Mode of antitumor action of recombinant human tumor necrosis factor on the sarcoma Meth A transplanted in the mouse

The mode of antitumor action of rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation with respect to time course, dose-response relationships and selectivity of the effects. The maximal cytotoxic effect on tumor cells revealed by inhibition of DNA synthesis and maximal lesional effect on tumor vasculature revealed by change in blood pool-size in the tissue were detected at 30 min and I h after administration of rHu-TNF, respectively. The dose-response relationship between cytotoxic and tumoricidal effects of rHu-TNF was irrespective of administration route. ED₅₀s of these antitumor effects afteri.v. administration of rHu-TNF were about 50 times as high as ED₅₀s afteri.t. administration. ED₅₀ ofi.t. given rHu-TNF for vascular effect was about 20 times as high as that for cytotoxicity while ED₅₀ ofi.v. rHu-TNF for vascular effect was only 2–3 times as high as that for cytotoxicity. The whole body autoradiographies with [¹²⁵I] HSA giveni.v. to see the blood influx into tumor tissue and [¹⁴C]thymidine given i.v. to see DNA synthesis in the whole body after administration of rHu-TNF revealed that the distribution of radioactivity was markedly changed in the tumor alone without any detectable change in other whole body tissues.In conclusion, thein vivo antitumor effect of rHu-TNF giveni.t. ori.v., appears to be exerted through the direct action on Meth A sarcoma rather than indirectly on tumor vasculature. Under present conditions, the effect of rHu-TNF in the whole body tissues seems rather selective on cells and vasculature of the tumor.     

Disposition of 125I-labeled recombinant human tumor necrosis factor in the tumor-bearing mouse

Disposition of [125I]rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation. After i.v. administration, [125I]rHu-TNF measured by radioactivity and immunoreactivity biphasically decreased in plasma. Tumor level of [125I]rHu-TNF was the maximum at 1 h, then decreased and finally remained essentially constant. After i.t. administration, plasma level reached the maximum at 1 h. Tumor level decreased quickly and then became essentially constant. [125I]rHu-TNF was suggested to be degraded to small fragments in the tumor. Significant distribution of [125I]rHu-TNF was found in the kidney, lung, liver and tumor. Most tissue levels decreased with time in parallel with plasma levels. [125I]rHu-TNF radioactivity was found in proximal convoluted tubules of kidney and in those areas of tumor consisting of degenerating cells with pyknotic nuclei. Urine contained most of administered radioactivity, which being neither immunoreactive nor protein-bound.     

Disposition of125I-labeled recombinant human tumor necrosis factor in the tumor-bearing mouse

Disposition of [¹²⁵I]rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation. Afteri.v. administration, [¹²⁵I]rHu-TNF measured by radioactivity and immunoreactivity biphasically decreased in plasma. Tumor level of [¹²⁵I]rHu-TNF was the maximum at 1 h, then decreased and finally remained essentially constant. After i.t. administration, plasma level reached the maximum at 1 h. Tumor level decreased quickly and then became essentially constant. [¹²⁵I]rHu-TNF was suggested to be degraded to small fragments in the tumor. Significant distribution of [¹²⁵I]rHu-TNF was found in the kidney, lung, liver and tumor. Most tissue levels decreased with time in parallel with plasma levels. [¹²⁵I]rHu-TNF radioactivity was found in proximal convoluted tubules of kidney and in those areas of tumor consisting of degenerating cells with pyknotic nuclei. Urine contained most of administered radioactivity, which being neither immunoreactive nor protein-bound.     

Antitumor effect of intrapleural administration of Lactobacillus casei in mice

Summary The antitumor effect of intrapleural (i.pl.) administration of Lactobacillus casei YIT 9018 (LC 9018) on Meth A sarcoma in BALB/c mice was examined. Inoculation of Meth A cells into the thoracic cavity of BALB/c mice caused growth of the cells and the mice died from the tumor with an increased amount of pleural fluid. LC 9018 was given i.pl. to BALB/c mice before or after i.pl. inoculation of Meth A cells and the survival of the mice was determined. The i.pl. administration of LC 9018 was effective in prolonging the survival of the mice after i.pl. inoculation of Meth A tumor, and pretreatment with LC 9018 i.pl. also prolonged survival. Moreover, i.pl. administration of LC 9018 not only increased the number of thoracic exudate cells (TEC) but also augmented both cytolytic activity of thoracic macrophages and natural killer cell activity of TEC. Furthermore, phagocytic activity of thoracic macrophages against sheep red blood cells was enhanced and Ia antigen-positive cells in TEC were increased by the i.pl. treatment with LC 9018. These results showed that TEC induced by i.pl. administration of LC 9018 had antitumor activity against Meth A tumor inoculated i.pl. into BALB/c mice.     

An extract of seeds fromAeginetia indica L., a parasitic plant,induces potent antigen-specific antitumor immunity in Meth A-bearing BALB/c mice

Summary The antitumor activity of an extract of seeds fromAeginetia indica L., a parasitic plant, was investigated. BALB/c mice, inoculated i.p. 1 × 10⁵ syngeneic Meth A tumor cells, were administered 2.5 mg/kgA. indica extract i.p. every 2 days from day 0. The untreated mice died of an ascitic form of tumor growth within 21 days, whereas all the treated mice completely recovered from tumor challenge without any side-effects. The extract did not exert direct cytotoxic activity against Meth A in vitro. Mice that survived after the first challenge as a result ofA. indica treatment overcame the rechallenge with homologous Meth A without additional administration of the extract. On the other hand, those mice could not survive after rechallenge with Meth 1 tumor cells, which were also established in BALB/c mice but were different in antigenicity from Meth A, suggesting the development of antigen-specific concomitant immunity in theA. indica-cured mice. In the induction phase of antitumor resistance in this system, CD4⁺ T cells appeared to be the main contributors, since in vivo administration of anti-CD4 mAb completely abolished such resistance. In contrast, anti-CD8 mAb administration did not influence the effect ofA. indica. The importance of CD4⁺ T cells in antitumor immunity was again clarified by Winn assay; that is, spleen and lymph node cells depleted of CD4⁺ T cells in vitro prior to assay abolished antitumor activity on co-grafted Meth A tumor cells in vivo.     

Augmentation of antitumor effect on syngeneic murine solid tumors by an interleukin 2 slow delivery system,the IL-2 mini-pellet

We evaluated the antitumor effect of an interleukin 2 (IL-2) slow delivery system, the IL-2 mini-pellet, in two murine solid tumor models, and also investigated the enhancement of its therapeutic effect by serial administration. The IL-2 mini-pellet contains 1 × 106 units of IL-2 and releases it slowlyin vivo. In our experiment, the IL-2 mini-pellet was administered subcutaneously near the tumor site in combination with the intravenous injection of lymphokine-activated killer (LAK) cells. When this regimen was given on days 8 and 11 after the subcutaneous inoculation of Meth A fibrosarcoma into BALB/c mice, tumor growth was significantly inhibited (p < 0.05) compared to tumor growth in untreated controls. Moreover, the IL-2 mini-pellet alone was also effective in inhibiting tumor growth. In another experiment, MH134 hepatoma was inoculated into C3H/He mice. Both administration of the IL-2 minipellet alone and in combination with LAK cells resulted in complete tumor regression in four of five mice. In a third experiment, serial administration of the IL-2 mini-pellet at 3- or 5-day intervals prolonged the suppression of Meth A fibrosarcoma growth in BALB/c mice. These results suggested that the IL-2 mini-pellet could be applied to cancer immunotherapy and that its antitumor effect could be prolonged by serial administration.     

In vivo antitumor effects of murine interferon- γ -inducing factor/interleukin-18 in mice bearing syngeneic Meth A sarcoma malignant ascites

Interferon-γ-inducing factor/interleukin-18 is a novel cytokine that reportedly augments natural killer (NK) activity in human and mouse peripheral blood mononuclear cell cultures in vitro and has recently been designated IL-18. In this study, IL-18 exhibited significant antitumor effects in BALB/c mice challenged intraperitoneally (i.p.) with syngeneic Meth A sarcoma when administered i.p. on days 1, 2 and 3 after challenge. Intravenous (i.v.) administration also induced antitumor effects in the tumor-bearing mice; however, subcutaneous (s.c.) administration did not. When mice were twice pretreated with 1 μg IL-18 3 days and 6 h before tumor challenge, all mice survived whereas control mice died within 3 weeks of challenge. Inhibitory effects on Meth A cell growth in vitro were not observed with either IL-18 or interferon γ. The effects of IL-18 pretreatment were abrogated by abolition of NK activity after mice had been injected with anti-asialo GM1 antibody 48 h before and, 24 h and 72 h after tumor challenge. Mice pretreated with IL-18 and surviving tumor challenge resisted rechallenge with Meth A cells but could not reject Ehrlich ascites carcinoma, and spleen cells from the resistant mice, but not control mice, exhibited cytotoxic activity against Meth A cells in vitro after restimulation with mitomycin C-treated Meth A cells for 5 days. The effector cells in the spleen cell preparations from resistant mice appear to be CD4⁺ cells because cytolytic activity was significantly inhibited after depletion of this subset by monoclonal antibodies and complement. In conclusion, IL-18 exhibits in vivo immunologically (primarily NK) mediated antitumor effects in mice challenged with syngeneic Meth A sarcoma and induces immunological memory and the generation of cytotoxic CD4⁺ cells. Received: 17 September 1996 / Accepted: 8 November 1996     

Augmentation of the generation of cytotoxic T lymphocytes against syngeneic tumor cells by recombinant human tumor necrosis factor

In order to clarify the effect of recombinant human tumor necrosis factor (rHu-TNF) on the antitumor T cell immune response, we examined the effect of rHu-TNF on the generation of cytotoxic T cells (CTL) against syngeneic tumor cells. Spleen cells from X5563 plasmacytoma-transplanted mice were stimulated in vitro with mitomycin C-treated X5563 cells in the presence or absence of rHu-TNF. The generation of CTL was augmented in a dose-dependent manner by the addition of rHu-TNF. The augmenting effect of rHu-TNF was more marked when indomethacin was added to the culture. The augmenting effect was observed only when rHu-TNF was added at the early stage of the generation of CTL. The cell surface phenotype of CTL generated was L3T4- and Lyt2+. The augmentation was shown not only by the chromium-51 release assay but also by the Winn assay. As to the specificity, the augmentation of CTL generation was observed by the addition of rHu-TNF when responder-primed spleen cells were stimulated with the tumor cells in vitro. On the other hand, augmentation was not observed when responder spleen cells were not stimulated with the tumor cells in vitro, or when responder spleen cells were obtained from normal mice. The CTL generated was not cytotoxic against other tumor cells of the same haplotype. Thus, rHu-TNF augmented the generation of CTL against syngeneic tumor cells in an antigen-specific manner. The in vivo effect of rHu-TNF was examined by administering rHu-TNF into X5563-bearing mice. The spleen cells of rHu-TNF-injected mice generated a much higher CTL activity against X5563 cells in vitro than did the spleen cells of uninjected mice. From these results, a possibility can be considered that in some cases, rHu-TNF may exert its antitumor activity by stimulating the immune system.     

Induction of delayed-type hypersensitivity and antitumor immunity by systemic BCG

Both delayed-type hypersensitivity (DTH) and antitumor resistance induced in mice by intravenous (i.v.) and local injection of highly immunogenic irradiated Meth A cells were potentiated by prior systemic BCG infection. DTH and antitumor immunity were not elicited by i.v. injection of poorly immunogenic irradiated mastocytoma cells, P 815 (MA), but were induced by the local injection of these cells when animals were systemically infected with BCG. The level of the potentiated response corresponded with the dose of immunogen up to an optimum, beyond which additional immunogen was suppressive. At all dose levels the subcutaneous (s.c.) route of immunogen inoculation was more effective than the i.v. route. Significant DTH was first detected 7 days after the local administration of immunogen and was correlated with antitumor immunity. Systemically administered BCG grew mainly in the liver and spleen until the development of maximal tuberculin sensitivity when the number of organisms decreased. However, the small number of mycobacteria that reached the peripheral lymph nodes remained constant after maximal tuberculin sensitivity but failed to augment the cell proliferation that occurred in these lymph nodes as a result of the local inoculation of irradiated tumor cells. Autoradiographs of such nodes revealed proliferation in the thymus-dependent areas whereas nodes from mice immunized with immunogen alone manifested B- as well as T-cell activity. Local immunization in both BCG-infected and uninfected hosts was also associated with a proliferative response in the red pulp of the spleen but the BCG-infected hosts differed conspicuously by virtue of the presence of tubercles and depletion of lymphoid cells from the periarteriolar sheath. Immunity generated by the local administration of immunogen in systemically infected mice was tumor specific and could be adoptively transferred with spleen cells.     

Characterization of the antitumor activities of human tumor necrosis factor-alpha and the comparison with other cytokines: induction of tumor-specific immunity

We have investigated the in vitro and in vivo antitumor activities of recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) against Meth A sarcoma. Meth A sarcoma cells were found to a) be relatively insensitive in vitro to rHuTNF-alpha, and b) express low numbers of TNF-alpha receptors. Intraperitoneally implanted Meth A sarcoma was insensitive to the antitumor effects of rHuTNF-alpha. In contrast, rHuTNF-alpha was highly efficacious against subcutaneously implanted Meth A sarcoma. Biodistribution studies with 125I- or 3H-labeled rHuTNF-alpha demonstrated that, after intravenous administration, the majority of the labeled rHuTNF-alpha localized in the kidney, lungs, and liver. Only low levels of radiolabel were found in subcutaneous Meth A implants. These results support the in vitro data on the low number of TNF-alpha receptors on Meth A sarcoma cells. The ability of rHuTNF-alpha to induce regression of established (7 days) subcutaneous Meth A implants, positively correlated with the degree of both macroscopic and microscopic tumor necrosis. In addition, recombinant human tumor necrosis factor-beta (lymphotoxin) and recombinant murine tumor necrosis factor-alpha induced similar levels of necrosis. Other lymphokines with known antitumor activities, recombinant human interferon-gamma, murine interferon-gamma, and human interleukin 1 alpha, failed to induce detectable necrosis of Meth A sarcoma. Mice which had rejected subcutaneous Meth A sarcoma implants after rHuTNF-alpha treatment and which were later challenged subcutaneously with Meth A sarcoma or other noncross-reacting chemically induced sarcomas were found to be specifically immune to Meth A sarcoma. In addition, low levels of cytotoxic antibodies reactive to Meth A sarcoma were detected in the sera of 21 of 30 Meth A immune mice. Histological evaluation of the hemorrhagic tumor necrosis induced by rHuTNF-alpha suggests that the primary lesion is vascular, possibly directly on the endothelial cells. The mechanisms involved in the generation of specific cell-mediated antitumor immunity in this model are at present unknown.     

Antitumor action of bovine seminal ribonuclease

Unlike the bovine pancreatic ribonuclease (RNAase A), bovine seminal ribonuclease (BS RNAase) displays various biological activities including antitumor cytotoxicity. To learn more about its antitumor activity, we investigated BS RNAase effect on athymic nude mice bearing various tumors. BS RNAase (250 μg per mouse per day) was administered to the mice with prostate carcinoma for three weeks by three different routes (intraperitoneally—i.p., subcutaneously—s.c., and intratumorally—i.t.). Administration i.p. was ineffective, while s.c. administration reduced significantly size of tumors and i.t. administration abolished half of the tumors in treated mice. The i.t. administration of BS RNase to nude mice bearing melanoma showed even better results. Eighty % of mice were without tumors and in the other mice the tumors were significantly diminished. The best antitumor effect was obtained in case of seminoma. All mice bearing this tumor were cured after ten doses of BS RNAase.     

Long-term infusions of p-boronophenylalanine for boron neutron capture therapy: evaluation using rat brain tumor and spinal cord models

Rat 9L gliosarcoma cells infiltrating the normal brain have been shown previously to accumulate only approximately 30% as much boron as the intact tumor after administration of the boronated amino acid p-boronophenylalanine (BPA). Long-term i.v. infusions of BPA were shown previously to increase the boron content of these infiltrating tumor cells significantly. Experiments to determine whether this improved BPA distribution into infiltrating tumor cells after a long-term i.v. infusion improves tumor control after BNCT in this brain tumor model and whether it has any deleterious effects in the response of the rat spinal cord to BNCT are the subjects of the present report. BPA was administered in a fructose solution at a dose of 650 mg BPA/kg by single i.p. injection or by i.v. infusion for 2 h or 6 h, at 330 mg BPA/kg h(-1). At 1 h after the end of either the 2-h or the 6-h infusion, the CNS:blood (10)B partition ratio was 0.9:1. At 3 h after the single i.p. injection, the ratio was 0.6:1. After spinal cord irradiations, the ED(50) for myeloparesis was 14.7 +/- 0.4 Gy after i.p. administration of BPA and 12.9 +/- 0.3 Gy in rats irradiated after a 6-h i.v. infusion of BPA; these values were significantly different (P < 0.001). After irradiation with 100 kVp X rays, the ED(50) was 18.6 +/- 0.1 Gy. The boron compound biological effectiveness (CBE) factors calculated for the boron neutron capture dose component were 1.2 +/- 0.1 for the i.p. BPA administration protocol and 1.5 +/- 0.1 after irradiation using the 6-h i.v. BPA infusion protocol (P < 0.05). In the rat 9L gliosarcoma brain tumor model, the blood boron concentrations at 1 h after the end of the 2-h infusion (330 mg BPA/kg h(-1); n = 15) or after the 6-h infusion (190 mg BPA/kg h(-1); n = 13) were 18.9 +/- 2.2 microg 10B/g and 20.7 +/- 1.8 microg 10B/g, respectively. The irradiation times were adjusted individually, based on the preirradiation blood sample, to deliver a predicted 50% tumor control dose of 8.2 Gy ( approximately 30 photon-equivalent Gy) to all tumors. In the present study, the long-term survival was approximately 50% and was not significantly different between the 2-h and the 6-h infusion groups. The mode of BPA administration and the time between administration and irradiation influence the 10B partition ratio between the CNS and the blood, which in turn influences the measured CBE factor. These findings underline the need for clinical biodistribution studies to be carried out to establish 10B partition ratios as a key component in the evaluation of modified administration protocols involving BPA.     

Pharmacological Effects of Carboplatin-Liposomes (CPL) in Mice: A Reviev of Present Knowledge

Abstract

Carboplatin was encapsulated in reverse phase evaporation vesicles (REV) consisting of hydrogenated egg phosphatidylcholine and cholesterol (HEPC:CH) in a molar ratio of 1 : 0.25. Both antitumor effects, influence on hematopoiesis and cytokine levels were measured in mice after i.p. or i.v. injection in comparison to the free drug. In the syngeneic murine P 388 leukemia and the Meth A sarcoma liposomal encapsulation resulted in a loss of antitumor activity. On contrary, in 3/6 breast carcinomas xenografted to nude mice CPL had a superior tumor inhibiting effect compared to free carboplatin, which could be further improved by using the combination of free and liposomal drug. As reported earlier CPL induced a five- or tenfold, at least 30 days lasting increase in peripheral white blood cells after only one single i.v. or i.p. injection, respectively. A second administration in a 7–10 weeks distance was able to a repeated stimulation. The colony forming activity and the percentage of cells in S-phase were elevated in spleen cells three days after treatment of mice with CPL while these parameters remained unchanged in the bone marrow. Serum taken from CPL-treated nude or normal mice induced significantly colony formation of bone marrow cells in a soft agar culture. Concerning side effects, CPL led to a 15 days lasting blockade of RES (measured by carbon clearance), to a 7 days lasting increase of serum glutamate oxalacetate transaminase and a diminuation of body weight while the blood urea levels as parameter of kidney toxicity were in normal range. A combination of CPL with either cyclophosphamide or free carboplatin prevented the cytostatic-induced leukopenia.     

MDL-646, a new synthetic E1-prostaglandin with local protective effects on the gastric mucosa

The gastric protection, diarrheogenic and arterial hypotensive effects of MDL-646, a PGE1 derivative, have been studied in rats. The compound administered p.o. or i.v. was able to inhibit the macroscopic damage to gastric mucosa produced by noxious stimuli (ethanol and indomethacin). In the stomach perfusion test with the anesthetized rat, intravenously administered MDL-646 reduced histamine- or pentagastrin-stimulated gastric secretion. After intraduodenal administration (i.d.) doses at least 40-50 times greater were necessary for an antisecretory effect. In conscious rats with chronic gastric fistulas, intragastrically administered (i.g.) MDL-646 affected both acid concentration and volume of unstimulated gastric secretion. In experimental models for gastric lesions, MDL-646 was much more potent after oral (p.o.) (15-30 times) than after i.v. administration. (ED50 micrograms/kg: vs. alcohol lesions, 0.05 p.o. and 0.7 i.v.; vs. indomethacin ulcers, 7.0 p.o. and 195 i.v.). Our data would fit the hypothesis that it was a local effect on the gastric mucosa. The mechanism of this effect is not known. The supposed local activity coupled with the antisecretory effects and the good tolerability make it interesting to test MDL-646 as an anti-ulcer agent in man.     

Comparison of the amounts of hCG and PMSG to induce ovulation in 50% of the animals, mice, Syrian hamsters and rats]

On the day of diestrus, female mice, syrian hamsters and rats, showing regular 4-day estrous cycles, were injected with hCG or PMSG and were inspected for the presence of ovulation the following day. The dose level expected to cause an effect in 50% of the animals (ED50) was calculated using the Van der Waerden method. When hCG was injected into i. v., i. p. and s. c., the ED50 values per animal and per body weight (kg) in parenthesis were as follows; 0.2 (7.7), 0.3 (11.5) and 0.7 (26.9) I. U. for mice, 1.0 (9.5), 1.8 (17.1) and 2.6 (24.8) I. U. for syrian hamsters and 1.3 (4.6), 3.5 (12.3) and 7.5 (26.3) I. U. for rats, respectively. In PMSG study, the ED50 values per animal and per body weight (kg) in parenthesis were as follows: i. v., 0.8 (30.8); i. p., 2.0 (76.9); s. c., 2.8 (107.7) I. U. for mice, i. v., 3.6 (34.3); i. p., 8.0 (76.2); s. c., 13.2 (125.7) I. U. for syrian hamsters and i. v., 6.0 (76.8); i. p., 20.8 (73.0); s. c., 76.8 (269.5) I. U. for rats, respectively. From these results, the intravenous ED50 value was lower than other routes in three rodents with hCG or PMSG. In all injection routes, the ED50 value for mouse was lower than others. However, there were not significant differences in the ED50 values per body weight (kg) among three rodents. In particular, subcutaneous ED50 of hCG and intraperitoneal ED50 of PMSG were almost same values among three rodents, respectively. Given that the ED50 value per body weight (kg) in one of three rodents is determined, its value may be possible to be extrapolated to remaining two rodents.     

Correlation between increase in Ia-bearing macrophages and induction of T cell-dependent antitumor activity by Lactobacillus casei in mice

Summary When Lactobacillus casei YIT 9018 (LC 9018) or Corynebacterium parvum, known to be immunomodulators possessing antitumor activity, were injected i.p. into BALB/c mice, peritoneal exudate macrophage Ia antigen detected by indirect immunofluorescence method was expressed on their cell surface, but it was not expressed following the injection of 10% proteose peptone, an inflammatory agent, or Lactobacillus fermentum YIT 0159 (LF 0159), which have no antitumor activity. The percentage and absolute number of Ia-positive peritoneal macrophages were maximum on the 7th day after the injection of LC 9018. Immunization by injection of Meth A fibrosarcoma cells treated with mitomycin C (MMC-Meth A) 7 days after LC 9018 injection suppressed the growth of Meth A implanted i.p. 14 days after MMC-Meth A injection. A shorter interval between the injections of LC 9018 and MMC-Meth A did not allow suppression of Meth A growth. These results showed that the increase in Ia-positive macrophages in the peritoneal cavity coincided with the effective interval for induction of the antitumor activity by LC 9018. The antitumor activity induced by injections of LC 9018 and MMC-Meth A did not affect the growth of RL l leukemic cells, syngeneic to BALB/c mice. Neutralization (Winn type) tests showed that peritoneal T lymphocytes possessed tumor cytotoxicity and that the antitumor capacity was reduced by in vivo treatment with anti I-A^d monoclonal antibody simultaneously with and 1 day prior to MMC-Meth A injection. These results indicate that LC 9018-induced Ia-positive macrophages, which first encounter a tumor antigen in the peritoneal cavity, play an important role in the in vivo induction of tumor specific T cell-mediated antitumor immunity.     

A new simple mouse model for the in vivo evaluation of cholecystokinin (CCK) antagonists: comparative potencies and durations of action of nonpeptide antagonists

A new simple mouse assay for the in vivo evaluation of CCK antagonists which is based upon visual determination of the gastric emptying of a charcoal meal is described. CCK-8 (24 micrograms/kg s.c.) but not various other peptide and nonpeptide agents effectively inhibited gastric emptying in this test system. The effect of CCK-8 was antagonized by established peripheral CCK antagonists but not representative agents of various other pharmacological classes. The rank order of potency of the CCK antagonists were: L-364,718 (ED50 = 0.01 mg/kg, i.v.; 0.04 mg/kg, p.o.) greater than Compound 16 (ED50 = 1.5 mg/kg, i.v.; 2.0 mg/kg p.o.) greater than asperlicin (ED50 = 14.8 mg/kg i.v.) greater than proglumide (ED50 = 184 mg/kg i.v.; 890 mg/kg, p.o.). Duration of action studies based upon ED50 values determined at various time intervals after oral administration showed that L-364,718 and proglumide are considerably longer acting than Compound 16. Asperlicin (ED50 greater than 300 mg/kg, p.o.) was ineffective as a CCK antagonist when administered orally. These data provide the first direct comparisons of the in vivo potencies of current CCK antagonists and demonstrate the utility of a new simple mouse assay for the in vivo characterization of peripheral CCK antagonists.     

Postoperative administration of interleukin-12 significantly enhances the anti-tumor immune response of MBT-2 bladder cancer bearing mice

This study, using the MBT-2 murine bladder tumor model, mainly investigated the role of interleukin-12 (IL-12) in the specific antitumor immune response of a tumor-bearing host when systemically administrated after surgery. Day 17 tumor-bearing mice (D17TBM) along with non-tumor bearing naive mice were treated with daily intraperitoneal (i.p.) injection of IL-12 (0.25 microg/mouse) from day 18 to day 24 for a total of 7 doses. Their splenocytes were obtained on Day 31 for natural killer cells (NK), lymphokine activated killer cells (LAK) and cytotoxic T lymphocyte (CTL) activity assay and lymphocyte subsets phenotypic analysis. The tumor suppression effect of systemic IL-12 administration was evaluated based on the tumor outgrowth of the higher number of tumor cells rechallenged 24 hours after resectioning of the primary tumor. After evaluation on Day 31, the result of in vitro cytotoxicity assay revealed that systemic administration of IL-12 mainly enhanced the splenic LAK and CTL activities in non-tumor-primed naive mice, and the NK activity in tumor-primed D17TBM, respectively. However, in vivo administration of IL-12 with or without IL-2 failed to upgrade the proportions of either CD4+ CD44+ or CD8+ CD44+ T cells subsets in the spleens and regional inguinal lymph nodes (LNs) of both the D17TBM and naive mice. However, the splenic CD8+ CD44+ T-cell subset in the IL-12-treated D17TBM increased prominently after further culturing in the presence of IL-2 400 units/ml plus IL-12 25 ng/ml for 4 days. The fact that systemic administration of IL-12 significantly suppressed the outgrowth of Day-18 challenged tumor cells, especially in D17TBM, clearly indicates that the established specific antitumor immunity in tumor-primed D17TBM was efficiently augmented. From the results of this study, we conclude that, after surgical resection of a primary tumor, systemic administration of IL-12 can be an effective adjuvant therapy because it demonstrates a significant augmentation effect on the tumor-specific immune response in the tumor-primed host.     

The improved effects of specific active immunotherapy on a rat fibrosarcoma by antitumor drugs

Summary We have tried to find out if the combination of a xenogenized tumor cell vaccine and antitumor drugs is able to induce a synergistic increase in the antitumor therapeutic effect. The degree of increase in the LTD₅₀ (50% lethal tumor dose) is expressed numerically, as a quantitative index designed to compare degrees of transplantation resistance to tumor cell challenge. A LTD₅₀ was achieved by an intradermal (i. d.) immunization with xenogenized tumor cells when challenged with tumor cells implanted intraperitoneally 2 weeks after the immunization: this LTD₅₀ value was 527 000 times higher than that of the non-immunized group. When we combined this type of immunization with appropriate doses of bleomycin (BLM) or cyclophosphamide (CY), which are able to augment antitumor immunity, the LTD₅₀ was 723 000–1190 000 times higher than that of the non-immunized group. This increase in the LTD₅₀ is definitely higher than that achieved by a single immunization with irradiated tumor cells (× 33 000) and combined with either BLM (× 93 000) or CY (×140 000). We also studied the therapeutic effect of a tumor cell vaccine combined with antitumor drugs BLM or CY in tumor-bearing rats. We observed a synergistic effect caused by BLM or CY after i. d. immunization with xenogenized tumor cells: this showed a significant increase when compared with the therapeutic effects obtained by chemotherapy alone (P <0.01). Nevertheless, there was no evidence that the above antitumor effects is superior to the effect achieved by irradiated tumor cells.     

Successful immunotherapy with intraperitoneal Corynebacterium parvum in a murine ovarian cancer model is associated with the recruitment of tumor-lytic neutrophils into the peritoneal cavity

The rejection of a murine ovarian teratocarcinoma (MOT) after i.p. injection of Corynebacterium parvum was investigated. Treatment with C. parvum (1400 micrograms) 24 hr after i.p. inoculation of a lethal number of tumor cells (10(5] induced an antitumor effect that cured 75 to 95% of the mice. Morphologic analysis and an in vivo cytotoxicity assay that measured the rate of disappearance of radioactivity from the peritoneal cavity after injection of 125IUdR-labeled tumor cells indicated that the antitumor effect was initiated during the first 24 hr after C. parvum injection. During this period of time, host effector cells retrieved from the peritoneal cavity prevented tumor growth in a Winn assay and lysed radiolabeled MOT targets in a 4-hr Cr-release assay. After separation of peritoneal inflammatory cells on a Percoll gradient, neutrophil-enriched fractions demonstrated significant in vitro tumor lysis, but neutrophil-depleted populations were ineffective. Microscopic analysis of lysis at the single cell level confirmed that neutrophils were binding to and lysing MOT targets. Further characterization of these tumor cytolytic neutrophils revealed that they are nylon wool-adherent, not generated in indomethacin-pretreated mice (but effectively generated in whole body-irradiated mice), and achieve lysis within 30 min after binding to MOT targets. These results indicate that neutrophils must be considered potential antitumor effectors that can be recruited by treatment with biologic response modifiers.