Cryopreserved Enterocytes for the Evaluation of Drug-Drug and Food- Drug Interactions

Transcription

1 Cryopreserved Enterocytes for the Evaluation of Drug-Drug and Food- Drug Interactions Albert P. Li, Ph. D. In Vitro ADMET Laboratories Inc. Columbia, MD and Malden, MA

2 Why Enterocytes Key cell type for oral bioavailability First pass metabolism before the liver Intestinal DDI with orally co-administered substances (foods; nutrient supplements; drugs) Intestinal DDI may not occur in the liver due to lower hepatic exposure (e.g. grapefruit juice) 2

3 3

4 4

5 As of now, primary enterocytes are not commercially available for drug metabolism evaluation Current commercially available enterocytes are cultured for multiple passages with little information on drug metabolizing enzyme activities

6 Isolation and cryopreservation of enterocytes from human small intestines 6

7 Cryopreservation of Human Enterocytes at IVAL Successful isolation and cryopreservation of enterocytes with high viability (>75%) and reproducible yield (1-3 million cells per vial) 7

8 Cell Specific Marker Gene Expression Enterocytes Sucrose Isomaltase (SI) Maltase Glucoamylase (MAGM) Hepatocytes Albumin (ALB) 8

9 Cell-type Specific Markers Gene name 2 -ΔCт enterocytes 2 -ΔCт hepatocytes Ratio of enterocyte to hepatocytes ALB* ND NA SI** ND NA MAGM** ND NA *ALB (Albumin) is a marker gene for hepatocytes **SI (Sucrose Isomaltase) and MAGM (Maltase Glucoamylase) are marker genes for enterocytes 9

10 P450 Isoform, UGT and SULT Gene Expression

11 CYP Pie Charts for Intestine and Liver 11

12 CYP Isoforms Gene name 2 -ΔCт enterocytes 2 -ΔCт Ratio of enterocyte hepatocytes to hepatocytes CYP1A CYP1A CYP2B CYP2C CYP2C CYP2C CYP2D CYP2J CYP2S CYP3A CYP3A CYP4F

13 Phase II DME Gene name 2 -ΔCт enterocytes 2 -ΔCт hepatocytes Ratio of enterocyte to hepatocytes UGT1A SULT1A GSTP CES CES

14 Transporter Gene Expression

15 Transporters Transporter 2 -ΔCт enterocytes 2 -ΔCт hepatocytes Ratio of enterocyte to hepatocytes NTCP ND NA OCT OAT OATP1B OATP1B OATP2B OATP1A PEPT OCTN OCTN MDR BSEP MRP MRP MRP BCRP Human hepatocytes Lot: PHH8006 (pool hepatocytes); Human enterocytes Lot: HE

16 Enterocytes vs Hepatocytes in Gene Expression Enterocytes had high expression of enterocyte marker genes, thereby confirming cell identity Significant differences between enterocytes and hepatocytes in metabolism and transporter gene expression 16

17 Drug Metabolizing Enzyme Activities

18 CYP3A4: Luciferin IPA Metabolism Human Hepatocytes (Pool of 30 donors) Human Enterocytes (HE3005) Km: 11.2 um Vmax: 27.6 pmol/10^6 cells/min Km: 5.9 um Vmax: 2.3 pmol/10^6 cells/min Vmax Km Human Enterocytes (multiple donors) Vmax Km HE HE HE3007` HE HE HE HE HE HE HE HE Average

19 DME Activities of Human Enterocytes Lot Number Gender Ethnicity Age CYP2C9 CYP2C19 CYP3A4 UGT SULT 2J2 CES2 HE3005 Male C HE3006 Female C HE3007 Male H HE3008 Male C HE3009 Female C HE3010 Male C HE3011 Female C HE3013 Female AA 57 NA NA 0.2 NA NA NA NA HE3014 Male AA HE3015 Male C HE3016 Male AA HE3019 Male C HE3020 Male C HE3021 Male AA HE3027 Female C HE3028 Male AA HE3029 Male C HE3031 Female C

20 Drug Metab Dispos 45: , June 2017 Human Enterocytes as an In Vitro Model for the Evaluation of Intestinal Drug Metabolism: Characterization of Drug-Metabolizing Enzyme Activities of Cryopreserved Human Enterocytes from Twenty-Four Donors 20

21 Comparison of Duodenum, Jejunum and Ileum in DME Activities (same donor) Duodenum (HE3052) Jejunum (HE3055) Ileum (HE3056) Marker Metabolite Ave. Std Dev Metabolite Activity (pmole/10 6 /min.) Ave. Std Dev Metabolite Activity (pmole/10 6 /min.) Ave. Std Dev Metabolite Activity (pmole/10 6 /min.) 4-OH Diclofenac ± ± ± OH S-Mephenytoin ± ± ± Hydroxymidazolam ± ± ± β-hydroxytestosterone ± ± ± Hydroxycoumarin Glucuronide 7-Hydroxycoumarin Sulfate ± ± ± ± ± ± 0.44 O-Demethyl Astemizole ± ± ± 0.09 SN ± ± ± 0.46 Postpyloric sphincter lengths used to aid regional identification: duodenum, 26 cm (9.84 inches); jejunum, 2.5 m (8.2 feet); and ileum, 3.5 m 21

22 MetMax Enterocytes 22

23 Enhanced DME Activities of MetMax Pooled Human Enterocyte Metabolite Activity (pmole/10 6 /min) Metabolic Pathway Substrate Marker Metabolite Before Re-Cryopreservation After Re-Cryopreservation MetMax Enterocytes CYP2C9 Diclofenac 4-OH Diclofenac 4.05 ± ± ± 1.13 CYP2C19 S-Mephenytoin 4-OH S- Mephenytoin 0.55 ± ± ± 0.32 CYP3A4-1 Midazolam 1-OH-midazolam 1.21 ± ± ± 1.22 CYP3A4-2 Testosterone 6βOH-testosterone 10.6 ± ± ± 14.5 UGT 7-OH-Coumarin 7-Hydroxycoumarin Glucuronide ± ± ± 79.5 SULT 7-OH-Coumarin 7-Hydroxycoumarin Sulfate 7.24 ± ± ± J2 Astemizole O-Demethyl Astemizole 0.92 ± 0.43 BLQ 5.14 ± 1.53 CES2 Irinotecan SN ± ± ± 0.27

24 Enteric Food-Drug Interactions

25 Grapefruit Juice Clinically significant DDI with orally administered drugs Inhibition of intestinal CYP3A4 Inhibition of P-gp efflux Minimal hepatic effects 25

26 Relative Activity Intestinal Drug-Drug Interactions: Grapefruit Juice Inhibition of Enterocyte CYP3A4 Activity 120 Grapefruit Juice % 0.07% 0.02% 0.06% 0.19% 0.55% 1.70% 5.00% 10.00% 20.00% Concentration (v/v) 26

27 Ln % Parent Remaining -Slope Time-Dependent Inhibition Assay with Human Enterocytes Time Dependent Inhibition of Enterocyte CYP3A4 Grapefruit Juice Time (min) Control 0.31% 0.63% 1.25% 2.50% 5.00% Grapefruit Juice TDI of Enterocyte CYP3A Concentration (v/v %) K inact =.083 min -1 K I = 3.6% 27

28 Drug-Drug Interaction Evaluation with Enterocytes Time-dependent inhibition assay developed with cryopreserved enterocytes Inhibition of CYP3A4 activity observed with grapefruit juice but not orange or apple juice Time-dependent and concentration dependent inhibition with GFJ observed, allowing the calculation of TDI inhibition constants 28

29 Enteric Herbal-Drug Interactions: CYP3A4 Inhibition in pooled donor cryopreserved human enterocytes Dietary Supplement Species Recommended Dosage Brand Relative N-CYP3A4 Activity Valerian Root Valeriana officinalis 450 mg (X5) Nature's Bounty Ginger Root Zingiber officinale 550 mg Spring Valley Black Cohosh Extract Cimicifuga racemosa 40 mg GNC Herbal Plus Grapefruit Juice Citrus paradisi 1 Simply Orange Juice Co Black Elderberry Sambucus nigra 575 mg (X2) Nature's Way Horehound Plant Marrubium vulgare 1 ml Botanic Choice Milk Thistle Silybum marianum 200 mg GNC Herbal Plus Tumeric Curcumin Curcuma longa 500 mg Spring Valley Cranberry Vaccinium macrocarpon 500 mg James Lake Farms Cinnamon Cinnamomum cassia 500 mg (X2) Spring Valley St. John's Wort Extract Hypericum perforatum 300 mg GNC Herbal Plus Ginseng Panex ginseng 600 mg GNC Herbal Plus Fenugreek Trigonella foenum-graecum 610 mg Spring Valley Red Yeast Rice Monascus purpureus 600 mg (X2) Spring Valley Ginkgo Biloba Ginkgo biloba 60 mg GNC Herbal Plus Guarana Paullinia cupana 250 mg GNC Herbal Plus Echinacea Extract Echinacea purpurea 500 mg GNC Herbal Plus Saw Palmetto Berries Serenoa repens 540 mg GNC Herbal Plus Evening Primrose Oil Oenothera biennis 1000 mg Spring Valley Spirulina Arthrospira platensis 200 mg (X2) Spring Valley Garlic Allium sativum 500 mg GNC Herbal Plus White Kidney Bean Phaseolus vulgaris 500 mg (X2) Nature's Way Soy Isoflavone Concentrate Glycine max 50 mg GNC Super Foods Flaxseed Oil Linum usitatissimum 1000 mg Spring Valley Aloe Vera Gel Aloe Barbadensis Miller 25 mg Spring Valley Rhodiola Rhodiola rosea 1 ml Nature's Answer Wheat Grass Juice Triticum aestivum 1 ml Sunny Green mean sd 29

30 MetMax Pooled Donor Human Enterocytes Herb-Drug Interactions 30

31 384-well HTS Enteric Herb-Drug Interaction Assay with MetMAX Enterocytes 0% 1.56% 3.12% 6.25% 12.5% 25% 50% 100% Black Cohosh Black Cohosh Black Cohosh Black Cohosh Black Cohosh Black Cohosh Black Cohosh Black Cohosh Black Elderberry Black Elderberry Black Elderberry Black Elderberry Black Elderberry Black Elderberry Black Elderberry Black Elderberry Cinnamon Cinnamon Cinnamon Cinnamon Cinnamon Cinnamon Cinnamon Cinnamon Echinacea Echinacea Echinacea Echinacea Echinacea Echinacea Echinacea Echinacea Garlic Garlic Garlic Garlic Garlic Garlic Garlic Garlic Ginger Ginger Ginger Ginger Ginger Ginger Ginger Ginger Ginkgo Ginkgo Ginkgo Ginkgo Ginkgo Ginkgo Ginkgo Ginkgo Ginseng Ginseng Ginseng Ginseng Ginseng Ginseng Ginseng Ginseng Grapefruit Juice Grapefruit Juice Grapefruit Juice Grapefruit Juice Grapefruit Juice Grapefruit Juice Grapefruit Juice Horehound Horehound Horehound Horehound Horehound Horehound Horehound Horehound Horehound Milk Thistle Milk Thistle Milk Thistle Milk Thistle Milk Thistle AX Milk Thistle Milk Thistle Milk Thistle St. John's Wort St. John's Wort St. John's Wort St. John's Wort St. John's Wort St. John's Wort St. John's Wort St. John's Wort Spirulina Spirulina Spirulina Spirulina Spirulina Spirulina Spirulina Spirulina Green Tea Green Tea Green Tea Green Tea Green Tea Green Tea Green Tea Green Tea 31

32 384-well HTS Enteric Herb-Drug Interaction Assay with MetMAX Enterocytes 0% 1.56% 3.12% 6.25% 12.5% 25% 50% 100% Green Tea Grapefruit Juice St. John's Wort Echinacea Ginger Horehound Spirulina Milk Thistle Black Elderberry Ginkgo Cinnamon Ginseng Garlic Black Cohosh

33 Cryopreservation of Human Enterocytes Successful isolation and cryopreservation of enterocytes from multiple donors viability (>80%) yield (1-3 million cells per vial) Gene expression confirmed expression of enterocyte specific genes Expression of enterocyte marker genes DME and transporter genes different from hepatocytes Retention of drug metabolizing enzyme activities MetMax enhancement of enterocytes DME activities Successful application in the evaluation of food-drug interactions CYP3A4 inhibition by grapefruit juice and herbal supplements 33

34 Enterocyte Applications Enteric metabolism Enteric metabolic stability (bioavailability) Fg determination Clint,ent Species comparison Enteric metabolite identification Optimization of enteric metabolic stability Enteric drug-drug, food-drug interactions Estimation of enteric DDI potential Enteric toxicity Estimation of enterotoxic potential 34

35 Regulatory Perspectives: Human enterocytes may be used to aid the identification of foods and herbs with drug-interaction potential, akin to the use of hepatocytes for DDI evaluation 35

36 Acknowledgment Carol Loretz: Cell culture David Ho: Analytical Chemistry Qian Yang: Molecular Biology 36

37 Thank you! IVAL Contacts: George Amaral, Kirsten Amaral, Albert P. Li, Ph.D.,