SOLVO Biotechnology

SOLVO Biotechnology 

"The Transporter Company" 

Bill Johnson Johnson, Ph Ph.D. D 

VP, Global Operations & Services

Presentation Topics 

SOLVO "The The Transporter Company" Company 

Membrane Transporters Transporters, Barriers 

RRegulatory l t BBackground k d 

Assay Service and Systems

"[Kelsey] prevented ... the birth of tho 

armless and legless children." The publi 

was swift and drug testing reforms were 

unanimously y by y Congress g a few months lat 

The drug testing reforms required "stric 

on the testing and distribution of new d 

to avoid similar problems. 

The amendments also, for the first time, 

recognized that "effectiveness [should b 

required to be established prior to mark 

Frances Kathleen Oldham Kelsey receiving the 

President's Award for Distinguished Federal 

Civilian Service from President John F. 

Kennedy, in 1962.

• Founded in Hungary in1999 

SOLVO Facts 

• 100+ transporter related products and 

services 

• 25 peer reviewed articles in transporter 

science i 

• 400+ clients in 35 countries 

• 80 employees, 60 scientists and 30 in 

R&D

Why SOLVO? 

Proven Scientific Expertise 

Topic Publications 

Impact cholesterol on transporter 

activity 

IVIVC 

BCRP 

MRP2 

MDR1 

Pal et al (2007), (2007) Kis et al (2009) 

MDR1: Sziraki et al (2011) 

MRP2: Heredi-Szabo (2009) 

BSEP Kis et al 2009 (2) 

Interaction with neutraceuticals and 

environmental toxicants 

Ozvegy et al (2001), Jani et al (2009), 

Kis et al (2009), Pál et al (2007), 

Glavinas et al (2007), ( ), 

Krajcsi et al (2005), Heredi-Szabo et al 

(2008) 

SSzeremy et t al l 2010, 2010 RRajnai j i et t al l 2010, 2010 V 

Richter et al 2009, Schmitt et al 2006 

Dreiseitel et al (2009), Oosterhuis et 

al (2008), Williamson et al (2007), 

Zh n t l (2007) L pin t l (2006)

Why SOLVO? 

Portfolio 

We offer the entire FDA and EMA set (and a 

lot more): ) 

• P P-gp (MDCKII or vesicles) i l ) 

• BCRP (MDCKII or vesicles) 

• BSEP (vesicles) (vesicles) 

• OAT1 (CHO cells) 

• OAT3 (CHO cells) 

• OCT1 (CHO cells) 

• OCT2 (CHO cells) 

• OATP1B1 (CHO cells) ll ) 

• OATP1B3 (CHO cells)

RRecent t Developments D l t at t SOLVO 

• Transporter Consultancy Service 

– How to study transporters - Experimental design, which transporters are 

important, etc. 

– When to study transporters - Let us help you in designing your transporter 

strategy 

– Why to study transporters - Help with regulatory requests, questions and 

submissions 

• Additional Services 

– Primary Renal Proximal Tubule cells (aProximateTM – Primary Renal Proximal Tubule cells (aProximate ) 

Package] 

[Kidney 

– Hepatocyte uptake assay (various protocols) 

Package] g 

[Liver 

– Excipient interaction assay [Absorption Package] 

– Solubility Assay [Absorption Package] 

– RBEC, , MBEC [BBB [ Package] g ] 

• US Expansion 

A il 2012 SOLVO ffi i B t MA d t bli h i l

Lack of adequate q superiority p y in 

efficacy the major reason related 

tto ddrug ffailure il

Role of Transporters: 

ADMET ADMETox and d DDrug-Drug D 

Interactions

Types of Transporters 

Uptake (SLC) transporters 

• Mediate active transport of 

compounds accross cell membranes 

• Energy source differs per family 

of transporters (sodium gradient gradient, 

proton gradient, etc) 

• Transport wide variety of 

molecules: peptides, organic anions 

and cations, cations bile acids acids, amino 

acids, fatty acids, etc 

• Expression in all major barrier 

organs 

H + , Na + , other 

extracellular 

S 

A 

L 

B 

C intracellular C 

ATP 

ADP + Pi 

Efflux (ABC) transporters 

• >1 >1,000 000 different transporters in 

the family (49 human) 

• Energy source: ATP 

• Functions: 

– Transport substrates 

accross cell membranes 

– Regulate(d) ion channels 

– Regulate multi-protein 

channel complexes 

(receptors) 

• Alter pharmacokinetics of drugs, 

nutrients and other molecules 

• An array of genetic diseases 

linked with the human transporters

EExpression i in i Barrier B i OOrgans 

Intestinal efflux 

Pgp, gp, MRP2, , MRP4, , 

BCRP 

CNS efflux 

PPgp, OAT3 OAT3, MRP1 MRP1, 

MRP2, BCRP, MRP4 

ATP dependent efflux transporters 

Exchange/co-transport transporters 

Intestinal uptake 

OATP2B1, OATP1A2 

PEPT-1, MRP3 

Biliary secretion 

Pgp, MDR3, BCRP 

MRP2 (BSEP) 

Hepatic uptake 

OATP1B1 OCT1 

OATP2B1 OAT2 

OATP1B3 NTCP 

Hepatic efflux 

MRP3, , MRP4, , 

MRP6 

Renal uptake 

OAT1,OAT3, OCT2, OATP4C1 

Renal re-uptake 

OATP2B1, PEPT-2 

Renal secretion 

Pgp, MRP4, MRP2, OAT4, MATE1, MATE2K

Role of Transporters p in 

ADMETox H extracellular 

+ , Na + , other 

• Intestinal absorption 

S 

A 

• Distribution 

L 

C 

B 

C 

• Modulation of metabolism 

• Excretion ATP 

– Intestinal 

– Liver 

– Kidney 

• Drug-drug interaction 

• Toxicity 

– interference with 

transport and d 

metabolism 

of endogenous 

b 

intracellular ADP + Pi

Role of Transporters p in 

• Intestinal absorption 

Drug Transporter Effect 

Paclitaxel MDR1 6 x increase in 

Mdr1a -/- mice 

(Van Asperen et 

al 2007) 

Digoxin MDR1 157% increase in 

AUC when codosing 

with Dronedarone 

Atorvastatin BCRP 71% increase in 

AUC in c.421AA 

genotypes 

Rosuvastatin BCRP 144% increase in 

AUC in c.421AA 

genotypes 

(M Niemi, 2010) 

ADMETox

Roles of Transporters p in 

• Distribution 

ADMETox 

– CNS (blood-brain barrier, blood – CSF barrier) 

– Fetus (blood-placenta barrier in 

syncytiotrophoblast) 

– Testis (blood-testis ( barrier) )

Effect of P-gp P gp on BBB 

permeability of verapamil 

Lee et al 2006, JPET 

B/D B/D: 11C 11C‐Verapamil V ilb brain i uptake tk 

in rhesus monkeys 

C/E: 11C 11C‐Verapamil Verapamil brain uptake + 

PSC833 

>4 fold increase in brain uptake in 

presence of PSC833

First 

generation ti 

SSedative d ti Side Sid Eff Effects t of f 

Anti-histamines 

Anti histamines 

Transporter 

(MDR1) 

interaction 

brain/plasma Sedative CNS 

distribution side effect 

Notes 

chlorpheniramine No 18.4 Yes High passive 

permeability 

diphenhydramine No 34.0 Yes High passive 

permeability 

Hydroxyzine 

No 4.30 Yes High passive 

(At (Atarax) ) 

permeability bilit 

Second 

generation 

FFexofenadine f di 

(Allegra, Aventis) 

YYes 00.018 018 NNo 

Acrivastine 

(Semprex, (Se p e , GSK) GS ) 

Yes 0.072 No 

Cetirizine (Zyrtec, 

Pfizer) 

Yes 0.367 No

Roles of Transporters in 

ADMETox 

• Transporters play an important role in drug absorption, 

disposition, excretion and toxicity 

• Increasing evidence for clinical relevance of transporters 

• Increasing number of publications from regulatory authorities 

• RRegulatory l t view i iis still till evolving l i and d many questions ti remain i 

unanswered 

• The passive permeability of a compound has a great effect on 

the success of a transporter assay 

• Assays should be chosen wisely

Building evidence that adverse drug 

reactions are attributed, at least in part, to 

the involvement of drug transporters 

Cellular accumulation depends on transport 

What role of passive permeability? 

Adverse reactions can involve transporters 

MDR3, OATP, cardiac 

Zolk and Fromm, Clin Pharmacol Ther, 2001

Challenge g of Drug g Transporters p 

Rapidly growing scientific area with many in 

vitro, it preclinical li i l and d clinical li i l publications 

bli ti 

• More than 30 transporters ‘involved’ in ADME 

• Few agreed clinical translation approaches 

• Limited tools/reagents relative to CYP enzymes 

• Measuring drug exposure in plasma may not reflect impact on a 

drug’s disposition (e.g., toxicity)- this is highlighted in 

the EMA DDI guidance document 

• Conflicting messages to prescribers prescribers, patients patients, and regulatory 

bodies 

Drug Transporter White Paper 

‘Membrane Transporters in Drug Development’, NRDD 9:215 - 236 (2010) 

EMA Guideline on the Investigations of Drug 

Int Interactions- r ti n Draft guidance published 22April2010 

Fast changing, dynamic

Mechanisms Mechanisms of of Drug Drug-Induced Drug Drug-Induced Induced Liver 

Transporter 

Disruption 

Immune Injury j y 

hepatocyte 

Injury 

j y 

Bile 

Canaliculus 

Reactive Metabolites 

hepatocyte 

Mitochondrial Disruption 

Loss of ion 

gradients 

Other 

targets 

Glutathion 

e depletion

Hepatic Drug Transporters and 

OATP 

1B1 

OATP 

1B3 

OATP 

2B1 

MRP3 

Metabolising Enzymes 

MRP4 

Phase I: 

MRP5 

CYP 450, 450 FMO, FMO MAO 

Esterases 

ADH, CR, XO 

Phase II: 

UGT UGT, GST 

SULT 

NTCP N 

OCT1 O 

OAT2 O 

BLOOD 

Causes of transporter related 

hepatotoxicity 

• Cellular accumulation via 

efflux or uptake 

• “toxic” xenobiotics/ 

BILE metabolites BSEP 

• Impaired p efflux 

• endogenous bioactive 

molecules (e.g. bile acids) 

• modification of nutrient 

gradient 

• candidate or concomitant 

meds 

• Inhibition or altered 

expression

Selected human transport proteins for drugs and endogenous substances 

Which Transporters Have the Most 

Attention in Early Discovery? 

•P-glycoprotein (ABCB1) 

•Breast Breast Cancer Resistance Protein 

(ABCG2) 

•Multi-drug Resistance Protein 2 

(ABCC2) ( ) 

•Organic Anion Transport Protein 

(SLCO1B1) 

•Bile Salt Export Pump (ABCB11)

Classic Example p of f Transporter p 

Inhibition and Heptatoxicity 

Bosentan 

Troglitazone 

(400 mg) 

PFIC 1 >ATP8B1 

PFIC 2 >BSEP 

PFIC 3 >ABCB11 

Analogous with 

Changes g in bile acid transporters p during g 

Gilbert’s syndrome 

cholestasis. 

ASBT = apical Na+ dependent bile salt transporter 

CFTR = cystic fibrosis transmembrane regulator

Effect of Drugs with Clinical Cholestasis on BSEP 

*Er-Jia Wang, Schering- 

Plough Res. Inst., The 

Impact of drug transporters 

on preclinical development, 

Boston, June 2004 

Compounds I max (%CSA)* IC 50 (µM)* 

Chl Chlorpromazine i 88±2 34±1 

Clofazimine 335±41 24±6 

Cyclosporin y p A 105±7 ± 8±2 ± 

Glibenclamide 366±4 147±2 

Ketoconazole 83±4 69±4 

Paclitaxel 97±6 29±3 

Reserpine 167±40 19±5 

Rifamycin SV 133±19 39±15 

Tamoxifen 152±8 23±1 

Troglitazone 211±16 66±8 

Valinomycin 29±2 7±3 

Verapamil 152±17 179±24 

Vinblastine 204±14 62±7 

Cimetedine NE ND

Relative distribution of human BSEP IC50 values 

200 compounds d 

Drug-Induced BSEP Inhibition and Hepatotoxicity 

BSEP inhibition has been implicated in drug-induced 

hepatotoxicity 

p y 

80.5% of compounds that inhibit BSEP at

Clinically Relevant Transporters 

: EMA 

:FDA/ 

EMA 

ITC whitepaper, NRDD 2010

Role of Oatp1a/1b transporters in paclitaxel (PTX) pharmacokinetics. 

van dde Steeg St E et t al. l Clin Cli Cancer C Res R 2011;17:294-301 

2011 17 294 301 

©2011 by American Association for Cancer Research 

OATP1A/1B polymorphisms may develop toxicity 

upon paclitaxel or methotrexate treatment 

Tumor OATP1A/1B expression may also affect 

tumor drug sensitivity. 

OATP1A/1B inhibition by (pre)treatment with ot 

drugs may affect drug toxicity and effi 

Mrp2 dominates hepatobiliary excretion, reduced by 80% in Mrp2 −/ 

In contrast, P-gp dominates intestinal excretion, with a minor 

The AUC oral of paclitaxel increased 8.5-x by Mdr1a/1b deficiency, 

However, in the absence of Mdr1a/1b P-gp, additional 

Mrp2 deficiency increased the AUC oral another 1.7-fold.

DDIs - Rosuvastatin – 

Cyclosporine Cyc o po e A 

Limited bioavailability 

Hepatic portal vein 

Cyclosporine 

OATP1B1 

Pgp? 

BCRP 

MRP2? 

Bile 

7X AAUC0-24 

10X Cmax

DDrug-drug d Interactions I i of f 

Cyclosporine A with Statins 

Affected drug Interacting drug 

PK impact 

(clinical) 

Pravastatin Cyclosporine AUC 890%, C max 678% 

Pitavastatin Cyclosporine AUC 360%, Cmax 560% 

Glyburide Rifampicin AUC 125% 

Bosentan Rifampicin C through 500% 

Bosentan Lopinavir/ritonavi 

r 

Cyprotex 7th North American Drug Discovery Workshop 

C through 4700%

DDabigatran bi t (P (Pradax, d BI) BI): 

Arecent ece t example e a p e of o a P-gp gp DDI 

• Dabigatran: not substrate of SLC or 

Effl Efflux Transporter 

T 

• Dabigatran-Etexilate (prodrug): 

– ER = 6 @ 0.6 0 6 μMM 

– ER = 1 @ 40 μM (saturation effect assumed) 

• 80% excretion i in i ffaeces after f oral l 

administration, 6% excretion in faeces 

after iv i.v. administration 

administration. 

• 2.5 increase in AUC after co-dosing with 

Verapamil 

• Not all inhibitors affect absorption of 

DE

Clinical Translation From Mechanistic 

Hepatotox Investigation 

In Vitro Mechanistic data In Vivo Mechanistic data 

• The in vitro/in vivo work 

identify a mechanism that 

at a minimum exacerbate 

hepatotoxicity and may be 

an independent risk factor 

• Role of transporters (BSEP) 

• IImportance of f metabolism b li 

(CYP3A4 and UGT enzymes) 

T t k t t d t d b th 

Transporters are a key aspect to understand both 

the disposition as well as the toxicity of 

L ti ib

Platinum transport in renal cells 

Source: Drug Resistance Updates 2011; 14:22-34 (DOI:10.1016/j.drup.2010.12.002 ) 

Copyright © 2011 Elsevier Ltd Terms and Coditions 

OCT2 in basolateral membrane of proximal tubule 

MATE1 & MATE2-K in brush-border membrane

Pl Platinum i transporters iin the h proximal i l 

tubular cells of the human kidney 

• At pH 7.4, about 40% of the commonly 

prescribed ib d ddrugs are organic i cations. ti 

• OCs are endogenous g (choline, , dopamine, p , 

histamine) or exogenous (drugs such as 

vecuronium vecuronium, procainamide procainamide, quinine quinine, 

cimetidine).

Ci Cisplatin l i 

nephrotoxicity 

• Dose-limiting nephrotoxicity is a major concern 

-Observed in 41% of patients treated with cisplatin 

-One-third O hi d of f patients i experience i llong term kid kidney 

dysfunction, despite extensive 

prophylaxis 

-20% 20% of f all ll acute t renal l ffailure il cases among 

hospitalized patients 

are due to cisplatin-containing chemotherapy 

• Renal damage limits its use and efficacy in cancer 

treatment 

• Major determinant of nephrotoxicity: tubular cell death, 

tissue damage, and the loss of renal function or acute 

renal failure 

Cimetidine inhibits many transporters , OCTs , OATs

Influence of Oct1/Oct2-deficiency 

on cisplatin-induced cisplatin induced changes in urinary NAG and overall survival. 

©2010 by American Association for Cancer Research 

Franke R M et al. Clin Cancer Res 2010;16:4198-4206

Cisplatin-induced (10 mg/kg, i.p.) changes in urinary NAG activity in mice administered saline 

(i.v.), cimetidine (30 mg/kg, i.v.), or no injection immediately prior (n = 6-12 per treatment 

group). 

Franke R M et al. Clin Cancer Res 2010;16:4198-4206 

©2010 by American Association for Cancer Research

Influence of cimetidine on cisplatin 

nephrotoxicity (in vivo; dose, 10 

mg/kg) g/ g) 

NAG = N-acetyl-β-D-glucosaminidase, a 

marker for acute renal proximal p 

tubular damage 

Urinary NAG >0.4 AU in mice is 

associated with 21-fold increased odds 

for severe (grade 4) nephrotoxicity 

(P=0.0017) 

Cimetidine protects against cisplatin 

nephrotoxicity to a degree similar to 

that h observed b d in i Oct1/2(-/-) O 1/2( / ) mice i 

treated with cisplatin alone 

Ci Cimetidine tidi currently tl iin clinical lii ltil trial 

as a modulator of cisplatin toxicity 

Ototoxicity 

OCT in cochlear ce

Notable species differences 

Rats and humans have distinct composition of bile salt pools 

IIn humans, h a conjugated j t d cytotoxic tt i bil bile salt lt comprises i 31% of f t 

pool but in rats the related conjugated cytotoxic bile salt c 

only 2.5% of the bile salt pool. 

Risk of misinterpreted in vitro studies 

OATP1A2 function is inhibited by y rosuvastatin, ,y yet, , imatinib a 

was not dependent on OATP1A2 variants nor by rosuvastatin.

Recent Regulatory 

Developments p 

Focus on transporters in Drug- 

Focus on transporters in Drug 

Drug Interactions and Safety

Regulatory Background 

Guidance documents 

FDA , February, 2012 

GGuidance id for f 

Efflux 

Industry 

P-gp ( (MDR1) ) 

BCRP 

Uptake 

OATP1B1 

OATP1B3 

OAT1 OAT1 

OAT3 

OCT2 

BSEP 

MATEs 

DRAFT GUIDANCE 

PRPs 

DDrug IInteraction i SStudies di — 

Study Design, Data Analysis, 

Implications for Dosing Dosing, and 

Labeling Recommendations

FDA Advisory y Committee for 

Pharmaceutical Science and Clinical 

Pharmacology 

Question 1: For evaluation of NMEs as potential substrates of 

transporters: 

Do you agree that P-gp, BCRP, OATP1B1/1B3, OAT1/3 and OCT2 are 

the major transporters that should be routinely evaluated 

based on the proposed flow chart (Figure 1) during drug 

development? 

VOTING VOTING: 12 YYes, 4 NNo, 0 Ab Abstain i 

Question 2: For evaluation of NMEs as potential inhibitors of 

transporters: 

Do you agree that P-gp, BCRP, OATP1B1/1B3, OAT1/3 and OCT2 are 

the major transporters that should be routinely evaluated 

based on the proposed flow chart (Figure 2) during drug 

Cyprotex development? 

7th North American Drug Discovery Workshop

ug Interaction Studies – Draft Guidance, FDA February 2012 

Determine P-gp and 

BCRP Substrate and 

Inhibitor 

interactions 

Study 

OATP1B1 and 

OATP1B3 

Sb Substrate 

and 

Inhibitor 

it ti 

All Investigational Drugs (IDs) 

Hepatic or Biliary 

Secretion Major? 

(>25%) 

Active Renal 

Secretion Major? 

(>25%) 

YES NO YES NO 

Study 

OATP1B1 and 

OATP1B3 

IInhibitor hibi 

interaction 

s 

Study OAT1, 

OAT3 and 

OCT2 

Substrate 

and 

Inhibitor 

interactions interactions 

Study OAT1, 

OAT3 and 

OCT2 

Inhibitor 

interaction 

s

Guidance documents 

EMA Guideline on the 

Investigation of Drug 

IInteractions t ti 

Efflux 

P P-gp (MDR1) 

BCRP 

BSEP 

Uptake 

OATP1B1 

OATP1B3 

OCT1 

OAT1 

OAT3 OAT3 

OCT2 

47 

RRegulatory l t Background 

B k d

EMA: Recent Developments 

• Current draft expected to be finalized 

in Q2 of 2012 

• Transporter list will be ‘living’ 

• Id Identify tif transporter t t instead i t d of f check- h k 

box screening 

• Cut-off for DDI potential: 

– Ki > [I] 

• Where I liver = 50 x Cmax x fu 

• Where I intestine = 10 x dose/250ml 

More discussion needed!

Overview of Drugs with 

Transporter Information on 

Label 

2004-2006 2007-2009 

Number of approved NCEs 67 56 

Drugs with transporter information on label 11 21 

Number of drugs with P-gp/MDR1 P gp/MDR1 on label 6 20 

In vitro P-gp/MDR1 data 10 16 

In vivo P-gp/MDR1 data 14 9 

Number of drugs with other transporters on 

label 

1 6 

Drugs with transporters mentioned in 

Highlights section of label 

1 7 

Number of drugs requiring PMCs and/or PMRs 

ddue 

to transporters 

Agarwal et al., ACCP Annual Meeting, Sept 2010, Baltimore, MD 

Huang et al., AAPS workshop, March 2011, Bethesda MD 

3 10

New ABCB1 drug labels 

Aliskiren 

Direct renin inhibitor 

F= 0.026 

Contraindications 

The concomitant use of aliskiren 

with ciclosporin, a highly potent 

PP-glycoprotein l i (P (P-gp) ) iinhibitor, hibi 

and other potent P-gp inhibitors 

(quinidine, verapamil), is 

contraindicated 

Dabigatran 

Direct thrombin inhibitor 

F= 0.072, 0 072 BCS class IV 

Contraindications 

concomitant treatment with quinidine 

P- glycoprotein inhibitors 

caution should be exercised with 

strong P- glycoprotein inhibitors. 

Th The PP- glycoprotein l i iinhibitor hibi 

quinidine is contraindicated 

P- glycoprotein inducers 

potent P- glycoprotein inducers such 

as rifampicin or St John’s wort 

(Hypericum perforatum), may reduce 

the systemic exposure of dabigatran 

dabigatran. 

Caution is advised when coadministering 

these medicinal 

products. 

50

DDrugs Approved A d bby FDA iin 2009 ( (prior i to 

publication of ITC White Paper) 

Drug Indication 

or Tx 

Area 

Route DDI in 

Label 

Transporter 

DDI in Label 

Pralatrexate (Folotyn) Cancer IV 

Romidepsin (Istodax) Cancer IV 

Telavancin (Vipativ) Antibacterial IV 

Iloperidone (Fanapt) Schizophrenia Oral 

Saxagliptin (Onglyza) 

Artemether/Lumefantrine 

(Coartem) 

Type II 

Diabetes 

Oral 

Malaria Oral 

Dronedarone (Multaq) Atrial Fib Oral 

Everolimus (Afinitor) Cancer Oral 

Febuxostat (Uloric) Gout Oral 

Milnacipran (Savella) Fibromyalgia Oral 

Pazopanib (Votrient) Cancer Oral 

Pitavastatin (Livalo) Cholesterol Oral 

Prasugrel (Effient) ACS Oral 

Tolvaptan (Samsca) Heart Failure Oral 

Vigabatrin (Sabril) Epilepsy Oral 

Asenapine (Saphris) Antipsychotic Sublingual

PHATSO: Pharmacologic and Therapeutic Studies in 

the Obese 

Michelangelo 

's David 

Visits 

Modern USA 

Diabetes Obesity 

A GROWING epidemic

Clinical Examples of Transporter 

Mediated DDIs 

Victim Drug Inhibitor 

Clinical 

Consequence 

Pravistatin ( ) 

(1) Cyclosporine AUC 890% 

Rosuvastatin (1) Cyclosporine AUC 610% 

Bosentan (1) Ritonavir C trough 4700% 

Cerivastatin (1) Gemfibrozil 

Rhabdomyolysis 

Rhabdomyolysis, 

Death 

Digoxin (2) Digoxin Dronedarone AUC 157% 

( ) Dronedarone AUC 157% 

Metformin (3) Cimetidine AUC 50% 

(1): OATP (2) P-gp/MDR1 (3): OCT

Efflux 

Regulatory Recommendations 

Category Transporters Assays 

Hepatic p uptake p 

Renal uptake 

• P-gp (MDR1) 

• BCRP 

• BSEP (only EMA) 

• OATP1B1 

• OATP1B3 

• OCT1 (only EMA) 

Monolayer or Vesicles 

• Inhibition and substrate assays 

Cellular uptake 

• Inhibition 

• Substrate based on PK 

• OAT1 

Cellular uptake 

• OAT3 

• OCT2 

• Inhibition 

• Substrate based on PK

Conclusions 

The identification of transporters that influence the 

di disposition, i i toxicity i i and d safety f of f drugs d is i a new challenge h ll 

for drug discovery and development programs. 

Our understanding of drug transporters is still emerging and 

it is likely that multiple ABC and SLC transporters will 

influence the disposition and toxicity of a compound. 

For future drugs, drugs transporters are a key 

aspect in understanding efficacious response, 

as well as the drug’s drug s disposition, disposition toxicity 

and interactions. Multiple approaches 

Extrapolating animal studies not t

SOLVO Services and Assays 

Systems y to Study y 

Transporters

In-vitro In-vitro Approaches for Studying 

Membrane – 

based assays 

Cell – 

based assays 

Transporters 

ATPase assay 

liberated Pi; inexpensive, non‐radioactive 

indication on the nature of interaction (substrate or inhibitor) 

Vesicular transport assay 

quick and easy; vesicles from any cell type 

flexible readout (LSc, Fluo, LC/MS; modeling of ic. concentration 

Calcein assay 

fluorescent measurement 

indirect measurement – modulation of dye transport 

Cellular uptake assay 

quick and easy assay 

flexible read‐out and various expression systems 

Monolayer assay 

single or double transfectants; good absorption model 

permeability can be limiting 

Primary cells 

suspended and sandwich‐cultured sandwich cultured hepatocytes hepatocytes, 

brain capillary endothelial cells

Cellular Uptake Assay 

Flexible readout (LSc, 

LC/MS) 

Services 

Permeability is not a factor 

(inhibition) 

Modeling of Cmax 

Various expression systems 

Substrate testing only for 

low permeability compounds 

Substrate 

assay 

Inhibition 

assay 

Investigated drug 

Inhibitor

Uptake Transporter Studies 

General High-throughput High throughput Screening 

Protocol 

1. Add cells to the wells of a 96- 

2. 

well plate 

Add expression inducer 

33. Incubate in CO CO2 thermostat for 24h 

4. Wash wells 

55. Add test compounds 

6. Add labeled compound 

7. Incubate at 37 °C 8. Stop transport by ice-cold 

washing mix, wash wells 

9. Dissolve cells in 0.1% NaOH 12. Measure radioactivity y in 

10. Add scintillation cocktail to the 

wells of a 96-well plate 

11. Transfer lysate from plate 1 

each well 

13. Calculate transport rates 

and relative transport rates

Cellular Uptake Reference Data 

OATP1B1 

trnanspport 

rate 

(pmol/ /mg/min) 

Uptake of Fluvastatin into OATP1B1 

expressing cells 

450 

400 

350 

300 

250 

200 

150 

100 

50 

0 

0 20000 40000 60000 80000 

Concentration Fluvastatin (pmol/ml)


OATP1B3


OATP2B1


NNon-specific ifi binding bi di (NSB) 

Ceellular 

accummulation 

(pmol/mmg) 

Standard assay conditions Addition of 10% human serum 

30000 

25000 

20000 

15000 

10000 

5000 

Cellular C accuumulation 

(pmol/ /mg) 

0 

-5000 

2 min 20 min 

0 

CHO‐OATP1B1 CHO OATP1B1 

2 min 

1107 1107.43 43 

20 min 

1886 1886.98 98 

CHO-OATP1B1 

CHO-K 

Transporter 

specific transport 

(pmol/mg) 

9887.65 

9032.43 

855.22 

22137.47 

22062.62 

74.85 

CHO‐K 

Transporter 

specific transport 

(pmol/mg) (p / g) 

425.38 

682.05 

490.89 

1396.09 

2500 

2000 

1500 

1000 

500

Uptake Assays at SOLVO 

Cell line Probe substrate 

OATP1B1-CHO (SLCO1B1)/OATP2, OATP-C 

3 H-Estrone-3-Sulfate 

Reference 

Inhibitor 

Pravastatin; 

rosuvastatin 

Planned 

substrate 

Pravastatin; 

rosuvastatin 

OATP1B3-CHO (SLCO1B3)/OATP8 Fluo-3 Digoxin; rosuvastatin Digoxin; rosuvastatin 

OATP2B1-MDCKII (SLCO2B1)/OATP-B 

rat Oatp1a1 (Slco1a1) 

OATP1A2-HEK293 (SLCO1A2) 

OATP2A1-CHO (SLCO2A1)/PGT 

NTCP-CHO (SLC10A1), rat Ntpc (Slc10a1) 

NEW! 

NEW! 

3 

HH-Estrone-3-Sulfate E t 3 S lf t 

Fluvastatin 

3 H-Estrone-3-Sulfate Ketoconazole 

3 H-Estrone-3-Sulfate Fexofenadine 

3 H-Prostaglandin-E2 Diclofenac 

3 H-Taurocholate TCDC Rosuvastatin 

PEPT1-CHO PEPT1 CHO (SLC15A1) Glycylsarcosine Tyr-Phe Tyr Phe 

PEPT2-CHO (SLC15A2) Glycylsarcosine Cefadroxil 

3 

OAT1-CHO OAT1 CHO (SLC22A6), Rat Oat1 H-PAH H PAH Benzbromarone 

OAT3-CHO (SLC22A8) 

OCT1-CHO (SLC22A1) 


OCTN1-CHO (SLC22A4) 

OCTN2-CHO (SLC22A5), rat Octn2 

NEW! 

NEW! NEW! 

NEW! 

NEW! 

3 H-Estrone-3-Sulfate Probenecid 

Valacyclovir; 

ampicillin 

Valacyclovir; 

ampicillin 

Tenofovir; zidovudine; 

MTX 

Ciprofloxacin; 

zidovudine 

14 C-TEA, 14 C-Metformin Verapamil Metformin; quinidine 

14 C-TEA, 14 C-Metformin Cimetidine Cimetidine 

14 C-TEA Verapamil Quinidine; verapamil 

3 H-Carnitine Verapamil Quinidine; verapamil

Monolayer Assay Services 

Gold standard for PP-gp gp 

Modeling of important 

pharmacokinetic barriers 

Tight cell layer grown on 

a porous support 

Caco-2 and single and 

double transfectants 

MDCKII cells 

Permeability of test article 

should be considered

Determination of the P and P AB BA 

values and the net flux ratio in the MA 

t 0 

t 1 

AB BA 

Donor 

RReceiver i 

chamber 

chamber 

Apical Apical 

c 0 

Receiver 

chamber 

Basal Basal 

t 0 

t 1 

c c0 Donor 

chamber 

h b

MDDCKII-BCCRP 

MDCKII 

Interpretation of Results 

= dQ/dt 

PAPP 

Efflux ratio: 

ER = P app,BA/P app,AB 

ER NET = ERT/ER T W 

dQ 

dt 

1 

AC 

ERT and ERW are the efflux 

ratios for the transfected and 

non-transfected t f t d li lines 

0

Bidirectional Permeability of Digoxin on MDCKII 

Monolayer Assay Reference Data 

and MDCKII-MDR1 Monolayers in the Presence of 

Verapamil


Bidirectional transport of Prazosin +/- / 1 μM Ko143 

throuhg MDCKII-BCRP and parental monolayers 

(10-6cm/secc) 

Papp 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

BCRP A-->B 

BCRP B-->A 

wt A-->B 

wt B-->A 

Prazosin 1uM / I. Prazosin 1uM / II. Prazosin 1uM + Ko143 1uM 

ER BCRP Parental 

Prazosin I 19 0.7 

Prazosin II 17 11.0 0 

Prazosin + Ko143 0.8 0.6

Appareent 

permeaability 

(10-6cm/sec) 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 


MDCKII-OATP2B1/BCRP 

Vectorial transport of E-3-S 

BCRP OATP-B OATP B BCRP / OATP-B OATP B MDCKII 

Cell line 

1 uM BA 

1 uM AB 

10 uM AB 

10 uM AB 

100 uM BA 

100 uM AB

MMonolayer l AAssays at t SOLVO 

Cell line 

TType of f CCell ll li line 

Service 

assay 

licensing 

PreadyPort TM * 

Caco-2 Substrate ID 

(MDR1, BCRP) 

- - 

Inhibitor ID 

MDCKII-MDR1 

MDCKII-BCRP 

MDCKII- 

BCRP/OATP2B1 

MDCKII-BSEP/NTCP 

Substrate ID 

(24-well plate) 

 

IInhibitor hibi ID 

(96-well (96 well plate) 

Substrate ID 

Inhibitor ID 

Substrate ID 

Inhibitor ID 

Substrate ID Under 

Inhibitor ID 

 

Under Development 

Development 

Under 

Development 


* In cooperation with

PREDEASY ATP ATPase AAssay 

IInexpensive, i nonradioactive 

Idi Indication ti on th the nature t of f 

interaction 

For P-gp P gp reasonable 

correlation with cellular 

assays y 

Indirect assay 

Not as widely accepted by 

regulatory agencies as 

cell based and vesicular 

assays

PREDEASY TM ATPase kit 

Advanced d d Pi ddetection 

Higher sensitivity 

M More robust b t 

Shorter incubations 

Easier automation 

Lower cost for each 

data da a point po generated 

g a d 

Ready-to use 

• 

• 

membrane b preparation, ti 

and all reagents 

Data evaluation Excel on 

CD 

• 

• 

• 

3-compound size 

6- compound size 

99-compound d size 

i

Main Steps of the Assay 

1. Preparation of the assay mix 

2. Preparation of membrane suspension in 

assay mix (4 g g / well) 

3. Add membrane suspension and test drug 

to 96-well plate 

4. Pre-incubation (5 min, 37 o C) 

5. Add MgATP (final concentration: 2 mM) 

6. Incubation (10 min, 37 o C) 

7. Stop reaction by adding developer, 

add blocker after 2 min. 

8. Incubate (30 min) 

9. Measure OD at 620nm 

10 10. CCalculation l l ti

CContents t t of f th the AAssay-Mix Mi 

Name Conc Conc. Purpose 

MOPS-Tris, pH 7 50 mM Buffer 

KCl 50 mM Osmotic pressure 

Na-Azide 5 mM 

Inhibitor of mitochondrial ATPases 

(Antimicrobial agent) 

DTT 2 mM Denaturing agent, not for ABC transporters 

EGTA-Tris, pH 7 0,1 mM 

MgATP 

ATP 10 mM Catalyzes reaction 

MgCl 10 mM Co-factor Co factor 

Inhibitor of Ca2+ Inhibitor of Ca dependent ATPase activity 

2+ dependent ATPase activity 

(P-type)

Explanation of Results 

Abssorption 

at 620 6 nm 

1.600 

1.400 

1.200 

1000 1.000 

0.800 

0.600 

0.400 

0.200 

0.000 

vanadate sensitive baseline 

activity (0%) 

Solvent 

(CTRL1) 

Vanadate 

(CTRL2) 

vanadate sensitive activated 

transporter activity (100%) 

Activated 

(CTRL3) 

Activated + 

Vanadate 

(CTRL4) 

-34% 

Activated + 

Inhibitor 

(CTRL5)

vanadate se ensitive ATPase e acitivty 

(nmo ol Pi/mg prot./min n) 

PREDEASY Reference curves 

Substrate 

MDR1; Prazosin 

55 

50 

activation 

45 

40 

inhibition 

35 

30 

IC IC50= 50= 309 礛 

25 

20 

1 115 

10 

5 

0 

EC EC50= 50= 26.1 礛 

0 10 -7 10 -6 10-5 10-4 10-3 15 

10 

5 

0 

0 10 -7 10 -6 10-5 10-4 10-3 Prazosin a os [ [M] ] 

vanadate sensit tive ATPase acitiv vty 

(nmol Pi/ /mg prot./min) 

vanadate e sensitive ATPase e acitivty 

(n 

nmol Pi/mg prot./mi in) 

60 

50 

40 

30 

20 

10 

Inhibitor 

MDR1; Cyclosporin A 

0 

0 10-8 10-7 10-6 10-5 10-4 10-3 0 

0 10-8 10-7 10-6 10-5 10-4 10-3 Cyclosporin A [M] 

50 

40 

30 

20 

10 

activation 

inhibition 

IC IC50=1.0 50=1.0 50 1.0 M M M M 

Slowly transported substrate 

MDR1; Ivermectin 

0 

0 10-8 10-7 10-6 10-5 10-4 10-3 0 

0 10 

Ivermectin [M] 

-8 10-7 10-6 10-5 10-4 10-3 Ivermectin [M] 

activation 

inhibition 

IC IC50=0.6 50=0.6 M M

vanadate e sensitive ATPa ase acitivty 

(n mol Pi/mg prot./ min) 

22 

20 

18 

16 

14 

12 

10 


Substrate 

MRP1; NEM-GS 

8 

0 10-5 10-4 10-3 10-2 10-1 8 

0 10-5 10-4 10-3 10-2 10-1 NEM-GS [M] 

activation 

inhibition 

EC EC50= 50= 2231 M M 

vanadate sensitiv ve ATPase acitivty y 

(nmol Pi/m mg prot./min) 

va 

anadate sensitive e ATPase acitivty y 

(nmol Pi/mg g prot./min) 

20 

15 

10 

5 

Inhibitors 

MRP1; Benzbromarone 

0 

0 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 0 

0 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 0 10 10 10 10 10 10 10 10 

Benzbromarone [M] 

10.0 

7.5 

50 5.0 

2.5 

MRP1; MK571 

0.0 

0 10-7 10-6 10-5 10-4 10-3 10-2 10-1 0 10-7 10-6 10-5 10-4 10-3 10-2 10-1 MK571 [M] 

activation 

inhibition 

IC IC50=17 50=17 50 17.11 11 M M M M 

EC EC50=49.88 50=49.88 M M 

activation 

inhibition 

IC IC50=68.9 50=68.9 M M 

50 

EC EC50=404 50=404 M M

vanadate se nsitive ATPase acitivty a 

(nmol (nmol l Pi/mg prot./min) 

70 

60 

50 

40 

30 

20 

10 


Substrate 

MXR-HAM; BCRP-HAM; Furosemid Furosemide 

0 

0 10-7 10-6 10-5 10-4 10-3 0 

0 10 

Furosemid [M] 

-7 10-6 10-5 10-4 10-3 Furosemid [M] 

activation 

inhibition 

EC EC50=28.2 50=28.2 M M 

vanadate sen nsitive ATPase acitiv vty 

(nmol Pi/mg prot./min) 

vanad 

date sensitive ATPa ase acitivty 

(nmol Pi/mg prot./ min) 

Inhibitor 

MXR-HAM; BCRP-HAM; Ko 143 143Ko134 

70 

activation 

60 

50 

40 

30 

20 

10 

0 

0 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 

0 

0 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 

Ko 143 [mM ] 

MXR MXR-HAM HAM , Hoechst BCRP BCRP-HAM; HAM 33342 HHoechst33342 h 33342 

50 

40 

20 

10 

inhibition 

IC 50=35.3 50=35.3 nM 

IC 50=9.9 50=9.9 99 nM M 

Slowly transported substrate 

activation 

inhibition 

30 IC 50=3.1 50=3.1 M M 

0 

0 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 

0 

0 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 

Hoechst 33342 [M ] 

IC 50=2.1 50=2.1 M M

PREDEASY PREDEASY ATPase Assays Available at SOLVO 

Transporter Activator 

MDR1-Sf9 Verapamil 

ratMdr1b-Sf9 ratMdr1b Sf9 Verapamil 

MRP1-Sf9 NEM-GS 

MRP2-Sf9 MRP2 Sf9 Sulfasalazine 

MRP3-Sf9 Benzbromarone 

BCRP-HAM-Sf9 / BCRP-M NEW! Sulfasalazine 

mouseBsep-HAM-Sf9 TCDC 

defMRP-Sf9 - 

defBCRP-Sf9 - 

defP-gp-Sf9 - 

All available as a kit and 

service

PREDIVEZ Vesicular Transport 

Quick and easy assay 

Assay 

Flexible l bl readout d ( (LSc, Fluo, l 

LC/MS) 

PPermeability bilit iis not t a ffactor t 

(inhibition) 

Modeling of IC concentration 

Vesicles from any cell type 

Substrate testing only for 

low permeability compounds

PREDIVEZ Assay Steps 

11.Plate Plate membrane suspension 

in assay mix (containing 

transported and reporter 

substrate) 

2. Equilibrate with probe, 

ttest t article ti l and d controls t l 

3. Start reaction with ATP 

44.Incubate Incubate 

5. Filter and wash vesicles 

6. Measure counts

PPractical ti l Information I f ti for f using i 

Equipment needed: 

Millipore Multiscreen Multiscreen HTS 

Vacuum Manifold (Cat No. 

MSVMHTS00) 

PREDIVEZ Kits ts 

Plate map: 

Pl Plate t Map M 

(for two 

parallels) 

CDCF calibration curve ATP + ATP - 

1 2 3 4 5 6 7 8 

A 20 pmol p CDCF 150.00 150.00 150.00 150.00 

B 10 pmol CDCF 50.00 50.00 50.00 50.00 

C 5 pmol CDCF 16.67 16.67 16.67 16.67 

D 2.5 pmol CDCF 5.56 5.56 5.56 5.56 

E 0 pmol CDCF 185 1.85 185 1.85 185 1.85 185 1.85 

F ATP + ATP - 0.62 0.62 0.62 0.62 

G ATP + ATP - 0.21 0.21 0.21 0.21 

H ATP + ATP - DMSO DMSO DMSO DMSO 

Alternative plate maps for assaying 2 

compounds + calibration curve + 

negative control membrane test or 3 

compounds

Time curve of TC uptake into BSEP 

vesicles 

transport (pmool/mg 

membrane 

prootein) 

200 

160 

120 

80 

40 

0 

PREDIVEZ Reference Curves 

ATP+ 

ATP- 

0 10 20 30 40 

time (min) 

Substrate saturation of TC uptake 

iinto t BSEP vesicles i l 40 

ATP A 

dependent traansport 

(pmol/mg membbrane 

protein/minn) 

160 

120 

80 

40 

0 

0 10 20 30 40 50 60 

taurocholate (uM) 

ATP A dependent 

transport 

(%) 

Inhibition of NMQ transport into P-gp 

vesicles i l with ith reference f P-gp P iinhibitors hibit 

120 

110 

Cyclosporin A 

100 

90 

80 

Ketoconazole 

Verapamil 

70 

60 

50 

LY335979 

Erythromycin 

40 

30 

20 

10 

0 

-10 

Digoxin 

00.0001 0001 00.001 001 001 0.01 01 0.1 1 10 100 1000 10000 100000 

[drug] / M

Vesicular Transport of 3 PREDIVEZ Reference Curves 

H-Taurocholate into BSEP expressing 

vesicles 

Time dependent transport 

BSEP Beta-Gal 

trransport 

(pmol/m g membrane 

proteinn) 

ATP dependent transport t 

(pmol/mg memmbrane 

protein/min) 

200 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0 

0 10 20 

time (min) 

30 40 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0 

ATP ATP+ 

ATP- 

ATP dependent transport t 

(pmol/mg memmbrane 

protein/miin) 

180 

160 

140 

120 

100 

Concentraton dependent p transport p 

0 10 20 30 40 50 60 


1/ /ATP dependent trransport 

(mg 

membrane m 

proteinn*min/pmol) 

80 

60 

40 

20 

0 

-20 0 10 20 30 40 50 60 

0,06 

0,05 

0,04 

0,03 

0,02 

0,01 


0 

-0,2 0 0,2 

1/taurocholate (uM) 

0,4 0,6

Effect of drugs on CDCF transport by MRP2-Sf9 

ndent trannsport 

(%) ) 

ATP depe 

125 

100 

75 

50 

25 

0 

-25 

00.0001 0001 00.001 001 001 0.01 01 0.1 1 10 100 

[drug] / mM 

PREDIVEZ Reference Curves 

Effect of drugs on CDCF transport by MRP3 

ive transport 

(%) 

Relat 

140 

120 

100 

80 

60 

40 

20 

0 

-20 

0.01 0.1 1 10 100 1000 10000 

[drug] / M 

■ - Probenecid ■ - Indomethacin 

▲ - Sulfasalazine 

▼ - Indomethacin 

▲ - MK571 

▼ - Benzbromarone

Vesicular Transport Assays Available at SOLVO 

Transporter Probe substrate 

* MDR1-K MDR1 K 3 3HH-NMQ NMQ or cold ld NMQ 

* ratMdr1b-Sf9 3 H-NMQ or cold NMQ 

* MRP1 MRP1-Sf9 Sf9 

3 3H-LTC4 H LTC4, B-GS B GS 

* MRP2-Sf9 

3H-E217bG, CDCF 

* ratMrp2-HEK293 

ratMrp2 HEK293 3H-E217bG, H E217bG, CDCF 

* MRP3-Sf9 

3H-E217bG, CDCF 

MRP4-LLC-PK1 

3H-DHEAS * MRP5-HEK293 

3H-cGMP, CDCF 

BSEP-Sf9 

3H-Taurocholate mouseBsep-Sf9 

3H-Taurocholate * BCRP-M, BCRP-HAM-Sf9 

3H-E3S, 3H-MTX, Lucifer Yellow 

mouseBcrp1-MDCKII 

* Available as a kit and 

service 


Fluorescent probe 

substrate

Cellular Models - Calcein assay 

Transporter specific 

Fluorescent measurement 

Potential for High 

throughput g p 

Least expensive method to 

screen for possible P‐gp 

related DDI 

Indirect measurement – 

modulation of dye 

transport 

Low permeability 

compounds may not get 

into the cells 

Cell line licensing available 

Cell lines used in dye efflux assays 

K562MDR (Calcein assay) 

HL60MDR1 (Calcein assay) 

Hl60MRP1 (Calcein assay) 

HL60MXR (Hoechst 33342 assay)

Cellular uptake assay 

Stably transfected cells 

Quick and easy assay method 

Permeability y is not a factor 

(inhibition) 

Address possible drug‐drug drug drug 

interaction risks 

Fl Flexible ibl readout d (LSc, (LS Fl Fluo, 

LC/MS) 

Inhibitor / substrate assessment 

studies 

S b i l f l bili 

Substrate testing only for low permeability 

compounds

PrediCell TM Cellular Uptake 

Kit 

Uptake transporter experiments without cell 

line development or licensing 

Preplated mammalian cells 

Active thermal insulation 

Protection against shear stress 

Customization 

Plate pattern, stably transfected and parental cell line 

Reference substrate & inhibitor, assay buffer are optionally 

included 

Radiochemicals must be purchased separately! 

I In The Th Box B 

Plates with cells 

Standard Timeline 

User’s Manual 

Day 1: Package Dispatch 

Product Data Sheet 

(typically Tuesdays) 

List of Items 

Days 2-3: Package Delivery 

Optional: Reagents 

Days 2-3: Transport Experiments

Activity of PrediCell TM Kit

UptakeTransport Assays Available at 

SOLVO 

Cell line Probe substrate 

OATP1B1-CHO (SLCO1B1)/OATP2, OATP-C 


Reference 

Inhibitor 

Pravastatin; 

rosuvastatin 

OATP1B3-CHO (SLCO1B3)/OATP8 Fluo-3 Digoxin; rosuvastatin 

OATP2B1-MDCKII (SLCO2B1)/OATP-B 

rat Oatp1a1 (Slco1a1) 

NEW! 


3 H-Estrone-3-Sulfate Ketoconazole 

3 

OATP1A2-HEK293 OATP1A2 HEK293 (SLCO1A2) H-Estrone-3-Sulfate H Estrone 3 Sulfate Fexofenadine 

OATP2A1-CHO (SLCO2A1)/PGT 

NTCP-CHO (SLC10A1), rat Ntpc (Slc10a1) 

NEW! ! 

3 H-Prostaglandin-E2 Diclofenac 

3 H-Taurocholate TCDC 

PrediCell TM , 

Licensingg 

PrediCell TM /Licens 

ing 

PrediCell TM 

/Licensing 

Fluvastatin Licensing 

PEPT1 PEPT1-CHO CHO (SLC15A1) Gl Glycylsarcosine l i TTyr-Phe Ph Li Licensing i 

PEPT2-CHO (SLC15A2) Glycylsarcosine Cefadroxil Licensing 

OAT1-CHO (SLC22A6), Rat Oat1 

OAT3-CHO (SLC22A8) 



OCTN1-CHO (SLC22A4) 

OCTN2-CHO (SLC22A5), rat Octn2 

MATE1-CHO (SLC47A1) 

NEW! 

3 H-PAH Benzbromarone 

3 H-Estrone-3-Sulfate Probenecid 

PrediCell TM /Licens 

ing, 

14 C-TEA, 14 C-Metformin Verapamil Licensing 

PrediCell TM 

14 14 

NEW! C-TEA, C-Metformin Cimetidine 

/Licensing 

NEW! 

NEW! 

14 C-TEA Verapamil 

3 H-Carnitine Verapamil 

14 C-TEA, 14 C-Metformin Quinidine

Single and double 

transfectants 

Gold standard for P-gp 

Good for most PP-gp gp and 

BCRP substrates 

GGood d absorption b ti model d l 

Ti Time consuming i cell ll 

culturing (Caco-2) 

Permeability can be 

limiting 

MMonolayer l AAssay

Preplated ready-to-use MDCKII monolayers, in 

cooperation ti with ith Ad AdvanCell C ll SA SA. 

Available under a limited single-use license 

without extra charge 

Patented Shipping Medium 

– Forms semi-solid protective layer at room 

temperature while liquefies at 37°C -> 

Patented Shipping Container 

– protection against physical shock 

Customization 

Adaptable to automation 

In The Box 

• Plates with cells 

• User’s Manual 

• AAspiration i ti manifold if ld (optional) ( ti l) 

• Product Data Sheet 

Standard Timeline 

• Day 1: Package Dispatch (typically 

Mondays) 

• Day 2-3: Package Delivery 

• Day 4: Replacement of Shipping Media 

• Day 7: QC Experiments, Media 

Replacement 

• Days 8-11: Transport Experiments

Activity of preplated ready-to-use ready to use MDCKII monolayers 

MDR1 t f t d MDCKII ll 

MDR1 tranfected MDCKII cells 

Each measurement was performed in 

triplicates at day 5 and at day 12. 

• Up to five days of 

transportation in 

semi-solid medium 

• Experiments can be 

performed up to 7 

days y after arrival

Using pre-plated pre plated MDCKII 

cells 

Mon Tue Wed Thu Fri Sat Sun 

QA check 

TEER + LY 

QA check 

TEER, 

change 

media 

di 

Plates 

Shipped 

Order 

Receival 

Days to perform assay(s) 

Cells 

Plated 

Change 

shipping 

medium

Monolayer Assays at SOLVO 

CCell ll li line 

Type yp of 

assay 

Caco-2 (MDR1, Substrate ID 

BCRP) IInhibitor hibi ID 

MDCKII-MDR1 

MDCKII-BCRP 

MDCKII- 

BCRP/OATP2B1 

MDCKII-BSEP/NTCP 

Substrate ID 

Inhibitor ID 

Substrate ID 

Inhibitor ID 

Substrate ID 

Inhibitor ID 

SService i 

SSubstrate b t t ID Under 

Inhibitor ID 

Cell line 

licensing 

P d P tTM PreadyPort * TM * 

- - 

 





Development 

Under 

Development 


* In cooperation with

RRecommendations d ti for f Choosing Ch i the th Optimal O ti l Assay A 

ompound assayed a 

P Papp 

of co 

Hiigh 

Low L 

ATPase / 

VT and 

uptake 

inhibitio 

n 

Reliable 

Reliable 

Monolayer 

efflux 

inhibition 

assay 

Reliable 

VT and 

uptake 

substrate 

Reliablee 

Monolayer 

efflux 

transport 

assay 

Application of different types of assays for Low-High permeability 

compounds 

d 

Reliable 

Reliablle

In In vitro vitro P-gp P gp assays – Correlation Analysis 

Von Richter 2008 Naun Schmied Arch Pharmacol 

Polli 2001 JPET 

Transporter effect in 

monolayer efflux assay 

No transporter effect in 

monolayer efflux assay 

● compounds that show ATPase activation 

◊ comounds that show no ATPase activation

Compound EAC 

Importance of Permeability 

MDCKII-MDR1 

AB 

permeability 

/ nm/sec* 

MDCKII- Digoxin g BA Calcein 

MDR1 

BA/AB* 

IC 50 / 

mM** 

assay IC 50 

/ mM 

MDR1 

vesicular i l 

transport 

IC50 / mM 

GF120918 NNY 2.12 ND 0.055 0.026 0.049 

Quinidine YYY 36.4 27.2 14.9 12.89 30.74 

Vinblastine YYY < 10 >23 17.8 26.00 0.44 

Cyclosporin 

e A 

Ketoconazol 

e 

YNY 15 15.9 9 99.61 61 11.6 6 00.49 49 00.201 201 

NYY 316 1.02 3.07 3.03 2.16 

Verapamil NYY 591 1.16 10.7 9.45 5.19 

Erythromyci 

n 

YYN 0.94 14.4 N/A N/A 36.2 

Digoxin YYN 3.07** 16 N/A N/A 162.5 

* ‐ Polli JPET 2001; ** ‐ Rautio DMD 2006

Additional Model 

Systems Offered at 

SOLVO

Wh Why SOLVO? 

Portfolio 

In vivo transporter services 

Blood-Brain Barrier 

• Brain penetration study - both in rats and 

mice 

Liver 

• Bile duct cannulated (BDC) rats 

Kidney 

• Renal excretion model - under 

development 

IIntestine t ti 

• Oral absorption model - under 

development

Rat Brain Endothelial Cell Culture 

Model 

Endothelial cells 

Pericytes 

At Astrocytes 

t 

Transport of Quinidine via RBEC monolayer 

in the presence and absence of PSC833 

Effflux 

ratio 

3 

2 

1 

0 

15 minn 

30 minn 

60 minn 

0.1 M QND 

0.1 MQND+ M QND 1 MPSC833 

M PSC833

Microdialysis (rats and 

mice) 

I. Determination of brain 

penetration of test molecules 

in rats (unbound) 

II. Determination of test molecule 

interactions with 

transporters; +/- / MDR11 or BCRP C 

specific inhibitors 

III.Determination of brain 

penetration of test molecules 

and simultaneous monitoring of 

neurotransmitter release in 

specified brain regions.

Dose-dependent Brain 

Penetration of Transporter 

Substrates b 

2007 AAPS-FDA workshop white paper: Chaurasia C.S. et al, 

Microdialysis principles, application and regulatory perspectives 

- Currently MD is the only tool available that explicitly provides 

data on the extracellular space

NNew LLaunch h 

Hepatocyte Uptake Assay 

Plated primary human and rat hepatocytes 

Simple uptake experiments 

Km/Vmax determination 

DDI studies 

Three probe substrates 

1. NTCP 

2. OATP 

3. OCT1-related uptake capacity

Biliary Excretion - rat 

Bile duct cannulated rats model 

Study the excretion of test compounds into 

the bile 

Study the effect of the test compound on 

the excretion of other (physiological) 

compounds ( (e.g. bile salts) ) 

Both standard and transporter specific 

study designs 

Measure inhibition of Mrp2, Bcrp and Bsep

Liver 

Kidney 

SOLVO Barrier Packages 

Individual 

Transporters 

OATPs, MRPs, 

BSEP, NTCP, OCT1, 

P-gp P-gp, BCRP 

Cellular Models In vivo models 

Hepatocyte uptake 

assay 

BDC rats 

OATs, OCTs, Renal Proximal 

MATEs, MRPs, P- Tubule cells Under development 

gp, BCRP (aProximateTM ) 

Absorption P-gp, P gp, BCRP Caco-2 Caco 2 

Blood-Brain 

Barrier 

PP-gp, BCRP 

RBEC, , MBEC, , 

MDCKII-MDR1 

In vivo 

absorption ( (PK) ) 

Mi Microdialyis 

di l i

Efflux 

Uptake 

Other 

Portfolio OOverview er ie 

Products Services 

• PREDEASYTM • PREDEASY ATPase kits • ATPase assays 

TM ATPase kits 

• ATPase assays 

• PREDIVEZTM VT kits 

• PREADYPORTTM Monolayer kits 

• ATPase membrane preparations 

• VT membrane preparations 

• VT substrate assays 

• VT inhibition assays 

• Dye efflux assays 

• MDCKII monolayers 

• MDCKII cell line licensing • Caco-2 monolayers 

• PREDICELLTM • Cellular uptake substrate assays 

kits 

• Cellular uptake inhibition assays 

• Cell line licensing • Double transfected monolayer assays 

• Isolated hepatocyte uptake studies 

• PSC833 (Pgp inhibitor) 

• Ko134 (BCRP inhibitor) 

• MDQ diagnostic kits 

•Solubility tests 

• In vivo microdialysis y studies 

- Pgp specific 

- BCRP specific 

• In vivo liver transporter studies 

• Rat Brain Endothelial Cell monolayer assay

Wh Why to choose h SOLVO? 

We are the h iindustry d lleader d iin the h fi field ld of f iin vitro i 

transporter services and products 

Unique expertise in the transporter science 

Widest drug-transporter assays portfolio 

Competitive pricing 

We not only y ship p a product, p , but provide p technical 

support whenever it is needed 

Continually expanding product and service portfolio 

with 10-15 10 15 new launches annually 

Provide assays for all efflux and uptake transporters 

assays recommended by the FDA / EMA 

1st 1 and only CRO offering transporter specific in 

vivo assays 

NOW! US operations providing direct customer 

support support, technical expertise expertise, and GLP assay services for 

our North American customers

Contact SOLVO 

• Regular Webinars, Newsletters and Science 

letters, Scientific Publications, Reviews 

• LinkedIn, Facebook, Twitter 

• CConferences f 

• Transporter Workshops and Symposiums 

• www.solvo.com 

• sales@solvo.com Please contact me directly if you have any 

questions! 

Kent Grindstaff, Ph.D. 

Senior Director BD and Operations, North 

America 

grindstaff@solvo.com 

May 14, 2012 

119

SOLVO Biotechnology

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