약물 상호작용 평가 | DDI Panel

Metabolism-mediated Drug Interaction (대사 매개 의약품 상호작용의 평가)

간에 존재하는 Phase I (Cytochrome P450)과 Phase II (GST, UGT..) 대사효소는 대부분의 의약품 대사에 관여합니다. 의약품은 이 효소들과 작용하여 효소의 활성을 방해하거나 (CYP inhibition), nuclear receptor를 활성화시켜 gene expression을 유도합니다. (CYP induction)

대사 매개 의약품 상호작용은 의약품이 대사 효소의 활성을 변경하여 ADME와 PK에 영향을 미칠 때 발생할 수 있습니다.

1) CYP enzyme substrate assessment assays

의약품이 CYP 효소에 대한 기질로 작용하는지, 즉 어떤 효소가 의약품을 대사 시키는지 평가
의약품을 CYP1A12, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4등의 CYP효소와 다양한 시간에서 인큐베이션한 후, 잔량을 HPLC-MS/MS로 정량 측정
의약품 소실 값을 통해 각 대사 효소에 대한 약물의 청소율 (Clearance) 확인

CYP Enzyme	Source	Substrate	Incubation
CYP1A2	Human recombinant CYP1A2	Test Compound	0, 15, 30, 45, 60 min, 37°C
CYP2B6	Human recombinant CYP2B6	Test Compound	0, 15, 30, 45, 60 min, 37°C
CYP2C8	Human recombinant CYP2C8	Test Compound	0, 15, 30, 45, 60 min, 37°C
CYP2C9	Human recombinant CYP2D9	Test Compound	0, 15, 30, 45, 60 min, 37°C
CYP2C19	Human recombinant CYP2C19	Test compound	0, 15, 30, 45, 60 min, 37°C
CYP2D6	Human recombinant CYP2D6	Test compound	0, 15, 30, 45, 60 min, 37°C
CYP3A4	Human recombinant CYP3A4	Test compound	0, 15, 30, 45, 60 min, 37°C

2) CYP enzyme inhibition assessment assays

의약품의 CYP 효소 (CYP1A, 3A, 2B6, 2C8, 2C9, 2C19, 2D6) 활성 억제 가능성을 평가
각 CYP enzyme probe substrate의 특정한 metabolite를 정량

a. 가역적 억제인지 평가 (10분간 37도 인큐베이션)

b. 시간의존적 억제 (Time-Dependant-Inhibition) 인지 평가

(테스트 의약품을 NADPH가 생성되는 조건과 생성되지 않는 조건에서 human liver microsome과 37도에서 30분동안 사전 배양한 후 substrate를 처리하고 10분간 배양)

3) CYP enzyme induction assessment assays

약물이 CYP 효소의 유도제로 작용하는지 여부 평가
CYP enzymes (CYP1A2, 2B6, 3A4, 2C8, 2C9 and 2C19)의 mRNA 레벨을 qPCR로 측정하거나 probe substrate의 metabolite를 정량함으로써 효소 활성 측정

a. CYP Induction Enzyme Activity

(3 donor로부터의 냉동 hepatocyte에서 compound와 함께 72시간 배양)

b. CYP Induction mRNA level

(3 donor로부터의 냉동 hepatocyte에서 compound와 함께 72시간 배양)

Transporter-mediated Drug Interaction (수송체 매개 의약품 상호작용의 평가)

수송체는 독소 및 약물과 같은 내인성 및 외인성 물질을 포함하여 이온 또는 분자의 막간 이동을 촉진하는 막 단백질 입니다. 수송체는 인체 전체의 여러 기관에서 발현되며 내인성 및 외인성 물질의 접근을 제어함으로써, 약물의 약동학과 약력학에 영향을 줍니다. 약물상호작용에 관여하는 수송체 종류는 다음과 같습니다.

ATP Binding cassette (ABC) transporter : P-gp, BCRP
Solute Carrier (SLC) transporter : OATP1B1, OATP1B3 (for hepatic clearance drugs), OAT1, OAT3, OCT2, MATE1, MATE2-K (for renal clearance drugs)

1) Transporter inhibition assay

규제 기관은 P-gp, BCRP, OATP1B1, OATP1B3, OAT1, OAT3, OCT2, MATE1 및 MATE2-K에 대한 연구 약물의 억제 가능성을 테스트할 것을 권장합니다.
Eurofins Discovery는 P-gp 또는 BCRP를 발현하는 HEK 세포에서 준비한 막 소포를 사용하여 P-gp 및 BCRP inhibition 분석을 제공합니다.

2) Transporter substrate assay

약물의 투과도나 용해도가 높지 않을 경우, P-gp나 BCRP의 substrate로서의 잠재성을 평가합니다.
약물이 간대사나 담즙분비를 통해 제거되거나, 간으로의 약물 흡수가 임상적으로 중요하다면, OATP1B1이나 OATP1B3의 substrate로서의 잠재성을 평가합니다.
약물이 신장을 통해 주로 제거된다면 OAT1, OAT3, OCT2, MATE1이나 MATE2-K의 substrate로서의 잠재성을 평가합니다.

Transporter	Cell line	Substrate	Incubation and Inhibitor
P-gp	Caco-2 cell line	Test compound	60 min A-B and 40 min B-A, with and without Verapamil (100 µM)
BCRP	Caco-2 cell line	Test compound	60 min A-B and 40 min B-A, with and without Ko143 (10 µM)
OATP1B1	OATP1B1-CHO	Test compound	2 min and 30 min, with and without Cyclosporine (5 µM)
OATP1B3	OATP1B3-CHO	Test Compound	2 min and 30 min, with and without Cyclosporine (5 µM)
OAT1	OAT1-CHO	Test Compound	2 min and 30 min with and without Probenecid (30 µM)
OAT3	OAT3-CHO	Test Compound	2 min and 30 min with and without Probenecid (30 µM)
OCT2	OCT2-HEK	Test Compound	2 min and 30 min, with and without Verapamil ( 50 µM)
MATE1	MATE1-HEK	Test Compound	2 min and 30 min, with and without Verapamil ( 50 µM)
MATE2-K	MATE2-K-HEK	Test Compound	2 min and 30 min, with and without Verapamil ( 50 µM)

DDI Panel 서비스 종류

CYP450 Inhibition Assays - Human Liver Microsomes (HLM)	Item #	Panel
CYP Inhibition (HLM; package of 8 assays)		G232
CYP3A Inhibition (HLM, midazolam substrate)	1770	P
CYP3A Inhibition (HLM, testosterone substrate)	1769	P
CYP1A Inhibition (HLM, phenacetin substrate)	2064	P
CYP2B6 Inhibition (HLM, bupropion substrate)	2065	P
CYP2C8 Inhibition (HLM, amodiaquine substrate)	4481	P
CYP2C9 Inhibition (HLM, diclofenac substrate)	2066	P
CYP2C19 Inhibition (HLM, omeprazole substrate)	1772	P
CYP2D6 Inhibition (HLM, dextromethorphan substrate)	1838	P
CYP450 Inhibition Assays - Time Dependent (+/- NADPH)	Item #	Panel
Time-dependent CYP Inhibition (HLM; package of 14 assays)		G234
Time-dependent CYP3A Inhibition (HLM; midazolam substrate)	G201	P
Time-dependent CYP1A Inhibition (HLM; phenacetin substrate)	G197	P
Time-dependent CYP2C9 Inhibition (HLM; diclofenac substrate)	G199	P
Time-dependent CYP2C19 Inhibition (HLM; omeprazole substrate)	G198	P
Time-dependent CYP2D6 Inhibition (HLM; dextromethorphan substrate)	G200	P
Time-dependent CYP2B6 Inhibition (HLM; bupropion substrate)	G413	P
Time-dependent CYP2C8 Inhibition (HLM; amodiaquine substrate)	G414	P
CYP450 Phenotyping Assays - Recombinant	Item #	Panel
CYP Phenotyping (recombinant, panel of 7 CYP's)		G230
Intrinsic clearance (CYP3A4)	1649	P
Intrinsic clearance (CYP1A2)	1645	P
Intrinsic clearance (CYP2B6)	2387	P
Intrinsic clearance (CYP2C8)	2384	P
Intrinsic clearance (CYP2C9)	1646	P
Intrinsic clearance (CYP2C19)	1647	P
Intrinsic clearance (CYP2D6)	1648	P
CYP Induction Assays - Human Hepatocytes (mRNA level)	Item #	Panel
CYP Induction Panel (human hepatocytes, mRNA level, 3 donors)		G345
CYP1A2 Induction (human hepatocytes, mRNA level, 3 donors)	3862	P
CYP2B6 Induction (human hepatocytes, mRNA level, 3 donors)	3863	P
CYP3A4 Induction (human hepatocytes, mRNA level, 3 donors)	3865	P
Drug Transporters - P-gp & BCRP Substrate Assessment	Item #	Panel
Caco-2 Efflux Transporter (P-gp & BCRP) Substrate Assessment Panel		G357
A-B Permeability (Caco-2, pH 7.4/7.4)	3319	P
B-A Permeability (Caco-2, pH 7.4/7.4)	3321	P
A-B Permeability (Caco-2, pH 7.4/7.4 + verapamil)	3325	P
B-A Permeability (Caco-2, pH 7.4/7.4 + verapamil)	3326	P
A-B Permeability (Caco-2 pH 7.4/7.4 + Ko143)	4482	P
B-A Permeability (Caco-2 pH 7.4/7.4 + Ko143)	4483	P
Drug Transporters - Liver & Renal Transporter Substrate Assessment	Item #	Panel
Liver Drug Transporter Substrate Assessment Panel		G382
OATP1B1 Substrate Assessment (OATP1B1-CHO)	5015	P
OATP1B3 Substrate Assessment (OATP1B3-CHO)	5016	P
Renal Drug Transporter Substrate Assessment Panel		G383
OAT1 Substrate Assessment (OAT1-CHO)	5017	P
OAT3 Substrate Assessment (OAT3-CHO)	5018	P
OCT2 Substrate Assessment (OCT2-HEK)	5094	P
MATE1 Substrate Assessment (MATE1-HEK)	5092	P
MATE2-K Substrate Assessment (MATE2-K-HEK)	5093	P
Drug Transporters - P-gp & BCRP Inhibition	Item #	Panel
P-gp Inhibition (P-gp-HEK Vesicles, N-methyl-quinidine Substrate)	5500
BCRP Inhibition (BCRP-HEK Vesicles, Estrone 3-sulfate Substrate)	5501
Drug Transporters - Liver & Renal Transporter Inhibition	Item #	Panel
Liver Drug Transporter Inhibition Panel - IC50		G380
OATP1B1 Inhibition IC50 (OATP1B1-CHO, Estradiol-17Beta-Glucuronide Substrate)	5038
OATP1B3 Inhibition IC50 (OATP1B3-CHO, Estradiol-17Beta-Glucuronide Substrate)	5040
Renal Drug Transporter Inhibition Panel - IC50		G381
OAT1 Inhibition IC50 (OAT1-CHO, P-aminohippurate Substrate)	5039
OAT3 Inhibition IC50 (OAT3-CHO, Ranitidine Substrate)	5041
OCT2 Inhibition IC50 (OCT2-HEK, Metformin Substrate)	5098
MATE1 Inhibition IC50 (MATE1-HEK, Metformin Substrate)	5090
MATE2-K Inhibition IC50 (MATE2-K-HEK, Metformin Substrate)	5096