In vitro profiling of the metabolism and drug–drug interaction of tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, using human liver microsomes, human hepatocytes, and recombinant human CYP

Figures & data

Figure 1. Radio-HPLC chromatogram of [¹⁴C]tofogliflozin after 5 h incubation with human hepatocytes at the concentration of 1 μM. Pooled human hepatocytes from 10 donors of mixed gender were used. The percent in the figure expresses the mean value of percent radioactivity obtained from two experiments.

Figure 2. Proposed metabolic pathways of tofogliflozin in human.

Table 1. Cofactor requirement and effect of 1-aminobenzotriazole on the metabolism of [¹⁴C]tofogliflozin.

	Radioactivity (%)
	NADPH (−)^a	Control^b	1-Aminobenzotriazole^c
Tofogliflozin	99.7	99.1	99.8
M2/M3	ND	0.5	ND
M4	ND	0.2	ND

The mixture containing 1 mg/mL human liver microsomes, 0.1 M phosphate buffer (pH 7.4), NADPH-generating system (1.3 mM NADP⁺, 3.3 mM glucose-6-phosphate, 3.3 mM MgCl₂, and 0.4 units/mL glucose-6-phosphate dehydrogenase), and 10 μM [¹⁴C]tofogliflozin. Radioactivity (%) represents the radioactivity ratio of each peak in the mixture after 1 h incubation. Data are expressed as the mean of two experiments.

ND, not detected.

^aA blank vehicle was added in place of the NADPH-generating system.

^bA blank vehicle was added in place of 1-aminobenzotriazole.

^cA non-specific CYP inhibitor, 1-aminobenzotriazole (final concentration: 1 mM), was added to the incubation mixture.

Table 2. Metabolism of [¹⁴C]tofogliflozin to hydroxylated derivatives after 1 h incubation with a set of 14 rhCYPs at 37 °C.

	Radioactivity (%)
rhCYP isoforms	M2/M3	M4	Tofogliflozin
CYP1A2	ND	ND	98.6
CYP2A6	ND	0.3	98.8
CYP2B6	ND	0.3	99.1
CYP2C8	0.2	0.3	98.5
CYP2C9	ND	ND	98.9
CYP2C18	8.2	8.4	74.4
CYP2C19	0.2	ND	98.7
CYP2D6	1.0	0.3	97.8
CYP2E1	ND	ND	99.1
CYP3A4	13.0	ND	84.8
CYP3A5	12.6	ND	85.8
CYP4A11	ND	1.8	97.6
CYP4F2	ND	ND	99.1
CYP4F3B	ND	1.6	97.7

ND, not detected.

Data are expressed as the mean of two experiments.

Table 3. CYP1A2 and CYP3A4 induction by tofogliflozin.

	CYP1A2 activity (pmol/mg protein/h)				CYP3A4 activity (pmol/mg protein/h)
		Tofogliflozin				Tofogliflozin
Hepatocytes batch	Control	0.5 μM	5 μM	50 μM	Control	0.5 μM	5 μM	50 μM
Lot. 704	1.2	1.2	1.2	0.8	3420	5130	3021	3357
Lot. LMP	5.9	8.3	7.7	7.6	1855	2179	2457	1546
Lot. BAD	1.8	1.5	1.5	2.6	1198	953	1108	677

The plated human hepatocytes from three donors were treated with test article, tofogliflozin (0.5, 5, and 50 μM) for 48 h in a CO₂ incubator set at 37 °C, 5% CO₂, and 95% humidity. In the control sample, blank vehicle was added in place of tofogliflozin. After the treatment, 7-ethoxyresorufin O-deethylation activity (CYP1A2) or testosterone 6β-hydroxylation activity (CYP3A4) in hepatocytes was evaluated. Data are expressed as the mean of two experiments.

Table 4. IC₅₀ values for reversible inhibition of CYPs by tofogliflozin and M1.

		Tofogliflozin		M1
Enzyme	Substrate	IC50 (μM)	Inhibition (%) at 50 μM	IC50 (μM)	Inhibition (%) at 50 μM
CYP1A2	Ethoxyresorufin	>50	6.0 ± 1.0	>50	5.3 ± 4.0
CYP2B6	Bupropion	>50	15.0 ± 3.0	>50	8.2 ± 4.1
CYP2C8	Amodiaquine	>50	−2.0 ± 3.0	>50	17.2 ± 2.4
CYP2C9	Diclofenac	>50	−3.0 ± 3.0	>50	3.7 ± 1.7
CYP2C19	S-Mephenytoin	>50	1.0 ± 7.0	27.1 ± 6.5	58.7 ± 3.9
CYP2D6	Dextromethorphan	>50	4.0 ± 1.0	>50	3.8 ± 1.8
CYP3A	Midazolam	>50	1.0 ± 1.0	>50	10.2 ± 6.5
CYP3A	Nifedipine	>50	4.0 ± 2.0	>50	26.4 ± 2.7
CYP3A	Testosterone	>50	9.0 ± 4.0	>50	6.4 ± 2.1

The inhibitory effect of tofogliflozin and M1 on human CYP activity in human liver microsomes was determined by evaluating ethoxyresorufin O-deethylation (CYP1A2), bupropion t-butyl hydroxylation (CYP2B6), amodiaquine N-deethylation (CYP2C8), diclofenac 4′-hydroxylation (CYP2C9), S-mephenytoin 4′-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), midazolam 1′-hydroxylation (CYP3A4), nifedipine oxidation (CYP3A4), or testosterone 6β-hydroxylation (CYP3A4) activity. Data are expressed as the mean ± SD of three experiments.

Table 5. Time-dependent inhibition of CYP activity by tofogliflozin and M1.

Enzyme	Test inhibitor	kobs (min^−1)
CYP1A2	Control	0.013
	Tofogliflozin	0.013
	Control	0.009 ± 0.003
	M1	0.010 ± 0.002
CYP2C9	Control	0.000
	Tofogliflozin	−0.006
	Control	0.000 ± 0.001
	M1	0.000 ± 0.003
CYP3A	Control	0.003
	Tofogliflozin	0.003
	Control	0.001 ± 0.009
	M1	−0.001 ± 0.004

The time-dependent inhibition of human CYP activity by tofogliflozin and M1 in human liver microsomes was determined by evaluating ethoxyresorufin O-deethylation (CYP1A2), diclofenac 4′-hydroxylation (CYP2C9), or midazolam 1′-hydroxylation (CYP3A4) activity. In control samples, blank vehicle was added in place of tofogliflozin or M1. The k_obs values of tofogliflozin and M1 at the concentration of 100 and 50 μM, respectively, are shown in this table. Data are expressed as the mean ± SD of three experiments, and values without standard deviations are from a single experiment.

Table 6. In vitro IC₅₀ values and in vivo inhibition potencies for SGLT2 of tofogliflozin and its metabolites.

	IC50 (μM)	IC50 ratio	AUC0-24h^a (ng h/mL)	fp^b	In vivo inhibition potency versus tofogliflozin^f
Tofogliflozin	0.0039	1	1814	0.17^c	–
M1	2.7	700	2215	0.45^d	0.005
M2	0.015	3.8	225	1^e	0.2
M3	0.014	3.5	225	1^e	0.2
M4	0.0049	1.3	ND	1	NC
M5	0.016	4.1	136	1^e	0.1

The cells expressing SGLT2 were incubated with 1 mM [¹⁴C]AMG and a test article (tofogliflozin or its metabolites) at 37 °C for 40 min. After incubation, SGLT2 activity was determined by measuring AMG uptake into the cells. IC₅₀ values for SGLT2 activity are expressed as the mean of two experiments.

NC, not calculated due to no AUC_0–24h data.

ND, not detected

^aZell et al. 2013.

^bf_P: free fraction in plasma.

^cSchwab et al. 2013.

^dThe f_p value of M1 at 0.1 μg/mL obtained in the present study.

^eThere were no unbound fraction data for M2, M3, and M5; thus, they were assumed to be 1 to calculate in vivo inhibition potency.

^fValues were calculated by the following formula:

Zell M, Husser C, Kuhlmann O, et al. (2013). Metabolism and mass balance of SGLT2 inhibitor tofogliflozin following oral administration to humans. Xenobiotica 44:369–78

 PubMed Web of Science ®Google Scholar

Schwab D, Portron A, Backholer Z, et al. (2013). A novel double-tracer technique to characterize absorption, distribution, metabolism and excretion (ADME) of [(14)C]tofogliflozin after oral administration and concomitant intravenous microdose administration of [(13)C]tofogliflozin in humans. Clin Pharmacokinet 52:463–73

 PubMed Web of Science ®Google Scholar