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Nogalamycin is an anthracycline antibiotic that has been shown to exhibit significant cytotoxicity. Its biological activity requires two deoxysugar moieties: nogalose and nogalamine, which are attached at C7 and C1, respectively, of the aromatic polyketide aglycone. Curiously, the aminosugar nogalamine is also connected through a C-C bond between C2 and C5''. Despite extensive molecular genetic characterization of early biosynthetic steps, nogalamycin glycosylation has not been investigated in detail. Here we show that expression of the majority of the gene cluster in Streptomyces albus led to accumulation of three new anthracyclines, which unexpectedly included nogalamycin derivatives in which nogalamine was replaced either by rhodosamine with the C-C bond intact (nogalamycin R) or by 2-deoxyfucose without the C-C bond (nogalamycin F). In addition, a monoglycosylated intermediate-3',4'-demethoxynogalose-1-hydroxynogalamycinone-was isolated. Importantly, when the remaining biosynthetic genes were introduced into the heterologous host by using a two-plasmid system, nogalamycin could be isolated from the cultures, thus indicating that the whole gene cluster had been identified. We further show that one of the three glycosyltransferases (GTs) residing in the cluster-snogZ-appears to be redundant, whereas gene inactivation experiments revealed that snogE and snogD act as nogalose and nogalamine transferases, respectively. The substrate specificity of the nogalamine transferase SnogD was demonstrated in vitro: the enzyme was able to remove 2deoxyfucose from nogalamycin F. All of the new compounds were found to inhibit human topoisomerase I in activity measurements, whereas only nogalamycin R showed minor activity against topoisomerase II., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A chiral synthesis of the aminohydroxy expoxybenzoxocin 6 is described. Enantioselective Friedel-Crafts coupling using a chiral titanium catalyst was employed to produce the optically active atrolactic ester 16a from the phenol 11 and l-menthyl pyruvate (12). The phenolic group in 16a was protected as the benzyl ether and the t-alcohol functionality as the MEM ether to give 20, which after sequential reduction/oxidation provided the aldehyde 22. Addition of the acetylide anion of propargyl aldehyde diethyl acetal (23) to aldehyde 22, followed by oxidation of the resultant diastereoisomeric carbinols, gave the acetylenic ketone 24. Lindlar reduction of 24 afforded the trans-enone 26. Reaction of 26 with thiophenylate anion furnished 27, which was then cyclized to the alpha-methyl pyranoside 29. Oxidation of 29 to the sulfoxide and subsequent thermolysis afforded the hexenulose 30. Sequential epoxidation of 30, reduction of the keto epoxide 31, and reaction of the resultant epoxycarbinol 32 with dimethylamine produced the aminohydroxy pyranose 33a. Debenzylation of 33a to the phenol 33b, followed by intramolecular cyclization, completed the fabrication of the optically active aminohydroxy epoxybenzoxocin 6. The 17-step sequence from the phenol 11 to 6 was achieved in 22% overall yield.

The nuclear magnetic resonance (NMR) solution structures of the 2:1 complexes of nogalamycin-d(CGTACG)2 (Ng-CGTACG) and disnogalamycin-d(CGTACG)2 (DNg-CGTACG) have been determined by a quantitative treatment of two-dimensional nuclear Overhauser effect (2D-NOE) crosspeak intensities. The 1.3 A resolution crystal structure of the 2:1 complex of Ng-CGTACG was used as a starting model for refinement using the procedure, SPEDREF [Robinson and Wang, Biochemistry 31 (1992) 3524-3533], which incorporates full matrix relaxation theory and simulated annealing minimization. The refined solution structures have R-factors of 16.1 and 19.6% between the observed and simulated NOEs for Ng-CGTACG and DNg-CGTACG, respectively. The refined NMR structures retain major features of the crystal structure in which the elongated aglycone chromophore is intercalated between the CpG steps with its nogalose and aminoglucose lying in the minor and major grooves, respectively. The root mean square deviation between the solution and crystal structure for the complexes is 1.01 A (Ng-CGTACG) and 1.20 A (DNg-CGTACG) for the drug, plus the three base pairs surrounding the drug, indicating a very similar local structure at the intercalation site. In the NMR structure, the two G:C Watson-Crick base pairs (C1:G12 and G2:C11) that wrap around the aglycone have large buckles, as do those seen in the crystal structure. There is a 22 degree bend at the T3-A4 step in the refined solution structure. This rearrangement of the solution conformation is likely due to the absence of crystal packing. Specific hydrogen bonds between the drug and G:C bases in both grooves of the helix are preserved in the solution structure. A separate study of the 2:1 complex at low pH showed that the terminal G-C base pairing is destabilized.

Menogaril (TUT-7) is a novel antitumor antibiotic belonging to anthracyclines. The pharmacokinetic parameters derived from plasma concentration-time profiles after repeated (for 14 days) or single oral administration of TUT-7 to rats were found to be not significantly different by either administration schedule. The rats with artificial liver dysfunction were obtained by subcutaneous application of carbon tetrachloride (CCl4, 1 ml/kg) for 3 days. After oral administration of TUT-7 to the rats with CCl4-induced liver toxicity (3 daily administrations of 1mg/kg, S.C.), the maximum plasma concentrations (Cmax) and AUC of both the unchanged drug and its metabolite N-Demethyl menogaril, were increased. Also over all elimination was slower in animals with liver dysfunction.

Background: Studies have shown that response to a given chemotherapy in previously untreated patients with extensive-stage small-cell lung cancer is superior to that in patients previously treated with other regimens. This finding raises the question of whether it is necessary and ethical to study the effects of new anticancer agents in untreated patients. Such studies appear to be the best test for drug development, but there has been no evaluation of whether survival of untreated patients, whose cancer is sensitive to established drugs, is adversely affected in trials of new drugs., Purpose: This randomized study of untreated patients with extensive-stage small-cell lung cancer was designed (a) to compare the survival of patients treated with either effective standard chemotherapy or an investigational anticancer drug as initial therapy and (b) to evaluate response rates and toxic effects of such therapies., Methods: Eighty-six patients were randomly assigned to receive, as initial therapy, either the standard CAV regimen--cyclophosphamide (1000 mg/m2), doxorubicin (50 mg/m2), and vincristine (1.4 mg/m2) every 3 weeks--or the phase II drug menogaril (200 mg/m2) every 4 weeks. Treatment after induction therapy varied, depending on patient response, but nonresponders and those with disease progression received salvage chemotherapy--etoposide (120 mg/m2 on days 1, 2, and 3) and cisplatin (60 mg/m2 on day 1), repeated every 3 weeks., Results: Of the 43 patients on CAV, 42% responded (eight complete responses and 10 partial responses); 5% of the 43 on menogaril responded (two partial responses) (P = .0001). Twelve (22%) of 54 patients responded to salvage chemotherapy (five complete responses and seven partial responses). Within 3 months from start of treatment, twelve patients died--3 patients in the CAV group and nine patients in the menogaril group (P = .12). The estimated median survival was 37 weeks with menogaril and 45 weeks with CAV (P = .28). At 6 months, survival was 76.7% for the CAV group and 67.4% for the menogaril group. At 12 months, survival rates were 24.4% and 27.9%, respectively. Confidence intervals (95%) for the differences between the proportions surviving in the two groups were -9%-28% at 6 months and -25%-14% at 12 months. Use of CAV resulted in significantly higher occurrence of severe and life-threatening treatment-related complications (P = .002)., Conclusion: The confidence intervals for the differences in survival are too wide to conclude that evaluation of a new drug in untreated patients with extensive-stage small-cell lung cancer is or is not harmful. The data do suggest, however, that use of this study design may have no adverse effect on survival.

Menogaril is a new semisynthetic anthracycline agent derived from the antitumor antibiotic Nogalomycin. Compared to doxorubicin it has similar or improved activity in anti-tumor cell line screening; human tumor cloning assays suggest modest anti-tumor activity as well. Menogaril is much less cardiotoxic than doxorubicin. We performed a phase II trial of this agent in 22 patients with advanced malignant mesothelioma. At a dose of 200 mg/m2 iv every 4 weeks (160 mg/m2 in previously radiated patients) only 1 of 22 (5%) evaluable patients had a partial remission lasting 4 months. (95% confidence limits 0.1-23%). The major toxic effects included pain at the site of infusion and granulocytopenia. While well tolerated, Menogaril has minimal activity in malignant mesothelioma. We do not plan further studies with Menogaril in this disease.

Twenty-eight patients were treated with oral menogaril daily x 14 every 4 weeks. Granulocytopenia was dose-limiting at 50-60 mg/m2 per day. Neutropenic fever occurred in one patient. Thrombocytopenia occurred in 3 of 6 patients treated with menogaril 60 mg/m2/day. Nausea and vomiting and other toxic effects were generally mild. No cardiac toxicity was seen. One partial remission and three minor responses were noted among 11 previously treated patients with carcinoma of the bladder. One minor response was noted among 3 patients with colorectal cancer. The dose recommended for phase II studies is 50 mg/m2 per day for 14 days. Phase II studies are recommended in carcinoma of the bladder. Gastrointestinal toxicity is substantially less on this schedule than with oral menogaril administered on a weekly or q4wk schedule, but thrombocytopenia may be more common.

Fifteen patients with relapsed multiple myeloma (MM) were treated with menogaril 160 mg/m2 intravenously (IV) every 28 days. No responses were seen: 8 patients had stable disease, 4 progressed after one course of therapy, and 3 patients were removed from study after 1 course for other reasons. Four of the 8 patients with stable disease had an improved performance status, and 3 had a decrease in analgesic use. The major toxicity was myelosuppression. The median progression-free interval was 3.0 months with a range of 0.7 to 22 months and median survival was 11.3 months with a range of 0.7 to 39+ months. Menogaril displays little activity in patients with previously treated MM.

The National Cancer Institute of Canada (NCIC) Clinical Trials Group conducted a phase II study of weekly oral menogaril as first-line therapy in 51 patients with incurable, metastatic or locally advanced breast cancer. While no prior chemotherapy for metastatic disease was permitted, prior adjuvant chemotherapy was allowed provided that no anthracycline or anthracene had been given. Forty-eight patients were evaluable for response. Two patients (4%) achieved complete remissions, 9 patients (19%) achieved partial remissions, 26 patients (54%) were stable and 11 patients (23%) failed. At the initial menogaril dose of 275 mg/m2 per week, 13 of 14 patients required a dose reduction and/or a treatment delay of one or more weeks. Therefore, the menogaril dose was reduced to 225 mg/m2 per week for the last 37 patients. At that those, 20 of 37 patients developed grade 3 or 4 granulocytopenia and 22 required dosage delays. At the initial starting dose, the average dose intensity actually delivered was 169 mg/m2 per week. At 225 mg/m2 the average dose intensity actually delivered was 197 mg/m2 per week. Toxic effects included mild to moderate nausea and vomiting, diarrhea, hair loss and occasional hyperpigmentation. In summary, menogaril is an anthracycline derivative that has modest activity when administered orally to minimally pretreated patients with breast cancer.

Fourteen patients with advanced/recurrent squamous cell carcinoma of the uterine cervix received menogaril, 200 mg/m2 by one hour intravenous infusion at four-week intervals. No objective regressions were observed. Median time to progression was less than two months and median survival was seven months. All patients experienced neutropenia. Platelet toxicity was negligible. Venous irritation and phlebitis occurred at the infusion site in 43% of patients. Menogaril as administered in this protocol is ineffective in treating previously irradiated advanced/recurrent squamous cell carcinoma of the uterine cervix and warrants no further investigation in this disease at the dosage and administration schedule used in this protocol.

Forty-one women with advanced, recurrent epithelial ovarian carcinoma (in whom prior chemotherapy with a platinum-based regimen failed) were treated with menogaril 200 mg/m2 intravenously every 4 weeks in a Phase II trial. Partial responses were seen in two of 19 (10.5%) measurable disease patients and three of 12 (25%) nonmeasurable but evaluable patients, an overall objective response rate of 16.1% (95% confidence interval, 5% to 34%). Median time to progression for all patients was 2 months and median survival, 5 months. Toxicities were acceptable and consisted primarily of leukopenia and gastrointestinal toxicity. Twenty-nine percent of the patients had venous irritation or painful phlebitis at the intravenous injection site. Menogaril, as administered in this protocol, had modest antineoplastic activity in previously treated ovarian carcinoma patients. The responses were of short duration, and there appeared to be no survival advantage with menogaril treatment.

Fifteen patients with advanced renal cell carcinoma were treated with Menogaril, 200 mg/m2 by one-hour, intravenous infusion at four-week intervals. No objective regressions were observed. Median time to progression was two months, and median survival was seven months. All patients experienced neutropenia. Platelet toxicity was negligible. Venous irritation and phlebitis at the infusion site was seen in 47% of patients. Menogaril as administered in this protocol is ineffective in advanced renal cell carcinoma.

The NCI Canada Clinical Trials Group conducted a phase II study of menogaril given intravenously every 4 weeks in low-grade non-Hodgkin's lymphoma. Fifteen of 26 eligible patients had had no prior therapy. Partial responses were seen in 9 patients (35%). Toxicity was moderate including myelosuppression, nausea, phlebitis, alopecia, and lethargy. This drug has only modest activity in this potentially responsive group of patients.

Forty-five patients with non-small cell lung cancer were treated in a phase II trial with menogaril 200 mg/m2 IV every twenty-eight days by a one-hour infusion. One partial response was noted while twenty-two patients had stable disease (51%). Progressive disease was noted in the remaining twenty-two patients. There was one fatal complication due to pancytopenia and pneumonia. Otherwise, the drug was reasonably well tolerated. At this dosage and schedule, menogaril has no substantial anti-tumor activity for patients with non-small cell lung cancer.

This review assimilates up-to-date information on the biochemical pharmacology of nogalamycin and selected derivatives that have shown good biological activities and/or received a relatively detailed investigation. The structure and chemical preparation of these derivatives from nogalamycin is described and the nomenclature which has been rather perplexing in the literature is clarified. The interaction of this class of compounds, particularly nogalamycin, with DNA is extensively reviewed. The biochemical mechanism of action of nogalamycin and its structurally closely-related derivatives is described. Among nogalamycin derivatives, menogaril showed distinct biochemical effects as well as superior cytotoxicity and antitumor activity and also proved to be effective against breast cancer clinically.

Doxorubicin is an essential component of the treatment of aggressive lymphoma, childhood solid tumors, bone and soft tissue sarcomas, and breast cancer and additional indications are emerging. On the other hand, daunorubicin has occupied the central position of interest in the treatment of acute leukemia. Epirubicin has a spectrum very similar to doxorubicin but lesser toxicity. The ability to protect against cardiotoxicity with ICRF-187 further enhances clinical interest in exploiting modifications in doze intensity to therapeutic advantage. Idarubicin has at least equivalent activity to daunorubicin and doxorubicin in leukemia. New areas of research in relation to anthracycline antibiotics include introduction of new the analogs, insight into mechanisms of resistance, the reversal of multidrug resistance in vitro, the protection of cardiac toxicity, and the study of other important biochemical reactions relevant to cytotoxicity. Orally active anthracyclines such as idarubicin and compounds which lack cross-resistance with the parent drugs or have other mechanisms for cytotoxicity are being developed. It is likely that these modifications will lead to an expanding therapeutic spectrum for these already widely useful drugs.

We have used both HPLC and flow cytometry to measure and compare the uptake of two anthracyclines, menogaril (MEN) and Adriamycin (ADR), in V79 Chinese hamster lung fibroblasts grown as monolayers and as 650 microns multicell spheroids. In order to compare intracellular drug accumulation in spheroid cells measured by the two methods, we converted mean channel fluorescence of the flow cytometer to drug uptake expressed as ng/10(6) cells by using a standard curve. The standard curve related the flow cytometric mean channel fluorescence, of monolayer cells exposed to either drug, to the intracellular drug accumulation determined by HPLC. This standard curve was then used to convert the mean channel fluorescence of cells from drug-exposed spheroids to ng/10(6) cells. Our results show that equal intracellular drug accumulation (determined by HPLC) in spheroids and monolayers does not result in equal cellular fluorescence emission (determined by flow cytometry) by these 2 cell populations. For example, monolayer cells with an intracellular MEN accumulation of 650 ng/10(6) cells, emit 40 units of fluorescence as measured by flow cytometry. However, spheroid cells with the same intracellular accumulation emit about 80 units of fluorescence. This results in the intracellular MEN uptake in spheroids measured by flow cytometry being as much as 2- to 3-fold higher than that measured by HPLC. Intracellular ADR accumulation measured by flow cytometry was also higher than that obtained by HPLC. In spite of the quantitative difference between the two methods, qualitatively both methods gave similar results. Thus, both techniques showed that at equal drug concentration in medium drug uptake in monolayers was much greater than in spheroids.(ABSTRACT TRUNCATED AT 250 WORDS)

Menogaril is an anthracycline presently in Phase II clinical trials. Menogaril-resistant mouse leukaemia P388 cells were developed in vitro by 4 months of exposure to step-wise increasing concentrations of menogaril after which resistant cells (P388/MEN) were cloned in 320 ng ml-1 menogaril. P388/MEN cells were 40-fold more resistant to menogaril in vitro compared to P388/O and were also resistant in vivo. Resistance to menogaril was stable for at least 2 months in the absence of the drug. The results indicate that P388/MEN, although resistant to an anthracycline, did not display the typical multidrug resistant phenotype. It was not cross-resistant to several structurally unrelated drugs such as actinomycin D, cisplatin, or vinblastine, but it was cross-resistant to the anthracycline, adriamycin. Uptake and efflux of menogaril was similar in sensitive and resistant cell lines. Also, resistance was not reversed by verapamil. No major karyotypic difference was noted between P388/O and P388/MEN. There was no significant amplification or overexpression of the mdr gene in P388/MEN compared to P388/O. In contrast to P388/MEN, P388 cells resistant to adriamycin displayed the typical multidrug resistant phenotype. Glutathione content of P388/MEN cells was similar to that of P388/O and depletion of glutathione did not potentiate menogaril cytotoxicity. Therefore, we conclude that glutathione is not likely to be involved in menogaril resistance to P388/MEN cells.

Eighteen women with metastatic breast cancer previously untreated with chemotherapy were entered on a phase II trial of intravenous menogaril, a new anthracycline derivative. Treatment was given at 140 mg/m2 on days 1 and 8 of each 28 day cycle. The most common toxicities were leukopenia in all patients and burning and phlebitis at infusion sites in 72%. Serial assessment of cardiac function by resting and stress gated blood pool scans showed temporary decrements in ejection fraction in only 2 patients (11%). The response rate to the therapy was 19% [95% CI 0-38%] including 1 complete and 2 partial responses. The median time to relapse among responders was 6.5 months. Mean survival in all patients entered was 15.8 months from date of entry. Menogaril at this dose and schedule has modest activity as first line therapy for metastatic breast cancer but also has significant marrow and local toxicity.

In this phase II trial, menogaril was administered to patients with metastatic colon cancer at a dose of 200 mg/m2 IV over one hour with cycles repeated every 28 days provided the absolute granulocyte count was greater than or equal to 2000 cells/microliters. Dose adjustments up or down were made depending upon nadir counts. Twenty-four patients were entered on this study with 21 eligible and evaluable for response. There was 1 CR lasting four and one-half months and 1 PR lasting three months for an overall CR + PR rate of 10% with a 95% confidence interval of 1% to 30%. Six patients (29%) had stable disease and 13 (62%) progressed. Median survival is 13.1 months. Toxicity was primarily hematologic with two cases of life-threatening leukopenia (less than 1000 cells/microliters) and one case of life-threatening granulocytopenia (less than 250 cells/microliters) among the 21 eligible patients, and one case of life-threatening leukopenia and granulocytopenia in one ineligible patient. There were no deaths due to treatment.

Menogaril, an anthracycline compound possessing a significant antitumor activity after both po and iv administration, has been introduced into clinical trials. However, its mechanism of action has not been clarified yet. This study revealed that its cytotoxicity correlated very well with the inhibition of macromolecular synthesis, indicating the involvement of interaction with DNA. The spectrophotometric study showed a weaker binding of this compound to calf thymus DNA when compared to that of doxorubicin (adriamycin). Despite the lower binding affinity of menogaril to DNA, pronounced DNA cleavage was observed in an intact cell system, indicating that the character of the interaction with DNA is different from intercalation. In contrast to doxorubicin, menogaril is extensively localized in the cytoplasm. The cytoplasmic localization prompted us to study its effect on cytoskeleton proteins. It was found that menogaril inhibited the initial polymerization rate of tubulin, indicating a possible contribution of this process to the overall cytotoxicity of menogaril.

We have carried out a phase II study of intravenous menogaril given every four weeks in a group of patients with breast cancer who had received no prior chemotherapy for metastatic disease. Myelosuppression, nausea and vomiting and local reactions were seen frequently. Six partial responses (median duration 154 days) were seen in 24 eligible patients. We conclude menogaril is active in breast cancer and recommend that because it can be delivered in high doses orally, future trials in this disease should focus on intense oral schedule.

We estimated antitumor activity of TUT-7 following p.o. administration using animal tumor models and human tumor xenografts. In mouse L 1210 leukemia system, antitumor activity of TUT-7 administered orally was as good as that by i.v. administration. Treatment involving schedules of every 4-days or daily administration was much more effective than single treatment. Therapiotic indices of this compound administered both p.o. or i.v. routes, were better than that of adriamycin administered i.v.. TUT-7 showed antitumor activities against various mouse tumors (L 1210 leukemia, P 388 leukemia, colon 38 adenocarcinoma, B 16 melanoma), LX-1 human tumor xenografts, and Yoshida sarcoma in rat. Base on above results, we concluded that oral administration is one of the useful route of TUT-7 administration.

Forty-seven patients with solid tumors were treated on a phase I study of menogaril administered by mouth once per week. Nausea and vomiting were excessive at weekly doses of 350 and 450 mg/m2/week but were tolerable and controlled reasonably well by antiemetics at lower doses. There appeared to be a relatively shallow dose-vs-granulocytopenia curve above a menogaril dose of 180 mg/m2/week. No patient receiving chronic dexamethasone for cerebral edema developed granulocytopenia, even at menogaril doses of 350-450 mg/m2/week. Two patients developed neutropenic infection. No patient developed thrombocytopenia. Mild arrhythmias were seen in 3 patients. Two patients suffered possible myocardial infarcts that may not have been related to treatment. Asymptomatic blood pressure fluctuations were common and were probably not related to treatment. Diarrhea was dose-related but was generally not severe. Alopecia and stomatitis occurred occasionally. Minor responses were seen in two patients with gliomas, and three of five evaluable prostate cancer patients experienced marked pain relief. The dose recommended for phase II studies is 250-300 mg/m2/week with antiemetic pretreatment. This schedule appears to allow an oral menogaril dose-intensity that is approximately double that attainable with other oral schedules that have been studied.

The antitumor activity of TUT-7, a new anthracycline compound, was compared to that of adriamycin in the screening system with rat ascites hepatomas. A marked antitumor effect was observed in most tumor lines, and the activity was assumed to be better than adriamycin.

The Southwest Oncology Group (SWOG) studied the response rate and toxicity of menogaril (200 mg/m2 i.v. q 28 days) in patients with advanced metastatic renal cell carcinoma. During the early stage of the trial two partial responses were seen in the first 20 treated patients, and an additional 36 evaluable patients were studied. Three of 56 (5%) evaluable patients achieved partial responses. Significant white cell toxicity was observed. Mild or moderate degrees of thrombocytopenia, gastrointestinal side effects, alopecia and phlebitis occurred. No cardiac toxicity was noted. The low response rate suggests that menogaril in this dose schedule has no role in the treatment of patients with advanced metastatic renal cancer.

The mechanisms of action and resistance to menogaril, a clinically active anthracycline antitumor drug, were evaluated in sensitive and doxorubicin-selected multidrug resistant human breast tumor (MCF-7) cell lines. While MCF-7/ADRR cells were highly resistant (250-500-fold) to doxorubicin, they displayed only marginal resistance (10-fold) to menogaril. In contrast to doxorubicin, the mechanism of resistance to menogaril in these cells does not involve differential inhibition of DNA synthesis as measured by thymidine incorporation. P-170-glycoprotein-dependent drug transport did not contribute to resistance as there was no difference in the accumulation and retention of menogaril by sensitive and resistant cell lines. However, there was a 2-fold decrease in oxygen free radical formation in the resistant cells, compared to sensitive cells, in the presence of menogaril. Since resistant cells contain 12-fold higher glutathione peroxidase activity than the parental sensitive cells, the detoxification of hydrogen peroxide may be responsible for the decreased free radical formation and thus, may play a role in the resistance to menogaril.

Eight human ovarian cancer lines grown in nude mice were used to compare the activity of doxorubicin, epirubicin, mitoxantrone and menogaril. The tumour lines were different in histological subtype, tumour doubling time and sensitivity to doxorubicin. The compounds were administered intravenously at the maximum tolerated dose twice with one week in between when tumours measured 50-150 mm3. Growth inhibition greater than 50% was obtained for doxorubicin in 8/8, for epirubicin in 4/8, for mitoxantrone in 5/8 and for menogaril in 2/8 tumour lines. In MRI-H-207, doxorubicin was the only drug able to induce complete remission. Compared with doxorubicin, mitoxantrone and menogaril were given in proportionally higher doses than those administered to patients, but did not result in superior antitumour activity.

Adriamycin and menogarol are anthracyclines which cause more than 100% increase in life span of mice bearing P388 leukemia and B16 melanoma. Unlike Adriamycin, menogarol does not bind strongly to DNA, and it minimally inhibits DNA and RNA synthesis at lethal doses. Adriamycin is a clinically active drug, and menogarol is undergoing preclinical toxicology at National Cancer Institute. In view of the reported mutagenicity of Adriamycin, we have compared the genotoxicity of the two drugs. Our results show that, although Adriamycin and menogarol differ significantly in their bacterial mutagenicity (Ames assay), they have similar genotoxic activity in several mammalian systems. Adriamycin is strongly mutagenic in the Ames assay with TA98 and TA100. Menogarol is nonmutagenic to TA98 and TA100. For the mammalian cell culture systems, V79 (Chinese hamster) cells are exposed for 2 hr to drug, following which cell survival, induction of sister chromatid exchanges, chromosome damage, and production of mutants resistant to 6-thioguanine are measured. The percentage of survival obtained with the two drugs ranges between 25 and 50% at 0.15 microgram/ml and 5 to 15% at 0.3 microgram/ml. At 0.15 microgram/ml, Adriamycin and menogarol increase the percentage of cells with chromosome damage from a background level of 8.8 to 30 and 22.5%, respectively. The same drug concentration causes a small but significant increase in sister chromatid exchange rate. Both drugs are equally active (increase mutation frequency about 3- to 6-fold above background) in producing 6-thioguanine-resistant mutants. The induction of micronuclei in polychromatic erythrocytes of rats is the most sensitive assay system. Both drugs cause 10- to 15-fold increase in micronuclei at nontoxic doses.

Nogalamycin is an anthracycline antibiotic which was markedly cytotoxic in vitro and was active against several tumor systems in vivo. We compare here the lethality of several nogalamycin analogs against Chinese hamster ovary (CHO), mouse leukemia (L1210), and mouse melanoma (B16) cells in culture. 7-con-O-Methylnogarol (7-con-OMEN) was the most lethal of all the analogs tested. Thus, for CHO cells exposed for two hr to the drug, the 50% lethal doses of 7-con-OMEN, nogalamycin, and dis-nogamycin were 0.25, 2.7, and 5.8 micrograms/ml, respectively. In general, CHO cells were less sensitive than B16 or L1210 cells to most compounds. All compounds gave dose-survival curves which consisted of a shoulder region followed by a region of exponential decline in survival. The nogalamycin analogs nogalamycin, dis-nogamycin, 7-con-O-methylnogalarol, and 7-con-OMEN were selected for further study because of their greater lethality in vitro and antitumor activity in vivo. The lethality of these compounds was compared to that of Adriamycin. 7-con-OMEN was more toxic to CHO cells than was Adriamycin but was less toxic to B16 and L1210 cells. All of these compounds (except 7-con-O-methylnogalarol which was not tested) were more lethal to exponentially growing cells than to plateau-phase cells. The survival response after different periods of exposure to these drugs was compared. In order to make valid comparisons of the time-survival response to different drugs, the drug concentrations chosen were such that they were equitoxic after a two-hr exposure. Under these conditions, the order of lethality after long-term exposure (8 hr to 24 hr) was nogalamycin > dis-nogamycin > 7-con-OMEN, Adriamycin > 7-con-O-methylnogalarol. With all the drugs, the rate of cell death increased with increasing drug concentrations.

Nogalamycin, an antitumor antibiotic, has been converted to a series of analogs by removal of the carbomethoxy group at C-10 and replacement of the neutral sugar at C-7 by other groups. Removal of the carbomethoxy group to give disnogamycin (6) followed by acidic alcoholysis gave pairs of isomeric 7-alkoxy compounds differing in configuration at C-7. Treatment of 6 with trifluoroacetic acid followed by nucleophiles gave a series of analogs having substituents at C-7 with a configuration at C-7 opposite to that of nogalamycin. Among the analogs prepared, 7-con-O-methylnogarol (7) is a highly active antitumor agent.

Menogaril is a new anthracycline analog of nogalamycin. When administered as a 72-hour continuous iv infusion the dose-limiting toxic effect of menogaril was venous irritation at dose levels that cause only mild leukopenia and minimal gastrointestinal toxicity. Pharmacokinetic studies showed that the rise in plasma concentration during infusion was first-order, with a half-life of 11.9 hours. Total-body clearance of menogaril was 204 ml/minute/m2. There were no detectable metabolites of menogaril in plasma. Urinary excretion of unchanged menogaril was 17.3% of the dose and N-demethylmenogaril was 0.5% over 72 hours. Since menogaril does not appear to be metabolized, a high degree of tissue binding is likely.

Nogalamycin (1) has been modified by changes at C-10 and C-7 and in the dimethylamino group to prepare an extensive series of analogues. The chemistry involved in the modifications and structure--activity relationships among these nogalamycin analogues are discussed, as well as comparisons with previously reported compounds 1, 7-con-O-methylnogarol (2), and disnogamycin (11).

Menogarol is a new anthracycline undergoing phase I clinical trial. We report here the lethality after 2 hr exposure to 2 drug combinations of menogarol and several antitumor agents. A new statistical procedure was used to identify synergistic combinations. Most of these combinations were additive, except for menogarol plus melphalan, which was synergistic. Adriamycin plus melphalan was also synergistic. The menogarol-melphalan combination wa studied in detail with regard to the effect of dose and drug-schedule, lethality for exponential and plateau phase cells and effect on cell cycle progression. Although the combination was synergistic for exponential cells it was additive for plateau phase cells. The combination exerted a synergistic effect in inhibiting progression of cells through the cell cycle. After 2 hr menogarol exposure cells were blocked in G2 for about 12 hr following which the block was reversed. This reversal was inhibited when menogarol was combined with melphalan. The uptake of menogarol or melphalan was not changed in the presence of the other drug.

Eighteen adult patients with solid tumors were treated with oral menogaril, a new anthracycline antibiotic active against human breast cancer after intravenous administration. The drug was given orally on 3 consecutive days every 4 weeks at doses ranging from 50 to 175 mg/m2/day. Reversible and dose-related leukopenia was the dose-limiting toxicity. At doses from 50 to 150 mg/m2/day, non-hematologic side effects of oral menogaril were unfrequent and mild and consisted of nausea and vomiting (1 patient), alopecia (2 patients), mucositis (2 patients) and liver function test abnormalities (3 patients). The only patient treated at a daily dose of 175 mg/m2 developed grade IV leukothrombocytopenia, with fever and gastrointestinal bleeding. This was followed by heart insufficiency and the patient died from multisystem organ failure. A dose of 150 mg/m2/day for 3 consecutive days is recommended for phase II trials with oral menogaril.

Menogaril, a semisynthetic derivative of nogalomycin, was brought to phase I clinical testing in patients with refractory solid tumors. Twenty-seven patients received 50 evaluable courses. Menogaril was given as a 1-2-hour iv infusion on 5 consecutive days, with courses repeated every 4 weeks, provided there was reversal of all drug-related toxic effects. The starting dose was 3.5 mg/m2/day X 5, with escalations in subsequent cohorts of patients to 56 mg/m2/day X 5. Neutropenia was dose dependent and dose limiting. At 56 mg/m2/day X 5, the median wbc count nadir was 1100/microliter, and two of four patients were hospitalized for fever and suspected bacteremia. At 50 mg/m2/day X 5, the wbc count nadir was 2300/microliter. Platelet toxicity was less severe. Nonhematologic toxicity consisted primarily of local urticaria and moderate to severe phlebitis at the infusion site, which were dose dependent and lasted up to 6 weeks. For phase II studies, the recommended dose of menogaril is 50 mg/m2/day for 5 consecutive days administered as a 2-hour intermittent infusion, repeated every 28 days.

7-con-O-Methylnogaril (menogaril, NSC-269148) is a new anthracycline antibiotic that has been evaluated in a Phase I clinical trial. The drug was administered in a single i.v. infusion over a period of 60 min given every 3 weeks. Twenty-four patients received 64 courses of the drug in a dose range of 16 to 256 mg/m2. Granulocytopenia was dose limiting and prolonged, requiring treatment delay in 5 of 9 patients treated at doses greater than or equal to 192 mg/m2. Concentration dependent phlebitis occurred in 12 patients, and was of minimal severity when the menogaril concentration was less than 1 mg/ml. Hair loss was experienced by 8 patients but was generally mild with only one patient developing total alopecia. Possible acute cardiac toxicity was noted in one patient who had a transient episode of atrial fibrillation following his fifth course of menogaril. Phase II studies of 7-con-O-methylnogaril are planned at a starting dose of 160 mg/m2 for patients with prior chemotherapy or radiotherapy, and 200 mg/m2 for those without prior therapy given at 28-day intervals.

Fifteen patients with advanced adenocarcinoma of the pancreas were treated with menogarol 150-225 mg/m2 i.v. every 3 weeks. All patients had bidimensionally measurable disease. This regimen and dosage schedule are well tolerated, with minimal toxicity that included myelosupression; median white blood cell (WBC) count nadir of 2,700 cells/mm3 (range 1,400-7,100 cells/mm3) and median platelet nadir of 162,000 cells/mm3 (range 53,000-390,000 cells/mm3). Anorexia occurred in one patient, nausea or vomiting in six, phlebitis in one, and alopecia in six patients. No patients responded. At this dosage and schedule, there is no role for menogarol in the treatment of advanced pancreatic adenocarcinoma.

Menogaril [menogarol, 7(R)-O-methylnogarol, 7-OMEN] is a new anthracycline agent which was chosen for clinical trials based on: broad spectrum activity against a panel of murine tumors, lower cardiotoxicity than doxorubicin in the chronic rabbit model, differences in biochemical effects from other anthracyclines suggesting a possible difference in mechanism of action, murine antitumor activity by oral as well as parenteral routes. Biochemical studies indicated that, in comparison to doxorubicin, menogaril is bound weakly to DNA, inhibits RNA synthesis less, and has different cell cycle phase-specific cytotoxicity. Pharmacology studies in the mouse and dog using HPLC analytical methodology have shown multiexponential clearance from plasma and metabolism of menogaril to a material which co-chromatographs with N-demethylmenogaril in addition to at least two other metabolites of unknown structure. Oral bioavailability studies in the mouse showed significant absorption of menogaril from the gastrointestinal tract followed by first-pass metabolism. In acute toxicity studies in the rat, the dog, and the monkey, dose-related myelosuppression and gastrointestinal toxicity predominated. Phase I clinical trails on menogaril are currently in progress on a variety of schedules.

A multidrug resistant variant (H69AR) of the human small cell lung cancer cell line NCI-H69 was obtained by culturing these cells in gradually increasing doses of Adriamycin up to 0.8 microM after a total of 14 months. H69AR expresses the multidrug resistant phenotype because it is cross-resistant to anthracycline analogues including daunomycin, epirubicin, menogaril, and mitoxantrone as well as to acivicin, etoposide, gramicidin D, colchicine, and the Vinca alkaloids, vincristine and vinblastine. H69AR is also similar to other multidrug resistant cell lines in that it displays little or no cross-resistance to bleomycin, 5-fluorouracil, and carboplatin. It has a slight collateral sensitivity to 1-dehydrotestosterone and lidocaine. H69AR has increased cell-cell adhesiveness compared to H69, but a similar growth rate in vitro and tumorigenicity in nude mice. When cultured in the absence of Adriamycin, there is a 40% decrease in resistance by 35 days of culture, compared to cells in continuous culture in drug, but no further decrease in resistance up to 181 days. Monoclonal antibodies to P-glycoprotein have no detectable reactivity with H69AR cells as determined by enzyme-linked immunosorbent assay and immunoblotting techniques. Thus, unlike most multidrug resistant cell lines, H69AR does not appear to express enhanced levels of P-glycoprotein. H69AR will provide a useful model for the study of multidrug resistance in human small cell lung cancer.