Slide1 l.jpg
This presentation is the property of its rightful owner.
Sponsored Links
1 / 29

oral absorption, brain uptake pharmacokinetic and metabolic properties PowerPoint PPT Presentation


  • 159 Views
  • Uploaded on
  • Presentation posted in: General

A NEW IN-VITRO MODEL TO PREDICT THE IN VIVO BEHAVIOR OF DRUGS BASED ON PARALLEL ARTIFICIAL MEMBRANE AND PLASMA PROTEIN BINDING. Roberto Bozic October 27-29, 2008. INTRODUCTION. Plasma Protein Binding: is a reversible association of a drug with the proteins of the plasma compartment of blood.

Download Presentation

oral absorption, brain uptake pharmacokinetic and metabolic properties

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Slide1 l.jpg

A NEW IN-VITRO MODEL TO PREDICT THE IN VIVO BEHAVIOR OF DRUGS BASED ON PARALLEL ARTIFICIAL MEMBRANE AND PLASMA PROTEIN BINDING.Roberto BozicOctober 27-29, 2008


Slide2 l.jpg

INTRODUCTION

Plasma Protein Binding:

is a reversible association of a drug with the proteins of the plasma compartment of blood.

Albumin is the major component of plasma proteins.

Lipophilicity :

is expressed as partition or distribution coefficient (Log P or Log D) between octanol/aqueous phases. It is an important physicochemical parameterinfluencing :

  • oral absorption,

  • brain uptake

  • pharmacokinetic and metabolic properties

PPB has influence on :

  • determination of margin in safety assessment/toxicology studies

  • the efficacy of a drug

  • drug metabolism and pharmacokinetics

  • drug-drug interactions (low influence)

  • blood-brain barrier penetration


Introduction l.jpg

Lipophilicity :

Shake flask (96 well plate)

RP-HPLC techniques

IAM (immobilized artificial membrane chromatography)

Liposome chromatography

pH-metric technique

Plasma Protein Binding:

Equilibrium dialysis (gold std method)

Ultrafiltration

Ultracentrifugation

Chromatographic techniques (immobilized-albumin support coupled with HPLC)

INTRODUCTION

Experimental methods:


Pampa basics l.jpg

Acceptor

Membrane

Donor

PAMPA Basics

ASSEMBLY

DIFFUSION PROCESS

Acceptor

Lipid membrane

Filter Support

Donor

PAMPA (Parallel Artificial Membrane Permeability Assay)


Slide5 l.jpg

PURPOSE

  • to develop an in vitro model to investigate the influence of a lipidic membrane on the protein binding of drugs, and to obtain a rank ordering of them.

  • To develop, compatibly with the needs of the modern drug discovery process, a highly automated process allowing the rapid turnaround of in vitro data using appropriate analytical, MS-based, methods to assess widely diverse compounds.


Assay principle l.jpg

Drug + HSA

Drug-HSA

Lipid membrane

Drug

Filter Support

ASSAY PRINCIPLE

  • Assay based on the 96-well plate format.

  • Human Serum Albumin (at physiological concentration, 600uM or 43 g/l in HBSS buffer pH=7.4);

  • it is not immobilized on any surface.

  • - Hydrophobic filter membrane impregnated with 15% soy lecithin in n-dodecane.


Sample preparation l.jpg

SAMPLE PREPARATION

Solution 10 mM in DMSO of drug

Dilution with HSA solution

(4.3% of HSA in HBSS buffer with 25 mM Hepes, pH=7.4)

Solution 10 μM of drug in HSA

Incubation for 3 h, at 37 °C, under shaking.

The solution was placed in a filter plate coated with a lipidic membrane (15% soy lecithin in n-dodecane)

An aliquot of this solution was collected at different time (range time profile: t= 0÷20 h)

It was purified by protein precipitation with MeCN

Vortex 30’’

Centrifugation 3700 rpm, 15’

LC-MS/MS analysis


How much does lipidic membrane affect ppb l.jpg

How much does lipidic membrane affect PPB?

Distribution kinetics of warfarine and propranolol.

propranolo

warfarin

(*) J. G. Hardman, L. E. Limbird, A. G. Gilman. Book “Goodman & Gilman'sThe Pharmacological Basis of Therapeutics”,9th ed. (1996).

(**) data obtained experimentally by PAMPA assay


Lc ms ms equipments l.jpg

LC-MS/MS EQUIPMENTS


Analytical procedure l.jpg

ANALYTICAL PROCEDURE

ANALYTICAL PROCEDURE

MASS PARAMETERS

OPTIMIZATION

SAMPLES ANALYSIS

BY LC-MS/MS

  • FIA (Flow injection Analysis)

  • Software: Automaton version 1.3 (PE Sciex)

  • run time: 1 min (the optimization of the mass parameters requires three consecutive runs, 3.0 min, for each compound)

  • Inj. Vol.: 20 uL

  • Isocratic Conditions (FIA): mobile phase 20%A / 80% B

  • flow rate: 200µl/min

  • Analytical Guard Column SB-C8 4.6 x 12.5 mm, 5-Micron (Zorbax - Agilent Technologies)

  • Software: Analyst version 1.4.1 (PE Sciex)

  • Inj. Vol.: 20 µL

  • Gradient Conditions:


Mass parameters optimizated l.jpg

MASS PARAMETERS OPTIMIZATED


Method validation precision l.jpg

Method validation: precision

Nicardipine

Intra-Assay

Inter-Assay


Compound selection l.jpg

COMPOUND SELECTION

The model has been applied to 11 commercial drugs:

  • with high Plasma Protein Binding

    PPB data taken from literature source (*)

  • with high membrane retention

    data obtained experimentally by PAMPA assay(Automated Parallel Artificial Membrane Permeability Assay)

  • Solubility @ pH=7 > 30 uM

    solubility data obtained experimentally

(*) J. G. Hardman, L. E. Limbird, A. G. Gilman. Book “Goodman & Gilman'sThe Pharmacological Basis of Therapeutics”, 9th ed. (1996).


Compound selection14 l.jpg

COMPOUND SELECTION

(*) J. G. Hardman, L. E. Limbird, A. G. Gilman. Book “Goodman & Gilman'sThe Pharmacological Basis of Therapeutics”,9th ed. (1996).

(**) data obtained experimentally


Non specific binding evaluation l.jpg

NON-SPECIFIC BINDING EVALUATION

  • The non-specific binding was investigated using the same

  • procedure above described but in absence of lipidic membrane.

  • Results show a negligible influence of non-specific binding.


Results and discussion l.jpg

RESULTS AND DISCUSSION

Distribution kinetics of 11 commercial drugs.

  • 11 commercial drugs: with high PPB and high

  • membrane retention.

  • These kinetic profiles show a different

  • behavior of these 11 compounds and allowed

  • their rank ordering.


Results and discussion17 l.jpg

RESULTS AND DISCUSSION

Distribution kinetics, comparison between acids, neutral and bases compounds.

  • - Acids showed a stronger protein binding than neutral or basic compounds and a lower trend to distribute

  • on lipidic membrane.

  • Neutral compound showed a higher trend to protein binding than bases.

  • - Bases resulted more absorbed on lipidic membrane

(a) Taken from ref 12. (b)predicted by ACDLABS. (c) Dominant species at physiological pH: n=neutral or anpholite, a=anion and c=cation.


Conclusions l.jpg

Conclusions

  • A LC-MS/MS-based medium throughput method has been development.

  • A good precision, compatible with drug discovery needs, was showed.

  • The model was able to rank order compounds with similar properties

  • in term of PPB and Lipophilicity.

Next steps:

  • To try out basic compounds in plasma (instead of HSA), to evaluate the contribution of 1-acid glycoprotein on bases protein binding.

  • To estimate the correlation between these data and pharmacokinetics properties of compounds.

  • Further development of the method in terms of high throughput, particularly automation of sample preparation.


References l.jpg

REFERENCES

(1)Kerns EH. J. Pharm. Sci. 2001, 90, 1838-1858

(2)Borchardt RT, Kerns EH, Lipinski CA, Thakker DR, Wang B. Book “PharmaceuticalProfiling in Drug Discovery for Lead Selection”, AAPS PRESS, Arington, VA, 2004, pp 127-182.

(3)Caron G, Ermondi G, Lorenti M. J. Med. Chem. 2004, 47, 3949-3961.

(4)Testa B, Crivori P, Reinst M, Carrupt PA. Book “The influence of Lipophilicity on the Pharmacokinetics Behaviour of Drugs: Concepts and Examples”. Kluvert Academic Publisher: Norwell, MA, 2000, pp 179-211.

(5)Borchardt RT, Kerns EH, Lipinski CA, Thakker DR, Wang B. Book “Pharmaceutical Profiling in Drug Discovery for Lead Selection”, AAPS PRESS, Arington, VA, 2004, pp 327-336.

(6)Testa , Kramer SD, Wunderli-Allespach H, Folkers G (Eds.). Book “Pharmacokinetic Profiling in Drug Research”, VHCA, Zurich, 2006, pp 119-141.

(7)Testa , Kramer SD, Wunderli-Allespach H, Folkers G (Eds.). Book “Pharmacokinetic Profiling in Drug Research”, VHCA, Zurich, 2006, pp 165-202.

(8)Banker MJ, Clark TH, Williams JA, J. Pharm. Sci. 2003, 92, 967-974.

(9)Schuhmecher J, Buhner K, Witt-laido J Pharm Sci, 2000, 89, 1008-1021.

(10)Loidl-Stahlhofen A, Hartmann T, Schottner M, Rohring C, Brodowsky H, Schmitt J, Keldenich J. Pharmaceutical Research, 2001, 18, 12, 1782-1788.

(11)Elisabet Lazaro, Philip J. Lowe, Xavier Briand, Bernard Faller. J. Med. Chem. 2008, 51, 2009-2017.

(12)Avdeef A., Book “Absorption and Drug Development”, Wiley-Interscience, a John Wiley & Sons, Inc., Publication, 2003.

(13)Joel Griffith Hardman, Lee E. Limbird, Alfred G. Gilman. Book “Goodman & Gilman'sThe Pharmacological Basis of Therapeutics”, 9th ed. (1996).


Back up slides l.jpg

Back-up slides


Slide21 l.jpg

SUMMARY

  • Setup of analytical method

  • Setup of sample preparation conditions

  • Method validation (reference compounds, reproducibility)

  • Non-specific binding evaluation

  • Method application on commercial drugs having high PPB and high membrane retention

  • Method application on acidic, basic and neutral compounds


Assay principle22 l.jpg

Drug + HSA

Drug-HSA

ASSAY PRINCIPLE

-----------------------------------------------------------Membrane

Drug


Method validation precision23 l.jpg

Method validation: precision

Clozapine

Intra-Assay

Inter-Assay


Pampa parallel artificial membrane permeability assay l.jpg

Acceptor chamber

Artificial Membrane on Filter

Donor chamber

PAMPA* (Parallel Artificial Membrane Permeability Assay)

* Kansy M et al. J. Med Chem, 1998, 41, 1007-1009


Pampa l.jpg

PAMPA

Low Membrane Retention

High Membrane Retention


Calcoli pampa l.jpg

Calcoli PAMPA

Il calcolo della permeabilità si basa su una versione modificata della seguente equazione11:

M = è la quantità totale di farmaco nel sistema meno la quantità di campione persa nella membrana.

CA (t) = è la concentrazione di farmaco nella cella accetrice al tempo t

CA (0) = è la concentrazione di farmaco nella cella accetrice al tempo 0

VA = è il volume della cella accetrice

VD = è il volume della cella donatrice

Pe = è la permeabilità effettiva

A = è l’area del filtro

t = è il tempo di permeazione

La ritenzione percentuale in membrana, %R, è calcolata mediante la seguente equazione:

M = è la quantità di farmaco in D (Donor), A (Acceptor) al tempo (0) e alla fine dell’esperimento (t).


P ion vs in house model l.jpg

P-ION vs In House Model


Simple model for passive diffusion l.jpg

Simple model for Passive Diffusion

SpH

Least soluble

Most soluble

Unionized drug molecule

Ionized drug molecule

pKa / pH

LogP

Least lipophilic, least permeable

Most lipophilic,

most permeable


Advantages disadvantages of pampa l.jpg

Advantages

Direct measurement of passive diffusion (Clean number)

Easy to set up

Higher Throughput

Highly Reproducible

Non cell based assay (less labor intensive)

Another Partition Coefficient (membrane retention)

Data more directly amenable to in silico modeling

Disadvantages

No passive paracellular info

No active uptake

Advantages & Disadvantages of PAMPA


  • Login