Non-enzymatic reduction of aliphatic tertiary amine N-oxides mediated by the haem moiety of cytochrome P450

1. The mechanism of reduction of aliphatic tertiary amine N-oxides to tertiary amines in liver microsomes was examined and a novel type of reduction by cytochrome P450 was found. 2. Rat liver microsomes exhibited a significant N-oxide reductase activity toward brucine N-oxide and imipramine N-oxide in the presence of both NAD(P)H and FAD under anaerobic conditions. These N-oxide reductase activities were inhibited by carbon monoxide or air. However, the activities were not abolished by boiling the microsomes; indeed, in the case of brucine N-oxide, the activity was enhanced. 3. The activity toward brucine N-oxide was also observed after the conversion of cytochrome P450 to cytochrome P420. Cytochrome P4502B1 alone exhibited the reductase activity in the presence of both NAD(P)H and FAD. After the removal of haem from cytochrome P4502B1, the activity was observed in the haem moiety, but not in the cytochrome P450 apoprotein. 4. Photochemically reduced FAD was effective in the reduction in place of NAD(P)H and FAD. 5. The N-oxide reduction appears to proceed non-enzymatically, catalysed by the haem group of cytochrome P450 in the presence of a reduced flavin.     

Enzymatic and non-enzymatic reduction of brucine N -oxide by aldehyde oxidase and catalase

1. Brucine N -oxide was reduced by aldehyde oxidase in rabbit liver cytosol in the presence of an electron donor, 2-hydroxypyrimidine, under anaerobic conditions. The flavoprotein purified from rabbit liver exhibited significant reductase activity in the presence of electron donors. 2. Brucine N -oxide was also reduced by rabbit liver cytosol and blood in the presence of both a reduced pyridine nucleotide and FAD under anaerobic conditions. The N -oxide reductase activities were inhibited by carbon monoxide and air. However, these activities were not abolished when liver cytosol and blood were boiled. Rabbit erythrocytes exhibited the reductase activity, but not plasma. 3. When liver cytosol or blood was separated by DEAE-cellulose column chromatography, the fractions with the reducing activity in the presence of both NADH and FAD also showed catalase activity. 4. Catalase catalysed the brucine N -oxide reduction in the presence of both NAD(P)H and FAD. Hematin also exhibited the reductase activity in the presence of both NAD(P)H and FAD. Photochemically reduced FAD was effective in the reduction instead of NAD(P)H and FAD. 5. Brucine N -oxide reduction proceeds via two routes in liver cytosol and blood. One is enzymatic reduction by aldehyde oxidase; the other is non-enzymatic reduction catalysed by the haem group of catalase in the presence of reduced flavin.     

Investigation of alternative compounds to poly(E-MA) as a polymeric surfactant for preparation of microcapsules by phase separation method

Various water-soluble polymers were used to examine an alternative emulsifier for poly(ethylene-alt-maleic anhydride), used in the preparation of crosslinked polyurea microcapsules. Microcapsules were successfully prepared by using the water-soluble polymers with large molecular weight alternating copolymers, namely poly(olefin-maleic anhydride), poly(olefin-maleic acid), and poly(acrylic acid). On the other hand, no microcapsule resulted from olefin-maleic acid with small molecular weight alternating copolymers. From these results, the following guidelines were obtained for the selection of polymeric surfactants suitable for crosslinked polyurea microcapsule. A polymeric surfactant must have maleic acid or a carboxyl group in order to form a crosslinked polyurea microcapsule membrane. Furthermore, to form a stronger capsule membrane it is desirable to have a maleic anhydride group. It is also important for membrane formation that the polymeric surfactant has a suitable molecular weight.     

Rizatriptan (Maxalt), a new entity of triptan for migraine: pharmacology and therapeutic relevance

Rizatriptan is a highly potent, selective serotonin 5-HT(1B/1D)-receptor agonist. Current theories on the mechanism of migraine suggest the central role of vasodilation of intracranial, extracerebral blood vessels and activation of perivascular trigeminal sensory nerves. There abundantly exist 5-HT(1B) receptors in meningeal blood vessels and 5-HT(1D) receptors in the trigeminal ganglia. The therapeutic activity of rizatriptan in migraine can most likely be attributed to agonist effects at 5-HT(1B/1D) receptors on these target sites. Two types of the 10 mg formulation, a tablet (Maxalt) tablet) and an orally disintegrating tablet (Maxalt)RPD tablet), are available. The latter may have a clinical relevance for patients who administer it without liquid. Pharmacokinetic study demonstrated the approximate T(max) of 1.0 or 1.1 h in tablets and 1.3 h in RPD tablets, resulting in early onset for headache relief and also pain free. Bioavailability was estimated to be about 45%. The efficacy and good tolerability and underlying profiles of pharmacokinetics of rizatriptan are almost similar between Japanese and other races, and a reduction in headache response up to 2 h can be attained in a large majority of patients. Several reports have described the favorable clinical profile of rizatriptan in comparison to other triptans. Rizatriptan is thus effective and provides migraine sufferers with an appropriate quality of life.     

Over-expressed Bcl-2 cannot suppress apoptosis via the mitochondria in buprenorphine hydrochloride-treated NG108-15 cells

We previously reported that the morphine alkaloid derivative buprenorphine hydrochloride (Bph) induces rapid apoptosis in NG108-15 nerve cells accompanied by the activation of caspase-3. Here, we found this kind of apoptosis was also accompanied by rapid loss of the mitochondrial membrane potential, followed by the efflux of cytochrome c from the mitochondria to the cytosol and the activation of caspases-9 and -3. Together, these results strongly suggested the Bph death signal was routed through the mitochondrial pathway in NG108-15 cells. In these cells, serum-starvation induces a different apoptosis, which we exploited to investigate Bcl-2's role as an apoptosis inhibitor. We made an NG108-15 transfectant, Bcl-2(P2), that stably expressed human Bcl-2, and used it to test Bcl-2's effect on the serum-starvation-induced apoptosis in NG108-15 cells. Cell viability, DNA-ladder formation, and efflux of cytochrome c from the mitochondria were all detected, showing that the human Bcl-2 functioned normally in the Bcl-2(P2) cells. Although the apoptotic events tested were identical in the parental cells and transformants, Bcl-2 expression completely failed to inhibit Bph-induced apoptosis in the Bcl-2(P2) cells.     

19F NMR spectroscopic study on the binding of triflupromazine to bovine and human serum albumins

The 19F NMR spectrum of triflupromazine hydrochloride (TFZ) in a buffer solution (pH 6.8) showed a single sharp signal of the TFZ CF3 group at 13.5 ppm from the external trifluoroacetic acid. The addition of 1 mM HSA or BSA to the sample solution caused a split of the CF3 signal into two broadened signals shifted to slightly lower (0.2 ppm) and higher (0.7 ppm) fields, respectively, from the original position. Denaturation of the albumins by guanidine hydrochloride (3M) restored the two broadened signals to a slightly broadened single signal, indicating that TFZ has at least two binding sites on HSA and BSA, respectively. From the competitive binding 19F NMR experiments using Warfarin (Site-I ligand), l-tryptophan (Site-II ligand), NaCl, and oleate, the signal at high field was assigned to the TFZ bound to Site II. Comparison of the signal intensity revealed that the affinity of TFZ for Site II on HSA was considerably higher than that on BSA. The low-field signal could be identified as a weight-averaged signal between nonspecifically bound TFZ to HSA (BSA) and free TFZ in the water phase. In the presence of physiological concentrations of NaCl, major binding of TFZ to HSA and BSA was considered to be nonspecific. The present work indicates that 19F NMR is very useful for obtaining important detailed information regarding the binding of fluorinated drugs to serum albumins.     

Behavioural and serological human immunodeficiency virus risk factors among female commercial sex workers in Cambodia

BACKGROUND: The spread of human immunodeficiency virus (HIV) in Cambodia is mainly caused by sexual transmission and the high-risk group in this country are female commercial sex workers (CSW). There are two types of CSW, direct CSW (DCSW) and indirect CSW (IDCSW), who are different from each other in sexual activities. This study was conducted in order to describe the risk factors on HIV for each type of CSW, and to establish effective preventive strategies against the HIV epidemic among CSW. METHODS: The participants, 143 DCSW and 94 IDCSW, were interviewed using a questionnaire to determine their demographic characteristics and behaviour. Blood samples were taken for serological tests on HIV, Chlamydia trachomatis and syphilis. The association between their behavioural pattern and their serological results was analysed. RESULTS: The questionnaire study showed that IDCSW had a riskier behavioural pattern than DCSW. The HIV seroprevalence rates of the DCSW and the IDCSW were 52.4% and 22.3%, respectively. Univariate logistic analyses showed a significant association between HIV antibody (HIV-Ab) and current age, age at commencement of commercial sex work, duration of commercial sex work, and the seropositivity of Chlamydia trachomatis-IgG antibody (CT-IgG-Ab) among the DCSW. The analyses also showed a significant relationship between HIV-Ab and CT-IgG-Ab among the IDCSW. CONCLUSIONS: Improving condom use rate is very important in order to prevent an HIV epidemic among the two types of CSW. This study also suggests it is important to prevent sexually transmitted disease (STD) such as Chlamydia trachomatis infection. The STD control programme could be efficient for HIV prevention, especially among DCSW.     

Early pregnancy blood lead and spontaneous abortion

Although evidence tends to suggest that high levels of lead exposure increase the risk of spontaneous abortion, we do not yet know whether moderate- to low-level exposure elevates risk. Among 351 women (aged 16 to 35 years, with single pregnancies) who were registered for a longitudinal study, 15 (4.3%) women experienced spontaneous abortion after the 12th week of gestation and before the 20th week. We collected participants' blood samples during the first trimester of pregnancy (8-12 weeks) for lead measurement by inductively coupled plasma-mass spectrometry. Mean ± standard deviation of blood lead was 3.8 ± 2.0 μg/dl (range 1.0-20.5 μg/dl) with a geometric mean of 3.5 μg/dl. Mean blood lead concentrations did not differ significantly between spontaneous abortion cases and ongoing pregnancies (3.51 ± 1.42 and 3.83 ± 1.99 μg/dl, respectively). The findings suggest that in apparently healthy women, low blood lead levels (mean < 5 μg/dl) measured in early pregnancy may not be a risk factor for spontaneous abortion.