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Herbert Gritsch,1 Gerald Schrenk,1 Nina Weinhappl,1 Björn Mellgård,2 Bruce Ewenstein,2 Peter L Turecek3,4 1Pharmaceutical Sciences, Baxalta Innovations GmbH, a Takeda Company, Vienna, Austria; 2Rare Genetics and Hematology, Research & Development, Takeda Development Center Americas, Inc, Cambridge, MA, USA; 3Department of Pharmaceutical Sciences, Division of Pharmacology, University of Vienna, Vienna, Austria; 4Plasma Derived Therapies, Research & Development, Baxalta Innovations GmbH, a Takeda Company, Vienna, AustriaCorrespondence: Peter L Turecek, Baxalta Innovations GmbH, a Takeda company, DC-Tower, Donau-City-Straße 7, Vienna, A-1220, Austria, Tel +43 1 20 100 247 2520, Fax +43 1 20 100 247 5733, Email peter.turecek@takeda.comAbstract: Background: Recombinant von Willebrand factor (rVWF, vonicog alfa) is a purified VWF concentrate produced from Chinese hamster ovary cells. rVWF is not exposed to the VWF-cleaving protease ADAMTS13 and so is not subject to proteolytic degradation of large (L) and ultra-large (UL) VWF multimers by that enzyme.Purpose: To compare the structure and function of rVWF with the human plasma-derived VWF [pdVWF] concentrates Haemate P®/Humate-P®, Voncento®, Wilate®/Eqwilate®, and Wilfactin®/Willfact®; to investigate the relationship between VWF multimeric pattern and VWF:ristocetin cofactor (VWF:RCo) activity through population pharmacokinetic (PK) modeling in patients with severe von Willebrand disease (VWD) treated with rVWF.Methods: Analyses included VWF:RCo activity, VWF:collagen-binding activity, VWF:platelet glycoprotein Ib receptor binding, factor VIII (FVIII) binding capacity, and VWF-mediated platelet adhesion under flow conditions. VWF multimeric structure was determined by agarose gel electrophoresis. Population PK models describing the activity-time profile of small, medium, and L/UL multimers following intravenous administration of rVWF in patients with severe VWD were developed.Results: Findings demonstrate that rVWF contains a non-degraded VWF multimer pattern including the UL multimers not present in pdVWF concentrates. rVWF displayed higher specific platelet-binding activity, and faster mediation of platelet adhesion to collagen under shear stress versus pdVWF concentrates. rVWF also demonstrated higher FVIII binding capacity than Haemate P®, Voncento® and Wilate®. Modeling provided evidence that VWF:RCo activity in patients with severe VWD treated with rVWF is associated with L/UL VWF multimers in the circulation.Conclusions: Findings suggest that the L and UL multimers preserved in rVWF contribute to high biological activity and might be important for providing hemostatic efficacy.Keywords: agar, electrophoresis, factor VIII, plasma, von Willebrand diseases, von Willebrand factor

Background: Our aim was to infect rats with Helicobacter pylori and to study the effects of the infection on the gastric mucosa in normal and in ulcer-operated rats. Methods: A mouse-adapted H. pylori (cagA, VacA) strain was inoculated into 23 rats. Another 20 uninfected rats served as controls. Two months later a gastric ulcer was induced in some rats. The animals were killed 3, 6, or 15 days after the ulcer operation. Tissues were taken for histology and for culture of H. pylori. Serum antibodies were determined. Results: All inoculated rats were infected by H. pylori after 2 months, mainly in the antrum. In these rats a mild to moderate chronic inflammation and a significantly increased frequency of apoptotic cells were observed in the antrum and in the ulcer margin, the ulcer healing was delayed, and the serum level of H. pylori-specific Ig was increased. Conclusions: H. pylori infection in rats was successful and was accompanied by a mild to moderate mucosal inflammation. Gastric ulcer healing was delayed in infected rats, probably due to the inflammation and the increased apoptosis in epithelium. [ABSTRACT FROM AUTHOR]

Background: Congenital thrombotic thrombocytopenic purpura (TTP) results from severe hereditary deficiency of ADAMTS13. The efficacy and safety of recombinant ADAMTS13 and standard therapy (plasma-derived products) administered as routine prophylaxis or on-demand treatment in patients with congenital TTP is not known., Methods: In this phase 3, open-label, crossover trial, we randomly assigned patients in a 1:1 ratio to two 6-month periods of prophylaxis with recombinant ADAMTS13 (40 IU per kilogram of body weight, administered intravenously) or standard therapy, followed by the alternate treatment; thereafter, all the patients received recombinant ADAMTS13 for an additional 6 months. The trigger for this interim analysis was trial completion by at least 30 patients. The primary outcome was acute TTP events. Manifestations of TTP, safety, and pharmacokinetics were assessed. Patients who had an acute TTP event could receive on-demand treatment., Results: A total of 48 patients underwent randomization; 32 completed the trial. No acute TTP event occurred during prophylaxis with recombinant ADAMTS13, whereas 1 patient had an acute TTP event during prophylaxis with standard therapy (mean annualized event rate, 0.05). Thrombocytopenia was the most frequent TTP manifestation (annualized event rate, 0.74 with recombinant ADAMTS13 and 1.73 with standard therapy). Adverse events occurred in 71% of the patients with recombinant ADAMTS13 and in 84% with standard therapy. Adverse events that were considered by investigators to be related to the trial drug occurred in 9% of the patients with recombinant ADAMTS13 and in 48% with standard therapy. Trial-drug interruption or discontinuation due to adverse events occurred in no patients with recombinant ADAMTS13 and in 8 patients with standard therapy. No neutralizing antibodies developed during recombinant ADAMTS13 treatment. The mean maximum ADAMTS13 activity after recombinant ADAMTS13 treatment was 101%, as compared with 19% after standard therapy., Conclusions: During prophylaxis with recombinant ADAMTS13 in patients with congenital TTP, ADAMTS13 activity reached approximately 100% of normal levels, adverse events were generally mild or moderate in severity, and TTP events and manifestations were rare. (Funded by Takeda Development Center Americas and Baxalta Innovations; ClinicalTrials.gov number, NCT03393975.)., (Copyright © 2024 Massachusetts Medical Society.)

International guidelines conditionally recommend long-term prophylaxis in patients with von Willebrand disease (VWD) and severe and frequent bleeding. As recombinant von Willebrand factor (rVWF; vonicog alfa) may reduce the frequency of treated spontaneous bleeding events (BEs), we investigated the efficacy and safety of rVWF prophylaxis in adults with severe VWD. Patients with BEs requiring VWF therapy in the past year (on-demand VWF therapy [prior on-demand group] or plasma-derived VWF prophylaxis [pdVWF; switch group]) were enrolled in a prospective, open-label, nonrandomized, phase 3 study. The planned duration of rVWF prophylaxis was 12 months; starting rVWF dose was 50 ± 10 VWF: ristocetin cofactor (VWF:RCo) IU/kg twice weekly (prior on-demand group) or based on prior pdVWF weekly dose/dosing frequency (switch group). The primary endpoint was annualized bleeding rate (ABR) of treated spontaneous BEs (sABR) during rVWF prophylaxis. Over the 12-month study period, treated sABR decreased by 91.5% on-study vs historical sABR in 13 patients in the prior on-demand group, and by 45.0% in 10 patients in the switch group (model-based analysis ratio, 0.085; 95% confidence interval [CI], 0.021-0.346 and 0.550; 95% CI, 0.086-3.523, respectively). No treated spontaneous BEs were recorded in 84.6% (11/13) and 70.0% (7/10) of patients, respectively. The safety profile of rVWF was consistent with the previously established profile, with no new adverse drug reactions identified. Findings suggest that rVWF prophylaxis can reduce treated spontaneous BEs in patients previously receiving on-demand VWF therapy and maintains at least the same level of hemostatic control in patients who switch from prophylaxis with pdVWF to rVWF, with a favorable safety profile. This trial was registered at www.clinicaltrials.gov (#NCT02973087) and www.clinicaltrialsregister.eu (#EudraCT 2016-001478-14)., (© 2022 by The American Society of Hematology.)

The glucocorticosteroid budesonide is one of the mainstay treatments in inflammatory bowel disease. The aim of this study was to investigate its effects on Caco-2 cells upon coculture with LPS stimulated macrophages, or with conditioned medium (CM) from the same. The mRNA expression of the chemokines IL-8 and ENA-78 were upregulated in Caco-2 cells, which was always more pronounced in the coculture, as compared to the CM situation, with the ENA-78 induction being higher than of IL-8. Addition of budesonide to the apical side of Caco-2 cells, decreased both IL-8 and ENA-78 mRNA levels in a dose dependent manner. A reduction in trans epithelial electrical resistance (TEER) and an increase in the apical-basolateral transport of fluoresceinated sulfonic acid (FS) were observed in the Caco-2 cells both in cocultures and CM experiments. The reduction in TEER was counteracted by budesonide, whereas it had no effect on the FS transport. We conclude that budesonide exert an anti-inflammatory effect directly on intestinal epithelial cells, which may contribute to the overall action of budesonide in the treatment of inflammatory bowel diseases.

Large-scale public data mining will become more common as public release of microarray data sets becomes a corequisite for publication. Therefore, there is an urgent need to clarify whether data from different microarray platforms are comparable. To assess the compatibility of microarray data, results were compared from the two main types of high-throughput microarray expression technologies, namely, an oligonucleotide-based and a cDNA-based platform, using RNA obtained from complex tissue (human colonic mucosa) of five individuals. From 715 sequence-verified genes represented on both platforms, 64% of the genes matched in "present" or "absent" calls made by both platforms. Calls were influenced by spurious signals caused by Alu repeats in cDNA clones, clone annotation errors, or matched probes that were designed to different regions of the gene; however, these factors could not completely account for the level of call discordance observed. Expression levels in sequence-verified, platform-overlapping genes were not related, as demonstrated by weakly positive rank order correlation. This study demonstrates that there is only moderate overlap in the results from the two array systems. This fact should be carefully considered when performing large-scale analyses on data originating from different microarray platforms.

Crohn's disease is a chronic inflammatory bowel disease characterized by inflammation of both the small and large intestines. Methotrexate (MTX), a classical dihydrofolate reductase (DHFR) inhibitor, has been used as a therapeutic agent in the treatment of patients with Crohn's disease in recent years. We sought to develop antifolates similar in structure to MTX that would be effective in reducing inflammation in a mouse disease model of colitis. Four classical DHFR inhibitors encompassing ester bridges in the central parts of the molecules were synthesized. These antifolates were efficient inhibitors of the DHFR enzyme derived from rat. They were also tested in vitro for their ability to inhibit induced proliferation of lymphocytes from mouse spleen. Inhibition of cell proliferation was achieved only in the micromolar range, whereas MTX was effective at low nanomolar concentrations. One of the DHFR inhibitors (1), with an IC(50) value for rlDHFR approximately 8 times higher than that of methotrexate, was selected for in vivo experiments in an experimental colitis model in mice. This compound demonstrated a clear antiinflammatory effect after topical administration, comparable to the effect achieved with the glucocorticoid budesonide. Three parameters were evaluated in this model: myeloperoxidase activity, colon weight, and inflammation scoring. A favorable in vivo effect of compound 1 (15 mg/(kg.day)) was observed in all three inflammatory parameters. However, the results cannot be explained fully by DHFR inhibition or by inhibition of lymphocyte cell proliferation, suggesting that other yet unidentified mechanisms enable reduction of inflammation in the colitis model. The mechanism of action of methotrexate analogues encompassing a bridging ester group is not well understood in vivo but seems to lend itself well to further development of similar compounds.

Colonization of the human stomach by Helicobacter pylori is associated with the development of gastritis, duodenal ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. H. pylori-antigen-binding single-chain variable fragments (ScFv) were derived from murine hybridomas producing monoclonal antibodies and expressed as a g3p-fusion protein on a filamentous M13 phage. The recombinant ScFv-phage reacted specifically with a 30-kDa monomeric protein of a H. pylori surface antigen preparation and by means of immunofluorescence microscopy the phage was shown to bind to both the spiral and coccoid forms of the bacterium. In vitro, the recombinant phage exhibited a bacteriocidal effect and inhibited specifically the growth of all the six strains of H. pylori tested. When H. pylori was pretreated with the phage 10 min before oral inoculation of mice, the colonization of the mouse stomachs by the bacterium was significantly reduced (P<0.01). The results suggest that genetic engineering may be used to generate therapy-effective phages.

We aimed to produce a unifying hypothesis to explain the different locations of peptic ulcer and gastritis observed in different populations. The pre-Helicobacter pylori literature on patterns of gastroduodenal disease was reviewed and compared with recent human and animal findings on H. pylori infection. Early observations revealed that duodenal and non-pre-pyloric ulcers tend to be mutually exclusive. In duodenal ulcer patients, gastritis is usually restricted to the antrum, while gastric ulcer patients experience more severe pangastritis. The manipulation of acid output by surgery or acid suppressive therapy alters the distribution of gastritis. Recent experimental evidence in humans and animals has shown that these changes parallel changes in the distribution and cellular responses to H. pylori infection. We propose that the most important factor in the ecology of the H. pylori-infected stomach is local acid production. Local acid production determines the location and severity of inflammation and the clinical outcome of this bacterial infection. Priority research areas should be the investigation of the in vivo behaviour of H. pylori in the acid and the non-acid producing areas of the stomach and the measurement of acid output in populations known to have different patterns of gastroduodenal disease.