Identification of human drug-metabolizing enzymes involved in the metabolism of SNI-2011.

In vitro studies were conducted to identify human drug-metabolizing enzymes involved in the metabolism of SNI-2011 ((+/-)-cis-2-methylspiro [1,3-oxathiolane-5,3'-quinuclidine] monohydrochloride hemihydrate, cevimeline hydrochloride hydrate). When 14C-SNI-2011 was incubated with human liver microsomes, SNI-2011 trans-sulfoxide and cis-sulfoxide were detected as major metabolites. These oxidations required NADPH, and were markedly inhibited by SKF-525A, indicating that cytochrome P450 (CYP) was involved. In a chemical inhibition study, metabolism of SNI-2011 in liver microsomes was inhibited (35-65%) by CYP3A4 inhibitors (ketoconazole and troleandomycin) and CYP2D6 inhibitors (quinidine and chlorpromazine). Furthermore, using microsomes containing cDNA-expressed CYPs, it was found that high rates of sulfoxidation activities were observed with CYP2D6 and CYP3A4. On the other hand, when 14C-SNI-2011 was incubated with human kidney microsomes, SNI-2011 N-oxide was identified as a major metabolite. This N-oxidation required NADPH, and was completely inhibited by thiourea, indicating that flavin-containing monooxygenase (FMO) was involved. In addition, microsomes containing cDNA-expressed FMO1, a major isoform in human kidney, mainly catalyzed N-oxidation of SNI-2011, but microsomes containing FMO3, a major isoform in adult human liver, did not. These results suggest that SNI-2011 is mainly catalyzed to sulfoxides and N-oxide by CYP2D6/3A4 in liver and FMOI in kidney, respectively.

Effective administration route for the deleted form of hepatocyte growth factor To exert its pharmacological effects.

The pharmacokinetics and the pharmacological effects of the deleted form of hepatocyte growth factor (dHGF) after intravenous (iv), subcutaneous (sc), or intramuscular (im) administration (0.25 and 2. 5 mg/kg) were studied in rats. After single iv administration (2.5 mg/kg), dHGF in serum rapidly decreased (alpha- and beta-phase half-life: 3.2 and 26.5 min, respectively). Two to four hours after single sc or im administration (2.5 mg/kg), the serum level of dHGF reached a maximum and then gradually declined (half-life: 2.7 h). The serum levels were not changed by repetitive iv administration, but were dramatically decreased by repetitive sc or im administration. Liver weight and serum levels of total protein, albumin, and HDL-cholesterol were significantly increased by iv administration of dHGF (twice daily for 4 days at 0.25 mg/kg). Sc or im administration of dHGF did not increase these parameters at the same dose, but did significantly at 2.5 mg/kg. These observations suggest that iv administration is the most effective in exerting the pharmacological effects of dHGF among three administration routes. dHGF after iv administration was distributed mainly and rapidly into liver (53.6% of the injected dHGF within 5 min) and was sustained at a higher level in the liver than in plasma. In infusion (0.5 mg/kg/3 h), dHGF level in plasma and liver reached a steady-state 15 and 60 min after starting the infusion, respectively. The steady-state level of dHGF was 7- to 9-fold higher in liver than in plasma, and the higher level in liver was sustained beyond the steady-state.

Preservation of Mycobacterium leprae in vitro for four years by lyophilization.

Although the viability of Mycobacterium leprae suspended in distilled water with or without 10% fetal calf serum was reduced approximately 10(-2) to 10(-4) from that of the starting material during the process of lyophilization, bacilli capable of multiplication in nude mouse foot pads were found in the lyophilized samples stored for 4 years at 4 degrees C. The multiplication rate of the lyophilized bacilli which were suspended in 10% serum-water was much higher than that of the bacilli suspended in water only. On the other hand, no reduction of the viability of M. leprae suspended in 10% skim milk-water was demonstrated during the process of lyophilization as well as storage for 2 years at 4 degrees C. From the results obtained here, it could be suggested that M. leprae might be preserved in vitro by means of lyophilized M. leprae was extremely stable during cryopreservation when the bacilli were suspended in 10% skim milk-water. Therefore, the composition of the solution for suspending the bacilli is definitely critical for the maintenance of M. leprae viability by means of lyophilization.

[Effects of partial nephrectomy on the pharmacokinetics and pharmacodynamics of recombinant human erythropoietin (SNB-5001) in rats].

The present study examined the pharmacokinetic and pharmacodynamic profiles of recombinant human erythropoietin (SNB-5001) in partially nephrectomized rats. The plasma level of SNB-5001 was measured by a 2-step enzyme immunoassay. The plasma disappearance curve after intravenous injection of SNB-5001 (50 U/kg) in these rats showed a biexponential pattern similar to that in non-treated rats, conforming to a two-compartment model. However, the total body clearance was reduced, the plasma half life was prolonged and the area under the concentration-time curve of SNB-5001 was increased by the partial nephrectomy. The distribution volumes of SNB-5001 were almost the same as those in non-treated rats. It is suggested that the kidney may contribute to the elimination of SNB-5001. Dose-dependent increases of reticulocytes, red blood cells, hemoglobin and hematocrits were observed after seven repetitive intravenous injections of SNB-5001 in both partially nephrectomized rats and non-treated rats. Hemopoietic responses were calculated by subtracting the initial values from the values after SNB-5001 injections of each hematological parameter (reticulocytes, red blood cells, hemoglobin and hematocrits). Hemopoietic responses in partially nephrectomized rats were apparently stronger than those in non-treated rats. These results suggest that the reduction of clearance by the partial nephrectomy may contribute to the hemopoietic responses, in addition to suggesting that the uremic conditions do not inhibit the effects of SNB-5001 in partially nephrectomized rats.

Effects of cyclosporin A on bacterial growth and immunological responsiveness in BALB/c mice infected with Mycobacterium leprae.

When BALB/c mice were infected with Mycobacterium leprae and orally treated 6 times weekly with a dose of 8 mg/kg cyclosporin A (CsA) for 19 months, the number of organisms was slightly higher at 19 months as compared with mice in which the dose of CsA was gradually decreased after 6 months and discontinued at the 8th month (p less than 0.01 for the 15th and 19th months). Lymphocyte blast transformation (LBT) showed that spleen cells from CsA-treated mice 4 weeks after infection with M. leprae and 3 weeks after CsA treatment was stopped responded to the sonicated supernatant of M. leprae suspension (SS), M. leprae (Ml), and concanavalin A (ConA) less than those cells from mice not treated with CsA. This response was dose-dependent. At week 15, 14 weeks after CsA administration was stopped, the LBT response to SS and Ml by cells from M. leprae-infected mice exceeded that of mice without CsA treatment, and the response to ConA in M. leprae-infected mice was less than that in uninfected mice without CsA-treatment. Thus, if CsA was administered, the T-cell functions were suppressed. However, when CsA treatment was discontinued for longer periods, the T-cell function was activated. From these results, we speculate that M. leprae would have the capability of growing more abundantly in mice treated with CsA 100 mg/kg for 1 week every month.

[Acid-fast bacilli isolated from foot pads of nude mice infected with leprosy bacilli].

When leprosy bacilli grown in nude mouse foot pad were used for culture experiments, cultivable acid-fast bacillus was sometimes isolated as a contaminant. Whenever bacilli were inoculated to nude mice, the same leprosy bacilli were killed by autoclaving and were inoculated in to foot pads of 5 nude mice for examination of this cause of the contamination. Acid-fast bacillus was cultivated on 3% Ogawa egg medium at 33 degrees C from homogenates of foot pads of nude mice infected with M. leprae after one year and a while of infection. Foot pad of nude mouse injected with leprosy bacilli was cut off, ground in mortar and passed through sterile absorbent cotton and the filtrate was centrifuged at 10,000 rpm for 30 minutes. The sediment was inoculated on 3% Ogawa egg medium after treating with a small amount of sterile 1 N sodium hydroxide. Acid-fast bacilli were isolated from 3 out of 41 mice inoculoted with heat killed bacilli. The isolated acid-fast bacillus did not be observed in the same experimental group inocudated with live bacilli, positive cases were scattered in another groups. Four out of 16 tubes were positive for acid-fast bacilli in mice infected with Kurume-naha and 5 out of 7 tubes in the Amami-KM infected mouse group. The two negative tubes were discarded due to contamination. Kurume-Oki strain which has yellow colonial morphology was isolated from one out of 6 culture tubes. Strains Kurume-naha and Amami-KM have the same characteristics as follows: slow grower with pale yellow smooth colonial morphology, strongly positive for niacin production and ureas; positive for nicotinamidase, pyradinamidase and 68 degrees C catalase; no growth at 45 degrees C, negative for nitrate reduction, hydrolysis of Tween 80, diamine oxidase, heat stable acid-phosphatase and arylsulphatase; resistant to streptomycin, isoniazid, rifampicin and B 663. Two isolates were identified as Mycobacterium simiae from these characteristics. Characteristics of a Kurume-Oki isolate was as follows: slow grower with yellow smooth colonial morphology, positive for urease, 68 degrees C catalase, hydrolysis of Tween 80 and arylsulfatase; no growth at 45 degrees C, negative for niacin production, nicotinamidase, pyradinamidase, nitrate reduction, daimine oxidase and heat stable acid-phosphatase; resistant to streptomycin, isoniazid, rifampicin and B. 663. This bacillus was identified as Mycobacterium gordonae from these characteristics.(ABSTRACT TRUNCATED AT 400 WORDS)

Overproduction, affinity purification and characterization of 65-kDa protein of Mycobacterium leprae in Escherichia coli.

The 65-kDa protein of Mycobacterium leprae was produced in an Escherichia coli strain carrying a plasmid harboring the recloned gene coding for the protein. The protein was purified through affinity chromatography prepared with the IgG fraction of a monoclonal antibody which was prepared against the 65-kDa protein. The purified 65-kDa protein also reacted immunologically with the monoclonal antibody IIIE9, which recognizes the epitope for M. leprae, prepared by Buchanan, et al. BALB/c mice were inoculated with M. leprae and 4 months later were skin tested with the purified 65-kDa protein. Gross changes were observed at the skin-test site. The role of the protein in protective immunity against M. leprae foot pad infection in mice was also studied.

Effect of lyophilization on viability of Mycobacterium leprae multiplied in nude mouse.

In an effort to preserve Mycobacterium leprae in vitro, the effect of freezing and drying, i.e., lyophilization, on viability of M. leprae was studied. The viability of the bacilli was quantitatively measured with foot-pad inoculation method using nude mouse. The results obtained demonstrate that the viability of M. leprae was reduced approximately 10(-2) to 10(-3) from that of the starting material, during the process of lyophilization; no viable bacilli were detected in the lyophilized sample containing less than 1.8 X 10(3) bacilli. On the other hand, the bacilli capable of multiplication in nude mouse foot-pads were found in the lyophilized sample with more than 10(5) bacilli. From the results obtained here, it could be suggested that there might be a possibility to preserve M. leprae in vitro by means of lyophilization.

Multiplication of Mycobacterium leprae in the nude mouse, and some applications of nude mice to experimental leprosy.

Two aspects of the immune deficiency of nude mice make these animals particularly useful tools for leprosy research. Nude mice are capable of supporting multiplication of M. leprae to levels approaching 10(10) per g in peripheral body tissues. In addition, nude mice may be inoculated with greater than 10(4) (in fact, with as many as 10(8) organisms per foot pad, without provoking an immune response that prevents multiplication of the organisms. Thus, the nude mouse should be particularly suitable for detecting persisting M. leprae in treated patients, and as a model of the patient for evaluating chemotherapeutic regimens.

Serum concentrations of rutoside metabolites after oral administration of a rutoside formulation to humans.

In the present study a highly specific and sensitive method by gas chromatography-mass spectrometry has been established for the determination of the blood levels of four metabolites of 5,7,3',4'-tetrahydroxyflavonol-3-rutinoside (rutoside, rutin), i.e. 3,4-dihydroxytoluene (DHT), 3-hydroxyphenylacetic acid (mPHAA), 3,4-dihydroxyphenylacetic acid (DHPAA), and 3-methoxy-4-hydroxyphenylacetic acid (homovanillic acid, HVA) after the oral administration of rutoside to healthy volunteers. By the established method the pharmacokinetics in the blood and the urinary excretion of those metabolites were investigated. Blood levels of DHT, mHPAA, DHPAA, and HVA started to increase at 4 to 8 h after the oral dosage of the rutoside formulation, Esberiven (further active ingredient: coumarin). At 8 to 12 h post-administration, blood levels reached a maximum level which was 2- to 3-fold the time 0 level. Blood levels decreased gradually afterwards and returned to the original level at 20 to 35 h. The sum of the four metabolites was at a maximum value at 8 h which then returned to the initial levels at 35 h yielding a half-life of 11 h. Total urinary excretion of metabolites was 50.5% of the dose in 48 h.

Identification of cat leprosy bacillus grown in mice.

Cat leprosy bacilli passaged in mice could be isolated on 1% Ogawa yolk medium. The isolated cat leprosy bacilli which were cultivated successively four times on 1% Ogawa yolk medium produced a leproma in mice. All characteristics of the isolated cat leprosy bacillus were the same as isolated murine leprosy bacillus, as follows: slow grower, light yellowish-white rough colony, production of much coproporphyrin on the medium, heat-resistant catalase negative, heat-resistant phosphatase negative, arylsulfatase negative, niacin negative, hydrolysis of Tween 80 negative, urease negative, nicotinamidase positive, pyrazinamidase positive, cytochrome b1 at 560 nm positive, cytochrome a2 at 630 nm positive, and cytochrome c at 550 nm negative. Cats are susceptible to both cat and murine leprosy bacilli; the bacilli produced a leproma in a newborn cat at 3 to 4 months and in an adult cat at 2 months after inoculation. Many globi of acid-fast bacilli (AFB) were observed in the histopathological sections and the smear preparations of the newborn cat's lepromas, especially in the necrotic areas of the lepromas. Many AFB and polymorphonuclear leukocytes were seen in the histopathological sections and the smear preparations of the adult cat's lepromas. These lepromas formed ulcers by autolysis and healed or absorbed without ulcer formation over the course of months. Large lepromas remained for a long time without ulcer formation and caseation in some cats. Secondary infections with cat and murine leprosy bacilli were done respectively to the right and left femoral subcutaneous regions of newborn cats carrying primary lepromas. After one month, granulomas in which many AFB were observed were produced in both infection sites. Cats are susceptible to infection with cat and murine leprosy bacilli; however, the bacilli did not invade progressively to internal organs or other subcutaneous areas. Cat leprosy bacilli which were passaged in the mouse are identical to murine leprosy bacilli.