Assessment of the Impact of Alternative Fixatives on HER2 Detection in Breast Cancer and Gastric Cancer Tumor Specimens.

The type of fixative used for preserving tumor specimens can significantly impact the performance of the immunohistochemistry and in situ hybridization assays used for assessing human epidermal growth factor receptor 2 (HER2) status. This study reports the prevalence of the use of alternative fixatives other than the guideline-recommended 10% neutral buffered formalin (NBF) during HER2 testing in a real-world setting. The effects of alternative fixatives [20% NBF and 10% unbuffered formalin (UBF) fixatives] on HER2 testing of breast cancer (BC) and gastric cancer (GC) cell lines and tissues are also assessed. Overall, 117,636 tumor samples received at a central laboratory from >8000 clinical trial sites across 60 countries were reviewed to determine the prevalence of alternative fixative usage. To investigate the impact of alternative fixatives, 27 cell lines (21 BC and 6 GC) and 76 tumor tissue samples (50 BC and 26 GC) were fixed in 10% NBF, 20% NBF, or 10% UBF, and evaluated for HER2 status by immunohistochemistry and in situ hybridization. Real-world data showed that 9195 (7.8%) tumor samples were preserved using an alternative fixative. In cell lines, overall percentage agreement, negative percentage agreement, and positive percentage agreement among the 3 fixatives were 100%. In tumor tissues, the agreement among 10% NBF, 20% NBF, and 10% UBF ranged between 94.7% and 96.6% for negative percentage agreement and 90.9% for overall percentage agreement compared with a range of 58.3% to 66.7% for positive percentage agreement. These results suggest that alternative fixatives may have the potential to convert HER2 status in tissues from positive to negative.

Evaluation of species difference in peripheral lymphocyte reduction effect of CS-0777, a sphingosine 1-phosphate receptor modulator, based on a pharmacokinetic/pharmacodynamic model analysis.

Pharmacokinetic (PK) and pharmacodynamic (PD) modeling was conducted for the reduction of peripheral lymphocytes after oral administration of CS-0777 to healthy rats, monkeys and experimental autoimmune encephalomyelitis (EAE) induced rats. The phosphorylated active metabolite of CS-0777, M1, is a selective sphingosine 1-phosphate receptor-1 modulator. A linear one- and two-compartment model with a reversible metabolism process characterized the time courses of CS-0777 and M1 concentrations in rats and monkeys, respectively. The relationship between lymphocyte counts and M1 concentrations in blood was well described by an indirect response model in all animals examined. An Imax of 0.815 and an IC50 of 6.58 nM in healthy rats, an Imax of 0.807 and an IC50 of 5.09 nM in the EAE rats, an Imax of 0.789 and an IC50 of 0.484 nM in monkeys were estimated by the indirect PD model. Since the IC50 values calculated in terms of the unbound plasma concentration in rats and monkeys were within a similar range, after correction of the IC50 in blood described above with the blood to plasma concentration ratio and the plasma free fraction of M1, it was revealed that there is no species difference in the essential activity of M1 against lymphocyte reduction. The sensitivity of the lymphocytes to M1 was not affected by the EAE status. Comparison of the simulated lymphocyte reduction in EAE rats after multiple dosing with CS-0777 and the actual EAE clinical scores implies that the significant suppressive effect on EAE did not require the elimination of all lymphocytes from the blood. Copyright © 2016 John Wiley & Sons, Ltd.

Component of Caramel Food Coloring, THI, Causes Lymphopenia Indirectly via a Key Metabolic Intermediate.

Caramel color is widely used in the food industry, and its many variations are generally considered to be safe. It has been known for a long time that THI (2-acetyl-4-(tetrahydroxybutyl)imidazole), a component of caramel color III, causes lymphopenia in animals through sphingosine 1-phosphate (S1P) lyase (S1PL) inhibition. However, this mechanism of action has not been fully validated because THI does not inhibit S1PL in vitro. To reconcile this situation, we examined molecular details of THI mechanism of action using "smaller" THI derivatives. We identified a bioactive derivative, A6770, which has the same lymphopenic effect as THI via S1PL inhibition. In the case of A6770 we observe this effect both in vitro and in vivo, and demonstrate that A6770 is phosphorylated and inhibits S1PL in the same way as 4-deoxypyridoxine. In addition, A6770 was detected in rat plasma following oral administration of THI, suggesting that A6770 is a key metabolic intermediate of THI.

Synthesis and SAR of 1,3-thiazolyl thiophene and pyridine derivatives as potent, orally active and S1P3-sparing S1P1 agonists.

We have previously disclosed 1,2,4-oxadiazole derivative 3 as a potent S1P(3)-sparing S1P(1) agonist. Although compound 3 exhibits potent and manageable immunosuppressive efficacy in various in vivo models, recent studies have revealed that its 1,2,4-oxadiazole ring is subjected to enterobacterial decomposition. As provisions for unpredictable issues, a series of alternative compounds were synthesized on the basis of compound 3. Extensive SAR studies led to the finding of 1,3-thiazole 24c with the EC(50) value of 3.4 nM for human S1P(1), and over 5800-fold selectivity against S1P(3). In rat on host versus graft reaction (HvGR), the ID(50) value of 24c was determined at 0.07 mg/kg. The pharmacokinetics in rat and monkey is also reported. Compared to compound 3, 24c showed excellent stability against enterobacteria.

Synthesis and evaluation of CS-2100, a potent, orally active and S1P(3)- sparing S1P(1) agonist.

Modulators of sphingosine phosphate receptor-1 (S1P(1)) have recently been focused as a suppressant of autoimmunity. We have discovered a 4-ethylthiophene-based S1P(1) agonist 1-({4-Ethyl-5-[5-(4-phenoxyphenyl)-1,2,4-oxadiazol-3-yl]-2-thienyl}methyl)azetidine-3-carboxylic acid (CS-2100, 8) showing potent S1P(1) agonist activity against S1P(3) and an excellent in vivo potency. We report herein the synthesis of CS-2100 (8) and pharmacological effects such as S1P(1) and S1P(3) agonist activity in vitro, peripheral blood lymphocyte lowering effects and the suppressive effects on adjuvant-induced arthritis and experimental autoimmune encephalomyelitis (EAE) in animal models. The pharmacokinetic data were also reported. CS-2100 (8) had >5000-fold greater agonist activity for human S1P(1) (EC(50); 4.0 nM) relative to S1P(3) (EC(50); >20,000 nM). Following administration of single oral doses of 0.1 and 1 mg/kg of CS-2100 (8) in rats, lymphocyte counts decreased significantly, with a nadir at 8 and/or 12 h post-dose and recovery to vehicle control levels by 24-48 h post-dose. CS-2100 (8) is efficacious in the adjuvant-induced arthritis model in rats (ID(50); 0.44 mg/kg). In the EAE model compared to the vehicle-treated group, significant decreases in the cumulative EAE scores were observed for 0.3 and 1 mg/kg CS-2100 (8) groups in mice. While CS-2100 (8) showed potent efficacy in various animal disease models, it was also revealed that the central 1,2,4-oxadiazole ring of CS-2100 (8) was decomposed by enterobacteria in intestine of rats and monkeys, implicating the latent concern about an external susceptibility in its metabolic process in the upcoming clinical studies.

Discovery of CS-2100, a potent, orally active and S1P3-sparing S1P1 agonist.

S1P(3)-sparing S1P(1) agonists have attracted attention as a suppressant of autoimmunity with reduced side effects. Our synthetic efforts and extensive SAR studies led to the discovery of 10b named CS-2100 with the EC(50) value of 4.0 nM for human S1P(1) and over 5000-fold selectivity against S1P(3). The in vivo immunosuppressive efficacy was evaluated in rats on host versus graft reaction and the ID(50) value was determined at 0.407mg/kg. The docking studies of CS-2100 with the homology model of S1P(1) and S1P(3) showed that the ethyl group on the thiophene ring of CS-2100 was sterically hindered by Phe263 in S1P(3), not in the case of Leu276 in S1P(1). This observation gives an explanation for the excellent S1P(3)-sparing characteristic of CS-2100.

Discovery of CS-0777: A Potent, Selective, and Orally Active S1P1 Agonist.

CS-0777 (3) is phosphorylated in vivo, and the phosphate of CS-0777 (CS-0777-P) (4) acts as a selective S1P receptor-1 (S1P1) modulator. We report herein the synthesis of CS-0777 and CS-0777-P, pharmacological effects such as S1P1 and S1P3 agonist activity in vitro, peripheral blood lymphocyte lowering effects and the suppressive effect on experimental autoimmune encephalomyelitis (EAE), and also the pharmacokinetics in rats. CS-0777-P had ∼320-fold greater agonist activity for human S1P1 (EC50; 1.1 nM) relative to S1P3 (EC50; 350 nM). Following administration of single oral doses of 0.1 and 1 mg/kg of CS-0777 in rats, lymphocyte counts decreased significantly, with a nadir at 12 h postdose and recovery to vehicle control levels by 5 days postdose. In the EAE model compared to the vehicle-treated group, significant decreases in the cumulative EAE scores were observed for the 0.1 and 1 mg/kg CS-0777 groups in rats. CS-0777 is currently in clinical trials for the treatment of multiple sclerosis (MS).

Ca2+-releasing effect of cerivastatin on the sarcoplasmic reticulum of mouse and rat skeletal muscle fibers.

We analyzed the effect of HMG-CoA reductase inhibitors on Ca(2+) release from the sarcoplasmic reticulum (SR) using chemically skinned skeletal muscle fibers from the mouse and the rat. Cerivastatin (>20 microM) released Ca(2+) from the SR, while pravastatin showed only a little effect. The rates of Ca(2+) release were increased by cerivastatin at all Ca(2+) concentrations tested. Cerivastatin-induced Ca(2+) release in the presence of Ca(2+) was affected by adenosine monophosphate, Mg(2+), and procaine in essentially the same way as for caffeine-induced Ca(2+) release. The Ca(2+)-uptake capacity of the SR was reduced after co-treatment with ryanodine and cerivastatin at pCa 6.0 to a much greater extent than with ryanodine alone. Thus, cerivastatin-induced Ca(2+) release in the presence of Ca(2+) must be a result of the activation of the Ca(2+)-induced Ca(2+) release (CICR) mechanism of the ryanodine receptor. However, even when CICR was maximally inhibited by Mg(2+) and procaine, or in the practical absence of Ca(2+) (pCa >8), cerivastatin still caused Ca(2+) release. These results indicate that cerivastatin causes Ca(2+) release also by activating some other mechanism(s) in addition to the activation of CICR. Either or both of these effects might be related to its adverse effect, rhabdomyolysis.