The effect of two root canal sealers on the retentive strength of glass fibre endodontic posts.

This study was undertaken to examine the effect of eugenol-containing and non-eugenol-containing root canal sealers on the retention strength of glass fibre endodontic posts (ParaPost Fibre White) luted with a resin cement (ParaPost cement). We also examined the mode of failure that occurred visually by using scanning electron microscopy. Seventy-two single rooted, recently extracted, premolar teeth were root canal treated and randomly divided into two groups. Group 1 was obturated with gutta percha and a calcium hydroxide-based sealer (Sealapex, Kerr). Group 2 was obturated with gutta percha and a eugenol-based sealer (Tubli-Seal Kerr). The teeth were stored for 1 week in distilled water at 37 degrees C and then prepared for 9 mm posts with a 1.40-mm drill. The matching glass fibre post was luted with a resin cement following the manufacturer's instructions. The samples were stored for 1 week and thermocycled. The posts were removed from the root canals using a calibrated testing machine in tensile mode. The mean dislodging force for group 1 was 190.46 N and for group 2 was 183.8 N, with standard deviations of 54.9 and 56.0 N respectively. The t-test indicated no significant difference between the two groups. Failure of the posts occurred mainly within the resin layer. This study showed that under experimental conditions there was no statistically significant difference between Sealapex sealer and Tubli-Seal sealer on the retention of glass fibre posts using a resin cement.

Oxindole-based inhibitors of cyclin-dependent kinase 2 (CDK2): design, synthesis, enzymatic activities, and X-ray crystallographic analysis.

Two closely related classes of oxindole-based compounds, 1H-indole-2,3-dione 3-phenylhydrazones and 3-(anilinomethylene)-1,3-dihydro-2H-indol-2-ones, were shown to potently inhibit cyclin-dependent kinase 2 (CDK2). The initial lead compound was prepared as a homologue of the 3-benzylidene-1,3-dihydro-2H-indol-2-one class of kinase inhibitor. Crystallographic analysis of the lead compound bound to CDK2 provided the basis for analogue design. A semiautomated method of ligand docking was used to select compounds for synthesis, and a number of compounds with low nanomolar inhibitory activity versus CDK2 were identified. Enzyme binding determinants for several analogues were evaluated by X-ray crystallography. Compounds in this series inhibited CDK2 with a potency approximately 10-fold greater than that for CDK1. Members of this class of inhibitor cause an arrest of the cell cycle and have shown potential utility in the prevention of chemotherapy-induced alopecia.

Prevention of chemotherapy-induced alopecia in rats by CDK inhibitors.

Most traditional cytotoxic anticancer agents ablate the rapidly dividing epithelium of the hair follicle and induce alopecia (hair loss). Inhibition of cyclin-dependent kinase 2 (CDK2), a positive regulator of eukaryotic cell cycle progression, may represent a therapeutic strategy for prevention of chemotherapy-induced alopecia (CIA) by arresting the cell cycle and reducing the sensitivity of the epithelium to many cell cycle-active antitumor agents. Potent small-molecule inhibitors of CDK2 were developed using structure-based methods. Topical application of these compounds in a neonatal rat model of CIA reduced hair loss at the site of application in 33 to 50% of the animals. Thus, inhibition of CDK2 represents a potentially useful approach for the prevention of CIA in cancer patients.

Preclinical development of eniluracil: enhancing the therapeutic index and dosing convenience of 5-fluorouracil.

Eniluracil (5-ethynyluracil, GW 776, 776C85) is being developed as a novel modulator of 5-fluorouracil (5-FU) for the treatment of cancer. Eniluracil is an effective mechanism-based inactivator of dihydropyrimidine dehydrogenase (DPD), the first enzyme in the catabolic pathway of 5-FU. By temporarily eliminating this prevalent enzyme, eniluracil provides predictable dosing of 5-FU and enables oral administration of 5-FU to replace intravenous bolus and continuously infused dosing. New DPD is synthesized with a half-life of 2.6 days. It also eliminates the formation of problematic 5-FU catabolites. Most importantly, in laboratory animals, eniluracil increases the therapeutic index and absolute efficacy of 5-FU. Accompanying reports in this journal indicate that eniluracil has promising clinical potential.

alpha-fluoro-beta-alanine: effects on the antitumor activity and toxicity of 5-fluorouracil.

We have shown previously that (R)-5-fluoro-5,6-dihydrouracil (FUraH(2)) attenuates the antitumor activity of 5-fluorouracil (FUra) in rats bearing advanced colorectal carcinoma. Presently, we found that alpha-fluoro-beta-alanine (FBAL), the predominant catabolite of FUra that is formed rapidly via FUraH(2), also decreased the antitumor activity and potentiated the toxicity of FUra. In rats treated with Eniluracil (5-ethynyluracil, GW776), excess FBAL, in a 9:1 ratio to FUra, produced similar effects when administered 1 hr before, simultaneously with, or 2 hr after FUra. FBAL also decreased the antitumor activity of FUra in Eniluracil-treated mice bearing MOPC-315 myeloma at a 9:1 ratio with FUra, but not at a 2:1 ratio. FBAL did not affect the antitumor activity of FUra in mice bearing Colon 38 tumors. We also evaluated the effect of thymidylate synthase (TS) and thymidine kinase (TK) from tumor extracts after FUra +/- Eniluracil +/- FBAL treatment. The activity of TK was similar among the three groups at both 18 and 120 hr. There was also no difference in TS inhibition ( approximately 35%) at 18 hr. However, significantly more TS inhibition was observed in the Eniluracil/FUra group than in the FUra-alone group at 120 hr. FBAL did not alter the effect of Eniluracil/FUra in TS inhibition. Neither FUraH(2) nor FBAL affected the IC(50) of FUra in culture. Thus, the effect of FBAL did not result from direct competition with FUra uptake or immediate anabolism. Either another downstream catabolite that is not formed in cell culture is the active agent, or the effect requires the complexity of a living organism or an established tumor.

Burn management. Psychosocial interventions: pharmacologic and psychologic modalities.

The devastating nature of a thermal injury drastically alters both the physical and psychologic elements of the survivor. Understanding the physiologic and psychologic processes of burn patients in all phases of recovery enables the caregiver to provide optimal holistic treatment to the patient. The pain of burns can be treated best with analgesic medication in conjunction with therapeutic psychologic interventions. Care of thermally injured patients requires dedicated and well-trained staff members who can effectively assist the patient in coping with the situation and working toward recovery.

The development of beta-lactamase as a highly versatile genetic reporter for eukaryotic cells.

We describe in this report that TEM-1 beta-lactamase has several desirable characteristics as a genetic reporter. First, it has no endogenous counterpart in eukaryotic cells and therefore provides a background-free measure of gene expression. Second, because of the uniqueness of the substrate cleavage reaction, a wide variety of substrates which are efficiently cleaved can be synthesized for beta-lactamase. Third, since the assays involve no more than addition of substrate to media, it is possible to continuously monitor a culture without destruction of the cells. Fourth, the enzyme is extremely versatile in that it can be fused to other proteins and retain activity. To demonstrate the versatility of beta-lactamase, we created three forms of the enzyme including secreted, intracellular, and membrane-bound forms of the enzyme, each form having distinct advantages as a reporter system. We also showed that levels of secreted beta-lactamase were proportional to both the levels of transfected DNA, beta-lactamase mRNA, as well as activity of the chloramphenicol acetyl transferase gene controlled by the same promoter, validating the reliability of this reporter. beta-Lactamase thus represents a novel and highly versatile genetic reporter.

Inhibition of uridine phosphorylase. Synthesis and structure-activity relationships of aryl-substituted 1-((2-hydroxyethoxy)methyl)-5-(3-phenoxybenzyl)uracil.

Structure-activity relationship studies on a series of 1-((2-hydroxyethoxy)methyl)-5-(3-(substituted-phenoxy)benzyl)uracils as inhibitors of murine liver uridine phosphorylase have led to compounds with IC50s as low as 1.4 nM. The two most potent compounds, 10j (3-cyanophenoxy) and 11f (3-chlorophenoxy) were tested in vivo for effects on steady-state concentrations of circulating uridine in mice and rats. Both compounds were substantially more efficacious than BAU (5-benzylacyclouridine) both in vitro and in vivo.

Dihydropyrimidine dehydrogenase inactivation and 5-fluorouracil pharmacokinetics: allometric scaling of animal data, pharmacokinetics and toxicodynamics of 5-fluorouracil in humans.

UNLABELLED: The pharmacokinetics of 5-fluorouracil (5-FU) in different animal species treated with the dihydropyrimidine dehydrogenase (DPD) inactivator, 5-ethynyluracil (776C85) were related through allometric scaling. Estimates of 5-FU dose in combination with 776C85 were determined from pharmacokinetic and toxicodynamic analysis. METHOD: The pharmacokinetics of 5-FU in the DPD-deficient state were obtained from mice, rats and dogs treated with 776C85 followed by 5-FU. The pharmacokinetics of 5-FU in humans were then estimated using interspecies allometric scaling. Data related to the clinical toxicity for 5-FU were obtained from the literature. The predicted pharmacokinetics of 5-FU and the clinical toxicity data were then used to estimate the appropriate dose of 5-FU in combination with 776C85 in clinical trials. RESULTS: The allometric equation relating total body clearance (CL) of 5-FU to the body weight (B) (CL = 0.47B0.74) indicates that clearance increased disproportionately with body weight. In contrast, the apparent volume of distribution (Vc) increased proportionately with body weight (Vc = 0.58 B0.99). Based on allometric analysis, the estimated clearance of 5-FU (10.9 l/h) in humans with DPD deficiency was comparable to the observed values in humans lacking DPD activity due to genetic predisposition (10.1 l/h), or treatment with 776C85 (7.0 l/h) or (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdUrd, 6.6 l/h). The maximum tolerated dose (MTD) of 5-FU in combination with 776C85 was predicted from literature data relating toxicity and plasma 5-FU area under the concentration-time curve (AUC). Based on allometric analysis, the estimated values for the MTD in humans treated with 776C85 and receiving 5-FU as a single i.v. bolus dose, and 5-day and 12-day continuous infusions were about 110, 50 and 30 mg/m2 of 5-FU, respectively. DISCUSSION: The pharmacokinetics of 5-FU in the DPD-deficient state in humans can be predicted from animal data. A much smaller dose of 5-FU is needed in patients treated with 776C85.

Detection of a R173W mutation in the porphobilinogen deaminase gene in the Nova Scotian "foreign Protestant" population with acute intermittent porphyria: a founder effect.

OBJECTIVES: Acute intermittent porphyria (AIP) is caused by mutations in the porphobilinogen deaminase (PBGD) gene that disrupt the function of the enzyme. Many mutations that lead to decreased PBGD activity have been described. An Arg to Trp substitution at codon 173 (CGG-->TGG in exon 10) and designated R173W, which leads to a CRIM-negative phenotype, has been reported in Swedish, Finnish, Scottish, and South African kindreds, and in a Nova Scotian proband with fatal AIP. In this work, we investigated the presence of this mutation in a Nova Scotian patient population presenting with AIP. DESIGN AND METHODS: Single-strand conformation polymorphism analysis and DNA sequencing by TA cloning and Sanger's dideoxy chain termination method, were used to confirm the maternal transmission of this mutation to the proband. The mutation also eliminates an Ncil (also Mspl) endonuclease restriction site, which allows for detection of the mutant allele by polymerase chain reaction amplification and restriction enzyme digestion. RESULTS: The family of the Nova Scotian proband and four other AIP kindreds showed the presence of the same mutation. These five families are descendants of German, Swiss, and French immigrants historically known as the "Foreign Protestants," who were recruited to Nova Scotia in the 1750s. CONCLUSION: In all these families, descent from one couple that settled in Nova Scotia in 1751 has been identified by genealogy research, consistent with a founder effect within this population. This is the first identified mutation in PBGD causing AIP that has been linked to a founder effect in descendants of an immigrant population to North America, and which could be traced to such a distant background, similar to the South African variegate porphyria mutation.

5-Ethynyluracil (776C85): effects on the antitumor activity and pharmacokinetics of tegafur, a prodrug of 5-fluorouracil.

We studied the effects of 5-ethynyluracil (776C85 and 776C), a potent mechanism-based inactivator of dihydropyrimidine dehydrogenase, on the antitumor efficacy and pharmacokinetics of tegafur (FT), a prodrug of 5-fluorouracil (5-FU), in rats with large s.c. colon carcinoma. Rats were dosed p.o. once daily for 7 days with either FT, FT and uracil in a 1:4 molar ratio (UFT), FT 1 h after 776C (776C/FT), or UFT 1 h after 776C (776C/UFT). 776C, which was dosed at 1 mg/kg, had neither intrinsic antitumor activity nor toxicity. The rank order in antitumor efficacy at the maximal tolerated dose of the FT (mg/kg/day) component was 776C/FT (5 mg/kg/day) > or = UFT (80 mg/kg/day) = 776C/UFT (5 mg/kg/day) >> FT (200 mg/kg/day). One-hundred % of rats treated with 776C/FT had complete and sustained tumor regression with no severe toxicity. The area under the plasma 5-FU concentration versus the time curve generated from UFT, FT, and 776C/FT at their maximum tolerated dose was 140, 50, and 27 microM.h, respectively. The area under the concentration in plasma versus time curve did not correlate with the rank order of antitumor efficacy. The vast majority of 5-FU derived from FT (alone) appeared to be rapidly catabolized. Furthermore, plasma exposure of 5-FU derived from UFT was more variable than that from 776C/FT. Each therapy also produced different levels of plasma uracil. Endogenous plasma uracil levels (1-3 microM) were not affected by FT but increased to 100 microM after dosing with 776C. Plasma uracil from UFT was 800 microM 1 h after dosing. These results suggest that moderately elevated uracil (776C/FT) may be beneficial, whereas uracil that is greatly elevated during the first 5 h (UFT) and 5-FU catabolites (FT alone) may interfere with antitumor efficacy. 776C, coadministered with FT, could provide once-a-day oral therapy for cancer patients.

Inhibition of uridine phosphorylase: synthesis and structure-activity relationships of aryl-substituted 5-benzyluracils and 1-[(2-hydroxyethoxy)methyl]-5-benzyluracils.

A series of 1-[(2-hydroxyethoxy)methyl]-5-benzyluracils were synthesized and tested for inhibition of murine liver uridine phosphorylase (UrdPase). Inhibitors of UrdPase are reported to enhance the chemotherapeutic utility of 5-fluoro-2'-deoxyuridine and 5-fluorouracil and to ameliorate zidovudine-induced anemia in animal models. We prepared a series of 5-aryl-substituted analogues of 5-benzylacyclouridine (BAU), a good inhibitor of UrdPase (IC50 of 0.46 microM), to develop a compound with enhanced potency and improved pharmacokinetics. The first phase of structure-activity relationship studies on a series of 32 aryl-substituted 5-benzyluracils found several 5-(3-alkoxybenzyl) analogues of 5-benzyluracil with enhanced potency. The acyclovir side chain, the (2-hydroxyethoxy)methyl group, was substituted on the more potent aryl-substituted 5-benzyluracils. The two most potent compounds, 10y (3-propoxy) and 10dd (3-sec-butoxy), were inhibitors of UrdPase with IC50s of 0.047 and 0.027 microM, respectively. Six compounds were tested in vivo for effects on steady-state concentrations of circulating uridine in rats. Plasma uridine levels were elevated 3-9-fold by compound levels that ranged from 8 to 50 microM.

The anesthetic management of a patient with chromosome 10qter deletion syndrome.

Terminal deletions of chromosome 10q are uncommon. The resulting syndrome includes cardiac and facial anomalies, urogenital abnormalities, limb defects, and mental retardation. Most affected infants require surgical correction of these anomalies. Presented are features inherent in the syndrome that will aid the anesthesiologist in the perioperative management of such patients.

Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase.

The gene encoding cytosine deaminase (CD) has been expressed in the human colorectal carcinoma cell line WiDr. Metabolism studies confirm that tumor cells expressing CD convert the very nontoxic prodrug 5-fluorocytosine (5FCyt) to 5-fluorouracil (5FUra) and 5FUra metabolites. Tumor xenografts composed of CD-expressing cells can selectively generate tumor levels of > 400 microM 5FUra when the host mouse is dosed with nontoxic levels of 5FCyt. The selective metabolic conversion of 5FCyt to 5FUra in CD-expressing tumor cells results in the inhibition of thymidylate synthase and incorporation of 5FUra into RNA. 5FUra is also liberated into the surrounding environment when CD-expressing tumor cells are treated with 5FCyt. The liberated 5FUra is able to kill neighboring, non-CD-expressing tumor cells in vitro and in vivo. Most importantly, when only 2% of the tumor mass contains CD-expressing cells (98% non-CD-expressing cells), significant regressions in all tumors are observed when the host mouse is dosed with nontoxic levels of 5FCyt.

5-Ethynyluracil (776C85): protection from 5-fluorouracil-induced neurotoxicity in dogs.

5-Ethynyluracil (776C85) is a potent mechanism-based inactivator of dihydropyrimidine dehydrogenase (DPD), the enzyme that catalyzes the rapid catabolism of 5-fluorouracil (5-FU). Because catabolism is the major route for 5-FU clearance, we studied the effect of 5-ethynyluracil on the pharmacokinetics and toxicity of continuous i.v. 5-FU infusion in the dog. 5-FU at 40 mg/kg/24 hr resulted in a steady-state plasma 5-FU concentration of 1.3 microM and was fatal with dogs dying from apparent neurotoxicity. 5-Ethynyluracil lowered the total clearance of 5-FU from 9.9 to 0.2 L/hr/kg and enabled 1.6 mg/kg/24 hr 5-FU to achieve a steady-state plasma 5-FU concentration of 2.4 microM with no apparent toxicity. 5-FU at 4 mg/kg/24 hr achieved a steady-state plasma 5-FU concentration of 5.3 microM and produced only mild gastrointestinal disturbances in 5-ethynyluracil-treated dogs. Thus, a catabolite of 5-FU appears to be responsible for the 5-FU-induced neurotoxicity in dogs.

5-Ethynyluracil (776C85): a potent modulator of the pharmacokinetics and antitumor efficacy of 5-fluorouracil.

5-Ethynyluracil (5-EU, 776C85) is a mechanism-based irreversible inhibitor of dihydropyrimidine dehydrogenase (EC 1.3.1.2), the rate-determining enzyme in 5-fluorouracil (5-FU) catabolism. In the present study, 5-EU was found to be a potent modulator of 5-FU catabolism in mice and rats. Liver extracts prepared up to 6 hr after a 5-EU dose (2 mg/kg) were > 96% inhibited in their ability to catalyze 5-FU degradation. 5-EU treatment increased the elimination t1/2 and the area under the plasma concentration-time curve of 5-FU. 5-FU oral bioavailability was approximately 100% in rats pretreated with 5-EU. Consequently, 5-EU induced a linear relationship between the area under the plasma concentration-time curve and the oral dose of 5-FU. As expected from the preservation of plasma 5-FU, 5-EU potentiated the antitumor activity and the toxicity of 5-FU in two mouse tumor models (Colon 38 and MOPC-315). However, 5-EU potentiated the antitumor activity to a greater degree and thereby increased the therapeutic index of 5-FU 2- to 4-fold.

Variation in human thymidylate synthase is associated with resistance to 5-fluoro-2'-deoxyuridine.

Two human colorectal tumor cell lines are differentially sensitive to growth inhibition by 5-fluorodeoxyuridine (FdUrd); cell line RCA is less sensitive to FdUrd than is cell line C. Thymidylate synthase (TS), a target of FdUrd, has been purified to homogeneity from both cell lines. Because of differences in the avidity for a folate ligand affinity matrix, TS forms from the cells were purified by two different procedures. Relative to the enzyme from C cells, the enzyme from RCA cells demonstrated higher Km values for the substrates deoxyuridylate and 5,10-methylene-tetrahydrofolate, a lower rate of association of the inhibitor 5-fluorodeoxyuridylate (FdUMP), a similar rate of FdUMP dissociation, and lower enhancement of covalent FdUMP binding by folate derivatives. The activities of the enzymes in situ and the catalytic efficiencies of the purified enzymes were similar. Thus, a cell line that is naturally resistant to FdUrd has been identified that expresses a TS with reduced affinity for FdUMP and 5,10-methylenetetrahydrofolate, relative to the enzyme expressed in a FdUrd-sensitive cell line.

Species-dependent differences in the biochemical effects and metabolism of 5-benzylacyclouridine.

The pharmacokinetics and biochemical effects of the uridine phosphorylase (UrdPase) inhibitor 5-benzylacyclouridine (BAU) were investigated in the mouse, rat and monkey. Following i.p. administration of BAU (30 mg/kg) in the mouse and i.v. administration in the rat and monkey, initial BAU plasma half-life values were 36, 36 and 25 min, and the areas under the plasma BAU concentration versus time curves (AUC) were 127, 80 and 76 microM.hr, respectively. Rats were also dosed p.o. and i.v. with BAU at 90 mg/kg, and a comparison of the AUC values showed an oral bioavailability of 70%. Analyses of plasma samples by HPLC indicated that the metabolism of BAU differed in these species. A major BAU metabolite was observed in monkeys. Its concentration was greater than or equal to that of BAU in almost every plasma sample, and its elimination paralleled that of BAU. Urinary recovery of the metabolite was 10-fold higher than the recovery of unchanged drug. The compound was identified as the ether glucuronide of BAU by its UV absorption spectrum, its co-elution with BAU after incubation with beta-glucuronidase, and liquid chromatography/mass spectrum analysis. A different metabolite was detected in rat plasma; its maximum concentration was 15% of the BAU level, and its elution position on the HPLC chromatogram was not affected by the action of beta-glucuronidase. BAU had equivalent potency against UrdPase in liver extracts from the three species, with Ki values of about 0.17 microM. However, the in vivo effects of BAU on plasma uridine concentrations were species dependent. In mice, a 30 mg/kg i.p. dose of BAU increased the plasma uridine concentration to 11 microM from a control level of 1.8 microM. In the rat, a 30 mg/kg i.v. dose of BAU increased plasma uridine to 2.1 from 1.1 microM control levels, and a 300 mg/kg oral dose resulted in a peak plasma uridine concentration of only 6 microM. In the monkey, BAU (30 mg/kg, i.v.) had no effect on plasma uridine despite the presence of 10-100 microM BAU levels in plasma for 1.5 hr. These data show that there are significant differences in the biochemical effects and metabolism of BAU in CD-1 mice, CD rats and cynomolgus monkeys.

Modulation of 5-fluoro-2'-deoxyuridine response by folinic acid in human colonic tumor cell lines: the role of thymidylate synthase.

Recent investigations have revealed a significant increase in cytotoxic response to (5-fluoropyrimidine, FP) agents in the presence of the folate folinic acid (CF). It has been suggested that CF provides a source of intracellular reduced folates which, in turn, enhances the inhibition of the cellular target thymidylate synthase (TS) by the FP metabolite 5-fluoro-2'-deoxyuridylate (FdUMP). The extent of variation in the response to FP-CF combinations is unknown but it is an important consideration in view of the utilization of these combinations for the therapy of colorectal carcinoma. In the present study, variation in the response to 5-fluoro-2'-deoxyuridine (FdUrd)-CF combinations was observed between two human colorectal tumor cell lines, RCA and C. The response of both cell lines to FdUrd increased with increasing CF, but the effect was more pronounced in cell line RCA. RCA was 4-fold less responsive than cell line C to FdUrd at low CF concentrations, whereas both cell lines exhibited similar sensitivity at high CF concentrations. RCA accumulated lower levels of TS folate cosubstrates after CF than did C; however, this was not the sole mechanism accounting for the differential response to FdUrd-CF. The two cell lines responded differently to equivalent intracellular levels of 5,10-methylenetetrahydrofolate (CH2H4PteGlu) derivatives, the folate ligands involved in tight-binding inhibition of TS by FdUMP. The differential response to CH2H4PteGlu was not due to lack of folate polyglutamation; the predominant CH2H4PteGlu derivative in both cells was the hexaglutamate form. The difference in response to CH2H4PteGlu was associated with a reduction in the affinity of the RCA TS for CH2H4PteGlu, relative to the C enzyme. Thus, a cell line has been identified that responds poorly to FdUrd at physiological levels of CF and that contains a variant TS enzyme.