Ro 22-3245

Role of receptor occupancy assays by flow cytometry in drug development

The measurement of the binding of a biotherapeutic to its cellular target, receptor occupancy (RO), is increasingly important in development of biologically-based therapeutic agents. Receptor occupancy (RO) assays by flow cytometry describe the qualitative and/or quantitative assessment of the binding of a therapeutic agent to its cell surface target. Such RO assays can be as simple as measuring the number of cell surface receptors bound by an antireceptor therapeutic agent or can be designed to address more complicated scenarios such as internalization or shedding events once a receptor engages the administered therapeutic agent. Data generated from RO assays can also be used to model whether given doses of an experimental therapeutic agent and their administration schedules lead to predicted levels of receptor occupancy and whether the receptor is modulated (up or down) on cells engaged by the therapeutic agent. There are a variety of approaches that can be used when undertaking RO assays and with the ability to measure distinct subsets in heterogeneous populations, flow cytometry is ideally suited to RO measurements. This article highlights the importance of RO assays on the flow cytometric platform in the development of biotherapeutic agents.

Anti-Ro/SSA and La/SSB antibodies

The Ro/La system is considered as an heterogeneous antigenic complex, constituted by three different proteins (52 kDa Ro, 60 kDa Ro and La) and four small RNAs particles. Anti-Ro/SSA are the most prevalent specificity among many autoimmune diseases, such as systemic lupus erythematosus (SLE), SS/SLE overlap syndrome, subacute cutaneous LE (SCLE), neonatal lupus and primary biliary cirrhosis. In contrast, anti-La/SSB is more associated with Sj?gren's syndrome (SS). The differences between 52 kDa, 60 kDa Ro and La could explain why different assays did not show equivalent performance in anti-Ro and anti-La autoantibodies detection. The RNA precipitation assay had the highest sensitivity and specificity, usually considered as the reference methods. CIE is considered the most reliable to detect anti-Ro/SSA antibodies in routine practice, performing better than immunoblotting (IB) and some ELISAs. It shows a high sensitivity (89%) and specificity (100%). ELISA is generally considered a safe, rapid, sensitive and specific tecnique. Therefore, its high sensitivity often corresponds to a very low clinical specificity and the assay can give false positive results. Therefore, it is very important to search anti-Ro and anti-La only in selected patients, using the assay with high specificity and good predictive value, in order to have clinically significant and true positive results.

Machine Learning for the Identification of a Common Signature for Anti-SSA/Ro 60 Antibody Expression Across Autoimmune Diseases

Objective: Anti-Ro autoantibodies are among the most frequently detected extractable nuclear antigen autoantibodies, mainly associated with primary Sj?gren's syndrome (SS), systemic lupus erythematosus (SLE), and undifferentiated connective tissue disease (UCTD). This study was undertaken to determine if there is a common signature for all patients expressing anti-Ro 60 autoantibodies regardless of their disease phenotype.
Methods: Using high-throughput multiomics data collected from the cross-sectional cohort in the PRECISE Systemic Autoimmune Diseases (PRECISESADS) study Innovative Medicines Initiative (IMI) project (genetic, epigenomic, and transcriptomic data, combined with flow cytometry data, multiplexed cytokines, classic serology, and clinical data), we used machine learning to assess the integrated molecular profiling of 520 anti-Ro 60+ patients compared to 511 anti-Ro 60- patients with primary SS, patients with SLE, and patients with UCTD, and 279 healthy controls.
Results: The selected clinical features for RNA-Seq, DNA methylation, and genome-wide association study data allowed for a clear distinction between anti-Ro 60+ and anti-Ro 60- patients. The different features selected using machine learning from the anti-Ro 60+ patients constituted specific signatures when compared to anti-Ro 60- patients and healthy controls. Remarkably, the transcript Z score of 3 genes (ATP10A, MX1, and PARP14), presenting with overexpression associated with hypomethylation and genetic variation and independently identified using the Boruta algorithm, was clearly higher in anti-Ro 60+ patients compared to anti-Ro 60- patients regardless of disease type. Our findings demonstrated that these signatures, enriched in interferon-stimulated genes, were also found in anti-Ro 60+ patients with rheumatoid arthritis and those with systemic sclerosis and remained stable over time and were not affected by treatment.
Conclusion: Anti-Ro 60+ patients present with a specific inflammatory signature regardless of their disease type, suggesting that a dual therapeutic approach targeting both Ro-associated RNAs and anti-Ro 60 autoantibodies should be considered.

Antiscalants in RO membrane scaling control

Reverse osmosis (RO) plays an important role in freshwater production. Mineral scaling is an inevitable problem in the RO desalination process. Various methods, including the pretreatment of feed water, the optimization of operational processes, the development of novel membrane materials, and the addition of antiscalants, have been developed to mitigate scale formation in RO systems. Among these methods, the addition of antiscalants is a relatively cost-effective and convenient technique for membrane scaling control. In the current work, various kinds of antiscalants, scale inhibition mechanisms, and their applications to RO membrane scaling control are reviewed. Weakness of existing antiscalants and challenge arising from their practical applications, such as membrane fouling caused by antiscalants, increased bacterial growth, dosing control, and the disposal of resultant concentrates, are also presented. To effectively alleviate scaling on RO membrane by using antiscalants, the development of novel, high-performance, and environment-friendly antiscalants on the basis of an in-depth study of the inhibition mechanisms and well-established structure-activity relationships is urgently necessary. The optimization of antiscalants and their combinations with other pretreatments in practical RO operations are essential in efficient scaling control.

[3H]Ro 22-1319 (piquindone) binds to the D2 dopaminergic receptor subtype in a sodium-dependent manner

Ro 22-1319, a novel pyrroloisoquinoline compound, is a potent antipsychotic agent with low potential for extrapyramidal effects and tardive dyskinesia. In this study, the specific binding of [3H]Ro 22-1319 to the rat striatal homogenates has been examined. The binding of [3H]Ro 22-1319 was found to be critically dependent on the presence of sodium in the incubation medium. There appeared to be a single saturable binding component for [3H]Ro 22-1319 with a high affinity. The binding sites showed a stereochemical specificity for (-)Ro 22-1319, (+)butaclamol, and (alpha)flupenthixol. Ro 22-1319 and three D2 antagonistic antipsychotics (sulpiride, metoclopramide, and molindone) exerted a more potent inhibition of [3H]Ro 22-1319 binding than of [3H]spiroperidol binding, whereas other classical antipsychotics were almost equipotent at both binding sites. The requirement for sodium to detect Ro 22-1319 binding was also verified by the use of [3H] spiroperidol binding. The competition curves of Ro 22-1319, sulpiride, metoclopramide, and molindone for [3H]spiroperidol binding were shifted to the right by the omission of sodium in the incubation medium, whereas spiroperidol, chlorpromazine, and domperidone were equiactive under both conditions. These results suggest that Ro 22-1319 has a sulpiride-like property and binds to a D2 dopaminergic receptor subtype in a sodium-dependent manner. Nineteen pyrroloisoquinoline derivatives were also tested for their inhibitory effects on [3H]Ro 22-1319 and [3H] spiroperidol binding. An interesting finding was that small changes in chemical structure modulated the potency at D2 dopaminergic receptor subtypes. Thus, the compounds having a nonlipophilic functional group on the basic nitrogen (Ro 22-1319, Ro 22-3822, etc.) showed a stronger potency at [3H]Ro 22-1319 receptors whereas the compounds having a lipophilic group (Ro 22-6600, etc.) were nonselective antagonists at both [3H]Ro 22-1319- and [3H]spiroperidol-binding sites. However, all pyrroloisoquinoline derivatives including Ro 22-6600 showed a sodium dependency for [3H]spiroperidol-binding sites, indicating that the functional moiety which displays a sodium dependency may be the pyrroloisoquinoline moiety itself.