# Presentation: 026463

## BI Experience/Opinion: Transporter Based Drug Interactions

-  Clinical Pharmacology Subcommittee of Advisory Committee Meeting for Pharmaceutical Science (ACPS)
- October 18, 2006
- Mitchell E. Taub,
- Boehringer Ingelheim, DMPK

##   Drug Transporters: Background

  Drug-Transporter Interactions

  P-gp and CYP3A4

  In Vitro Techniques and Data

  Reports: Clinical Transporter-Based DDIs

-   Drug-Transporter Interactions
-   P-gp and CYP3A4
-   *In Vitro* Techniques and Data
-   Reports: Clinical Transporter-Based DDIs

**Outline**

## Drug Transporters: Overview

-  Transporters contribute to the absorption, distribution and elimination of
-    drugs, metabolites, various endogenous molecules, vitamins, and nutrients
-  Tissue entry of drugs can be either facilitated or hindered by transporters

## The Importance of Transporters

- In addition to AD/E; transporters can also facilitate the access of certain drugs to metabolizing enzymes (e.g. liver)
- Understanding PK, PD of certain drugs requires knowledge of drug transporter interactions
- As with CYP450s, interactions with transporters differ between species (consideration: prediction of clinical outcome)
- DDI: variable exposure, potential toxicity, and therapeutic failures can originate from drug transporter interactions
- What do we need to predict whether (and to what extent) the biological fate of a drug is influenced by drug transporters?
- _Challenge_: It is likely that a compound will interact with multiple transporters; likelihood increases for newer drugs structurally related to those already known to interact with transporters

## The Most Relevant Transporters for Consideration in R&D Programs

_**Drug Transporters**_** (alternative names)****	****	**_**CYP450 Enzymes**_
**P-gp (MDR1, ABCB1)****	****	****	****	****CYP3A4****	****/3A5**
- OATP-C	(LST-1, OATP-2, SLC21A6, OATP1B1)	CYP2C9
- BCRP (MXR, ABCG2)				CYP2D6
- MRP2 (cMOAT, ABCC2)				CYP1A2
- OATP-B	(SLC21A9, OATP2B1)			CYP2C19
- OATP-8	(SLC21A8, OATP1B3)			[CYP2C8, CYP2B6]

- How many transporters exist?
- Identified to date: 48 ABC genes, 300 solute carriers
- Which transporters should be evaluated?
- General consensus: similar to CYP450s, not all transporters are relevant

## P-gp and CYP3A4: Similarities

- Effects and consequences on pharmacokinetics by CYP3A4 or P-gp can only be fully understood with the help of investigations covering the enzyme _and_ the transporter

- Atypical (sigmoidal) kinetics
- ex: cooperativity/activation

## Permeability Experiments: Transwells with Monolayers of MDCK-MDR1, Caco-2, L-MDR1 

**BL to AP P****app**
**Secretory Transport**

**AP to BL P****app**
**Absorptive Transport**

**BL-AP / AP-BL ~1**
**Not an Efflux Pump Substrate**
**BL-AP / AP-BL ****> 1-2**
**Efflux Pump Substrate**

## P-gp Considerations/Complexities:Selecting Probe Substrates and Inhibitors

- Multiple binding sites (4+) on P-gp: selective probes for each P-gp binding site not yet identified
- Taub *et al.*, DMD (November, 2005) Vol. 33, No. 11
- In MDCK-MDR1 cells, ketoconazole activates P-gp at low concentrations and inhibits P-gp at high concentrations
- Would a compound with similar properties as KETO have a differential effect on P-gp in the intestine (high conc.) compared to the effect on P-gp at the BBB (lower conc.)?
- Substrate cooperativity and allosteric binding can complicate determination of secretory transport of P-gp substrates in P-gp expressing cell lines such as MDCK-MDR1 and Caco-2
- Inhibition of P-gp can potentially alter the PK and possibly the PD profile of a drug – what about activation?
- Possibly due to expression of other transporters, need to be cautious comparing data between cell lines that express P-gp

## Clinical Study: P-gp Mediated DDI InvolvingLoperamide and Quinidine

- Sadeque, Wandel, He, Shah, and Wood, CPT (2000) **68:**231-237

-  LOP: potent opiate/anti-diarrheal; no CNS effects at normal doses
-  When LOP (16 mg) given with QND (600 mg) AUC increased ~2.5 fold
-  Respiratory depression produced by LOP only when co-administered with QND
-  Authors conclude: QND inhibited the P-gp mediated efflux of LOP at the BBB
-  Example of transporter mediated DDI with potential for toxic effect in humans

## -

**Expression of OATP-X within 16-20 hrs post-infection by vaccinia virus**

**OATP-X expressing**
**HeLa cell**

**T7 Polymerase**

**Plasmid with OATP-X cDNA**
**(negatively charged)**

**Lipofectin**
**(positively charged)**

**HeLa Cell Membrane (net negative charge)**

**Expression of OATPs in HeLa Cells Using Vaccinia-Based Transfection System**

- Utilizes the highly efficient bacteriophage T7 RNA polymerase
- “Modular” system for evaluating uptake transporters; only one cell line to passage
- No need to establish range of stably transfected cells for each transporter

## Expression of OATP-A in HeLa Cells Using Vaccinia-Based Transfection System

**Expression of OATP-A in HeLa Cells Using Vaccinia-Based Transfection System**

**Figure courtesy of Richard B. Kim**

## OATP-A Mediated DDI:Fexofenadine and Grapefruit Juice

**Fig 1.** Mean plasma drug concentration–time curves of 120 mg fexofenadine for individuals (N = 12) administered water or grapefruit juice (GFJ), 300 mL or 1200 mL (300 mL with drug followed by 150 mL every 0.5 hour until 3.0 hours). *Bars* represent SEM.

- Dresser, Kim, and Bailey
- CPT (2005) **77:**170-177

## ABC Transporters in Hepatocytes

| High expression level: | MRP2, MDR1, MDR3 |
| --- | --- |
| Lower expression level: | MRP1, MRP3 (inducible)  |

**Illustration courtesy of SOLVO Biotechnologies**

**Notes:**
-Most important ones: Mrp2/cMOAT and BSEP
-MRP3 has similar substrate specificity as MRP2, MRP3 exp goes up is MRP2 function is compromised - importance in removing toxic substrates, metabolites from hepatocyte,

Millenium Boehringer: BSEP is important, liver package:
Human: MRP2, MRP3 and BSEP
Rat: ratMrp2,  ratBsep

## BCRP-Expressing Sf9 Vesicles: Inhibition of [3H]Methotrexate Uptake in by Sulfasalazine

## BCRP Mediated DDI:Topotecan and GF120918

**BCRP Mediated DDI:****Topotecan and GF120918**

- Kruijzer *et al.*, J. Clinical Oncology
- (2002) **20:**2943-2950

-  When co-administered with 1000 mg GF120918, the AUC of oral topotecan increased >2-fold
-  Fpo of topotecan increased from 40% to >97% when co-administered with GF120918
-  GF120918 had a slight effect on IV administered topotecan AUC and CL, but no effect on t1/2

## Clinical Relevance of Transporter Mediated Effects: P-gp, Other Transporters

**Survey of University of Washington DDI database:**

**Results of a recent literature survey, including the UW DDI Database:**
- P-gp inhibition: ~120 studies published; max. effect ~18-fold increase of AUC
- P-gp induction: ~40 studies published; max. effect ~ 80% reduction of AUC
- Some results due to combined effects of P-gp and CYP3A4 induction or inhibition
- P-gp effects often exceed the 2-fold increase/decrease of exposure that may be considered as acceptable PK variability
- What is the current regulatory perspective on the design and implementation of clinical studies to investigate potential transporter-based DDI?

## Concluding Remarks

- CYP3A4 and P-gp demonstrate many similarities and are both integrally important to consider in most R&D programs
*Examples:* CNS, cancer, liver-targeted indications
- To what extent/frequency do clinical DDIs or toxic effects involving transporters occur?  Careful consideration in R&D programs is necessary
- The selection of appropriate transporter probe substrates and inhibitors is a critical issue; this area is still not well-defined for many transporters
*In vitro* methods for ascertaining drug-transporter interactions vary considerably from one laboratory to another.....  standardization?
- Legal barriers exist (patents) restricting FTO for the mechanistic evaluation of certain transporters

## Are the criteria for determining whether an investigational drug is an inhibitor of P-gp and whether an in vivo drug interaction study is needed (Fig. 1) appropriate?

- How relevant is the [I]/Ki relationship, originally established for CYP450 inhibition, to transporter interactions?
- This area is not as well defined for transporters (even P-gp) as it is for the CYP450s.
- Should [I] be the plasma Cmax or an estimated GI concentration of drug?
- [I] for CYPs  microsomes, [I] for P-gp  cells......comparable?
- In a previous version of this document, there was a cutoff IC50 value of <10 μM for classification of a compound as an inhibitor of P-gp.  While it could be argued that this is an arbitrary value, is an [I]/IC50 (or Ki) > 0.1 any less arbitrary? (consider statement above)
- Some of the most potent inhibitors of P-gp are compounds that are not commercially available and may not be suitable for evaluation in humans.
- Examples: LY335979, Valspodar (PSC833), Elacridar (GF 120918)
- Concerning ritonavir and cyclosporine and proposed inhibitors, these compounds have been shown to inhibit many transporters.  At this point it is not clear how this lack of specificity would affect results of a clinical DDI study.

## Are the criteria for determining whether an investigational drug is a substrate of P-gp and whether an in vivo drug interaction study is needed (Fig. 2) appropriate?

- A reasonable concern may be that flux ratios ≥ 2 could represent a value that is too liberal and will lead to too may positive results.
- Need to present a consensus opinion representing PhRMA members
- Would it be expected that any Development compound with a flux ratio ≥ 2 be evaluated clinically with P-gp inhibitors to determine potential DDI?
- Consideration of the transcellular passive permeability of a compound in relation to the efflux ratio may be the most important issue.
- General concern: Many open questions still exist regarding the complexity of the transporter field and how to appropriately link *in vitro* data to the potential for clinical outcome.
- Even for the CYP450 area, for which the IVIVC for DDIs is better characterized, we are not always able to correctly predict DDI.
- Current knowledge base does not yet support the recommendation of drug interaction studies involving other transporters such as OATP1B1, MRP2, BCRP, OCTs, and OATs.

## Acknowledgements

**Laboratory Work – Transporter Group:**
- Lalitha Podila
- Diane Ely
- Rucha Sane
- Susan Lazos
- Iliana Almeida
**Helpful Advice and Consultation:**
- Donald Tweedie
- Richard Kim
- Naoki Ishiguro