Bioanalysis of ibrutinib, and its dihydrodiol- and glutathione cycle metabolites by liquid chromatography-tandem mass spectrometry.

Ibrutinib is a targeted covalent inhibitor frequently used for the treatment of various lymphomas. In addition to oxidative metabolism, it is metabolized through glutathione coupling. The quantitative insight into this kind of metabolism is scarce, and tools for quantitation are lacking. The non-oxidative metabolism could prove a more prominent role when oxidative metabolism is impaired. Also, in-vitro studies could over-estimate the effect of CYP450-inhibition. To gain quantitative insight into this relatively unknown biotransformation pathway of the drug we have developed a validated simple, fast and sensitive bio-analytical assay for ibrutinib, dihydrodiol-ibrutinib, and the glutathione, cysteinylglycine and cysteine conjugates of ibrutinib in human plasma. The method emphasizes on simplicity, the thiol-conjugates were synthesized by a simple one step synthesis, followed by LC-purification. Sample preparation was done by a simple protein crash with acetonitrile containing labeled internal standards, evaporation of solvents, and reconstitution in eluent. Finally, the compounds were quantified using UHPLC-MS/MS. The assay was successfully validated in a 0.5-500nM calibration range for all compounds, and also a lower range of 0.05-50 nM was demonstrated for ibrutinib to accommodate for even the lowest trough levels. This assay has a considerably higher sensitivity than previous published assays, with the previous lowest LLOQ being 1.14 nM. Both, ibrutinib, dihydrodiol-ibrutinib and the cysteine conjugate were deemed stable under refrigerated or frozen storage conditions. At room temperature, the glutathione conjugate showed rapid degradation into the cysteinylglycine conjugate in plasma. Finally, the applicability of the assay was demonstrated in patient samples.

Cytomegalovirus induces interstitial lung disease in allogeneic bone marrow transplant recipient rats independent of acute graft-versus-host response.

Interstitial lung disease (ILD) after allogeneic bone marrow transplantation (BMTx) is an important clinical problem in terms of diagnosis, therapy, and pathogenesis. Graft-versus-host disease (GvHD) and cytomegalovirus (CMV) infection seem to be major risk factors for ILD, but their role in the pathogenesis is not well established. We previously reported CMV-induced ILD in allogeneic bone marrow recipient rats, which was prevented by antiviral drug treatment. In this paper, we describe the pathology of ILD in allogeneic bone marrow transplant recipient rats and the relation of ILD with CMV infection and GvHD. Brown Norway rats received an allogeneic (Lewis) BMTx and rat CMV infection, after an allogeneic (Lewis) lung transplantation and immunosuppression. CMV infection was recorded by the amount of infectious virus and viral antigens in the lung. ILD was monitored by the presence of diffuse histopathologic changes in the alveolar septal wall. GvHD was scored by the relative splenic weights and the presence of perivascular infiltrates in the lung. Cells expressing CMV antigens were more numerous in the alveolar septa of the allogeneic recipient lungs than in the syngeneic donor lungs (p < 0.05). The high viral load in the recipient lung of allogeneic BMTx recipient rats was accompanied by diffuse ILD, marked by extensive microvascular damage and congestion of the alveolar septa. ILD was observed neither in the syngeneic donor lung nor in both lungs of mock-infected animals. GvHD was not observed in rat-CMV-infected or in mock-infected animals. Our results indicate that CMV induces microvascular damage, resulting in ILD. This process is independent of GvHD.

The presence of cytomegalovirus nucleic acids in arterial walls of atherosclerotic and nonatherosclerotic patients.

The presence of cytomegalovirus (CMV) nucleic acids was demonstrated in abdominal aortas and femoral arteries of patients with and without atherosclerosis by dot blot and in situ DNA hybridization using a DNA probe derived from immediate early genomic regions. Viral antigens could not be detected by immunohistochemistry and infectious virus could not be recovered from the arterial wall by virus isolation techniques. The high percentage (55%) of vascular wall specimens containing CMV nucleic acids, in atherosclerotic as well as in control material and the location of CMV-containing cells in arteries without gross changes indicative of atherosclerosis suggest that the human arterial wall may be a site of latency for this virus.

The development and characterization of monoclonal antibodies against rat cytomegalovirus induced antigens.

In this paper the development of a battery of approximately 70 mouse monoclonal antibodies (McAbs) to RCMV-induced antigens and their characterization is discussed. Their reactivity with the whole scala of ca. 30 virus specific proteins was tested in an enzyme linked immunoassay (ELISA) whereas their ability to detect RCMV-antigens at different locations of in vitro infected cell cultures and at different stages of infection was tested by immunofluorescence. In order to determine to what specific (viral) protein each of these McAbs is directed against we used an immunoprecipitation technique, followed by SDS-PAGE. Furthermore, neutralizing capacity of each McAb was tested, as well as the immunoglobulin class they belong to. In this manner we defined six categories of monoclonal antibodies on the basis of immunofluorescence aspect. The six categories identify most important viral structural proteins.

Studies on rat cytomegalovirus induced structural and non-structural proteins present at (immediate-)early and late times of infection.

Radioactive-labelled virions and nucleocapsids of rat cytomegalovirus (RCMV) were purified from the supernatant and subcellular fractions of infected rat embryo fibroblasts (REF) and analyzed by SDS-polyacrylamide gel electrophoresis. Nuclear nucleocapsids contain one major protein of 138 kD, which is considered to be the basic invariant structural element of RCMV. Enveloped virions consist of 28 protein species, five of which were clearly identified as glycoproteins (58, 64, 76, 112 and 118 kD). Using pulse labelling procedures on RCMV-infected REF cells, after removal of a previously established translation block by cycloheximide, two RCMV-induced immediate-early (IE) protein species (71 and 85 kD) were both detected in the nuclear and cytoplasmic cell fractions. When pulse-labelling was performed for 16 hours in presence of Actinomycin D, only the 85 kD IE protein was detected in the nucleus. The results indicate that the 85 kD IE polypeptide is of importance in early transcriptional events of viral genes. Protein metabolism studies revealed that late in the course of RCMV infection (at 3 days p.i.) protein synthesis has been dramatically changed. Some cellular proteins are greatly suppressed while other cellular proteins are clearly enhanced. Moreover, active synthesis of 8 new cytoplasmic proteins and 9 new nuclear proteins occurs. Most of these proteins were identified as structural constituents of virions.

Rat cytomegalovirus induces cellular purine and pyrimidine nucleoside kinases in rat embryo fibroblasts and TK- rat-2 cells. Correlations with the antiviral activity of Acyclovir.

Rat cytomegalovirus (RCMV) induces a cytosol thymidine kinase (TK) in G0-phase rat embryo fibroblasts (REF), but not in a TK deficient rat cell line (R-2), though virus titers in both cell types reached comparable levels. The results indicate that TK is neither virus-coded nor is required for a productive infection in R-2 cells. A deoxycytidine kinase (dCK) is induced in either growing or RCMV-infected REF and R-2 cells, suggesting that dCK is essential for both host-cell and viral DNA synthesis. A deoxyguanosine kinase (dGK) is detectable in low concentrations in either growing or G0-phase REF and R-2 cells suggesting that this enzyme is cell-cycle independent. In contrast, RCMV induces high persisting levels of dGK, particularly in R-2 cells, indicating that this enzyme is of crucial importance for viral DNA synthesis. By comparison of thermostabilities and electrophoretic mobilities (Rf for TK, dCK and dGK were 0.12; 0.97; and 0.54, respectively) the enzymes were found to be substrate specific but of cellular origin. In contrast to TK and dCK, only dGK is inhibited by Acyclovir (Ki = 320 microM). It is suggested that RCMV inducable dGK is an important enzyme determining the in vitro anti-CMV activity of Acyclovir.

Rat cytomegalovirus: studies on the viral genome and the proteins of virions and nucleocapsids.

The nucleocapsids (N-capsids) isolated from the nuclei of rat cytomegalovirus (RCMV)-infected rat embryo fibroblasts (REF) are composed of three major proteins: 142 X 10(3) (142K), 40K and 32K mol. wt. Nucleocapsids isolated from the cytoplasmic fraction (C-capsids) are composed of proteins found in N-capsids and five major and seven minor new protein species. Most of the proteins present in C-capsids are found in the extracellular enveloped virions, although the ratios vary. Proteins that are abundantly present, particularly in virions (mol. wt. 125K, 116K, 87K, 79K, 71K, 68K, 62K, 50K, 43K and 28K), are probably the major constituents of the viral envelope. The DNA recovered from extracellular virions was purified to homogeneity and by equilibrium centrifugation in CsCl one density class of 1.716 (+/- 0.001) g/ml was found. Contour length measurements showed one size class of a linear double-stranded DNA corresponding to an average mol. wt. of 144(+/-9) X 10(6) which is in good agreement with data obtained by restriction endonuclease analysis (REA), which yielded mol. wt. values of 132(+/-9) X 10(6) (HindIII), 138(+/-2) X 10(6) (EcoRI) and 137 X 10(6) (BglII). The REA patterns also revealed the presence of 0.25 M and 0.5 M fragments, which might indicate, in analogy with other cytomegalo- and herpesviruses, the existence of four different configurations of the RCMV genome. The infectivity of RCMV DNA was determined in subconfluent REF monolayers. A cytopathic effect characteristic of RCMV was observed 6 days post-transfection and up to 60 plaques/microgram DNA were obtained. Using DNA-DNA filter hybridization the degree of homology between the genomes of RCMV and murine or human CMV was examined. Under stringent conditions (50% formamide) values of 12(+/-2)% and 3(+/-1)% were found whereas under non-stringent conditions (20% formamide) values of 21(+/-2)% and 6 (+/-1)% were obtained, respectively.

Isolation of a cytomegalovirus-like agent from wild rats.

In 8 of 10 wild rats trapped in The Netherlands, an infectious viruslike agent was isolated predominantly from the salivary glands and could be serially passed in laboratory rats. In rat embryo cells a typical cytomegalo-like cytopathic effect was produced. The morphologic and cultural characteristics of the isolated agent were comparable with those of the mouse cytomegalovirus (MCMV). The virus-nucleocapsid had a size of 92 nm and was not ether-resistant. The extracellular nucleocapsids were often enclosed by an outer layer of very variable shape and size. The formation of Fc receptors on cells infected with the rat virus could be demonstrated. The wild rats possessed neutralizing antibodies to the isolated agent. The rat agent grew only in rat embryo fibroblast cells while MCMV grew in rat and mouse embryo cells. The rat agent gave plaques in REF monolayers. Electron microscope studies showed the presence of nucleocapsids in the nucleus.