The effect of the triazene compound CT913 on ovarian cancer cells in vitro and its synergistic interaction with the PARP-inhibitor olaparib.

OBJECTIVES: Extending the therapeutic spectrum of PARP-inhibitors (PARPi) beyond BRCA1-deficiency and/or overcoming PARPi-resistance is of high clinical interest. This is particularly true for the identification of innovative therapeutic strategies for ovarian cancer, given the recent advances in the use of PARPi in clinical practice. In this regard, the combination of PARPi with chemotherapy is a possible strategy for defining new therapeutic standards. In this study, we analyzed the therapeutic effect of novel triazene derivatives, including the drug CT913 and its metabolite CT913-M1 on ovarian cancer cells and describe their interaction with the PARPi olaparib. METHODS: In vitro assays for drug characterization including RNA-Seq were applied in a selected panel of ovarian cancer cell lines. RESULTS: CT913 treatment conferred a dose-dependent reduction of cell viability in a set of platinum-sensitive and platinum-resistant ovarian cancer cell lines with an IC50 in the higher micromolar range (553-1083 µM), whereas its metabolite CT913-M1 was up to 69-fold more potent, especially among long-term treatment (IC50 range: 8-138 µM). Neither of the drugs sensitized for cisplatin. CT913 conferred synthetic lethality in BRCA1-deficient ovarian cancer cells, indicating that its effect is augmented by a deficiency in homologous recombination repair (HR). Furthermore, CT913 showed a synergistic interaction with olaparib, independently of BRCA1 mutational status. CT913 strongly induced CDKN1A transcription, suggesting cell cycle arrest as an early response to this drug. It moreover downregulated a variety of transcripts involved in DNA-repair pathways. CONCLUSIONS: This is the first study, suggesting the novel triazene drug CT913 as enhancer drug for extending the therapeutic spectrum of PARPi.

Drug-induced xenogenization of tumors: A possible role in the immune control of malignant cell growth in the brain?

In recent years, immune checkpoint inhibitors (ICpI) have provided the ground to bring tumor immunity back to life thanks to their capacity to afford a real clinical benefit in terms of patient's survival. Essential to ICpI success is the presence of tumor-associated neoantigens generated by non-synonymous mutations, since a direct relationship between mutation load of malignant cells and susceptibility to ICpI has been confidently established. However, it has been also suggested that high intratumor heterogeneity (ITH) associated with subclonal neoantigens could not elicit adequate immune responses. Several years ago we discovered that in vivo treatment of leukemic mice with triazene compounds (TZC) produces a marked increase of leukemia cell immunogenicity [a phenomenon termed Drug-Induced Xenogenization (DIX)] through point mutations able to generate strong tumor neoantigens (Drug-Induced Neoantigens, DIN). Immunogenic mutations are produced by TZC-dependent methylation of O6-guanine of DNA, that is suppressed by the DNA repair protein methyl-guaninemethyltransferase (MGMT). This minireview illustrates preclinical investigations conducted in animal models where DIN-positive murine leukemia cells were inoculated intracerebrally into histocompatible mice. The analysis of the literature indicates that the growth of xenogenized malignant cells is controlled by anti-DIN graft responses and by intra-cerebral or intravenous adoptive transfer of anti-DIN cytotoxic T lymphocytes. This survey reminds also that PARP inhibitors increase substantially the antitumor activity of TZC and can be administered with the intent of suppressing more efficiently tumor load and possibly reducing ITH through downsizing the polyclonality of xenogenized tumor cell population. Finally, the present report illustrates a hypothetical clinical protocol that could be considered as an example of future development of DIXbased tumor immuno-chemotherapy in brain malignancies. The protocol involves oral or intravenous administration of TZC along with loco-regional (i.e. intracerebral "wafer") treatment with agents able to increase tumor cell sensitivity to the cytotoxic and xenogenizing effects of TZC (i.e. MGMT and PARP inhibitors) without enhancing the systemic toxicity of these DNA methylating compounds.

Triacsin C reduces lipid droplet formation and induces mitochondrial biogenesis in primary rat hepatocytes.

Intracellular long-chain acyl-CoA synthetases (ACSL) activate fatty acids to produce acyl-CoA, which undergoes ß-oxidation and participates in the synthesis of esterified lipids such as triacylglycerol (TAG). Imbalances in these metabolic routes are closely associated with the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Triacsin C is one of the few compounds that inhibit TAG accumulation into lipid droplets (LD) by suppressing ACSL activity. Here we report that treatment of primary rat hepatocytes with triacsin C at concentrations lower than the IC50 (4.1 µM) for LD formation: (i) diminished LD number in a concentration-dependent manner; (ii) increased mitochondrial amount; (iii) markedly improved mitochondrial metabolism by enhancing the ß-oxidation efficiency, electron transport chain capacity, and degree of coupling - treatment of isolated rat liver mitochondria with the same triacsin C concentrations did not affect the last two parameters; (iv) decreased the GSH/GSSG ratio and elevated the protein carbonyl level, which suggested an increased reactive oxygen species production, as observed in isolated mitochondria. The hepatocyte mitochondrial improvements were not related to either the transcriptional levels of PGC-1α or the content of mTOR and phosphorylated AMPK. Triacsin C at 10 µM induced hepatocyte death by necrosis and/or apoptosis through mechanisms associated with mitochondrial permeability transition pore opening, as demonstrated by experiments using isolated mitochondria. Therefore, triacsin C at sub-IC50 concentrations modulates the lipid imbalance by shifting hepatocytes to a more oxidative state and enhancing the fatty acid consumption, which can in turn accelerate lipid oxidation and reverse NAFLD in long-term therapies.

Exogenous nitric oxide enhances the prophylactic effect of aminoguanidine, a preferred iNOS inhibitor, on bleomycin-induced fibrosis in the lung: Implications for the direct roles of the NO molecule in vivo.

OBJECTIVE: Inducible nitric oxide synthase (iNOS) aggravates and endothelial nitric oxide synthase (eNOS) ameliorates fibrosis in the lung. Our previous study demonstrated that aminoguanidine (AG), a preferred iNOS inhibitor, prevents bleomycin-induced injury and fibrosis in the lung. The diethylenetriamine nitric oxide adduct (DETA/NO) is a slow-release NO donor. Here, to clarify the exact role of the nitric oxide (NO) molecule in the pathogenesis of pulmonary fibrosis in vivo, we observed the effects of inhalation of aerosolized DETA/NO on fibrosis in the lungs of bleomycin-exposed rats with AG treatment, including the effects on the myofibroblast number, collagen deposition, peroxynitrite anion (ONOO-) formation, and injury in the lung. DESIGN AND METHODS: Rats received a single intratracheal instillation of bleomycin or normal saline (NS) on day 0, followed by a daily intraperitoneal injection of AG or NS from day 1 to day 13. Each group was additionally given a daily inhalation of DETA/NO or placebo from day 1 to day 13. On day 14, half of the rats in each group was euthanized, and plasma nitrite and nitrate (NOx), myofibroblasts, type I collagen, ONOO- and injury in the lung were estimated by the Griess reaction, western blotting, immunohistochemical staining, sirius red staining, and hematoxylin and eosin (HE) staining, respectively. On day 28, the other half of the rats in each group was euthanized, and the total collagen of the lung was evaluated by hydroxyproline assay. RESULTS: ① At the day 14 time point, AG reduced the plasma NOx level in bleomycin rats, while this drug had no significant effect on sham rats. Inhalation of aerosolized DETA/NO increased the plasma NOx level of bleomycin + AG rats, sham rats and sham + AG rats. However, due to large areas of airspace obliteration in the lungs of bleomycin rats, DETA/NO inhalation had no significant effect on the plasma NOx level in these rats. ② At the day 14 time point, AG reduced ONOO- formation (marked by nitrotyrosine, NT), injury, myofibroblast number, and type I collagen deposition in the lungs of bleomycin rats, while this drug had no significant impact on the above parameters in the lungs of sham rats. Interestingly, DETA/NO inhalation enhanced the preventive effects afforded by AG on myofibroblast number and type I collagen deposition, but had no significant impact on ONOO- and injury in lung. ③ At the day 28 time point, because rats were not exposed to DETA/NO after day 13, there was no significant difference of the plasma NOx level in sham rats, sham + AG rats, bleomycin rats, and bleomycin + AG rats between DETA/NO inhalation and placebo inhalation. Interestingly, rats administered both DETA/NO and AG still showed a reduction in total collagen of the entire lung compared to rats administered AG alone at this time point. CONCLUSIONS: Exogenous NO enhances the prophylactic effect afforded by AG on the myofibroblast number and collagen deposition in the lungs of bleomycin-treated rats in vivo. These results suggest that NO has a direct antifibrotic effect in lungs, except for the formation of ONOO- in the development of pulmonary fibrosis in vivo.

The Role of Nitric Oxide in the Antidepressant Actions of 5-Aminoimidazole-4-Carboxamide-1-ß-D-Ribofuranoside in Insulin-Resistant Mice.

OBJECTIVE: Depression and Type 2 diabetes mellitus are interrelated conditions, but the underlying neurobiology is insufficiently understood. The current study compared the effects of a pharmacological manipulation with 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) that targets neurobiological processes by adenosine 5'-monophosphate-activated protein kinase activation versus exercise on depression-like behavior and nitric oxide (NO)-related measures. METHODS: A mouse model of a depression-like and insulin-resistant state, induced by the co-treatment of high-fat diet and corticosterone administration, was used to examine the antidepressant action of AICAR and exercise. RESULTS: Data showed that AICAR was a putative antidepressant in the depression-like and insulin-resistant mice (total ambulatory distance in the open-field test was 5120.69 ± 167.47 cm, mobility duration in the forced swim test was 17.61 ± 1.54 seconds, latency to feed in the novelty suppressed feeding test was 255.67 ± 37.80 seconds; all p values < .05). Furthermore, the antidepressant actions of AICAR required endothelial nitric oxide synthase activity with increased NO production in the prefrontal cortex, whereas corticosterone-induced expression of neuronal nitric oxide synthase and NO production may increase the risk of depression. In contrast to the traditional antidepressants such as ketamine and imipramine, AICAR interfered with the effects of insulin in skeletal muscle in the context of high-fat diet, consistent with the potential antidepressant effects of AICAR. Exercise also resulted in activation of adenosine 5'-monophosphate-activated protein kinase, nitric oxide synthase, and NO production (all p values < .01), which in turn may be implicated in the antidepressant effects of exercise. CONCLUSIONS: These findings suggest that NO is an essential signal mediating the antidepressant actions of AICAR. Ultimately, the concurrent effects of AICAR on brain insulin action and mitochondrial function suggest a potential of neural insulin resistance, which may contribute to our understanding of the comorbidity of depression and Type 2 diabetes.

Hepatoselective Nitric Oxide (NO) Donors, V-PYRRO/NO and V-PROLI/NO, in Nonalcoholic Fatty Liver Disease: A Comparison of Antisteatotic Effects with the Biotransformation and Pharmacokinetics.

V-PYRRO/NO [O(2)-vinyl-1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate] and V-PROLI/NO (O2-vinyl-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate), two structurally similar diazeniumdiolate derivatives, were designed as liver-selective prodrugs that are metabolized by cytochrome P450 isoenzymes, with subsequent release of nitric oxide (NO). Yet, their efficacy in the treatment of nonalcoholic fatty liver disease (NAFLD) and their comparative pharmacokinetic and metabolic profiles have not been characterized. The aim of the present work was to compare the effects of V-PYRRO/NO and V-PROLI/NO on liver steatosis, glucose tolerance, and liver fatty acid composition in C57BL/6J mice fed a high-fat diet, as well as to comprehensively characterize the ADME (absorption, distribution, metabolism and excretion) profiles of both NO donors. Despite their similar structure, V-PYRRO/NO and V-PROLI/NO showed differences in pharmacological efficacy in the murine model of NAFLD. V-PYRRO/NO, but not V-PROLI/NO, attenuated liver steatosis, improved glucose tolerance, and favorably modified fatty acid composition in the liver. Both compounds were characterized by rapid absorption following i.p. administration, rapid elimination from the body, and incomplete bioavailability. However, V-PYRRO/NO was eliminated mainly by the liver, whereas V-PROLI/NO was excreted mostly in unchanged form by the kidney. V-PYRRO/NO was metabolized by CYP2E1, CYP2C9, CYP1A2, and CYP3A4, whereas V-PROLI/NO was metabolized mainly by CYP1A2. Importantly, V-PYRRO/NO was a better NO releaser in vivo and in the isolated, perfused liver than V-PROLI/NO, an effect compatible with the superior antisteatotic activity of V-PYRRO/NO. In conclusion, V-PYRRO/NO displayed a pronounced antisteatotic effect associated with liver-targeted NO release, whereas V-PROLI/NO showed low effectiveness, was not taken up by the liver, and was eliminated mostly in unchanged form by the kidney.

[Five-year visual outcomes of typical age-related macular degeneration and/or polypoidal choroidal vasculopathy patients who received photodynamic therapy (PDT) as initial treatment in comparison with patients prior to the PDT era].

PURPOSE: To evaluate five-year visual outcomes of typical age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) in patients who received photodynamic therapy (PDT) as initial treatment compared with the outcomes of patients prior to the PDT era. SUBJECTS AND METHODS: Twenty-three eyes observed for 5 years before PDT was available (group A: typical AMD/PCV 16 eyes/7 eyes) and 61 eyes which had been observed for 5 years after PDT with additional treatment as needed (group B: typical AMD/PCV 25 eyes/36 eyes). The visual changes in these groups were retrospectively compared. RESULTS: In group A of typical AMD patients, the mean visual acuity (VA, logMAR) was significantly worse at the 3-year visit and later. In group B of typical AMD patients, the VA was stabilized after 2 years and no significant mean VA deterioration was observed for 5 years. More patients in group B retained a logMAR of less than 1.0 (43% vs. 25%) than in group A. Those patients in group B with PCV, maintained the VA for one year, but it gradually worsened thereafter. CONCLUSION: The PDT shortened the duration of VA deterioration in typical AMD patients from 5 to 2 years with no significant VA decrease for 5 years. The positive effect of PDT on PCV eyes was temporary.

Endothelial nitric oxide synthase plays an obligatory role in the late phase of ischemic preconditioning by activating the protein kinase C epsilon p44/42 mitogen-activated protein kinase pSer-signal transducers and activators of transcription1/3 pathway.

BACKGROUND: The role of endothelial nitric oxide synthase (eNOS) in ischemic preconditioning (PC) and cardioprotection is poorly understood. We addressed this issue using a genetic, rather than pharmacological, approach. METHODS AND RESULTS: In the nonpreconditioned state, eNOS-/- mice exhibited infarct sizes similar to those of wild-type mice. A sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles (ischemic PC) induced late PC in wild-type mice; genetic deletion of eNOS abrogated the cardioprotection induced by late PC. In wild-type mice, ischemic PC induced membranous translocation of protein kinase C (PKC) epsilon and an increase in pSer-MEK-1/2 and pTyr-p44/42 mitogen-activated protein kinase, nuclear pSer-signal transducers and activators of transcription (STAT)1 and pSer-STAT3, and nuclear STAT1/3 DNA binding activity, followed by upregulation of cyclooxygenase-2 protein and activity 24 hours later. All of these changes were abrogated in eNOS-/- mice. The NO donor diethylenetriamine/NO recapitulated the effects of ischemic PC. CONCLUSIONS: In contrast to previous reports, we found that basal eNOS activity does not modulate infarct size in the nonpreconditioned state. However, eNOS is obligatorily required for the development of the cardioprotective effects of late PC and acts as the trigger of this process by activating the PKC epsilon-MEK-1/2-p44/42 mitogen-activated protein kinase pathway, leading to Ser-727 phosphorylation of STAT1 and STAT3 and consequent upregulation of STAT-dependent genes such as cyclooxygenase-2. The effects of eNOS-derived NO are reproduced by exogenous NO (NO donors), implying that nitrates can upregulate cardiac cyclooxygenase-2.

Effect of chemotherapy and immunotherapy on tumor-specific immunity in melanoma.

The effects of chemotherapy, with nitrosoureas or dimethyl-triazeno-imidazole-carboxamide (DTIC), or immunotherapy with Bacillus Calmette-Guérin (BCG), on cell-mediated immunity (CMI), and serum blocking factor (BF) to melanoma cells were studied in 23 patients. Studies were performed with autologous or allogenic melanoma target cells obtained from recent biopsy, in 16 mm diameter plastic wells. Assays for lymphocyte-mediated cytotoxicity and BF were performed at weekly intervals over the course of 3-4 mo, with some studies extending beyond 3 yr. The specificity of cytotoxicity was good with these methods. Nine patients given nitrosoureas, predominantly methyl-chloroethyl-cyclohexyl-nitrosourea, showed a transient decline in CMI from 42.2 to 14% 3 wk after administration of a single dose of the agent, with a rapid recovery within 1 week. 10 patients given 5-day courses of DTIC at 3-wk intervals showed no decline in CMI after two courses, and 7 of the 10 had no decline even after three courses. Three of the four patients who achieved a remission lost BF previously present: BF reappeared in both patients studied during a subsequent relapse. BCG intradermally or intralesionally elevated CMI within 2 mo after initiation of therapy, but despite continuation of the injections CMI returned to base line in all but two of the nine patients studied. These results indicate that chemotherapy for melanoma with nitrosoureas or DTIC at these schedules is not profoundly immunosuppressive towards tumor-specific immunity, as measured by our procedures. Putative immunotherapy with BCG at these schedules was likewise only transiently stimulatory.

Tumor immunotherapy: drug-induced neoantigens (xenogenization) and immune checkpoint inhibitors.

More than 40 years ago, we discovered that novel transplantation antigens can be induced in vivo or in vitro by treating murine leukemia with dacarbazine. Years later, this phenomenon that we called "Chemical Xenogenization" (CX) and more recently, "Drug-Induced Xenogenization" (DIX), was reproduced by Thierry Boon with a mutagenic/carcinogenic compound (i.e. N-methyl-N'-nitro-N-nitrosoguanidine). In both cases, the molecular bases of DIX rely on mutagenesis induced by methyl adducts to oxygen-6 of DNA guanine. In the present review we illustrate the main DIX-related immune-pharmacodynamic properties of triazene compounds of clinical use (i.e. dacarbazine and temozolomide).In recent years, tumor immunotherapy has come back to the stage with the discovery of immune checkpoint inhibitors (ICpI) that show an extraordinary immune-enhancing activity. Here we illustrate the salient biochemical features of some of the most interesting ICpI and the up-to-day status of their clinical use. Moreover, we illustrate the literature showing the direct relationship between somatic mutation burden and susceptibility of cancer cells to host's immune responses.When DIX was discovered, we were not able to satisfactorily exploit the possible presence of triazene-induced neoantigens in malignant cells since no device was available to adequately enhance host's immune responses in clinical settings. Today, ICpI show unprecedented efficacy in terms of survival times, especially when elevated mutation load is associated with cancer cells. Therefore, in the future, mutation-dependent neoantigens obtained by appropriate pharmacological intervention appear to disclose a novel approach for enhancing the therapeutic efficacy of ICpI in cancer patients.

Antimetastatic action and hematological toxicity of p-(3,3-dimethyl-1-triazeno)benzoic acid potassium salt and 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide used as prophylactic adjuvants to surgical tumor removal in mice bearing B16 melanoma.

The treatment of mice bearing i.m. B16 melanoma with equitoxic dosages of the clinically used 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) and of its benzenoid water-soluble analogue p-(3,3-dimethyl-1-triazeno)benzoic acid potassium salt (DM-COOK) prior to surgical tumor removal results in a remarkable proportion of cures, even when the treatment is started on already palpable tumors for which surgery alone is ineffective. The survival time of mice which are not cured is also significantly increased with DM-COOK. At the same time, DM-COOK does not affect artificial metastases or spontaneous metastases in mice undergoing surgery and treated with DM-COOK postoperatively. Inhibition of i.m. tumor growth in surgical experiments, and of s.c. tumors in mice not treated with surgery, is significant, although not as pronounced as is necessary to obtain significant prolongation of the life span of the host; the survival time of mice with s.c. tumors treated with both drugs is indeed not significantly increased. DM-COOK thus appears to exert selective antimetastatic effects, unrelated to cytotoxicity for tumor cells, against B16 melanoma in addition to those reported for Lewis lung carcinoma and M5 ovarian reticular cell sarcoma; its therapeutic usefulness is evidenced in adjuvant surgical experiments. DM-COOK, unlike DTIC, is devoid of hematological toxicity for the host. Since, in leukemic mice, it is at least as active as DTIC in increasing the life span of the treated animals, it appears to be an advantageous substitute for DTIC that could undergo preliminary clinical trial.

Chemotherapy of a human malignant melanoma transplanted in the nude mouse.

The effects of single agent therapy with 1-(-chloroethyl)-3-cyclohexyl-1-nitrosourea, 5-fluorouracil, and 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide on a human malignant melanoma transplanted and passed serially in thymusless nude mouse were studied. Tumor response varied. A single dose of 1-(-chloroethyl)-3-cyclohexyl-1-nitrosourea induced initial tumor regression, but thereafter growth resumed at a rate similar to that in the untreated control animals. Wehn 1-(-chloroethyl)-3-cyclohexyl-1-nitrosourea was given in divided dosage at an interval of 8 days, marked and persistent tumor regression was observed. 5-Fluorouracil had no effect. Treatment with 5-(3,3 dimethyl-1-triazeno)-imidazole-4-carboxamide was always reflected by almost total regression of tumors, an effect that was independent of dose within the range tested in this study. The results resemble those reported from clinical practice in patients with disseminated malignant melanomas treated with the same agents. This suggests that the pattern of drug susceptibility is preserved after transplantation of tumors in the nude mouse. The human tumor-nude mouse system is advocated as a new in vivo model for determination of individual tumor response to chemotherapeutic agents, and its potential as a model for the proving of new chemotherapeutic agents is suggested.

Relationships between the formation of O6-methyldeoxyguanosine by 1-p-carboxyl-3,3-dimethylphenyltriazene in DNA and O6-alkylguanine-DNA alkyltransferase in human peripheral leukocytes.

There is increasing experimental evidence to indicate that O6-methyldeoxyguanosine (O6-MedG) formation in DNA is a critical cytotoxic event following exposure to certain antitumor alkylating agents and that the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (ATase) can confer resistance to these agents. We recently demonstrated a wide interindividual variation in the depletion and subsequent regeneration of ATase in peripheral blood lymphocytes of patients treated with 24-h continuous infusion of 1-p-carboxyl-3,3-dimethylphenyltriazene (CB10-277) for metastatic melanoma. We have now measured the formation of O6-MedG in the DNA of peripheral leukocytes of nine patients receiving this treatment regimen. This lesion could be detected in DNA within 1 h and a progressive increase in adduct levels occurred during the CB10-277 infusion and for 24 h after completion. Considerable interindividual variation was observed in the peak O6-MedG levels, with values ranging from 3.0 to 23.8 mumol O6-MedG/mol deoxyguanosine (mean, 12.3 +/- 6.4 mumol O6-MedG/mol deoxyguanosine) following the first treatment cycle, possibly as a consequence of differences in the capacity of patients to metabolize CB10-277 to a methylating agent. There was, nevertheless, a clear temporal relationship between the progressive formation of leukocyte O6-MedG and lymphocyte ATase depletion. Repeated-measures regression showed that this was statistically significant (P < 0.001) during the CB10-277 infusion. A significant inverse correlation was also seen between pretreatment lymphocyte ATase activity and peak O6-MedG levels in leukocyte DNA (r = -0.73) and the area under the leukocyte O6-MedG concentration-time curve (r = -0.76). Metabolism of CB10-277 to a methylating agent could be one factor that combines with DNA repair capacity to determine clinical response, because the two responses observed in this series occurred in the two patients with the highest leukocyte O6-MedG levels and also the lowest pretreatment ATase activity. Hematological toxicity developed in the same two patients.

Discovery and Clinical Evaluation of MK-8150, A Novel Nitric Oxide Donor With a Unique Mechanism of Nitric Oxide Release.

BACKGROUND: Nitric oxide donors are widely used to treat cardiovascular disease, but their major limitation is the development of tolerance, a multifactorial process to which the in vivo release of nitric oxide is thought to contribute. Here we describe the preclinical and clinical results of a translational drug development effort to create a next-generation nitric oxide donor with improved pharmacokinetic properties and a unique mechanism of nitric oxide release through CYP3A4 metabolism that was designed to circumvent the development of tolerance. METHODS AND RESULTS: Single- and multiple-dose studies in telemetered dogs showed that MK-8150 induced robust blood-pressure lowering that was sustained over 14 days. The molecule was safe and well tolerated in humans, and single doses reduced systolic blood pressure by 5 to 20 mm Hg in hypertensive patients. Multiple-dose studies in hypertensive patients showed that the blood-pressure-lowering effect diminished after 10 days, and 28-day studies showed that the hemodynamic effects were completely lost by day 28, even when the dose of MK-8150 was increased during the dosing period. CONCLUSIONS: The novel nitric oxide donor MK-8150 induced significant blood-pressure lowering in dogs and humans for up to 14 days. However, despite a unique mechanism of nitric oxide release mediated by CYP3A4 metabolism, tolerance developed over 28 days, suggesting that tolerance to nitric oxide donors is multifactorial and cannot be overcome solely through altered in vivo release of nitric oxide. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01590810 and NCT01656408.

Clinical comparison of adriamycin and a combination of methyl-CCNU and imidazole carboxamide in disseminated malignant melanoma.

The effects of adriamycin were compared to a combination program of methyl-CCNU and imidozole carboxamide (DTIC) in 44 patients with disseminated malignant melanoma. There were objective clinical responses in 6 of 21 patients with the combination of DTIC and methyl-CCNU who received this program as primary treatment and none in 23 patients receiving adriamycin as primary treatment. Secondary responses were not observed with either treatment regimen. Toxicity with the combination program included leukocyte depression (less than 3,000/cu mm) in 25% and platelet depression (less than 100,000/cu mm) in 40% compared to 52% leukocyte depression and 16% platelet depression after adriamycin. There were no responses after the combination treatment program in the absence of myelosuppression. There was nausea and vomiting in virtually all patients, which was moderately severe in one third of the patients receiving the combination and in only 10% of those receiving adriamycin. Alopecia developed in all who received adriamycin but in only 15% of the combination treatment group. The combination treatment response of 28% was of the same order as most response rates previously reported in this disease. This randomized controlled clinical trail found adriamycin without clinical benefit and not worthy of further trial in patients with disseminated malignant melanoma.

Effects of DTIC, DM-COOK and ICRF-159 on the number of circulating Lewis lung carcinoma cells detected by flow cytometry.

Circulating tumor cells can be detected by means of flow cytometry in the blood of mice bearing i.m. Lewis lung carcinoma. This technique can be applied in the case of aneuploid tumors and does not require either concentration of nucleated cells or other processing of the blood samples. It offers the advantages of simplicity and speed and allows quantitative measurement of the number of circulating tumor cells. It can be applied to the study of the effects of drug treatment on the number of circulating tumor cells, for those drugs which do not cause alterations in the nuclear DNA content of normal diploid blood cells. The number of circulating tumor cells determined by flow cytometry is markedly reduced by treatment with ICRF-159, by dimethyltriazene DM-COOK, and also by its clinically used analog, DTIC.

Effects of antimetastatic, antiinvasive and cytotoxic agents on the growth and spread of transplantable leukemias in mice.

The effects of cytotoxic (cyclophosphamide, CCNU, GANU), antiinvasive (vincristine, vinblastine) and antimetastatic (ICRF-159, DM-COOK) agents have been compared in mice-bearing P388 and L1210 leukemias, and TLX5 lymphoma. The drugs tested increase the survival time of the treated mice in a manner consistent with a cytotoxic action in the case of cyclophosphamide, CCNU, GANU, vincristine and vinblastine. Leukemic infiltration of the brain after i.p. tumor implantation has been determined by bioassay of this organ, and is reduced by treatment with all of the drugs tested, with the exception of ICRF-159. DM-COOK appears to increase the life-span of the treated animals by the inhibition of leukemic spread rather than by a cytotoxic action. The marked cytotoxicity of vincristine and vinblastine is sufficient to account for failure to detect any antimetastatic effects of these agents. The lack of antidisseminative effect observed for ICRF-159 under the experimental conditions employed might be connected with the observation that the antimetastatic action of this drug on solid tumors is due to its effects on tumor blood vessels.

Preparation and antitumor activity of 1-aryl-3,3-dimethyltriazene derivatives.

Several 1-aryl-3,3-dimethyltriazene derivatives have been synthesized and tested for their antitumor activity against the TLX5 lymphoma in mice. These compounds are characterized by the presence of a carbonyl group bound to the benzene nucleus in the para position to the triazene funciton. Three p-sulfamoyl derivatives have also been included and proved to be inactive. Among the carbonyl derivatives compounds 1 and 20, which can be used as reference, cause ILS of about 50%, respectively, at four and three dose levels. Compound 16, the o-nitro-phenylhydrazone of the hydrazide 1, is active at all six dose levels studied. The adduct 19, obtained from the same hydrazide and p-nitrobenzaldehyde, is active at four dose levels, and the ILS values at two optimum doses are significantly greater than those caused by compound 1.

Antitumor 1-(X-aryl)-3,3-dialkyltriazenes. 1. Quantitative structure-activity relationships vs. L1210 leukemia in mice.

Quantitative structure-activity relationships (QSAR) have been formulated for phenyl-, pyrazolyl-, and imidazolyltriazenes acting L1210 leukemia in mice. All three sets of congeners have the same ideal lipophilicity (log Po approximately 1). Electron releasing substituents increase potency; ortho substitution decreases activity. The synthesis of a number of new triazenes and some of their partition coefficients are reported.

Antitumor 1-(X-aryl)-3,3-dialkyltriazenes. 2. On the role of correlation analysis in decision making in drug modification. Toxicity quantitative structure-activity relationships of 1-(X-phenyl)-3,3-dialkyltriazenes in mice.

A series of 11 triazenes (X-C6H4N=NNRCH3) was characterized for toxicity in mice (LD50). The quantitative structure-activity relationship (QSAR) obtained for toxicity was compared with the QSAR for antitumor activity. The close correspondence of the two QSAR leaves essentially no means for the synthesis of more potent, less toxic triazenes.