Antitumor and chemopreventive effects of a clomiphene analog, MDL 103,323, in mammary carcinoma.

MDL 103,323, an enclomiphene analog, was tested for binding to the estrogen receptor, inhibition of human tumor xenografts and prevention of carcinogen-induced mammary tumors. MDL 103,323 had 5-6 fold greater affinity for the human estrogen receptor than did either tamoxifen or enclomiphene. Consistent with enhanced binding affinity, MDL 103,323 was more potent against MCF-7 cell proliferation and MCF-7 xenografts in nude mice were inhibited almost completely by MDL 103,323 doses > or = 0.02 mg/mouse/day (ED50 l 0.01 mg/mouse/day). N-methylnitrosourea induced rat mammary carcinomas were inhibited by > or = 50% at 0.003 mg/kg daily and by 90% at 0.1 mg/kg/day. The antitumor potency and efficacy of MDL 103,323 are striking and further evaluation of the compound for potential clinical utility is warranted.

In vitro and in vivo inhibition of glioblastoma and neuroblastoma with MDL101731, a novel ribonucleoside diphosphate reductase inhibitor.

We examined the effects of MDL101731, a novel ribonucleoside reductase inhibitor, against human glioblastomas and neuroblastoma, both in vitro and in xenograft models, to determine its activity against malignant brain tumors. MDL101731 produced a concentration-dependent inhibition of both glioblastoma cell lines (HS683 and J889H) and neuroblastoma (SK-N-MC) in nanomolar concentrations (IC50, 30-90 nM). s.c. xenografts of human glioblastoma (D54) in athymic mice increased to five times their initial volume at a median of 7.4 days in control animals, while tumor regression occurred in 12 of 12 animals treated with MDL101731 (100 mg/kg, i.p., two times/week) during 22 days of treatment (P < 0.0001). Intracerebral implants of D54 carried a median survival of 20 days in control animals, whereas animals receiving MDL101731 (100 mg/kg, i.p., two times/week, days 10-35) had a median survival of 46.5 days (P < 0.0001). Intracerebral xenografts of SK-N-MC in athymic mice resulted in a median survival of 23 days in control animals and 26 days in animals treated with carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea 20 mg/kg/week, i.v. x 2; difference not significant). There was 90% survival in animals treated with MDL101731 (200 mg/kg, i.v., two times/week, days 7-35) up to 90 days after implant. These studies indicate that MDL101731 has potent antiproliferative activity against human malignant brain tumors.

Antiretroviral activity of mechanism-based irreversible inhibitors of S-adenosylhomocysteine hydrolase.

S-Adenosylhomocysteine hydrolase (AdoHcy-nase) is a key enzyme in transmethylation reactions. The objective of the present study was to examine the potential antiretroviral activities of novel mechanism-based irreversible AdoHcy-nase inhibitors. (Z)-4',5'-didehydro-5'-deoxy-5'-fluoroadenosine (ZDDFA), (E)-4',5'-didehydro-5'-deoxy-5'-fluoroadenosine (EDDFA), (Z)-4',5'-didehydro-5'-deoxy-5'-chloroadenosine (ZDDCA) and 5'-deoxy-5'-acetylenic adenosine (DAA) inhibited AdoHcy-nase activity with Ki values of 0.55, 1.04, greater than 10.0 and 3.30 microM, respectively. These four compounds were tested for antiviral activity in vitro against Moloney leukemia virus (MoLV) in the XC-plaque assay. MoLV replication in murine fibroblasts (SC-1) was inhibited by ZDDFA, EDDFA and DAA with IC50 values of 0.05, 0.25 and 3.30 micrograms/ml, respectively. ZDDCA did not inhibit MoLV infection at the concentrations tested. Antiviral activity correlated with the ability of the individual compounds to maintain sustained elevations in intracellular S-adenosylhomocysteine (AdoHcy) concentrations in the SC-1 cells. ZDDFA, the most potent inhibitor of AdoHcy-nase and MoLV was also the most active in maintaining sustained elevations in intracellular AdoHcy levels. The antiviral activity of ZDDFA was also examined in murine C3H1OT1/2 fibroblasts which constitutively produce MoLV. Pretreatment with ZDDFA (1.0 microgram/ml) for 24 hr inhibited virus production by 88%. Similar to the SC-1 cells, and concomitant with enzyme inhibition, there was a 300-fold increase in AdoHcy levels in ZDDFA (1.0 microgram/ml) treated C3H1OT1/2 cells. Incorporation of a [3H]methyl group from tritiated S-adenosylmethionine into total RNA in C3H1OT1/2 cells was inhibited by ZDDFA without affecting cell viability. These results suggest that mechanism-based inhibitors of AdoHcy-nase, such as ZDDFA, may have potential as antiretroviral agents.

4',5'-unsaturated 5'-halogenated nucleosides. Mechanism-based and competitive inhibitors of S-adenosyl-L-homocysteine hydrolase.

The design and synthesis of (E)- and (Z)-5'-fluoro-4',5'-didehydro-5'-deoxyadenosine (6 and 13, respectively), a new class of mechanism-based inhibitors of S-adenosyl-L-homocysteine (SAH) hydrolase, is described. A number of analogues of 6 and 13 were synthesized in order to determine the structure-activity relationship necessary for inhibition of the enzyme. Substitution of chlorine for fluorine in 6 (i.e. 44), addition of an extra chlorine to the 5'-vinyl position (i.e. 51 and 52), modification of the 2'-hydroxyl group to the deoxy (34 and 35) and arabino (36 and 37) nucleosides provided competitive inhibitors of SAH hydrolase. Nucleosides 6 and 13, as well as 5'-deoxy-5',5'-difluoroadenosine (14) proved to be time-dependent inhibitors of SAH hydrolase. All three compounds are postulated to inhibit through the potent electrophile derived from oxidation of the 3'-hydroxyl of 6 or 13 to the ketone (i.e. 3 and/or the E-isomer). Consistent with the proposed mechanism of inactivation of SAH hydrolase by 6, 13, and 14 was the observation that incubation of purified rat liver SAH hydrolase with 6 resulted in release of 1 equiv of fluoride ion (by 19F NMR) and incubation with 14 resulted in release of 2 equiv of fluoride ion. The general synthetic route developed for the synthesis of the title nucleosides utilized the fluoro Pummerer reaction for the introduction of fluorine into the requisite precursors. Preliminary antiretroviral data from Moloney leukemia virus (MoLV) is presented and correlates with SAH hydrolase inhibition. Antiviral activity (IC50 against MoLV) ranged from 0.05 to 10 micrograms/mL.

Participation of T-lymphocytes in the curative effect of a novel synthetic polyamine analogue, N,N'-bis[3-(ethylamino)propyl]-1,7-heptanediamine, against L1210 leukemia in vivo.

We have recently established that combination therapy with N,N'-bis[3-(ethylamino)propyl]-1,7-heptanediamine (BEPH), a synthetic polyamine analogue, and N,N'-bis-2,3-butadieneyl-putrescine, a polyamine oxidase inhibitor, eradicated L1210 leukemia in mice and induced resistance to a subsequent L1210 challenge. We now demonstrate that BEPH treatment alone, given on a more frequent schedule (5 mg/kg, day 3, 4, 5) or at a higher dose (10 mg/kg, day 3, 4), cures 100% of L1210 leukemic mice. These treated animals were subsequently immune to a second challenge with L1210 tumor cells. However, mice cured with BEPH did not reject P388 leukemic cells, although their mean survival time was slightly prolonged. In an in vivo tumor neutralization assay, splenocytes from cured mice and L1210 cells were injected into naive mice; 80% did not develop L1210 leukemia. Coculturing lymphocytes from cured mice with L1210 cells in vitro generated a potent tumor-specific cytolytic response against L1210 target cells, whereas lymphocytes from naive mice did not generate any significant cytolytic activity. Both the in vitro and in vivo activities were completely eliminated by pretreating the splenic lymphocyte population with anti-Thy-1.2 monoclonal antibodies and complement, indicating T-cells as the effector population. In T-cell-deficient nude mice BEPH treatment was not curative, increasing survival time by approximately 2-fold. We conclude from these studies that T-cell-mediated immunity plays a pivotal role in the mechanism by which synthetic polyamine analogues, such as BEPH, prevent neoplastic growth.

Antitumor activity of a novel synthetic polyamine analogue, N,N'-bis-[3-(ethylamino)-propyl]-1-7-heptane diamine: potentiation by polyamine oxidase inhibitors.

The requirement of the natural polyamines, putrescine, spermidine and spermine, for cell growth suggests that appropriate structural analogues of these compounds could serve as potential antiproliferative agents acting via polyamine antagonism. In this investigation, the antiproliferative activity of N, N'-Bis[3-(ethylamino)-propyl]-1-7-heptane diamine (BEPH), a synthetic polyamine analogue, was investigated employing HeLa cells in culture and L1210 leukemia in mice. BEPH inhibited the growth of HeLa cells with an IC50 of 0.25 microM during a four day culture period. This concentration of the compound was cytotoxic to the cells as evidenced by an 80% reduction in cloning efficiency. Only marginal changes in intracellular polyamine concentrations were observed during incubation with 0.25 microM BEPH. In both HeLa cells and L1210 cells in culture, incorporation of radioactive precursors into DNA, RNA and protein were reduced by BEPH. Inhibition of protein synthesis was discernible prior to inhibition of RNA and DNA in these cells. In mice inoculated i.p. with 10(5) L1210 cells on day 0, i.p. administration of 10.0 mg/kg of BEPH qd(X5) beginning on day 1 prolonged the survival time by 84% compared to controls. The same dose of the compound, in combination with 10.0 mg/kg of N,N'-bis-2-3-butadienylputrescine, an inhibitor of the polyamine catabolizing enzyme polyamine oxidase (PAO), produced a 100% cure rate. Similar results were obtained when BEPH was combined with N-methyl-N'-2-3-butadienylputrescine, another PAO inhibitor. Furthermore, animals cured of the leukemia by the combination chemotherapy were resistant to a subsequent challenge with L1210 cells, indicating the development of tumor "immunity". The striking antitumor activity along with the development of tumor immunity indicate that synthetic polyamine analogues have potential for development as antineoplastic agents.

Polyamine analogues with antitumor activity.

A series of tetraamines derived from 1,8-diaminooctane was prepared and tested as antitumor agents. The reaction of 1,8-diaminooctane with acrylonitrile gave N,N'-bis(cyanoethyl)-1,8-diaminooctane, which was reduced to tetraamine 20. Alkylation of the terminal nitrogen atoms of the tetra-Boc derivative of this compound by methyl or ethyl halide followed by removal of the Boc groups gave the bis(alkyl)polyamines 26a and 26b, respectively. These three compounds exhibit promising antitumor activity in the mouse L1210 leukemia model. Coadministration of a polyamine oxidase inhibitor potentiated the antitumor activity.

Antitumor activity of norspermidine, a structural homologue of the natural polyamine spermidine.

The structural specificities of the natural polyamines putrescine (Put), spermidine (Spd) and spermine (Spm) for cell growth are rather stringent, suggesting that appropriate structural analogues of these polycations could serve as potential antineoplastic agents via polyamine antagonism. Norspermidine (Nspd), a homologue of spermidine, had significant antitumor activity against L1210 leukemia, 3LL carcinoma and EL4 lymphoma in mice. The observed antitumor activity of the compound was potentiated by administration of a - difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase. DFMO treatment alone, or in combination with Nspd reduced tumoral Put and Spd levels by greater than 50% in all three tumor models. In animals receiving both Nspd and DFMO, Nspd accumulation in the tumor cells was increased by 50% or more compared to cells from animals receiving Nspd only. Co-administration of Spd, but not Put, abolished the antitumor activity of L1210 observed with DFMO and Nspd treatment, and also reduced the tumoral accumulation of Nspd. These results indicate that appropriate structural analogues of the natural polyamines may be useful as antineoplastic agents.

Effect of inhibition of polyamine biosynthesis by DL-alpha-difluoromethylornithine on the growth and melanogenesis of B16 melanoma in vitro and in vivo.

The objective of the present study was to investigate the effect of polyamine depletion by alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase, on the growth and differentiation of B16 melanoma cells grown in culture and also as solid tumors in mice. Polyamine depletion by DFMO (2.5 mM) resulted in a complete inhibition of cell growth in culture and a 90% inhibition of viability of melanoma cells as determined by clonogenic assay at the end of 7 days after DFMO treatment. These results indicate that polyamine depletion induced by DFMO is cytotoxic to B16 melanoma cells in culture. Furthermore a 2- to 5-fold increase in tyrosinase activity and 10-fold accumulation of melanine were observed in polyamine depleted cells compared to control cultures. These effects of DFMO could easily be reversed by the addition of putrescine simultaneously with DFMO. Administration of different doses of DFMO in drinking water to B16 melanoma tumor bearing mice also resulted in an increase in tyrosinase activity and a dose dependent inhibition (86-90%) of tumor growth. Although one cannot rule out the possibility of induction of differentiated phenotype as a result of antiproliferative activity of DFMO, the data presented indicate that the unique sensitivity of melanoma to DFMO may be due to a combination of cell growth inhibition and concomitant induction of differentiation upon polyamine depletion. The results of the present study indicate that polyamines play an important role in growth and differentiation of melanoma and also provide an example of inhibition of tumor cell growth by induction of cellular differentiation.

Potentiation by alpha-difluoromethylornithine of the activity of 3,4-dihydroxybenzylamine, a tyrosinase-dependent melanolytic agent, against B16 melanoma.

Continuous exposure for 96 hr of B16 melanoma cells in culture to 2.5 mM alpha-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase, resulted in a marked increase in the activity of the enzyme tyrosinase, and also 20% cell kill as assessed by clonogenic assay. A 4-hr exposure to 0.4 mM 3,4-dihydroxybenzylamine (DHBA), a compound which is melanolytic due to its conversion to a cytotoxic quinone by the tumor specific enzyme tyrosinase, was found to be approximately equitoxic to 2.5 mM DMFO. However, a combination of DFMO (2.5 mM) and DHBA (0.4 mM) produced greater than 95% cell kill. This observed cytotoxicity with the combination suggests that induction of tyrosinase by DFMO sensitizes B16 melanoma cells to the melanolytic activity of DHBA. Oral administration of DFMO to mice bearing subcutaneous B16 melanomas also resulted in marked increases in the activity of tyrosinase in the tumor tissue. In mice inoculated intraperitoneally with 10(5) B16 melanoma cells, administration of DFMO via the drinking water (2%) increased the survival time by 8.5 days, whereas intraperitoneal administration of 300 mg/kg of DHBA for 14 days resulted in an increase in life span of 4.5 days compared to untreated controls. A combination of DFMO and DHBA prolonged the survival time by 14.6 days. These results indicate that DFMO in combination with an appropriate tyrosinase-dependent melanolytic agent might be useful in the chemotherapy of malignant melanomas.

Potentiation of antitumor and antimetastatic activities of alpha-difluoromethylornithine by interferon inducers.

The objective of the present investigation was to study the potentiation of antitumor and antimetastatic activities of DL-alpha-difluoromethylornithine (DFMO) by inducers of interferon, namely, tilorone and polyriboinosinic:polyribocytidilic acid complex [poly(l) X poly(C)]. The results of this study indicate that these interferon inducers enhance the antitumor activity of DFMO against B16 melanoma and Lewis lung carcinoma in mice. In B16 melanoma, DFMO, tilorone, or poly(l) X poly(C), when administered alone, showed 85, 39, and 39% of inhibition of tumor growth, respectively. However, a combination of DFMO and tilorone or poly(l) X poly(C) resulted in 98 and 95% inhibition of growth, with about 20% of animals showing no detectable tumors. This potentiation appears to be related to the ability of the compounds to induce interferon, since an analogue of tilorone, MDL 10,842, neither induced interferon nor potentiated the antitumor activity of DFMO. The data also indicate that this combination is particularly effective when the tumor burden is relatively low. When tilorone was given 7 days after tumor inoculation, it did not show any potentiation of antitumor activity of DFMO. The studies with Lewis lung carcinoma also showed that the interferon inducers potentiated both the antitumor and antimetastatic activities of DFMO. DFMO or tilorone administered alone showed 28 and 46% inhibition of tumor growth and 80 and 58% inhibition of metastases, respectively. Poly(l) X poly(C) by itself did not have any effect on the tumor growth and metastases. However, a combination of DFMO and tilorone brought about 78% inhibition of tumor growth and 99.5% inhibition of metastases, with 87% of the animals free of any metastases. A combination of DFMO and poly(l) X poly(C) also showed a potentiation of both antitumor activity (58% inhibition) and antimetastatic activity (94% inhibition), with 62% of the animals free of any detectable metastases. The mechanism underlying this tumor suppression by combination of DFMO and interferon inducers is not yet known. Enhancement of host immune response or interferon-mediated cytotoxicity could account for the observed marked suppression of tumor growth. Previous studies using interferon and the data reported here with interferon inducers, along with the relatively nontoxic nature of DFMO, suggest a potential use for the inhibitors of polyamine biosynthesis in combination with interferon or interferon inducers in cancer chemotherapy and other proliferative states.

Combination chemotherapy involving alpha-difluoromethylornithine and 1-beta-D-arabinofuranosylcytosine in murine L1210 leukemia.

Administration of 2% aqueous alpha-difluoromethylornithine (DFMO) as the sole drinking-fluid to C57BL X DBA/2 F1 (hereafter called BD2F1) mice for 5 days resulted in significant reduction of labeling index, percentage of cells in S phase, and intracellular putrescine and spermidine concentrations of bone marrow cells. DFMO treatment also resulted in moderate leukopenia. The decreases in body weight, peripheral leukocyte and erythrocyte counts, and subsequent recovery following administration of 1-beta-D-arabinofuranosylcytosine (ara-C), an S-phase-specific cytotoxic agent, to animals receiving DFMO was comparable to those observed in animals which received ara-C alone. In animals inoculated i.p. with L1210 cells, DFMO treatment produced a marginal increase in survival time and significant reductions in putrescine and spermidine concentrations of the tumor cells. However, in contrast to normal bone marrow cells, depletion of polyamines in tumor cells resulted in the progressive accumulation of these cells in S phase; by Day 5 following DFMO treatment, greater than 70% of the tumor cells were found to be in S phase compared to only 55% for cells from untreated animals. A significant synergistic increase in survival time with a combination of DFMO and ara-C was observed when the ara-C therapy was initiated at the time of maximum accumulation of tumor cells in S phase. The implications of these findings for the management of neoplasia are discussed.

Role of polyamines during chromosome condensation of mammalian cells.

The objective of the present study was to investigate the role of polyamines in the process of chromosome condensation. The phenomenon of premature chromosome condensation (PCC) involving fusion between mitotic and interphase cells was used as the assay system. The factors present in the mitotic cells would bring about the breakdown of the nuclear membrane and condensation of the interphase chromatin into chromosomes, similar to that which occurs during the initiation of mitosis. Alpha-difluoromethyl ornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase was used to deplete both mitotic and interphase cells of polyamines. The results indicate that the polyamine depleted mitotic cells have a diminished ability to induce PCC. This inhibition could easily be reversed by exogenous addition of polyamines at the time of fusion. Furthermore, exogenously added polyamines hastened the entry of exponentially growing cells into mitosis. These observations suggest an essential role for polyamines during the process of chromosome condensation of mammalian cells.

Tumor suppression with a combination of alpha-difluoromethyl ornithine and interferon.

alpha-Difluoromethyl ornithine and mouse type 1 interferon, when administered simultaneously, were highly toxic to B16 melanoma cells in culture. Oral administration of alpha-difluoromethyl ornithine suppressed B16 melanoma development in mice 85 percent whereas interferon given subcutaneously inhibited tumor growth only 24 percent. Total or near total suppression of tumor growth was observed in mice receiving both treatments.

Tissue-and metal-specific effects of thiolacrylic acids in rats.

Kidney copper increased 12- to 18-fold above the normal level in rats administered alpha-mercapto-beta-(2-furyl)acrylic acid (MFA). Kidney zinc increased twofold; plasma zinc increased more than 10-fold and liver zinc increased 30-50%. No other changes in copper, iron, and zinc concentrations were found in these tissues or in bone, brain, heart, lung, skeletal muscle, spleen, or testis. Related compounds produced similar effects, although MFA and its disulfide were the most potent of the compounds tested. These increases in tissue copper and zinc were largely complete after 2-5 d of daily administration of compound. Increased plasma zinc returned toward normal with a half-life of 1.0 d for the process, after dosing was ended; albumin was identified as the species binding the excess zinc in plasma. Kidney copper and zinc, which had increased in the ratio of 3 Cu/Zn, returned to normal levels after dosing was stopped with half-lives of 2.1-2.5 d. Consistent with the observations of highly tissuespecific effects of MFA, copper and zinc balances over 8 weeks of trials were found to be not greatly affected by administration of the compound. Thus, it was not established whether excess metal in affected organs derived from enhanced retention of dietary metal or redistribution from other tissues. Kidney copper and zinc and serum zinc increased even in zinc-deficient rats administered MFA.

Effects of DL-alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, on the rat mammary tumour induced by 7,12-dimethylbenz[a]anthracene.

1. The effects of DL-alpha-difluoromethylornithine (RMI 71782; DFMO) on the tumours induced in female rats by a single oral administration of 20 mg 7,12-dimethyl-benz[a]anthracene (DMBA) have been investigated. 2. Treatment with DFMO (2% aqueous solution as sole drinking fluid) starting 30 days after administration of DMBA resulted in markedly fewer animals with tumours and greater than 90% reduction in the total number of tumours. 3. In rats bearing at least one palpable tumour, treatment with DFMO (2% in the drinking water) slowed significantly the rate of appearance of new tumours but affected to only a minimal extent the growth of existing tumours. Tumour ornithine decarboxylase activities and putrescine concentrations were reduced by treatment with DFMO; the activity of S-adenosyl-L-methionine decarboxylase was increased and the concentration of spermine either remained unchanged or increased depending on the length of treatment. 4. Cyclophosphamide, 100 mg/kg, injected once then repeated after 10 days, altered neither the rate of appearance of new tumours nor the growth of the existing tumours. Combined treatment with DFMO plus cyclophosphamide resulted in regression of the majority of tumours existing at the start of treatment and a marked reduction in the rate of appearance of new tumours. 5. In conclusion, DFMO has clear antitumoral activity against the rat mammary tumour induced by DMBA. The effects are manifested principally as a decreased rate of tumour appearance but meaningful effects on tumour growth are observed if the drug is administered during early tumour development or in combination with cyclophosphamide.