Optimization of cell-wall skeleton derived from Mycobacterium bovis BCG Tokyo 172 (SMP-105) emulsion in delayed-type hypersensitivity and antitumor models.

Cell-wall skeleton prepared from Mycobacterium bovis BCG (BCG-CWS) is known as a potent adjuvant and has been shown to possess antitumor activity in many non-clinical and clinical studies. As there are no approved BCG-CWS formulations for cancer therapy, we investigated the potential for cancer immunotherapy of SMP-105, our originally produced BCG-CWS. For optimizing SMP-105 emulsion, we compared the effects of drakeoland squalane-based SMP-105 emulsions on IFN-γ production in rats and evaluated their ability to induce skin reaction in guinea pigs. Both emulsions had the same activity in both experiments. We selected squalane as base material and produced two types of squalane-based formulations (vialed emulsion and pumped emulsion) that can easily be prepared as oil-in-water emulsions. Although the vialed emulsion showed the same pattern of distribution as a usual homogenized emulsion, the pumped emulsion showed more uniform distribution than the other two emulsions. Whereas both emulsions enhanced strong delayed type hypersensitivity (DTH) reaction in a mouse model, the pumped emulsion induced slightly smaller edema. Data on oil droplet size distribution suggest that few micrometer oil droplet size might be appropriate for oil-in-water microemulsion of SMP-105. The antitumor potency of SMP-105 emulsion was stronger than that of some of the launched toll-like receptor (TLR) agonists (Aldara cream, Picibanil, and Immunobladder). Aldara and Picibanil showed limited antitumor effectiveness, while Immunobladder had almost the same effect as SMP-105 at the highest dose, but needed about 10 times the amount of SMP-105. These findings first indicate that SMP-105 has great potential in cancer immunotherapy.

Arabino-mycolates derived from cell-wall skeleton of Mycobacterium bovis BCG as a prominent structure for recognition by host immunity.

Arabino-mycolates are components of the cell-wall skeleton of Mycobacterium bovis BCG (BCG-CWS). It is known that synthesized arabinomycolates induce the production of tumor necrosis factor alpha (TNF-α) in murine macrophage cell lines at an intensity similar to that of BCG-CWS. However the immunological activity of natural arabino-mycolates isolated from BCG has not been investigated, probably due to the complexity of the molecule. In this paper, we investigated the immunostimulatory activity of arabino-mycolates isolated from BCG-CWS by acid hydrolysis. Arabino-mycolates obtained by acid hydrolysis from the originally prepared CWS (SMP-105) of M. bovis BCG Tokyo 172 strain consisted mainly of mono-arabinose mono-mycolate, penta-arabinose tetra-mycolate and hexa-arabinose tetramycolate fractions. Arabino-mycolates significantly induced TNF-α production with an intensity comparable to that of CWS and enhanced delayed type hypersensitivity (DTH) reactions against inactivated tumor cells. Arabino-mycolates-induced TNF-α production was completely dependent on TLR2 and MyD88 pathways. These findings indicate that isolated natural arabino-mycolates possess potent adjuvant immunostimulatory activity.

Phagocytosis plays a dual role in activating dendritic cells; digestive production of active Toll-like receptor ligands and cooperation with Toll-like receptor signaling.

Phagocytosis is an initial step in innate immunity, which is also stimulated by signals via Toll-like receptors (TLRs); however, the cooperation of phagocytosis with signals through TLRs to establish acquired immunity is unknown. We found that phagocytosis is an essential process to induce an immune reaction against an insoluble TLR ligand. Cell-wall skeleton prepared from Mycobacterium bovis BCG (BCG-CWS), an insoluble TLR2 ligand, activated and matured murine splenic dendritic cell (DC) line BC-1 as well as a soluble TLR2 ligand, Pam3CSK4. Surprisingly, BC-1 maturation with BCG-CWS was completely suppressed by inhibiting phagocytosis, while that with Pam3CSK4 was not affected. Moreover, BCGCWS induced intense delayed-type hypersensitivity (DTH) reactions against mitomycin C-inactivated Lewis lung carcinoma cells but Pam3CSK4 did not. These results suggested that the phagocytosis process enables the insoluble TLR2 ligand to activate DCs via TLR2 comparable to a soluble TLR2 ligand in vitro, and stimulating TLR2 alone is not sufficient to establish T cell-mediated immunity in vivo. It is therefore conceivable that the process of phagocytosis induces additional effects on TLR2-stimulated DCs to activate cellmediated immunity in vivo.

Immune response against cell-wall skeleton of Mycobacterium bovis BCG at the inoculation site and peripheral lymphoid organs.

We reported in the previous paper that highly purified cell-wall skeleton of M. bovis BCG (SMP-105) eliminated lymph node metastases and primary implanted tumor, presumably by generating tumor immunity, employing guinea pigs. In this paper, we investigated the immune reactions to elucidate the mechanisms of antitumor activity. Twenty-four hours after intradermal injection, inflammatory cells were seen migrating to the inoculation site. Massive infiltrations of lymphocytes were observed on day 7, when a large amount of SMP-105 was still observed in the dermis. Several chemokines attracting neutrophils and monocytes, detected by TaqMan RT-PCR, were induced rapidly and declined 72 h post-injection, but most increased again on day 7, consistent with the pathological findings of lymphocyte infiltration. Activation of lymph node cells was investigated using mice. Upon stimulation by SMP-105 in vitro, the draining lymph node cells collected from mice treated with SMP-105 produced interferon-γ (IFN-γ), whereas, lymph node cells did not release IFN-γ when prepared from mice treated with OK-432. This evidence prompted us to assume that SMP-105 functioned as T cell antigens. Intracellular cytokine analysis demonstrated that IFN-γ was mainly attributable to CD4-CD8+αßT and CD4-CD8-αßT cells. In conclusion, oil-in-water emulsion of SMP-105 resided for a long time at the inoculation site and activated T cells, probably recognizing SMP-105 itself. The strong tumor eliminating activity of SMP-105 may be explained by the boost of generating tumor immunity via positive feed-back from T cells reacting to it, and CD4-CD8+αßT and CD4-CD8-αßT cells may distinguish SMP-105 from other synthetic adjuvants.

Injection of cell-wall skeleton of Mycobacterium bovis BCG draining to a sentinel lymph node eliminates both lymph node metastases and the primary transplanted tumor.

Based on recent developments in innate immunity, we focused on a microbial immunostimulator for cancer immunotherapy. If innate immunity is properly activated, tumor antigens distributed endogenously in cancer patients will be exploited to activate tumor immunity. We chose the cell-wall skeleton of M. bovis BCG (BCGCWS) and investigated the potential of monotherapy without exogenous tumor antigens. We used strain 2 guinea pigs bearing syngenic line 10 hepatoma, which is an excellent disease model of spontaneous lymph node metastasis, and examined the tumor-eradicating activity of highly purified BCG-CWS (SMP-105), excluding the effect of local inflammation on tumor growth. SMP-105 eliminated both established metastases and the implanted tumor, when injected into different but not distant sites from the tumor, whereas, when injected into the opposite side, neither metastases nor the primary tumor was eradicated. SMP-105 was observed in the draining lymph node engulfed by phagocytes, presumably macrophages or dendritic cells, but was not detected in distant lymph nodes or the spleen. It took about 2 weeks until the tumor-eliminating effect was observed. Taken together it is considered that macrophages or dendritic cells were activated by SMP-105 and encountered tumor cells in the sentinel lymph node to generate tumor immunity during the lag time. In conclusion, we suggested the potential of mono-therapy with a strong immunostimulator and that SMP-105 is a most promising agent for cancer immunotherapy. Separate injection from tumor draining to a sentinel lymph node using classical guinea pig models will be a useful method for investigating immunostimulators.

Distribution of viral antigens and development of lesions in chicken embryos inoculated with nipah virus.

An isolate of Nipah virus was injected into fertile eggs via the allantoic cavity or yolk sac. Allantoic inoculation resulted in considerable pathological variation and only partial mortality. Dead embryos showed severe necrosis in the brain and congestion in the kidney and the subcutis of limbs. In contrast, yolk sac inoculation led to uniform infection and mortality, the dead embryos exhibiting the same lesions as those described above but without the subcutaneous congestion. Histological lesions in dead embryos inoculated by either route were similar and particularly severe in the central nervous system. Viral antigens were detected mainly in the vasculature and neurons. The results indicated that Nipah virus is highly pathogenic to chicken embryos, and that the route of inoculation is an important determinant of the course of disease. The findings also suggested that yolk sac inoculation can be used for viral titration, and that the chicken embryo represents a useful model for studying the vascular and neuronal tropisms of Nipah virus.

Monoclonal antibody-based immunohistochemical diagnosis of Malaysian Nipah virus infection in pigs.

Formalin-fixed, paraffin wax-embedded tissues of three Malaysian farm pigs naturally infected with Nipah virus were used to investigate the value of anti-Nipah virus mouse monoclonal antibodies (Mabs) and rabbit polyclonal antibody for immunohistochemical diagnosis. Mabs 11F6 and 12A5 gave intense immunolabelling in lung tissue that had been fixed in 10% neutral buffered formalin for about 4 years, whereas the reactivity of Mabs 13A5 and 18C4 and polyclonal antibody was reduced significantly by long-term formalin fixation. Immunohistochemical examination of Malaysian farm pig samples with Mab 11F6 confirmed the affinity of Nipah virus for respiratory epithelium, renal glomerular and tubular epithelium, meningeal arachnoidal cells, and systemic vascular endothelium and smooth muscle. In addition, Nipah virus antigens were identified in laryngeal epithelial cells, Schwann cells of peripheral nerve fascicles in the spleen, and endothelial cells in the atrioventricular valve. The study demonstrated the value of Mabs 11F6 and 12A5 for the immunohistochemical diagnosis of Nipah virus infection in pigs.

Preliminary studies by ELISA on the antigen and antibody dynamics in the early stages of experimental infections with Trypanosoma evansi in cattle.

Six 6-month-old bulls were experimentally infected with five different isolates of Trypanosoma evansi; two received the same isolate and the other four received different isolates. The parasitaemias and serum antigen levels were monitored regularly by the haematocrit centrifuge technique (HCT) and antigen-detection ELISA (Ag-ELISA), respectively. Trypanosomal antigen was demonstrated by the Ag-ELISA by 10-14 days post inoculation in four cattle, while parasitaemias were first found to be positive in individual cattle over a longer period of time post inoculation (6-28 days). In two cattle, the Ag-ELISA values were also positive when the animals were found to harbour trypanosomes by the HCT and only turned negative 3 days after treatment, while the ELISA values fluctuated during the experiment in another two bulls. The remaining two cattle never produced positive ELISA results despite positive parasitological results. The antibody titres in all six cattle started to rise around 10 days post inoculation and then stayed high throughout the experiment. It was concluded that the Ag-ELISA would produce some false negative results in the early stages of T. evansi infection owing to variations in the balance of parasitaemia and antibody levels in the circulation, and in the pathogenicity of parasite strains.

Postnatal neosporosis in dairy cattle in northeast Thailand.

A total of 83 dairy cows in Loei Province (Muang) and Nong Bua Lamphu (NBL) Province, northeast Thailand were sampled three times within 6 months in 1998 and their sera were examined for antibodies to Neospora caninum at a dilution of 1:100 in the indirect fluorescent antibody test (IFAT). In Muang the seroprevalence of N. caninum was 37.5% (June), 60% (August), and 62.5% (November). In NBL the prevalence was 50% (August) and 70% (November). In both areas abortions were observed between 1 and 3 months after the introduction of these cattle from another area. Nine of 14 and seven of 17 calves were descendants of seropositive dams, of which only two calves from Muang and two calves from NBL were positive for N. caninum antibodies. These findings suggest postnatal N. caninum transmission.

Development of new drug delivery system for protein drugs using silicone (I).

A novel technique, by which protein drugs effective in small doses can be released over a long period, was developed using silicone and a water-soluble substance. In this study, interferon (IFN) was used as a model of the protein drugs. The IFN-silicone formulation released IFN over long periods of time in vitro and suppressed tumor growth in nude mice for about 100 days after a single administration. This indicates that physiologically active IFN is released over a prolonged period of time from the IFN-silicone formulation in vivo. Silicone formulations are expected to be a practically feasible sustained-release formulation.

A preliminary comparative study of a dipstick colloidal dye immunoassay and two antigen-detection ELISAs for diagnosis of Trypanosoma evansi infection in cattle.

A dipstick colloidal dye immunoassay (DIA) was developed for the field diagnosis of Trypanosoma evansi infection using affinity-purified polyclonal antibodies (PcAbs) and the monoclonal antibody (McAb) 8B9. PcAbs were adsorbed onto Palanil Red dye particles and used as dye reagents. Dipsticks were dotted with four different antibodies; normal rabbit and mouse IgGs as negative controls, and anti-T. evansi PcAb and McAb 8B9, which capture trypanosome antigens in the tested samples. Since the dye reagent bound to the captured antigens, the presence of coloured dots on the dipstick identified trypanosome infections. The sensitivity of the DIA was compared with two antigen detection ELISAs (Ag-ELISA); one was PcAb-based and the other was based on a combination of the same Mc- and PcAbs as were employed for the DIA. With a positive serum, the DIA detected trypanosomal antigen up to a dilution of 1:500 for both the PcAb and McAb dots, at which dilution the PcAb- and combination-based Ag-ELISA gave positive OD readings of 0.13 and 0.36, respectively. When 124 field sera were tested, circulating antigens were detected in 51 (41%) samples by the DIA, and 76 (61%) and 49 (40%) samples by the PcAb- and combination-based Ag-ELISAs respectively, of which 48 (63%) and 34 (69%) were also positive by the DIA.

Influence of Trypanosoma evansi infection on milk yield of dairy cattle in northeast Thailand.

Effect of subclinical Trypanosoma evansi infection on the milk yield of newly introduced Holstein Friesian dairy cattle were investigated. Five hundred pregnant heifers were introduced in Loei Province, northeast Thailand and a total of 168 blood samples were collected at 20 farms during 6 visits over 2 years. Trypanosomes were found in cattle in June and November 1996, after which the parasite was rarely seen. On the other hand, the infection prevalences by antigen-detection ELISA (Ag-ELISA) were around 40% from the first sampling through October 1997; then, antigenemic cattle decreased to 20% by June 1998. Milk yields of the cattle with detectable parasitaemia in June and November 1996 were significantly lower than those of the non-infected cattle by Student's t-test. Similarly, the milk yields of Ag-ELISA positive cattle were lower than those of negative cattle at every sampling and significant differences were observed during the first year and in February, 1998 (tested by 2-way ANOVA; T. evansi status and herd as factors). This study suggested that subclinical trypanosomosis caused decrease in milk yield of newly introduced dairy.

Seroprevalence of antibodies to Neospora caninum and associated abortion in dairy cattle from central Thailand.

A total of 904 sera from dairy cattle in 11 provinces of central Thailand were tested for antibodies to Neospora caninum employing the indirect fluorescent antibody test (IFAT). Fifty four (6%) cattle were positive in IFAT, titres of 1:200 (16 cattle), 1:400 (9 cattle), 1:800 (14 cattle), 1:1600 (7 cattle), 1:3200 (6 cattle) and two positives. No significant difference was observed among the provinces. The seropositivity for Toxoplasma gondii by a commercial latex agglutination test was 4% (2 out of 50) in positive sera, 2.9% (2 out of 69) in negative sera for anti-Neospora antibodies and 3.4% (4 out of 119) in total. The results of the IFAT were not associated with the presence of antibodies to T. gondii in bovine sera. Furthermore, the cause of abortions experienced in neighbouring three areas in the northeast, where pregnant heifers were newly introduced into small-scale farms from the central region, was investigated. The positive rates for anti-N. caninum antibody were 12, 28 and 44% at a cut-off titre of 1:200, and cattle were suspected to be infected after the introduction. In the area with the highest rate, seven out of eight aborting cattle were positive for antibodies to N. caninum while other two areas had similar abortion rates in both negative and positive cattle. However, in the latter two areas, positive rates for Trypanosoma evansi antigen along with parasitaemic animals were observed by an antigen-detection ELISA, but not for the former area. Considering the endemic diseases of the areas, Neospora was presumed to be responsible for the abortions in the former area while the examination results pointed out T. evansi as the most probable cause in the latter two areas. This is the first report of Neospora-associated abortion in Southeast Asia.

Biologic and binding activities of IFN-alpha subtypes in ACHN human renal cell carcinoma cells and Daudi Burkitt's lymphoma cells.

Nine interferon-alpha subtypes, IFN-alpha1, IFN-alpha2, IFN-alpha5, IFN-alpha7, IFN-alpha8, IFN-alpha10, IFN-alpha14, IFN-alpha17, and IFN-alpha21, were separated from purified human lymphoblastoid IFN. We tested their inhibitory effects on cell growth and replication of Semliki Forest virus (SFV) and vesicular stomatitis virus (VSV) and their induction of 2',5'-oligoadenylate synthetase (2', 5'-OAS) in ACHN renal cell carcinoma cells. In terms of all three activities, the nine subtypes had similar relative activities, with IFN-alpha10 the most active and IFN-alpha1 the least. Their relative effects on cell growth were similar in two other human cell lines, SK-LU-1 lung cancer cells and KU-2 renal cell carcinoma cells, whereas cells of the Daudi Burkitt lymphoma line behaved quite differently, being highly sensitive to all the nine subtypes. The relative effects with ACHN cells correlated well with their relative binding affinities. However, each of the subtypes bound to both ACHN and Daudi cells to almost the same extent. This suggests that their profound inhibitory effects on the growth of Daudi cells are amplified at some stage in the signal transduction pathway or in the expression of genes that results from binding to the IFN-alpha receptor.

Haemoparasite infections in newly introduced dairy cattle in Loei Province, Thailand: Trypanosoma evansi antigen levels by ELISA referring to abortion.

An antigen-detection enzyme-linked immunosorbent assay (Ag-ELISA) based on affinity-purified polyclonal antibody was utilised as an immunodiagnosis for Trypanosoma evansi infections in cattle. Five hundred pregnant heifers were introduced in Loei province, Thailand and a total of 271 samples were collected at 37 farms during four visits over a year commencing a month after the introduction. Each time the haematocrit centrifuge technique (HCT) was carried out as a field diagnosis for T. evansi, and sera were examined for trypanosomal antigen levels by Ag-ELISA. At the first sampling over 80% of the cattle were positive for trypanosome antigens by Ag-ELISA although the titres were low. Soon after, aborted cases at the late stage of pregnancy were reported and at the second sampling in the rainy season, 25.5% of the cattle sampled were found to harbour T. evansi by HCT. This time the infection rate by Ag-ELISA was 52.9% with high antigen levels. Between the first and second sampling nine cattle out of 51 aborted, which was suspected to be due to T. evansi. As soon as treatment with a trypanocidal drug was started, abortion cases decreased. However, the infection rate remained high during the rainy season when Tabanus flies were active. As the climate became cool and dry, the antigen levels in the area lowered and the positive rate by Ag-ELISA dropped to 32.3%.

Effect of anti-immunoglobulin antibodies produced in cattle infected with Trypanosoma evansi on antigen detection ELISA.

The possibility of interference with the antigen-detection ELISA for trypanosomosis by anti-rodent IgG antibodies produced in cattle infected with Trypanosoma evansi was investigated. Two different ELISA for detection of trypanosome antigen and three different systems for anti-rodent IgG antibody detection were established. The former two were respectively polyclonal antibody-based and a combination of monoclonal and polyclonal antibody-based assays. The latter three were also adapted for detection of anti-mouse IgG, anti-rabbit IgG and anti-IgG antibodies cross-reactive with both rabbit and mouse IgGs. A total of 170 samples were collected from a dairy cattle farm where an outbreak of T. evansi infection was reported. One hundred and two cattle (59%) were found to be positive for trypanosome antigens by the polyclonal antibody-based assay and 86 (51%) were positive by the combination-based system. On the other hand, 51 (30%) and 10 (6%) of cattle had anti-rabbit and anti-mouse IgG antibodies respectively but none had antibodies cross-reactive with both IgGs. Of the 102 cattle positive for trypanosome antigens in the polyclonal antibody-based ELISA, 48 (47%) were also anti-rabbit IgG antibody positive. It is concluded that antigen detection ELISA based on a single-species immunoglobulin for capture and detection might misdiagnose T. evansi infection. Results indicate that this bias will be avoided if reagents for capture and detection are derived from different species.

Regulation of IFN-alpha/beta genes: evidence for a dual function of the transcription factor complex ISGF3 in the production and action of IFN-alpha/beta.

BACKGROUND: Efficient production of interferons (IFNs) in virally infected cells is an essential aspect of the host defence. The transcription factor complex ISGF3 (IFN-stimulated gene factor 3) was originally identified as a critical mediator of the IFN signal; it is formed upon IFN receptor (IFNR) stimulation and binds to ISREs (IFN-stimulated response elements) to activate IFN-inducible genes. It has recently been shown that the DNA binding component of ISGF3, p48 (ISGF3gamma) also binds to virus-inducible elements in the IFN-alpha/beta genes, suggesting a potential new role of p48 in IFN production. RESULTS: Primary cells from mice with a targeted disruption of the p48 gene show severe defects in virus-induced IFN-alpha/beta gene expression. A similar defect was also observed in cells lacking type I IFNR or Stat1, further demonstrating the role of IFN signalling in the induction of these IFN genes. ISGF3 in fact binds to the virus-inducible elements within the IFN-alpha/beta promoters. We also provide evidence showing that these elements are additionally controlled by an unidentified factor(s) which presumably triggers the primary phase of IFN gene induction. CONCLUSIONS: Our results demonstrate that the IFN signal transducing complex ISGF3 plays a crucial role in IFN production and suggest that ISGF3 may participate directly in the activation of IFN-alpha/beta promoters. This dual function of ISGF3 may insure the efficient operation of this cytokine system in the host defence.

Essential and non-redundant roles of p48 (ISGF3 gamma) and IRF-1 in both type I and type II interferon responses, as revealed by gene targeting studies.

BACKGROUND: Interferons (IFNs) are a class of cytokines which confer cellular resistance against viral infections. Type I (IFN-alpha and -beta) and type II (IFN-gamma) IFNs utilize distinct receptors, the stimulation of which results in the induction of downstream target genes. These target genes usually contain within their promoter region an IFN responsive element, termed ISRE (IFN stimulated response element) which binds a heterotrimeric transcription factor, ISGF3 (IFN-stimulated gene factor 3) consisting of p48 (ISGF3 gamma), Stat1 (Signal transducers and activators of transcription-1; alpha or beta), and Stat2. The ISRE sequence overlaps with that of IRF-E which binds another IFN-inducible factor, IRF-1 (IFN regulatory factor-1). RESULTS: We generated mice lacking p48 by gene targeting. We show that p48 plays an essential role in both type I and type II IFN responses; activation of IFN-inducible genes and establishment of the antiviral state by IFN-alpha or -gamma are both severely impaired, and ISRE-binding activities induced by both IFNs are absent in the p48-negative embryonic fibroblasts (EFs). Furthermore, we generated mice deficient for both p48 and IRF-1 and found that at least one IFN-inducible gene is dependent on both factors. CONCLUSIONS: p48 and IRF-1 do not perform redundant functions in the cell, but rather complement one another in both type I and II IFN responses.

A multiple antigen detection dipstick colloidal dye immunoassay for the field diagnosis of trypanosome infections in cattle.

Monoclonal antibodies (McAbs) were developed against aspartate aminotransferase purified from Trypanosoma brucei rhodesiense bloodstream form (bf) soluble extracts using a combination of anion-exchange and hydrophobic interaction chromatography. McAb 1A1 was Trypanozoon and Nannomonas specific while 2F1 was Trypanozoon bloodstream form specific. A dipstick colloidal dye immunoassay (DIA) was employed as a field diagnostic test for African trypanosome infections and designed using affinity purified polyclonal antibodies (PcAbs) raised against T. b. rhodesiense bf and the two McAbs, 1A1 and 2F1. PcAbs were adsorbed onto Palanil Red dye particles and used as dye reagents. Dipsticks were dotted with the three different antibodies, which captured trypanosomal antigens in samples tested, while the dye reagent bound to the captured antigens; the presence of coloured dots on the dipstick identified trypanosome infections. A field trial of the DIA was carried out in southeastern Uganda. A total of 1686 cattle from seven areas were bled and tested by DIA and haematocrit centrifuge technique (HCT). A total of 798 cattle (47.3%) were found to be trypanosomal antigen positive by DIA while only 162 (9.6%) were revealed to harbour trypanosomes by HCT, of which 151 (93%) were also positive by DIA.

Antitumor activities of a novel 9-aminoanthracycline (SM-5887) against mouse experimental tumors and human tumor xenografts.

The antitumor effects of SM-5887, a totally synthetic 9-aminoanthracycline derivative, were evaluated in six murine experimental tumor systems (P388, Ehrlich carcinoma, sarcoma 180, Lewis lung carcinoma, B16 melanoma and colon 38) and nine human tumor-nude mouse systems (one breast cancer, two lung cancers and six gastric cancers). Characteristically SM-5887 showed excellent antitumor activities, superior to adriamycin (ADR), against human tumor xenografts, although its activities against murine experimental tumors were almost equal to those of ADR. When the human tumors were implanted sc in female athymic mice (BALB/c, nu/nu) and their volume reached 100-300 mm3, SM-5887 and ADR were injected iv. All nine human tumors tested showed statistically significant responses to SM-5887, and 7 of them were strongly suppressed in their growth by SM-5887 so that minimum T/C values were less than 30% at the maximum tolerated dose (MTD, 25 mg/kg) with a single iv injection. Compared with ADR, SM-5887 was statistically more effective in five tumors (one breast, one lung and three gastric), equal in two tumors (two gastric), and less potent in two tumors (one lung and one gastric). In addition, the 10-day-interval repeated iv treatments with SM-5887 at the MTD (25 mg/kg) resulted in remarkably potent antitumor effects (including complete regression) against human gastric cancer, 4-1ST, implanted in nude mice without enhancement of toxic effects. SM-5887 was also effective against ip-inoculated P388 by oral administration as well as iv injection.