We have established a single catheter technique of percutaneous isolated liver perfusion using a 4-lumen-2-balloon (4L - 2B) catheter for treatment of unresectable malignant liver tumors. Herein reported are the technique, safety and pharmacokinetics of the system in comparison with the original double-balloon technique. This study included 19 patients with malignant liver tumors treated by adriamycin at a dose of 100 mg/m2. Seven patients had the double-balloon technique (group D), in which filtered hepatic effluent and the rest of the inferior vena caval blood were separately drawn and returned to the left axillary vein. The other 12 patients had single catheter technique (group S). In group S, hepatic effluent was solely isolated and directed to CHP filters. All patients except for one in group S showed good hemodynamic stability. The hepatic venous flow rate of group S was significantly higher than in group D (p < 0.05). Although the mean area under the time concentration curve at systemic serum was significantly lower in group S compared to group D, the rate of side effects was similar in both groups. A 4L. 2B single catheter allowed safe and repeated percutaneous isolated liver perfusion for technical simplification of the treatment.

In order to determine the optimal drug administration route in patients with metastatic liver tumor, we studied the pharmacokinetic parameters after intra-arterial or intra-portal adriamycin (ADM) administration. Four patients with metastatic liver tumors were included in this investigation. One catheter was inserted into the gastroduodenal artery for arterial infusion and the other catheter was inserted into the portal vein via mesenteric vein for portal infusion under laparotomy. ADM 30 mg was infused and blood samples were collected from peripheral and hepatic veins. The concentration of ADM was determined with HPLC technique and the pharmacokinetic parameters were analyzed with a three-compartment open model. The parameters analyzed were blood concentration at t0, half-life, rate of elimination, total body clearance, volume of distribution and area under the blood concentration versus time curve. There were no significant differences of the pharmacokinetic parameters between intra-arterial infusion and intra-portal infusion. Also, no differences were observed between the data from peripheral venous blood and those from hepatic venous blood. These data suggest that the drug distributions were almost similar between intra-arterial and intra-portal drug administration in patients with metastatic liver tumor.

Twenty patients with unresectable hepatoma, recurrent hepatoma or metastatic liver cancer were treated by intra-hepatic arterial injection of mitoxantrone, and its effectiveness and side effects were studied. In 35.5% of the patients, complete or partial responses were seen. The survival intervals after the beginning of therapy were from one to 21 months (mean, 9.4 months) and one-year survival ratio was 56.3%. Red blood cell, leukocyte, and platelet counts diminished significantly one week later after administration of mitoxantrone compared with the pre-administration levels. Severe hepatic dysfunction has not been experienced.

It is difficult to administer an effective dose of cyclosporin A (CsA), a potent inhibitor of P-glycoprotein, to prevent multiple drug resistance due to its side effect. We herein evaluated the efficacy of concomitant use of this agent and complete hepatic venous isolation and charcoal hemoperfusion (HVI.CHP). Dogs were divided into two groups; group I (n = 4), intraarterial infusion of only adriamycin (ADM) and group II (n = 4), intraarterial infusion of CsA and ADM. In both groups, ADM was intraarterially administered for 10 minutes under HVI.CHP. In addition, in group II, CsA infusion (0.3 mg/min.kg) was initiated 20 min prior to the start of ADM infusion and maintained for 30 min. The AUC (micrograms.min/ml) of ADM were 21.2 +/- 8.6 (mean +/- SD) and 28.4 +/- 10.3 in groups I and II, respectively, at prefilter (hepatic venous level) and 8.1 +/- 4.6 and 4.8 +/- 3.8, respectively, at postfilter, showing an effective drug elimination in both groups. The Cmax (micrograms/ml) were 14.1 +/- 2.2 at prefilter, 2.4 +/- 0.5 at postfilter, and 3.4 +/- 1.2 in systemic level. These results indicated that HVI.CHP allowed the high-dose CsA infusion required for P-gp inhibition in the liver and could reduce extraregional CsA leakage.

Human renal adenocarcinomas do not adequately respond to cancer chemotherapy. Their multidrug resistance is mainly conferred by the P-glycoprotein (P-gp). In this study, we analyzed effects of P-gp modulators on enhancement of anticancer activities against human renal cell carcinomas.

Ovarian cancer is one of the significant and deadly disease. Since 1980 when cisplatin was introduced in the chemotherapy, about 30% of the patients with advanced disease have achieved 5-year survival. However, remaining patients have had progressive disease or recurrence after achieving NED. Forty-seven% of recurrent disease was discovered as distant metastasis, while at initial therapy. In the recurrent disease, distantly metastatic lesions were encountered more frequently than those in primary disease. In the recurrent tumor, expression of immunohistochemical markers of malignancy, such as p53 protein and CD44v6 antigen were increased. These clinical data suggest that recurrent ovarian cancer which are exposed to anticancer agents attain increased metastatic potential. In order to assure that anticancer agent contribute to this increment, an experimental system using two human ovarian cancer cell lines (HRA, KF) and nude mice in which cancer cells were exposed to cisplatin in vivo was introduced. Cancer cells exposed to cisplatin in vivo (treated cells) made spontaneously more metastatic nodules in the mouse lung than those exposed to PBS (untreated cells). This result suggest that cisplatin induce the increase of metastatic potential of cancer cells in vivo. Treated cells showed higher invasiveness compared with untreated cells when inoculated in the footpad. Three major factors which were generally proposed to be necessary for cancer cell to give rise to invasion, such as attachment to extracellular matrix, production of proteolytic enzyme, and cellular mobility. For all of these factors, treated cells were superior to untreated cells. These results obtained suggests that cisplatin could increase the metastatic potential of cancer by enhancing potential of invasion. To investigate the mechanism of this phenomenon from the standpoint of genetic mutation, clonal analysis of experimental cancer in vivo was performed using southern blot method. Cancer cells before inoculation to the mice consisted of multiple clones. In 5 week after inoculation, tumor was wholely occupied by only one clone which showed one band on the lane. At this point cisplatin were administered. In 6 week, new single clone appeared with different band pattern from that of the clone at the administration of cisplatin. Furthermore, the cisplatin-induced new clone metastasized to the lung, while no metastasis was observed in the mouse with PBS-treated tumor during the same period. These data suggest that increased metastatic potential after cisplatin treatment is due not to selection but to creation of highly metastatic clone caused by potential of genetic mutation of cisplatin. In conclusion, chemotherapeutic agent has a potential to create highly malignant cancer cells as well as a potential to kill cancer cells.

The effective and less side effect retention time of intravesical instillation therapy with Pirarubicin (THP) was investigated for the treatment of urinary bladder tumor. Fifty-seven cases of urinary bladder tumor were treated by intravesical instillation therapy with THP (20 mg/40 ml) a total of 6 times, with the first instillation at the time of surgery and the other 5 at a rate of three times a week thereafter. The retention time was 30 minutes, and it was allowed to last 10 or 60 minutes for comparison in some cases. Tumor and normal tissue were examined in the first instillation, and normal tissue in the sixth instillation. Infiltration to the bladder wall of THP was observed under fluorescence microscopy. Although the amount of tumor uptake was larger than normal tissue in the first instillation, no satisfactory infiltration was obtained. Repeated administration of THP with the retention time of 30 minutes enhanced the uptake and increased the infiltration in many cases, and side effects were scarcely noted. Retention time of 10 minutes was unsatisfactory, while the retention time of 60 minutes was discontinued in the early stage of treatment due to severe irritative bladder symptoms. Therefore, a retention time of 30 minutes is adequate in the case of repeated administration of THP in a short period.

With a rapid progress in biotechnology, a variety of endogenous macromolecular substances have become a novel class of therapeutic agents. This review will focus on the development of delivery systems for macromolecular drugs. Current status and future perspectives in this research field are reviewed mainly based on the results obtained in our laboratory. First of all, we studied pharmacokinetic characteristics of macromolecules in relation to their physicochemical properties such as molecular weight and electric charge. Based on this information, we first developed macromolecular prodrugs as a delivery system for low molecular weight drugs. An antitumor antibiotic, mitomycin C (MMC) were covalently conjugated with dextran and various types of macromolecular prodrug of MMC were developed for tumor targeting. Secondly, delivery systems for protein drugs such as soybean trypsin inhibitor, uricase, and recombinant superoxide dismutase (SOD) were developed. In particular, successful targeting of SOD to the liver, kidney and blood circulation was achieved by chemical modification of the protein drug. Finally, we have been trying to develop delivery systems for nucleic acid drugs involving antisense oligonucleotides and plasmid DNA. Prior to the development of delivery systems, we found that the pharmacokinetics of the nucleic acid drugs are decided by their physicochemical properties as polyanions even if these materials contain genetic information. Several approaches were tested to control the in vivo behavior of the oligonucleotides and plasmid DNA based on the finding. Thus, we have established the strategy for rational design of delivery systems for various types of macromolecular drugs based on the pharmacokinetic considerations. This methodology can be a formidable tool for the development of clinically applicable macromolecular drugs.

The acquired immunodeficiency of the host plays an essential role in the occurrence of infections even with low pathogenic bacteria. The increase of cases with MRSA and/or pseudomonas infection is one of the serious problems in hospital management in Japan for the elderly as well as pediatric patients. In the present study, mitomycin C (MMC)-treated hosts were prepared in young, adult and old mice to test the immunopotentiating action of the promising Chinese herbal medicine, Tohki-Rikuoh-Toh (TRT), Hotyu-Ekki-Toh (HET) and Juzen-Taiho-Toh (JTT). The effect of these herbal medicines on organ structure and its function in the MMC-treated hosts is clarified and discussed for medical use. 4-5, 8-10 and over 50 week old male C57BL/6 (Clea Japan Inc.) were injected with MMC at a dosage of 3 to 5 mg/kg to inhibit the bone marrow, thus creating a mouse model with reduced immunopotential. A powder extract of TRT, HET and JTT was administrated orally at a dosage of 500 mg/kg/day for seven consecutive days. The white cell number and the subset analysis were carried out by the FACS method. The bactericidal effect of the host was monitored by NBT reduction test. Peritoneal macrophages were prepared by the adherence technique. The macrophage phagocytic activity was examined by an ACAS system. After the administration of TRT, HET and JTT, the body weights recovered as much as 90%, especially in young animals which had been reduced to 75% of their normal values. After MMC-treatment, with the herbal medicines, HET was good for young mice while JTT was effective for the old ones. As for the effect on B cells, the plaque-forming cells (PFC) of spleen cells were compared among the groups. As a result, PFC in the HET group was 184% and the other two were 80 approximately 95% as compared to 76% in the MMC-treated ones. The number of white blood cells in the MMC-treated mice returned to 80% of their normal value. In addition, the phagocytic activity of macrophages increased to 50% although that of the non-treated group was only 20%. The phagocytic activity also recovered in the JTT and TRT of 131% to 95%, respectively compared to 11% in the MMC-treated control. When TRT, HET and JTT were administered orally to mouse models whose immunopotential had been inhibited, the herbal medicines activated both quantitatively and qualitatively, showing themselves to be effective interstitial medicines. In addition, the data from the animal models showed no side effects, confirming the complete efficacy of the drug. Moreover, there was no direct anti-bactericidal effect from these medicines, suggesting that the immunomodulating action of this medicine is host-mediated. It is interesting that quantitative and qualitative recovery were seen when HET was administered to MMC-treated young hosts while JTT was good for the old. With this investigation, the effective components are still unknown for different generations, and we need to clarify this aspect for better understanding of the efficacy of herbal medicines.

The present study was designed to investigate the contribution of proteases in anticancer agents-induced apoptosis of human leukemia HL-60 cells. A serine protease inhibitor TPCK and an ICE-like protease inhibitor VAD-FMK prevented etoposide, camptothecin and ara-C-induced internucleosomal DNA cleavage. Using a cell-free system, we have examined the inhibitory mechanism of these inhibitors on etoposide-induced internucleosomal DNA cleavage. We found that cell lysates prepared from etoposide-treated HL-60 cells undergoing apoptosis contain the significant activity to induce internucleosomal DNA fragmentation in isolated nuclei. On the other hand, we could not detect such activity in the cell lysates from untreated HL-60 cells. Treatment of the cell lysates with a serine protease inhibitor TPCK abrogated the DNA fragmenting activity. An ICE-like protease inhibitor VAD-FMK had no effect on this DNA fragmenting activity in vitro. However, the formation of TPCK-sensitive DNA fragmenting activity in etoposide-treated cells was blocked by the VAD-FMK. These data indicate that serine and ICE-like proteases may be involved in etoposide-induced apoptosis at the different stages, and especially a serine protease may be closely associated with the final step for induction of DNA fragmentation during apoptosis in HL-60 cells.

Many anticancer agents induce apoptosis in human leukemia cells. Among the various leukemia cells, especially HL-60 cells and U937 cells are very sensitive to apoptosis upon anticancer agents treatment. A serine protease inhibitor TPCK and an ICE-like protease inhibitor VAD-FMK prevented etoposide, camptothecin and ara-C-induced internucleosomal DNA cleavage in human myeloid leukemia HL-60 and U937 cells. Using a cell-free system, we have examined the inhibitory mechanism of these inhibitors on anticancer agent-induced internucleosomal DNA cleavage. Our data indicate that serine and ICE-like proteases may be involved in anticancer agent-induced apoptosis at the different stages, and especially a serine protease may be closely associated with the final step for induction of DNA fragmentation during apoptosis in human myeloid leukemia HL-60 and U937 cells.

Bropirimine is an antitumor agent currently used in clinical trials on bladder cancer. It is known to induce IFN, to activate NK cells and to inhibit the growth of tumor cells. In this study, we examined the in vitro and in vivo efficacy of bropirimine on human and murine bladder cancer. Bropirimine showed in vitro antiproliferative activity on the human bladder cancer cell lines, T 24 and KoTCC-1. This activity was not: affected by the neutralizing antibodies against IFN-alpha, TNF-alpha or IL-1 beta, indicating it is the direct activity of bropirimine without involvement of cytokine production by tumor cells. Bropirimine was active at the concentrations comparable to those in serum or urine attained in clinical trials, which suggests that this direct antiproliferative activity is one of the important antitumor mechanisms of bropirimine. In in vivo experiments, bropirimine reduced the growth of transplanted murine MBT-2 and human KoTCC-1 bladder cancers by oral administration every 4 days starting on day 1, but did not show efficacy when the drug treatment was started on day 8. The antitumor activity of bropirimine was dependent on the timing for drug treatment initiation.

LY188011 (Gemcitabine hydrochloride) is a new derivative of deoxycytidine. Phase I study was carried out by a cooperative study group. LY188011 was administered weekly for 3 consecutive weeks starting with an initial dose of 60 mg/m2 (1n) and then increasing the dosage to 1,000 mg/m2 (16.7n). Dose limiting factor was found to be myelosuppression (decreases of WBC, neutrophils and platelet), and MTD was considered to be 1,000 mg/m2. The nadir of WBC and platelet were observed after about 1-3 weeks. It took 1-2 weeks for their recovery. Other adverse reactions included fever, fatigue, anorexia, nausea/vomiting, anemia and transient elevations of GOT and GPT. However, those adverse reactions were mild. T1/2 rho of plasma concentration was about 19 min and the C5min was dependent on the dose. Anti-cancer effects were observed in one gastric cancer and two colon cancer patients. It is recommended that the dosing schedule for an early phase II study is 800 mg/m2 weekly for 3 weeks with 1 week of rest as one cycle, in multiple cycles.

To establish a method for reasonable clinical use of adriamycin (ADM) and pirarubicin (THP) in the intravesical chemotherapy for superficial bladder cancer, intracellular concentrations of these drugs were examined in culture cell lines (T-24, T-24/ADM and FHS736b1) with variable durations of contamination. The intracellular concentration of THP showed a plateau at 15-30 min. contamination in T-24, and in T-24/ADM, and showed the time dependence of contamination in FHS736b1, human normal bladder mucosa cell line. The intracellular concentration of ADM showed the time dependence of contamination in T-24, T-24/ADM and FHS736b1. And these concentrations of THP were 20 times higher than those of ADM. In conclusion, it seems better that THP was retained for 5-15 min. in the bladder in the intravesical chemotherapy, from the point of view of drug efficacy and preventing side effects. And it seems good that ADM was retained for more than 30 min. in the case with drug sensitive tumors.

We conducted a randomized controlled study to evaluate the clinical usefulness of (2"R) -4'-O-Tetrahydropyranyladriamycin (THP)-based combination therapy subsequent to induction therapy which was consisted with THP, 5'-DFUR, and CPA in the treatment of advanced or recurrent breast cancer. In maintenance therapy, Arm C received CPA and TAM, and Arm T received these two drugs plus THP. Survival time of 50% for all cases in which maintenance therapy was conducted was 26.9 months in Arm T and 20.9 months in Arm C, showing no significant difference by the log-rank test (p = 0.64). Survival time in all cases in which therapy had been completed was 54.6 months in Arm T and 28.1 months in Arm C, showing a significant difference by the log-rank test (p = 0.03) although the number of cases was few. A few cases showed a decrease in total leukocyte count to below 2,000/mm3 at the time of induction therapy, but this was transient in all cases. No significant difference in count was noted between two arms at the time of maintenance therapy. However, many cases in Arm T showed decreased total leukocyte count and hemoglobin content, and thrombocytopenia. These results suggest that combination therapy including THP conducted as maintenance therapy after induction is useful in the prolongation of survival time in patients with advanced or recurrent breast cancer.