In vitro evaluation of experimental agents for anti-HIV activity.

This unit presents an assay that has proven useful as an initial screening test is an HIV cytopathic effect (CPE) inhibition assay in which immortalized T cell lines (e.g., ATH8 or MT2) that are profoundly sensitive to the cytopathic effect of certain strains of HIV are utilized as target cells. Additional protocols assess the anti-HIV activity of certain candidate agents by measuring inhibition of syncytium formation or p24 gag protein production by ELISA. Calculation of the 50% inhibitory concentration (IC(50)) is also presented.

The role of low dose cisplatin plus 5-fluorouracil for treatment of recurrent and/or advanced squamous cell carcinoma of the head and neck.

To improve survival rate in advanced head and neck cancer, we scheduled 90 patients to receive low dose cisplatin plus 5-fluorouracil regimen as neoadjuvant(NAC), concurrent(CC), adjuvant(AC), and second line chemotherapy (SC) setting. Our regimen consisted of cisplatin (CDDP 5 mg/m2/1 hr infusion on days 1-5, 8-12, 15-19, 22-26) and 5-fluorouracil (5-FU 200 mg/m2/24 hr infusion or oral administration of tegaful-uracil (UFT-E) 400 mg/body on days 1-28). The concurrent chemoradiotherapy consisted of conventional irradiation with 1.6-2.0 Gy/day on five days per week up to a total dose around 60Gy, and CDDP 3 mg/m2 by intravenous infusion over 1 hour plus 5-FU 150 mg/m2 by intravenous infusion over 24 hours per day on five days per week. For SC, 24 patients evaluable for response, 4 CR and 6 PR with RR of 42% were achieved. For NAC, 14 patients were evaluated for response, 2 CR and 7 PR were achieved. CC was indicated for locally unresectable cases. Of the 33 patients evaluable for response were 17 CR and 9 PR with RR of 79%. Dose limiting toxicities for chemotherapy were anemia and leukopenia and chemoradiotherapy was mucositis. Our treatment modality showed marginal toxicity and good response. Moreover, our regimen could be given in an outpatient setting safely so quality of life for patients was identical. We concluded that for advanced head and neck cancer, these treatment options were effective for second line and adjuvant setting. Chemoradiotherapy with this regimen also gave a impact for improving local control and survival period for locally unresectable cases.

Synthesis of the optical isomers of 4-[1-(4-tert-butylphenyl)-2-oxo- pyrrolidine-4-yl]methyloxybenzoic acid (S-2) and their biological evaluation as antilipidemic agent.

The enantiomers of (+/-)-4-[1-(4-tert-butylphenyl)-2-oxo-pyrrolidine- 4-yl]methyloxybenzoic acid (S-2), a new antilipidemic agent having dual action on the plasma triglyceride (TG) and cholesterol (Cho) lowering effects, were prepared via separation by Chiralcel OJ column chromatography of their methyl ester and also by the same method as the described racemate's synthesis from optically active 1-(4-tert-butylphenyl)-2-oxo-pyrrolidine-4-carboxylic acid respectively. These optically active carboxylic acids were prepared by the resolution of diastereomeric N-[(S)-(-)-[4-methyl-(alpha-methyl)benzyl]]-1-(4-tert-butylphenyl)-2-oxo - pyrrolidine-4-carboxyamide using silica gel column chromatography, followed by deamination with N2O4. The absolute configurations for the enantiomers of S-2 were indirectly determined using X-ray analysis of the 4-bromo-2-fluorobenzamide of the (+)-4-[1-(4-tert-butylphenyl)-2- oxo-pyrrolidine-4-yl]-methyloxybenzoic acid. S-2 and its enantiomers showed an essentially equipotent activity on the fatty acid- and sterol-biosynthesis inhibition in vitro. On the other hand, in the in vivo activity, (S)-(+)-4-[1-(4-tert-butylphenyl)-2-oxo-pyrrolidine- 4-yl]methyloxybenzoic acid (S-2E) was superior in the lowering abilities of the plasma TG and phospholipid(PL) and was chosen as a candidate for a novel antilipidemic agent. The difference in the in vivo activity among S-2 and its enantiomers was explained from the pharmacokinetics after administration p.o.

[Biochemical modulation of 5-FU--effect of low dose CDDP].

A pilot study of continuous or intermittent low dose 5-FU and cisplatin chemotherapy (low-dose FP therapy) was conducted at the Department of Surgery of Sapporo Medical University School of Medicine (Group A) and Sapporo Tsukisamu Hospital, and at the Department of Internal Medicine of the Kochi Prefectural Center Hospital (Group B). The cases with esophageal cancer, stomach cancer, pancreatic cancer, hepatocellular carcinoma or colonic cancer co-existing with their inoperable lesion(s) were considered in this chemotherapy. The rates of complete and partial response and of side effects were studied. Also, the effects of low-dose FP on the prognosis of the patients with pancreatic or colonic cancers were investigated. The procedure consisted of continuous 5-FU 320 mg/m2 i.v. with daily CDDP 2.5 mg/m2 i.v. for five days/week rescue was performed for at least four weeks as a rule. The rates of complete response and partial response were 64% (Group A) and 56% (Group B) in esophageal cancer, 62% (Group A and B) in stomach cancer, 48% (Group A) and 57% (Group B) in colonic cancer, and 8% (Group A) and 21% (Group B). The overall response rate was 57.8%. The frequencies of severe side effect(s) (grades 3 and 4) were within three to eight percent, and no death from side effect(s) was experienced. The effects of low-dose FP therapy on the prognosis of stage IV colonic cancer and stage IV b pancreatic cancer were studied retrospectively. It is suggested that this chemotherapy might contribute to the survival of patients with these two cancers. Otherwise, the chemotherapy of intermittent administration (day by day) of 5-FU 750 mg/m2 i.v. and CDDP 2.5 mg/m2 i.v. was selected in order to decrease the rate of side effects and their severity. The pilot study encountered no severe side effects, no cases with grade 4 side effect were experienced but the remission rates were mostly similar to that of sequential low-dose FP therapy. However, the side effect of low grade ones as symptoms in gastrointestinal tract were observed in more patients. We concluded that sequential or intermittent 5-FU/CDDP therapy might be fairly effective, and since the adjuvant chemotherapy of choice for advanced or recurrent gastrointestinal cancer, their FP therapy might be one of the adjuvant treatments.

Development of a novel form of an oral 5-fluorouracil derivative (S-1) directed to the potentiation of the tumor selective cytotoxicity of 5-fluorouracil by two biochemical modulators.

We have focused our attention on the development of a novel form of a tegafur-based [FT; a prodrug of 5-fluorouracil (5-FU)] antitumor agent. We have used two biochemical and pharmacological modulators of 5-FU to improve its overall activity. To potentiate the antitumor activity of FT, 5-chloro-2,4-dihydroxypyridine (CDHP) was used as a potent reversible inhibitor of 5-FU degradation. The reduction of gastrointestinal (GI) toxicity, induced in the host by 5-FU, was modulated by potassium oxonate (Oxo), an inhibitor of orotate phosphoribosyltransferase that catalyzes the phosphorylation of 5-FU, a process believed to be responsible for the toxic effects of 5-FU. When CDHP and FT were simultaneously given orally to Yoshida sarcoma-bearing rats in various molar ratios, the antitumor effect of FT was significantly potentiated by the combination consisting of at least a 0.2 versus 1 molar ratio of CDHP to FT, respectively. This augmentation of an antitumor activity was supported by potent and prolonged inhibition of dihydrouracil dehydrogenase activity (5-FU degrading activities) in the liver of tumor-bearing rats after oral CDHP (0.2:0.8 molar ratio) and furthermore by elevation and over 12 h retention of 5-FU levels in the tumors following combined administration of FT and CDHP at a molar ratio of 1:0.4, respectively. Moreover, to reduce the severe GI injury and subsequent loss of body weight, observed in parallel with an increased antitumor efficacy, Oxo was given orally to Yoshida sarcoma-bearing rats and nude rats xenografted with H-81 human gastric carcinoma, during consecutive administration of the FT-CDHP mixture. Combined treatment with Oxo and FT (1:2 molar ratio) supplemented with 0.4 molar CDHP resulted in protection of body weight loss without affecting the high antitumor efficacy of the FT-CDHP mixture. When [2-14C]FT plus CDHP was administered with Oxo, the 14C-labeled fluoronucleotide content was objectively decreased in the GI tract of the tumor-bearing rats but not in the tumor and bone marrow, which supports our initial hypothesis. Based on these promising data, we propose a suitable formulation of a FT-based anticancer drug, called S-1, and consisting of FT, CDHP and Oxo at a 1:0.4:1 molar ratio and showing tumor-selective cytotoxicity of 5-FU.

Feasibility study on protracted infusional 5-fluorouracil and consecutive low-dose cisplatin for advanced gastric cancer.

In this phase II trial 31 patients with advanced gastric cancer (21 with metastatic cancer and 10 with locally advanced cancer) were treated with a continuous 24-hour infusion of 5-fluorouracil (5-FU) 330 mg/m2/day plus low-dose cisplatin (CDDP) 6 mg/m2/day by bolus infusion on days 1-5. The regimen with a combination of 5-FU and low-dose CDDP (FLDP) was repeated weekly for two to four courses according to response and tolerance. In 24 (77%) of the 31 patients, four courses of this regimen were administered. The overall response rate was in 14/31 (45%) patients with measurable disease, including one complete response and 13 partial responses. An especially high response rate of 60% was seen in 10 patients with liver metastasis. Median survival time was 11 months (range 6-27+) in the 10 cases of locally advanced cancer and 11 months (range 6-24+) in the 21 cases of metastatic cancer. Toxicity was primarily hematologic. Leukopenia and thrombocytopenia of World Health Organization (WHO) grade 3 or 4 occurred in 4/31 (13%) and 4/31 (13%) of patients, respectively. Renal dysfunction, which is a major toxicity associated with CDDP, was not observed without hydration. The patients were able to eat during therapy and preserved a good quality of life. A randomized trial including the FLDP regimen is needed to compare it with other active regimens, particularly the use of high-dose CDDP.

[Combined chemotherapy with 5-FU and low dose CDDP for advanced or recurrent cancer of the digestive system and home anti-cancer chemotherapy].

We have performed combined chemotherapy with 5-FU, a biochemical modulator, and low dose CDDP for advanced or recurrent cancer of the digestive system. The therapy was effective in 37% of all cases and in 45.5% and 41.6% of esophageal and gastric cancer cases, respectively. In addition, few patients developed adverse side effects including renal disorders, one of the major side effects of CDDP. Therefore, we considered home anti-cancer chemotherapy feasible. For 27 outpatients with advanced cancer of the digestive system including 15 cases of esophageal cancer, 4 cases of gastric cancer, 3 cases of colon cancer, 4 cases of pancreatic cancer and 1 case of gall bladder cancer, 4 to 6 week home adjuvant chemotherapy was performed. The regimen comprised 1 week of oral administration of 300 mg/body/day of UFT-E granules and 5 days of continuous intravenous infusion of 25 mg/body/day of CDDP using an infusor pump. During the follow-up, 3 cases of catheter obstruction, 3 cases of catheter sepsis and 1 case of pneumothorax appeared. These complications all resulted from the catheter, and safe home anti-cancer chemotherapy could be continued because 5-FU and CDDP did not cause severe side effects.

Emergence of human immunodeficiency virus type 1 variants with resistance to multiple dideoxynucleosides in patients receiving therapy with dideoxynucleosides.

A set of mutations [Ala-62-->Val(A62V), V75I, F77L, F116Y, and Q151M] in the polymerase domain of reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) confers on the virus a reduced sensitivity to multiple antiretroviral dideoxynucleosides and has been seen in HIV-1 variants isolated from patients receiving combination chemotherapy with 3'-azido-3'-deoxythymidine (AZT) plus 2',3'-dideoxycytidine (ddC) or 2',3'-dideoxyinosine (ddI). The IC50 values of AZT, ddC, ddI, 2',3'-dideoxyguanosine, and 2',3'-didehydro-3'-deoxythymidine against an infectious clone constructed to include the five mutations were significantly higher than those of a wild-type infectious clone. The K1 value for AZT 5'-triphosphate determined for the virus-associated RT from a posttherapy strain was 35-fold higher than that of RT from a pretherapy strain. Detailed analysis of HIV-1 strains isolated at various times during therapy showed that the Q151M mutation developed first in vivo, at the time when the viremia level suddenly increased, followed by the F116Y and F77L mutations. All five mutations ultimately developed, and the viremia level rose even further. Analyses based on the three-dimensional structure of HIV-1 RT suggest that the positions where at least several of the five mutations occur are located in close proximity to the proposed dNTP-binding site of RT and the first nucleotide position of the single-stranded template.

Changes in folate concentration in Yoshida sarcoma after administration of leucovorin or cisplatin.

Both leucovorin (LV) and cisplatin (cis-dichlorodiammine platinum II, CDDP) act as modulators of 5-fluorouracil (5-FUra) by increasing the intracellular concentration of reduced folate. We measured intracellular folate levels following the administration of LV or cisplatin in tumor-bearing rats to determine the optimal schedules for their use as 5-FUra modulators. Donryu rats were inoculated with Yoshida sarcoma cells on the right flank. Seven days after tumor inoculation, the animals were injected with LV or CDDP. The kinetic and dose-related changes in intracellular folate concentration were analyzed by means of a binding assay. Folate levels in the tumor tissues were significantly higher than baseline 1 and 2 h after administration of LV and remained significantly high until 8 h after administration. Folate levels in the tumor tissues were significantly higher than baseline 1 and 2 h after cisplatin administration, then decreased to a rather low level 8 h after, and to a significantly lower level than baseline 24 h after administration. The folate levels in the tumor tissue increased in proportion to the dose of LV, but did not increase when the dose of cisplatin was increased from 1 mg/kg to 8 mg/kg. Repeat high-dose administration of LV and repeat low-dose administration of cisplatin are advocated when they are used as modulators of 5-FUra.

[The clinical effect of chemotherapy with 5-FU and low-dose CDDP in patients with advanced gastric cancer].

A trial of FP therapy, a novel systemic chemotherapy consisting of 5-FU and low-dose CDDP, was carried out in patients with advanced gastric cancer and the clinical effects were evaluated. Five hundred mg/body/day of 5-FU was continuously administered via the central venous catheter for 7 consecutive days and 10mg/day of CDDP was rapidly administered with 500ml saline on days 1-5. The regimen was repeated for at least 4 weeks. The FP therapy was carried out in 22 cases, including several patients who underwent FP as an adjuvant therapy; 1 CR, 2PR's 1MR and 2NC's were obtained among the 6 patients with clinically evaluable lesions, resulting in a response rate of 50%. In four cases, the FP therapy was given as a neoadjuvant therapy, and histopathological examination of the resected specimen revealed histological effects exceeding Grade 2 in all 4 cases. Bone marrow suppression was the most commonly observed side effect, but renal dysfunction and nausea were not found. FP therapy is considered an effective therapy against advanced gastric cancer, from the viewpoint of both the clinical and adverse effects, in comparison with conventional regimen consisting of 5-FU and a single and large dose of CDDP.

High-level resistance to zidovudine but not to zalcitabine or didanosine in human immunodeficiency virus from children receiving antiretroviral therapy.

Human immunodeficiency virus type 1 (HIV-1) isolates from children receiving long-term therapy with an alternating regimen of zidovudine and zalcitabine, or with didanosine monotherapy, were evaluated for resistance to zidovudine, zalcitabine, and didanosine, and for mutations known to be associated with zidovudine or didanosine resistance. HIV-1 from four of six patients receiving zidovudine with zalcitabine developed high-level resistance to zidovudine. A mutation in the HIV-1 reverse transcriptase that is highly associated with zidovudine resistance was identified in all four zidovudine-resistant posttherapy isolates. In contrast, none of the HIV-1 isolates from the seven patients receiving didanosine developed high-level resistance to this agent, despite the identification of a didanosine-associated mutation in six of these posttherapy isolates, although small decreases in sensitivity to didanosine were observed. These results indicate that nucleoside analog-associated mutations in HIV-1 occur frequently in children receiving long-term antiretroviral therapy and that alternating combination therapy does not prevent the development of resistance to zidovudine. They also suggest that there may be differences in the degree of resistance conferred by mutations that result from therapy with different nucleoside analogs. These findings underscore the need for studies to define the clinical importance of these mutations, and for treatment strategies to overcome the emergence of viral resistance in vivo.

Changes in drug sensitivity of human immunodeficiency virus type 1 during therapy with azidothymidine, dideoxycytidine, and dideoxyinosine: an in vitro comparative study.

Human immunodeficiency virus type 1 (HIV-1) strains were isolated from nine patients before and after prolonged therapy with either an alternating regimen of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC) (AZT/ddC) or 2',3'-dideoxyinosine (ddI) alone. All strains obtained from four patients who received AZT/ddC for up to 41 mo were highly insensitive to AZT in vitro. Only one strain obtained after AZT/ddC therapy showed reduced susceptibility to ddC in addition to AZT and had previously unreported amino acid substitutions in the viral polymerase-encoding pol region, whereas three other strains had one or more of the five previously reported AZT-related mutations. In five HIV-1 strains from patients who received ddI for up to 29 mo, no appreciable decrease in sensitivity to ddI was detected. Two strains isolated after ddI therapy had no significant amino acid mutations, although three strains had a mutation reportedly associated with ddI administration. These data suggest that HIV-1 develops reduced susceptibility to AZT more readily than to ddC and ddI and/or that the reduced susceptibility to ddC and ddI is modest in degree. Moreover, the present data suggest that an alternating regimen of AZT and ddC does not block the emergence of AZT-insensitive variants. It should be noted, however, that the current results do not provide a basis for concluding that AZT/ddC or ddI is inferior, equivalent, or superior to AZT as therapy of AIDS.