Biomedical subjects
Publications and source records attributed to M Rapp.
To assess the role of the cyclic amide moiety in indolocarbazole and bis-indole protein kinase C inhibitors, five amides in these series were synthesized in which the amide group is acyclic. These new compounds have no inhibitory effect on protein kinase C, indicating that the rigid cyclic structure of the amide group is compulsory for biological activity.
K252c-related compounds were synthesized with different framework flexibilities and different functions (imide, amide and amide-alcohol) on the non-indolic heterocycle. The inhibitory activities towards protein kinase C and protein kinase A are compared.
The efficacy and safety of cefixime, the first oral third-generation cephalosporin, were evaluated in a multicenter clinical trial involving 118 adult patients with acute sinusitis or acute exacerbations of chronic sinusitis. Patients received a single daily dose of 400 mg of cefixime for a mean duration of 10 days; 106 patients completed a course of therapy. Clinical cure and improvement were achieved in 90% of these patients (61% cured and 29% improved). Among the patients evaluated again 2 weeks after therapy, 91% had a sustained clinical cure or improvement. Sinus exudate specimens were obtained from all patients by transantral puncture before therapy. Pathogens were isolated from 76 patients (66%), the most common pathogens being Haemophilus influenzae, alpha-hemolytic streptococci, and Streptococcus pneumoniae. Eighty-six percent of pathogens were presumed eradicated. Three patients discontinued therapy because of side effects. The most frequently reported adverse effects were gastrointestinal, with 20% of patients reporting diarrhea. Cefixime was effective in the treatment of bacterial sinus infections in adults and was well tolerated.
1. The disposition of stiripentol labelled with 14C and 3H on two positions has been studied in the pregnant and non-pregnant female rat after p.o. administration of a 200 mg/kg dose. 2. For both labelled species radioactivity was eliminated mainly in the faeces (69% within 72 h). Urinary excretion was rather low (22% within 72 h). No significant difference was found between the disposition of the two labelled species. 3. For both labelled species concentrations of radioactivity reached a plateau in the plasma and tissues between 1 and 6 h after administration. The liver, fat, mammary gland and adrenal gland were the most extensively-labelled organs. The affinity for the mammary gland was significantly greater in pregnant rats and for the adrenal gland was significantly greater in the non-pregnant rats. The fact that the concentration in the placenta was higher than in the foetus demonstrated that this membrane acts as a barrier for the penetration of the drug in the amniotic fluid. 4. Chromatographic analysis of the faeces and urine showed that an important portion of the dose remained unabsorbed through the gastrointestinal tract. The absorbed fraction undergoes an extensive first-pass metabolism involving mainly the oxidative cleavage of the methylenedioxy ring. Comparison with the results of other work conducted on the non-pregnant rat demonstrated that pregnancy did not affect the disposition and metabolic process.
Since 201Tl, 99mTc-sestamibi and 99mTc-teboroxime concentrate in cardiac cells through different mechanisms, we compared their uptake in cultured normal cells and carcinoma cell lines in order to define their possible use for tumor evaluation in vivo. Four lines of normal cells from animals, including myocytes from newborn rats, and four lines of human carcinoma cell lines were incubated for 1 hr with 37 kBq of either tracer. Results, expressed in percent of the total activity taken up by 1 million cells, showed a 9% difference between the uptake of teboroxime by normal and carcinoma lines (24.6% +/- 2.8% versus 22.5% +/- 2.1%, respectively, p < 0.05). Mean uptake was 80% higher in tumor than in normal cells for 201Tl (5.39% +/- 1.33% versus 3.00% +/- 1.08%, respectively, p < 0.001) and nearly 4 times higher for sestamibi (5.37% +/- 2.34% versus 1.44% +/- 1.88%, p < 0.001). For both agents, uptake by the myocytes and carcinoma cells was comparable (5.14% +/- 0.11% for 201Tl and 5.28% +/- 1.03% for sestamibi). When the myocytes are excluded from the group of normal cells, the uptake is 112% higher in tumor than in normal cells for 201Tl (5.39% +/- 1.33% versus 2.54% +/- 0.44%, p < 0.001) but it becomes nearly nine times higher for sestamibi (5.37% +/- 2.34% versus 0.60% +/- 0.23%, p < 0.001). It is concluded that these experiments show that the uptake of sestamibi was the most discriminant to separate between normal and malignant cells, while teboroxime was the less discriminant. Potential clinical applications for tumor visualization based on differences in sestamibi and teboroxime uptake could be envisioned.
Fluoxetine is a potent and specific serotonin re-uptake inhibitor and an effective antidepressant drug. Male mice were treated with either fluoxetine (8 mg/kg body weight per day) or saline. Wheel running activity was monitored for 2 weeks in a 12:12 LD cycle followed by 2 weeks in constant darkness (DD). Fluoxetine significantly shortened free-running circadian period for wheel running activity (23.93 +/- 0.08 h for fluoxetine treated mice versus 24.17 +/- 0.07 h for saline treated mice; p less than 0.03). These results are consistent with a role for serotonin in the regulation of circadian period in mice.
Several cationic or neutral technetium-nitrido complexes of Schiff bases [for which 5-methyl 3-(2-hydroxyphenyl methylene) dithiocarbazate (L1) was a prototype], bis-aminoethanethiol (BAT-TM) and macrocyclic amines were prepared. We report here, the synthesis and isolation of these TcNLn complexes from reaction mixtures by high pressure liquid chromatography or sephadex G25 column. In vivo tissue distribution studies were performed on tumor bearing mice (B16, EMT6, 3LL) after i.v. injection of 10-20 microCi of TcNLn. Although pharmacokinetic differences appeared between the three studied ranges, we did not obtain proof of any particular specificity to tumor tissues. Nevertheless, the complexes were very stable and some of the ligands could be used as chelators after linking side chains or groups inducing specific tumor localization.
The gene encoding glycoprotein B (gB) of murine cytomegalovirus (MCMV) strain Smith was identified, sequenced, and expressed by recombinant vaccinia virus. The gB gene was found adjacent to the polymerase gene, as it is in the genome of human cytomegalovirus (HCMV). The open reading frame consists of 2,784 nucleotides capable of encoding a protein of 928 amino acids. Comparison with gB homologs of other herpesviruses revealed a high degree of homology. The similarity between the MCMV gB and the HCMV gB is most prominent, since 45% of the amino acids are identical. In addition, all cysteine residues are at homologous positions, indicating a similar tertiary structure of the two proteins. In contrast to HCMV, the MCMV gB mRNA is a true late transcript. A recombinant vaccinia virus expressing the MCMV gB gene has been constructed (Vac-gB). Antibodies raised against the Vac-gB recombinant precipitated proteins of 130, 105, and 52 kDa from MCMV-infected cells. The identity of the MCMV gB with the major envelope glycoprotein of MCMV described by Loh et al. was shown (L. C. Loh, N. Balachandran, and L. F. Qualtiere, Virology 166:206-216, 1988). Immunization of mice with the Vac-gB recombinant gave rise to neutralizing antibodies.
Urine samples were collected from five Brown Swiss cows during the 18 days prior to and 11 days after parturition and were analysed for 19-nortestosterone using an enzyme immunoassay. Nortestosterone concentrations ranged from 70 to 130 nmol/l in all samples taken before parturition. The levels declined within two days, and 11 days post partum no nortestosterone was detectable. In urine from newborn calves, maximal nortestosterone concentrations were determined during the first day of life (10.9-120 nmol/l), declining below 7.3 nmol/l until day 3 in most animals and remaining below the detection limit (less than 3.6 nmol/l) after day 8 in all animals. There was no obvious difference between cows carrying a male or a female calf nor between newborn male or female calves. Using the combined methods high performance liquid chromatography/enzyme immunoassay and high performance liquid chromatography/gas chromatography-mass spectrometry, the immunoreactivity in urine was identified to be 19-nortestosterone-17 alpha. Although there is unequivocal evidence for the endogenous production of nortestosterone in pregnant cows, its function for placenta physiology, pregnancy anabolism and parturition remains unclear. However, new threshold levels for residue control of nortestosterone need to be fixed in accordance with the endocrine status of the animals.
Different biological aspects of a novel 2-chloroethyl nitrosourea derived from cysteamine, N'-(2-chloroethyl)-N-[2-(methylsulfinyl)ethyl]-N'- nitrosourea (CMSOEN2), were studied. Drug-induced cytotoxic effects, uptake kinetics, DNA damage, and O6-alkylguanine-DNA alkyltransferase activity were determined in 3 melanoma cell lines: the murine B16 and 2 human metastatic-derived cell lines (M4 Beu and M3 Dau). We found that radioactivity uptake and incorporation in acido-precipitable material was inversely proportional to cell drug viability. The highly CMSOEN2-sensitive B16 line showed the lowest total radioactivity uptake. In fact, among the melanoma cell parameters studied, 3 of them were well correlated: (a) cytotoxicity as reflected by the colony-forming assay; (b) DNA cross-link frequency estimated by the alkaline elution technique; and (c) O6-alkylguanine-DNA alkyltransferase activity (Mer phenotype), defined as the ability of cell extracts to remove O6-methylguanine from N-methyl-N-nitrosourea-alkylated DNA. The 2 human cell lines (M4 Beu and M3 Dau), the most resistant to the cytostatic drug effects, showed little or no ability to form DNA lethal cross-links. These results correspond to the higher O6-alkylguanine-DNA alkyltransferase activity found in human-derived cell lines compared with that present in murine B16 cell lines. This study confirms that the cell content in this repair DNA protein is certainly one of the important factors implicated in the variability of response to 2-chloroethyl nitrosourea treatment observed in a number of established malignant cell lines. It has been shown that pretreatment of derived cell lines with methylating agents (N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine) or O6-methylguanine used as a free base, increased cytotoxic effects of this class of anticancer agents, likely by saturating receptor sites (sulfhydryl groups) of this specific DNA repair enzyme. Nevertheless, in preliminary Phase I and II clinical trials, 2 patients who had been treated with multiple chemotherapies including alkylating agents [1-(2-chloroethyl)-3- cyclohexyl-1-nitrosourea, 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide, platinum derivatives], presented complete or partial remission after CMSOEN2 treatment. Our results raise the question of the exact relation between the Mer phenotype determined in derived murine or human cultured cells and that directly observed on surgically excised tumors in cancer patients. The original Mer phenotype could be modified by cell culture conditions since it has been shown that O6-alkylguanine-DNA alkyltransferase activity is widely distributed between normal and tumoral tissues without any real difference.
Kidney fat samples from animals either untreated or after different treatments with 19-nortestosterone (NT) were cleaned up and NT-17 beta and 19-norandrostenedione (NA) were purified using high-performance liquid chromatographic (HPLC) systems that allowed their complete separation from other steroids and interfering physiological compounds. HPLC fractions and urine samples were analysed by an enzyme immunoassay that measures both steroids equally well (antigen: NT-17 beta-hemisuccinate-bovine serum albumin). In samples from untreated animals no NT metabolites were detectable (less than 10 pg/g NT-17 beta and less than 10 pg/g NA in fat; less than 0.25 pg/ml NT-17 beta + NT-17 alpha in urine). After adding NT to animal feed the urine became positive, whereas in the fat no residues were detectable. After different injections of NT both the fat and urine were positive. Only after parenteral application did free steroids enter the circulation and adipose tissues.
Certain milk replacers were reported to cause 19-nortestosterone (NT)-positive urine samples after feeding them to veal calves. In order to find the possible source of contamination, milk replacers and crude fat and meat meal from homogenized veal calves and commercial crude fat from a rendering plant were analysed for NT plus its metabolites and constituents of illicit 'cocktails' (NT esters, estradiol benzoate and medroxyprogesterone acetate). The steroids were separated using different high-performance liquid chromatographic systems and measured by specific immunoassays. The results show that animal food processed from carcasses of treated animals contains hormone concentrations that may cause positive urine samples in animals fed on such feed.
In connection with investigations of the anabolic action of estrogens, we examined skeletal muscle of veal calves for estradiol receptors. The high speed supernatant of muscle homogenate was incubated with .5 nM 3H-estradiol and for the determination of nonspecific binding with .5 nM 3H-estradiol plus 13 nM estradiol at 0 C overnight. After treatment with charcoal two times, the supernatant was analyzed by agar gel electrophoresis. Specific binding was found in the typical position of cytosolic estradiol receptor. Ninety percent of 3H-estradiol binding was suppressed by estradiol-17 beta, zeranol, estrone or diethylstilbestrol, but was not affected by testosterone, dihydrotestosterone, trenbolone or progesterone. The specific binding activity varied between .3 and 2.0 fmol/mg protein and the dissociation constant of the receptor was Kd = 60 pM. After an enrichment up to 42 fmol/mg cytosolic protein using heparin sepharose, the receptor remained unchanged as determined by agar gel electrophoresis. Although uterine tissue generally contains 1,000 times more estradiol receptors, these results clearly demonstrate that skeletal muscle also contains estradiol receptors with identical properties. This indicates that one possible component of the anabolic action of estrogens may be the direct stimulation of the muscle via the estradiol receptor.
In the hybrid mouse strain BDF1, some 35--40% of spermatocytes had unpaired X and Y choromosomes in stages ranging from diplotene to first meiotic metaphase. This phenomenon varied significantly from mouse to mouse. In pooled material from Swiss Albino and CF1 mice, the corresponding frequency was 5.7%. In C57 BL/6 mice, one of the parent strains of BDF1 mice, the X and Y were separate in 7.7% of the spermatocytes. Based on the behavior of the X and Y in the BDF1 strain, it is concluded that they do not pair, rather than initially pairing and then precociously separating. The factor interfering with the pairing of the X and Y does not affect the autosomes; possibly it is an incompatibility of the two sex chromosomes, which come from different inbred lines.
The effects of the cancer drug methylhydrazine derivative procarbazine (PC) on the chromosomes of normal and malignant cells in the mouse strains Swiss Albino, CF1, and BDF1 have been analyzed. PC broke chromosome in the hypotetraploid Ehrlich and the diploid P388 ascites tumors. Practically all breaks seemed to be of the G2 type and, as far as could be determined, took place in the quinacrine-dark or the corresponding Giemsa-light regions. The vast majority of the broken ends had rejoined to form chromatid translocations. No increase in chromosome breaks were seen in any of the normal tissues treated in vivo: spleen, bone marrow, spermatocytes, or spermatogonia (in this tissue the cells were not counted). The chromosomes of the Ehrlich ascites tumor have been studied by means of both Q-banding and G-banding. Very few, if any, chromosomes seem to have an exact homologue or to correspond to any of the normal mouse chromosomes.
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As part of a cartilage targeting program based on the affinity of the quaternary ammonium (QA) moiety for cartilage, QA derivatives of D-glucosamine (DG), an antirheumatic drug exhibiting a natural tropism for cartilaginous tissues, were designed and evaluated by pharmacokinetic studies. Two QA-DG conjugates were synthesized and labeled with (14)C by cross-linking the QA entity (trimethylammonium or pyridinium) to [(14)C]DG via an amide bond in a two-step procedure. After intravenous injection to male Sprague-Dawley rats, the two (14)C-labeled conjugates exhibited similar pharmacokinetic profiles, but their behavior clearly differed from that of unconjugated DG in several ways. (i) The tissue distribution for the conjugates was more restricted, with a decreased radioactivity level for whole tissues except for kidney, cartilage, and skin. (ii) The radioactivity concentrated more rapidly and strongly in cartilage for the conjugates than for DG for the short times after injection; on the other hand, 1 h after administration, the radioactivity level in cartilage was higher for DG, this result being consistent with the tropism already observed for this compound. (iii) Both conjugates were eliminated predominantly by the urinary route (85%); the radioactivity level in urine for DG was lower (45% of the injected dose), and significant (14)CO(2) was found in expired air, indicating metabolization and utilization of DG for energy-consuming processes. (iv) Blood and plasma kinetics studies displayed an enterohepatic cycle for DG, whereas for the QA conjugates, a rapid disappearance was observed. (v) HPLC analyses of plasma and urine indicated a low degree of metabolization for the conjugates, most of the radioactivity recovered in urine and plasma corresponding to the unchanged molecule. This study demonstrates that the introduction of the QA moiety on DG modifies its biodistribution and lends it a greater specificity for cartilage, at least for short times after injection. These findings justify further work on QA derivatives of other antirheumatic agents.
Procedures for the determination of medroxyprogesterone acetate (MPA) in adipose tissue collected at time of slaughter allowing the control of MPA application to veal calves are described. Screening radioimmunoassays after sample clean-up were sufficient for a first survey of MPA treatments in livestock herds. Validation of all positive samples was performed by two-dimensional (silica gel diol phase and RP-18 phase) HPLC/RIA immunograms. Megestrol acetate and melengestrol acetate with cross-reactivities of 31% and 0.3% respectively were clearly separated by the RP HPLC. With an absolute detection limit of 4 pg MPA/tube (90% relative binding) negative control samples did not exceed 6 pg/tube, equivalent to 6 pg/g fat in the validating method. Seventeen days after intramuscular (i.m.) injection of 24 mg MPA only 32 pg MPA/g fat were found, while i.m. injection of 60 mg MPA and a waiting period of 19 days resulted in 2700 pg MPA/g fat. After feeding two calves 20 micrograms MPA per head daily for 1 week followed by 200 micrograms MPA per head daily for 2 weeks 359 and 468 pg MPA/g fat were measured. In plasma as well as in adipose tissue more than 80% of the whole immunoreactive material was MPA itself, without indications for the presence of cross-reacting MPA metabolites as confirmed by HPLC/RIA immunograms. Based on day of slaughter ratios of accumulation of MPA from plasma into fat of MPA-fed veal calves were 52 and 72 respectively. In urine MPA was only detectable a few days after injection; as compared to a plasma concentration of 950 pg MPA/ml the amount in urine was only 37 pg MPA/ml and also 325 pg unidentified MPA-equivalents/ml.