Metabolism and mass balance of SGLT2 inhibitor tofogliflozin following oral administration to humans.

1. Tofogliflozin is a novel and selective SGLT2 inhibitor increasing glucosuria by inhibition of glucose re-absorption in the kidney for the treatment of type 2 diabetes mellitus. 2. In this study, the metabolism and the mass balance of tofogliflozin was evaluated following administration of a single oral dose of 20 mg [(14)C]-tofogliflozin to six healthy subjects. 3. Tofogliflozin underwent mainly oxidative metabolism in the ethylphenyl moiety, but also minor glucuronide conjugates of metabolites and the parent drug were formed. 4. In plasma, the parent drug and its major phenyl acetic acid metabolite M1 accounted for 42% and 52% of the total drug-related material, respectively. The hydroxyl metabolites and their successor ketone metabolite showed an exposure well below 5%, along with an acyl glucuronide of M1. 5. Tofogliflozin was completely absorbed with subsequent predominate metabolic clearance and a small contribution of direct urinary elimination. Approximately, 76% of the dose was excreted in urine and 20% in faeces within 72 h. The high absorption of tofogliflozin was exemplified by the small trace of parent drug in faeces. The phenyl acetic acid metabolite M1 was the major component excreted in urine and faeces accounting for more than half of the dose. Tofogliflozin demonstrated a high metabolic turnover.

Method development and validation of the simultaneous determination of a novel topoisomerase 1 inhibitor, the prodrug, and the active metabolite in human plasma using column-switching LC-MS/MS, and its application in a clinical trial.

A robust and sensitive method using liquid chromatography-tandem mass spectrometry was developed and validated for the simultaneous determination of a novel topoisomerase 1 inhibitor CH0793076 (3076), the prodrug CH4556300 (TP300), and the active metabolite CH0793011 (3011) in human plasma. All plasma analyzed with this method was acidified with 1M HCl and 46% citric acid solution in a ratio of 100:10:1 (v:v:v) to avoid the pH-based degradation of TP300 and to shift the equilibria of 3076 and 3011 between the lactone and carboxylate forms towards the lactone forms. After the plasma proteins were precipitated with methanol:acetonitrile:HCl 1M (50:50:1, v:v:v) containing stable isotopic internal standards, the analytes were trapped on an Xterra MS C18 column (10×2.1 mm i.d., 5 µm) and separated on a Gemini C18 column (50×2.0 mm i.d., 5 µm) using column-switching liquid chromatography. Electrospray ionization in the positive-ion mode and multiple reaction monitoring were used to quantify the analytes with transitions m/z 587.2>441.2 for TP300, 459.1>415.2 for 3076, and 475.1>361.1 for 3011. The inter- and intra-day precisions were below 12%, and the accuracy was between -16% and 16% at the lower limit of quantitation (LLOQ) and between -11% and 14% at the other quality controls. The LLOQs of TP300, 3076, and 3011 were 0.8, 0.04, and 0.04 ng/mL, respectively. The validated method was successfully applied to clinical sample analysis and incurred sample reanalysis was also conducted.

Prediction of human blood-to-plasma drug concentration ratio.

The objective of this study was to predict R(b) (blood/plasma ratio) in humans using a simple method. Human and rat R(b) and free fraction in plasma (f(p)) values were obtained from the literature. The ratio of total red blood cell concentration to the free concentration in plasma (K(b)) was calculated using f(p) and R(b). Four methods were used for the prediction of R(b): (A) use of rat R(b); (B) use of R(b) calculated from rat K(b) and human f(p); (C) correlation of human log ((1-f(p))/f(p)) and human log K(b); and (D) correlation of log D with human log K(b). The R(b) of 96 compounds in humans ranged from 0.52 to 2.00, with an average of 0.89. A significant correlation was observed among human log K(b), human log ((1-f(p))/f(p)), and log D; however, no obvious correlation was observed among human R(b), human log ((1-f(p))/f(p)), and log D. The errors within 1.25-fold for methods A-D were 68.3%, 77.6%, 61.5% and 64.8%, respectively. All predictive methods considered here were superior to the use of the average value of human R(b) or R(b)=1. Rat R(b) corrected by human f(p) improved the accuracy of the prediction. Method B was the most accurate of the four methods.

Estimation of serum protein binding of compounds metabolized in serum using matrix inhibition.

It is difficult to evaluate the serum protein binding of compounds that are metabolized in rat serum, even when using ultrafiltration. Protein binding was estimated using matrix inhibition, a method that uses the change in metabolic velocity achieved by changing the free fraction of a compound in the incubation mixture by diluting the serum with phosphate buffered saline. The T(1/2) of phenyl nicotinate, benzyl nicotinate, octyl nicotinate, hexyl nicotinate, butyl nicotinate and [(3)H] compound A were 0.165, 0.780, 2.62, 3.94, 5.22 and 135 min, respectively, with protein binding values of 82.1%, 91.6%, 98.8%, 98.5%, 85.5% and 96.9%. The protein binding value of compound A estimated by ultrafiltration was 93.4%, indicating that the two methods give similar values. The matrix inhibition method is thus applicable for the evaluation of compounds metabolized in serum, and provides a simple, useful method to determine protein binding.

New method for the simultaneous estimation of intrinsic hepatic clearance and protein binding by matrix inhibition.

The purpose of this study was to develop a method for estimating the hepatic clearance (CL(h)) without using a protein binding test. This method allows the simultaneous evaluation of the intrinsic hepatic clearance (CL(int)) with a correction for microsomal binding, and the free fraction in the serum (fu). It uses the decrease in metabolic velocity achieved by decreasing the free fraction of a compound in the incubation mixture (fu(inc)) by the addition of serum, and by changing the microsomal protein concentration. This method is denoted as the 'matrix inhibition method', because it uses the inhibition of the metabolic velocity by the incubation matrix. The metabolic rates of eight compounds (diazepam, imipramine, warfarin, and compounds A-E) were evaluated under several incubation conditions using rat serum and microsomes. The correlation of CL(int) evaluated using the method and using equilibrium dialysis after the CL(int) was corrected for microsomal binding was r = 0.968. The correlation of fu . CL(int) was r = 0.996. Although the method required a high enough fu and fu(microsomes) difference among the reaction conditions for each compound, it could evaluate CL(int) and fu simultaneously and easily by adding additional reaction conditions to the metabolic stability tests performed in ADME screening.