Atea Pharmaceuticals : Low Risk of Drug Drug Interactions for Ruzasvir Based Upon In Vitro Metabolism and Transporter Interaction Studies (SAT 412)

Low Risk of Drug-Drug Interactions for Ruzasvir Based Upon In Vitro Metabolism and Transporter Interaction Studies

SAT-412

Alex Vo,1* Jocelyn Yabut,2 Michael J. Hafey,2 Hui Wan,2 Nancy Agrawal,1 Jean-Pierre Sommadossi1

1Atea Pharmaceuticals, Inc., Boston, MA, USA; 2Merck & Co., Inc., West Point, PA, USA

INTRODUCTION

• Approximately 50 million people globally are living with chronic HCV infection,

with 1.0 million new infections occurring per year and 242,000 deaths per year1

• New HCV treatment regimens with direct-acting antivirals have become the

standard of care, with sustained virological response (SVR) rates exceeding

95% and treatment duration reduced to 8-12 weeks depending on the

regimen and patient population

• Despite high efficacy rates with existing therapies, better treatment options

are needed for certain patient populations that include those with severe liver

decompensation, active hepatocellular carcinoma, genotype 3 HCV infection,

treatment failure due to resistance requiring at least 12 weeks of treatment,

often with adjunctive ribavirin treatment, and those with comorbid conditions

RESULTS

Table 1. Recovery of radioactivity at 72 hours in urine, bile and feces following administration of [3H]-RZR to BDC male Wistar Han rats and Beagle dogs

Species, (n)​	Dose (mg/kg),		Percent of dose recovered​			Total percent
route	Urine	Bile	Feces	Cage wash​
Rat, (n=3)​	2, IV	0.24 ± 0.048	62	± 12	20 ± 9.1	0.053	± 0.023	83 ± 2.7
Dog, (n=3)​	1, IV	0.25 ± 0.080	51	± 16	35 ± 13	0.091	± 0.081	86 ± 2.6
Dog, (n=3)​	5, PO	0.21 ± 0.051	3.3	± 1.9	78 ± 5.9	0.38	± 0.28	82 ± 4.4

• RZR (10 µM) inhibited CYP3A4 38% with midazolam as substrate but had no significant impact on the rest of CYPs tested
• When assessed in hepatocytes from 3 human donors, RZR did not induce mRNA expression or enzyme activity of CYP3A4
• Moreover, RZR did not induce mRNA expression of CYP2B6 and CYP1A2
• However, in one donor, a slight increase in CYP2B6 activity (15.2% of positive control) and a slight increase in CYP1A2 activity (12-23% of positive control) was observed

receiving concomitant medications leading to drug-drug interactions (DDI)

• Atea Pharmaceuticals, Inc. is developing bemnifosbuvir (BEM) in

combination with ruzasvir (RZR, also known as AT-038, formerly known as

MK-8408) for the treatment of HCV

• BEM is a novel, oral NS5B polymerase inhibitor; RZR is a novel, oral NS5A

phosphoprotein inhibitor. RZR is a small molecule inhibitor of HCV

nonstructural protein 5A (NS5A), an essential protein for HCV replication

• Both have individually demonstrated potent, pan-genotypic, antiviral activity

against HCV2,3

• The combination of BEM-RZR, which has demonstrated a substantially

greater inhibition of HCV replication in vitro than the sum of the activities of

both agents alone, has the potential to offer a differentiated, short duration,

pan-genotypic, protease inhibitor-sparing regimen for HCV-infected patients

with or without cirrhosis

• For HCV patients with decompensated cirrhosis, the combination of BEM and

RZR may have the additional potential for treatment without the co-administration

of ribavirin, which can cause a wide range of serious side effects

METHODS

CYP450 inhibition using human liver microsomes (HLM)

Excretion summary

• Small amounts of hydrolytic and oxidative metabolites were detected in bile and feces
• Unchanged [3H]-RZR was the majority of drug-related radioactivity in circulation and in bile
• Since there was very little radioactivity observed in urine, substrate evaluation studies with kidney specific transporters were not conducted (OAT1, OAT3, OCT2, MATE1, MATE2-K)
• Labeled RZR was primarily recovered in bile and feces as unchanged drug in rats and dogs (Table 1)

CYP450 phenotyping

• In vitro, RZR was metabolized primarily by CYP3A4, resulting in the formation of mono-oxidative metabolites M6/7 (Figure 1)

Figure 1. RZR (1 µM) was incubated with HLM and known CYP inhibitors or inhibitory monoclonal antibody against CYP3A (A), and with recombinant human P450 enzymes (0.2 nmol/mL) and insect microsomes for 60 min at 37°C (B, C). The metabolites M6/7 (A, B) and the parent RZR (C) was then measured by LC-MS/MS in triplicate

Reversible CYP450 and UGT1A1 inhibition

Table 2. CYP and UGT1A1 inhibitory potential of RZR in pooled HLM. Enzyme activities were compared to known inhibitors (control)

CYP	Reaction	IC50 (µM)
Control inhibitor	RZRa
CYP1A2	Phenacetin	0.013​	>10​
O-deethylation	(α-napthoflavone)	(4.6 ± 0.77%)​
CYP2B6	Bupropion	0.43​	>10​
hydroxylation	(ticlopidine)​	(4.8 ± 0.10%)​
CYP2C8	Amodiaquine	0.32​	>10​
N-deethylation	(montelukast)​	(9.5 ± 1.2%)​
CYP2C9	Diclofenac	0.82​	>10​
4'-hydroxylation	(sulfaphenazole)​	(7.9 ± 1.0%)​
CYP2C19	S-mephenytoin	0.22​	>10​
4'-hydroxylation	(benzylnirvanol)​	(11 ± 0.46%)​

Transporter interaction

Table 3. Uptake and inhibition of RZR was evaluated in human hepatocytes and stably transfected cells expressing the transporter of interest in vitro, using known transporter inhibitors or substrates as controls

Transporter	Substrate potential	Inhibition IC50 (µM)
P-gp	Yes	0.05	± 0.03
BCRP	Inc	0.27	± 0.02
BSEP	No	0.37	± 0.02
OATP1B1	Inc	0.092	± 0.004
OATP1B3	Inc	0.052	± 0.002
OAT1	ND	No inhibition*
OAT3	ND	No inhibition*
OCT1	No	No inhibition*
OCT2	ND	No inhibition*
MATE1	ND	No inhibition*

• For direct CYP inhibition, RZR was pre-incubated in triplicate at 37ºC with HLM (reversible inhibition) and probe substrate in the absence of NADPH, followed with the addition of pre-warmed NADPH and incubation at 37ºC for 3-30 min depending on the individual CYP isoform
• For time-dependent inhibition, the test article was incubated at 37ºC with HLM in buffer for a duration ranging from 0 to 30 min in the absence of NADPH, followed by NADPH and probe substrate addition and incubation
• For UGT1A1 (uridine 5′-diphospho-glucuronosyltransferase enzyme 1A1) evaluation, the test article was incubated at 37°C with HLM for 20 min in a reaction mixture containing estradiol, UDPGA, and alamethicin
• Analyses were measured by LC-MS/MS

CYP450 induction in human hepatocytes

• Human cryopreserved hepatocytes from three donors were incubated in culture media spiked with RZR for 48 hours in triplicate
• Hepatocyte cultures were also treated in parallel with vehicle control or control compounds
• Positive controls included omeprazole (50 μM) for CYP1A2, phenobarbital (1000 μM) for CYP2B6, and rifampicin (10 μM) for CYP3A4
• Both mRNA expression and enzymatic activity were measured for each CYP

Transporter interaction studies

• For transcellular efflux assays, cell lines were cultured on semi-permeable inserts
• Transport measurements were performed at Day 3 or 4 after seeding to allow formation of confluent monolayers
• Samples were quantified using LC-MS/MS
• Transporter inhibition assays to investigate the interaction with the human BCRP, MDR1 (also known as P-gp), MATE1, MATE2-K, OATP1B1, OATP1B3, OAT1, OAT3, OCT1, and OCT2 transporters were conducted using inside-out membrane HEK293 vesicles and transporter-expressed MDCKII and HEK293 cells

A. Inhibition of metabolite formation in pooled HLM

CYP2D6

Dextromethorphan

0.18​

>10​

O-demethylation

(quinidine)​

(0%)b​

100

(M6/7)

80

CYP3A4M

Midazolam

0.029​

>10​

60

1'-hydroxylation

(ketoconazole)​

(38 ± 7.3%)​

inhibition

40

CYP3A4T

Testosterone

0.039​

>10​

6β-hydroxylation

(ketoconazole)​

(11 ± 1.2%)​

%

20

0

Estradiol-3-

2.69 ± 0.20​

Furafylline

Montelukast

Sulfaphenazole

Benzylnirvanol

Quinidine

Ketoconazole

Troleandomycin

MAb-3A

UGT1A1

2.76 ± 0.70​

(1A2)

(2C8)

(2C9)

(2C19)

(2D6)

(3A4/5)

(3A4)

glucuronidation

(nicardipine)

B. Formation of metabolite following RZR incubation with recombinant P450 enzymes	aValue in parenthesis represents the percent inhibition (mean ± SD, n=3) at 10 µM RZR;
0.03			bValue in parenthesis represents the percent inhibition (mean, n=2) at 10 µM RZR.

(M6/7)									• RZR was not a time-dependent inhibitor of CYP3A4 (Figure 2)
response	0.02
								Figure 2. TDI Inhibition of CYP3A4 in HLM
Metabolite	0.01
0.00								100
	CYP2B6	CYP2C8	CYP2C9	CYP2C19	CYP2D6	CYP3A4	Insect control	remaining
	CYP1A2							Rate constant
C.															(min-1)
													DMSO	0.004
	120
															MK-8408 (10 μM)	0.011
	100								10							MK-8408 (50 μM)	0.012
															Mifepristone (10 μM)	0.062
parentremaining	80								Percentactivityof
													Mifepristone (50 μM)	0.064
60
40
%	20
	0								0	5	10	15	20	25	30	35
	CYP2B6	CYP2C8	CYP2C9	CYP2C19	CYP2D6	CYP3A4	Insect control			Preincubation time (min)
	CYP1A2

MATE2-K

ND

No inhibition*

Inc, inconclusive; ND, substrate potential for urinary transporters were not determined because the urinary elimination of RZR was insignificant (<1%).

*No inhibition up to highest concentration tested (0.5 µM for OAT1/OAT3/OCT1/OCT2 and 2 µM for MATE1/MATE2-K).

CONCLUSIONS

• RZR is metabolized primarily by CYP3A4 in vitro, however the extent of in vivo metabolism was insignificant
• The main route of elimination of RZR in preclinical species was GI secretion and biliary excretion of the unchanged parent
• RZR exhibited weak direct inhibition of CYP3A4 in vitro, and no time-dependent inhibition of CYP3A4 was observed
• RZR was a substrate of P-gp and potentially of BCRP
• RZR inhibited P-gp, BCRP, BSEP, OATP1B1 and OATP1B3, however, because RZR was highly protein-bound (>99.9%), the risk of potential DDI is likely minimal

References

1. World Health Organization, Hepatitis C Fact Sheet. Available at: https://www.who.int/news-room/fact-sheets/detail/hepatitis-c (Accessed May 2024);
2. Good SS, et al. PLoS One 2020;15:e0227104;
3. Asante-AppiahE, et al. Antimicrob Agents Chemother 2018;62:e01280-18.

Acknowledgements

This study was funded by Atea Pharmaceuticals (Boston, MA, USA). We thank

Dr. Kerry-Ann da Costa for her excellent assistance in preparing this poster presentation.

Disclosures

The authors are employees of Atea Pharmaceuticals or Merck & Co.

Poster presented at The European Association for the Study of the Liver (EASL) 2024 Congress, 5-8 June, Milan, Italy.