Immunotherapy of Madison 109 lung carcinoma and other murine tumors using lentinan.

Lentinan was evaluated initially against the Lewis (LL) and Madison 109 (M109) lung carcinomas implanted in the footpads of syngeneic mice. Activity in these tumor models was assessed by both reduction in early tumor growth rates and increases in life span and cures, relative to untreated control mice with tumors. Lentinan given i.p. to mice bearing LL footpad tumors caused a reduction in tumor growth rate in only one of three experiments and an increase in life span of 48% at one dose level on another occasion. In contrast, lentinan given i.p. to mice bearing M109 footpad tumors was consistently curative (50 to 70%) in three experiments despite the lack of an effect upon early tumor growth rate. In subsequent experiments, syngeneic mice were implanted s.c. with M109 or LL and treated with lentinan. Although lentinan had no substantial effect upon LL, 25 to 75% of mice bearing s.c. M109 tumors were cured in three separate experiments following early treatment initiation. Delayed lentinan therapy, initiated when s.c. M109 tumors were greater than 100 mg, also resulted in complete tumor regression and cure of 29 to 63% of the mice in three experiments. Surgical adjuvant immunotherapy of s.c. M109 using lentinan also improved survival rates over those obtained using surgery alone. Mice cured of s.c.-implanted M109 using lentinan, or surgery plus lentinan, but not surgery alone, survived a rechallenge with M109. The therapeutic effects of lentinan in mice implanted s.c. with B16 melanoma were inconsistent.

Antitumor activity and toxicity in animals of RR-150 (7-cysteaminomitosane), a new mitomycin derivative.

The experimental antitumor activity of a new mitomycin derivative, 7-cysteaminomitosane (RR-150), was evaluated in mice. When administered i.p. to mice bearing i.p.-implanted tumors, RR-150 was superior to mitomycin C (MMC) in increasing the life span of animals bearing P388 leukemia, B16 melanoma, and a line of L1210 leukemia partially resistant to MMC. RR-150 appeared comparable to MMC in increasing life span of mice bearing Madison 109 lung carcinoma, Colon 26 carcinoma, or parental (nonresistant) L1210 leukemia. Mice immunosuppressed with 550 rads whole-body irradiation prior to i.p. implantation of B16 still benefited (e.g., 40% cure rate) following optimal RR-150 therapy when compared to nonirradiated, B16-implanted mice given RR-150 (e.g., 70% cure rate). RR-150 had inconsistent activity in the treatment of s.c.-implanted tumors. In toxicity evaluations, RR-150 was comparable to MMC in suppression of total while blood cell counts but appeared to be less neutropenic. RR-150 also caused less cumulative leukopenia than did MMC in a weekly chronic dose experiment. Based on serum chemistries, RR-150 did not have significant nephrotoxicity, but there was evidence of possible liver toxicity at doses near the 50% lethal dose. Because of the balance of favorable antitumor and toxicity properties of RR-150, work is under way to prepare a more bioavailable form for advanced evaluation.

Antitumor activity and toxicity in animals of BMY-25282, a new mitomycin derivative.

BMY-25282, 7-N-(dimethylamino methylene)mitomycin C, is one of a novel series of amidino mitomycin derivatives. Some of these were discovered as intermediates in a synthetic program being conducted to find improved procedures for modifying the structure of mitomycin C (MMC). Markedly superior in vivo antitumor effects have been observed with BMY-25282 compared to MMC in initial tests against i.p.-implanted P388 leukemia and B16 melanoma. When administered i.v. to mice bearing s.c. B16 melanoma, BMY-25282 was also superior to MMC. The derivative was fully active against a line of L1210 leukemia which was partially resistant to MMC treatment but had little or no activity against a line of L1210 fully resistant to MMC. It is also 2 to 4 times more potent than MMC based on a comparison of doses required to achieve optimum antitumor effects. The superior antitumor efficacy of BMY-25282 over MMC against both i.p. and s.c. B16 melanoma was maintained when the drug was given in pluronic acid formulation. Against P-388 leukemia, however, the efficacy of the drugs was equivalent when BMY-25282 was administered in the pluronic vehicle. In an in vitro clonogenic assay involving freshly explanted human tumors, BMY-25282 was consistently more potent in cytotoxic effects than MMC. With human colorectal carcinoma samples, BMY-25282 was 13.8 times more potent than MMC. The i.v. 50% lethal dose values of BMY-25282 and MMC in C57BL/6 X DBA/2 F1 mice were 2.1 mg/kg and 8.6 mg/kg, respectively. Leukopenic effects of the drugs in mice were comparable at doses up to their respective 50% lethal dose values. Hematology studies in ferrets revealed a similar pattern of depression and recovery of lymphocytes, neutrophils, and platelets for BMY-25282 and MMC; however, with BMY-25282 there was earlier recovery of platelet counts. BMY-25282 is being further developed toward possible clinical trial.

Animal models for evaluating the myelosuppressive effects of cancer chemotherapeutic agents.

An important objective of new anticancer drug discovery programs is identification of agents that are less myelosuppressive than those currently available. We have developed several animal models to evaluate these drugs for myelosuppression. Our screening model measures changes in neutrophil counts in mice as an indicator of myelosuppression. This model correctly predicted the myelosuppressive effects of 13 (76%) of 17 known agents. Cisplatin, carboplatin, spiroplatin, and marcellomycin caused no reduction in the neutrophil counts, representing four (24%) of 17 false negatives. Our secondary evaluation system is the more labor-intensive murine CFU-C assay on femoral bone marrow cells from drug-treated mice. Known myelosuppressive drugs such as mitomycin C, doxorubicin, and BCNU, as well as the false negatives from the mouse neutropenia model (cisplatin, carboplatin, spiroplatin, and marcellomycin) caused marked inhibition of colony formation 24 h after dosing; bleomycin was inactive. Advanced evaluations are performed using ferrets in which neutrophil counts can be monitored in the same animal for 28 days after treatment. Mitomycin C, doxorubicin, and BCNU caused significant reductions in the neutrophil counts whereas bleomycin had no effect. Importantly, cisplatin and marcellomycin also caused significant reductions in the neutrophil counts. Although the mouse neutropenia model is a rapid assay, there is potential for false-negative predictions. It is important that other test systems be used for more advanced evaluation of drugs identified by this model as being less myelosuppressive than reference drugs. The mouse CFU-C and ferret hematology models are suitable for this purpose in that they can identify the false-negative predictions as well as identify less myelosuppressive drugs such as bleomycin.

Mitomycin C: a clinical update.

Mitomycin C was reviewed in this journal 25 years ago and an update of its clinical usefulness is appropriate. The current review is based on representative publications covering clinical trials performed throughout the world. Single agent activity in each of the major neoplastic diseases has been reassessed when possible and the most important combinations evaluated. It is concluded that mitomycin C has a definite place in the treatment of localized bladder cancer, is active, but needs to be redefined, in the context of newer regimens for breast, head and neck, and non-small cell lung cancers, is active in, but is being displaced by, other drugs in cervical, gastric and pancreatic cancers, and is probably no longer of therapeutic value in colon cancer. It is also recognized that as many newer treatments have clinical success, the therapeutic role of mitomycin C will require continuing re-investigation.

Preparation and antitumor activity of olivomycin A analogues.

Novel analogues of olivomycin A were prepared by selective reactions involving the carbonyl and hydroxyl groups of the aglycon moiety. Electrophilic substitution of the aglycon also was successful. Of 11 analogues, all but two were active in the P-388 murine leukemia assay. One compound, the 2'-methoxime, showed superior activity to olivomycin A based on its wider dose range and greater potency. The methyl imine and the 8-O-methyl ether were equal to olivomycin A in potency and efficacy. Most of the other analogues were slightly less potent or effective.

Preparation and antitumor activity of 7-substituted 1,2-aziridinomitosenes.

7-Methoxy-1,2-aziridinomitosenes were prepared from mitomycin A and its N-methyl homologue by catalytic reduction followed by air oxidation. Treatment of these products with amines, including ammonia, ethylenimine, 2-methylethylenimine, propargylamine, and furfurylamine gave the corresponding 7-(substituted amino) derivatives. Screening of these compounds against P-388 leukemia in mice revealed some good activities. The more easily reduced compounds gave prolongation of life span comparable to that of mitomycin C, but their optimal doses were higher. Among these compounds, a methyl group on the aziridine nitrogen increased potency. The 7-amino derivatives, which were difficult to reduce to hydroquinones, were essentially inactive. The aziridinomitosenes were subjected to a Hansch-type analysis, but no statistically significant correlation was found.

Synthesis and antineoplastic activity of mitosene analogues of the mitomycins.

A series of 1-substituted mitosene analogues of the mitomycin antitumor antibiotics was prepared by total synthesis and screened for activity against P388 leukemia in mice. In general, analogues with moderately good leaving groups (mostly esters) at the 1 position were active, whereas analogues without such substituents were inactive or barely active. These results lend support to the idea that mitosenes with leaving groups at position 1 are capable of bifunctional alkylation of DNA in a manner similar to that of mitomycin C. The most active mitosenes were equal in potency (minimum effective dose) to a corresponding aziridinomitosene, but they were less effective in prolonging life span.

Preparation and antitumor activity of additional mitomycin A analogues.

On the basis of qualitative structure-activity relationships developed in the preceding article, a series of 32 new mitomycin A analogues were prepared and tested in antitumor screens. Seven of them gave greater prolongation of life (ILS) than mitomycin C in the mouse P388 leukemia assay. They included examples with 7-O substituents such as cyclic ethers and nitrogen heterocycles. A Hansch analysis was attempted with log P and MR as the independent variables, but no statistically significant correlation could be made. Seven compounds, chosen mainly for their good potency (MED), were tested in the subcutaneous B16 melanoma assay in mice and four of them showed greater ILS than mitomycin C.

Synthesis and biological activity of 6-substituted mitosene analogues of the mitomycins.

A series of 1-acetoxymitosene analogues, in which the substituent at C-6 was varied, was prepared by total synthesis and screened for activity against P388 leukemia in mice and induction of lambda phage in Escherichia coli. Among the 6-substituents prepared, none was as effective as the methyl group in conferring biological activity. However, certain N-methylcarbamates were more active than the unsubstituted carbamates.

Metal complexes of mitomycins.

The preparation of stable complexes between the N7-[2-(2-pyridyl)ethyl] and N7-(2-piperazinylethyl) derivatives of mitomycin C and metal ions such as Cu(II), Zn(II), and Pt(II) was accomplished. Mitomycin C did not form stable complexes, but it rearranged to a mitosene capable of complex formation. Some of these complexes had antitumor activity in mice. However, they were less active than mitomycin C. Weak associations between mitomycin C and metal ions were demonstrated by 13C and 15N NMR spectrometry.

Preparation and antitumor activity of new mitomycin A analogues.

A series of 26 mitomycin A analogues including 23 new ones was prepared by a variety of methods. The most useful methods were alkoxide exchange on mitomycin A and treatment of 7-hydroxymitosane with 3-substituted 1-phenyltriazenes. Many of the new analogues were superior to mitomycin C in the P388 leukemia assay and the more stringent subcutaneous B16 melanoma assay both in mice. Four of them gave long-term survivors in the latter assay. Quantitative correlations between log P and antitumor activity were not possible, but some guidelines for future analogue development are proposed.

Mitomycin C and porfiromycin analogues with substituted ethylamines at position 7.

A series of 7-(2-substituted-ethyl)amino analogues of mitomycin C and porfiromycin was prepared and screened in standard antitumor systems. Certain of these analogues showed better activity than mitomycin C against P-388 leukemia, L-1210 leukemia, and/or B-16 melanocarcinoma in mice. Compounds also tested for their leukopenic effects in mice, the limiting toxicity of mitomycin C. Some of them were less leukopenic and some were more leukopenic than this clinical agent. No statistically significant correlations could be made between physicochemical properties and antitumor activities of the analogues.

Preparation and antitumor activities of some derivatives of 5-methoxysterigmatocystin.

A series of derivatives of 5-methoxysterigmatocystin (3a,12c-dihydro-8-hydroxy-6,11-dimethoxy-7H-furol[3',2':4,5]furo[2,3-c]xanthen-7-one) has been prepared and evaluated for antitumor activity. The potency of the parent compound has been associated with the intact bisfurano ring system and with the double bond in the terminal furan ring. It has been shown that new substituents can be introduced in the xanthone portion of the molecule and that the antitumor activity is in some cases preserved.

Development of new mitomycin C and porfiromycin analogues.

New mitomycin C and porfiromycin analogues were prepared by treating mitomycin A and N-methylmitomycin A with a variety of amines, including aziridines, allylamines, propargylamines, chloroalkylamines, hydroxyalkylamines, glycine derivatives, aralkylamines, and heterocyclic amines. All analogues were evaluated against P-388 murine leukemia and selected ones were examined for their leukopenic properties. Certain analogues were found to be superior to mitomycin C in potency, efficacy, and therapeutic ratio in the P-388 assay. The most active substituents at the mitosane 7 position included aziridine, 2-methylaziridine, propargylamine, furfurylamine, methyl glycinate, and 3-aminopyridine. Mitomycin A and the 7-aziridino, 7-(2-methylaziridino), and 3-aminopyridine analogues were less leukopenic than mitomycin C. Certain other analogues, including propargylamino and methyl glycinate, were highly leukopenic. The three compounds tested against B-16 melanoma in mice were significantly more effective than mitomycin C in this assay. Previously established structure--activity relationships were found inadequate to account for all of the new data.

Bicyclic and tricyclic analogues of anthramycin.

As analogues of pyrrolo[2,1-c][1,4]benzodiazepine antitumor antibiotics, such as anthramycin and tomaymycin, several benzo[1,4]diazepine imines and carbinolamine ethers were prepared and tested in vivo against P388 leukemia. Two different synthetic approaches, namely, a reduction of an aromatic nitro group with a concomitant cyclization and a reduction of a lactam, were employed to generate an imine or a carbinolamine moiety. Bicyclic analogues 6a', 6f, and 6g were found to be active, indicating that the pyrroline ring of anthramycin is not an absolute necessity for the antitumor activity. Compound 6g, 3,4-dihydro-9-hydroxy-4-propargyl-5H-1,4-benzodiazepin-5-one, was at least as active as neothramycin although it was 5 times less potent. Among the tricyclic analogues, compounds 5, 7a, and 8b were active against P388 leukemia, and they generally appear to be more potent than bicyclic analogues.

Reductive amination of 3-ketoanguidin and antitumor activity of the products.

Amine-containing trichothecanes were prepared by reductive aminations of 3-ketoanguidin. In in vivo tests against P388 leukemia, most of them showed an improved activity compared to anguidin though their potency was decreased. 3-Ketoanguidin also produced some unexpected structures: oxazoline 5, dioxalane 7, and alpha-amino nitrile 13.

Mitomycin C analogues with increased metal complexing ability.

Twenty-three new mitomycin C analogues designed to have increased metal complexing ability were synthesized and tested against P388 leukemia in mice. Their ability to complex Cu(II) was revealed by the shifts in their UV absorption spectra caused by this metal. One analogue was clearly more active than mitomycin C in the antitumor assay and two others had good activity. Correlation between antitumor activity and Cu(II) complexing ability was ambiguous. The most active compounds were either not complexers or they were complexers limited to the 2-(2-pyridyl)alkyl type substituent on N7. A variety of amino acid substituents on N7 showed only weak antitumor activity.

Mitomycin C analogues with secondary amines at position 7.

A series of mitomycin C analogues with secondary amines at position 7 was prepared from mitomycin A. Eleven of the 20 new compounds in this series were more active than mitomycin C against P-388 murine leukemia, and 2 of these 11 were significantly less leukopenic. The two substituents conferring these superior properties were 4-formylpiperazine and 2-cyanoaziridine. No quantitative correlations could be made among antitumor activities and physicochemical properties of the analogues, although the relative ease of quinone reduction might be related to the good potencies (minimum effective doses) of many of them.

Mitomycin C analogues with aryl substituents on the 7-amino group.

A series of 30 different N7-phenyl-substituted mitomycin C analogues, including 25 new compounds, was prepared from mitomycin A. Seven of these compounds were clearly superior to mitomycin C in activity against P-388 murine leukemia. The para- and the meta-substituted derivatives were subjected to Hansch analysis, which revealed that the lipid-water distribution coefficient pi was the only significant factor in determining antitumor potency (MED). The substituent electronegativity factor sigma was statistically insignificant in determining potency, despite the good correlation of sigma p with the polarographic quinone-reduction potential. These results suggest that diffusion into the tumor cell or access to the receptor is more important than bioreductive activation in determining antitumor potency for this particular group of mitosanes . Fifteen new mitomycin C analogues with heterocycles on the 7-amino group also were prepared. Two of them, containing pyrazolyl and aminopyridyl substituents, were more active than mitomycin C against P-388 murine leukemia. No broad correlations could be made among the antitumor potencies and physicochemical properties for this type of analogue.