Antitumor activity of a novel podophyllotoxin derivative (TOP-53) against lung cancer and lung metastatic cancer.

We synthesized a potent new antitumor podophyllotoxin derivative (4beta-aminoalkyl-4'-O-demethyl-4-desoxypodophyllotoxin; TOP-53) in our search for a drug that has strong activity against lung cancer and lung metastatic cancer. TOP-53 exhibited twice the inhibitory activity of etoposide (VP-16) against topoisomerase II and induced DNA strand breaks but showed no inhibitory activity against tubulin polymerization. The in vitro cytotoxic activity of TOP-53 assessed as IC50 was 0.016-0.37 microg/ml and 0.26-8.9 microg/ml against marine tumor and human non-small cell lung cancer (NSCLC) cell lines, respectively. TOP-53 exerted significant efficacy equivalent to that of VP-16 on s.c.-implanted murine solid tumors (Colon 26, B16-BL6, and Lewis lung carcinoma) at doses 3-5 times lower than that of VP-16. In human tumor xenografts using NSCLC, TOP-53 was active for four of five tumors, whereas VP-16 was active for two of five tumors. Potent inhibitory activity of TOP-53 was also found against a lung tumor (Lewis lung carcinoma) and four lung metastatic tumors (NL-22 and NL-17 colon cancer, UV2237M fibrosarcoma, and K1735M2 melanoma). TOP-53 appeared to be more active against four of them than VP-16. Thus, TOP-53 is not only active against s.c.-implanted lung cancers but also strongly active against lung localized tumor and metastatic tumors in the lungs. The high selectivity of TOP-53 was attributed to its high distribution into the lung and its persistence. TOP-53 is expected to be highly effective against lung cancer including NSCLC and various lung metastatic tumors in the clinical field.

Specific distribution of TOP-53 to the lung and lung-localized tumor is determined by its interaction with phospholipids.

We have investigated the mechanism of TOP-53 distribution to the lung and lung-localized tumor. In contrast to etoposide (VP-16), TOP-53 contains a basic aminoalkyl group that may predispose it to interact specifically with phospholipids, consequently leading to an increase of drug accumulation in the tissues. Therefore, we have studied its interaction with phospholipids in vitro using an organic solvent-water partition system. TOP-53 appeared to have the most potent binding affinity (Ka = 563 x 10(-2) microM) to phosphatidylserine (PhS), whereas VP-16 showed no interaction with any phospholipid tested. PhS content determined after HPLC separation varied among tested tissues; however, large quantities were found in normal lung and lung cancer tissues far exceeding those present in the liver and kidney. The predicted tissue-to-plasma partition coefficient values, estimated based on PhS content and its binding affinity, resembled those experimentally determined. We concluded that tissue distribution of TOP-53 is determined by PhS content in the tissues and by binding affinity. As a result of specific accumulation in the lung, TOP-53 appeared to show a strong antitumor activity (increase of life span = 171%) against cancer metastasizing to the lung, whereas VP-16 was less effective (increase of life span = 78%). These results suggest that TOP-53 may have an advantage over VP-16 in the treatment of lung cancers in patients.

UFT and its metabolites inhibit the angiogenesis induced by murine renal cell carcinoma, as determined by a dorsal air sac assay in mice.

UFT, an anticancer agent that is composed of tegafur (FT) and uracil at a molar ratio of 1:4, is widely used in clinical practice in Japan to treat cancer patients requiring a long-term chemotherapy, and it is associated with few side effects, if any. In this study, we have evaluated the inhibitory effect of UFT against RENCA cell-induced angiogenesis by a dorsal air sac assay. Marked angiogenesis is induced by implantation of a chamber containing RENCA cells into mice. In this model, UFT showed a strong angiogenesis-inhibitory effect, whereas 5-fluorouracil (5-FU) and doxifluridine were less effective. Additional experiments revealed FT to be effective component of UFT; uracil remained ineffective in the inhibition of angiogenesis. Moreover, we have found that gamma-hydroxybutyric acid and gamma-butyrolactone, the metabolites of FT, possess a potent angiogenesis inhibitory effect that is amplified when the compounds are administered by a continuous infusion. This may reflect a transition in blood concentration of each metabolite resulting from the administration of UFT. Similar results were also obtained with respect to 5-FU. It was suggested that UFT has a stronger angiogenesis-inhibitory effect than did other fluorinated pyrimidines, partly due to its pharmacokinetic properties characterized by maintaining of higher and long-lasting blood levels of 5-FU and partly due the inhibitory effects derived from gamma-hydroxybutyric acid and gamma-butyrolactone, UFT-specific metabolites.

TAC-101, a benzoic acid derivative, inhibits liver metastasis of human gastrointestinal cancer and prolongs the life-span.

We examined the anti-tumor effect of a novel benzoic acid derivative, TAC-101 (4-[3,5-bis(trimethylsilyl) benzamide] benzoic acid) on models with liver metastasis. Oral administration of TAC-101 significantly inhibited spontaneous liver metastasis of AZ-521 (human gastric cancer ) by orthotopic implantation to athymic nude mice. It also inhibited both the liver metastasis of AZ-521 induced by intrasplenic injection and the secondary lung metastasis from the liver. In addition, TAC-101 inhibited the proliferation of Co-3 (human colon adenocarcinoma) that formed a single nodule in the liver of athymic nude mice by intrahepatic implantation. The growth inhibitory effect of TAC-101 on AZ-521 experimental liver metastasis was observed when treatment was started on day 7, 14, or 21 which may correspond to the progressive stage of liver metastasis in clinical settings. Multiple administration of TAC-101 (8 mg/kg/day) significantly prolonged survival time of the animals with liver metastasis by intrasplenic injection of AZ-521 (T/C = 230%) and A549 (human lung adenocarcinoma; T/C = 186%). These effects of TAC-101 were stronger than those of 5-FU, CDDP or ATRA. Furthermore, TAC-101 inhibited the binding of AP-1 to DNA on electrophoretic mobility shift assay using nuclear extract of AZ-521 cells, although ATRA did not inhibit. These findings suggested that TAC-101 may be a candidate for a new class of anti-cancer agents for liver metastasis.

TAT-59, a new triphenylethylene derivative with antitumor activity against hormone-dependent tumors.

The antiestrogenic action of TAT-59 [(E)-4-[1-[4-[2-(dimethylamino)ethoxy]-phenyl]-2-(4-isopropyl) phenyl-1-butenyl]phenyl monophosphate] was characterized and compared with that of Tamoxifen (TAM). Its active metabolite, 4-OH-TAT-59, had a high binding affinity to estrogen receptor (ER), present in the cytosol of the uterus of immature rat, similar to estradiol. TAT-59 and 4-OH-TAT-59 inhibited in vitro estrogen-stimulated proliferation of MCF-7 cells at a lower concentration than TAM. In the absence of estradiol, TAT-59 and 4-OH-TAT-59 were effective at a lower concentration than that of 4-OH-Tamoxifen (4-OH-TAM), the active metabolite of TAM. In uterine growth inhibition, the effective dose of TAT-59 was about 3-6-fold lower than that of TAM, in various administration schedules. The minimum effective dose of TAT-59 against in vivo MCF-7 cells was about 3-fold lower than that of TAM. In DMBA-induced rat mammary tumors, TAT-59 inhibited the growth of existing tumors at about a 10-fold lower dose than TAM. Especially in the tumors with low ER levels (10-20 fmol/mg protein), TAT-59 showed a significantly stronger inhibitory effect than TAM. These experiments showed that TAT-59 was more effective in lower doses than TAM, even against the tumors with low ER content.

Antitumor agents. 3. Synthesis and biological activity of 4 beta-alkyl derivatives containing hydroxy, amino, and amido groups of 4'-O-demethyl-4-desoxypodophyllotoxin as antitumor agents.

A series of 4 beta-alkyl (7-10), 4 beta-aminoalkyl (12a-y), and 4 beta-amidoalkyl derivatives (14a-g) of 4'-O-demethyl-4-desoxypodophyllotoxin have been synthesized, and their cytotoxicity, inhibition of DNA topoisomerase II (Topo II), and tubulin polymerization were evaluated. All derivatives of 12a-y and 14a-g did not inhibit tubulin polymerization. Many compounds exhibited cytotoxicity and inhibition of Topo II. In particular, 12o, 12s, 12t, and 12u strongly inhibited Topo II (IC50 (microM) 32.5, 60.9, 58.8, and 33.6, respectively) and were strong cytotoxicity against P388 cells (IC50 (M) 1.0, 4.1, 3.3, and 3.0 x 10(-9), respectively), compared with VP-16 (IC50 (microM) 59.2, IC50 (M) 1 x 10(-8), respectively). These compounds were nearly equal to or superior to VP-16 in antitumor activity in vivo (L1210, P388, and Lewis lung) and were more cytotoxic against various human cell lines in vitro than VP-16.

Interaction of DP-TAT-59, an active metabolite of new triphenylethylene-derivative (TAT-59), with estrogen receptors.

DP-TAT-59, (Z)-2-(4-(1-(4-hydroxyphenyl)-2-(4-isopropylphenyl)-1-butenyl) phenoxy)-N, N-dimethylethylamine, has been reported to inhibit estrogen-stimulated growth of MCF-7 cells as well as rat uterus at lower concentrations than the hydroxymetabolite of tamoxifen (4-OH-TAM). In the present study, the growth of mouse Leydig cell tumor, B-1F cells were also more effectively inhibited by DP-TAT-59 than 4-OH-TAM. Additionally, the expression of estrogen responsive element ligated CAT gene transfected into B-1F cells was also suppressed by DP-TAT-59. Thus, the interaction of DP-TAT-59 with estrogen receptor (ER) was characterized and compared with that of 4-OH-TAM using immature rat and bovine uteri. The dissociation constant of DP-TAT-59 to ER of immature rat uterus was 0.24 nM and was similar to that of 4-OH-TAM (Kd = 0.20 nM) and estradiol (Kd = 0.29 nM). Using sucrose density gradients, the sedimentation constant of DP-TAT-59 with bovine uterus was 4.9S, which was similar to that of estradiol (5.1S) and 4-OH-TAM (5.3S). However, the elution profile of the DP-TAT-59-ER complex from a DEAE-Sephadex column was different for both estradiol-and 4-OH-TAM-ER complexes. These results suggest that ER forms different complexes with DP-TAT-59 than estradiol or 4-OH-TAM, while the ER binding affinity of these compounds are similar to each other.

Substrate phage as a tool to identify novel substrate sequences of proteases.

Combinatorial phage peptide libraries have been used to identify the ligands for specific target molecules. These libraries are also useful for identification of the specific substrates of various proteases. A substrate phage library has a random peptide sequence at the N-terminus of the phage coat protein and an additional tag sequence that enables attachment of the phage to an immobile phase. When these libraries are incubated with a specific enzyme, such as a protease, the uncleaved phage is excluded from the solution with tag-binding macromolecules. This provides a novel approach to define substrate specificity. The aim of this review is to summarize recent progress on the application of the substrate phage technique to identify specific substrates of proteolytic enzymes. As an example, some of our own experimental data on the selection and characterization of substrate sequences for thrombin, a serine protease, and membrane type-1 matrix metalloproteinase (MT1-MMP) will be presented. Using this approach, the canonical consensus substrate sequence for thrombin was deduced from the selected clones. As expected from the collagenolytic activity of MT1-MMP, a collagen-like sequence was identified in the case of MT1-MMP. A more selective substrate sequence for MT1-MMP was identified during a substrate phage screen. The delineation of the substrate specificity of proteases will help to elucidate the enzymatic properties and the physiological roles of these enzymes. Comprehensive screening of very large numbers of potential substrate sequences is possible with substrate phage libraries. Thus, this approach allows novel substrate sequences and previously unknown target molecules to be defined.

Estrogen agonistic/antagonistic effects of miproxifene phosphate (TAT-59)

PURPOSE: We evaluated miproxifene phosphate (TAT-59) to elucidate its efficacy in antiestrogen therapy for breast cancer patients and to assess its tissue-selective estrogenic/antiestrogenic activity. METHODS: Using DP-TAT-59, a major and active metabolite of TAT-59, an in vitro cell growth inhibition test was performed. Antitumor activity was determined using TAT-59 against human tumor xenografts of the MCF-7 and the Br-10 cell lines and MCF-7-derived tamoxifen-resistant cell lines, R-27 and FST-1. The antitumor activity of DP-TAT-59 and DM-DP-TAT-59, major metabolites of TAT-59 found in human blood following a TAT-59 dose, was also examined after intravenous administration to experimental animals. The residual estrogenic activity of TAT-59, evaluated in terms of bone and lipid metabolism in ovariectomized rats, was then compared with that of tamoxifen. RESULTS: DP-TAT-59 significantly inhibited the proliferation of estrogen receptor-positive MCF-7 and T-47D tumor cells in the presence of 1 nM estradiol. TAT-59, given to mice bearing MCF-7 or Br-10 xenografts, at the dose level of 5 mg/kg, exerted a significant growth inhibitory effect that was stronger than that of tamoxifen. Moreover, R-27 and FST-1 tumors, which show a resistance to tamoxifen, responded strongly to TAT-59, suggesting that TAT-59 might be effective against tumors resistant to tamoxifen. The metabolites of TAT-59, DP-TAT-59 and DM-DP-TAT-59, showed similar antitumor activity. Both TAT-59 and tamoxifen suppressed the decrease in bone density and reduced the blood cholesterol levels in ovariectomized rats, suggesting that the estrogenic activity of TAT-59 is comparable to that of tamoxifen. CONCLUSIONS: On the basis of the above results, one may expect TAT-59 to become an effective drug in patients with tumors less sensitive to tamoxifen, while its estrogenic activity as determined by bone and lipid metabolism is similar to that of tamoxifen.

Potentiation of the antitumor activity of 5-trifluoromethyl-2'-deoxyuridine by the use of depot forms of the parent compound.

5-Trifluoromethyl-2'-deoxyuridine (CF3dUrd), an antitumor agent, is known to be short-lived in human plasma. Since its rapid elimination from the bloodstream seems to have descouraged the clinical evaluation of this drug, we explored the potential use of masked derivatives of CF3dUrd as "depot" forms of the parent compound. First, we observed that the toxicity of CF3dUrd against HeLA cells in culture was 10(4) times greater for a 24-h treatment as compared with a 1-h treatment at identical concentrations of the drug, which suggests the importance of using a prolonged treatment period. In fact, the divided dosing of CF3dUrd to L1210-bearing mice was markedly more effective than its single administration. 5'-O-Hexanoyl-, N3-p-butylbenzoyl-, 5'-O-benzyloxy-methyl-, and 3'-O-benzyl-CF3dUrd were found to be effective in maintaining the CF3dUrd concentration in plasma. The oral doses of these agents required to achieve 50% growth inhibition (ED50) in mice bearing sarcoma 180 tumors were 19, 34, 10, and 13 mg kg-1 day-1, respectively, whereas that of CF3dUrd was 63 mg kg-1 day-1. The ED50 values for these compounds were inversely correlated with the residence time of CF3dUrd in plasma. The therapeutic indices of these compounds, calculated as the dose producing a 50% inhibition of body-weight gain (IB50) divided by the ED50 value (1.89, 1.21, 1.40, and 2.15, respectively), were significantly higher than that of CF3dUrd (0.78). Consequently, these depot forms of CF3dUrd, particularly 3'-O-benzyl-CF3dUrd, are expected to be more useful than the parent compound as antitumor agents.

Influence of insulin on pharmacokinetics of mannosulfan in rats.

The purpose of the present study was to investigate the possibilities of potentiation of the antitumor action of mannosulfan after its administration together with insulin. The pharmacokinetics of mannnosulfan were investigated after its administration separately and together with insulin. Concentrations of mannosulfan in the plasma were determined by gas chromatography. Insulin enhanced the rate of absorption of mannosulfan from the peritoneal cavity and prolonged its elimination from the body. It may be assumed that insulin enhances not only passage of mannosulfan from the peritoneal cavity to blood, but also from blood to tissues. Since increased antitumor effectiveness of mannosulfan was accompanied by its decreased toxicity, it may be concluded that insulin causes selective cummulation of the cytostatic only in some tissues, among others, in the tumor.

The effect of sulfhydryl compounds on 5-fluorouracil toxicity and distribution.

The investigations performed revealed that sulfhydryl compounds (reduced glutathione, cysteine and mesna) reduced the acute and subacute toxicity of 5-fluorouracil in mice. The above compounds changed pharmacokinetics of 5-fluorouracil increasing its accumulation in tissue compartment. The studies on 5-Fu distribution revealed its increased concentration in certain organs after administration of only certain sulfhydryl-containing compounds and only after certain doses.

Influence of some sulfhydryl compounds on the antineoplastic effectiveness of 5-fluorouracil and 6-mercaptopurine.

The influence of three chosen model sulfhydryl groups containing compounds, characterized by increasing size of molecule--cysteine, glutathione and insulin on antineoplastic activity of 5-fluorouracil (5-FU) and 6-mercaptopurine (6-MP) against murine leukemia L-1210 5-FU and 6-MP and murine sarcoma Sa 180, was studied. Sulfhydryl compounds failed to change in most cases the therapeutic (antineoplastic) effect of 5-FU and 6-MP. However, certain doses of these sulfhydryl compounds enhanced the cytostatic effect of 6-MP in respect to Sa-180.

Anticancer effect of 4-[3,5-bis(trimethylsilyl)benzamido] benzoic acid (TAC-101) against A549 non-small cell lung cancer cell line is related to its anti-invasive activity.

We examined the effects of TAC-101 on the invasion and metastasis of human non-small cell lung cancer (NSCLC) cell lines. TAC-101 showed an ability to inhibit in vitro invasiveness of NSCLC at a non-cytotoxic concentration range of 3-10 microM; such concentration levels were easily achievable following oral administration of therapeutically effective doses. The inhibition of cell invasion at 10 microM of TAC-101 accounted for 58-69% when compared with control cells. Oral administration of TAC-101 (4 mg/kg/day) to mice bearing lung implanted A549 lung cancer resulted in significant life-prolonging effect (T/C: 143%). More pronounced life-prolonging effect was observed in the experimental liver metastasis model of A549, where T/C of 215% was observed following administration at 4 mg/kg/day of TAC-101. However, TAC-101 did not show the direct anti-tumor effect against the established A549 tumor xenografts after subcutaneous implantation. These findings suggest that TAC-101 interferes with cell-to-cell interaction processes leading, for instance, to the inhibition of the invasion of NSCLC cells. Taking into account the pharmacological properties of TAC-101, it is expected that TAC-101 may be a suitable candidate drug for the treatment of lung cancer patients, especially those with a predictable metastasizing potential.

UFT and its metabolites inhibit cancer-induced angiogenesis. Via a VEGF-related pathway.

Treatment with UFT for spontaneous lung metastasis of murine renal carcinoma (RENCA) after resection of the primary tumor has resulted in significant prolongation of the life span of tumor-bearing animals. UFT inhibited the growth of metastatic nodules in the lung, apparently via decreased density of microvessels in the metastatic foci. Subsequent experiments used dorsal air sac assay to directly trace newly forming microvessels. UFT abrogated the process of angiogenesis, induced by the RENCA cells, in a dose-dependent manner. The inhibitory effect appeared to originate from tegafur, a component of UFT, and from its known metabolites: fluorouracil (5-FU), gamma-hydroxybutyric acid (GHB), and gamma-butyrolactone (GBL). The inhibition of angiogenesis by UFT appeared to be a common phenomenon, also observed in other human cancer cell lines characterized by an excessive production of vascular endothelial growth factor (VEGF)--such as gastric, lung, and colon cancers. In vitro analysis revealed that 5-FU and gamma-hydroxybutyric acid regulated VEGF-dependent responses of human umbilical vein endothelial cells. Dorsal air sac assay revealed that UFT, 5-FU, and gamma-hydroxybutyric acid strongly inhibited the angiogenesis induced by recombinant human VEGF. These data suggest that the antiangiogenic activity of UFT is at least partially associated with an ability of the metabolites of UFT to interfere with VEGF-dependent responses of vascular endothelial cells.

Tubulin as a major determinant of tissue distribution of vincristine.

The tissue distribution of vincristine was correlated with the tubulin content in different mouse tissues. The concentration of tubulin in the mouse tissues was determined by estimation of the tissue binding capacities for colchicine. Significant differences in the tubulin concentration were observed among the tissues. The comparison of the apparent tissue-to-plasma partition coefficients (KPapp) of vincristine (taken from the literature) with the tubulin concentration (expressed by the binding capacities for colchicine) showed a good correlation. These results suggest that the in vivo distribution of vincristine is predicted by the tissue tubulin concentration.

Mechanism of action of a new macromolecular antitumor antibiotic, C-1027.

C-1027 is a new antitumor protein antibiotic containing a non-protein chromophore. The active moiety and the mechanism of action of this antibiotic were studied. C-1027 and its chromophore inhibited the growth of KB carcinoma and L1210 leukemia cells, even at extremely low concentrations. C-1027 inhibited DNA synthesis of L1210 cells and cleaved cellular DNA in a drug concentration-dependent manner. C-1027 and chromophore caused directly DNA single strand breaks in the purified DNA without any supplement of reducing agents. These results suggest that C-1027 chromophore may inhibit cell growth by causing DNA breakage with subsequent inhibition of DNA synthesis.