Diels-Alder reactions of benzylidenecyanomethyl-1,3-benzothiazoles 17 and -1,3-benzoxazoles 18 as 1-aza-1,3-butadienes are described. The dienes 17 and 18 featuring stabilized imine moieties in the form of heteroaromatic rings react with both electron-deficient and electron-rich dienophiles to give corresponding cycloadducts regioselectively. The cycloadditions of the intramolecular systems 34c,d and 35c,d proceeded smoothly via the exo-transition state, stereoselectively affording polycyclic compounds 36c,d and 37c,d in good to excellent yields. The diene systems of 17 and 18 were extended to dienes 19a-c with ester groups at diene-4-positions. Dienes 19a-c exhibited high Diels-Alder reactivities with electron-rich alkenes. Dienes 19a-c also reacted with allyl alcohols 55—58 in the presence of stanoxane catalyst 53 to give cycloadducts 59—62 via transesterification and intramolecular cycloaddition. Although α-alkoxycarbonylnitrones 64 have been very attractive nitrones for the syntheses of amino acids, the nitrones 64 exist as equilibrating mixtures of (E)-64 and (Z)-64. To solve this problem, three methods were explored: 1) sequential transesterification and intermolecular cycloaddition of nitrones 64 with allyl alcohols; 2) use of chiral and geometry-fixed nitrone 84; and 3) selective activation of (Z)-64 by Eu(fod)₃. These methods were applied to syntheses of nikkomycins, clavalanine, and β-substituted α-amino acids. The reactions of photoinduced carbonyl ylides from α,β-unsaturated γ,δ-epoxy nitriles were studied. Direct irradiation (λ=254nm) of (E)-129 led selectively to products arising from the carbonyl ylide XXV or the carbene intermediate XXVI. The carbonyl ylides generated from (E)-129, (E)-139, and (Z)-143 were trapped with MeOH in the presence of amine, affording the corresponding acetals in moderate yields (Schemes 42 and 43). Photocyclization reactions of δ-hydroxyalkyl epoxy nitriles 148a-e led to spiro acetals arising from the carbonyl ylides (Scheme 45). The photoinduced carbonyl ylides from the epoxy dinitriles 158 and 160—163 underwent 1,3-dipolar cycloaddition with enol ethers, leading to a tetrahydrofuran system (Schemes 49 and 50, Table 14). Electrocyclization of 3-butadienylindoles 184 to intermediary dihydrocarbazoles XXXII followed by elimination of MeOH gave 3-oxyganated carbazoles 185, which were transformed to carbazole alkaloids hyellazole 168, 4-demethoxycarbazomycin B 170 and carazostatin 171, respectively. Claisen rearrangement of 3-(1-amino-1-vinyloxy)indolines derived from 3-hydroxyindolines 192 and amide acetal 193 gave indol-4-ylacetamides 194, which was reduced to afford 4-(2-aminoethyl)indoles 198, which has a framework of biologically active 4-substituted indole compounds. Claisen rearrangement of 3-allyloxyindoles produced in situ by condensation of indolin-3-ones 202 with allyl alcohols 203 and 206—211 gave 2-allylindolin-3-ones 204, 205 and 212—220. The domino reactions, Horner-Wadsworth-Emmons olefination of 2-allyloxyindole 233, isomerization, and Claisen rearrangement produced 3-allylindolin-2-one 234, which was derivatized to 3a-allylpyrrolo[2,3-b]indole alkaloid, flustramine C 221. Reverse aromatic Cope rearrangement of 2-allyl-3-indolidene acetonitriles 241—243, formed by Horner-Wadsworth-Emmons reaction of 2-allylindolin-3-ones 238—240, afforded indoles 244—246.

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Successful gene therapy depends largely on vectors that can efficiently deliver the therapeutic genes into the target tissues and cells. Recombinant adenovirus (Ad) vectors continue to be the preferred vectors for gene therapy because they can easily be grown to high titers and can efficiently transfer genes into both dividing and nondividing cells. However, there are some limitations such as the time-consuming and labor-intensive procedures for vector construction, coxsackievirus-adenovirus receptor (CAR)-dependent gene transfer, immunologic side effects, lack of tissue specificity, lack of regulation of gene expression, etc. In this paper, I review our approach to the development of advanced recombinant Ad vectors. The next generation of Ad vectors have not only become promising vectors for gene therapy but also important tools for gene transfer into mammalian cells.

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The multidrug-resistant transporter encoded by the MDR1 gene belongs to the ATP-binding cassette superfamily of membrane transporters. It is involved not only in the acquisition of multidrug-resistance phenotypes in cancer cells but also in normal tissues such as the brain, kidneys, liver, and intestines. This transporter has the potential to export unnecessary or toxic exogenous substances or metabolites, and in the intestine it is thought to play a role in limiting the oral absorption of a number of structurally unrelated drugs. In 2000, Hoffmeyer et al. performed a systemic screening for MDR1 polymorphisms and suggested that a single-nucleotide polymorphism (SNP) in exon 26 of the MDR1 gene (C3435T) was associated with a lower level of intestinal MDR1 expression, and thereby with lower plasma concentrations of digoxin after oral administration. At present, over 20 SNPs have been found in the MDR1 gene. Clinical studies on the effects of C3435T on MDR1 expression and function in the tissues, and consequently on the pharmacokinetics, have been performed worldwide. In this review, the latest reports concerning the relationship of MDR1 genotypes with pharmacokinetics and MDR1 expression are summarized. Our experimental results demonstrate the importance of genetic polymorphisms at positions 3435 and 2677 in the MDR1 gene on pharmacokinetics and intestinal MDR1 expression. In the future, haplotype analysis of the MDR1 gene and subsequent classification of subjects are needed for individualized pharmacotherapy based on MDR1 genotyping.

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Drug-plasma protein binding analysis is indispensable for drug development and clinical use. However, conventional methods for binding analyses were not suitable for small amounts of proteins because of large sample requirements. On the other hand, high-performance frontal analysis/capillary electrophoresis (HPFA/CE) consumes very small sample volumes, and is useful for ligand-binding study of small amounts of proteins. In this study, HPFA/CE was used in a drug-binding study of α₁-acid glycoprotein (AGP) subtypes in which plasma concentrations change dynamically to elucidate the effects of structural variation on drug binding. Binding study on desialyrated AGP revealed that (S)-enantiomer selectivity in propranolol-AGP binding was caused by sialic acid residues, while neither sialic acid nor galactose caused the enantioselectivity of verapamil binding to AGP. Biantennary glycans slightly suppressed disopyramide binding to AGP, whereas the glycans did not have any influence on propranolol and verapamil binding. Disopyramide and verapamil were selectively bound to the A variant rather than the F1S variant. The A variant showed larger enantioselective binding to disopyramide, but not to verapamil.

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One of the most indispensable biological functions for all living organisms is the circadian clock, which acts like a multifunctional timer to regulate the homeostatic system including sleep and wakefulness, hormonal secretions, and various other body functions in a 24-hour cycle. Recent molecular dissections of the circadian biological clock system have revealed that oscillation in the transcription of specific clock genes plays a central role in the generation of circadian rhythms. Several drugs can affect the expression of clock genes, resulting in alteration of the 24-hour rhythms in physiology and behavior. Here, we report the disruptive effect of interferon (IFN) on the core circadian oscillation mechanism. Treatment of cultured hepatic cells with IFN-α caused a significant reduction in Clock and Bmal1 mRNA levels, which are positive regulators of circadian output rhythm, leading to a decrease in their protein levels. The continuous administration of IFN-α significantly decreased CLOCK and BMAL1 protein levels in the suprachiasmatic nucleus and liver of mice, thereby preventing oscillations in the expression of clock and clock-controlled output genes. These findings reveal a possible pharmacologic action of IFN-α on the core circadian oscillation mechanism and indicate that the disruptive effect of IFN-α on circadian output function is the underlying cause of its adverse effects on 24-hour rhythms in physiology and behavior. Furthermore, the alteration of clock function, a new concept of adverse effects, can be avoided by altering the dosage schedule of IFN-α to minimize the adverse drug effect on clock gene expression. One approach for increasing the efficacy of pharmacotherapy is administering drugs at the time of day when they are best tolerated. Attention should be paid to the alteration of clock gene expression, and it should be considered an adverse effect when it leads to altered circadian organization of the molecular clockwork which is a serious problem affecting basic function of living organisms.

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We studied the cause of cracking of clinically used polyurethane (PU) catheters during the constant infusion of etoposide (VP-16) injection (Lastet^® inj.) without dilution. After the vehicles used for VP-16 injection, ethanol or polyethylene glycol 400 (PEG400), were infused into the PU catheters at a constant infusion rate (30ml/h) for 24h, obvious degradation of the internal wall of the catheter was observed under an electron microscope. When the PU catheter was immersed in ethanol for 24h, condensed polymers of 1,4-butanediol (BD), contained in PU catheters as an elasticizer, were detected in the ethanol elute using the ESI/MS method. Moreover, time-dependent elution of BD from PU catheters with the infusion of ethanol into the catheter for 24h at 30ml/h was observed using the GC/MS method. The cumulative amount of BD eluted from the PU catheter with ethanol vehicle for 24h was 130μg. In conclusion, degradation and subsequent cracking of PU catheters during the infusion of VP-16 injection were caused by ethanol and PEG400 contained in the injection solution. Furthermore, to prevent the elution of BD from PU catheters, we suggest that PU catheters should not be used for the administration of VP-16 injection without dilution in consideration of safety and efficacy.

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From 2001 to the summer of 2002, more than 800 cases of liver damage were reported in Japan among people taking Chinese diet aids. The Japanese Ministry of Health, Labor and Welfare has recently announced that N-nitrosofenfluramine was the hepatotoxic compound contained in the diet aids based on animal experiments performed by the National Institute of Health Sciences. Although N-nitrosofenfluramine is a derivative of fenfluramine, a previously used antiobesity drug, neither pharmacologic nor toxicologic properties have been reported for N-nitroso fenfluramine.
It should be noted that N-nitrosofenfluramine has two optical isomers, although it is not yet known which isomer damages the liver and other organs. The Japanese Ministry of Health, Labor and Welfare has not commented on this point. Pursuing this question, 10 types of Chinese slimming aid samples including those obtained from patients with fulminating hepatitis were analyzed by NMR, GC/MS, and a newly established HPLC method using a chiral separation column. It was found that the N-nitrosofenfluramine in all of the toxic diet aids was the (S)-isomer form. No (R)-isomer was detected. These results strongly suggest that the nitroso-compound in the diets must be prepared from pharmacologically active (S)-fenfluramine (dexfenfluramine). Thus the pharmacologic and toxicologic properties of each isomer should be investigated.

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Midazolam and its active metabolites have a depressant effect on respiration and consciousness level, and therefore their effects should be considered in all patients for whom brain death testing is contemplated. The concentrations of midazolam and its active metabolites were measured in critically ill patients on a ventilator during and after continuous intravenous infusion of midazolam. Three days after cessation of midazolam infusion, the concentrations of midazolam and 1-hydroxymidazolam decreased to below the therapeutic range (100—1000ng/ml) in all patients, although the concentrations of 1-hydroxymidazolam glucuronide remained extremely high in a patient who showed deteriorating renal function. The concentrations of 1-hydroxymidazolam glucuronide (19,497—29,761ng/ml) were measured in this patient. When it is impossible to confirm factors consistent with irreversible brain death, such as the lack of cerebral blood flow, until 3 days after cessation of midazolam infusion, monitoring of the concentration of these substances should be carried out in all patients in whom suspicion exists prior to the evaluation of brain death. It is particularly imperative that monitoring of the 1-hydroxymidazolam glucuronide concentration be carried out in patients with poor renal function.

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We prospectively studied and compared the usefulness of Kampo medicine (Sino-Japanese traditional herbal medicine) “Toki-shakuyaku-san” and an oral iron preparation in the treatment of hypochromic anemia associated with uterine myoma. The study subjects consisted of 25 patients who were diagnosed as having hypochromic mild to moderate anemia associated with menorrhagia attributable to uterine myoma. They were divided into the Toki-shakuyaku-san group (n=10) and the oral iron group (n=15). We monitored the blood counts, subjective symptoms, and occurrence of side effects after oral administration of either preparation for 4 and 8 weeks in these subjects. In regard to the blood counts and improvement of the laboratory parameters of anemia, while marked improvement was observed in the oral iron group, no significant improvement was noted in the Toki-shakuyaku-san group. On the other hand, in terms of improvement of the signs and symptoms of anemia, such as facial pallor, spoon-shaped nails and dizziness, the latter group also showed significant improvement. In addition in the Toki-shakuyaku-san group, resolution of symptoms such as hypermenorrhea, dysmenorrhea, feeling cold, dizziness, headache and shoulder stiffness was also noted. While side effects were encountered in 80.0% of patients in the oral iron group, no significant side effects were observed in the Toki-shakuyaku-san group. From these findings, it is considered that Toki-shakuyaku-san may be useful for resolving the symptoms of mild or moderate anemia associated with uterine myoma.

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