Alf Lamprecht Laboratory of Pharmaceutical Engineering (EA3924), University of Franche-Comté,...

Alf Lamprecht

Laboratory of Pharmaceutical Engineering (EA3924),University of Franche-Comté, Besançon, France

Contact: [email protected]

Nanoparticulate drug delivery systems

for different therapeutic applications

“Le Menu”

A. Nanoparticles for the absorption of drugs with low bioavailability (oral heparin)

B. Nanoparticles for the selective drug targeting (inflammatory bowel disease)

C. Nanocapsules for intracellular drug therapy (cancer)

drug intestinal barrier organs cells, receptors

B CA

Nanoparticles fororal delivery of macromolecules

Objective:

enhanced absorption of the drugtransport across biological barrier

avoidance of repetitive injections

polymeric nanoparticles and theirtherapeutic applications

Heparin

- anionic glycosaminoglycans- treatment and prophylaxis

of venous thrombosis

Nanoparticles for oral absorption

• parenteral administration• no oral absorption

do not pass the intestinal barrier

NANOPARTICLES:

• drug entrapment (protection)• emulsion techniques• biocompatible polymers • diameter: 300-500nm

FormulationsFormulationsCmax Cmax

(s)(s)Tmax (h)Tmax (h)

AUC/KgAUC/Kg

(s.h/Kg)(s.h/Kg)

Absolute FAbsolute F

(%)(%)

RL/PCLRL/PCL 20-2720-27 66 28.3 ± 8.228.3 ± 8.2 42.1 ± 12.342.1 ± 12.3

RS/RL/PLGARS/RL/PLGA 20-2420-24 4-54-5 29.4 ± 12.629.4 ± 12.6 43.9 ± 18.843.9 ± 18.8

RS/PLGARS/PLGA 22-2722-27 6-76-7 21.9 ± 18.421.9 ± 18.4 32.7 ± 27.432.7 ± 27.4

RLRL 19-2719-27 3-63-6 26.3 ± 12.326.3 ± 12.3 39.2 ± 18.339.2 ± 18.3

IV UFH solutionIV UFH solution NDND NDND 67.1 ± 29.267.1 ± 29.2 100100

Pharmacokinetic parameters after oral administration of UFH-loaded NP (2000 IU) in rabbits (n=6)


Cell culture model for adsorption studies:

1. Absorption barrier (cells)

2. Mucus ???

no influence on absorption kinetics of small drugsbut macromolecules ???

adhesion studies on the cells, but adhesion to the mucus ???

mucoadhesion is the starting point!!!


0.01 0.1 1 100

500

1000

1500 blank RS-NP, x0=0.47

RS/PLG-NP, x0=1.66

PLG-NP, x0= /

resp

onse

[arc

sec

onds

]

NP conc. [mgpolymer

/ml]

0.01 0.1 1 100

50

100

150

+ LMWH RS-NP, x0=1.14

RS/PLG-NP, x0=1.49

PLG-NP, x0= /

resp

onse

[arc

sec

onds

]

NP conc. [mgpolymer

/ml]

resonant mirror system:NP adhesion differs with surface charge


Aqueous boundary layer (ca. 40 µm)

Epithelialcells

Basolateral compartment

Mucuslayer

• mucin coated Caco-2 cells• confluent but not homogeneous mucin layer• stained with astra blue


1.563 0.781 0.391 0.195 0.0980

5

10

15

20

254h - mucin

bin

din

g [

%]

NP conc. [mg/ml]

RS+LMWH RS/PLGA+LMWH PLGA+LMWH LMWH

• cationic NP adhesion• LMWH adhesion correlates!!!

0.01 0.1 1 100

5

10

15

20 RS RS/PLGA PLGA RS+LMWH RS/PLGA+LMWH PLGA+LMWH

part

icle

adh

esio

n [%

]

NP conc. [mg/ml]


0.1 1 10

2

4

6

8

fluo 4h: 1.71±0.48%fluo 24h: 2.18±0.51%

fluorescein RS 4h RS/PLGA 4h PLGA 4h RS 24h RS/PLGA 24h PLGA 24h

% d

rug

tran

spor

ted

NP [mg/ml]0.1 1 10

2

4

6

8

LMWH 4h: 0.29±0.18%LMWH 24h: 0.40±0.09%

LMWH RS 4h RS/PLGA 4h PLGA 4h RS 24h RS/PLGA 24h PLGA 24h

% d

rug

tran

spor

ted

NP [mg/ml]

LMWH transport across mucin coated Caco-2 cell layers


Conclusion

Particle adhesion to Caco-2 cells

Objective:

local drug delivery in IBD treatmenttargeting on tissue level

lower adverse effects by a selective drug release

Nanoparticle targetingin inflammatory bowel disease

Inflammatory bowel disease

• chronic, relapsing inflammation of the colon• unknown etiology• swelling and ulceration of mucosa and submucosa

therapeutic principles:• induce remission of outbreaks• prevent outbreaks during remission

drug treatmentoral (standard)a) rectal (limited use)b) intravenous (recent)

surgery ulcerative colitis Crohn’s disease

Histology of the inflamed colonic mucosa(TNBS-model for the rat)

healthy control colitis group increasesmucus production

colitis group withulcerated tissue

Nanoparticles in colitis

Localization of 100nm particles in the colon

TNBS intrarectal; 5 days particles orally

healthy control mucus layer ofcolitis group

ulcerated tissue ofcolitis group


• higher deposition in inflamed colon selectivity• size dependent accumulation• no influence on the deposition in other gut regions

0.1µm 1µm 10µm 0.1µm 1µm 10µm0

5

10

15

20

25

30

#

*

*

colitis groupcontrol group

part

icle

bin

ding

(%

)

0.1µm 1µm 10µm0

5

10

15

20

25

*

part

icle

bin

ding

[%]

particle size


0 2 4 6 8 10 12 14

0

1

2

3

TNBS

**

**

**

***

*

FK506

TN

BS

clin

ical

act

ivity

sco

reday


tacrolimus:(also FK-506 or Fujimycin)

• use in organ transplant • immunosuppressive• use in IBD

0

10

20

30

40

0.0000

0.0063

0.0125

0.0188

0.0250

***

*

B

***

**

**

TNBS-rectal

colo

n/b

od

y w

eig

ht

rati

o

MP

O a

ctiv

ity

[U/m

g t

issu

e]

healthy untretaed healthy FK506 colitis control NP control FK506 FK506-NP

0

10

20

30

40

0.0000

0.0063

0.0125

0.0188

0.0250

*

D

***

OXA-rectal

colo

n/b

od

y w

eig

ht

rati

o

MP

O a

ctiv

ity

[U/m

g t

issu

e]

healthy untreated healthy FK506 colitis control NP control FK506 FK506-NP


NP efficient after rectal administration

significant drug loss during intestinal transit


NP increase drug tissue penetration selectively

solution:

inflamed

healthy

NP:

inflamed

healthy

NP tissue penetration into inflamed tissue

into healthy tissue

CYP

P-gp

0

1

2

3

FK506 formulations

***

rel.

pene

trat

ion

inde

x[c

oliti

s/he

alth

y]

FK506 FK506+amio FK506+tro FK506-NP


Conclusion

Nanoparticles target the inflamed tissue in IBD

A) accumulate the drug in the inflamed tissue:

B) increase and selectivity of drug tissue penetration

I) high therapeutic efficiency

II) reduction of adverse effects

Nanocapsules in thetreatment of cancer

Objective:

local drug delivery of anticancer drugstargeting on cell level

higher efficiency by a selective drug release

Lipid nanocapsules and theirtherapeutic applications

• controlled drug release• controlled surfactant release (P-gp inhibitor) • slower release for higher drug loading

0 24 48 72 96 1200

20

40

60

80

100

0

200

400

600

800

1000

PE

G-H

S r

elea

se [µ

mol

]

etop

osid

e re

leas

e [%

]

time [h]

PEG-HS (LNC25) PEG-HS (LNC50) PEG-HS (LNC100) eto (LNC25) eto (LNC50) eto (LNC100)

Lipid nanocapsule preparation

basal activity

vinblastine

verapamil

2.5µmol

25nmol

1.09µmol

10.9nmol

0.63µmol

6.3nmol

0.0

0.5

1.0

1.5

2.0

*****

*

rela

tive

AT

Pas

e ac

tivity

of P

-gp

LNC25 LNC50 LNC100

in-vitro testing of LNC on glioma cell cultures:

Nanocapsules in cancer

• etoposide LNC up to 80-fold more potent• more effective in F98 cells

0.01 0.1 1 10 100 1000 100000

20

40

60

80

100

120

F98

viab

ility

[%]

etoposide [µmol]

eto LNC25 eto LNC50 eto LNC100 eto sol. eto + PEG-HS sol. blank LNC25 blank LNC50 blank LNC100

0.01 0.1 1 10 100 1000 100000

20

40

60

80

100

120

9L

viab

ility

[%]

etoposide [µmol]

eto LNC25 eto LNC50 eto LNC100 eto sol. eto + PEG-HS sol. blank LNC25 blank LNC50 blank LNC100

Confocal laser scanning microscopy

• LNC uptake in F98 (a) and 9L (b) cells• etoposide + blank LNC no effect

cellular uptake required !!!


Proposed mechanism of action:



LNC - an promising concept in cancer treatment ?

- intracellular uptake - drug release - PEG-HS release- potentially blocking the P-gp

overcoming the multidrug resistance ???

Acknowledgments

Nancy

P. MaincentN. Ubrich

Y. Meissner

Gifu

Y. KawashimaH. TakeuchiYamamoto

Saarbrücken

C.M. LehrU. Schäfer

D. NeumannT. Betz

Angers

J.P. BenoîtF. Lacoeuille

E. Garcion

Acknowledgements

- Japanese Society for the Promotion of Science- Alexander-von-Humboldt Foundation- Monbugakushō- Egide- Fujisawa Pharmaceutical Co. Ltd.

Date post:	17-Dec-2015
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