In vivo Anti-tumor Activity of a Novel Indolocarbazole Compound, J-107088, on Murine and Human Tumors Transplanted into Mice

J-107088 (6- N -(1-hydroxymethyl-2-hydroxy)ethylamino-12,13-dihydro-2,10-dihydroxy-13-(β-D-glu-copyranosyl)-5H-indolo[2,3- a ]-pyrrolo [3,4- c ]carbazole-5,7(6H)-dione) is a derivative of NB-506, an indolocarbazole compound previously reported as an anti-tumor agent targeting topoisomerase L The optimal administration schedule of J-107088 was found to be intermittent injections. The GID₇₅ (75% growth inhibiting total dose) values of J-107088 against LX-1 lung cancer and PC-3 prostate cancer when given by intermittent injection (twice a week for 2 consecutive weeks) were 200 and 15 mg/m², respectively, whereas the 10% lethal dose (LD₁₀) values of J-107088 against LX-1- and PC-3-bearing mice were 578 and 1200 mg/m². The ratio of LD₁₀/GID₇₅ indicates the therapeutic window of an anti-tumor agent. Although the ratios of doxorubicin, paclitaxel and cisplatin against PC-3 were <0.3, <0.5 and <0.2, J-107showed the widest therapeutic window among the anti-tumor drugs tested. J-107088 was also effective on cells that had acquired resistance related to P-glycoprotein. Furthermore, J-107088 was found to be highly effective in inhibiting proliferation of micro-metastases of tumors to the liver in mice. Therefore, J-107088 is considered to be a promising candidate as an anti-tumor drug for treatment of solid tumors in humans.     

Murine Liver Metastasis Model Using L5178Y-ML Lymphoma and the Effect of Antitumor Agents on the Metastasis

A reproducible tumor model for liver metastasis has been developed from murine L5178Y lymphoma line by sequential cycles of subcutaneous inoculation of liver tumor cells, that were originally generated in livers of female (BALB/c × DBA/2)F₁ mice by injecting the parental cells into the tail vein. This variant (L5178Y-ML) metastasized predominantly to the liver after intravenous or subcutaneous injection. The livers of the animals killed 9 days after intravenous implantation of 5 × 10⁵ tumor cells were about 3 times the weight of control livers. All tumor-bearing mice died 10 to 12 days after inoculation. Subcutaneous implantation of L5178Y-ML in the side flank of mice induced metastatic nodules spontaneously in the livers. The tumor cells proliferated more in livers than in the implanted sites, compared with the parental L5178Y cells. The effects of 5-fluorouracil, mitomycin C, cis -platinum and doxorubicin on the liver metastasis of L5178Y-ML were examined at subtoxic doses; 5-fluorouracil was the most effective in both inhibiting the tumor growth in livers and prolonging the survival period of mice. This model provides a useful tool for the experimental therapy of hepatic tumors in mice.     

Rapid Growth and Spontaneous Metastasis of Human Germinal Tumors Ectopically Transplanted into Scid (Severe Combined Immunodeficiency) and Scid-nudestreaker Mice

Xenograft acceptance, growth and spontaneous metastasis of ectopically transplanted human germinal tumors were compared among scid mice, athymic nude mice and F₂ hybrids constructed from scid and nude mice, in relation to the impairments of T and B cell functions in these mice. In scid mice which are deficient in T and B cell functions, human yolk sac tumor (YST-2) that originated from the ovary grew to enormous sizes in 100% of the animals after both subcutaneous and intraperitoneal transplantation, while only half (59.1% and 51.9%) of the subcutaneous and none of the intraperitoneal transplants were accepted in usual athymic nude mice (BALB/c- nu/nu and CDl- nu/nu ). The YST-2 grew rapidly in scid mice, developing 3 to 10 times larger tumors compared to nude-streaker (AKR/J- nu^str/nu^str ) and usual nude mice, respectively. Furthermore, ectopically transplanted tumors spontaneously metastasized to distant organs (mostly to the lung) in scid mice (but less frequently in leaky scid mice), while metastases have never been found in nude mice. Although a xenograft of human classic (typical or pure) seminoma of the testis has never been established in nude mice, it grows slowly in one-third (36.4%) of scid mice and very rapidly in all of scid-nu ( scid/scid; nu^str/nu^str ) double mutant mice. Spontaneous metastases of xenografted seminomas were also observed in distant organs (lymph node, lung, liver, spleen, and kidney). The metastastic distribution of the two human germinal tumors in scid and scid- nu^str mice mimics that found in human. These results (xenograft acceptance, growth of transplanted tumors and degree of metastatic spread) were compatible with the level of T and B cell impairments indicated by FACS analysis, as well as mitogen responses, serum IgG and morphological features of the thymus.     

Augmentation of Cancer Chemotherapy by Preinjection of Human Macrophage Colony-stimulating Factor in L1210 Leukemic Cell-inoculated Mice

Human macrophage colony-stimulating factor (hM-CSF) is a potent stimulator of the effector functions of monocytes/macrophages. We investigated the antitumor effects of this factor in CDF₁ male mice inoculated with L1210 cells, a mouse B-cell leukemia line. Mice preinoculated with various numbers of L1210 cells on day 0 were given intravenous injections of vehicle (human serum albumin; HSA) (100μg/kg/day) or hM-CSF (20μg/kg/day) for 3 days from day 1. In mice preinoculated with 10² L1210 cells but not with 10³ or more L1210 cells, a marked increment in survival rate was observed with hM-CSF treatment. We next examined the effect of hM-CSF treatment combined with chemotherapy on the survival of mice that had been preinoculated with 10⁵ L1210 cells. In our system, the administration of 4.9 mg/kg adriamycin (ADM) alone slightly prolonged survival of the tumorbearing mice, but all of the mice died within 20 days. When hM-CSF was injected for 3 days before this ADM treatment, the invasion and proliferation of tumor cells in the liver and spleen were markedly inhibited and 50% of the mice were still alive at day 50. We detected inhibitory activity toward L1210 growth in serum of mice administered with hM-CSF, and the degree of the inhibitory activity was correlated with the level of nitrite (NO₂) in the serum. When L1210 cells were co-cultured with peritoneal macrophages from mice intraperitoneally injected with hM-CSF, the uptake of [³H]thymidine in L1210 cells was inhibited. The inhibition was abolished by the addition of N^G-monomethyI-L-arginine, an inhibitor of NO₂− synthesis, suggesting that the reactive nitrogen oxide intermediate is involved in hM-CSF-induced inhibition of L1210 growth.     

Enhanced Liver Metastatic Potential of Alpha-fetoprotein-producing Human Gastric Carcinoma after Carbon Tetrachloride-induced Liver Damage in Nude Mice

The liver metastatic potential of alpha-fetoprotein (AFP)-producing human gastric carcinoma (NSC-3) was examined in male, BALB/c, nude mice. Metastatic nodules in the liver were produced by intrasplenic (IS) injection of tumor cell suspension prepared by trypsinization from subcutaneous NSC-3 tumor. The serum AFP level increased exponentially after IS injection along with the growth of metastatic nodules in the liver, and a positive correlation was observed between the estimated weight of metastatic nodules and serum AFP level. To investigate the effect of liver damage by carbon tetrachloride (CCI₄) on the metastatic potential of NSC-3 cells injected intrasplenlcally, the mice were divided into 4 groups: Group 1 received IS injection of 1×10⁶ of NSC-3 cells without CCI₄, treatment; Groups 2, 3 and 4 received IS injection 7 days, 2 days and 1 day after CCI₄ treatment, respectively. All mice were killed 64 days after IS injection. The incidence of liver metastasis was 80% in Group 1, but 100% in Groups 2, 3 and 4. The mean numbers of metastatic nodules per liver were 4.2 in Group 1, 16.8 in Group 2, 18.0 in Group 3 and 44.5 in Group 4. Significant differences in the mean numbers of metastatic nodules were observed between Group 4 and the other groups. It was clearly demonstrated that the metastatic potential of AFP-producing human gastric carcinoma cells (NSC-3) is enhanced in the situation prevailing after liver parenchymal cells are damaged by CCI₄.     

Epidermal Growth Factor Prolongs Survival Time of Tumor-bearing Mice

We observed that human epidermal growth factor (hEGF) alone prolonged the survival time of mice bearing various murine syngeneic tumors as well as athymic nude mice bearing human xenografts. No changes in the subcutaneous solid tumor mass volume were observed. Prolongation of survival time by hEGF was observed in mice bearing murine epidermoid carcinoma (BSC) and human gastric carcinoma (KATO III), but not in murine epidermoid carcinoma (KLN205) or human epidermoid carcinoma (A431). Human tumor cells such as A431, KATO III, and murine tumor cells, KLN205, BSC had roughly 2 × 10⁶, 3 × 10⁴, 1.3 × 10³ and 1 × 10³ EGF receptors/cell, respectively. Although KLN205 and BSC tumor cells maintained nearly the same number of EGF receptors, the effects of hEGF were very different. Although A431 tumor cells had nearly 100 times more receptors than KATO III cells, the prolongation of survival time of mice bearing A431 by hEGF was no better than that of mice bearing KATO III. Accordingly, it appears that this prolongation of survival time by hEGF is independent of the number of EGF receptors on tumor cells. In addition, hEGF was shown to inhibit experimental pulmonary metastasis of murine BSC tumor, but was ineffective with murine KLN205 tumor. These results suggest that prolongation of survival time by hEGF may result from the inhibition of tumor cell metastasis and EGF may play a role in preventing the metastasis of certain malignant neoplasms unrelated to its effects through the EGF receptor on tumor cells.     

Metastatic dissemination of B16 melanoma: evidence that metastases can result from nonspecific trapping of disseminated tumor cells

Spontaneous metastasis from tumor transplants of two representative mouse B16 melanoma clones, G3.5 and G3.12, was examined experimentally to determine whether initial dissemination to the lungs, or secondary systemic spread from established lung metastases, resulted from organ-specific tropism or from nonspecific trapping of circulating tumor cells in capillary beds. In parabiosed mice, subcutaneous tumors metastasized extensively within hosts, but guests remained metastasis-free except following the rare involvement of the parabiotic junction during secondary spread. Intrasplenic tumor transplants metastasized to the liver, whereas intrarenal transplants metastasized to the lungs, reflecting patterns of venous drainage. Subcutaneous implants of neonatal lung and kidney in the flank opposite from the site of tumor initiation acquired metastases only during secondary systemic spread, and there was no evidence of organ selectivity. Metastases from various organs, and derived cell lines, when transplanted subcutaneously grew into tumors that initially metastasized exclusively to the lungs. These results indicate that both initial and secondary metastases of these B16 melanoma transplants occurred by nonspecific trapping of tumor cells in the first capillary bed encountered. In contrast, organ colonization following intravenous injection of tumor cells frequently proceeded beyond the first capillary bed.     

Relationship between the Pharmacokinetics of Irinotecan and Diarrhea during Combination Chemotherapy with Cisplatin

Two phase I trials of irinotecan (CPT-11) in combination with cisplatin were conducted. In both cases, the dose-limiting toxicities were leukopenia and/or diarrhea. During these trials the pharmacokinetics of CPT-11 and its active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), were investigated to evaluate the relationship between pharmacokinetic parameters and diarrhea, since this is an unpredictable and severe toxicity of combination chemotherapy using CPT-11 and cisplatin. Twenty-three previously untreated patients with advanced lung cancer were evaluated in the pharmacokinetic study. Ten patients received CPT-11 at 80 or 90 mg/m² plus cisplatin at 60 mg/m². The other 13 patients received CPT-11 at 80 or 90 mg/m² plus cisplatin at 80 mg/m² with the granulocyte colony-stimulating factor support (2 μg/kg × 16 days). CPT-11 was given as a 90-min intravenous infusion on days 1, 8, and 15. Cisplatin was given on day 1. The pharmacokinetics of CPT-11 and SN-38 were analyzed on day 8 during the first course of treatment. The maximum tolerated dose of CPT-11 was 90 mg/m² in both phase I trials. The severity of diarrhea was best correlated with the peak plasma concentration of SN-38 among the pharmacokinetic parameters tested. In addition, patients with a plasma SN-38 level > 12.4 ng/ml at 1.75 h after the start of CPT-11 infusion had a higher incidence of Eastern Cooperative Oncology Group grade 3–4 diarrhea than those with a lower SN-38 level ( P =0.0003). Stepwise logistic regression analysis identified the SN-38 concentration as a significant contributor to the development of diarrhea ( P =0.0021). We conclude that there is a clear relationship between the SN-38 concentration and diarrhea during chemotherapy with CPT-11 plus cisplatin.     

Epidermal Growth Factor Receptor-mediated Selective Cytotoxicity of Antitumor Agents toward Human Xenografts and Murine Syngeneic Solid Tumors

Severe toxic side effects of antiproliferative agents limit their clinical usefulness as anti tumor drugs. Recently we observed that the anti tumor efficacy of various anti tumor agents (5-fluorouracil, tegafur, adriamycin, mitomycin C, cyclophosphamide, and cisplatin) against experimental solid tumors was enhanced by prior or simultaneous administration of human epidermal growth factor (EGF). However, coadministration of EGF did not enhance the toxicity of anti tumor agents as measured by LD₅₀ and body weight loss. The above selective potentiation of efficacy of the anti tumor agents by human EGF can be characterized as follows. In a dose-dependent manner, human EGF enhanced the efficacy of an antitumor agent (5-FU) treatment against human epidermoid carcinoma A431 transplanted sc in atliymic nude mice [ED₅₀=2.9 (0.2–49.7, 95% confidence interval) μg/kg, sc]. Various degrees of enhancement were also observed against other experimental tumors transplanted sc. The degrees of enhancement were directly proportional to the numbers of human EGF binding sites present on tumor cell plasma membrane (threshold of binding site density=1.5×10³ sites/cell) using 5-FU or cisplatin as an anti tumor agent, thus suggesting that the binding of EGF to the receptors on tumor cells is an essential process in enhancing the susceptibility of tumor cells to anti tumor agents. Normal cells including intestinal epithelial and bone marrow cells are endowed with fewer EGF binding sites (less than 10³ sites/cell). This may explain partially the absence of EGF-enhanced cytotoxicity by antitumor agents toward normal cells.     

Roles of a prostaglandin E-type receptor, EP3, in upregulation of matrix metalloproteinase-9 and vascular endothelial growth factor during enhancement of tumor metastasis

Cyclooxygenase (COX)-2 is known to correlate with poor cancer prognosis and to contribute to tumor metastasis. However, the precise mechanism of this phenomenon remains unknown. We have previously reported that host stromal prostaglandin E₂ (PGE₂)–prostaglandin E2 receptor (EP)3 signaling appears critical for tumor-associated angiogenesis and tumor growth. Here we tested whether the EP3 receptor has a critical role in tumor metastasis. Lewis lung carcinoma (LLC) cells were intravenously injected into WT mice and mice treated with the COX-2 inhibitor NS-398. The nonselective COX inhibitor aspirin reduced lung metastasis, but the COX-1 inhibitor SC560 did not. The expression of matrix metalloproteinases (MMP)-9 and vascular endothelial growth factor (VEGF)-A was suppressed in NS-398-treated mice compared with PBS-treated mice. Lungs containing LLC colonies were markedly reduced in EP3 receptor knockout (EP3^−/−) mice compared with WT mice. The expression of MMP-9 and VEGF-A was downregulated in metastatic lungs of EP3^−/− mice. An immunohistochemical study revealed that MMP-9-expressing endothelial cells were markedly reduced in EP3^−/− mice compared with WT mice. When HUVEC were treated with agonists for EP1, EP2, EP3, or EP4, only the EP3 agonist enhanced MMP-9 expression. These results suggested that EP3 receptor signaling on endothelial cells is essential for the MMP-9 upregulation that enhances tumor metastasis and angiogenesis. An EP3 receptor antagonist may be useful to protect against tumor metastasis. ( Cancer Sci 2009; 100: 2318–2324)     

The Changing Pattern of the Splenic Lymphocyte Subsets in Tumor-bearing Mice after Oral Treatment with OK-432

The aim of this study was to investigate the influence of oral administration of OK-432 on the tumor growth of tumor-bearing mice. In addition, the changing pattern of the splenic lymphocyte subsets of tumor-bearing mice was evaluated by flow cytometry. OK-432 at a dose of 0.1, 1 or 10 KE was administered orally every 3 days or every other day for 30 days to subcutaneously Meth A tumor-inoculated mice. The tumor growth was significantly inhibited in the 1 KE every 3 days group, in the 1 KE every other day group and in the 10 KE every 3 days group. In the 10 KE every other day group, OK-432 inhibited the tumor growth on days 10 and 20, while the agent did not show a marked inhibitory effect on day 30. The percentages of splenic L₃T₄-positive cells and splenic asialo GM₁-positive cells were significantly increased in the 1 KE every other day group, while the Lyt2⁺/ Thyl.2⁺ratio was decreased. On the other hand, in the 10 KE every other day group, OK-432 showed no effect on the percentages of splenic L₃T₄-positive cells and Lyt2⁺/Thyl.2⁺ratio on days 20 and 30. Our results suggest that the antitumor effect of oral administration of OK-432 may be correlated with the changing pattern of L₃T₄-positive cells and Lyt2⁺/Thyl.2⁺ratio.     

Invasion and metastasis of Lewis lung carcinoma in the mouse uterus

Patterns of Lewis lung carcinoma (LLC) cell invasion, colonization and metastasis were studied in C57 mouse uteri. LLC cells (5 X 10(4) in 0.05 ml) were infused nonsurgically into nonpregnant and pregnant uteri, 3 days postcoitum. The fate of cells was studied histologically on days 2, 5 and 18 posttreatment (PT). Despite a large degeneration of LLC cells in the lumina of both the pregnant and nonpregnant mice, in 38% of these uteri, tumor cells had invaded the endometrium by day 2 PT. However, subsequent distribution of tumor cells differed remarkably between the pregnant and the nonpregnant uteri. By day 5 PT, in 66% of the nonpregnant mice, tumor cells were common in the endometrium and metastases in other organs were seen in 40% of the animals. But in the pregnant mice only 40% uteri showed tumor cells and no metastasis was recorded. On day 18 PT tumor cells were rare in the nonpregnant uteri, but significantly, 75% of those animals showed lung and liver metastases. In pregnant mice, tumor cells neither survived in the uteri nor metastasized to other organs. LLC cells, infused into nonpregnant uteri, promptly metastasized to lungs without colonizing the uteri: this unique system may provide insight into the effects of organ-specific host factors on the growth and metastatic potential of tumor cells.     

Prospective Evaluation of the Feasibility of Cisplatin-based Chemotherapy for Elderly Lung Cancer Patients with Normal Organ Functions

A study was conducted to examine the feasibility of cisplatin-based chemotherapy in elderly patients (75 years old) with advanced non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC). Thirty-four patients were enrolled between September 1993 and December 1994. Patients with normal organ function and good performance status (PS) received cisplatin-based chemotherapy (cisplatin 80 mg/m² on day 1 and vindesine 3 mg/m² on days 2 and 8 for NSCLC, or cisplatin 80 mg/ m² on day 1 and etoposide 100 mg/m² on days 2 to 4 for SCLC). Ten patients (29%) were eligible for this study, 7 with NSCLC and 3 with SCLC. Reasons for exclusion were ischemic heart disease in 14, poor PS (2) in 11, reduced creatinine clearance (Ccr) in 10, abnormal electrocardiogram without ischemia in 9 and noncompliance with the protocol in 2 patients. Eight patients had two or more reasons. Nine of the 10 eligible patients were able to tolerate 2 or more courses of chemotherapy. All 3 patients with SCLC responded (1 complete response and 2 partial response), but only 1 of the patients with NSCLC achieved partial response. Toxicity was evaluated according to Japan Clinical Oncology Group criteria. All but one patient experienced grade 4 neutropenia, and 6 patients had infectious episodes requiring antibiotics. Grade 3 anemia and thrombocytopenia were observed in 1 and 2 patients, respectively. Non-hematological toxicities were mild. Only 10 of 34 patients (29%) satisfied our eligibility criteria and they experienced severe myelotoxicity. We conclude that chemotherapy should be given carefully to elderly patients even if they appear to have normal organ function.     

Sequential Involvement of Two Distinct CD4+ Regulatory T Cells during the Course of Transplantable Tumor Growth and Protection from 3–Methylcholanthrene-induced Tumorigenesis by CD25–depletion

The involvement of two phenotypically different regulatory T cells in different stages of tumor growth was investigated. Treatment of BALB/c mice with anti-CD25 monoclonal antibody (mAb) (PC61), but not anti-CD4 mAb (GK1.5) before RL male 1 or Meth A inoculation caused tumor rejection. On the other hand, treatment of BALB/c mice with anti-CD4 mAb (GK1.5) but not anti-CD25 mAb (PC61) on day 6 after inoculation of the same tumors caused rejection. The findings suggest that CD4⁺CD25⁺ T cells downregulated the rejection response in the early stage of tumor growth. On the other hand, putative CD4⁺CD25⁻ T cells downregulated the tumor rejection response in the late stage. Both CD4⁺CD25⁺ and putative CD4⁺CD25-T cells appeared to inhibit the efficient generation of cytotoxic T lymphocytes (CTL). The present study also demonstrated that the treatment of BALB/c mice with anti-CD25 mAb (PC61) at 4 or 6 weeks after 3–methylcholanthrene (3–MC) inoculation retarded tumor occurrence and prolonged survival.     

Murine liver metastasis model using L5178Y-ML lymphoma and the effect of antitumor agents on the metastasis

A reproducible tumor model for liver metastasis has been developed from murine L5178Y lymphoma line by sequential cycles of subcutaneous inoculation of liver tumor cells, that were originally generated in livers of female (BALB/c x DBA/2)F1 mice by injecting the parental cells into the tail vein. This variant (L5178Y-ML) metastasized predominantly to the liver after intravenous or subcutaneous injection. The livers of the animals killed 9 days after intravenous implantation of 5 x 10(5) tumor cells were about 3 times the weight of control livers. All tumor-bearing mice died 10 to 12 days after inoculation. Subcutaneous implantation of L5178Y-ML in the side flank of mice induced metastatic nodules spontaneously in the livers. The tumor cells proliferated more in livers than in the implanted sites, compared with the parental L5178Y cells. The effects of 5-fluorouracil, mitomycin C, cis-platinum and doxorubicin on the liver metastasis of L5178Y-ML were examined at subtoxic doses; 5-fluorouracil was the most effective in both inhibiting the tumor growth in livers and prolonging the survival period of mice. This model provides a useful tool for the experimental therapy of hepatic tumors in mice.     

Establishment and Characterization of BALB/c Retroperitoneal Sarcoma with Spontaneous Liver Metastases

The objective of this study was to examine the identity and characteristics of a spontaneously occurring murine retroperitoneal tumor of BALB/c mouse origin that selectively metastasized to the liver. From the primary tumor, a permanent cell line, termed LMFS (liver metastasis from sarcoma) was established in vivo and in vitro . After a subcutaneous injection of more than 1 × 10⁵ cells in the side back of mice, the LMFS cells proliferated at the inoculation site (100% take) and induced metastatic nodules spontaneously in the liver, but not in the lung. By the limiting dilution technique, a cloned cell line, LMFS-1, was established in vitro . The LMFS-1 cell line had similar morphological characteristics to the LMFS cells both in vitro and in vivo . The doubling time of the LMFS-1 cell line was 10 h in passage 60. The number of chromosomes ranged from 71 to 108 and 93% of metaphases showed near-tetraploidy. In microscopic examination, no specific arrangement of the LMFS tumor cells was seen; the LMFS cell had medium- to large-sized atypical nuclei and clear and large cytoplasm. Electronmicroscopy showed that the cytoplasm of the LMFS cell had a moderate amount of rough-surfaced endoplasmic reticulum but no desmosomes or microvilli. Immunohistochemically, the LMFS cells were positive for vimentin, but showed no reaction for keratin or cytokeratin. Therefore, the LMFS tumor was considered to be an undifferentiated sarcoma. The LMFS cell line should be a useful tool not only for studies of metastasis, but also for experiments on the therapy of hepatic tumors.     

The Roles of CD8+ and CD4+ Cells in Tumor Rejection

In vivo administrations of anti-Lyt-2.2 (CDS) mAb and anti-L3T4 (CD4) mAb selectively eliminated CD8⁺ cells amd CD4⁺ cells, respectively. The relative potencies of CD8⁺ cells and CD4⁺ cells and their roles in primary tumor rejections were studied by investigating the effects of these mAbs on tumor growth. CD8⁺ cells were themselves fully capable of mediating rejection in 5 different tumor rejection systems: two radiation leukemia virus (RadLV)-induced leukemias, B6RV2 and BALBRVD, a radiation-induced leukemia BALBRL♂1, and a plasmacytoma BALBMOPC-70A in CB6F₁ mice, and a Friend virus-induced leukemia B6FBL-3 in B6 mice. On the other hand, CD4⁺ cells were capable of resisting tumor growth of B6FBL-3, but not of the other four tumors. Furthermore, for efficient rejection of CB6F₁UV+^˚l sarcoma by CB6F₁ mice, synergy of CDS⁺ and CD4⁺ cells was necessary. Blocking of UV+^˚ 1 rejection was abrogated by delayed administration of anti-L3T4 (CD4) mAb but not anti-Lyt-2.2 (CDS) mAb, indicating the involvement of CD4⁺ cells in only the initial phase of rejection.     

Phase I Study and Clinical Pharmacological Evaluation of Daily Oral Etoposide Combined with Carboplatin in Patients with Lung Cancer

Twenty-eight patients with inoperable or relapsed lung cancer were given a combination of oral etoposide, administered once a day at doses ranging from 40 to 60 mg/m²/day (d) for 21 consecutive days, and carboplatin, administered intravenously over 1 h at doses ranging from 300 to 400 mg/m² on day 1 to determine the appropriate doses of this combination. In addition, pharmacokinetic and pharmacodynamic analyses were performed. All the patients had a performance status of 0 to 1. Serum etoposide and free platinum (Pt) concentrations were measured using high-performance liquid chromatography and atomic absorption, respectively. Myelosuppression, nausea and vomiting were the dose-limiting toxicities of this schedule. The maximum tolerated dose (MTD) was 50 mg/m²/d oral etoposide for 21 days and 400 mg/m² i.v. carboplatin on day 1. For heavily pretreated patients, the MTD was 40 mg/m²/d oral etoposide for 21 days and 350 mg/m² i.v. carboplatin on day 1. No cumulative increase in the area under the concentration-time curve (AUC) for oral etoposide over time was observed. There were significant correlations between the free Pt serum level (6, 8, 12, 24 h post-dose) and etoposide AUC level (days 1, 10 and 21) for graded hematological toxicity, and the percentage decreases and nadir counts of hemoglobin, leukocytes, neutrophils and platelets. Several pharmacodynamic models were developed to predict the hematological toxicity. In order to facilitate pharmacodynamic evaluations in future studies, a limited sampling model for oral etoposide was also developed and validated.     

Selective Suppression of the Generation of Anti-tumor L3T4+ but Not of Lyt-2+ T Cell-mediated Immunity in the Tumor-hearing State

C3H/He mice hyperimmune against syngeneic MH134 hepatoma were prepared by intradermal (id) inoculation of viable tumor cells followed by surgical resection of the tumor and by repeated id challenges with viable tumor cells. Winn assays performed utilizing spleen cells from these mice have revealed that both Lyt-2⁺ and L3T4⁺ T cell subsets from MH134-hyperimmune mice produced complete tumor protection. The in vivo tumor-neutralizing activity was also found in spleen cells from tumor-bearing mice at various times after id implantation of MH134 tumor cells. However, in contrast to comparable tumor-neutralization by Lyt-2⁺ and L3T4⁺ T subsets from hyperimmune mice, only the Lyt-2⁺ T cell subset from tumor-bearing mice was capable of mediating the in vivo protective immunity. L3T4⁺ T cell-mediated immunity was not detectable in the tumor-bearing state irrespective of the length of the sensitization period with a primary growing tumor, but emerged in the mice which resisted the first tumor challenge after the resection of the primary tumor. These results indicate that the emergence of L3T4⁺ T cell-mediated anti-tumor immunity is stage-dependent and the Lyt-2⁺ T cells represent the main functional subset in the tumor-bearing state, although both subsets of T cells are potentially capable of effecting anti-tumor in vivo immunity. The results are discussed in relation to the selective suppression of the L3T4⁺ but not of Lyt-2⁺ T cell function in the tumor-hearing state.