Search

High-dose methotrexate therapy(HDMTX)is effective against lymphoid malignancies. However, delayed elimination of methotrexate(MTX)after HDMTX administration may lead to severe adverse drug reactions. We surveyed the drugs coadministered with MTX and the incidence of delayed MTX elimination in patients treated with HDMTX in a clinical setting. We analyzed the plasma MTX concentration in 110 samples after 55 cycles of HDMTX in 33 patients. Delayed MTX elimination was defined as a plasma MTX concentration ≥1.0 mmol/L at 48 h after the start of HDMTX administration or ≥0.1 mmol/L at 72 h after the start of HDMTX administration. The incidence of the combined use of drugs affecting MTX excretion and drugs that exhibited typical renal excretion was 84.8%(n=28). The incidence of delayed MTX elimination was 39.4%(n=13). MTX-induced acute kidney injury occurred in 9 patients, all of whom also exhibited delayed MTX elimination. Therefore, when prescribing HDMTX, it is important to monitor adverse events, including acute kidney injury, which may be induced by prolonged MTX blood concentrations.

We evaluated the efficacy of gemcitabine-cisplatin (GC) therapy as a second line chemotherapy for recurrent urothelial cancer previously treated with or resistant to methotrexate, vinblastine, doxorubicin and cisplatin (M-VAC) therapy. Four patients who had recurrent cancer after adjuvant M-VAC therapy and five patients with resistant lesions to M-VAC were treated by GC. Of the nine patients, three completely responded to GC and three obtained partial response. These complete responders were cancer-free for 34, 32 and 24 months. In one partial responder, the metastatic masses have been decreasing in size for 12 months after completion of GC therapy. Our findings suggested that GC would be useful as a second line chemotherapy for urothelial cancer previously treated with M-VAC.

A pharmaco dynamic study of Methotrexate (MTX) during intraperitoneal MTX/5-FU sequential therapy was carried out after gastric surgery. A comparative study of the route of MTX administration and its dose was also done. 1) Comparative study of the serum concentration of MTX between i.p. and i.v. administration revealed a similar MTX concentration except immediately after administration. 2) A comparative study of the serum concentration of MTX administered i.p. between patients with and without malignant ascites was conducted. Immediate elevation of the serum concentration of MTX was observed in patients without malignant ascites. On the other hand, the MTX concentration was slowly elevated and washed out in patients with malignant ascites. 3) The MTX concentration in the intraperitoneal fluid was compared between patient with and without malignant ascites. In patients without malignant ascites, MTX disappeared quickly from the intraperitoneal fluid. However, the MTX concentration lasted long in the malignant ascites cases. These results were similar with a low-dose MTX (30 mg/body) or moderate dose (100 mg/body). MTX/5-FU sequential i.p. therapy can thus be an effective treatment for patients after gastric surgery, though clearance of MTX was slow in cases with malignant ascites.

The combination effect of cisplatin (CDDP) and methotrexate (MTX) on the cytotoxicity against normal fibroblast and malignant cell line were investigated utilizing MTT assay. In case of normal fibroblast, the optical density (OD) value in CDDP + MTX was significantly higher and remained at a consistently higher value in various mixture rates than that in CDDP alone. Such low cytotoxicity may be due to MTX because the addition of Leucovorin resulted in an apparent decrease in the OD value. The combination of the two drugs was thought to alleviate the toxicity of CDDP on fibroblast, in contrast to its increase on cancer cells.

A 49-year-old woman was diagnosed with local recurrence and cervical lymph node and bone metastases 55 months after surgery for breast cancer. She was treated with goserelin acetate and tamoxifen but the disease was assessed as progressive after 8 months. Five courses of CMF therapy were performed but lung, pleural and mediastinal lymph node metastases were detected. Then, five courses of CAF therapy were carried out, but a contralateral breast metastasis was detected and the patient complained of shortness of breath. The CAF therapy was assessed as PD. We attempted administration of doxifluridine (5'-DFUR) and mitomycin C (MMC) on an outpatient basis. After 6 months, no progressive disease was detected and she was relieved of her shortness of breath. The combination therapy was assessed as long NC. Combination therapy with 5'-DFUR and MMC is thus a useful treatment for adriamycin- and methotrexate-resistant breast cancer, especially in terms of quality of life.

The first clinical application of biochemical modulation (BCM) of 5-fluorouracil (5-FU) was the sequential MTX/5-FU regimen proposed in 1977 by Bertino for the treatment of colorectal cancer. In Japan, sequential MTX/5-FU therapy was mainly used as a new method of treating gastric cancer, and attracted a great deal of attention because it proved effective in many cases of advanced gastric cancer that had been unresponsive to the previous chemotherapy, particularly scirrhous gastric cancer with poor prognosis. Its therapeutic efficacy varied according to histologic type, it was effective in cases of peritoneal dissemination and disseminated intravascular coagulopathy (DIC), it was associated with fewer adverse effects, and it was a multidrug chemotherapy based on a clear rationale. With sequential MTX/5-FU therapy as a starting point, fundamental studies of BCM and its clinical applications have expanded rapidly in Japan. This paper provides an outline of sequential MTX/5-FU therapy from the aspects of its mechanism of action, indications, therapeutic efficacy, relevance to adjuvant therapy, counter-measures to adverse effects, and emergence of resistance to the drugs involved. The high therapeutic efficacy of this therapy in certain histologic types is also discussed, and its combined use with other forms of BCM, as in triple BCM (LV/5-FU + CDDP/5-FU + MTX/5-FU), is introduced.

We studied pharmaco-dynamic changes of intraperitoneal sequential MTX (30 mg)/5-FU (750 mg) therapy in gastric cancer patients with malignant ascites and those who had undergone a gastrectomy. The serum and ascites levels of MTX in patients with malignant ascites decreased much slower than in patients without ascites. In patients with ascites, the serum MTX concentration peaked 8 hours later and then gradually decreased. The ascites MTX level decreased as low as 1/10 2 days after the intraperitoneal administration. The serum and ascites 5-FU levels revealed a minor prolongation of 5-FU concentration in patients with ascites. Gastrectomy did not affect the pharmaco-dynamics of MTX in post-operative patients with sequential intraperitoneal administration of MTX/5-FU on 1, 8 and 15 POD.

Antimetabolic anticancer agents possess their own target enzymes: that of methotrexate is dihydrofolate reductase; 5-fluorouracil and ZD1604, thymidylate synthase; hydroxyurea, ribonucleotide reductase; 2'-deoxycoformycin, adenosine deaminase; N-(phosphonacetyl)-L-aspartate, aspartate transcarbamylase. Overproduction of each target enzyme has been observed with various animal and human cell lines which acquired resistance to all these agents. These facts suggest that this is a common mechanism for resistance to these agents. Most of these resistant cells showed amplification of the corresponding genes in double minute chromosome or homogeneously stained region of the chromosome. The relation between the degree of resistance and those of enzyme overproduction, the expression and amplification of the gene coding for each enzyme protein in various resistant cell lines are demonstrated and discussed.

An antifolate first introduced into the clinic about half decades ago, Methotrexate is effective against a variety of human cancers, when administered alone or in combination with leucovorin rescue at wide-ranging doses and by many different schedules. We know much more about MTX and its molecular and cellular pharmacology and mechanisms of anti-cancer action in light of current knowledge of the biochemistry of folate. This understanding has stimulated novel approaches to the clinical application of the drug and has greatly enhanced its therapeutic efficacy.

Unlabelled: To investigate the effects of disease modifying antirheumatic drugs (DMARDs) and DEX on production of IL-1 beta, IL-6 and TNF-alpha, synovial cells were observed after IL-1 beta administration in vitro., Materials and Methods: Synovial tissue was obtained aseptically from 8 rheumatoid arthritis patients during joint surgery. The dissected tissue was treated with collagenase and adherent cells were passaged before using as samples. They were stimulated with IL-1 beta (1 ng/ml) and cultured with DMARDs and DEX in serum-free media. After 24 hours' incubation, the production of IL-1 beta, IL-6 and TNF-alpha in the supernatants was measured., Results: DEX inhibited the production of IL-6. GST inhibited the production of IL-1 beta and IL-6., Conclusion: DEX and GST may modulate the disease activity by inhibiting the cytokine production from synovial cells.

In the current study, we ran a chemosensity test on 7 human undifferentiated carcinoma cell lines against 10 anticancer compounds using MTT assay. Efficacy was estimated by comparing fifty percent inhibitory concentration (IC50) with the peak plasma concentration. The results showed that the 7 cell lines could be divided into two groups which had different (high or low) chemosensitivity, and that in the high sensitivity group, ACD, VCR, and etoposide were indicated as useful drugs. Our results suggest that it may be impossible to rescue all undifferentiated carcinoma patients by chemotherapy alone because the tumor may consist of 2 clones which have a different chemosensitivity.

MTT assay, a sensitivity test of anti-tumor agents was performed, and its clinical usefulness, was discussed. In 15 surgical specimens, the cell suspensions were prepared aseptically, and divided into three processing; namely, Papanicolaou (Pap) smears to confirm the malignant cells, MTT assay, and cell cultures in chamber slides. MTT assay was evaluated only when tumor cells in the chamber slide were observed 50% or more by Pap staining. Drugs judged to be effective were applied for patients, resulting in 64.2% of predictive accuracy in the sensitivity. Conclusions; 1) MTT assay was developed for sensitivity test of anti-tumor agents, 2) Strict assessment was carried out by the confirmation of cancer cells using chamber slides, 3) On clinical usefulness, predictability for the sensitivity was 64.2%, that for resistance was 100%, and over all predictability was 66.7% 4) MTT assay was useful for the determination of effective and for elimination of ineffective drugs.

A new dosage format (MTX-CH) of methotrexate (MTX) comprises MTX adsorbed onto fine activated carbon particles which has strong affinity to lymphatics. MTX-CH was tested for its therapeutic effects on lymph node metastases in mice experimental model. It is known that metastases have been established in the regional lymph nodes of all mice 7 days after subcutaneous inoculation with P388 leukemia cells at 5 x 10(5) cells/mouse in left hind foot pad, and that assay mice survival time becomes short in proportion to log [P388 cell number inoculated intraperitoneally]. Therefore, drug treatment was carried out on day 7, and the regional nodes were extirpated and transferred intraperitoneally to assay mice on day 10. The survival time of mice to whom transferred were the nodes of mice treated with MTX-CH was significantly longer compared with those treated with MTX aqueous solution.

Two new cell lines were established from a single human uterine cervical adenocarcinoma which had been successfully transplanted into nude mice. They were subcultivated more than 70 times over a period of 46 months and designated as TCO-1 and TCO-2, respectively. Growth characteristics, modes of chromosomes and specific tumor makers were different in TCO-1 and TCO-2. The plating efficiency of TCO-1 was greater and its doubling time was shorter than those of TCO-2. Modes of chromosomes were 77 in TCO-1 and 94 in TCO-2. Their chemosensitivities to 13 anticancer drugs were studied by MTT assay. Only the mitomycin C (MMC) was an effective agent for both TCO-1 and TCO-2 cell lines which were characteristically different from each other. The results suggest that MMC would be a promising agent for chemotherapy of human uterine cervical adenocarcinoma. The influences of 6 hormones (estradiol, progesterone, dihydrotestosterone, medroxyprogesterone acetate, human menopausal gonadotropin, human chorionic gonadotropin) on their proliferation were studied by MTT assay and dye exclusion assay. TCO-1 and TCO-2 showed no significant changes in proliferation in any of them. It is suggested that hormone replacement therapy could be indicated even for postoperative patients with cervical adenocarcinoma.

III Purine antagonists Biosynthesis of guanine nucleotides has been reported to be up regulated in tumor cells. In guanine nucleotide synthesis, there are 2 rate-limiting enzymes, i.e. inosine monophosphate dehydrogenase for de novo synthesis and hypoxanthine guanine phosphoribosyltransferase for the salvage pathway. Therefore, agents acting on these 2 enzymes to inhibit guanine nucleotide synthesis could be expected to have a superior effect on tumor proliferation. The main antitumor agents belonging to this class are thiopurines [including 6-mercaptopurine (6 MP), 6-thioguanine (6 TG) and 6-thioinosine (TIR)], thiazofurin (TZF), and arabinofuranosylfluoroadenine (F-ara-A). In the activation of 6MP to its ribotide. PRPP is the rate limiting factor. After the ribotide is produced, it is metabolized to another active form by enzymes catalyzing purine nucleotide metabolism. The antitumor effect of TZF is enhanced by the combination of TZF with allopurinol, which increases the plasma hypoxanthine level and subsequently inhibits recovery of the reduced guanine nucleotide pool by TZF. F-ara-A induces DNA strand damage as well as inhibiting DNA synthesis and is expected to have a significant antitumor effect on slowly growing tumors. These agents are mainly effective for treating hematological malignancies. IV Antifolic agents Among the antifolates, methotrexate (MTX) is the most useful drug for both hematologic malignancies and solid tumors. MTX primarily inhibits one-carbon transfer through the inhibition of dihydrofolate reductase and thus blocks the biosynthesis of both purine and pyrimidine nucleotides. Formyl polyglutamate synthetase catalyzes folate to its polyglutamate, both the active and retention forms. It is also important as an activating enzyme as well as being a target of MTX. MTX directly inhibits thymidylate synthetase, which could be the main target during high-dose therapy. High-dose MTX therapy with leucovorin (LV) rescue is effective even for tumors which are resistant to conventional treatment. During clinical use, not only MTX levels but also those of its inactive metabolites [7-hydroxy-MTX and 2, 4-diamino-N10-methylpteroic acid(DAMPA)] should be monitored. High-dose MTX therapy with LV rescue requires precise monitoring and LV rescue should be continued until the MTX level falls below 5 x 10(-8) M. MTX is also known as the safest drug which can be directly administered to into the central nervous system. Many other antifolates are under development, among which trimetrexate might be the most promising. Studies on antimetabolites have developed side by side with research on nucleotide tumor cell metabolism, which has produced a number of the antitumor agents now available for cancer chemotherapy.(ABSTRACT TRUNCATED AT 400 WORDS)

Five anticancer agents (MTX, 5FU, MMC, ADR and VCR) and an antiestrogenic agent (TAM) were investigated to observe the effects on the growth of MCF-7 cells and the changes on the cell cycle distribution of them by flow cytometry. The growth-inhibitory effects of these agents were divided into three manners. The growth rate by ADR was affected in a concentration-dependent manner; MTX in a time-dependent; 5FU, MMC and VCR in both time and concentration-dependent manners. The effects on cell kinetics were divided into three categories. MCF-7 cells were accumulated on S phase by MTX, G0G1 phases by TAM and G2M phases by the other agents. The combined effect of two agents was the most prominent when MTX was first administered and followed by an agent which affected on G2M phases. In the combination of TAM and MMC, the strongest effect was obtained when MMC was first administered and followed by TAM. These results indicated that the combined effect of cell inhibition was obtained on cell kinetic basis by employing the technique of analytical flow cytometry.

We performed an in vitro chemosensitivity test with 8 malignant brain tumor cell lines, which were established in our Department. It was shown that ACNU was moderately tumoricidal against only one cell (ONS-86) line. IFN-beta (interferon-beta) was more active against 4 cell (ONS-6, -20, -76, and -81) lines. VCR (vincristine), MTX (methotrexate), and Ara-C (cytosine arabinoside) and FK 973 were most effective against all 8 cell lines. There were some difference in the drug sensitivities among the tumors with the same pathological diagnosis. Since IFN-beta was tumoricidal against to the cells, co-culture of IFN-beta and one of other antitumor agents seemed to induce more antitumor effects. With regard to the side effects of IFN-beta, the combined therapy with IFN-beta and other drugs induced more antitumor effects against malignant brain tumor cells and seemed to reduce the side effects.

The combined effects of pirarubicin (THP) and various antitumor drugs on HeLa S3 human uterine cervix carcinoma and K562 human myelocytic leukemia cells were determined by enhancement of their cytotoxic activities. The combination of 0.15 microgram/ml THP with cisplatin (CDDP), mitomycin C (MMC), peplomycin (PEP), 5-fluorouracil (5-FU), methotrexate (MTX), enocitabine (BH-AC) or etoposide showed synergistic effects on HeLa S3 cells. Also, the combination of 0.01 microgram/ml THP with CDDP, BH-AC or etoposide showed synergistic effects on K562 cells. Especially, the combined effects of THP with MMC or MTX were remarkable, and the combination under almost all concentrations of MMC or MTX showed synergistic effects on HeLa S3 cells. On the other hand, the combination of adriamycin (ADM) with MMC or MTX did not show such a remarkable effects on HeLa S3 cells.

Choriocarcinoma cells grown in the presence of MTX have developed resistance in two ways. The HCCM derived sublines (relatively high MTX resistant) produced enhanced levels of DHFR and had relatively unimpaired transport of MTX, though altered transport was the primary determinant of response in the CC1 derived sublines (low MTX resistant). Since the selection procedure used was identical, it was assumed that altered MTX transport was insufficient to account entirely for various degrees of resistance. Increased DHFR activity was necessary for the development of high MTX resistance. The overproduction of DHFR was the consequence of amplification of the DHFR gene sequence. The incidence of DMs in metaphases paralleled the degree of resistance. Since DMs were also present in the cells not showing DHFR gene amplification, mechanisms other than DHFR gene multiplication were responsible for the de novo synthesis of DMs.

Three choriocarcinoma cell lines (BeWo, GCH-2 and SCH) cultured in MTX or MTX-free medium were investigated with respect to cell growth and production of hCG, beta-hCG and specific beta-1-glycoprotein (SP-1). MTX was administered at the concentration of 10(-8), 10(-7), 10(-6), 10(-5)M for 24 hours, 72 hours and 168 hours from the fourth day of culture. Each cell line demonstrated that the cell growth and production of hCG and beta-hCG were dose, rather than time, dependent. 10(-6)M or more of MTX had a distinct effect, but 10(-7) or less of MTX had little effect. SP-1 was produced at a low level during culture regardless of the medium used. The ratio of the extra- to the intracellular hCG level was shown to be about 100:1 after the administration of MTX to BeWo and GCH-2. It is, therefore, possible that the temporary elevation of the hCG level observed in vitro after treatment with MTX is the result of an increase in secretion of hCG, rather than cellular damage.

With a view to examining the possible clinical applicability of pulsing magnetic fields (PMF), we investigated the effects of weak, non-heat inducing, PMF on DNA synthesis and sensitivity of cancer cells to antitumor agents. Leukemic T-cells (Molt-4) and a pancreatic ductal adenocarcinoma (solid tumor) transplanted in a Syrian golden hamster were used for the in vitro experiment and in vivo experiment respectively. In order to evaluate the effects of PMF on the DNA synthesis of cancer cells and the incorporation of antitumor agent into cancer cells, cultured cells or solid tumor were exposed to PMF generated by a solenoid coil immediately after 3H-or 14C-thymidine and 3H-methotrexate administration respectively. Thymidine uptake was found to increase by exposure to PMF, as did also 3H-methotrexate uptake by leukemic T-cells. Following exposure to PMF immediately after administration of methotrexate or mitomycin C, antitumor activity in both cells was increased. From these results it appears that the incorporation of antitumor agents into the cells increases by eddy current stimulation induced by PMF, and that the cell cycle shifts from the non-proliferative to proliferative phase, resulting in increased antitumor activity.

With advances in the understanding of intracellular nucleic acid metabolism, the biochemical modulation of the action of antimetabolites by MTX was discussed as an approach towards the development of more selective chemotherapy. Using high-performance liquid chromatography, the sequential changes occurring in the acid-soluble intra cellular nucleotide pools of L1210 mouse leukemic cells were analysed after treatment with MTX, and a reduction of the dTTP pool to 46% of the control level was observed. Declines in the dCTP, ATR and GTP pools were also reduced to 36%, 30% and 24%, respectively, of the control level after treatment with MTX. Using MTX-5-FU combination chemotherapy, pretreatment with MTX enhanced the intracellular level of FUTP which is one of the active metabolites of 5-FU. There was, however, no significant difference in the value of dTTP levels between MTX-5-FU and 5-FU alone. One of the mechanisms of the synergistic interaction between MTX and 5-FU might therefore be stimulated phosphorylation of 5-FU with an increased PRPP level, which occurs as a result of the MTX-blocked purine de novo pathway. Using MTX-ara C combination chemotherapy, MTX enhanced the intracellular production of ara-CTP, which occurs as a result of MTX reducing the intracellular dCTP pools, following activation of deoxycytidine kinase. More clarification of the effects on intracellular nucleic acid metabolism will aid the development new and increasingly effective drug combinations involving thiopurines. The methods of combination chemotherapy will become more sophisticated as the biochemical modulation effect of the drug is clarified.

The "cellular effect" i.e. temporary elevation of HCG titer may not be infrequently experienced during course of chemotherapy against choriocarcinoma. Present paper is to clarify real view of the effect and mechanism of its manifestation by using a cell-line (GCH-nu). 1) Cellular growth was suppressed moderately by 10(-7), 10(-6) and 10(-5)M MTX. 2) During 2-4 days of administration at these concentrations of MTX HCG per ml medium was elevated 5 times. HCG in cells per mg protein were also higher 5-7 times. 3) HCG-beta in media was enhanced 5-7 times in MTX group. 4) Estrone, estradiol and progesterone were not altered. 5) Choriocarcinoma cells developed marked enlargement of nuclei and cells, multinucleation and cytoplasmic vacuolization. 6) Cytochemistry of heat stable ALP revealed increased activity especially in cells with morphological changes. ALP examined by biochemical method was enhanced 5 times. 7) Amount of protein per cell increased 10 times after giving MTX. Real view of so-called "cellular effect" includes elevations of HCG-beta and ALP activity as well as HCG, whereas sex steroids are not apparently related. The data in addition to morphological and cytochemical changes reveal that MTX may induce differentiation of choriocarcinoma cells into those of syncytial type, which then sustain the "cellular effect".

Treatment of choriocarcinoma should be eradicated of trophoblastic cells which undergo destruction in both of primary and metastatic sites and Methotrexate (MTX) has a most effective value. While MTX is being administration to the patients, there is no determination of clear relationship between proliferation of trophoblastic cells and production of human chorionic gonadotropin (hCG). In this report, we attempted to clarify the changes of the dividing compartments in cell cycle of human choriocarcinoma in vitro in the administration of MTX (concentration in 5 x 10(-6) M and 5 x 10(-7) M) to the experimental cells. Cell samples were stained by propidium iodide (PI) and fluorescein isothiocyanate (FITC), then were measured nucleic acid and protein content by means of flow microfluorometry (FMF). MTX was remarkably increased the intensity of FITC associated with protein production, in dose that is not only killing effect to the cells but inhibition of DNA synthesis. Moreover, the most difference in both subjects were as follows; Ever when MTX in low concentration was removed after 48 hours, 50% of experimental cells recovered to the level of controls in contrast to the persistence of remarkable inhibition of DNA synthesis in high concentration.

Cis-platinum (CDDP) was investigated in vitro and in vivo for its ability to inhibit the growth of Methotrexate (MTX)-resistant choriocarcinoma cell line (BeWo) and was compared with the effect of MTX and Actinomycin D (ACD). Each drug was added into medium (RPMI 1640 containing 10% FBS) for 1 hr. at the concentration of peak plasma level in clinical use (CDDP 7 X 10(-6)M, MTX 10(-6)M, and ACD 8 X 10(-8)M), or for 48 hrs. at one-tenth of the level. CDDP inhibited the cell growth to 40% of control at 7 X 10(-6)M for 1 hr., and 25% at 7 X 10(-7) M for 48 hrs. And ACD suppressed the cell growth to 10% at both 8 X 10(-8)M for 1 hr. and 8 X 10(-9)M for 48 hrs. But MTX did not inhibit the cell growth at 10(-6)M for 1 hr., and inhibited to 60% of control at 10(-7)M for 48 hrs. On the other hand, BeWo transplanted to athymic nude mice (CD-1(ICR) nu/nu) was treated with intraperitoneal injections of CDDP (1.4 mg/kg/day for 4 consecutive days in a week and repeated for 2 and 4 weeks), MTX (2mg/kg/day for 4 consecutive days in a week and repeated for 2 weeks), and ACD (90 micrograms/kg/day for 4 consecutive days in a week and repeated for 2 and 4 weeks). The suppression of the tumor growth was seen in CDDP-treated group (TRW/CRW = 6.5%) and ACD-treated group (TRW/CRW = 29%) after 2 courses of treatment, but no apparent suppression was shown in the MTX-treated group.(ABSTRACT TRUNCATED AT 250 WORDS)

The necessity to assess the influence of chemotherapeutic agents on cell cycle kinetics has been stressed for development of effective cancer chemotherapy. Effects of these agents on cell cycle progression in cultured human lung cancer cells, PC-3 (adeno) and PC-6 (small cell), were studied by flow cytometry (FCM) with computer analysis. DNA distributions of the cells stained with propidium iodide were expressed numerically. Firstly the influence of adriamycin (ADR), then methotrexate (MTX), and finally, sequential combined chemotherapy with ADR and MTX on cell cycle kinetics were examined. The results are summarized as follows. Exposure to ADR induced concentration dependent cell killing effect in PC-3 and PC-6, while exposure to MTX showed mainly time dependent cell growth inhibition. By FCM, difference of effect of ADR between those two cell types was certified. Namely, in PC-3 low ADR concentration lead to G2 accumulation with decrease of G0/G1, polyploization, and high ADR concentration to S accumulation due to S-G2/M delay. Furthermore, in the highest ADR concentration (10 micrograms/ml), G1, G1-S boundary accumulation were observed. On the contrary, exposure of PC-6 to ADR remained to show only G2 accumulation with a slight decrease of G0/G1. Exposure of PC-6 to MTX lead to G2/M decrease with delay of G0/G1-S progression followed by S accumulation which was ascribed to delay of S-G2/M progression. Sequential combined chemotherapy showed superiority of MTX-ADR to ADR-MTX and other regimens in cytocidal effect, and the difference might be attributable to induction of partial synchronization to G0/G1-S boundary with MTX exposure. It was emphasized that FCM is a indispensable method for analysis of the influence of chemotherapeutic agents on cell cycle kinetics.

Subrenal Capsule Assay (SRCA) as a chemosensitivity test was performed on 14 esophageal squamous cell carcinomas in order to select a more effective form of chemotherapy. Of the 14 assays, 12 were evaluable. Mice were treated with anticancer agents (e.g. Cisplatin, Bleomycin, Methotrexate, Vindesine) on days 1 and 3 after transplantation, and on day 6, the sensitivities were determined. Fresh esophageal cancers yielded an evaluable assay rate of 74%. The implant grew progressively for six days in the remaining group of control mice. Histologically, host cell infiltration at the border of the implant was observed from day 3 after transplantation, and cells had degenerated or had been partially replaced by scar tissue by day 6. The results of chemosensitivity tests differed according to the anticancer agent used or from case to case. Clinically, correspondence between the assay results and clinical results was obtained in 5 out of 7 cases. SRCA is a new promising chemosensitivity test which is clinically useful, and the present results indicated the feasibility of its use in developing an effective chemotherapy for esophageal cancer.

The efficacy of anticancer drugs and the development of drug resistance after chemotherapy for advanced breast cancer has been evaluated in vitro using the human tumor clonogenic assay (in vitro colony assay technique.) The rates of in vitro chemosensitivity to various anticancer drugs in untreated patients were similar or somewhat higher than the clinical efficacy reported for single clinical use of the drugs. The results of in vitro chemosensitivities to the drugs against previously treated patients showed decreased chemosensitivities to the drugs which had been used, especially adriamycin and mitomycin C. In contrast to this, in vitro chemosensitivities to 5-FU, methotrexate and L-PAM were unchanged after exposure to combination chemotherapy consisting of these drugs. This result suggests that the development of resistance to these drugs is minimal at the doses used in our protocol. The overall chemosensitivity rate for anthracyclines including adriamycin in vitro was only 5% (5/93) for previously treated patients, compared with 41% (35/83) for vinca alkaloids (p less than 0.005). This finding indicates that combination chemotherapy including vinca alkaloids is the appropriate selection for second line chemotherapy for advanced breast cancer patients who have been treated previously with adriamycin.

The subrenal capsule (SRC) assay for cancer chemotherapy was tested according to Bogden's methodology. Of 37 patients providing tumor tissue for assay, 29 cases were considered suitable for evaluable assays. Fourteen patients had clinically evaluable diseases and 10 cases were evaluable for SRC assays. Correspondence between sensitive assay and clinical sensitivity was seen in 2 cases, and that between resistant assay and clinical resistance was seen in 4 cases. Discordance between sensitive assay and clinical resistance was seen in 4 cases. In histological studies, cancer tissues implanted in the subrenal space in immunocompetent mice did not show marked proliferation and were replaced by prominent leukocyte infiltration and fibrosis on day 6 after inoculation. The degree of leukocyte infiltration in the xenografts in the mice administered some anti-cancer drugs was slight in comparison with that in untreated control mice, which showed a remarkable trend in xenografts treated with 5-fluorouracil and cyclophosphamide, respectively. Our study suggests that there are many problems involved in the SRC assay methodology of Bogden, and that careful examination of this aspect will be required.

FK 973, a new substituted dihydrobenzoxazine, was obtained by chemical modification of a novel antibiotic which was isolated from the fermentation products of streptomyces sandaensis No. 6897. FK 973 had cytotoxic effects against in vitro cultured human and murine glioma cells. The concentration of FK 973 required to inhibit cell growth by 50% was 0.06-5 micrograms/ml, after 2-day exposure of this drug against human glioblastoma (ONS-6, 12, 23, and ONS-12/ACNU), human medulloblastoma (ONS-76, 81), human neuroblastoma (ST), and murine glioblastoma (RSV-M glioma). FK 973 showed antitumor efficacy in the meningeal gliomatosis models by RSV-M glioma cells. The median survival time (MST) of models treated by FK 973 (i.t.) was 30 days. However, the MST of control group was 23 days. In the in vitro neurotoxicity test, FK 973 proved to be slightly more toxic than ACNU and MTX, but it had no crucial problems, compared with ADM.

Methotrexate (MTX), a folic acid antagonist, is among the most active known drugs for clinical cancer therapy. The mechanisms of resistance to MTX have been extensively studied, and several mechanisms have been reported so far. Those are decreased cell permeability, increased synthesis of dihydrofolate reductase (DHFR), which is the target enzyme of MTX, decreased polyglutamation of MTX, and decreased binding capacity of MTX to DHFR. Recent basic studies revealed that an increase of DHFR synthesis is the result of a proportional amplification of DHFR gene, and that polyglutamation of MTX plays an important role in its action. High-dose methotrexate (HDMTX) therapy has a good rationale for overcoming MTX resistance. However, many clinical trials of HDMTX during past decade have not demonstrated any apparent clinical efficacy except childhood ALL and ostosarcoma. Further research would be necessary for the improvement of the therapy using MTX.

Five murine tumor cells (sarcoma 1509a, Gc-4, YAC-1, Dunn osteosarcoma, Lewis lung carcinoma) were exposed to methotrexate (MTX) at concentrations of 10(-1)-10(-9) mg/ml for 4-72 hours in vitro and then assayed for their viability. The effect of cell killing by MTX was dependent on both exposure time and concentration of the agent. MTX at a concentration of 10(-5) mg/ml killed the entire cell population in 72 hours, whereas the agent was not particularly effective even at a concentration of 10(-1) mg/ml on exposure for 4 hours. There was a close relation between doubling time of tumor cells and exposure time to MTX for killing entire populations of the tumor cells. The longer doubling time of the tumor cells was, the longer exposure time to MTX was needed to kill them. Lewis lung carcinoma was less sensitive to MTX than other sarcomas. The effect of citovorum factor (CF) on tumor cell killing by MTX was studied on sarcoma 1509a. After exposure to MTX sarcoma 1509a populations could not be rescued by the treatment of CF.

The relationship between the growth potential and hCG secretion in 2 kinds of human choriocarcinoma cell lines was studied by using 5 kinds of anticancer drugs in strict in vitro conditions. The results are as follows. The hCG secretion per cell was enhanced when the DNA synthesis and growth of the cells were suppressed, irrespective of the human choriocarcinoma cell lines or anticancer drugs used. The hCG secretion per cell was suppressed when the rate of cell death increased. These results indicate that the transient rise in blood or urinary hCG values during chemotherapy ("cellular effect") is due to the suppression of DNA synthesis and growth of the cells and is not due to the result of the cell death.

After serial transplantation in nude mice, we had established three new human choriocarcinoma cell lines (NaUCC-1,2 and 3). These three cell lines and BeWo were examined for sensitivity to Act-D, MTX and the combined agents (Act-D + MTX) 3H-Act-D uptake and 3H-MTX uptake, and were compared for each treatment. NaUCC-1 showed low sensitivity to MTX (p less than 0.05), but showed high sensitivity to Act-D (p less than 0.05). BeWo showed low sensitivity to Act-D (p less than 0.05). In examining sensitivity to the combined agents (Act-D+ MTX), the sensitivity of NaUCC-2 to Act-D was decreased (p less than 0.05) by MTX added at the same time. In the 3H-Act-D uptake experiment, NaUCC-1 did uptake a relatively larger amount of 3H-Act-D (p less than 0.01) and BeWo a smaller amount of it (p less than 0.05). In the 3H-MTX uptake experiment, NaUCC-1 did uptake a relatively smaller amount of 3H-MTX (p less than 0.05), but NaUCC-2 uptook a larger amount of it (p less than 0.005). NaUCC-1 established from the patient in whom tumor cells were resistant to treatment, had a low response to MTX. NaUCC-2 was established from the patient in whom it was found that MTX inhibits the Act-D effect on tumor cells. In the study of combined agents in NaUCC-2, the growth inhibition effect of Act-D was suppressed by the MTX added.

Each anticancer agent has its own pharmacologic activities. Effect of anticancer agent may be influenced by the concomitant administration of another drug. In addition, patients with advanced cancer occasionally require other medications as well. These drugs can alter both the effects and adverse reactions of anticancer agents. Therefore, much attention must be placed on the interaction with anticancer agents. This paper summarizes drug interactions between anticancer agents and other drugs. Furthermore, clinical significance of these interactions will be discussed. It is hoped that this paper will provide more attention for the study of this problem.

The sensitivity to methotrexate (MTX) of ten kinds of human choriocarcinoma cell lines was studied in vitro. The cell line most sensitive to MTX was HCCM-5 and it was fed with a medium containing increasing concentrations of MTX. A resistant subline (HCCM-5R) which could proliferate in the medium containing 5 X 10(-7)M MTX was obtained after about 80 weeks of feeding. The properties of HCCM-5R cells were compared with those of the parent HCCM-5 cells. i) There were no apparent differences in morphology between the HCCM-5 and HCCM-5R lines. The population doubling time was almost the same for both cell lines. ii) The hCG level in the culture fluid of the HCCM-5R cells was about twice as high as that of the HCCM-5 cells. iii) The concentration of MTX which inhibited 50% of DNA synthesis in HCCM-5R cells was 10,000 times higher than that for HCCM-5 cells. iv) Each X and Y chromosome was identified in 90% of the HCCM-5 cells, while no Y chromosome was detected in the HCCM-5R cells. v) The DNA distribution pattern for HCCM-5R cells consisted of a large fraction of each cell with DNA G1 phase content and distributed in the tetraploid range, whereas that of HCCM-5 showed no particular cell cycle pattern. vi) The incorporation of 3H-MTX in the HCCM-5R cells was one tenth of that in the HCCM-5. vii) The intracellular DHFR activity of the HCCM-5R cells was about 5 times higher than that of HCCM-5. These results suggest that MTX-resistant cells among the HCCM-5 cells were selected in long-term contact with MTX, and sensitivity to MTX was concerned with both the MTX transport and intracellular DHFR levels.

Cancer chemosensitivity test by Bogden's SRCA (subrenal capsule assay) method was experimentally evaluated using DMBA induced rat ovarian cancer. Cancer tissues were implanted beneath the renal capsule of BDF1 mice, and factors affecting development of the xenografts were examined. To observe the effect of the host-mediated immunologic reaction, the mice were put into immunosuppressive situations by applying cyclophosphamide (CPA) or cyclosporin A (CsA), and the growth of the tumor in each condition received macroscopic and histological evaluation. Under the most favorable conditions, drug sensitivity tests were performed. The results obtained were as follows: 1) In non-treated mice, the cancer cells of the grafted tumor were rejected, until the 7th day following the implantation, by host reaction. 2) In a CPA-induced immunosuppressive condition, the tumor tissue was damaged by the cytotoxic effect, although the size of the tumor was on the increase until the 9th day. 3) CsA brought about a favorable condition for tumor development, showing a linear growth curve from the 3rd to the 9th day with viable cancer cells. 4) Chemosensitivity tests indicated that methotrexate and CDDP were effective, while mitomycin was not effective in treating the implanted tumors. 5) The results obtained with SRCA were much the same in experimental therapeutic trials using rats with primary ovarian cancer induced by DMBA application.

The metabolism of methotrexate was investigated in rabbits with six hours infusion of (3'5'7-3H) methotrexate (50mg/kg). Methotrexate and its metabolites were analyzed by reverse-phase high-pressure liquid chromatography and by counting of radioactivity. 7-Hydroxymethotrexate was found to be the major metabolite in the plasma, urine, bile and tissues of rabbits. Cumulative production of 7-Hydroxymethotrexate during the first six hours was 31.8% of the total dose. The 7-Hydroxymethotrexate concentration in plasma was reached to peak level in six hours after the initiation of the infusion, and was declined slower than that of methotrexate. The highest total radioactivity was found in the kidney after six hours, which was 7.24 times higher than that in plasma. Ratio of 7-Hydroxymethotrexate/methotrexate increased in the liver, small intestine, lung, kidney and testis. This data suggested that conversion of methotrexate to 7-hydroxymethotrexate took place in lung and kidney as well as in liver.

High-dose methotrexate (100 mg/kg) and citrovorum factor rescue enhanced syngeneic sarcoma (S1509a) specific killer T cell (cytotoxic T lymphocyte) in tumor bearing mice, although high-dose MTX suppressed killer T cell against S1509a in immunized mice. It was suggested that tumor specific suppressor T lymphocyte played an important role in enhancement of killer T cell. Enhancement of killer T cell was induced at day seven after administration of MTX. From the clinical point of view, weekly high-dose (100 mg/kg) MTX therapy may be necessary to enhance tumor specific immunity of the patients with sarcoma. High-dose MTX therapy is a unique one not only in practical point of view, but also in immunological point of view.