using radioactive nitrosoureas as substrates and HPLC to detect the denitrosated products, a sensitive assay has been developed. The activities towards additional substrates, 1-chloro-2,4-dinitrobenzene (CDNB) and trans-stilbene oxide (tSBO) were also measured in the various cytosol fractions. The apparent Vmax and Km values for the denitrosation of TCNU in rat liver cytosol were calculated to be 830 pmol/min/mg protein and 8.4 mM respectively. The rate of denitrosation of TCNU was found to be highest in the mouse, followed by the rat and the dog. The induction of GSTs by phenobarbital in rats increased the denitrosation of TCNU similar to the activities found towards CDNB and tSBO as substrates. The highest denitrosation activity by GST was determined towards BCNU, followed by CCNU and TCNU. Our results suggest that the denitrosation of various nitrosoureas via GST may play an important role in the deactivating process occurring in vivo and for the efficacy of nitrosoureas used in chemotherapy.

The initial histological changes of leukemia were investigated in rats to which 1-ethyl-1-nitrosourea and 1-butyl-1-nitrosourea were orally administered. The appearance of orthochromatic erythroblasts in the peripheral blood was used as the index of the initial stage of leukemia. The rat leukemia progressed from solitary lesions to scattered and further diffuse lesions. These leukemias are thought to begin as one, or only a few nodular foci, mainly in the bone marrow and partly in the spleen.

Chemoresistance to O(6)-alkylating agents is a major barrier to successful treatment of melanoma. It is mainly due to a DNA repair suicide protein, O(6)-alkylguanine-DNA alkyltransferase (AGT). Although AGT inactivation is a powerful clinical strategy for restoring tumor chemosensitivity, it was limited by increased toxicity to nontumoral cells resulting from a lack of tumor selectivity. Achieving enhanced chemosensitization via AGT inhibition preferably in the tumor should protect normal tissue. To this end, we have developed a strategy to target AGT inhibitors. In this study, we tested a new potential melanoma-directed AGT inhibitor [2-amino-6-(4-iodobenzyloxy)-9-[4-(diethylamino) ethylcarbamoylbenzyl] purine; IBgBZ] designed as a conjugate of O(6)-(4-iododbenzyl)guanine (IBg) as the AGT inactivator and a N,N-diethylaminoethylenebenzamido (BZ) moiety as the carrier to the malignant melanocytes. IBgBZ demonstrated AGT inactivation ability and potentiation of O(6)-alkylating agents (cystemustine, a chloroethylnitrosourea) in M4Beu highly chemoresistant human melanoma cells both in vitro and in tumor models. The biodisposition study on mice bearing B16 melanoma, the standard model for the evaluation of melanoma-directed agents, and the secondary ion mass spectrometry imaging confirmed the concentration of IBgBZ in the tumor and in particular in the intracytoplasmic melanosomes. These results validate the potential of IBgBZ as a new, more tumor-selective, AGT inhibitor in a strategy of melanoma-targeted therapy.

According to the "combi-targeting" concept, the EGFR tyrosine kinase (TK) inhibitory potency of compounds termed "combi-molecules" is critical for selective growth inhibition of tumor cells with disordered expression of EGFR or its closest family member erbB2. Here we report on the optimization of the EGFR TK inhibitory potency of the combi-molecules of the nitrosourea class by comparison with their aminoquinazoline and ureidoquinazoline precursors. This led to the discovery of a new structural parameter that influences their EGFR TK inhibitory potency, i.e., the torsion angle between the plane of the quinazoline ring and the ureido or the nitrosoureido moiety of the synthesized drugs. Compounds (3'-Cl and Br series) with small angles (0.5-3 degrees ) were generally stronger EGFR TK inhibitors than those with large angles (18-21 degrees ). This was further corroborated by ligand-receptor van der Waals interaction calculations that showed significant binding hindrance imposed by large torsion angles in the narrow ATP cleft of EGFR. Selective antiproliferative studies in a pair of mouse fibroblast NIH3T3 cells, one of which NIH3T3/neu being transfected with the erbB2 oncogene, showed that IC(50) values for inhibition of EGFR TK could be good predictors of their selective potency against the serum-stimulated growth of the erbB2-tranfected cell line (Pearson r = 0.8). On the basis of stability (t(1/2)), EGFR TK inhibitory potency (IC(50)), and selective erbB2 targeting, compound 23, a stable nitrosourea, was considered to have the structural requirements for further development.

Red meat consumption is associated with endogenous metabolic generation of mutagenic N-nitroso compounds (NOC) and may be implicated in causation of colorectal cancer. Assessment of a biologically relevant dose of NOCs is hampered by imperfect understanding of NOC interactions with other dietary components. This study tests the hypothesis that NOC effects upon mutational biomarkers in mouse colon may be modulated by a non-genotoxic diet-related compound. N-methyl-N-nitrosourea (MNU) and undegraded lambda carrageenan (lambdaCgN) were selected as test chemicals, representing a NOC and a non-genotoxic agent, respectively. Study end-points included (i) DNA adduct formation and (ii) metallothionein (MT) crypt restricted immunopositivity indices (MTCRII) which are considered representative of crypt stem cell mutations. Frequency and size of MT immunopositive foci as well as total number of MT immunopositive crypts were assessed. Biologically effective doses of MNU and lambdaCgN were determined in model validation studies and the agents were then tested alone and in combination. Continuous lambdaCgN treatment for 10 weeks induced significantly greater colonic mucosal injury than a drinking water control. In combined treatment regimens, lambdaCgN treatment did not significantly affect MNU-induced DNA adduct formation. However, combinations of lambdaCgN with MNU significantly increased MTCRII in excess of those induced by MNU alone. Recurrent or continuous lambdaCgN regimens had greater interactive effects with MNU upon MTCRII than short-term lambdaCgN treatment. This study has shown that exposure to a non-genotoxic diet-related compound (lambdaCgN) modulates the effective NOC dosimetry for induction of MT crypt restricted immunopositivity.

Physicochemical properties, such as half life time (tau0.5), alkylating and carbamoylating activity and in vivo antimelanomic effects against B16 melanoma of spin labeled (containing nitroxyl free radical moiety) amino acid nitrosoureas, synthesized in our laboratory, have been studied and compared to those of the antitumor drug N'-cyclohexyl-N-(2-chloroethyl)-N-nitrosourea (lomustine, CCNU). We have shown that the introduction of amino acid moieties and the replacement of cyclohexylamine with nitroxyl moiety leads to a faster decomposition, higher alkylating, lower carbamoylating activity, better antimelanomic activity and lower general toxicity, when compared to those of CCNU. It was also established that spin labeled triazenes, previously synthesized by us, were more stable in phosphate saline than their nonlabeled analogue, 5-(3,3-dimethyltriazene-1-yl)-imidazole-4-carboxamide (dacarbazine, DTIC). A higher cytotoxicity to B16 melanoma cells than to YAC-1 and lymphocytes was demonstrated for all spin labeled triazenes, in comparison with DTIC. An assumption has been made to explain the lower general toxicity of the spin labeled nitrosoureas compared to that of CCNU. Based on the results presented, we accept that a new trend for synthesis of more selective and less toxic nitrosourea and triazene derivatives as potential antimelanomic drugs might be developed.

Adverse pulmonary reactions resulting from the use of chemotherapeutic agents are a life-threatening complication of modern cancer therapy. Their recognition and differentiation from lung injuries with infectious or other causes present a continuing diagnostic challenge to physicians treating patients with cancer. Many commonly used chemotherapeutic agents cause pulmonary injury and fibrosis. Until now, bleomycin has been the prototype, with fibrosis occurring in approximately 10 percent of patients receiving this agent, in whom it is fatal in roughly 10 percent.1 Adverse pulmonary reactions have also been associated with other cytotoxic agents, including busulfan, cyclophosphamide, chlorambucil, methotrexate, cytarabine, and the nitrosourea compounds; . . .

A relatively simple, high-sensitivity gas chromatographic assay is described for nitrosourea compounds, such as BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea] and MeCCNU [1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea], in small biopsy samples of brain and other tissues. After extraction with ethyl acetate, secondary amines in BCNU and MeCCNU are derivatized with trifluoroacetic anhydride. Compounds are separated and quantitated by gas chromatography using a capillary column with temperature programming and an electron capture detector. Standard curves of BCNU indicate a coefficient of variance of 0.066 +/- 0.018, a correlation coefficient of 0.929, and an extraction efficiency from whole brain of 68% with a minimum detectable amount of 20 ng in 5-10 mg samples. The assay has been facile and sensitive in over 1000 brain biopsy specimens after intravenous and intraarterial infusions of BCNU.

In the present study 34 agents, related to carcinogenesis in different ways, were investigated with respect to their recombinogenic activity in mammalian cells. The induction of intrachromosomal recombination was studied using the spontaneous mutant clone SP5 derived from V79 Chinese hamster cells, which exhibits a duplication of exon 2 and its flanking regions in the hprt gene, which was found to be inserted between the two EcoR1 sites of intron 1. Earlier studies on the removal of this insertion fragment in the SP5 clone indicated that such loss involved intrachromosomal recombination and was detectable by using a reversion mutation assay. The categories of agents investigated here included monofunctional alkylating agents, polyaromatic hydrocarbons giving rise to bulky adducts, chlorinated compounds giving small cyclic adducts, intercalating agents, DNA cross-linkers, UV and ionizing radiation, inhibitors of DNA synthesis and topoisomerases, DNA bases and base analogues, radical formers and tumour promotors. Statistically significant enhancements in the frequency of reversion in SP5 cells were observed after treatment with aflatoxin B1, 9-aminoacridine, benzo[a]-pyrene-7,8-dihydrodiol, benzo[a]pyrene-7,8-diol-9,10-epoxide, camptothecin, dimethylbenzanthracene, dimethyl-nitrosamine, ethidium bromide, ethylmethanesulfonate, N-ethyl-N'-nitrosourea, fluorodeoxyuridine, ICR 191, N-methyl-N'-nitrosoguanidine, mitomycin C and UV irradiation. Only slight inducing effects were indicated in the case of methylmethanesulfonate, N-methyl-N'-nitrosourea and gamma irradiation, although not statistically significant. Negative results were found after treatment with 3-amino-benzamide, 5-azacytidine, bleomycin, 5-bromodeoxyuridine, 1,2-dichloroethane, ethylene oxide, etoposide, formaldehyde, hydrogen peroxide, methotrexate, propylene oxide, quercitin, sodium azide, 12-O-tetradecanoylphorbol-13-acetate, thymidine and a complex mixture consisting of a cigarette smoke condensate. Our results on chemically or physically induced recombinogenic effects in the endogenous SP5 hprt gene are in agreement with data obtained in non-endogenous gene systems based on transgenic cell lines containing integrates of tandem mutated tk or hygromycin resistance genes, but not completely consistent with findings on integrates based on the neo genes. This suggests that many factors influence the recombination process, including a difference in the mechanisms underlying inter- and intrachromosomal recombination. Consequently, the endogenous SP5/V79 system is suggested to be more representative than integrated systems for investigating induction of recombination, as well as for mechanistic studies of recombination at the molecular level, e.g. intrachromosomal recombination.

Previously, we demonstrated point mutations of the H-ras gene in N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT)-induced rat urinary bladder carcinomas. In this study, ras oncogene activation was examined in urinary bladder carcinomas induced by N-(4-hydroxybutyl)nitrosamine (BBN) or N-methyl-N-nitrosourea (MNU) administration followed by uracil treatment. In the first experiment, MNU (20 mg/kg body wt) was i.p. injected into 11 male F344 rats twice a week for 4 weeks, followed by feeding 3% uracil for 20 weeks (MNU/uracil group). Ten rats were given only 3% uracil without MNU pretreatment. In the second experiment, 20 male F344 rats were given 0.05% BBN in the drinking water for 4 weeks, then fed 3% uracil for 20 weeks (BBN/uracil group). Another 20 rats were fed 3% uracil without the BBN pretreatment. Transitional cell carcinomas were induced in the urinary bladder of all rats in the MNU/uracil and BBN/uracil groups. Papillomas and hyperplasias were present in the rats given uracil without prior BBN or MNU. DNA and protein were extracted from the tumors (MNU/uracil or BBN/uracil groups) or from the scraped bladder epithelium (uracil alone groups). Sequences around codons 12, 13 and 61 of H-, K- and N-ras genes were examined by direct sequencing after polymerase chain reaction, and p21 was examined by Western blotting. No mutation was found within the examined sequences and p21 showed no changes in mobility. There was no difference in the level of p21 expression between rats treated with MNU/uracil or BBN/uracil compared to corresponding uracil alone groups. These results indicate that the ras oncogene was not activated in urinary bladder carcinomas induced by BBN or MNU in combination with uracil treatment, in contrast to previous findings with FANFT.

Tauromustine (TCNU) a new taurine-based nitrosourea in phase III clinical trials against colon cancer, has been tested and compared with 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (CCNU) for mutagenic potential in Salmonella typhimurium TA98 and TA100 and, for its ability to cause chromosome aberrations and sister chromatid exchanges (SCE) in human lymphocytes. A dose dependent increase in the number of mutations in S. typhimurium TA100 but not in TA98 was found from 1.6 to 1000 micrograms/plate for both TCNU and CCNU. In addition, in the presence of Aroclor activated liver microsomes, CCNU caused a further increase in the mutation frequency which was not found for TCNU. A dose dependent increase in the chromosome aberration rate in human lymphocytes was induced by both TCNU and CCNU at concentrations ranging from 3.75 to 30 micrograms/ml after 24 h treatment during the last cell cycle. When lymphocytes were treated in G0 both TCNU and CCNU induced a higher frequency of aberrations at 15 and 30 micrograms/ml than during the last cell cycle and, unexpectedly, chromosome-type, i.e. dicentrics appeared. The difference in the frequency of aberrations in these two phases may be related to the different levels of O6-methyl guanine transferase present in resting contra cycling lymphocytes. TCNU at 0.8-8 micrograms/ml also induced a dose dependent increase in the number of SCE in human lymphocytes. Neither the number of chromosomal aberrations nor the number of SCE were affected by the addition of 1000 micrograms/ml taurine to the culture medium.

Two cases of serpentine supravenous hyperpigmentation developing in the area of fotemustine infusions are reported. Histological features showed an increased melanin synthesis and the presence of melanophages without focal degeneration of basal cells or dermal inflammatory infiltrate. Perls' strain was negative. Hypotheses concerning the mechanisms of increased melanin synthesis over the veins are discussed.

Reaction products with DNA and blood proteins have been used for monitoring human exposure to electrophilic compounds and metabolites. The formation of products with nucleophilic sites in DNA after treatment with such reagents has been well characterized (especially for alkylating agents). It is therefore of great importance to collect corresponding data for nucleophilic groups in proteins. The formation of reaction products in hemoglobin (Hb) and DNA was studied after in vitro treatment with ethylene oxide (EtO) and N-(2-hydroxyethyl)-N-nitrosourea (HOEtNU). In DNA N-7-(2-hydroxyethyl)guanine was the main product of EtO, whereas O6-(2-hydroxyethyl)guanine was much lower (0.5% of the alkylation of guanine-N-7). For HOEtNU O6-(2-hydroxyethyl)guanine was found to be 63% of N-7-(2-hydroxyethyl)guanine. These relative reactivities are in agreement with what has been reported for related alkylating agents. The main reaction products in Hb were 2-hydroxyethylations of cysteine, N-terminal valine, the two imidazole nitrogens in histidine and carboxylic groups. The reactivities of human, mouse and rat Hb towards EtO were compared. The main difference between species was the 12 and 170 times higher reactivity of cysteine in mouse and rat Hb, respectively, than in human Hb. As expected from relative reactivities of alkylating agents with model nucleophiles, products with cysteine dominated for EtO (except in human Hb) whereas for HOEtNU products with carboxylic groups were far larger than any other. Relative amounts of cysteine and carboxylic group alkylation in mouse Hb were compared with corresponding data for other alkylating agents. The comparison showed that these amounts were close to what could be expected from known reactivities of these compounds with model nucleophiles, but that sterical and other modifying factors have to be taken into account. Each compound gives therefore a species-specific pattern that might be used for tracing human exposure of unknown origin.

Fotemustine (S 10036) is a new anti-tumor nitrosourea characterized by a phosphonoalanine carrier group coupled to the nitrosourea moiety, which potentially increases the cellular penetration of the drug. Using human tumor cell lines, the activity of S 10036 was compared with that of the more established nitrosoureas BCNU and CCNU. Growth-inhibiting effects were evaluated by the [3H]-thymidine incorporation test. In a panel of 12 human cancer cell lines [melanoma (4), ovary (2), head and neck (3), lung (1), bladder (1), breast (1)], the dose-response curves of S 10036 (0-100 microM) were similar to those obtained with equimolar concentrations of BCNU and CCNU; they indicated a moderately more marked effect for two and an equal effect for six melanoma cell lines with S 10036 as compared with BCNU. Moderate but significant synergistic combinations were obtained when S 10036 (0-80 microM) and CDDP (0-100 microM) or DTIC (250-6,500 microM) were combined in melanoma cell lines. In conclusion, the new nitrosourea S 10036 shows promising activity, particularly against human melanoma cell lines.