Inhibition of topoisomerases by fatty acids.

The inhibitory effects of various fatty acids on topoisomerases were examined, and their structure activity relationships and mechanism of action were studied. Saturated fatty acids (C6:0 to C22:0) did not inhibit topoisomerase I, but cis-unsaturated fatty acids (C16:1 to C22:1) with one double bond showed strong inhibition of the enzyme. The inhibitory potency depended on the carbon chain length and the position of the double bond in the fatty acid molecule. The trans-isomer, methyl ester and hydroxyl derivative of oleic acid had no or little inhibitory effect on topoisomerases I and II. Among the compounds studied petroselinic acid and vaccenic acid (C18:1) with a cis-double bond were the potent inhibitors. Petroselinic acid was a topoisomerase inhibitor of the cleavable complex-nonforming type and acted directly on the enzyme molecule in a noncompetitive manner without DNA intercalation.

[Comparison of the families of terminal stage cancer patients who underwent HPN and the nurses who cared those patients in their thoughts on home care].

In the home care for terminal stage cancer patients, the quality of life of patients has been regarded as more important than the QOL of their family. Now, however, we think the QOL of the family of the patients is as important as that of the patients themselves. We investigated the opinion of the families and nurses by questionnaire in the HPN care for terminal stage cancer patients and compared them. The family answered positively about HPN, but they had a negative opinion in that they do not want to undergo HPN because they felt a heavy burden and responsibility in caring for patients, and they do not want to cause these feelings in others in their family. The nurses also answered positively about HPN, and they answered that they want to undergo HPN if they have terminal stage cancer. These results suggest that the family feels a greater burden and responsibility in caring for patients at home than the medical staff realize. The medical staff should support the family as well as terminal stage cancer patients and should not force our own opinions as medical staff.

[How the lifestyle of families who care for home parenteral nutrition patients changes].

The role of families who care for home parenteral nutrition (HPN) patients is extremely important. They face many problems not only in caring for patients but also in their own lifestyle. We investigated by questionnaire the changes in the family of terminal stage cancer patients receiving HPN. Replies submitted to the questionnaire were obtained from 20 out of 32 families who underwent HPN in the Osaka Prefectural General Hospital. Ten out of 20 people answered that they changed their lifestyle during HPN. The changes were decreased opportunity to communicate with other people and decreased time for to spend on a hobby or other activity. The care of the HPN patients affects the wife, daughter-in-law and children more than the husband of the patient, because the former have to manage housekeeping as well as patient care. This result suggests that the quality of life of the family of terminal stage cancer patients at home with HPN declined because they had to change their own lifestyle in order to spare time to care for the patients.

Stimulatory effect of enkephalins on calcium efflux from bovine adrenal chromaffin cells in culture.

The effects of leucine- and methionine-enkephalin, opiate peptides, on Ca2+ efflux from cultured bovine adrenal chromaffin cells were examined. These enkephalins stimulated the efflux of 45Ca2+ from cells in a concentration-dependent manner (10(-8) M-10(-6) M). Leucine-enkephalin did not increase the intracellular free Ca2+ level, 45Ca2+ uptake, catecholamine secretion, cAMP level or cGMP level. The peptide-stimulated 45Ca2+ efflux was not inhibited by incubation in Ca2+-free medium, but was inhibited by incubation in Na+-free medium. These results indicate that enkephalins stimulate extracellular Na+-dependent 45Ca2+ efflux from cultured bovine adrenal chromaffin cells, probably by stimulating membrane Na+/Ca2+ exchange.

[Evaluation of dissolution behavior for the products containing tegafur and uracil].

Generic drugs are widely used with the object of cost saving in many Japanese therapeutic scenes now. The products containing the same active ingredient(s), even if they are innovative drugs or generic ones, must be designed to possess the equivalent quality. In this report, we observed the dissolution behavior patterns of three generic drugs that contain Tegafur and Uracil, drugs A, B, and C, and compared them with that of an innovative product, UFT. Drugs B and C were similar to UFT in the dissolution rate of Tegafur, but drug A was not. On the dissolution rate of Uracil, all the generic products, drugs A, B and C, did not amount to the level equivalent to that of UFT. Therefore, these generic products did not indicate the same dissolution behavior pattern as UFT. It was suggested that the pharmaceutical technology used in the manufacture was not equivalent even if the products of the same dosage form contain the same kind and content of the active ingredient(s).

Inhibition of topoisomerases by antitumor prostaglandins.

Topoisomerase II from human placenta was strongly inhibited by prostaglandins such as delta 12,14-PGJ2, delta 12-PGJ2, PGA2, and PGA1, which have an antitumor activity. The topoisomerase II inhibitions by these prostaglandins were dose-dependent and IC50 values of delta 12,14-PGJ2, delta 12-PGJ2, PGA2, and PGA1 were 22 microM, 74 microM, 75 microM, and 98 microM, respectively. Topoisomerase I from calf thymus gland was inhibited by delta 12,14-PGJ2, delta 12-PGJ2, and PGA2, but not inhibited by other prostaglandins even at high concentrations. Only the prostaglandins having the antitumor activity inhibited topoisomerase II. The results suggest that the antitumor activity of prostaglandins may be related, at least in part, to inhibition of topoisomerase II in tumor cells.

[Current status and problems of home parenteral nutrition for terminal cancer patients from the viewpoint of indication].

Since June 1993, we have been performing home parenteral nutrition (HPN) for end-stage cancer patients. We studied how many patients could be treated with HPN, why they were not able to be treated with HPN in the terminal stage cancer patients, 158 cases, who admitted and died in our ward from June 1993 to April 1997. Eighty-six patients (54.4%) were considered to not be indicated for HPN, due to general weakness (28 cases), dyspnea (17 cases), need for medical care other than HPN (34 cases), and inability to understand this treatment (1 case). Fifty-six patients (35.4%) were considered to have been able to be treated with HPN and could have had returned home. Fifteen patients (26.8%) were given HPN. Four cases wanted to stay in the hospital after they knew the truth and the prognosis of their disease. Another four patients became weak while preparing for HPN and could not go home. Eighty percent of patients could not to be treated with HPN because of the factors of their family. Eight patients had no family to cared for them. Twelve patients were refused to the truth of their disease. The families of 13 cases refused to accept and care for them in their home. We may conclude that co-operation among patients, their family and the medical staff is the most important factor in providing HPN for end-stage cancer patients.

Research to develop a predicting system of mammalian subacute toxicity, (3). Construction of a predictive toxicokinetics model.

A new predictive toxicokinetics model was developed to estimate subacute toxicity (target organs, severity, etc.) of non-congeneric industrial chemicals, where the chemical structures and physico-chemical properties are only available. Thus, a physiological pharmacokinetics model, which consists of blood, liver, kidney (these were experimentally found as major toxicological targets), muscle and fat compartments, was established to simulate the chemical concentrations in organs/tissues with pharmacokinetic parameters by means of Runge-Kutta-Gill algorithm. The pharmacokinetic parameters, i.e. absorption rate, absorption ratio, hepatic extraction ratio of metabolism and renal clearance were calculated by using separately established Quantitative Structure-Pharmacokinetics Relationship equations. The developed predictive model was then applied to simulations of 43 non-congeneric industrial chemicals. The chemical concentrations in organs/tissues after single oral administration were simulated, and their maximum concentrations (Cmax's) and area under the concentration-time curves (AUC's) were calculated. Fast Inverse Laplace Transform was newly applied for the purpose of simulation of 28-day repeated dose toxicity. Simulated concentrations of 28 days repeated dose were, however, found to be the same as those of simple repetitions of a single administration per day because of the short half-lives of non-congeneric industrial chemicals. A comparison of subacute toxicity data with Cmax's and AUC's in a single dose scenario suggested that the organs/tissues with relatively high concentrations of tested chemical substances were the most sensitive targets within a chemical. Chemical concentrations in liver, for instance, were correlated with the severity of hepatotoxicity among the chemicals. It was also suggested that to improve and widen the present approach, data of metabolite and reactivity of non-congeneric industrial chemicals to organs/tissues, receptors, etc. should be incorporated into the model.

A research to develop a predicting system of mammalian subacute toxicity. I. Prediction of subacute toxicity using the biological parameters of acute toxicities.

Predicting equations of subacute toxicity were developed by analyzing rat acute and subacute toxicity data of 56 chemicals of various structures. Minimum or 10% effect level in acute or subacute toxicity was estimated as a "biological parameter". Good regression equations were established between the geometrical means ("combined parameters") of any two of the parameters of acute and subacute toxicities and introduction of log P to the equations improved the correlations with a statistically significant multiple regression coefficient. The lowest predicted effect level of the subacute toxicity, which is selected from the data calculated by the above several correlations, can predict the upper limit of the no observed effect level. In recent years, for research and development of new chemical substances, it becomes one of the important factors that these have a lower environmental load in nature. Eventually, it becomes essential to evaluate not only their acute effects on human or environments, but also their chronic influences when they are to be exposed for a long period of time, and the cost for such verification is becoming a breaking factor for research and development. Thus, the development of a new technique which estimates the environmental load of a chemical substance including toxic effects on human with lower cost is now being attempted. For example, the development of in vitro new alternative methods using cultured cells, the utilization of a data base or software which relates mammalian and environmental toxicities and so on are internationally carried forwards. As for the latter, quantitative structure-activity relationship (QSAR) techniques have been applied practically to decide appropriate toxicity tests needed for regulatory purposes by EPA/TSCA, ITC/TSCA and FDA/FDAA in the USA. In addition, it was concluded recently in a joint meeting of EU and US-EPA that the approach by QSAR techniques was useful to specify the new chemical substances which are to be required toxicological examinations. In these QSAR techniques, however, while there are some considerations about common mechanisms of toxicity among chemicals with a similar structure (Congeneric chemicals, Congeners) for establishing of correlation formulas, for chemicals with various structures (Non-congeneric chemicals, Non-congeners) there often lack such common considerations. In addition, biological or physiological factors which are basic toxic indices are often ignored. In a previous study, we researched acute and subacute oral toxicities of industrial common chemicals in rats and reported the followings; 1) their subacute toxicological spectrum in target organs/tissues and morphologic changes was very limited and specific, 2) the important targets were liver, kidneys, blood (spleen) and stomach and these are considered the sites of dominant exposure due to the kinetics of chemical substances, 3) the morphological changes were hepatocellular hypertrophy, deposition of substance in renal tubules, extramedullary hematopoiesis in spleen and mucous lesion in stomach, and these are implying adaptation such as induction of drug-metabolizing enzymes, overload to renal function, anemia from erythrocyte destruction, and direct reaction, respectively, 4) there seems to occur a series of direct and adaptive reactions to exclude the "foreign compounds" which do not show any specific biological activities, 5) it is also considered that there is a possibility to establish a correlation between toxicological findings or target organs/tissues of both acute and subacute toxicities by their continuity. Therefore, in the present study, a predicting equation of subacute (28-day repeated dose) toxicity is attempted to develop from acute (single dose) toxicity data by considering both common mechanisms and biological factors for non-congeneric industrial chemical substances.

A research to develop a predicting system of mammalian subacute toxicity. II. Single dose detailed toxicity studies.

"Single dose detailed toxicity study" in rats was conducted with 6 chemical substances to establish a predicting system of subacute toxicity of non-congeneric industrial chemicals. In the detailed examinations, new and previously reported biological parameters were developed and utilized for the establishment and confirmation of the correlations for both qualitative and quantitative prediction of target organs and effects in repeated dose toxicity studies of non-congeners; the new parameters were concerned with liver, kidneys and blood and the established relating equations for prediction had a correlation coefficient of 0.83 (liver), 0.49 (kidneys) and 0.94 (blood), while the old parameters effectively confirmed the previous correlations. The serial changes in the study gave a biological explanation to the established relationships between acute and subacute toxicities in terms of extrapolation.

Immunoaffinity purification of 11-dehydro-thromboxane B2 from human urine and plasma for quantitative analysis by radioimmunoassay.

11-Dehydro-thromboxane B2 is now considered to be a reliable parameter of thromboxane A2 formation in vivo. An immunoaffinity purification method was developed for radioimmunoassay of this compound contained in human urine and plasma. Monoclonal anti-11-dehydro-thromboxane B2 antibody was prepared and coupled to BrCN-activated Sepharose 4B. Human urine or plasma was applied to a disposable column of the immobilized antibody. After the column was washed with water, 11-dehydro-thromboxane B2 was eluted with methanol/water (95/5) with a recovery of more than 90%. The purified extract was subjected to a radioimmunoassay utilizing 11-[3H]dehydro-thromboxane B2 methyl ester and the monoclonal anti-11-dehydro-thromboxane B2 antibody. The detection range of the assay was 10-600 fmol (IC50 = 90 fmol). The cross-reactivities of the antibody with thromboxane B2, 2,3-dinor-thromboxane B2, and other arachidonate metabolites were less than 0.05%. These compounds were efficiently separated from 11-dehydro-thromboxane B2 by the immunoaffinity purification. This procedure also allowed the separation of 11-dehydro-thromboxane B2 from unidentified urinary and plasma substances which interfered with the radioimmunoassay. Validity of the results obtained by the radioimmunoassay was confirmed by GC/MS employing selected ion monitoring for quantification.

A heterologous enzyme immunoassay of prostaglandin E2 using a stable enzyme-labeled hapten mimic.

A sensitive heterologous enzyme immunoassay for prostaglandin E2 was developed using 9-deoxy-9-methylene-prostaglandin F2 alpha as a stable prostaglandin E2 mimic. beta-Galactosidase was conjugated to the hapten mimic. Anti-prostaglandin E2 IgG was bound to a polystyrene tube. The enzyme-labeled hapten mimic mixed with unlabeled prostaglandin E2 was allowed to react in a competitive manner with the immobilized antibody. Then, the beta-galactosidase specifically bound to the antibody was assayed fluorometrically, and the enzyme activity was correlated with the amount of unlabeled prostaglandin E2. According to the calibration curve thus obtained, prostaglandin E2 could be determined in a range of 1.2-430 fmol. Prostaglandin E2 was extracted from human urine by the use of an octadecylsilyl silica column. The crude extract contained a substance(s) which disturbed the enzyme immunoassay and gave an apparently high content of prostaglandin E2. The interfering substance was separated from prostaglandin E2 by reverse-phase high-performance liquid chromatography. The purified urinary extract was examined by the enzyme immunoassay for prostaglandin E2, and the validity of the results was confirmed by gas chromatography-selected ion monitoring.

A solid-phase enzyme immunoassay of thromboxane B2.

A solid-phase enzyme immunoassay for thromboxane B2 was developed using a conjugate of thromboxane B2 and beta-galactosidase. Anti-thromboxane B2 IgG was bound to a polystyrene tube, and the enzyme-labeled and unlabeled thromboxane B2 were allowed to react in a competitive manner with the immobilized antibody. Then, the specifically bound beta-galactosidase was assayed fluorimetrically, and the enzyme activity was correlated with the amount of unlabeled thromboxane B2. By using a calibration curve, thromboxane B2 was determined in the range of 20 fmol-14 pmol. 2,3-Dinor- and 2,3,4,5-tetranor-thromboxane B2 cross-reacted with thromboxane B2 to the extents of 18.6% and 0.4%, respectively. Most prostaglandins and their metabolites tested showed cross-reactivities of less than 1%. In application of the method to human blood and urine, an octadecylsilyl silica column was utilized for extraction and concentration of thromboxane B2. The crude extract contained a substance(s) which disturbed the enzyme immunoassay and gave an apparently high value of thromboxane B2, and the interfering substance was separated from thromboxane B2 by reverse-phase HPLC. Various amounts of authentic thromboxane B2 added to the purified material from human plasma could be determined by the enzyme immunoassay with a recovery of about 80% and the results correlated well with the values obtained by radioimmunoassay (r = 0.979). When the extract from human urine was analyzed by reverse-phase HPLC, the 2,3-dinor metabolite rather than thromboxane B2 was the predominant compound detected by the enzyme immunoassay.

Radioimmunoassay of bencyclane in human serum.

A radioimmunoassay of bencyclane in human serum was developed. Male rabbits were immunized with p-(3-carboxy-propoxy)bencyclane-bovine serum albumin conjugate, giving antisera with high titers. 125I-p-Hydroxybencyclane with a high specific activity was prepared as a labelled antigen by a chloramine-T method. In the radioimmunoassay procedure, a mixture of serum sample, diluted antiserum and 125I-antigen solution were incubated at 4 degrees C for 18 h, and bound-free separation was carried out by a dextran-coated charcoal method. The detection limit of bencyclane in human serum was 1.0 ng/ml, and the cross-reactivity of the antiserum with metabolites was found to be very low. Serum samples from healthy volunteers dosed orally with bencyclane fumarate were analyzed by both of the radioimmunoassay and gas chromatography-mass spectrometric methods. An excellent correlation was observed between the values obtained by both methods.