The Novel Phosphate and Bile Acid Sequestrant Polymer SAR442357 Delays Disease Progression in a Rat Model of Diabetic Nephropathy.

As a gut-restricted, nonabsorbed therapy, polymeric bile acid sequestrants (BAS) play an important role in managing hyperlipidemia and hyperglycemia. Similarly, nonabsorbable sequestrants of dietary phosphate have been used for the management of hyperphosphatemia in end-stage renal disease. To evaluate the potential utility of such polymer sequestrants to treat type 2 diabetes (T2D) and its associated renal and cardiovascular complications, we synthesized a novel polymeric sequestrant, SAR442357, possessing optimized bile acid (BA) and phosphate sequestration characteristics. Long-term treatment of T2D obese cZucker fatty/Spontaneously hypertensive heart failure F1 hybrid (ZSF1) with SAR442357 resulted in enhanced sequestration of BAs and phosphate in the gut, improved glycemic control, lowering of serum cholesterol, and attenuation of diabetic kidney disease (DKD) progression. In comparison, colesevelam, a BAS with poor phosphate binding properties, did not prevent DKD progression, whereas losartan, an angiotensin II receptor blocker that is widely used to treat DKD, showed no effect on hyperglycemia. Analysis of hepatic gene expression levels of the animals treated with SAR442357 revealed upregulation of genes responsible for the biosynthesis of cholesterol and BAs, providing clear evidence of target engagement and mode of action of the new sequestrant. Additional hepatic gene expression pathway changes were indicative of an interruption of the enterohepatic BA cycle. Histopathological analysis of ZSF1 rat kidneys treated with SAR442357 further supported its nephroprotective properties. Collectively, these findings reveal the pharmacological benefit of simultaneous sequestration of BAs and phosphate in treating T2D and its associated comorbidities and cardiovascular complications. SIGNIFICANCE STATEMENT: A new nonabsorbed polymeric sequestrant with optimum phosphate and bile salt sequestration properties was developed as a treatment option for DKD. The new polymeric sequestrant offered combined pharmacological benefits including glucose regulation, lipid lowering, and attenuation of DKD progression in a single therapeutic agent.

Triantennary GalNAc Molecular Imaging Probes for Monitoring Hepatocyte Function in a Rat Model of Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease that can lead to irreversible liver cirrhosis and cancer. Early diagnosis of NASH is vital to detect disease before it becomes life-threatening, yet noninvasively differentiating NASH from simple steatosis is challenging. Herein, bifunctional probes have been developed that target the hepatocyte-specific asialoglycoprotein receptor (ASGPR), the expression of which decreases during NASH progression. The results show that the probes allow longitudinal, noninvasive monitoring of ASGPR levels by positron emission tomography in the newly developed rat model of NASH. The probes open new possibilities for research into early diagnosis of NASH and development of drugs to slow or reverse its progression.

Utility of the RIG-I Agonist Triphosphate RNA for Melanoma Therapy.

The pattern recognition receptor RIG-I plays an important role in the recognition of nonself RNA and antiviral immunity. RIG-I's natural ligand, triphosphate RNA (ppp-RNA), is proposed to be a valuable addition to the growing arsenal of cancer immunotherapy treatment options. In this study, we present comprehensive data validating the concept and utility of treatment with synthetic RIG-I agonist ppp-RNA for the therapy of human cancer, with melanoma as potential entry indication amenable to intratumoral treatment. Using mRNA expression data of human tumors, we demonstrate that RIG-I expression is closely correlated to cellular and cytokine immune activation in a wide variety of tumor types. Furthermore, we confirm susceptibility of cancer cells to ppp-RNA treatment in different cellular models of human melanoma, revealing unexpected heterogeneity between cell lines in their susceptibility to RNA agonist features, including sequence, secondary structures, and presence of triphosphate. Cellular responses to RNA treatment (induction of type I IFN, FasR, MHC-I, and cytotoxicity) were demonstrated to be RIG-I dependent using KO cells. Following ppp-RNA treatment of a mouse melanoma model, we observed significant local and systemic antitumor effects and survival benefits. These were associated with type I IFN response, tumor cell apoptosis, and innate and adaptive immune cell activation. For the first time, we demonstrate systemic presence of tumor antigen-specific CTLs following treatment with RIG-I agonists. Despite potential challenges in the generation and formulation of potent RIG-I agonists, ppp-RNA or analogues thereof have the potential to play an important role for cancer treatment in the next wave of immunotherapy.

Development and Validation of an Image Analysis System for the Measurement of Cell Proliferation in Mammary Glands of Rats.

Reliable detection and measurement of cell proliferation are essential in the preclinical assessment of carcinogenic risk of therapeutics. In this context, the assessment of mitogenic potential on mammary glands is crucial in the preclinical safety evaluation of novel insulins. The existing manual counting is time-consuming and subject to operator bias. To standardize the processes, make it faster, and resistant to errors, we developed a semiautomated image analysis system (CEPA software, which is open-source) for counting of proliferating cells in photomicrographs of mammary gland sections of rats labeled with Ki-67. We validated the software and met the predefined targets for specificity, accuracy, and reproducibility. In comparison to manual counting, the respective mean differences in absolute labeling indices (LIs) for CEPA software were 3.12% for user 1 and 3.05% for user 2. The respective regression analysis revealed a good correlation between the CEPA software user and manual counting. Moreover, the CEPA software showed enhanced reproducibility between independent users. The interuser variability is centered around 0 and the absolute difference was about 0.53% LI. Based on validation data, our software has superiority to the manual counting and is a valid and reliable tool for the routine analysis of cell proliferation in mammary glands from rats exposed to insulin analogs.

Regulatory Forum Opinion Piece: Review-Toxicological Pathology Profile and Regulatory Expectations for Nonclinical Development of Insulins and Insulin Analogues.

The toxicological profile of insulins is exclusively due to exaggerated pharmacology resulting in hypoglycemic findings. Insulin analogues displaying modifications and aimed at improving pharmacokinetics do not induce different toxicity. The main target is the brain displaying neuronal necrosis. Wallerian degeneration of nerves occurs rarely after severe hypoglycemia. These findings are of potential human relevance; nevertheless, these changes are induced in normoglycemic animals whereas diabetic patients suffer from hyperglycemia. Therefore, it is usually not difficult to achieve a therapeutic window for subsequent use in patients. Based upon this and in the absence of classical toxicity, there has been no scientific need for diabetic animal models. A greater challenge is the mitogenicity already inherent with regular insulin. Thus, the focus for preclinical safety evaluation of analogues is to demonstrate that modifications in regular insulin do not result in enhanced mitogenicity. The approaches used to assess the mitogenic potential of insulin analogues have changed over time driven by scientific progression and changes within the regulatory environment. Therefore, in vitro and in vivo evaluation of cell proliferation has become common practice, and to date there has been no evidence that the mitogenic potential of insulin analogues may be increased compared to regular insulin.