Assay of zofenopril and its active metabolite zofenoprilat by liquid chromatography coupled with tandem mass spectrometry

Zofenopril is a pro-drug designed to undergo metabolic hydrolysis yielding the active free sulfhydryl compound zofenoprilat, which is an angiotensin converting enzyme (ACE) inhibitor, endowed also with a marked cardioprotective activity. A simple, highly sensitive specific LC–MS–MS method was developed for the determination of zofenopril and zofenoprilat in human plasma. In order to prevent oxidative degradation of zofenoprilat and its internal standard, their free sulfhydryl groups were protected by treatment with N-ethylmaleimide (NEM), which produced the succinimide derivatives. The compounds and their corresponding fluorine derivatives, used as internal standards, were extracted from plasma with toluene. The reconstituted dried extracts were chromatographed and then monitored by a triple-stage-quadrupole instrument operating in the negative ion spray ionization mode. The method was validated over the concentration range of 1–300 ng/ml for zofenopril and 2–600 ng/ml for zofenoprilat. Inter- and intra-assay precision and accuracy of both zofenopril and zofenoprilat were better than 10%. The limit of quantitation was 1 ng/ml with zofenopril and 2 ng/ml with zofenoprilat. Extraction recovery proved to be on average 84.8% with zofenopril and 70.1% with zofenoprilat. Similar recoveries were shown by the above two internal standards. The method was applied to measure plasma concentrations of zofenopril and zofenoprilat in 18 healthy volunteers treated orally with zofenopril calcium salt at the dose of 60 mg.     

Hierarchy of effects of the MHC and T cell receptor beta-chain genes in susceptibility to Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Intracerebral inoculation of susceptible mice with Theiler's murine encephalomyelitis virus induces a demyelinating disease that is similar to human multiple sclerosis. This murine model for human multiple sclerosis is apparently immune-mediated and the genes involved in the immune response influence the outcome of disease susceptibility as observed with human multiple sclerosis. These genes include the MHC and TCR genes. However, the functional relationships among these genes on the disease susceptibility has not yet been studied. In this study, we demonstrate that the effect of the H-2s genotype from susceptible SJL/J mice overrides the resistant effect of the BALB/c TCR beta-chain gene in CXJ recombinant-inbred and BALB.S congenic mice. These results strongly suggest the presence of a hierarchy of genes involved in the immune response in Theiler's murine encephalomyelitis virus-induced demyelinating disease.     

Specific immune regulation of chronic-relapsing experimental allergic encephalomyelitis in mice

These studies were designed to examine immunologic means of regulating the clinical course of murine chronic-relapsing experimental allergic encephalomyelitis (R-EAE). We asked whether induction of specific immune tolerance to the major CNS myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), could inhibit the development of R-EAE. Neuroantigen-specific tolerance was induced in SJL/J mice in a dose-dependent manner by the i.v. injection of mouse spinal cord homogenate-coupled syngeneic splenocytes (MSCH-SP) on day -7 relative to immunization on days 0 and +7. Sham-tolerized controls developed significant MBP- and PLP-specific DTH responses before the onset of clinical R-EAE. In contrast, MSCH-SP tolerized mice exhibited a dramatically reduced incidence of clinical and histologic signs of disease which correlated with the failure to develop MBP- and PLP-specific DTH responses. In 10 separate experiments, 118/149 (79%) of control mice, but only 22/137 (16%) of tolerized mice developed clinical R-EAE. Tolerance took time to develop and lasted at least 4 wk as mice injected with Ag-coupled splenocytes on day -1 relative to immunization remained susceptible to R-EAE, whereas mice injected on days -7, -14, or -28 were resistant. Tolerance induction required neuroantigens as injection of splenocytes coupled with a syngeneic mouse kidney homogenate failed to significantly alter the incidence of R-EAE or the development of neuroantigen-specific DTH responses. Thus, induction of R-EAE can be specifically and significantly regulated after the i.v. injection of splenocytes coupled with a crude, heterogeneous mixture of neuroantigens (i.e. MSCH).     

Immunocytochemical localization of collagen types I,III, IV,and fibronectin in the human dermis

Summary The distribution of collagen types I, III, IV, and of fibronectin has been studied in the human dermis by light and electron-microscopic immunocytochemistry, using affinity purified primary antibodies and tetramethylrhodamine isothiocyanate-conjugated secondary antibodies. Type I collagen was present in all collagen fibers of both papillary and reticular dermis, but collagen fibrils, which could be resolved as discrete entities, were labeled with different intensity. Type III collagen codistributed with type I in the collagen fibers, besides being concentrated around blood vessels and skin appendages. Coexistence of type I and type III collagens in the collagen fibrils of the whole dermis was confirmed by ultrastructural double-labelling experiments using colloidal immunogold as a probe. Type IV collagen was detected in all basement membranes. Fibronectin was distributed in patches among collagen fibers and was associated with all basement membranes, while a weaker positive reaction was observed in collagen fibers. Ageing caused the thinning of collagen fibers, chiefly in the recticular dermis. The labeling pattern of both type I and III collagens did not change in skin samples from patients of up to 79 years of age, but immunoreactivity for type III collagen increased in comparison to younger skins. A loss of fibronectin, likely related to the decreased morphogenetic activity of tissues, was observed with age.     

Effect of liposome encapsulation on antigen presentation in vitro. Comparison of presentation by peritoneal macrophages and B cell tumors

We have studied the effect of liposome encapsulation of pigeon cytochrome-c (PCC) on its processing and presentation by peritoneal exudate cells and B-cell hybridomas to antigen specific T cells in vitro. Encapsulation of PCC in liposomes modestly increased the presentation when the macrophage was the presenting cell but totally eliminated presentation when the B cell hybridoma was the presenting cell as determined by the IL-2 response. Using radiolabeled PCC, the increased presentation by the macrophage was correlated with an increase in the uptake and rate of processing of the liposomal antigen. The elimination of presentation by the B cell was due to the inability of this cell type to internalize and degrade the encapsulated PCC. The results support the concept that the macrophage is the primary cell type involved in the initial stages of an immune response to a liposome encapsulated protein Ag in vivo.     

Twenty-Four-Hour Variations in the Sensitivity of Rat Aorta to Vasoactive Agents

The presence of time-dependent variations in the in vitro sensitivity of aorta preparations to either vasoconstricting or relaxing agents was investigated in rats maintained in light from 08: 00 to 20: 00 and in darkness from 20: 00 to 08: 00. Rat thoracic aorta rings were obtained from animals sacrificed at four different times of the day. The rat aorta was found to be more sensitive to the constricting effect of phenylephrine at 15: 00, and of 5-hydroxytryptamine at 21: 00. On the other hand, both endothelium-dependent and -independent relaxations were more remarkable at 03: 00 than at other times of the day. These variations represented significant circadian rhythms when analyzed by analysis of variance. Different in vitro responsiveness to these agents might reflect changes in the sensitivity and/or number of related receptors in vascular preparations. In conclusion, the circadian time of animal sacrifice to obtain vascular preparations constitutes an important aspect of the research method and a key determinant of findings. (Chronobiology International, 13(6), 465-475, 1996)