Prediction of Oral Absorption: 6-Fluoroquinolonesmbermejo/Garmisch01.pdf · STUDY OF THE INTESTINAL ABSORPTION OF CIPROFLOXACIN IN RATS. Rodríguez M et al. 1999 AAPS annual meeting

1

March, 20011

Prediction of Oral Absorption: 6-Fluoroquinolones

Mª del Val Bermejo Sanz, Ph.D.Depto. Farmacia y Tecnología Farmacéutica

Facultad de Farmacia. Universidad de ValenciaValencia, España

Strategies for Oral Drug DeliveryOral Absorption Regulation and Evaluation

March 11-16, 2001 Garmisch Partenkirchen, Germany

March, 20012

Prediction of Oral Absorption: 6-Fluoroquinolones

“Bioavailability prediction in drug development: fluoroquinolones.” CICYT (SAF 96-1710)

Director: Prof. JM Plá Delfina

•Absorption-partition relationships

•Bioavailability predictions

2

March, 20013

Absorption-Partition Correlations

ka1

membrane

ka

Aqueous layer

lumen

Plasma

ka2aqueous pathway

ka

P

da

a

PCCkp

PBPkm

Ka+

⋅++⋅=

membrane

ka

Aqueous layer

lumen

Plasma

P

ka

a

a

PBPkm

Ka+⋅=

J. Pharmacokin. Biopharm. 14(6),615-33 (1986)J. Pharmacokin. Biopharm. 15(6),633-43 (1987)

March, 20014


P

ka

dPME

dPCKa

⋅⋅+

⋅=

1

Higuchi- Ho

membrane

ka

Aqueous layer

lumen

Plasma

kmemkaqka111 +=

• Correlations in Colon or

• in Small intestine with Compounds MW>250

Journal of Pharmaceutical Sciences 84, 777-782 (1995).

3

March, 20015


F

N

O

COOH

N

NR

F

N

O

COOH

N

NR

R= H, Norfloxacin

R= H, Cyprofloxacin

R= CH3 , CH3-(CH2 )n - n=1-5

Homologous CompoundsCENAVISA S.A. Spain

N

F

NN

CH3

R

COOHO

CNV 97104-(CH2)3-CH3

CNV 97103-(CH2)2-CH3

CNV 97102-CH2-CH3

CNV 97101-CH3

CNV 97100H

NameR

March, 20016

F

N

O

COOH

OCF

N

O

O H

N

O

OCF

N

O

O H

N

S

CENAVISA 8804 CENAVISA 8919

Flumequine


HN

N N

O

F

F

NH2 O

OH

HN

NN

O

F

F

O

O H

FSparfloxacin Sarafloxacin

4

March, 20017

N-octanol

buffer

Saturation

Quinolonesolution

Aliquot

24 h 50 r.p.m

22ºCSplit

A. Ruiz ©


March, 20018

Partition Coefficient:

Organic Phase: n-Octanol

Aqueous Phase: aqueous buffer solution at pH. 7.00

Phase Volume adjusted for each compound

•Saturation

•Dissolution of quinolone in aqueous phase

•Equilibrating during 24 h at 22ºC

•Analysis of the aqueous concentration by HPLC


( )aaf

oafai

a

oV/Q

V/QQCCP −==

5

March, 20019

Absorption studies:

•Solutions of compounds on saline buffered at pH. 7.00

•Concentration of each compound far enough of its Cs

•Perfused Volume: 10 mL


March, 200110


Closed Loop Perfussion Technique: Doluisio’s Method

Cannula

Stopcock

Syringe

Gut

Samples•Whole small intestine

•Bile ductclosed•Water reabsorption measurement

6

March, 200111



SamplesSamplesSamplesSamplesSamplesSamples

March, 200112



SamplesSamplesSamples

7

March, 200113



March, 200114


0

0

VV

AA

tkVV

tc

t

⋅=

⋅−=

exp

Water reabsorption correction

8

March, 200115


Absorption rate constant:

ka, mean value of six animals

tkaeAA ⋅−⋅= 0

time

LnA

March, 200116

Absorption-Partition CorrelationsAnalysis: HPLC fluorimetric detection

Method validation

Stability of compound in luminal fluid

9

March, 200117


0.01 0.1 1 10 100 1000 100000

1

2

3

4

5

6

7

0.01 0.1 1 10 100 1000 100000

1

2

3

4

5

6

7

Norfloxacin homologous Cyprofloxacin homologous

Partition coefficient, PPartition coefficient, P

Abs

orpt

ion

rate

cons

tant

h-1

Abs

orpt

ion

rate

cons

tant

h-1


March, 200118

Absorption-Partition CorrelationsKaq (h-1) Kmem (h -1)

7.073 0.1956.944 0.3976.923 3.3226.801 4.0706.782 4.7956.667 6.2106.649 14.8136.541 15.3596.524 30.2676.422 33.7616.405 79.7016.309 85.3876.293 181.0756.201 233.9916.186 389.1646.099 545.0160.01 0.1 1 10 100 1000 10000

0.01

0.1

1

10

100

1000

Abs

orpt

ion

rate

cons

tant

h-1

Partition coefficient, P

kaq

kmem

kmemkaqka111 +=

10

March, 200119


Compound MIC90 Compound MIC90Norfloxacin 0.1 Ciprofloxacin 0.025N’-Methylnorfloxacin 0.1 N’-Methylciprofloxacin 0.025N’-Ethylnorfloxacin 0.2 N’-Ethylciprofloxacin 0.05N’-Propylnorfloxacin 0.4 N’-Propylciprofloxacin 0.1N’-Butylnorfloxacin 0.4 N’-Butylciprofloxacin 0.2N’-Pentylnorfloxacin 0.4 N’-Pentylciprofloxacin 0.2N’-Hexylnorfloxacin 1.6 N’-Hexylciprofloxacin 0.4N’-Heptylnorfloxacin 3.2 N’-Heptylciprofloxacin 0.4

MIC90 of the compounds against E. Coli ATCC 25922


March, 200120


Coeficiente de reparto en n-Octanol

1e-3 1e-2 1e-1 1e+0 1e+1 1e+2 1e+3 1e+4 1e+5 1e+6

Con

stan

te d

e V

eloc

idad

de

Abs

orci

ón, k

a (h

-1)

0

2

4

6

8

CNV 97102

CNV 97101

CNV 97104

CNV 97103

CNV 97100

Wagner-Sedman Fit

Higuchi-Ho Fit

Ana Ruiz-García. Ph.D Dissertation. University of Valencia. November 2000

11

March, 200121

Quinolone ka-pred HH ka exp. ER%97100 1.06 1.24 -14.997101 1.91 3.54 -46.097102 3.06 3.12 -2.197103 4.35 4.5 -3.297104 5.20 5.16 0.8


Ana Ruiz-García. Ph.D Dissertation. University of Valencia. November 2000

March, 200122

Journal of Pharmaceutical Sciences (4) 398-405, 1998.


n-Octanol Partition Coefficient

1x10-3 10x10-3 100x10 -3 1x100 10x100 100x100 1x103 10x103 100x103 1x106

Pef

f (c

m/s

)

0.00

5.00e-5

1.00e-4

1.50e-4

2.00e-4

CNV 97100

Flumequine

CNV 8804CNV 8919

Norfloxacin derivatives

Ciprofloxacin derivatives

12

March, 200123

CH3-COOH

CH3-CH2-CH2-CH3

CH3-CH-CH3

CH3

1

2

3

FO-A1-B11-C1

A2 B12Traze

Vector A1 B11 C1B12

0 1 0 0 0

A2

1 0 1 0 1

1 0 1 1 0

Absorption-Partition Correlations : QSAR methodologies in drug absorption

Vizet P. PATQSAR©. Version 2.55. 1997

Instituto de Topología y Dinámica de Sistemas ITODYS. Université Paris VII. France


March, 200124

C1

C2

C3

C4C6

C5C7

N N

O

COOHF

N

CH2

S N

O N

CH30.00

1.82

0.27

0.54

F0 : 0.96

-2.56

0.00

0.81

0.54

C0

*C1 : 0.00

0.54

0.54

0.54

0.54

Lipophilicity -structure correlation



13

March, 200125

C1

C2

C3

C4C6

C5C7

N N

O

COOHF

N

CH2

S N

O N

CH30.35

0.64

0.140.140.00

0.00 0.00 0.14

F0 : 0.82

-1.13

0.00

0.00

0.06

C0

Absorption-structure correlation



March, 200126

C1

C2

C3

C4

C5

C6

C7Fo : 1.03

0.00

CH2

N

F

NN

O

COOH

-0.28 -0.28

-0.28

-0.28

0.00 -0.28

0.56

C1

C2

C3

C4

C5

C6

C7Fo : 0.33

0.00

CH2

N

F

NN

O

COOH

-0.28

-0.28

-0.28

-0.28

-0.28

0.28

0.00

Structure- antibacterial activity correlation



14

March, 200127


Concentration (µg/ml)

0.05 0.5 5 50 500

Ka

(h-1

)

0

1

2

CNV 97 100

Gonzalez I. et al.. V SEFIG meeting, Valencia 2001

Conc.(µg/mL) ka(h-1) DE500 1.215 0.10750 1.081 0.1105 0.757 0.050

0.5 0.523 0.0400.05 0.242 0.010

0.5+V 1.114 0.113

Conc(µg/mL) 500 50 5 0.5 0.05 0.5+V

500 - - - - - -50 NS - - - - -5 S S - - - -

0.5 S S S - - -0.05 S S S S - -

0.5+V NS NS S S S -

March, 200128

n-Octanol partition coefficient, P1e-3 1e-2 1e-1 1e+0 1e+1 1e+2 1e+3 1e+4 1e+5 1e+6

ka

in s

itu

(h-1

)

0

2

4

6

8

Sarafloxacin

Sparfloxacin


Fernández C. et al. V SEFIG meeting, Valencia 2001

15

March, 200129


Sarafloxacin

Fernández C. et al. V SEFIG meeting, Valencia 2001

Concentration (µg/ml)

0.5 1 5 25 50

Ka

(h-1

)

0.0

0.2

0.4

0.6

0.8

1.0

Conc(µg/mL) ka(h-1) DE50 0.399 0.04425 0.512 0.0165 0.513 0.0141 0.789 0.025

0.5 0.816 0.026

Conc(µg/mL)

50 25 5 1 0.5

50 - - - - -25 S - - - -5 S NS - - -1 S S S - -

0.5 S S S NS -

March, 200130

STUDY OF THE INTESTINAL ABSORPTION OF CIPROFLOXACIN IN RATS.

Rodríguez M et al. 1999 AAPS annual meeting

0.0

.1

.2

.3

.4

.5

.6

.7

.8

.9

1.0

0.5 5 500.5+V

Ciprofloxacin concentration (µg/mL)

Absorption rate constant, ka, h

-1

n=6

n=6

n=6n=9

Figure 1

Time (min)0 5 10 15 20 25 30

Concentration in lum

en (µg/m

L)

0

1

2absence of V presence of V

Figure 2

16

March, 200131

INFLUENCE OF P-GLYCOPROTEIN ON THE INTESTINAL ABSORPTION OF GREPAFLOXACIN.

Sánchez-Castaño G. et al. 1999 AAPS annual meeting Grepafloxacin donated by GLAXO

Absorption rate constant (ka, h

-1

0.0

.5

1.0

1.5

2.0

2.50.3 mg/mL Grepafloxacin0.01 mg/mL Grepafloxacin0.01mg/mL Grepafloxacin+ 0.2 mM Verapamil

Whole intestine

Proximal fraction

Mediumfraction

Distalfraction

n=6

*

*

* Statistical differences

Figure 1

March, 200132

Bioavailability prediction in drug development

)()( hg EEFaF −⋅−⋅= 11Eg= gut or liver first pass effects

Eh= biliary excretion

17

March, 200133


TPMC

PB

a

Tkaa

a

a

eF

eF

⋅

+⋅+⋅

−

⋅−

−=

−=

11

1

March, 200134


jugularVein

Blood Samples

Administration(I.V.)

Cannula

Oral Administration

18

March, 200135


Oral administration

Group A (8-12 rats)

I.V administration

(bolus or perfusion)

Group B (8-12 rats)

Plasma sampling and

Analysis

AUC0∞

calculation

AUC0t + AUCt

∞

(Trapezoidal rule) ( Ct/k)

March, 200136

Time (min)

0 100 200 300 400

Norfloxacin

plasm

a levels (mg

/L)

0.01

0.1

1

10

100Div=2 mgDoral=4 mg

(1)

19

March, 200137

Time (min)

0 100 200 300 400

Cip

roflo

xacin p

lasma levels (m

g/L

) 0.01

0.1

1

10

100DiV=4 mgDoral=4 mg

March, 200138

Time (min)

0 100 200 300 400

4N'-

Pro

pyl-ciprofloxacin plasm

a levels(mg/L)

0.01

0.1

1

10

100D

iV=4 mg

Doral

=4 mg(3)

20

March, 200139

Time (min)

0 100 200 300 400 500 600

Peflo

xacinp

lasma levels (m

g/L

)

0.01

0.1

1

10

100DiV=8 mgDoral=8 mg

(5)

March, 200140

Substituents in the formula

Molecular

Weight

(M)

P (n-octanol) ka (h-1 ) in situTested

quinolone

R1 R2 R3

Norfloxacin Ethyl H H 319 0.028±0.002 0.42±0.07

Pefloxacin Ethyl Methyl H 333 1.883±0.050 1.92±0.18

4N’-Propyl-

norfloxacinEthyl Propyl H 361 11.27±0.27 4.07±0.40

Ciprofloxacin Cyclopropyl H H 331 0.075±0.003 0.63±0.08

4N’-Propyl-

ciprofloxacinCyclopropyl Propyl H 373 11.80±0.20 4.80±0.52

3’-Methyl-

ciprofloxacinCyclopropyl H Methyl 345 0.209±0.001 1.24±0.07

FlumequineOccupied by

a 1-8 ring

Without 7N-piperazinyl

substituent261 9.40±0.90 6.87±1.09

N N

F

N

O

COOH

R1

R2

R3

21

March, 200141

ka in situ

0 1 2 3 4 5 6 7 8

F in

viv

o

0.2

0.4

0.6

0.8

1.0

1.2

Norfloxacin

Ciprofloxacin

CNV97100

Pefloxacin

CNV97102

Propil-Nor

Propil-Cipro

CNV97104

Flumequine

Journal of Pharmaceutical Sciences. 89(11)1395-1403(2000)European Journal of Biopharmaceutics andPharmacokinetics. 48,253-258 (1999).Ana Ruiz-García. Ph.D Dissertation. University of Valencia. November 2000

March, 200142

Pre

dict

ed F

Correlation with ka values

Experimental F

0.0 0.2 0.4 0.6 0.8 1.0 1.20.0

0.2

0.4

0.6

0.8

1.0

1.2

Pre

dict

ed F


22

March, 200143

Experimental F

0.0 0.2 0.4 0.6 0.8 1.0 1.2

Pre

dict

ed F

0.0

0.2

0.4

0.6

0.8

1.0

1.2Predictions from Higuchi-Ho eq.

1.2 TaPMC

aPB

a eF⋅

+⋅+

⋅−−= 11

P- Lipophilicity

1e-3 1e-2 1e-1 1e+0 1e+1 1e+2 1e+3 1e+4 1e+5 1e+6

ka

(h-1

)

0

2

4

6

8


March, 200144

Conclusions

1. Lipophilicity seems to be the main factor governing quinolone absorption

2. A Partition coefficient in n-Octanol over 10 produces an absorption constant ~ 2 h-1 Peff ~ 1* 10-4 cm/s

3. Quinolones with a in situ permeability over 2*10-4 cm/s have a bioavailability aprox. 90%.

23

March, 200145

Conclusions

4. The absorption-partition relationship based on Higuchi-Ho equation has a good predictive performance at least with homologous quinolones; for heterologues compounds, the model is still able to predict the partition coefficient for maximum absorption.

5. The absorption rate constant ka determined in situ in rat is a good predictive parameter of oral bioavailability for the studied compounds.

6. The efflux processes observed in situ and in vitro have not a significant influence in vivo in rats at the oral doses used.

March, 200146

Work in progress

1. In vitro Permeability studies (Caco-2):

•Double Log-linear relationship Peff in situ-Peff in vitro.

•Efflux process for some quinolones.

2. In situ absorption experiments of 97100 and 97101 at different concentrations: apparent active mechanism?

3. In vivo relevance of 97100 efflux.

4. In vitro metabolism of 97103.

24

March, 200147

Research Team

•Ma del Val Bermejo Sanz. Ph.D.

•Teresa Garrigues Pelufo. Ph.D.

•Virginia Merino Sanjuán. Ph.D.

•Gloria Sánchez Castaño Ph.D

•Ana Ruíz García Ph.D

Chairman: José M. Plá-Delfina. Ph. D.

Professors Teaching assistants

Ph.D candidates

Carlos Fernández

Isabel González

Ricardo Nalda

Margarita Rodríguez

Documents

Prediction of Oral Absorption: 6-Fluoroquinolonesmbermejo/Garmisch01.pdf · STUDY OF THE INTESTINAL ABSORPTION OF CIPROFLOXACIN IN RATS. Rodríguez M et al. 1999 AAPS annual meeting