Enzymatic kinetics regarding reversible metabolism of CS-0777, a sphingosine 1-phosphate receptor modulator, via phosphorylation and dephosphorylation in humans.

1. CS-0777, a candidate compound for autoimmune diseases, becomes phosphorylated active metabolite, M1, by fructosamine 3-kinase (FN3K), FN3K-related protein (FN3K-RP); and M1 is reverted back to CS-0777 by alkaline phosphatase (ALP) in the body. We performed enzyme kinetic analysis of phosphorylation of CS-0777 by FN3K, FN3K-RP, human erythrocytes and human platelets; and dephosphorylation of M1 by various ALP isozymes and human liver, kidney, lung and small intestine microsomes. 2. The Michaelis constants of human FN3K, FN3K-RP and erythrocytes for CS-0777 phosphorylation were in the range from 498 µM to 1060 µM. FN3K inhibitor, 1-deoxy-1-morpholinofructose, suppressed only about 20% of CS-0777 phosphorylation activity in human erythrocyte lysate. Immunodepletion of FN3K and FN3K-RP decreased M1 formation activity by about 25% and 50%, respectively, in human erythrocyte lysate. 3. The Michaelis constants of four human ALPs and microsomes were in the range from 10.9 µM to 32.1 µM. The ALP inhibitor, levamisole, suppressed over 50% of M1 dephosphorylation activity in liver, kidney and lung microsomes. 4. FN3K-RP is expected to take a prominent role in the phosphorylation of CS-0777 in human erythrocytes; dephosphorylation of M1 was observed in all ALPs and human tissue microsomes examined, with a similar affinity towards M1 among them.

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.

Identification of activating enzymes of a novel FBPase inhibitor prodrug, CS-917.

CS-917 (MB06322) is a selective small compound inhibitor of fructose 1,6-bisphosphatase (FBPase), which is expected to be a novel drug for the treatment of type 2 diabetes by inhibiting gluconeogenesis. CS-917 is a bisamidate prodrug and activation of CS-917 requires a two-step enzyme catalyzed reaction. The first-step enzyme, esterase, catalyzes the conversion of CS-917 into the intermediate form (R-134450) and the second-step enzyme, phosphoramidase, catalyzes the conversion of R-134450 into the active form (R-125338). In this study, we biochemically purified the CS-917 esterase activity in monkey small intestine and liver. We identified cathepsin A (CTSA) and elastase 3B (ELA3B) as CS-917 esterases in the small intestine by mass spectrometry, whereas we found CTSA and carboxylesterase 1 (CES1) in monkey liver. We also purified R-134450 phosphoramidase activity in monkey liver and identified sphingomyelin phosphodiesterase, acid-like 3A (SMPADL3A), as an R-134450 phosphoramidase, which has not been reported to have any enzyme activity. Recombinant human CTSA, ELA3B, and CES1 showed CS-917 esterase activity and recombinant human SMPDL3A showed R-134450 phosphoramidase activity, which confirmed the identification of those enzymes. Identification of metabolic enzymes responsible for the activation process is the requisite first step to understanding the activation process, pharmacodynamics and pharmacokinetics of CS-917 at the molecular level. This is the first identification of a phosphoramidase other than histidine triad nucleotide-binding protein (HINT) family enzymes and SMPDL3A might generally contribute to activation of the other bisamidate prodrugs.

Pharmacokinetics and disposition of CS-0777, a sphingosine 1-phosphate receptor modulator, in rats and monkeys.

1. Disposition and metabolism of CS-0777 (1-{5-[(3R)-3-amino-4-hydroxy-3- methylbutyl]-1-methyl-1H-pyrrol-2-yl}-4-(4-methylphenyl) butan-1-one), a selective sphingosine 1-phosphate receptor-1 modulator under development for autoimmune conditions was investigated following oral and/or i.v. bolus administration to rats and monkeys. 2. After oral administration of [14C]CS-0777, CS-0777 was well absorbed in rats and monkeys with total recoveries of over 90% of the dose, majorly in feces. CS-0777 and phosphorylated pharmacologically active metabolite of CS-0777 (M1) were highly bound to plasma proteins among rats, monkeys and humans (>93%). 3. The structures of 12 metabolites were identified and phosphorylation and two hydroxylation pathways were proposed as primary metabolism. In the blood of rats and monkeys, the major metabolite was M1 and a few phosphorylated metabolites were also detected. Meanwhile, in urine and feces of rats and monkeys, not phosphorylated, but oxidized CS-0777 metabolites and/or those various conjugated metabolites were observed. This suggests that CS-0777 and its oxidized metabolites would be phosphorylated in the body, but their phosphorylated metabolites would revert back to their dephosphorylated form again then be further metabolized and finally eliminated from the body. 4. Pharmacokinetic analysis using a reversible metabolism model revealed that the clearance of phosphorylation was larger than the clearance of dephosphorylation and elimination.

Identification of a metabolizing enzyme in human kidney by proteomic correlation profiling.

Molecular identification of endogenous enzymes and biologically active substances from complex biological sources remains a challenging task, and although traditional biochemical purification is sometimes regarded as outdated, it remains one of the most powerful methodologies for this purpose. While biochemical purification usually requires large amounts of starting material and many separation steps, we developed an advanced method named "proteomic correlation profiling" in our previous study. In proteomic correlation profiling, we first fractionated biological material by column chromatography, and then calculated each protein's correlation coefficient between the enzyme activity profile and protein abundance profile determined by proteomics technology toward fractions. Thereafter, we could choose possible candidates for the enzyme among proteins with a high correlation value by domain predictions using informatics tools. Ultimately, this streamlined procedure requires fewer purification steps and reduces starting materials dramatically due to low required purity compared with conventional approaches. To demonstrate the generality of this approach, we have now applied an improved workflow of proteomic correlation profiling to a drug metabolizing enzyme and successfully identified alkaline phosphatase, tissue-nonspecific isozyme (ALPL) as a phosphatase of CS-0777 phosphate (CS-0777-P), a selective sphingosine 1-phosphate receptor 1 modulator with potential benefits in the treatment of autoimmune diseases including multiple sclerosis, from human kidney extract. We identified ALPL as a candidate protein only by the 200-fold purification and only from 1 g of human kidney. The identification of ALPL as CS-0777-P phosphatase was strongly supported by a recombinant protein, and contribution of the enzyme in human kidney extract was validated by immunodepletion and a specific inhibitor. This approach can be applied to any kind of enzyme class and biologically active substance; therefore, we believe that we have provided a fast and practical option by combination of traditional biochemistry and state-of-the-art proteomic technology.

Purification and identification of activating enzymes of CS-0777, a selective sphingosine 1-phosphate receptor 1 modulator, in erythrocytes.

CS-0777 is a selective sphingosine 1-phosphate (S1P) receptor 1 modulator with potential benefits in the treatment of autoimmune diseases, including multiple sclerosis. CS-0777 is a prodrug that requires phosphorylation to an active S1P analog, similar to the first-in-class S1P receptor modulator FTY720 (fingolimod). We sought to identify the kinase(s) involved in phosphorylation of CS-0777, anticipating sphingosine kinase (SPHK) 1 or 2 as likely candidates. Unlike kinase activity for FTY720, which is found predominantly in platelets, CS-0777 kinase activity was found mainly in red blood cells (RBCs). N,N-Dimethylsphingosine, an inhibitor of SPHK1 and -2, did not inhibit CS-0777 kinase activity. We purified CS-0777 kinase activity from human RBCs by more than 10,000-fold using ammonium sulfate precipitation and successive chromatography steps, and we identified fructosamine 3-kinase (FN3K) and fructosamine 3-kinase-related protein (FN3K-RP) by mass spectrometry. Incubation of human RBC lysates with 1-deoxy-1-morpholinofructose, a competitive inhibitor of FN3K, inhibited ∼10% of the kinase activity, suggesting FN3K-RP is the principal kinase responsible for activation of CS-0777 in blood. Lysates from HEK293 cells overexpressing FN3K or FN3K-RP resulted in phosphorylation of CS-0777 and structurally related molecules but showed little kinase activity for FTY720 and no kinase activity for sphingosine. Substrate preference was highly correlated among FN3K, FN3K-RP, and rat RBC lysates. FN3K and FN3K-RP are known to phosphorylate sugar moieties on glycosylated proteins, but this is the first report that these enzymes can phosphorylate hydrophobic xenobiotics. Identification of the kinases responsible for CS-0777 activation will permit a better understanding of the pharmacokinetics and pharmacodynamics of this promising new drug.

Use of an exposure-response model to aid early drug development of an oral sphingosine 1-phosphate receptor modulator.

Pharmacokinetic (PK) and exposure-response modeling of a selective sphingosine 1-phosphate receptor-1 modulator (CS-0777) was conducted in an iterative process to guide early clinical development decisions. A model based on preclinical data from monkeys was extrapolated to humans to support a single ascending dose (SAD) study. The model was updated after each cohort, providing guidance on both maximal inhibition and time to recovery for lymphocyte counts. A 2-compartment PK model with first-order absorption and elimination was found to describe the monkey and human datasets. The relationship between lymphocyte counts and active metabolite (M-1) concentrations was modeled via an indirect response model, whereby M-1 inhibited the reentry of lymphocytes to the circulation. The indirect-response model based on SAD data had an Imax of approximately 85% and an IC50 of 0.24 ng/mL. Additionally, based on SAD data, similar models were developed for lymphocyte subsets, including CD4 cells. Subsequently, simulations were utilized to design a multiple ascending dose study with adaptive dosing regimens that would meet targeted pharmacodynamic (PD) response thresholds (eg, minimum 40% reduction in lymphocytes) while maintaining CD4 counts above a reasonable safety threshold. In conclusion, model-based development and use of adaptive designs for dose optimization can reduce the time and number of subjects needed in early clinical development.