A phase I trial of carboxyamido-triazole and paclitaxel for relapsed solid tumors: potential efficacy of the combination and demonstration of pharmacokinetic interaction.

PURPOSE: Preclinical and clinical investigation of the combination of the antiangiogenesis/anti-invasion agent carboxyamido-triazole (CAI) administered with the cytotoxic agent paclitaxel (PAX). EXPERIMENTAL DESIGN: Colony-forming assays were used to test the activity of CAI plus PAX on A2780 human ovarian cancer. The sequence of CAI followed by PAX (CAI>Pax) was modeled in nude mice to test for potential additive toxicity. The Phase I clinical dose escalation schema tested p.o. administered CAI in PEG-400 (50-100 mg/m(2)) or micronized CAI (250 mg/m(2)) for 8 days followed by a 3-h infusion of PAX (110-250 mg/m(2)) every 21 days. Patients were assessed for toxicity, pharmacokinetics of CAI and PAX, and disease outcome. RESULTS: In preclinical studies, CAI>Pax was additive in A2780 human ovarian cancer cell lines when CAI (1 or 5 microM) preceded subtherapeutic doses of PAX. CAI did not reverse PAX resistance and collateral resistance to CAI was documented in PAX-resistant cells. CAI>PAX administration had no overt additive toxicity in nude mice. Thirty-nine patients were treated on a dose-escalation Phase I trial using daily oral CAI for 8 days followed by the PAX infusion. Pharmacokinetic analysis revealed that PAX caused an acute increase in circulating CAI concentrations in a dose-dependent fashion. No additive or cumulative toxicity was observed, and grade 3 nonhematological toxicity was rare. Three partial responses and two minor responses were observed. CONCLUSIONS: The sequential combination of CAI and PAX is well tolerated, and the activity observed suggests that further study of the combination is warranted.

Phase I trial of 72-hour continuous infusion UCN-01 in patients with refractory neoplasms.

PURPOSE: To define the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of the novel protein kinase inhibitor, UCN-01 (7-hydroxystaurosporine), administered as a 72-hour continuous intravenous infusion (CIV). PATIENTS AND METHODS: Forty-seven patients with refractory neoplasms received UCN-01 during this phase I trial. Total, free plasma, and salivary concentrations were determined; the latter were used to address the influence of plasma protein binding on peripheral tissue distribution. The phosphorylation state of the protein kinase C (PKC) substrate alpha-adducin and the abrogation of DNA damage checkpoint also were assessed. RESULTS: The recommended phase II dose of UCN-01 as a 72-hour CIV is 42.5 mg/m(2)/d for 3 days. Avid plasma protein binding of UCN-01, as measured during the trial, dictated a change in dose escalation and administration schedules. Therefore, nine patients received drug on the initial 2-week schedule, and 38 received drug on the recommended 4-week schedule. DLTs at 53 mg/m(2)/d for 3 days included hyperglycemia with resultant metabolic acidosis, pulmonary dysfunction, nausea, vomiting, and hypotension. Pharmacokinetic determinations at the recommended dose of 42.5 mg/m(2)/d for 3 days included mean total plasma concentration of 36.4 microM (terminal elimination half-life range, 447 to 1176 hours), steady-state volume of distribution of 9.3 to 14.2 L, and clearances of 0.005 to 0.033 L/h. The mean total salivary concentration was 111 nmol/L of UCN-01. One partial response was observed in a patient with melanoma, and one protracted period ( > 2.5 years) of disease stability was observed in a patient with alk-positive anaplastic large-cell lymphoma. Preliminary evidence suggests UCN-01 modulation of both PKC substrate phosphorylation and the DNA damage-related G(2) checkpoint. CONCLUSION: UCN-01 can be administered safely as an initial 72-hour CIV with subsequent monthly doses administered as 36-hour infusions.

Pharmacokinetics, distribution, metabolism and excretion of.

PURPOSE: To evaluate the metabolic fate of UCN-01, a signal transduction inhibitor, blood and plasma concentrations, distribution, metabolism and excretion were investigated in rats and dogs after intravenous administration of [3H]UCN-01. METHODS: The radioactivity in plasma, blood and tissues was measured after intravenous administration of UCN-01. In addition, the radioactivity excreted in bile, urine and feces was also determined. RESULTS: The radioactivity in rat and dog plasma disappeared triphasically with terminal half-lives of 21.3 and 27.2 h, respectively. The ratios of the blood-to-plasma concentrations ranged from 0.82 to 1.13 in rats and 0.81 to 1.73 in dogs. From 0.5 to 4 h after giving [3H]UCN-01 to rats, the radioactivity in all tissues except the brain and testes was higher than in plasma. The highest concentration was observed in the lungs followed by the liver and kidneys. The radioactivity was mainly excreted in feces, reaching 96.0% of the radioactivity dose in rats and 78.4% in dogs up to 168 h after injection. Since the biliary excreted radioactivity was 67.2% over 48 h in bile duct-cannulated rats, most of the radioactivity excreted in feces was from biliary radioactivity. There were several metabolites in bile samples, but little UCN-01. CONCLUSIONS: UCN-01 is mainly eliminated by the liver, and there are high concentrations of radioactivity derived from [3H]UCN-01 in all tissues except the brain and testes.

Physiological modeling of altered pharmacokinetics of a novel anticancer drug, UCN-01 (7-hydroxystaurosporine), caused by slow dissociation of UCN-01 from human alpha1-acid glycoprotein.

PURPOSE: The extremely low clearance and small distribution volume of UCN-01 in humans could be partly due to the high degree of binding to hAGP. The quantitative effects of hAGP on the pharmacokinetics of UCN-01 at several levels of hAGP and UCN-01 were estimated in rats given an infusion of hAGP to mimic the clinical situation and a physiological model for analysis was developed. METHODS: The plasma concentrations of UCN-01 (72.5-7250 nmol/kg i.v.) in rats given an infusion of hAGP, 15 or 150 nmol/h/kg, were measured by HPLC. Pharmacokinetic analysis under conditions assuming rapid equilibrium of protein binding and incorporating the dissociation rate was conducted. RESULTS: The Vdss and CLtot of UCN-01 (725 nmol/kg i.v.) in rats given an infusion of hAGP, 150 nmol/h/kg, fell to about 1/250 and 1/ 700 that in control rats. The Vdss and CLtot following 72.5-7250 nmol/kg UCN-01 to rats given 150 nmol/h/kg hAGP were 63.9-688 ml/kg and 3.18-32.9 ml/h/kg, respectively, indicating non-linearity due to saturation of UCN-01 binding. The CLtot estimated by the physiological model assuming rapid equilibrium of UCN-01 binding to hAGP, was six times higher than the observed value while the CLtot estimated by the model incorporating k(off), measured using DCC, was comparable with the observed value. CONCLUSIONS: These results suggest that the slow dissociation of UCN-01 from hAGP limits its disposition and elimination.

Pharmacokinetics and pharmacodynamics of a novel protein kinase inhibitor, UCN-01.

PURPOSE: 7-Hydroxystaurosporine (UCN-01) is a potent protein kinase inhibitor and is being developed as a novel anticancer agent. We describe here its pharmacokinetics and pharmacodynamics in experimental animals. METHODS: The pharmacokinetics of UCN-01 were studied following intravenous (i.v.) administration to mice, rats and dogs at doses of 1-9, 0.35-3.5 and 0.5 mg/kg, respectively. We also studied the pharmacodynamics of UCN-01 (9 mg/kg per day) during and after five consecutive i.v. administrations to nude mice bearing xenografted human pancreatic tumor cells (PSN-1). The concentrations of UCN-01 in plasma and tumor were measured by HPLC using a fluorescence detector. RESULTS: UCN-01 in plasma after i.v. administration was eliminated biphasically in mice and rats, and triphasically in dogs. The elimination half-lives in mice, rats and dogs were 3.00-3.98, 4.02-4.46 and 11.6 h, respectively. The total clearance (Cl(total)) values in mice, rats and dogs were high (1.93-2.64, 2.82-3.86 and 0.616 l/h per kg, respectively). The hepatic clearance (Cl(hepatic)) in rats represented 54.0-81.3% of Cl(total). The volumes of distribution at steady-state in mice, rats and dogs were large (7.89-8.42, 13.0-16.9 and 6.09 l/kg, respectively). These pharmacokinetic parameters were dose-independent in mice and rats. UCN-01 produced significant inhibition of tumor growth during five consecutive i.v. administrations in mice bearing the xenografted PSN-1 cells, and the inhibitory effect continued for 3 days after the final administration. UCN-01 concentrations in tumor tissue were much higher than those in the plasma, and the ratio of tumor to plasma concentrations was about 500 at 24 h after five consecutive doses. CONCLUSIONS: The pharmacokinetic studies showed that UCN-01 has a high clearance and large distribution volume in various experimental animals, and its disposition is linear over the range of doses tested. The pharmacodynamic study showed that UCN-01 is distributed at much higher concentrations in tumor than those in plasma and that it significantly inhibits tumor growth. The high distribution of UCN-01 into tumor cells may contribute to the potent inhibition of tumor growth in vivo.

Altered pharmacokinetics of a novel anticancer drug, UCN-01, caused by specific high affinity binding to alpha1-acid glycoprotein in humans.

The large species difference in the pharmacokinetics/pharmacodynamics of 7-hydroxystaurosporine (UCN-01) can be partially explained by the high affinity binding of UCN-01 to human alpha1-acid glycoprotein (AGP) (Fuse et al, Cancer Res., 58: 3248-3253, 1998). To confirm whether its binding to human AGP actually changes the in vivo pharmacokinetics, we have studied the alteration in its pharmacokinetics after simultaneous administration of human AGP to rats: (a) the protein binding of UCN-01 was evaluated by chasing its dissociation from proteins using dextran-coated charcoal. The UCN-01 remaining 0.1 h after adding dextran-coated charcoal to human plasma or AGP was approximately 80%, although the values for other specimens, except monkey plasma (approximately 20%), were <1%, indicating that the dissociation from human AGP was specifically slower than from other proteins; and (b) the pharmacokinetics of UCN-01 simultaneously administered with human AGP has been determined. The plasma concentrations after i.v. administration of UCN-O1 with equimolar human AGP were much higher than those after administration of UCN-01 alone. The steady-state distribution volume and the systemic clearance were reduced to about 1/100 and 1/200, respectively. Human AGP thus reduced the distribution and elimination of UCN-01 substantially. On the other hand, dog AGP, which has a low binding affinity for UCN-01, did not change the pharmacokinetics of UCN-01 so much. Furthermore, human AGP markedly reduced the hepatic extraction ratio of UCN-01 from 0.510 to 0.0326. Also, human AGP (10 microM) completely inhibited the initial uptake of UCN-01 (1 microM) into isolated rat hepatocytes, whereas the uptake of UCN-01 was unchanged in the presence of human serum albumin (10 microM). In conclusion, the high degree of binding of UCN-01 to human AGP causes a reduction in the distribution and clearance, resulting in high plasma concentrations in humans.

Development of a sensitive liquid chromatography-electrospray ionization tandem mass spectrometry method for the measurement of 7-cyanoquinocarcinol in human plasma.

A sensitive method for the determination of an anti-cancer agent, DX-52-1 (7-cyanoquinocarcinol, I) and quinocarmycin (II) which is formed from I either by metabolism or degradation, in human plasma has been developed utilising liquid chromatography electrospray-ionization tandem mass spectrometry (LC-ESI-MS-MS). The procedure involves solid-phase extraction at pH 2 and low temperature (4-6 degrees C) to prevent the decomposition of I to II, the separation by reversed-phase HPLC and the multiple reaction monitoring (MRM) by ESI-MS-MS. The mean precision and accuracy at the lower limit of quantitation (LLOQ) of I, 0.25 ng ml(-1), were 8.7% and - 10.8%, respectively. Since an interfering peak eluting slightly earlier than II was observed on the HPLC of blank plasma, the LLOQ of II was set at 5 ng ml(-1) where the mean precision and accuracy were 15.6% and -9.8%. The results suggested that the method is useful for the simultaneous monitoring of I and II in the clinical trials of I.

Clinical pharmacology of UCN-01: initial observations and comparison to preclinical models.

UCN-01 (7-hydroxystaurosporine; NSC 638850) is a protein kinase antagonist selected for clinical trial based in part on evidence of efficacy in a preclinical renal carcinoma xenograft model. Schedule studies and in vitro studies suggested that a 72-h continuous infusion would be appropriate. In rats and dogs, maximum tolerated doses produced peak plasma concentrations of approximately 0.2-0.3 microM. However, concentrations 10-fold greater are well tolerated in humans, and the compound has a markedly prolonged T1/2. Specific binding to human alpha1-acidic glycoprotein has been demonstrated. These findings reinforce the need to consider actual clinical pharmacology data in "real time" with phase I studies.

Unpredicted clinical pharmacology of UCN-01 caused by specific binding to human alpha1-acid glycoprotein.

The pharmacokinetics of UCN-01 after administration as a 72- or 3-h infusion to cancer patients in initial Phase I trials displayed distinctive features that could not have been predicted from preclinical data. The distribution volumes (0.0796-0.158 liters/kg) and the systemic clearance (0.0407-0.252 ml/h/kg) were extremely low, in contrast to large distribution volume and rapid systemic clearance in experimental animals. The elimination half-lives (253-1660 h) were unusually long. In vitro protein binding experiments demonstrated that UCN-01 was strongly bound to human alpha1-acid glycoprotein. The results suggest that unusual pharmacokinetics of UCN-01 in humans could be due, at least in part, to its specifically high binding to alpha1-acid glycoprotein.

Development of a highly sensitive high-performance liquid chromatographic method for measuring an anticancer drug, UCN-01, in human plasma or urine.

We have established a highly sensitive high-performance liquid chromatographic method for the determination of an anticancer drug, UCN-01, in human plasma or urine. Using a fluorescence detector set at an excitation wavelength of 310 nm and emission monitored at 410 nm, there was a good linearity for UCN-01 in human plasma (r=0.999) or urine (r=0.999) at concentrations ranging from 0.2 to 100 ng/ml or 1 to 400 ng/ml, respectively. For intra-day assay, in plasma samples, the precision and accuracy were 1.8% to 5.6% and -10.0% to 5.2%, respectively. For inter-day assay, the precision and accuracy were 2.0% to 18.2% and 2.4% to 10.0%, respectively. In urine samples, the intra- and inter-day precision and accuracy were within 3.9% and +/-2.7%, respectively. The lower limit of quantification (LLOQ) was set at 0.2 ng/ml in plasma and 1 ng/ml in urine. UCN-01 in plasma samples was stable up to two weeks at -80 degrees C and also up to four weeks in urine samples. This method could be very useful for studying the human pharmacokinetics of UCN-01.

Design and synthesis of potent antitumor 5,4'-diaminoflavone derivatives based on metabolic considerations.

Recently, we reported that 5,4'-diaminoflavone (1) exhibits potent and specific growth-inhibitory activity against the estrogen receptor (ER)-positive human breast cancer cell line MCF-7. However, when compound 1 was incubated with S-9 mix, its metabolites were observed. Moreover, addition of S-9 mix to the medium caused the drastic decrease in activity of compound 1. Since the 6-, 8-, and 3'-positions were considered to be metabolized oxidatively in vivo from MO calculations, a series of 5,4'-diaminoflavone derivatives substituted at such putative metabolic positions with various functional groups were synthesized aiming at the metabolically stable derivatives. Among them, 5,4'-diamino-6,8,3'-trifluoroflavone (14d) exhibited strong growth-inhibitory activity against MCF-7 cells even in the presence of S-9 mix. Moreover, orally administered compound 14d completely suppressed the growth of MCF-7 inoculated into nude mice, and the effect was more potent than that of compound 1. In addition to ER-positive breast cancer cells, compound 14d exhibited growth-inhibitory activity against a panel of human cancer cell lines including a part of ER-negative breast, endometrial, ovarian, and liver cancers. From these results, fluorine introduction to the putative metabolic positions of compound 1 was elucidated to be effective in the enhancement of the in vivo antitumor activity, probably due to the block of the metabolic deactivation.

[Study of disposition of adriamycin and mitomycin C in liver by determination of plasma concentrations in hepatic vein and artery during intravenous constant infusion in rats].

The hepatic extraction ratios (EH) of adriamycin (ADR) and mitomycin C (MMC), which were administered clinically by an intra-hepatic arterial route, were measured in rats to clarify the disposition of ADR and MMC in liver. EH values of ADR and MMC were determined by comparing the femoral arterial and hepatic venous plasma concentrations at steady state during continuous intravenous administration. The EH value of ADR in rats at each infusion rate of 2, 10 and 50 micrograms/kg/min, was 0.290, 0.286 and 0.251, respectively. There was no significant difference between the EH values (p > 0.05). The systemic clearance (CLtot) at each infusion rate was 108, 77.6 and 72.9 ml/min/kg, respectively. The EH value of MMC in rats at each infusion rate of 2.5, 7.5 and 25 micrograms/kg/min, was 0.332, 0.358 and 0.360, respectively. There was no significant difference between the EH values, the same as for ADR. The systemic clearance (CLtot) at each infusion rate was 38.3, 36.1 and 35.3 ml/min/kg.

Disposition of DX-52-1, a novel anticancer agent, after intravenous administration to mice and dogs.

DX-52-1 is a new derivative of a quinocarmycin analogue. The disposition of [3H]-DX-52-1 was investigated in mice and dogs after intravenous administration (4 and 0.15 mg/kg, respectively). The plasma concentration of non-volatile radioactivity was 7.4 micrograms eq./ml 3 min after administration to mice, then declined biphasically until 2 h. The distribution of non-volatile radioactivity into blood cells was 20% 3 min after administration, being maintained until 30 min. The plasma concentration of unchanged drug was almost equal to that of the radioactivity 3 min after administration and the unchanged drug ratio decreased rapidly. High radioactivity was found in the gall bladder, kidney, liver, and lung 15 min after administration. No radioactivity was detected in most tissues 24 h post-administration. The cumulative excretion of total radioactivity into urine and feces after administration was 68 and 28% within 96 h, respectively. The plasma concentration of non-volatile radioactivity was 0.65 micrograms eq./ml 3 min after administration to dogs. The distribution of non-volatile radioactivity into blood cells was about 20% 3 min after administration and this level tended to increase with time. The cumulative excretion of total radioactivity into urine and feces after administration was 62 and 24%, respectively.

Hepatic extraction ratio of 5-fluorouracil in rats. Dose dependence and effect of uracil and interleukin-2.

The hepatic extraction ratios (EH) of 5-fluorouracil (FUra) in rats were studied to clarify the disposition of FUra in the liver. The EH of FUra in rats at infusion rates ranging from 0.375 to 3 mg/kg/min decreased from 0.750 to 0.225. The EH of values of tegafur, a pro-drug of FUra, were 0.076 to 0.103 over the range of infusion rates, 0.577 to 4.616 mg/kg/min, and were much lower than those of FUra. The EH of FUra at an infusion rate of 0.375 mg/kg/min combined with uracil (0.323 mg/kg/min) was 0.646, which was significantly lower than that of FUra alone, 0.750 (P < 0.001). The EH of FUra combined with interleukin-2 (IL-2) at an infusion rate of 7500 U/kg/min was significantly higher than that of FUra alone (P < 0.01). The dose dependence of the EH of FUra and the effects of uracil and IL-2 on the EH of FUra corresponded with clinical findings. These results suggest that this experimental model in rats may be useful for predicting the clinical pharmacokinetics and efficacy of FUra. We also studied the effect of IL-2 on the EH of mitomycin C (MMC). The EH of MMC combined with IL-2 was higher than that of MMC alone, but the difference was not significant.

Evaluation of the efficiency of targeting of antitumor drugs: simulation analysis based on pharmacokinetic/pharmacodynamic considerations.

Antitumor drugs can be classified into two groups; cell cycle phase nonspecific (type I) and specific (type II) drugs. The cytotoxic activity of type I drugs depends on the time-concentration product (AUC), whereas that of type II drugs is time-dependent. Therefore, not only the AUC in the target organ, but also the exposure time is an important factor for evaluating the efficiency of any delivery system for antitumor drugs. In the present study, we examined the factors governing the cytotoxicity of drugs in tumors based on a hybrid perfusion model. It is suggested that the increase in tumor tissue binding of drug results in an increased unbound drug mean residence time (MRTT,U), leading to the increased activity of type II drugs. In contrast, the cytotoxic activity of type I drugs is unaffected by the alteration in the tissue binding since the intracellular AUC defined for unbound drugs (AUCT,U) is unaffected by the extent of drug binding. We also found that the symmetrical increase in the permeability-surface area products (PS) for drug influx (PSinf) and efflux (PSeff) across the tumor plasma membrane results in the unaltered and reduced antitumor activity for the type I and type II drugs, respectively, due to the unaltered AUCT,U and to the reduced MRTT,U. The kinetic analysis suggests that the increase in PSinf/PSeff ratio results in the increased cytotoxic activity of both type I and type II drugs. Collectively, optimization of the antitumor activity can be attained by increasing the tissue binding for type II drugs and by increasing PSinf and/or by decreasing PSeff type I and type II drugs. The present simulation study was carried out by considering the pharmacodynamic features of antitumor drugs and was a method of predicting how the antitumor activity may change on altering each process (tissue binding and membrane permeability for the influx and efflux processes) which governs the characteristics of drug distribution to tumors.

Application of pharmacokinetically guided dose escalation with respect to cell cycle phase specificity.

BACKGROUND: In 1986, the concept of pharmacokinetically guided dose escalation (PGDE) was proposed to predict the maximum tolerated dose (MTD) of an antitumor drug in humans from animal data. We have previously shown that antitumor drugs can be classified into two types, depending on their cytotoxic mechanisms: type 1 drugs, which are cell cycle phase-nonspecific agents, i.e., area under the curve for drug concentration in the plasma versus time (AUC)-dependent drugs; and type 2 drugs, which are cell cycle phase-specific agents, i.e., those that are time dependent. PURPOSE: The validity of the assumption that the AUC at the dose lethal for 10% of mice administered drug (LD10) is equal to the AUC at MTD for humans, the premise on which PGDE is based, was examined for type 1 and 2 drugs. METHODS: Findings in the literature, including those of Collins and co-workers, were retrospectively analyzed. The human/mouse ratios for the AUC were compared with each other and with the human/mouse dose ratios, based on milligram per meter square of body surface area, the measurement currently used in clinical trials of antitumor drugs. For six of the type 1 drugs, the human/mouse ratio for the AUC of total drug (AUC) and that of unbound drug (AUCu), which has been considered a determinant of pharmacologic and toxicologic effects, were also compared. RESULTS: There was an excellent correlation between log AUC at LD10 for mice and log AUC at MTD for humans for type 1 drugs (r = .898), but not for type 2 drugs (r = .677). For type 1 drugs, the correlation between mouse AUC at LD10 and human AUC at MTD was better for unbound drug (r = .961) than for total drug (r = .892). CONCLUSIONS: PGDE is useful for type 1 drugs; differences in protein binding between species should, however, be considered when using this method.

Adenosine A1 antagonists. 3. Structure-activity relationships on amelioration against scopolamine- or N6-((R)-phenylisopropyl)adenosine-induced cognitive disturbance.

The effects of a variety of adenosine A1 and A2 antagonists on N6-((R)-phenylisopropyl)adenosine (R-PIA)- and scopolamine-induced amnesias were investigated in rodents in order to clarify the role of adenosine receptors in learning and memory. Some of the selective adenosine A1 antagonists exhibited antiamnesic activities at several doses where they did not induce an increase of spontaneous locomotion. These results suggest that the blockade of A1 receptors is more important than that of A2 receptors in learning and memory. Detailed studies of structure-activity relationships of adenosine A1 antagonists in two amnesia models demonstrated that there were three types of adenosine A1 antagonists: (A) Compounds 3-5 (8-substituted 1,3-dipropylxanthines) ameliorated the shortened latency in both models. (B) Compounds 7-11 (8-substituted 1,3-dialkylxanthines) and 19-21 (imidazo[2,1-i]purin-5(4H)-one derivatives) ameliorated the shortened latency in the (R)-PIA-induced amnesia model but not in the scopolamine-induced amnesia model. (C) Compounds 14-16 ameliorated the shortened latency in the scopolamine model but not in the (R)-PIA model. Aminophenethyl-substituted compounds C did not exhibit adenosine A1 antagonism in vivo presumably due to rapid metabolism. The dramatic change in the activities of A and B could not be explained by their simple pharmacokinetic differences because both types of compounds showed clear blockade of central adenosine A1 receptors in the (R)-PIA model. 8-(3-Dicyclopropylmethyl)-1,3-dipropylxanthine (5) (KF15372) was chosen for further studies and is currently under preclinical development as a cognition enhancer.

A silicone model method to compare three methods of preparing the root canal.

To compare the ability of three methods of preparing root canals to provide a properly shaped canal, silicone models were used. The canals of extracted teeth were prepared and filled with a silicone impression material. The teeth were dissolved, and the remaining models were studied and photographed. The hand instrument group and the SET angle were equal. The subsonic instrument did not shape a canal as well as the other two.

Effects of margarine on hepatocarcinogenesis by N,N'-2, 7-fluorenylenebisacetamide in mice.

The effect of a safflower margarine on hepatocarcinogenesis by N,N'-2, 7-fluorenylenebisacetamide was compared with that of safflower oil from which the margarine was made. A high-dextrin diet was used as a control for the two high-fat diets. The number of surviving mice in the margarine group at the end of the experiment was significantly lower compared with both the dextrin and the oil groups. A significantly high incidence of hepatic nodules type 2 was observed in the margarine group, whereas most liver tumors in both the dextrin and the safflower oil groups were of HN type 1 (p less than 0.05). Hepatocellular carcinomas were induced in some mice in all the groups. However, no statistical difference was found among the three dietary groups. Thus the difference in the types of the hepatic nodules among the three groups was not reflected in the incidence of hepatocellular carcinoma.