Comparative analysis of rule elements for transportation of cell therapy products among regulations and standards.

Aim: This study aimed to identify the elements involved in the transportation of cell therapy products by conducting a comparative analysis of four related international standards for temperature-controlled delivery and good distribution practice (GDP). Methods: An analytical framework was constructed to cover the entire transportation process. The descriptions of each element in the Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-operation Scheme (PIC/S) GDP, International Organization for Standardization (ISO) 21973, Foundation for the Accreditation of Cellular Therapy Common Standards for Cellular Therapies and ISO 23412 were compared. Results: The study identified some elements that were present in the PIC/S GDP and other standards but were absent in ISO 21973, and vice versa. These elements are crucial in view of the increasing opportunities to transport allogeneic cells in the future. Conclusion: The study identified the necessary elements that should be included in the development of transport regulations for cell therapy products.

The quality of cell therapy products needs to be ensured during transportation to the hospital, just like during their manufacture. However, cell therapy products are living cells, and ensuring their quality during transportation is challenging. The International Organization for Standardization (ISO) has published ISO 21973 to address this issue, but transport regulations for cell therapy products have not been modified yet. To create draft guidance for the transportation of cell therapy products, it is necessary to analyze the relationship between ISO 21973, good distribution practice, Foundation for the Accreditation of Cellular Therapy Common Standards and ISO 23412. We compared these regulations and standards and identified some elements that are necessary for transporting cell therapy products. These elements are crucial in view of the increasing opportunities to transport allogeneic cells in the future. This study proposed elements to be included in the development of transport regulations for cell therapy products.

Genome-wide siRNA screening in mouse bone marrow-derived macrophages revealed that knockdown of ribosomal proteins suppresses IL-10 and enhances TNF-α production.

Macrophages play a central role in the immune response, and their diverse functions are attributed to the spectrum of their functional states. To elucidate molecules involved in modulating the balance between the anti-inflammatory cytokine IL-10 and the pro-inflammatory cytokine TNF-α, we conducted genome-wide siRNA screening. First, we established an siRNA screening system using mouse bone marrow-derived macrophages, which are a suitable model for studying functional states of macrophages in vitro. In the primary screen and the subsequent reproducibility assay, 112 siRNA pools demonstrated enhancement of IL-10 production and 497 siRNA pools suppressed IL-10 production. After a deconvolution assay for IL-10-up-regulating siRNA pools, 8 genes were identified as IL-10 repressors, including Cnot1 and Rc3h1, components of the CCR4-NOT complex known to degrade cytokine mRNAs. On the other hand, siRNA pools targeting ribosomal proteins were frequently found among those that down-regulated IL-10 production and up-regulated TNF-α production. Four pools were assayed using deconvoluted siRNAs and identified as high-confidence hits. Thus, we found that the genome-wide knockdown of 19 ribosomal proteins resulted in decreased IL-10 and increased TNF-α production.

Discovery and in vivo effects of novel human natriuretic peptide receptor A (NPR-A) agonists with improved activity for rat NPR-A.

Natriuretic peptide receptor A (NPR-A) agonists were evaluated in vivo by optimizing the structure of quinazoline derivatives to improve agonistic activity for rat NPR-A. A 1,4-Cis-aminocyclohexylurea moiety at 4-position and hydroxy group of d-alaninol at 2-position on the quinazoline ring were found to be important factors in improving rat NPR-A activity. We identified potent quinazoline and pyrido[2,3-d]pyrimidine derivatives against rat NPR-A, with double-digit nanomolar EC50 values. The in vivo results showed that compound 56b administered at 1.0 mg/kg/min significantly increased plasma cGMP concentration and urine volume in rats. We discovered novel potent NPR-A agonists that showed agonistic effects similar to those of atrial natriuretic peptide.

Discovery and SAR of a novel series of Natriuretic Peptide Receptor-A (NPR-A) agonists.

Novel thienopyrimidine compounds 2 and 3 were discovered from high-throughput screening as Natriuretic Peptide Receptor A (NPR-A) agonists. Scaffold hopping of a thienopyrimidine ring to a quinazoline ring, introduction of the basic functional group and optimization of the substituent on the 6-position of the benzene ring of quinazoline led to improved agonistic activity. We discovered compound 48, which showed potent agonistic activity for NPR-A with an EC50 value of 0.073µM, indicating 350-fold potency compared to the hit compound 3.

Discovery and dimeric approach of novel Natriuretic Peptide Receptor A (NPR-A) agonists.

Novel agonists of the Natriuretic Peptide Receptor A (NPR-A) were obtained through random screening and subsequent structural modification of triazine derivatives. The key structural feature to improve in vitro activity was the dimerization of triazine monomer derivatives. The non peptide derivative 7c and 13a showed highly potent NPR-A agonistic activity in vitro and diuretic activity in vivo. These results implied that non-peptidic small molecules open the possibility of new therapy for congestive heart failure.

Activity of beta3-beta4 loop of the PH domain is required for the membrane targeting of SWAP-70.

SWAP-70 translocates to the plasma membrane in a phosphoinositide 3-kinase (PI 3-kinase)-dependent manner and contributes to membrane ruffling. It binds to phosphatidylinositol trisphosphate (PtdIns(3,4,5)P(3)) through its PH domain, which is essential for the membrane translocation after EGF stimulation. We examined the behavior of the SWAP-70s which have mutations in the beta3/beta4 loop of the PH domain. The two mutants fused to green fluorescent protein (GFP) carrying the mutations failed to translocate to the plasma membrane. The sole PH domains carrying the same mutations behaved similarly. The PtdIns(3,4,5)P(3) binding activity of two mutants was comparable to that of the wild-type protein. These results suggest that translocation of SWAP-70 largely depends on the activity of the PH domain, and that not only PtdIns(3,4,5)P(3) binding activity, but also some additional activity of the PH domain is required for the translocation.