Contribution of UGT1A1, CYP2A6, CYP2C9 and CYP3A4 in Metabolism of Belinostat

Contribution of UGT1A1, CYP2A6, CYP2C9 and CYP3A4 in Metabolism of Belinostat Gang Luo1, Michael Schicker1, Deborah Lee1, Sara Leitz1, Donald McKenzie1, Daniel Albaugh1 and Guru Reddy2 1Covance Laboratories Inc., Madison, WI; 2Spectrum Pharmaceuticals, Inc., Irvine, CA

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Abstract t 4 s o

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Samples were analyzed for C‑belinostat and its metabolites 1 8 6 2 1 X

Belinostat, a potent inhibitor of class I and class II histone deacetylase B e X ( P d d P

500.0 i t e ( c t a

using an HPLC with radiometric detection. Separation of t a e l A s d t i o enzymes, has been approved for the treatment of patients with relapsed y a n h t m i t l s e e A belinostat and metabolite reference standards by HPLC using o t n B M i a l or refractory peripheral T-cell lymphoma. Previous work demonstrated 400.0 t s B e n o

UV detection was demonstrated in Figure 2. Remaining i l that UGT1A1-mediated glucuronidation was a significant metabolic d e 14C‑belinostat and formed metabolites were expressed as a B e 300.0 u r o n i

pathway of belinostat. A separate report suggests that belinostat was u c l A G B t percent of total radioactivity. Metabolic rates were calculated S a t s metabolized by recombinant human CYP enzymes. In the present - A o 3 n

200.0 i based on the formation of metabolites as needed. l

study, contribution of UGT1A1, CYP2A6, CYP2C9, and CYP3A4 in B e metabolism of 14C-belinostat was assessed using in vitro test systems. Data Analysis 100.0 14C‑Belinostat was extensively metabolized by human liver microsomes Figure 2. HPLC The data fitting was performed with SigmaPlot (version 12.5) UV detection at 240 nm. profile of belinostat (HLM) in the presence of both uridine 5′-diphosphoglucuronic acid 0.0 for enzyme kinetics. and metabolites. UV (UGPDA) and dihydronicotinamide-adenine dinucleotide phosphate 0.00 10.00 20.00 30.00 40.00 50.00 min detection at 240 nm. (NADPH). Identified metabolites include belinostat glucuronide (major), belinostat amide (minor) and belinostat acid (minor). For example, belinostat glucuronide, belinostat amide, and belinostat acid accounted Results for 54.6 to 55.1, 2.68 to 2.71 and 1.45 to 1.78% of the total radioactivity, ▶ 14 C‑Belinostat was metabolized in HLM in a linear fashion respectively, after 14C-belinostat (10 and 100 μM) was incubated with in the presence of UDPGA and NADPH (Figure 3). human hepatic microsomes (1 mg/mL) for 60 minutes. Km and Vmax of ▶ Identified metabolites included belinostat glucuronide glucuronidation were determined to be 72.8 µM and 2160 pmol/min/mg, (major), belinostat amide (minor) and belinostat acid respectively. The projected hepatic clearances obtained from time- (minor), as shown in Figure 4. dependent total metabolism of belinostat (9.67 to 10.8 mL/min/kg) and Glucuronidation of 14C‑belinostat was determined in six from Km/Vmax (12.9 mL/min/kg) of belinostat glucuronidation were ▶ equivalent to human in vivo clearance (estimated as 12.6 to UGT1A1 genotyped donors (*1*1, *1*28, and *28*28), as 14.7 mL/min/kg). Rates of belinostat glucuronidation by HLM prepared shown in Figure 5. from six individual UGT1A1 genotyped donors were significant different ▶ Belinostat glucuronidation and estradiol glucuronidation by between allelic variants of *1*1 and *28*28) and highly correlated with HLM from six individual UGT1A1 genotyped donors were 2 that of estradiol glucuronidation (r = 0.9613). Belinostat amide and acid highly correlated (r2 = 0.9613), as shown in Figure 6. 14 generated in HLM from all six donors were minor, and more importantly, Figure 3. Kinetics of C-belinostat disappearance in HLM in the presence of UDPGA and NADPH. UGT1A1 inhibitor troglitazone markedly decreased their amounts were almost identical among the different UGT1A1 ▶ belinostat glucuronidation, but CYP2A6, CYP2C9 and CPM 1600 t

genotyped donors. Therefore, it was concluded that UGT1A1 plays a a t

s ther Metabolism

CYP3A4 inhibitors did not change production of belinostat o

1000 n i predominant role whereas CYP enzymes play little or no role in l

e Glucuronidation B amide and belinostat acid (Figure 7). - C e 4 d metabolism of belinostat. 1 i )

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800 u c Major contribution of UGT1A1 and minor contribution of m /

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CYP enzymes were confirmed by using recombinant a m t / s l o n o i 600 l

human enzymes (Table 1). e 800 m e - B p

Introduction d C ( i

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d i t

K and V of glucuronidation were determined to be i c ▶ a

m max

Belinostat, a potent inhibitor of class I and class II histone deacetylase t i A s

400 t o c a n i 72.8 µM and 2160 pmol/min/mg, respectively, as shown in t l s A e enzymes, has been approved for the treatment of patients with relapsed o 400 n - B i l C e

Figure 8. 4 1 or refractory peripheral T-cell lymphoma. The chemical structure and - B C

200 4 metabolic pathway of belinostat are presented in Figure 1. This study 1 1600 0 aimed to assess contribution of UGT1A1, CYP2A6, CYP2C9 and ) g 0 *1*1 (n=2) *1*28 (n=2) *28*28 (n=2) 14 m CYP3A4 in metabolism of C-belinostat using in vitro test systems. y = 0.4607x + 281.15 n / i UGT1A1 Genotyped Donors R² = 0.9613 0.00 10.00 20.00 30.00 40.00 50.00 min m / l 1200 14 14 o Figure 4. Metabolism of C-belinostat (10 µM) by HLM Figure 5. Metabolism of C-belinostat by H

p m H N N ( (0.5 mg/mL) in the presence of UDPGA and NADPH for UGT1A1 genotyped donors. S S n

H o NH 2 � 30 minutes.

d a 800 n i o

Belinostat amide Belinostat acid u r u c 1000 Glucuronide Formation 1000 Amide Formation 1000 Acid Formation

G l (pmol/min/mg) (pmol/min/mg) (pmol/min/mg)

a 400 Phase I t

Phase I n o s i l 800 800 e 800 H H N N B S H S N NH 0 H Phase II 0 800 1600 2400 3200 600 600 600 Estradiol 3ß-D-Glucuronida�on (pmol/min/mg) Belinostat Methyl belinostat Figure 6. Correlation between 14C-belinostat and estradiol Phase II glucuronidation. 400 400 400 Phase II (UGT1A1)

H H

H H 2000 200 200 200

H H H H N N g ) S S H H 1500 0 0 0 N N m H H

n / K = 72.8 µM Figure 7. Metabolite formation i m 14

m of C-belinostat in the / Vmax = 2160 pmol/min/mg

o l absence and presence of Belinostat glucuronide Belinostat glucoside 1000

p m inhibitors. (

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Figure 1. In vivo metabolic pathways of belinostat. a 500 R Table 1. Metabolism of 14C-Belinostat (100 µM) by Recombinant Human Enzymes Remaining Radioactiity ( of the total) Methods 0 0 200 400 600 800 1000 Enzyme Belinostat Glucuronide Amide Acid General Incubation Procedure Belinostat (µM) Vector Control 95.8 0 1.82 1.12 Incubation mixtures containing 14C-belinostat with microsomal protein or 14 UGT1A1 32.1 63.2 1.60 0 recombinant UGT1A1, CYP3A4, CYP2C9 and CYP2A6 in assay buffer Figure 8. Kinetics of C-belinostat glucuronidation by HLM (0.5 mg/mL) for 30 minutes. CYP2A6 95.5 0 1.92 1.01 were warmed at 37°C for 5 minutes. The reactions were initiated with CYP2C9 95.6 0 1.56 1.01 the addition of warmed (37ºC) UDPGA (2 mM) and/or NADPH CYP3A4 94.3 0 1.72 1.04 (1 mM) and terminated by the addition of stop solution followed by vortex Summary mixing. Supernatants obtained from centrifugation were transferred to a separate plate and stored at approximately 4°C prior to analysis. ▶ UGT1A1 mediated glucuronidation was the predominant metabolic pathway for belinostat. ▶ CYP2A6, CYP2C9 and CYP3A4 did not play a significant Presented at ISSX 2018 role in metabolism of belinostat.