Inhibitors of aromatase are of interest in the treatment of advanced estrogen-dependent breast cancers. In addition, the inhibitors are promising to play as conformational and catalytic probes for the active site of this enzyme, aromatase. There fore, we synthesized a number of steroidal aromatase inhibitors, including suicide substrates, and also studied the mechanism for a time-dependent inactivation of aromatase by the suicide substrates. The mechanism for the aromatase inactivation by 6-oxo-androstenedione (AD) (1), one of the first discovered suicide substrates, was explored using the 19-substituted analogs 2-5 as well as stereo- and/or regio-specifically labeled [3H, 14C]- compound 1. The results indicated that the 4β, 5β-epoxy-19-oxo derivative 7 is a reactive electrophile that irreversibly binds to the active site of aromatase. Studies on the aromatase inhibition by regioisomers of AD, 4-en-6-one 17, 5-en-4-one 18 and 5-en-7-one 19, revealed that the C-3 carbonyl function is not essential for the tight binding of an inhibitor to the active site. 3-Deoxy AD (22) and its 6α, 7α-cyclopropano steroid 24 as well as some of 6-alkyl-ADs are among the most potent competitive inhibitors reported so for (Km for AD/Ki>6). Structure-activity relationships of the 6-alkyl-ADs and their 3-deoxy-, Δ1-, Δ6-, and Δ1, 6-analogs as aromatase inhibitors showed that aromatase has a hydrophobic binding pocket with a limited accessible volume in the active site in the region corresponding to the β-side rather than the α-side of the C-6 position of the substrate. The 6-alkyl-ADs and their Δ1-analogs were converted into the corresponding estrogens with human placental aromatase, whereas the 3-deoxy steroids 22 and 25 were metabolized to the corresponding 19-oxygenated compounds. The relative apparent Km values for the androgens are different from the relative Ki values, indicating that there is a difference between the ability to serve as an inhibitor and that to serve as a substrate. Moreover, it seems likely that the alignment of the substrate AD analogs in the active site would be markedly different from that of the 3-deoxy steroids.
This review summarizes the principle and the features of high-performance frontal analysis (HPFA), a novel chromatographic method to determine the concentrations of unbound drugs under drug-protein binding equilibrium conditions, and its application to the study on the plasma protein binding. HPFA uses a "restricted-access" type HPLC column which retains a small molecule in the drugs in the micropores, while a large molecule in the plasma protein is size-excluded. After direct and continuous injection of a sample, the drugprotein binding equilibrium in the sample solution is regenerated in the column, and the constant concentration zone of the unbound drug appears from the equilibrium zone. The unbound drug is eluted as a trapezoidal peak with a plateau. The concentration in this plateau region is equal to that of the unbound drug in the sample solution, and the concentration of the unbound drug can be determined by subsequent on-line HPLC analysis. HPFA allows a simple analysis following direct injection of a sample, and does not cause undesirable drug adsorption on the membrane nor leakage of the bound drug from the membrane, which are often encountered in the conventional ultrafiltration or dialysis method. HPFA allows the simultaneous determination of the concentrations of total and unbound drugs in a single analysis. HPFA can be easily incorporated into the on-line HPLC system. By coupling HPFA with a chiral HPLC column, the concentration of an unbound racemic drug can be determined enantioselectively. The detection limit can be improved dramatically by coupling HPEA with a preconcentration column. Frontal analysis in capillary electrophoresis format (CE/FA) allows us an ultramicro binding assay. The concentration of the unbound racemic drug can be determined stereoselectively by coupling HPFA with a chiral CE technique.
Laminin-1, a major component of basement membranes, has multiple biological activities including promotion of cell adhesion, spreading, migration, growth, neurite outgrowth and tumor metastasis. Several active sites on laminin-1 have been identified previously. We modified these biologically active peptides to enhance their activities. The multimeric YIGSR (Tyr-Ile-Gly-Ser-Arg) peptides assembled on a branched lysine core were found to strongly enhance the activity of YIGSR in inhibiting tumor growth and metastasis. We also found the all-D-configuration peptide segment containing the IKVAV (Ile-Lys-Val-Ala-Val) sequence had similar biological activities to the native all-L-peptide in vitro and in vivo. These results suggest that these modified compounds are potentially useful for clinical applications. We have identified new active sequences from the laminin α1 chain carboxyl-terminal globular domain (G domain). Using a systematic screening for cell binding sites with 113 overlapping synthetic peptides, we found five peptides (AG-10, AG-22, AG-32, AG-56, and AG-73) showed cell attachment activities with cell-type specificities. AG-10 and AG-32 were found to interact with integrins. AG-73 caused metastases of B16-F10 mouse melanoma cells to the liver colonization in mice. Additionally AG-73 was found to promote neurite outgrowth. Moreover, this peptide inhibited laminin mediated acinar-like development of a human submandibular gland cell line. The AG-73 domain on laminin-1 could be one of the most important biologically active sites. These active peptides may useful for study of the molecular mechanism of laminin-receptor interactions and for development of therapeutic reagents for tumor metastasis and angiogenasis.
We studied the cause of cracking of a clinically used polyurethane (PU) catheter during the constant infusion of etoposide (VP-16) injection (Lastet【○!R】), administered without dilution to patients as a part of combination high-dose chemotherapy. After VP-16 injection was infused into the PU catheter at a constant infusion rate (30ml/h) for 24h, a decrease in the elasticity (36% of untreated) and on increase in the length of the catheter (3.7%) were observed. These changes were significantly higher than those treated with the control saline. The similar changes of the PU catheter were observed after treatment with a basal solution containing polyethylene glycol 400 (PEG 400), polysorbate 80 and ethanol, which is the vehicle of the VP-16 injection, and with ethanol alone. Moreover, obvious degeneration of the internal wall (occurrence of spots like melting) and cutting face (micro-cracking) of the catheter was observed with an electron microscope after treatment with the vehicle. On the other hand, the elasticity or extension of the PU catheter were not changed after treatment with saline or PEG 400. From these findings, it was suggested that the degeneration and subsequent cracking of the PU catheter during the infusion of VP-16 injection was caused by ethanol contained in its injection solution. No cracking or morphological changes of polyvinyl chloride (PVC) and silicone catheters were found after treatment with the vehicle solution. However, since it has been reported in previous reports that di(2-ethylhexyl)phthalate was leached from PVC bags, the high dose chemotherapy with the dilution-free VP-16 injection should be achieved safely and effectively using a silicon catheter, rather than the PU catheter.
The order system to manage various works on injections carried out in the hospital established in July 1994, was found to be quite successful in the appropriate use of drugs for injections as well as in integrating various operations for injection including prescription, inventory management and reimbursement. However, dispensing has still been dependent on the works by hospital pharmacists, and also requires much time for them to complete their tasks. Therefore we introduced an automatic dispenser for injections prepared according to the order system of materials needed for injections. This integrated system has been operating since November 1997, and was successful in reducing the work load of staffs in the pharmacy department in spite of providing all wards with drugs for injections. The reduction of the work load will contribute to pharmacists to provide additional services such as inpatient compliance education.
Convenient synthesis of angular pyranocoumarin from hydroxycoumarin and 1, 1-diethoxy-3-methyl-2-butene according to the North's method was investigated. Both 5- and 7-hydroxycoumarins afforded corresponding pyranocoumarins in a comparatively good yield. Moreover, effects of additives and solvents were examined, and in the presence of cesium fluoride (CsF) in diethylaniline, 6-hydroxycoumarin provided the corresponding pyranocoumarin in a 62% yield.
The effects of shikonnin (SK) and its optical isomer alkannin (AK) on the hydroxyl radical (HO·) generation system including iron ions were evaluated using the spin trap method by ESR spectroscopy. 5, 5-Dimethyl-1-pyrroline-1-oxide (DMPO) was used as a spin trap agent and HO· was generated by a reaction between an iron ion and hydrogen peroxide, which is called Fenton reaction system. SK inhibited the HO· spin adduct (DMPO-OH) yielded in a dose-dependent manner. In this effect no difference was observed between SK and AK. When different concentrations of DMPO were used for the confirmation of its competitive reaction, no difference was also observed in the concentration of SK required to reduce the amount of the DMPO-OH by 50% (ID50). These findings suggested that the inhibitory effect of SK against the thus yielded DMPO-OH was not generated by the scavenging for HO·, but by the inhibition on the Fenton reaction system. The mechanism of the inhibition on this system may be based on the formation of a complex between SK and the iron ion. The molar ratio of SK to the iron ion in the complex was considered 2 to 1 (2 : 1), because the concentrations of the observed ID50 and the used iron ion exhibited the same value. In addition, the same result was also obtained from the study using spectroscopic analysis.
The effects of ascites fluids and sera of tumor-bearing mice on the mycelial growth of Candida albicans were examined. When the ascites fluids or the sera obtained from mice inoculated with MM46 mammary carcinoma were added to the culture medium, mycelial growth of C. albicans was strongly inhibited. The molecular size of the growth inhibitory factor in the ascites fluids was estimated to be approximately 80 K dalton by gel-filtration chromatography. Ferric chloride (6 μM) neutralized the anti-Candida activity. On the basis of these results including morphological observation, a possible role of a transferrin-like molecule was discussed.