Gastric proton pump inhibitors (PPIs) are substituted benzimidazole prodrugs that require an acid-induced activation. Its rate depends on the reactivity of the molecule relative to the environmental pH and determines the drug’s tissue selectivity. Factors affecting the exposure of moderately acidic tissues to the activated PPI are the area under the serum concentration-time curve (AUC), serum protein binding, the partition coefficient logP and the serum elimination half-life relative to the chemical activation half-life at a critical tissue pH of about 5. These parameters have therefore been determined in a comparative fashion in the present study. The data shows that pantoprazole is less likely to undergo unwanted activation at moderately acidic targets as opposed to the parietal cell, compared to omeprazole. Actually, although 40 mg pantoprazole (steady state) gave a slightly higher serum AUC of the total parent compound than 40 mg omeprazole (10.5 vs. 7.1 µmol × h × l–1), a higher serum protein binding of pantoprazole versus omeprazole (98 vs. 96%) reversed the AUC values for the free drug in favor of a lower value for pantoprazole (0.19 vs. 0.28 µmol × h × 1–1). It is the free parent compound that equilibrates across cell membranes to be activated in acidic tissue compartments. At pH 5.1, the activation half-life of pantoprazole was 4.7 versus 1.4 h for omeprazole, the latter being in the order of the common serum elimination half-life determined in an intraindividual comparison (1.24 vs. 1.25 h). Thus, pantoprazole is eliminated faster from blood than it is activated at a pH of about 5, while omeprazole is as quickly activated at this pH as it is eliminated from blood. Biological in vitro experiments confirmed that pantoprazole displays a lower liability to interfere with unwanted biological targets. This has been demonstrated in vitro for inhibition of both renal Na+/K+-ATPase, lysosomal acidification and the production of reactive oxygen species by neutrophils.
BackgroundRecombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs.ResultsWe explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs) were targeted by Flp recombinase mediated cassette exchange (RMCE). The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context.ConclusionRMCE provides a powerful method to specifically design vectors for optimized gene expression with high accuracy. Upon considering the specific requirements of chromosomal sites this method provides a unique tool to exploit such sites for predictable expression of biotechnologically relevant proteins such as antibodies.
The present review will verify by intra-study rank orders, and their comparison between studies, that the different gastric proton pump inhibitors (PPIs) display similar dose-response relationships with similar potencies and efficacies on a milligram basis, i.e., at the same milligram doses. This is in line with their basic pharmacology which suggests that, primarily, the serum AUCs of the free pro-drugs and their chemical activation half lives at pH 1 relative to their serum elimination half lives determine the efficacies of PPIs. According to the literature, these drug characteristics are similar for all PPIs. Although PPIs have been introduced into the therapy of acute peptic ulcer disease at different daily, oral doses of 20 mg (omeprazole and rabeprazole), 30 mg (lansoprazole) and 40 mg (pantoprazole), the data suggest that the optimal dose of lansoprazole, omeprazole and pantoprazole, with respect to the acute treatment of peptic ulcers and moderate to severe gastroesophageal reflux disease (GERD), is about 30–40 mg daily. The data base of rabeprazole appears to be too small at present to make any definite statement. Lower daily doses of the PPIs of about 15–20 mg are sufficient in less severe cases of GERD and in maintenance therapy. It appears that different dose recommendations were based on different strategies to balance optimal drug dosage and safety, rather than on real differences in milligram-related efficacies.
The novel antiulcer drugs omeprazole, lansoprazole, and pantoprazole are members of the class of substituted benzimidazoles. They potently inhibit the gastric proton pump by a common mechanism which depends on the acid-induced conversion of the parent compounds to the pharmacologically active principles: thiophilic cyclic sulfenamides. This transformation takes place in the luminal compartment of the secreting parietal cell. However, while the three proton pump inhibitors belong to the same chemical class, their two ring systems bear different functional substituents. This leads to essential modification of the physicochemical, metabolic, and pharmacokinetic properties of these drugs, possibly resulting in differences in tissue selectivity and thereby, in the long term, drug safety. Both preclinical and clinical data have accumulated that point to advantages of pantoprazole related to the above parameters: pantoprazole shows a higher stability at moderately acidic pH values and less inhibitory potential against cytochrome P450 than the other two drugs. In addition, pantoprazole displays linear pharmacokinetics with a high bioavailability.
CD4+CD25+ regulatory T cells (Tregs) represent a specialized subpopulation of T cells, which are essential for maintaining peripheral tolerance and preventing autoimmunity. The immunomodulatory effects of Tregs depend on their activation status. Here we show that, in contrast to conventional anti-CD4 monoclonal antibodies (mAbs), the humanized CD4-specific monoclonal antibody tregalizumab (BT-061) is able to selectively activate the suppressive properties of Tregs in vitro. BT-061 activates Tregs by binding to CD4 and activation of signaling downstream pathways. The specific functionality of BT-061 may be explained by the recognition of a unique, conformational epitope on domain 2 of the CD4 molecule that is not recognized by other anti-CD4 mAbs. We found that, due to this special epitope binding, BT-061 induces a unique phosphorylation of T-cell receptor complex-associated signaling molecules. This is sufficient to activate the function of Tregs without activating effector T cells. Furthermore, BT-061 does not induce the release of pro-inflammatory cytokines. These results demonstrate that BT-061 stimulation via the CD4 receptor is able to induce T-cell receptor-independent activation of Tregs. Selective activation of Tregs via CD4 is a promising approach for the treatment of autoimmune diseases where insufficient Treg activity has been described. Clinical investigation of this new approach is currently ongoing.
Shewanella oneidensis COAG, a hyper-aggregating mutant of MR-1, was isolated from a rifampicin-challenged culture. Compared to the wild-type, COAG exhibited increased biofilm formation on glass carrier material. The role of surface-located proteins in the process of COAG auto-aggregation was confirmed by different proteolytic treatments of the aggregates. All of the tested proteolytic enzymes resulted in deflocculation within 3 h of incubation. In order to examine the altered expression of outer-membrane proteins in COAG, membrane-enriched cell preparations were analysed by proteomics and the protein pattern was compared to that of MR-1. From the proteomics results, it was hypothesized that the agglutination protein AggA, associated with the secretion of a putative RTX protein, was involved in the hyper-aggregating phenotype. These results were confirmed with a DNA microarray study of COAG versus MR-1. An insertional mutation in the S. oneidensis COAG aggA locus resulted in loss of the hyper-aggregating properties and the increased biofilm-forming capability. The insertional mutation resulted in strongly decreased attachment during the initial stage of biofilm formation. By complementing this mutation with the vector pCM62, expressing the aggA gene, this effect could be nullified and biofilm formation was restored to at least the level of the MR-1 wild-type.
To define the basic antisecretory profile of a potassium-competitive antagonism of the gastric acid pump relative to other classes of acid-inhibitory drugs, they were compared to each other against all three major stimuli of acid secretion. Pumaprazole is an imidazo-pyridine derivative that was used in this investigation as an example of reversibly binding acid pump antagonists (APAs). It differs from covalently binding proton pump inhibitors (PPIs), such as omeprazole, both with respect to chemical structure and mode of interaction with the gastric H+/K+-ATPase (i.e., the acid or proton pump). The present data show that a single dose of pumaprazole is able to elevate intragastric pH in the dog with gastric fistula under pentagastrin or carbachol stimulation from pH 1 to about pH 7 while still displaying a dose-dependent, well-controlable duration of action of a few hours. Ranitidine at the same oral dose also shows a short duration of action, but combined with a far lower efficacy. By contrast, a single oral dose of the PPI omeprazole elevates intragastric pH for a longer time period, but this pH elevation is far lower compared to that of the APA. Regarding the less stringent parameter of inhibition of total acid output in the Heidenhain pouch dog, the modified Shay rat or the Ghosh-Schild rat, pumaprazole is, overall, slightly more efficacious than ranitidine. The M1-muscarinic antagonist pirenzepine is ineffective (against histamine stimulation) or far less effective than pumaprazole (against pentagastrin-stimulation), but as effective as pumaprazole against carbachol stimulation in the Ghosh-Schild rat. Basal acid output in the same model is more effectively inhibited by pumaprazole than by ranitidine. In conclusion, our data demonstrate the exceptional ability of a reversibly binding APA to elevate intragastric pH up to neutrality even upon a first administration while still displaying a limited, dose-dependent duration of action.
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