Hexobarbital, a short-acting hypnotic, is metabolized to 3′-hydroxyhexobarbital by cytochrome P450, and then to 3′-oxohexobarbital by liver cytosolic dehydrogenase. New methods of separation for hexobarbital and its metabolites by TLC have been developed and applied to study the metabolism of hexobarbital enantiomers and stereoselective metabolism of hexobarbital. (+)-Hexobarbital preferentially was transformed into β-3′-hydroxyhexobarbital and the (−)-enantiomer preferentially transformed into α-3′-hydroxyhexobarbital by rat liver microsomes. Glucuronidation and dehydrogenation of 3′-hydroxyhexobarbital were also stereoselective and the S-configuration at the 3′-position was preferred. α-3′-Hydroxyhexobarbital from (−)-hexobarbital and the β-isomer from (+)-hexobarbital were shown to be preferentially conjugated with glucuronic acid in rabbit urine, and to be preferentially dehydrogenated to form 3′-oxohexobarbital by rabbit and guinea pig 3-hydroxyhexobarbital dehydrogenases. A new metabolic pathway of hexobarbital was found in which 3′-oxohexobarbital reacts with glutathione to form 1,5-dimethylbarbituric acid and a cyclohexenone-glutathione adduct, a novel metabolite. 1,5-Dimethylbarbituric acid was excreted into the urine and the cyclohexenone-glutathione adduct into the bile of rats dosed with hexobarbital. 3-Hydroxyhexobarbital dehydrogenases that dehydrogenate 3-hydroxyhexobarbital into 3′-oxohexobarbital were purified from the liver cytosol of rabbits, guinea pigs, goats, rats, mice, hamsters, and humans and characterized. These enzymes were monomeric proteins and had molecular weights of about 34500—42000, and used NAD+ and NADP+ as cofactors, except for the human enzyme that had a molecular weight of about 58000 and used NAD+ alone. Each enzyme exhibited its own characteristics. Substrate specificity demonstrated that 3-hydroxyhexobarbital dehydrogenases dehydrogenate not only α,β-unsaturated cyclic and acyclic secondary alcohols but also some 17β-, 3α-hydroxysteroids or both, except for the human enzyme. The amino acid sequence of the hamster enzyme indicated that it belongs to the aldo-keto reductase superfamily and hydroxysteroid dehydrogenase subfamily.
This paper describes research performed in the Laboratory of Organic Chemistry, Showa Pharmaceutical University. Oxidation reactions involving the oxidase can be divided roughly into two kinds of reactions: The first involves electron removal from an aromatic ring or an active CH-bond. The other reaction involves hydrogen abstraction from an inactive CH-bond. The oxidase models, Fe(DMF)3Cl21+ and Fe(AN)63+/AN, which we have synthesized, have been shown to work by the former mechanism, and the models Fe(AN)63+-IO4-/AN, Fe(AN)62+-Ac2O-H2O2/AN, Fe(AN)62+-2PAH-5Py-Ac2O-H2O2/AN, Fe(PA)3(OH2)-H2O2/AN and Fe(PA)3(OH2)-O2-electrolysis/AN do so by the latter mechanism. Further, we found some iron(II or III)picolinate-H2O2/AN complexes have the 7α-hydroxylase-like activity.
To characterize energy metabolism in brown adipose tissue (BAT), differential screening of a cDNA library of rat BAT with a cDNA probe of rat white adipose tissue was carried out. We isolated one novel cDNA clone encoding a protein of 88.2 kDa consisting of 772 amino acids. The deduced amino acid sequence showed the highest homology (62.6%) with that of rat liver carnitine palmitoyltransferase I (CPTI). The transcript corresponding to this cDNA was abundantly expressed not only in BAT but also in the heart and skeletal muscle. CPTI is a protein necessary for the β-oxidation of long-chain fatty acids in mammalian mitochondria, and it has been suggested that at least two isoforms, the liver type and muscle (M-CPTI) type, exist. Based on these observations, we concluded that the novel cDNA clone isolated from rat BAT encodes M-CPTI. Isolation and characterization of a genomic DNA clone revealed that the gene for human M-CPTI consists of two 5′-noncoding exons, 18 coding exons, and one 3′-noncoding exon spanning approximately 10 kbp, and a gene encoding choline/ethanolamine kinase-β (CK/EK-β) was located about 300 bp upstream from the M-CPTI gene with the same strand direction. Furthermore, we found atypical transcripts containing exons of both CK/EK-β and M-CPTI genes in humans and rodents. The physiologic role(s) of these transcripts is still unknown. However, it is interesting that such transcripts are produced from two tightly arranged and functionally unrelated genes in mammalian tissues.
Owing to the increasingly globalized nature of the cyclodextrin (CyD)-related science and technology, development of the CyD-based pharmaceutical formulation is rapidly progressing. The pharmaceutically useful CyDs are classified into hydrophilic, hydrophobic, and ionic derivatives. Because of the multi-functional characteristics and bioadaptability, these CyDs are capable of alleviating the undesirable properties of drug molecules through the formation of inclusion complexes or the form of CyD/drug conjugates. This review outlines the current application of CyDs in drug delivery and pharmaceutical formulation, focusing on the following evidences. 1) The hydrophilic CyDs enhance the rate and extent of bioavailability of poorly water-soluble drugs. 2) The amorphous CyDs such as 2-hydroxypropyl-β-CyD are useful for inhibition of polymorphic transition and crystallization rates of drugs during storage. 3) The delayed release formulation can be obtained by the use of enteric type CyDs such as O-carboxymethyl-O-ethyl-β-CyD. 4) The hydrophobic CyDs are useful for modification of the release site and/or time profile of water-soluble drugs with prolonged therapeutic effects. 5) The branched CyDs are particularly effective in inhibiting the adsorption to hydrophobic surface of containers and aggregation of polypeptide and protein drugs. 6) The combined use of different CyDs and/or pharmaceutical additives can serve as more functional drug carriers, improving efficacy and reducing side effects. 7) The CyD/drug conjugates may provide a versatile means for the constructions of not only colonic delivery system but also site-specific drug release system, including gene delivery. On the basis of the above-mentioned knowledge, the advantages and limitations of CyDs in the design of advanced dosage forms will be discussed.
Excessive production of reactive oxygen species (ROS) may lead to oxidative stress, loss of cell function, and cell death by apoptosis or necrosis. Recently, ROS have gained attention as important second messengers. ROS lifetimes can be very short, and many types of ROS cannot penetrate organelle membranes. It is therefore thought that only ROS signal molecules that are generated locally in an organelle are transduced when cells are stimulated. Lipid hydroperoxides are one type of ROS of which the biological function has not yet been clarified. The phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) is a unique antioxidant enzyme and separately distributed to the mitochondria, nucleus, nucleoli, and cytosol, where it regulates phospholipid hydroperoxide and fatty acid hydroperoxide as signal molecules. This review focuses on the structure and biological functions of PHGPx in mammalian cells. Overexpression of different types of PHGPx in the RBL2H3 cell line provides a useful model system with which to clarify the ability of different types of PHGPx to modulate cellular function and the importance of lipid hydroperoxides as signal molecules. Transformant studies show that lipid hydroperoxide is an activator of lipoxygenase and cyclooxygenase and participates in inflammation, cardiolipin hydroperoxide is the signal molecule for the release of cytochrome c during apoptotic cell death, and PHGPx is a signal regulator in the IgE receptor-mediated signaling pathway. It is becoming clear that PHGPx has an important role in spermatogenesis, sperm function, and embryonic development, and its deficiency is implicated in human infertility and in embryonic lethality of PHGPx knockout mice.
Exposure of cells or organs to sublethal stress induces the expression of some heat-shock proteins (Hsp), including Hsp70. In porcine renal LLC-PK1 cells, heat stress (HS) elevates Hsp70 expression and Na+-dependent glucose transport. We examined whether Na+-dependent glucose transporter (SGLT1) interacts with Hsp70 to elevate SGLT1 activity and whether SGLT1 activation is involved in the recovery from HS injury. HS (42°C for 3 h) elevated SGLT1 activity and expression of SGLT1 in the apical membrane fraction. HS increased the maximal transport rate (Vmax), but did not affect the apparent affinity constant (Km) for glucose. The HS-induced SGLT1 activation was inhibited by anti-transforming growth factor (TGF)-β1 antibody. Furthermore, transfection of anti-Hsp70 antibody into the cells inhibited SGLT1 activation. These results suggest that HS induces TGF-β1 secretion, and then Hsp70 forms a complex with SGLT1 and increases the distribution of SGLT1 in the apical membrane. Next, we examined the effect of HS on plasma membrane integrity. Accumulation of calcein, a membrane-impermeable fluorescent dye, was decreased by HS and then returned to basal level. This recovery was inhibited by phloridzin, a selective SGLT inhibitor, and nonmetabolizable glucose analogues. Anti-TGF-β1 antibody also inhibited the recovery of calcein accumulation. Taken together, the present results show that HS elevates SGLT1 activity mediated via the TGF-β1 signaling pathway and that the increase in glucose uptake is necessary to repair plasma membrane injury.
In order to design an injectable formulation of E5531, a novel synthetic disaccharide analog of novel lipid A, for the treatment of septic shock, a 'pH-jump method' was developed. In this method, E5531 was dispersed in 0.003 mol/l NaOH (pH 11.0, above pKa2) at 50°C (above phase transition temperature) and then mixed with a buffer to neutralize the pH to 7.3. E5531 was dispersed as particles, and the size was approximately 20 nm. The structure of the particles was vesicular. After dispersal, the solution was sterilized using a filter, filled aseptically into vials, and lyophilized. The size of the particles did not change before and after lyophilization. The relationship between the physicochemical properties of the particles and the pharmacokinetics in rats after intravenous administration was investigated. The membrane fluidity of the particles was affected by the dispersal methods, the dispersal time in 0.003 mol/l NaOH in the pH-jump method, and the addition of Ca2+ to the solution. The membrane fluidity was correlated with the pharmacokinetics in rats.
Recently, combination treatment with cisplatin has been recommended as chemotherapy for lung cancer. However, no clinical pathway for safe and efficient use of anticancer agents has been established. We devised a clinical pathway satisfying evidence-based medicine (EBM) criteria by analyzing case records and the relevant literature. We analyzed 73 case records of hospitalized patients who had undergone chemotherapy for lung cancer on the internal medicine ward of the Showa University Hospital. Grade 3 or higher toxicities of leukopenia, thrombocytopenia, anemia, vomiting, and diarrhea occurred in 30%, 51%, 14%, 5%, 8%, and 1% of patients, respectively. Therefore the checklists for these toxicities were included in the clinical pathway. The National Cancer Institute Common Toxicity Criteria were used for the evaluation of toxicities. According to the guidelines of the American Society of Clinical Oncology and the US Infection Society, the indicated agents and criteria for their use were chosen for supportive cancer treatment. Pharmacists, physicians, and nurses collaborated in making the clinical pathway safe and sufficiently easy for practical use. The final version of the clinical pathway is compatible with EBM and includes items required for safe chemotherapy, which could be helpful in risk management.
When Escherichia coli is cultured in nutrient broth at 1×103 cells/ml, 7.5 h are needed to detect cell growth as turbidity. The time to detect E. coli in this medium is reduced using the alamar blue method. Alamar blue is an oxidation-reduction indicator that changes color in response to cell growth. E. coli can grow in nutrient broth containing red blood cells, but the detection of E. coli based on turbidity is difficult because the red blood cells muddy the medium. As the change in color in the alamar blue method is not affected by red blood cells, the growth of E. coli contaminating red blood cells is detectable. These results suggest that a cell growth indicator such as alamar blue is useful for the detection of bacteria contaminating blood for transfusion.
We conducted a randomized, controlled study to evaluate whether pharmacists' advice on smoking cessation would result in a higher smoking cessation rate using Nicorette (nicotine gum preparation). Fourteen pharmacies in Tokyo, Kanagawa, and Nagano participated. Smokers who visited pharmacies to buy Nicorette from March 1, 2002, through August 31, 2002, were recruited and randomly assigned to two groups. For the intervention group (A), pharmacists provided both regular instructions on Nicorette use and smoking cessation advice at the first sale and then gave follow-up advice just before starting a cessation and 1, 3, and 8 weeks and 3 months thereafter. For the control group (B), pharmacists provided regular instructions alone. The primary outcome measure was the self-reported smoking cessation rate and the secondary outcome measure was the relationship between the smoker's egogram and effectiveness of intervention. Twenty-eight smokers were enrolled and randomized into group A (n=11) or group B (n=17). The absolute abstinence rate in groups A and B at 3 months was 45.5% and 31.2%, respectively. The odds ratio was 1.83, which was not statistically significant. There was no difference in egogram score between absolute abstinence subjects and nonabstinence subjects in group A. The egogram scores in Adapted Child of absolute abstinence subjects in group B were significantly higher than in nonabstinence subjects. In conclusion, instructions and advice given by pharmacists may improve the smoking cessation rate in smokers receiving nicotine replacement therapy.
With the recent rapid shift in pharmaceutical education to the development of clinical experts, emphasis on education in humanism and communication has increased. However, there is a lack of experience in these fields of pharmaceutical education in Japan, and there have been few studies on the curriculum, from admission to the pharmaceutical science to the stage before on-the-job training. Also our previous survey of communication-related education revealed there is no consensus on the interpretation of communication-related education. In this study, therefore, we investigated communication-related education is incorporated before on-the-job training at 46 schools of pharmaceutical science in Japan. Communication-related education was carried out at 26 (56.5%) of the 46 schools, and role-playing was incorporated in the program at 23 (88.5%) of these 26 schools. However, SP (simulated patient/standardized patient) was adopted at 12 (46.2%) of these 26 schools. There was a psychologist or a communication specialist on the staff at only 10 (38.5%) of these 26 schools, revealing the lack of instructors in these fields. Interest in education related to communication was generally weak at national and public universities, and marked differences in the approach to pharmaceutical education among university types were observed. The preparation of basic guidelines and textbooks for stepwise communication education from lower to higher grades and the training of instructors are urgently needed.