estudios de permeabilidad en - as-lat.com€¦ · estudios de permeabilidad en cÉlulas caco-2...

© 2011 Absorption Systems

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ESTUDIOS DE PERMEABILIDAD EN CÉLULAS CACO-2

Ismael J. Hidalgo, Ph.D.

Absorption Systems, Exton, PA


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Outline Morphology & Barrier Properties of the Intestinal Mucosa

What is Permeability?

Techniques for Studying Permeability

Description of the Caco-2 cell assay system -Origin of Caco-2 cells

-Cell Culture

-Permeability Assay

-Inter-laboratory Variability

Applications of the Caco-2 permeability assay -Absorption potential of drug candidates

-Biopharmaceutics classification systems (BCS): class determination

-Transporter-mediated drug-drug interactions

Final Thoughts

Acknowledgements


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Morphology


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Morphology


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Apical membrane Insert

Well

Cell Monolayer

Microporous filter

Basolateral membrane


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3 D

ays p

ost-s

eed

ing


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16 D

ays P

ost-s

eed

ing


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15 D

ays

Po

st-s

ee

din

g


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Human Jejunal Mucosa


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Why Caco-2?

Caco-2 cells express the most common intestinal efflux and uptake transporters

DM β-actin P-gp BCRP MRP2 1B1 1B3 2B1 MRP1 DM

Intestinal Enterocytes C2BBe1 Monolayer


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Origin of Caco-2 Cells

• Heterogeneous cell line derived from a colon adenocarcinoma removed from a 74 years old Caucasian male and deposited at the Sloan-Kettering Memorial Institute in NY in 1974.

• When grown in culture they develop polarity and undergo spontaneous differentiation into an enterocyte-like phenotype.

• Functionally, they mimic the barrier properties of the small intestine and express numerous intestinal enzymes and transporter proteins in the correct cellular domain.

• They have become the pharmaceutical industry standard for in vitro drug absorption/permeability studies.


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Permeability Coefficient (Apparent Permeability Coefficient, Papp)

• An in vitro parameter used to assess the rate at which compounds traverse a barrier (e.g., the intestinal epithelial mucosa). Referred to as apparent because it represents the combined effect of all permeation pathways and not the permeation across any single barrier such as the unstirred water layer, cell membrane or tight junctions.


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Permeability Assay: Transwell Device


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Permeability Assay: Integrity of Cell Monolayers


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Transepithelial electrical resistance (TEER) measurement

Transwell

Tray

EVOM Epithelial

Voltohmmeter

Endohm


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Caco-2 Permeability Assay Work Flow

• Cells are cultured in tissue culture flasks and split every 3-5 days.

• Cells seeded on collagen-coated polycarbonate filters (Transwell) at 60,000 cells/cm2 and cultured for 3 weeks in humidified CO2 incubator.

• When ready, the transepithelial electrical resistance (TEER) is measured.

• If TEER is acceptable, determine Papp values for control compounds.

• Monolayers are used in permeability experiments.


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API concentrations

are determined in

the apical and

basolateral dosing

compartments by

sampling the

solutions at

various times after

dosing.


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Permeability Assay: Transport vs Time Profiles

19

Cum

ula

tive T

ransport

Time


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Apparent Permeability Coefficient (Papp)

• An in vitro parameter used to assess the rate at which compounds traverse a barrier (e.g., the intestinal epithelial mucosa). Referred to as apparent because it represents the combined effect of all permeation pathways and not the permeation across any single barrier such as the unstirred water layer, cell membrane or tight junctions.

• Papp = (dQr/dt)/(A x C0)

dQr/dt: Cumulative amount of compound that appears in the

receiver compartment vs time

A: permeation surface area (e.g., cell monolayer or tissue)

C0: Initial compound concentration in the donor compartment


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Is In Vitro Papp a predictor of Fabs in Humans?

0

20

40

60

80

100

1.0E-07 1.0E-06 1.0E-05 1.0E-04Papp (cm/s)

Fab

s (

%)

BBRC (1991) 175:880-85


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Inter-laboratory Variability: Caco-2 Papp vs Fabs data from 5 laboratories

0

20

40

60

80

100

1.0E-07 1.0E-06 1.0E-05 1.0E-04

Papp (cm/s)

Fab

s (

h)

Curr. Top. Med. Chem. 1: 385, 2001


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• Biologic

– Seeding density

– Passage number

– Attachment factors

– Days in culture

– Cell viability

– Permeable support

– Trypsinization process

– Culture conditions

– Uptake Transporters

– Efflux Transporters

– Metabolizing enzymes

• Study Design

– Stirring vs stagnant

– Sampling schedule

– Transport Buffer

– pH

– Drug concentration

– Analytical methods

– Data analysis

– Co-solvents

What Factors Can Cause Variability?


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Caco-2 cell assay: Quality Control

Caco-2 QC Strategy

Batch QC of randomly

selected monolayers

Screen each monolayer

prior to use

by measuring TEER

Check each monolayer

after use by

measuring marker

dye permeability

A Batch consists

of about 20

Transwell plates

or 240 individual

monolayers


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Caco-2 QC chart for control compounds

1.00E-07

1.00E-06

1.00E-05

1.00E-04

1.00E-03

1.00E-02

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

QC assay #

Pa

pp

(c

m/s

ec

)

atenolol

pindolol

propranolol

Permeability Assay: Continuous Monitoring of Monolayer Barrier Properties


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Applications of the Caco-2 Permeability Assay

1. Determination of the absorption potential of new chemical entities (NCEs)

2. Drug classification according to the BCS

3. Support of Formulation Development

4. Transport-mediated drug-drug interaction(s)


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1. Absorption Potential Determination

Low

Medium

High

High

Low

•Drug Discovery

•Assign to a Bin

•Large numbers of NMEs


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2. Biopharmaceutics Classification System (BCS)

Class III Ranitidine Cimetidine Atenolol Vancomycin

Class IV Furosemide Hydrochlorothiazide

High

Hig

h

Per

mea

bili

ty

Class II Ketoprofen Naproxen Carbamazepine

Class I Propranolol Verapamil Metoprolol

Solubility

Low

Low


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2. BCS Classification

• BCS Based Biowaiver

• Regulatory acceptance of in vitro testing as a reliable surrogate for in vivo

• bioequivalence study


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Multiple Validated pH Levels


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BCS Study Design


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Caco-2 Cell Monolayers

Permeability and P-gp

Function

• Days in culture: 21-28

• Passage number: 58-76

• Donor pH: 6.5 and 7.4

• Multiple operators


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Caco-2 cell assay: Quality Control

Caco-2 QC Strategy

Batch QC of randomly

selected monolayers

Screen each monolayer

prior to use

by measuring TEER

Check each monolayer

after use by

measuring marker

dye permeability

A Batch consists

of about 20

Transwell plates

or 240 individual

monolayers


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34

Permeability Classification

Caco-2 Cell Monolayers

Criteria for Biowaiver

► Rapid Dissolution

► Dose-Relevant Solubility

► High Permeability

Phase 1A: Eligibility

Screen

Phase 1B: Protocol

Optimization

Phase 2: Pivotal Classification


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Phase 1A: Eligibility Screen

Determine tolerable concentration range

Determine magnitude of non-specific binding

Is efflux ratio less than three?

Is permeability greater than minoxidil?


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Phase 1B: Protocol Optimization

Impact of Control Compounds

Concentration Range Mass Balance

Obtaining Quality Permeability Data


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Phase 2: Pivotal Study

GLP Analytical Validation

Multiple Concentrations

(0.01X, 0.1X, 1X)

Additional Replicates

(N=4)

GLP Classification


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BCS Permeability Classification

Cum

ula

tive T

ransport

Time

HP, IS

LP, IS

Test Compound


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Minoxidil C

ac

o-2

Pap

p (

x 1

0-6

cm

/s)

Antipyrine: Papp ~63

Metoprolol: Papp ~28

Pindolol: Papp ~17

Minoxidil: Papp ~4

Absolute BA is 98%

• pH 7.4


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Why Minoxidil?

• At least 95% fraction

absorbed

• Passive transport

• No known food effects

or DDI

• No pH effect

How do you justify using minoxidil

as your high permeability internal

standard?


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FDA Experience with Biowaivers

Mehta, Sept. 2010; AAPS Webinar: Application of Biopharmaceutical Classification System (BCS) in Regulatory Submissions

Davit, June 2011, ‘BCS Classification Workshop, Canadian Society for Pharmaceutical Sciences ‘FDA Experience with Biopharmaceutics Classification System (BCS) Biowaivers

>75%

Caco-2

Types of Data Submitted New Drugs vs. Generics


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Applications of Biowaivers

• Approval for Generic Medicinal Products

• Formulation Development for New Drug Products

• Line Extensions Based on Proportional Similarity

• Post-approval Changes: Innovator and Generics


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Our Experience

Number of BCS Studies per Year at Absorption Systems


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BCS Biowaivers

• Independent of Therapeutic Class or PK


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Waive Complex BE Studies

•Therapeutic Area and PK Independent

Special Population

Special Population with

Reference Scaled Approach

Fed/Fasted


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Compound Indication FDA Product Recommendation

Tested at Absorption

Systems (AS) Pemeability Classification at AS (High, Low or Not Performed)

BCS Classification by AS Based on Permeability in

Caco-2

BCS Classification based on human PK data or Mass

Balance

Buspirone Anxiety Buspirone Recommendation Yes High Class I Class I

Capecitabine Colorectal and breast cancers Capecitabine Recommendation Yes High Class I Class I

Cefadroxil Hemihydrate Antibiotic Cefadroxil Recommendation No NP NP Class I

Donepezil HCl Alzheimer-related dementia Donepezil Recommendation Yes High Class I Class I

Emtricitabine# HIV Emtricitabine Recommendation No NP NP Class I

Galantamine HBr Alzheimer’s dementia Galantamine Recommendation Yes High Class I Class I

Granisetron HCl Antiemetic Granisetron Recommendation Yes High Class I Class I

Levetiracetam Epileptic seizures Levetiracetam Recommendation Yes High Class I Class I

Levofloxacin Antibiotic Levofloxacin Recommendation Yes High Class I Class I

Linezolid Antibiotic Linezolid Recommendation Yes High Class I Class I

Memantine HCl Alzheimer-related dementia Memantine Recommendation Yes High Class I Class I

Milnacipran HCl Fibromyalgia Milnacipran Recommendation Yes Low* Class III* Class I

Palonosetron HCl Chemotherapy-induced nausea Palonosetron Recommendation Yes High Class I Class I

Pramipexole Dihydrochloride Parkinson’s disease Pramipexole Recommendation Yes High Class I Class I

Pregabalin Epileptic seizures Pregabalin Recommendation Yes Low** Class III ** Class I

Propranolol HCl Hypertension, tremors, migraine Propranolol Recommendation Yes High Class I Class I

Protriptyline HCl Depression Protriptyline Recommendation Yes High Class I Class I

Ramelteon Insomnia Ramelteon Recommendation Yes High Class I Class I

Rivastigmine Tartrate Alzheimer-related dementia Rivastigmine Recommendation Yes High Class I Class I

Sotalol HCl Cardiac arrhythmia Sotalol Recommendation Yes Low*** Class III *** Class I

Tapentadol HCl Pain Tapentadol Recommendation Yes High Class I Class I

Temozolomide Glioblastoma Temozolomide Recommendation Yes High Class I Class I

Tiagabine HCl Epileptic seizures Tiagabine Recommendation Yes High Class I Class I

Varenicline Tartrate Smoking cessation Varenicline Recommendation Yes High Class I Class I

Venlafaxine HCl Depression Venlafaxine Recommendation Yes High Class I Class I

23 of the 25 have been tested at Absorption Systems

http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM194596.pdf



http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm083276.pdf




























































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Conclusions

Caco-2 Permeability assays can be used to: • Investigate mechanisms of intestinal drug absorption (e.g.,

paracellular vs transcellular or passive diffusion vs transporter-mediated)

• Determine the absorption potential of NMEs

• Rank order sets of compounds to help prioritize resources

• Classify compounds according to BCS to apply for biowaver or guide formulation strategy

• Help identify clinically important transporter-mediated drug-drug interactions (development)

• Evaluate the role of specific excipients in drug absorption


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• THANK YOU!


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Obtaining Quality Permeability Data

• Mass balance

• Replicates

• Time points

• Relevant concentrations

• Lack of interactions between test

compound and co-dosed controls


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BCS Studies by Year at Absorption Systems

Unique Compounds Classified: 85 Highly Permeable: 35 (41%)


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Use of BCS by the Pharmaceutical Industry

From 2004-2010, 45 drug products were submitted to the FDA for classification

Adapted from: Mehta, Sept. 2010; AAPS Webinar: Application of Biopharmaceutical Classification System (BCS) in Regulatory Submissions

0

5

10

15

20

25

2004 2005 2006 2007 2008 2009 2010

BE

BCS

Adapted from PPB Open Forum 2010 FIP PSWC/AAPS Annual Meeting, Lawrence Yu


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BCS Data Received by FDA for Biowaiver Applications

Caco-2 accounts for ~80% of non-clinical experimental data submitted.

Adapted from: Mehta, Sept. 2010; AAPS Webinar: Application of Biopharmaceutical Classification System (BCS) in Regulatory Submissions


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ADVANTAGES OF IN VITRO BCS STUDIES OVER CLINICAL BE STUDIES

• In vitro studies reduce drug development costs.

• In vitro studies assess drug absorption more directly than human BE studies, for which the approach is indirect and the interpretation often complicated.

• In vitro permeability data is inherently more precise than clinical data. This is especially true for highly variable drugs for which a large number of subjects may be required to adequately power a clinical BE study.

• It is unethical to expose healthy volunteers to NCEs, about which very little is known, considering that a validated in vitro alternative is available.


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•“…when the in vivo dissolution of an IR solid oral dosage form is rapid

in relation to gastric emptying and the drug has high permeability, the

rate and extent of drug absorption is unlikely to be dependent on

drug dissolution and/or gastrointestinal transit time. Under such

circumstances, demonstration of in vivo BA or BE may not be

necessary for drug products containing class I drug substances…”

•FDA Guidance for Industry: Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate-Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System, CDER 2000.

Rationale for Biowaivers


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Clinical Waiver of Transporter Studies

• EMA Perspective

“…If the permeability in the absence of transporters is high

(> the permeability constant of the highly permeable drug

metoprolol) the effect of active drug transport will be

negligible as compared to the passive concentration-

gradient driven absorption of the drug.”

FDA Perspective

“For drugs that are highly permeable and highly

soluble, the intestinal absorption is not a rate-limiting

step, and, therefore it may be appropriate to exempt

such drugs from the in vivo evaluation with a P-gp or

BCRP inhibitor.”


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What Does the IPR Mean?

Firm can explore a BCS waiver if the firm believes that a drug product meets BCS class I criteria

Does not mean:

• FDA classifies this as BCS I

• IPR can be used as a basis for requesting a waiver of in vivo BE studies

You need data.


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Aqueous Solubility

Intestinal Absorption

Gastric Dissolution

Scientific Framework for BCS

• Gastric emptying

• Luminal contents

• GI transit time

•Intrinsic membrane

permeability

• Drug dissolution, solubility, and GI permeability are

the fundamental parameters controlling rate and

extent of absorption.

• pH range


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How is a Biowaiver Possible?

Dn = tres/tdiss Do = Mo/CS x Vo An = Peff x tres / R

• High Permeability

• High Solubility

• Rapid Dissolution

• Solubility does not

limit dissolution or

absorption

• Absorption is not

limited by GI

transit time

• Absorption/gastric

emptying not limited by

dissolution


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Choice of High Permeability Internal Standard (HPIS)

How did you choose your

high permeability internal

standard?

• Common Issues with HPIS:

• High Papp

• Variations with pH

• Misclassification

HPIS AB Papp pH

6.5

AB Papp pH

7.4

%F

Antipyrine 63.44 62.54 100

Metoprolol 11.65 27.88 95


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pH Considerations: Asymmetry

pH

6.5/7.4

Efflux Ratio

Without

CsA

6.30

With

CsA

7.34

pH gradient may result in pKa-related artifacts

Why use pH 7.4 in the

apical and basolateral

compartments?


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Symmetrical Permeability


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Mass Balance

0

/

CA

VdtdCP rr

app

)(

)(

BAapp

ABapp

P

PR

Underestimated Papp

Exaggerated efflux ratio

FDA expects >80% recovery

Ca

co

-2 P

ap

p (

x 1

0-6

cm

/s)

Provide

intracellular

accumulation data

to demonstrate

mass balance


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Time Points


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Relevant Concentration Range

Test

Compound

Concentration

Efflux Ratio

Permeability

Classificatio

n

10 µM 6.2 Similar

32 µM 1.9 High

100 µM 1.2 High

320 µM 0.9 High • Consider:

• Highest dose per administration vs. highest dose

strength

• Tolerability

• Analytical sensitivity


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Lack of Interaction with Internal Standards

• Phase 1A:

– Test compound codosed

with controls

• Phase 1B:

– Test compound dosed alone

•

Does the test compound impact

permeability of internal standards

and vice versa?


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Papp 33.6

Minoxi

dil

4.33

Atenol

ol

0.26

Efflux

Ratio

1.1

Recove

ry

89%

Extensive Metabolism, High

Variability

• Rapid absorption

• Extensive metabolism

– 29-63% in urine

– 18-38% in feces

• Plasma concentrations of unchanged drug very

low and highly variable (Cmax 1-6 ng/mL, 40-90

min)


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Stability

• Phase 1 and Phase 2 Metabolism

• SGF/SIF

• Pro-drugs

• Additional matrices may be required

• Intestinal brush border membrane

//upload.wikimedia.org/wikipedia/commons/0/0f/Normal_Villus_Illustration.png


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Acceptance of In Vitro Permeability Techniques


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Compound Indication FDA Product Recommendation

Tested at Absorption

Systems (AS) Pemeability Classification at AS (High, Low or Not Performed)

BCS Classification by AS Based on Permeability in

Caco-2

BCS Classification based on human PK data or Mass

Balance

Buspirone Anxiety Buspirone Recommendation Yes High Class I Class I

Capecitabine Colorectal and breast cancers Capecitabine Recommendation Yes High Class I Class I

Cefadroxil Hemihydrate Antibiotic Cefadroxil Recommendation No NP NP Class I

Donepezil HCl Alzheimer-related dementia Donepezil Recommendation Yes High Class I Class I

Emtricitabine# HIV Emtricitabine Recommendation No NP NP Class I

Galantamine HBr Alzheimer’s dementia Galantamine Recommendation Yes High Class I Class I

Granisetron HCl Antiemetic Granisetron Recommendation Yes High Class I Class I

Levetiracetam Epileptic seizures Levetiracetam Recommendation Yes High Class I Class I

Levofloxacin Antibiotic Levofloxacin Recommendation Yes High Class I Class I

Linezolid Antibiotic Linezolid Recommendation Yes High Class I Class I

Memantine HCl Alzheimer-related dementia Memantine Recommendation Yes High Class I Class I

Milnacipran HCl Fibromyalgia Milnacipran Recommendation Yes Low* Class III* Class I

Palonosetron HCl Chemotherapy-induced nausea Palonosetron Recommendation Yes High Class I Class I

Pramipexole Dihydrochloride Parkinson’s disease Pramipexole Recommendation Yes High Class I Class I

Pregabalin Epileptic seizures Pregabalin Recommendation Yes Low** Class III ** Class I

Propranolol HCl Hypertension, tremors, migraine Propranolol Recommendation Yes High Class I Class I

Protriptyline HCl Depression Protriptyline Recommendation Yes High Class I Class I

Ramelteon Insomnia Ramelteon Recommendation Yes High Class I Class I

Rivastigmine Tartrate Alzheimer-related dementia Rivastigmine Recommendation Yes High Class I Class I

Sotalol HCl Cardiac arrhythmia Sotalol Recommendation Yes Low*** Class III *** Class I

Tapentadol HCl Pain Tapentadol Recommendation Yes High Class I Class I

Temozolomide Glioblastoma Temozolomide Recommendation Yes High Class I Class I

Tiagabine HCl Epileptic seizures Tiagabine Recommendation Yes High Class I Class I

Varenicline Tartrate Smoking cessation Varenicline Recommendation Yes High Class I Class I

Venlafaxine HCl Depression Venlafaxine Recommendation Yes High Class I Class I

23 of the 25 have been tested at Absorption Systems
































































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Minoxidil C

ac

o-2

Pap

p (

x 1

0-6

cm

/s)

Antipyrine: Papp ~63

Metoprolol: Papp ~28

Pindolol: Papp ~17

Minoxidil: Papp ~4

Papp Recove

ry

Test

Compound

11.8 92.5%

Pindolol 13.9 96.1%

Atenolol 0.21 93.1%

Pindolol is Inadequate Standard

Biowaiver Recommendation in IRP

Absolute BA is 98%

• pH 7.4


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Why Minoxidil?

• At least 95% fraction

absorbed

• Passive transport

• No known food effects

or DDI

• No pH effect

How do you justify using minoxidil

as your high permeability internal

standard?


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Multiple Validated pH Levels


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Absorption: Solubility and Permeability

J = D ∆C

∆x

J= drug flux (mass/area/time)

P= membrane permeability

D = diffusion coefficient

∆x = width of membrane

k = partition coefficient

C = concentration available for transport

P = k • D

∆x

= Co • P

Absorption

Solubility Permeability


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Question… • For a rapidly dissolving IR product with

High permeability

• High solubility

Adequate stability

• Will absorption always be complete?


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• Direct

• Human intestinal perfusion

Methods for Permeability Classification

• Indirect

• PK: Absolute BA/Mass

Balance

• Rat intestinal perfusion

• Epithelial cell monolayers

• Excised human or animal

intestinal tissues

estudios de permeabilidad en - as-lat.com€¦ · estudios de permeabilidad en cÉlulas caco-2...

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