Inhibition of murine tumor growth by an interferon-inducing imidazoquinolinamine.

The low-molecular-weight imidazoquinolinamine derivative, 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine (imiquimod, previously described as R-837), induced alpha-interferon (IFN-alpha) in mice. IFN induction was identified at oral doses as low as 3 mg/kg. The 10% lethal dose for daily treatment with imiquimod was 200 mg/kg. Oral treatment with 30 mg/kg imiquimod once every three days significantly inhibited MC-26 colon carcinoma. Delay of treatment from day 1 to day 5, when tumors were easily palpable, did not reduce benefits. Ten daily treatments were slightly more effective than five. However, delivery of the same total dose of imiquimod either once every day for 20 days, once every 4 days, once every 7 days, or once every 10 days inhibited tumor growth to the same level. The antitumor effects of imiquimod were significantly abrogated by an antiserum to murine IFN-alpha, suggesting that the antitumor effect was to a substantial extent mediated by IFN induction. Imiquimod also significantly reduced the number of lung colonies in mice inoculated i.v. with MC-26 tumor cells. Combination of treatment with imiquimod and cyclophosphamide was significantly (P less than 0.01) better than treatment with either drug alone. Combination treatment with cyclophosphamide led to cures in some of the mice inoculated either s.c. or i.v. with MC-26 cells. Treatment with imiquimod also inhibited the growth of RIF-1 sarcoma and Lewis lung carcinoma but was ineffective for P388 leukemia. Imiquimod is an oral IFN-alpha inducer with antitumor effectiveness for transplantable murine tumors.

Protection from carcinogen-induced murine bladder carcinoma by interferons and an oral interferon-inducing pyrimidinone, bropirimine.

Interferons (IFNs) have established activities as antivirals and inhibitors of viral and transplantable tumors. To establish whether IFNs or their inducers can affect induction of carcinogenesis in vivo, the bladder-specific carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) was administered in the diet at 0.11 or 0.13% (w/w) to female C3H/He mice beginning at 7 weeks of age. Mice treated with the IFN-inducing bropirimine [2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone] i.p. twice a week for 14 weeks starting on day 30 of start of FANFT feeding developed fewer transitional cell carcinomas (TCC) than mice treated with the vehicle. Bropirimine (200 mg/kg twice a week) orally resulted in even greater effectiveness: 6 of 43 bladders with TCC for bropirimine-treated mice versus 24 of 39 for control glycine buffer-treated mice (P less than 0.01, x2 test). Mice treated i.p. daily on days 29 through 210 with 5,000 units of beta interferon (specific activity, 2.0 x 10(8) units/mg) had 0 of 15 TCC while control mice had 7 of 13 TCC (P less than 0.001). Bladders of untreated mice were also significantly heavier than those of beta interferon- or bropirimine-treated mice. This dose of IFN treatment was confirmed as effective in a second experiment, in which mice were treated daily on days 30-223 with 5,000 units alpha/beta interferon (specific activity, 1.2 x 10(7) units/mg). This resulted in 4 of 25 bladders with TCC versus 24 of 39 for control mice (P less than 0.001). A higher dose of IFN (50,000 units alpha/beta interferon daily) was toxic; 24 of 30 mice died within 2 months. IFN and an IFN inducer, bropirimine, inhibited development and progression of FANFT-induced bladder TCC in vivo and thus may have roles as chemopreventive modalities.

Schedule-dependent variations in the response of murine P388 leukemia to cyclophosphamide in combination with interferons-alpha/beta.

Positive therapeutic effects of interferons (IFNs) in combination with other therapies will depend on defining modalities, doses, and timing of treatment in the setting of varied tumor burdens. When 10(4) P388 leukemia cells were inoculated i.p. on day 0 in BALB/c x DBA/2 F1 mice, all mice died within 18 days if left untreated. Murine IFN-alpha/beta (5 x 10(5) units) injected daily i.p. on days 5-9 resulted in 20% increase in life span (ILS) (P less than 0.0001). Cyclophosphamide (CY) (100, 33, or 15 mg/kg) was injected i.p. once 2 days before start (day 3), simultaneously with start (day 5), or 2 days after cessation of IFN treatment (day 11). When 100 mg/kg CY alone were injected on day 3 or 5, all mice survived more than 90 days and were considered cured. When IFN was given after this curative dose of CY, more tumor deaths occurred; up to 100% of the mice died when 100 mg/kg CY on day 3 were combined with IFN on days 5-9. Increased mortality with the combination was not due to added toxicity of CY and IFN since the mice developed abdominal tumors and ascites. Mice not inoculated with tumor cells and treated similarly suffered only a transient weight loss, had only moderate white count depression, and did not die. When IFN was injected before CY on days 1-5 (instead of days 5-9), IFN did not alter the effectiveness of CY (100 mg/kg on day 5). In contrast to these results, when CY (100 mg/kg) was administered on day 11, after IFN (days 5-9), an augmented survival occurred with 119% ILS and 40% cures (CY alone on day 11 resulted in 69% ILS but no cures). In addition, when CY at a lower dose of 15 mg/kg was injected in combination with IFN, survival was consistently augmented by IFN; e.g., CY alone on day 3 caused 40% ILS and with IFN (days 5-9) 60% ILS (P less than 0.0001). Qualitatively similar findings were obtained when P388 leukemia cells were inoculated s.c. and the drugs delivered i.p. Inhibition by IFN of antitumor effects of a second alkylating agent, 1,3-bis(2-chloroethyl)-1-nitrosourea, was also identified. Thus, IFN-alpha/beta potentiated suboptimal CY effects for P388 leukemia, had neutral effects when injected before CY treatment, and inhibited antitumor activity of curative CY or nitrosourea schedules.

Inhibition of angiogenesis by interferons: effects on tumor- and lymphocyte-induced vascular responses.

Interferons (IFNs) have established antitumor action; the mechanism underlying this effect is, however, not yet clear. To probe the possible contribution of inhibition of angiogenesis, we have assessed angiogenesis in the mouse initiated by either human or murine tumor cell lines. Whether test cells were inoculated in the dermis or tumor fragments were grafted onto the cornea, tumor-induced angiogenesis (TIA) was inhibited by IFNs. TIA was also inhibited by the potent IFN inducer polyriboinosinic-polyribocytidylic acid. The effect of IFN was species specific; human IFNs inhibited human tumors and mouse IFNs inhibited murine tumors. This effect suggested that in contrast to other angiogenesis inhibitors, IFNs modulated the signal for angiogenesis produced by the tumor cells. Tumor cells treated in vitro with homologous IFN were significantly (P less than 0.005) less competent to initiate angiogenesis than were untreated cells. Inhibition of angiogenesis was achieved whether vascular response was assessed 1 or 3 days after tumor cell inoculation, suggesting that antiangiogenesis activity was independent of the antiproliferative effects of IFNs. To further substantiate this, L1210 leukemia cells, resistant to the antiproliferative effects of IFNs, were treated with 500 units/ml IFN-beta. IFN had no effect on their proliferation, but in four separate experiments, L1210R cells were impaired in their ability to induce angiogenesis. Thus, inhibition of TIA by IFNs was species specific, occurred at least partly by modulation of the signal inducing angiogenesis, and was expressed in the absence of antiproliferative effects. IFNs also inhibited immunologically induced angiogenesis, whether initiated by allogeneic lymphocytes (LIA) or by the mouse's own T-cells in response to an exogenous antigen (sheep RBC). LIA was markedly suppressed by treatment of host mice with homologous IFN-beta. For example, mean vessel counts induced by allogeneic mouse lymphocytes were decreased from 22.8 +/- 1.4 (SE) to 12.5 +/- 0.8 (P less than 0.0001); mouse IFN-beta had no corresponding effect on xenogeneic human lymphocytes (mean vessel counts decreased to 21.7 +/- 2.6 from 22.7 +/- 2.0). Treatment with human IFN-alpha, -beta, or -gamma in vitro or host mice in vivo reduced the ability of inoculated human peripheral blood lymphocytes to initiate xenogeneic LIA. Inhibition of LIA required a lower dose and/or a shorter incubation period than that needed to modulate TIA. Treatment of the donor of the allogeneic spleen cells in vivo with murine IFN or inducers also resulted in lesser LIA.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of interferon alpha, interferon beta, and interferon gamma on the in vitro growth of human renal adenocarcinoma cells.

Interferon-alpha, interferon-beta, and interferon-gamma differ in their antiproliferative effects for several cell lines. Interferons were thus assessed for their activity in inhibiting proliferation of three renal cell carcinoma cell lines. The malignant epithelial phenotype of each of these cell lines was confirmed by electron microscopy, histology, karyotype and tumorigenicity. When compared on an anti-viral unit basis, naturally produced interferon-beta was more effective than natural interferon-alpha for all cell lines and clones. Proliferation of each of the cell lines was inhibited by interferon-gamma. In all cases, removal of interferons from culture media resulted in resumption of the rate of cell growth after a variable delay of 6-10 days. If the antiproliferative effects of interferons predominate in mediating tumor regression, clinical response may depend upon the type of interferon to which the tumor is exposed.

Inhibitory effects of interferon-inducing pyrimidinones on the growth of transplantable mouse bladder tumors.

Pyrimidinones are low-molecular-weight compounds which are inducers of interferon in several animal species. They have established antiviral, immunomodulatory, and antitumor effects. Four pyrimidinones as well as another potent interferon inducer, polyriboinosinic-polyribocytidylic acid, and beta-interferon were tested for effects on growth of the transplantable mouse bladder tumor (MBT-2). The pyrimidinones 2-amino-5-bromo-6-phenyl-4(3H)pyrimidinone (ABPP) and 2-amino-5-bromo-6-(3-fluorophenyl)-4(3H)pyrimidinone (ABMFPP) significantly inhibited MBT-2 growth in a dose-dependent manner and with equal potency when injected i.p. every 4 days starting 1 day after tumor cell inoculation. Administration of ABPP p.o. was as effective as i.p. injections. Direct intravesical application of ABPP to transplantable tumors growing in the bladder may be more effective in inhibiting MBT-2 growth than the same dose introduced p.o. Although ABPP (100 mg/kg) has an inhibitory effect comparable to 5000 units of beta-interferon, both pyrimidinones even at 500 mg/kg were less inhibitory of tumor growth than 10 mg of polyriboinosinic-polyribocytidylic acid per kg. The pyrimidinones 2-amino-5-bromo-6-(2,5-difluorophenyl)pyrimidine-4(3H)one (ABDFPP) and 2-amino-5-iodo-6-(2,3-difluorophenyl)pyrimidin-4(3H)one (AIDFPP) were also of comparable potency in inhibiting MBT-2 growth and were more effective on mg/kg basis than both ABPP and ABMFPP. Treatment with ABDFPP or AIDFPP also resulted in long-term cures of up to 40% of mice. In this respect these latter two compounds were superior to treatment with 10 mg of polyriboinosinic-polyribocytidylic acid per kg, a treatment which reduced tumor size but had no effects on tumor incidence. The data suggest that tumors of bladder origin may be particularly sensitive to treatment with pyrimidinones.

Antitumor effects of polyribonucleotides for mouse transitional cell carcinoma enhanced by cyclophosphamide.

Mouse bladder tumor (MBT-2), derived from a carcinogen-induced transitional cell carcinoma of the bladder, has proven a useful model for study of pathogenesis and prediction of cytotoxic drug sensitivity of human bladder carcinoma. To define optimal conditions for activity of the potent interferon inducer polyriboinosinic-polyribocytidylic acid [poly(I) X poly(C)] in this model, studies of dose, timing, and combinations with a cytotoxic drug were initiated. Poly(I) X poly(C) inhibited MBT-2 growth when 10(5) or 10(6) tumor cells were implanted. Tumor growth reduction was relatively more pronounced in mice inoculated with higher numbers of MBT-2 cells (10(6] than in mice inoculated with an intermediate dose (10(5] or small dose (10(4]. In mice inoculated with 10(5) MBT-2 tumor cells, poly(I) X poly(C) (2.5 or 10 mg/kg i.p.) on Days 5 to 19 every other day reduced tumor size markedly. It had no effect, however, on tumor incidence or the time of their first detection. Treatment for a shorter period (alternate days from Days 11 to 19) resulted in less inhibition of tumor growth. Once treatment was discontinued, tumors grew progressively. Polyriboadenylic:polyribouridylic acid [poly(A) X poly(U)] (10 mg/kg) which inhibited tumor growth but to a lesser degree than poly(I) X poly(C) induced lower, less sustained levels of serum interferon. Cyclophosphamide, injected i.p. on Day 1, resulted in inhibition of tumor incidence and growth in direct proportion to the dose administered (25 to 200 mg/kg), but it was curative only at greater than or equal to 30% lethal doses. When combined with poly(I) X poly(C) (2.5 or 10 mg/kg), cyclophosphamide (50 mg/kg) had an additive antitumor effect. Optimal inhibition of MBT-2 tumor growth occurred by combining cyclophosphamide (100 mg/kg) with poly(I) X poly(C) (2.5 mg/kg); eight of 14 mice were tumor free on Day 60.

Monoclonal antibody against angiotensin-converting enzyme: its use as a marker for murine, bovine, and human endothelial cells.

A monoclonal antibody has been prepared against rat angiotensin-converting enzyme (ACE). By selection for antibody binding to endothelial cells of bovine rather than rat origin we have obtained a reagent that has broad cross-species binding properties and that can at the same time serve as a useful marker for the surface of endothelial cells. The IgM-producing clone that we have established, alpha-ACE 3.1.1, has been grown in ascites form to yield ascites fluid that binds selectively to immobilized ACE at a greater than 1:10,000 dilution. By use of enzyme-linked immunosorbent assays, immunofluorescence histology, and flow cytometry, we have demonstrated the presence of ACE on endothelial cells of murine, bovine, and human origin. By means of a fluorescence-activated cell sorter (FACS-IV) we have been able to selectively isolate viable endothelial cells from a mixture of endothelial cells and fibroblasts. We believe the antibody will be useful not only for the selection and in vitro cultivation of endothelial cells but also as a tool for the identification and pharmacological study of ACE.

Nature of the stimulus leading to lymphocyte-induced angiogenesis.

Experiments are described that characterize the nature of the stimulus leading to lymphocyte-induced angiogenesis (LIA), a reaction previously shown to reflect a local in vivo graft-vs-host reaction. The studies demonstrate that circulating cells of the host animal provide the stimulation essential for activation of donor lymphocytes and that the major allogeneic stimulus in congenic lines of mice is correlated with I-region disparity, primarily associated with IA-controlled determinants. The results are readily compatible with the hypothesis that is proposed that LIA is in large part of the consequence of the release of soluble mediators or lymphokines that may act either directly on endothelial cells or indirectly by activating macrophages, which in turn generate the vascular reaction.

Regional differences in the incidence and growth of mouse tumors following intradermal or subcutaneous inoculation.

Tumor cells inoculated intradermally or s.c. into more cranial regions of the lateral trunk show strikingly greater tumor growth and development than do similar cells injected more caudally. At low tumor cell doses the incidence anteriorly may be double that found posteriorly and tumors become detectable more rapidly anteriorly; at higher cell doses the anterior:posterior ratio of tumor weight may be 4:1. The effect appears to be independent of the type of tumor used (mastocytoma, sarcoma, teratoma, lymphoma, or adenocarcinoma) and of the strain of mouse host; it does not appear to be influenced by the sex of the host animal, the immunogenicity of the tumor, or the immunological competence of the tumor recipient. The results are discussed both in terms of practical considerations for developing adequate tumor transplantation and treatment protocols and in terms of the biological significance in relation to spontaneous or induced oncogenesis.

Lymphocyte-induced angiogenesis in tumor-bearing mice.

The presence of a growing tumor can lead to a significant curtailment of a graft-versus-host reaction as measured by the ability of allogeneic spleen cells to induce a host vascular response. This interference with the normal pattern of immunological reactions may be a reason for the survival of tumors in an immunologically alien environment.

Lymphocyte-induced angiogenesis: a quantitative and sensitive assay of the graft-vs.-host reaction.

A new and sensitive assay for the effect of intracutanous administration of immunocompentent lymphocytes into the skin of irradiated unimmunized mice is described. The assay, which we have termed lymphocyte-induced angiogenesis (LIA) involves enumeration of new vascular branches induced by the action of these competent cells. As is the case for the previously described normal lymphocyte transfer reaction, LIA is a manifestation of the graft-vs.-host reaction, as shown by experiments utilizing appropaiate genetic combinations. The reaction is dose-dependent, and within the dose range of 2 times 10 minus 5 -4 times 10-6 cells the mumber of vessels induced correlates with the mumber of immunocompetent cells injected. At these dose levels spleen, lumph node, and hydrocortisone-resistant thymocytes are effective; bone marrow and thymus cells are not. Spleen cells from nude mice are incapable of inducing LIA, while mitomycin-C and irradiated lymphocytes can initiate but not maintain the reaction. The relationship between lymphocyte-induced angiogenesis has been discussed as have the implications of these findings to delayed hypersensitivity, inflammation, and vascular pathology.