Real-time observation of spherical aberration-free phase image using high-speed image processing CCD video camera.

Spherical aberration-free phase images have been observed at a time resolution of 1/30 s for the first time by using a defocus-image modulation processing electron microscope (DIMP-EM) combined with a newly developed high-speed image processing CCD video camera. In a profile image of the Au (011) crystal surface, no extra interference fringes were observed outside the crystal region, which clearly demonstrates successful correction of spherical aberration in real-time. The novel real-time DIMP-EM enabled the dynamic observation of surface and interface phenomena at an atomic level.

Role of desethylamiodarone in the anticoagulant effect of concurrent amiodarone and warfarin therapy.

BACKGROUND: The concurrent use of amiodarone and warfarin inhibits metabolism of S-warfarinby cytochrome P450 (CYP) 2C9, thereby increasing the anticoagulant effect of warfarin. Amiodarone primarily inhibits CYP1A2 and CYP3A4, and desethylamiodarone primarily inhibits CYP2C9. We investigate whether a relationship exists between the plasma concentration of desethylamiodarone and anticoagulation when amiodarone is administered to patients receiving warfarin therapy. METHODS AND RESULTS: The correlation between the plasma concentration of either amiodarone or desethylamiodarone, and prolongation of prothrombin time-international normalized ratio/dose of warfarin (Delta INR/Dose) on day 7 of amiodarone administration was studied in 25 patients (22-74 years old) with structural heart disease and refractory arrhythmias receiving stable warfarin therapy. RESULTS: No correlation was found between the plasma concentration of amiodarone and Delta INR/Dose, but a correlation was found between the plasma concentration of desethylamiodarone and Delta INR/Dose. CONCLUSIONS: It was suggested that inhibition of CYP2C9 by desethylamiodarone, the active metabolite of amiodarone, plays an important role in the interaction of warfarin and amiodarone.

Effects of antitumor agents on subcutaneous implants and hepatic metastases of colon carcinoma 26 in mice.

We investigated the responses of experimentally produced hepatic metastases of colon carcinoma 26 tumor and subcutaneously (SC) implanted colon carcinoma 26 tumor in mice to 17 clinically-used and one under-development antitumor agents using same dose regimen. In intravenous administrations on days 7 and 14, there were no significant differences in their responses to most of the tested agents. However, there were big differences in the responses to some of the agents. Nimustine more effectively prolonged the lifespan of SC implanted tumor-bearing mice than of mice bearing hepatic metastases. Mitomycin C was, however, considerably more effective on hepatic metastases than on SC implanted tumor. ME2303, a new fluorinated anthracycline derivative, showed a similar effect to doxorubicin on both tumors. However, administrations of ME2303 on days 7, 11 and 15 showed more marked antitumor effect only on hepatic metastases than administrations on days 7 and 14. Doxorubicin was less active against both tumors for administrations on days 7, 11 and 15 than for those on days 7 and 14. These results suggest the importance of the site of tumor growth for the action of some drugs. ME2303 may be active against hepatic metastases if it is administered by multiple injections.

In vivo antitumor effects of fluoropyrimidines on colon adenocarcinoma 38 and enhancement by leucovorin.

Antitumor effect and active metabolites of fluoropyrimidines were examined in mice with transplantable colon adenocarcinoma 38 (Co 38). 5-Fluoro-2'-deoxyuridine (FUdR) treatment resulted in a much higher level of free 5-fluoro-2'-deoxyuridine-5'-monophosphate in the tumor than 5-fluorouracil (5-FU) did, and thymidylate synthase was almost completely inhibited after FUdR treatment, but FUdR showed weaker antitumor activity than 5-FU did. Moreover, 5-fluorouridine (FUR) also hardly inhibited tumor growth. A more marked tumor inhibition was obtained when FUdR and FUR were administered together. The antitumor activity of 5-FU was similar to that of the combination of FUdR and FUR. In combination with 2,2'-anhydro-5-ethyluridine, a uridine phosphorylase inhibitor, FUdR lost its antitumor activity, but that of FUR was somewhat potentiated. On the other hand, in combination with leucovorin (LV), 5-FU showed markedly potentiated antitumor activity, while the antitumor activity of FUdR or FUR was not potentiated. Addition of LV to the combination of FUdR and FUR enhanced the inhibitory effect of the drugs. From these results, the combination of FUdR and FUR together with LV, and the combination of 5-FU and LV seem to be highly efficacious against Co 38.

Effects of anthracycline derivatives on hepatic neoplastic nodules of Lewis lung carcinoma and colon adenocarcinoma 26.

Five anthracycline derivatives, i.e. doxorubicin, epirubicin, pirarubicin, aclarubicin and a new fluorinated anthracycline derivative (ME2303), were tested for antitumour activity in mice with hepatic neoplastic nodules of Lewis lung carcinoma and colon adenocarcinoma 26. Intravenous administrations of pirarubicin and ME2303 on day 4 or days 4, 8 and 12 in mice with hepatic neoplastic nodules of Lewis lung carcinoma rendered more than 50% of mice tumour-free over wide ranges of nontoxic doses, whereas a few mice were cured by treatment with doxorubicin and no mice were cured by treatment with epirubicin or aclarubicin. Moreover, when ME2303 was administered at 50 mg kg-1 on days 7, 11 and 15 to six mice bearing more advanced hepatic tumours, five were cured, while pirarubicin and doxorubicin never achieved cure. Furthermore, in mice with hepatic neoplastic nodules of colon adenocarcinoma 26, ME2303 also showed a marked antitumour effect compared to pirarubicin or doxorubicin. Two or three injections of ME2303 starting from day 7 conferred a greater antitumour effect than did more fractionated or single-dose regimens.

Activation of quiescent ovaries by administration of PMSG after LH-RH analogue treatment in heifers.

The effect of pregnant mare serum gonadotrophin (PMSG) treatment on activation of quiescent ovaries was examined in heifers. Groups of thirteen, twenty and twelve heifers which showed ovulation within 2 d and corpus luteum (CL) development after injection with a luteinizing hormone releasing hormone analogue (LH-RH-A) were supplementally injected with 500 IU of PMSG (Group I); 500 IU of PMSG and 500 mug of Prostaglandin F(2alpha) analogue (PGF(2alpha)-A; Group II); and 500 mug of PGF(2alpha)-A (Group III) on Day 6 after the injection of 200 mug of LH-RH-A (Day 0), respectively. Estrus appeared in 33.3 to 45.0% of the heifers of the respective groups after the treatment. Ovulation occurred at a significantly (P<0.01) higher rate in Groups I (100%) and II (90.0%) than in Group III (41.7%). The ovarian cyclic activity was initiated in all the heifers that ovulated. Plasma progesterone levels decreased significantly (P<0.05) to about 1 ng/ml on Day 8 and Day 7 in Group I and Groups II and III, respectively. Plasma estradiol-17beta (E(z)) levels increased significantly (P<0.05), reaching a peak on Days 7 to 7.5 in Groups I and II but not in Group III. It is concluded that PMSG treatment stimulates maturation and E(z) secretion of a follicle, thus promoting ovulation and the onset of ovarian cyclic activity.

Therapeutic activity and tissue distribution of ME2303, a new anthracycline containing fluorine, and its metabolites in mice bearing hepatic metastases of Lewis lung carcinoma.

Doxorubicin (DXR) and ME2303, a new fluorine-containing (C'-2) anthracycline derivative, were studied for their tissue distributions--particularly in the plasma, liver and bone marrow--following administration at the maximum tolerated doses to normal mice and mice bearing hepatic metastases of Lewis lung carcinoma. ME2303 was rapidly metabolized and disappeared rapidly from the plasma, liver and bone marrow. Its metabolites--the product of esterolysis (M1) and its reduced derivative at the C-14 position (M2)--remained for a long period except in bone marrow. On the other hand DXR remained in the analyzed tissues for a long period; an especially large amount of DXR was found in the bone marrow even at 24 h after administration of the drug while, in the case of ME2303, by this time even its metabolites had disappeared. The concentrations of M1 and DXR in the liver at 2 h were about 50- and 300-fold higher than their plasma concentrations. The tissue distributions in the normal mice and hepatic-metastases-bearing mice showed no significant differences. Regarding the antitumor effects of ME2303, M1, M2 and DXR in the hepatic-metastases-bearing mice, ME2303 was the most effective compound, and M1 was also active; DXR showed only a marginal effect, and M2 showed no effect.

Effect of recombinant interleukin-1 alpha, recombinant interleukin-2, recombinant interferon-beta, and recombinant tumor necrosis factor on subcutaneously implanted adenocarcinoma 755 and Lewis lung carcinoma.

The antitumor activity of recombinant human interleukin-1 alpha (rHIL-1 alpha), recombinant human tumor necrosis factor, and recombinant murine interferon-beta (rIFN-beta) was augmented by concomitant administration of recombinant human interleukin-2 (rHIL-2) in the treatment of adenocarcinoma 755. Especially when a divided dose (two doses/day) of rHIL-1 alpha or rIFN-beta was combined with a divided dose of rHIL-2, the antitumor effect was markedly potentiated. However, only a marginal effect was seen by combination of rHIL-1 alpha and/or rIFN-beta and rHIL-2 against Lewis lung carcinoma, a tumor that is resistant to cytokines.

Differential effects of 2,2'-anhydro-5-ethyluridine, a uridine phosphorylase inhibitor, on the antitumor activity of 5-fluorouridine and 5-fluoro-2'-deoxyuridine.

2,2'-Anhydro-5-ethyluridine (ANEUR), a potent inhibitor of uridine phosphorylase, markedly potentiated the antitumor activity of fluorouridine (FUR) against murine mammary adenocarcinoma 755 in BDF1 mice and human colon adenocarcinoma LS174T in athymic-nude mice. Whereas ANEUR annihilated the antitumor activity of 5-fluoro-2'-deoxyuridine (FUdR) and 5'-deoxy-5-fluorouridine (DFUR) in the murine adenocarcinoma 755 system, it did not alter the antitumor activity of FUdR in the human adenocarcinoma LS174T system. In vitro, ANEUR proved inhibitory to the phosphorolytic cleavage of both FUR and FUdR by uridine phosphorylase, and this could explain why in vivo conversion of FUR and FUdR to 5-fluorouracil was suppressed. FUR can be held directly responsible for the antitumor effects observed in the murine adenocarcinoma 755 system, whereas in the activity against human adenocarcinoma LS174T may be mediated by both FUR and FUdR.

Effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine on life-span and 5-fluorouracil metabolism in mice with hepatic metastases.

5-Fluorouracil (5FU) is rapidly metabolised in the liver by dihydrouracil dehydrogenase. Bromovinyluracil is formed in the liver from (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) by pyrimidine nucleoside phosphorylase and is a potent inhibitor of dihydrouracil dehydrogenase. The co-administration of 5FU (intravenously) and BVDU (orally) was investigated in normal BDF1 mice and in those bearing liver metastases of Lewis lung carcinoma. 5FU alone rapidly disappeared from plasma and liver within 60 min of dosing. Administered with BVDU, 5FU persisted in plasma and liver for 60-180 min. The combination also significantly enhanced the life-span of tumour-bearing mice. 5FU plus BVDU may have therapeutic potential in the treatment of primary and secondary liver tumours.

Effects of interleukin-2 and interferon-beta treatment on lymphocytes in various tumor-bearing mice.

Treatment with a combination of recombinant human interleukin-2 (rHIL-2) and recombinant mouse interferon-beta (rIFN-beta) had a significant antitumor effect against subcutaneous (s.c.) adenocarcinoma-755 and colon-38, but this combination performed no better than rHIL-2 alone against s.c. Lewis lung carcinoma in C57BL/6 mice. Injecting a combination of rHIL-2 and rIFN-beta into mice with adenocarcinoma-755 or colon-38 tumors resulted in a marked increase in L3T4+, Lyt-2+ and asialo GM1+ cells in the peritoneal cavity. On the other hand, the treatment of mice with Lewis lung carcinoma with rHIL-2 and rIFN-beta produced almost no change of each subset in the peritoneal cavity compared to cytokine alone. Thus, sensitive tumors (adenocarcinoma-755 and colon-38) in combined treatment with rHIL-2 and rIFN-beta markedly increased L3T4+, Lyt-2+ and asialo GM1+ cells in the peritoneal cavity, but the insensitive tumor (Lewis lung carcinoma) did not.

Enhancing effect of bromovinyldeoxyuridine on antitumor activity of 5'-deoxy-5-fluorouridine against adenocarcinoma 755 in mice. Correlation with pharmacokinetics of plasma 5-fluorouracil levels.

5'-Deoxy-5-fluorouridine (DFUR), whether or not combined with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was pursued in BDF1 mice from both a pharmacokinetic viewpoint, following a single oral dose administration, and an anticancer viewpoint, following 5 daily oral doses in mice inoculated subcutaneously with adenocarcinoma 755 tumor cells. Half-life (t1/2) values for the elimination of DFUR and 5-fluorouracil (5-FU) from plasma following DFUR (100 mg/kg) administration were about 0.80 and 0.39 hr, respectively. Plasma 5-FU AUC (area under the curve) values following oral DFUR (100 mg/kg) was 0.224 micrograms.hr/ml. If DFUR (100 mg/kg) was combined with BVDU (10 mg/kg) the t1/2 and AUC values for 5-FU increased from 0.39 to 1.24 hr, and from 0.224 to 1.699 micrograms.hr/ml, respectively. Thus, BVDU significantly increased the plasma levels of 5-FU. It had no effect on the plasma levels of DFUR. At 100 mg/kg, DFUR did not show a significant antitumor activity. At 500 mg/kg it effected a 90% inhibition in tumor growth. When combined with BVDU (10 mg/kg), DFUR at 100, 200 and 300 mg/kg reduced tumor growth by 96, 100 and 100%, respectively. The antitumor activity achieved by DFUR, in the presence or absence of BVDU, correlated highly significantly with the AUC values for plasma 5-FU.

Changes in lymphocyte subsets following multiple administration of recombinant interleukin-2 plus recombinant interferon-beta or -gamma in tumor-bearing mice.

Treatment with a combination of recombinant human interleukin-2 (rHIL-2) and recombinant mouse interferon-beta (rIFN-beta) or -gamma (rIFN-gamma) showed a significant antitumor effect against sc adenocarcinoma 755 in mice, although treatment with either one alone had almost no effect. The combination of rHIL-2 and rIFN-beta caused regression of the tumor but the combination of rHIL-2 and rIFN-gamma did not. Injection of tumor-bearing mice with the combinations of rHIL-2 and rIFN resulted in marked increases in the total number of peritoneal lymphocytes, and the frequency of Lyt-2+ cells was more markedly increased by the combination of rHIL-2 and rIFN-beta than by the combination of rHIL-2 and rIFN-gamma. In Winn assay, elimination of the Lyt-2+ population abolished the protective capacity of the peritoneal cells. The subsets of thymocytes were drastically changed when mice were bearing a tumor or were treated with cytokines. In particular, Lyt-2+/L3T4+ cells were decreased in tumor-bearing mice, but many Lyt-2+/L3T4+ cells were maintained in the thymus by treatment with a cytokine alone. When treated with rHIL-2 and rIFN-beta, the Lyt-2+/L3T4+ cells were markedly decreased, while Lyt-2+/L3T4- T-cells were increased, but these subsets were little changed by treatment with rHIL-2 plus rIFN-gamma. Thus, injections of rHIL-2 and rIFN-beta into tumor-bearing mice resulted in a high frequency of Lyt-2+/L3T4- cells in the peritoneal cavity, together with changes in the T-cell subsets in the thymus. These results suggest that maturation of T-cells in the thymus may be an important step in the pathway by which cytokine treatment brings about regression of tumors.

Effects of interleukin-2 and interferon-alpha A/D treatment on lymphocytes from tumour-bearing mice.

The in vivo antitumour activities of recombinant human interleukin-2 (rHIL-2) and recombinant human hybrid interferon alpha A/D (rIFN-alpha A/D) were tested in relation to adenocarcinoma 755. The tumour growth, following s.c. inoculation of tumour cells, was inhibited to a greater extent in mice treated with the combination of cytokines than in mice treated with either one alone. Pretreatment with these cytokines did not affect the tumour growth. Injection of tumour-bearing mice with a combination of these cytokines resulted in a marked increase in the total number of lymphocytes in the peritoneal cavity. Among them, Lyt-2+/L3T4- and asialo GM1+ cells were markedly enhanced by the combination of cytokines, and the frequencies of these marker cells were closely correlated with the antitumour activity. In tumour-bearing mice, the size of the thymus was decreased while that of the spleen was increased compared to non-tumour-bearing (normal) mice. Treatment with rHIL-2 caused the thymus, spleen and liver to be larger compared to untreated tumour-bearing mice, but when treated with a combination of rHIL-2 and rIFN-alpha A/D these organs were smaller than when rHIL-2 was administered alone. Thymocytes were drastically changed when mice were bearing a tumour or were treated with a cytokine. Especially immature T-cells, Lyt-2+/L3T4+, were drastically decreased in tumour-bearing mice, but were maintained following administration of rHIL-2 or rIFN-alpha A/D. When treated with rHIL-2 plus rIFN-alpha A/D, Lyt-2+/L3T4+ T-cells were decreased while Lyt-2+/L3T4- T-cells were increased. Frequency of immature T-cells, Lyt-2-/L3T4-, was not changed. On the other hand, T-cell subsets of splenocytes were markedly decreased in tumour-bearing mice compared to normal mice, but all the subsets of splenocytes were almost unchanged even when tumour-bearing mice were treated with rHIL-2 plus rIFN-alpha A/D. Thus, injection of rHIL-2 and rIFN-alpha A/D to tumour-bearing mice resulted in induction of Lyt-2+/L3T4- and asialo GM1+ cells in the peritoneal cavity, and the frequencies correlated with the observed antitumour activity in vivo in this murine model. The increase in Lyt-2+/L3T4- T-cells in the peritoneal cavity may be related to changes in the T-cells in thymus.

Enhanced antitumor effect of combination therapy with interleukin-2 and polyribonucleotides against adenocarcinoma 755.

Human recombinant interleukin-2 (IL-2) and interferon (IFN) inducers in combination were evaluated for their in vivo antitumor efficacy in relation to s.c.-implanted adenocarcinoma 755 in C57BL/6 mice. Two IFN inducers, polyinosinic-polycytidylic acid [poly(I)poly(C)] and polyadenylic-polyuridylic acid [poly(A)poly(U)], induced high IFN production in various tissues for a long time compared to treatment with murine interferon-beta. Especially, poly(I)poly(C) at 5 mg/kg, the maximum tolerated dose, produced the highest level of IFN in the tumor, but the tumor did not show regression. Poly(I)poly(C), however, brought about marked regression of the tumor when administered together with IL-2. This combination resulted in cure of some mice when both drugs were administered intraperitoneally. Intraperitoneal injection of IL-2 in combination with poly(I)poly(C) was more effective than intravenous injection of IL-2. The combination of IL-2 and poly(A)poly(U) also showed an enhanced antitumor effect. Thus, endogenous IFN produced by IFN inducers as well as exogenous IFN as reported previously potentiated the antitumor effect when administered together with IL-2. The degree of potentiation by combination of IL-2 and IFN inducers may depend on the level of IL-2 and IFN at the injection site.