drug release from liposomes: role of mechanism based...

Drug Release from Liposomes: Role of Mechanism Based Models

Bradley D. Anderson

University of Kentucky

Why liposomes? l i p o s o m e

Drug solubilization Tumor targeting – EPR effect Controlled release potential Safety/reduction of drug toxicity Commercial products

N

N

O

OH

Si

O

OOH

Xiang and Anderson, Adv. Drug Delivery Rev. 58, 357–1378 (2006)

AR-67

Rakesh K. Jain et.al. Nature Reviews Cancer 2, 266-276 (2002)

EPR effect (enhanced permeability & retention) Particle size cut-off for extravasation

Pegylation and particle size effects on liposome clearance and tissue uptake

Ishida et al., Int. J. Pharm., 190, 49-56 (1999)

Tunable release kinetics?

• Nanoparticle accumulation in tumor may require up to12-24 hrs to reach optimum

• Free drug concentration profile in tumor determines efficacy

• What release rate is optimal? • Can mechanistic models enable the

design of optimal release rates?

NCI Alliance for Nanotechnology in Cancer

t1/2 = 57 h

t1/2 = 14 h

Vehicle CPT-11 50 mg/kg

Liposomal CPT-11 50 mg/kg

Drummond et al., Cancer Res., 66, 3271 (2006)

NCI Alliance for Nanotechnology in Cancer

Antitumor efficacy of CPT-11 in a colon cancer model depends on liposome

release kinetics

“As long as the release process is still not fully understood it is difficult and speculative to make improvements to the existing formulation or devise new compounds.”

Predictive Models Must Account for the Driving Force, Membrane Permeability

and Environmental Factors • Prediction of: Driving Force Contributions

What species are transported? pH gradients and their role? Membrane binding Drug precipitation & self-association Role of environmental factors (pH, temperature, media composition)

Membrane Permeability Barrier domain for lipid bilayer transport. What governs membrane selectivity to permeant structure?

How do we predict chemical selectivity? How do we predict permeant size selectivity?\ Temperature effects

from the Doxil® package insert

• 2 mg/mL doxorubicin HCl • 16 mg/mL lipid

– 3.19 mg/mL cholesterol

– 9.58 mg/mL HSPC – 3.19 mg/mL MPEG-

DSPE • ~0.6 mg/mL NH4SO4

• pH 6.5 buffer(histidine) • sucrose for isotonicity

Equilibria and kinetic processes governing DXR release rates

from actively loaded liposomes using dynamic dialysis OH

OH3C

OHNH3

OH

O

OH

OH

OHO

OOCH3

Cl

Doxorubicin (DXR)

DXR actively-loaded - low intravesicular pH

Extravesicular pH 7.2-7.8

Physical processes included in a mechanistic model for Doxil

• Acid-base dissociation equilibria (i.e., DXR pKa) for DRX inside and outside the liposomes;

• Acid-base dissociation equilibria (i.e., pKa) for Ammonia inside and outside the liposomes;

• Acid-base dissociation equilibria (pKa) for sulfate inside and outside (if present) the liposomes;

• Precipitation equilibrium (i.e., Ksp) for DRX+ and SO42- inside the liposomes;

• Binding equilibria for DRX and DRX+ onto outer and inner bilayer leaflets;

• Forward/reverse rate constants for DRX bilayer and dialysis membrane transport

• Forward and reverse rate constants for ammonia bilayer and dialysis membrane transport

Csuhai et al., J. Pharm. Sci., 104, 1087 (2015)

Separation of drug-loaded liposomes by

gel filtration

Liposomal fraction

Unentrapped ‘free’ drug

PD MiniTrap G25 Gel filtration columns

-2

0

2

4

6

8

10

12

14

0 5 10 15

Dox

con

c( µ

g/m

l)

Volume (ml)

ENCAP

FREE

Gel filtration: Dox spiked (90%) human plasma/pH 7.4 buffer (10%)

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 20 40 60 80

2 ug/mL Dox5 ug/mL Dox10 ug/mL Dox15 ug/mL Dox20 ug/mL Dox

Enc

apsu

late

d D

ox (

ug/m

L)

Time (h)

Representative blank liposomal uptake profiles (pH 5.5,3 mg/ml lipid w/250 mM (NH4)2SO4)

Which is the permeable DXR species?

Rate constant vs pH for DXR monomer uptake into blank liposomes containing 250 mM (NH4)2SO4 at 37 °C.

pKa = 8.13 ± 0.20

)1( npnn fPfPSAk −+=

)/][1/(1 an KHf ++=

Suspension concentration: 3 mg lipid/mL (HPLC with ELSD detection)

Pn = 8.5 x 10-4 cm/h Pp ~ 0

Neutral form is the permeable species Csuhai et al., J. Pharm. Sci., 104, 1087 (2015)

DXR self-association

DXR uptake vs. time at varying DXR concentrations in 242 mM Na2SO4 at pH 6.5 (10 mM phosphate), 37 ºC. Dotted lines represent simultaneous fits to the isodesmic self-association model.

Top view

Side view

Anti parallel stacking

Agrawal et al., Eur. J. Med. Chem., 44, 1437 (2009)


Rate of liposomal uptake (- - -) and fraction of DXR monomer ( ̶̶̶ ̶ ̶ ) vs.

DXR concentration (3 mg lipid/mL, 240 mM Na2SO4 pH 6.5, 37°C)

DXRmon = DXRTotal{2/[1 + (4KnDXRTotal + 1)0.5]}2

Kn = 7030 ± 900 M-1


Ksp determination for (DXR)2SO4

DXR Solubility (mg/mL)*

T (°C) 250 mM (NH4)2SO4 150 mM (NH4)2SO4 100 mM (NH4)2SO4

50 mM (NH4)2SO4 Apparent Ksp (M3) (Eq. 7)

5 ± 1 0.17 ± 0.01 0.23 ± 0.01 0.27 ± 0.01 0.36 ± 0.02 2.1± 0.2 x 10-9

25 ± 1 0.23 ± 0.01 0.29 ± 0.01 0.35 ± 0.01 0.46 ± 0.01 3.6 ± 0.3 x 10-8

37 ± 0 0.63 ± 0.01 0.81 ± 0.04 1.00 ± 0.05 1.31 ± 0.03 2.9 ± 0.2 x 10-7

*n=3; average values ± SD

Pooled DXR solubility data at 5 ± 1°C, 25 ± 1°C, and 37 ± 0°C, in the presence of 250, 150, 100, or 50 mM (NH4)2SO4.

DXR solubility vs pH and (NH4)2SO4 concentration at 37 ± 0 °C. Error bars represent standard deviations.


Influence of membrane binding on liposomal uptake rate constant

DXR uptake versus lipid concentration at 10 µg/mL DXR, 37◦C, pH 6.5, with 250 mM intraliposomal (NH4)2SO4. The model fit assumes only the monomeric, neutral species is membrane permeable.

A: liposomal surface area per ml P1: neutral monomer permeability fn: fraction neutral monomer fubo: fraction unbound c & d: aqueous & outer bilayer leaflet volume ratios

Intravesicular membrane binding is amplified due to the high internal surface/volume ratio.

OH

OH3C

OHNH3

OH

O

OH

OH

OHO

OOCH3

Cl

Doxorubicin (DXR)

DXR actively-loaded - low intravesicular pH

Extravesicular pH 7.2-7.8

• Mechanistic model accounts for all equilibria and kinetic processes below

• Dynamic intravesicular pH calculation is critical.

Implicit eqn. for pH calculation (from charge balance

Fugit et al., J. Control. Release, 217, 82 (2015)

Mechanistic model simulations in various release conditions (e.g., external pH and [NH3])

Intravesicular pH governed by external [NH3]

Low internal pH driven by NH3 release when [NH3] = 0 in media


Intravesicular pH modulates the driving force for DXR release –

model simulations pH 7.4 0 mM NH3

pH 7.0 50 mM NH3

pH 7.4 50 mM NH3

pH 7.8 50 mM NH3


DXR release vs time (expt. vs. prediction)

0

20

40

60

80

100

0 12 24 36 48 60 72

% D

XR R

emai

ning

Time (hours)

1 2 3 4 56 7 8 9 1011 12 13 14 15

0

20

40

60

80

100

0 12 24 36 48 60 72

% D

XR R

emai

ning

Time (hours)

1 2 3 4 56 7 8 9 1011 12 13 14 15

Experimental Prediction

NH

4+ 0-1

00 m

M

x1

x2

x3

pH 7.0 – 7.8


-15

0

15

30

45

60

75

1.5 2.5 3.5 4.5 5.5 6.5 7.5

% D

XR R

elea

sed

Afte

r 19

hr

Calculated Intravesicular pH

Observed Simulated

Empirical model Mechanistic model

Superiority of the mechanistic model


Dox uptake vs. Temperature pH 6.0, 30 mM phosphate,

265 mM NaCl, 10 ug/mL Dox, 3 mg/mL lipid

y = 0.1815x + 0.0404

y = 1.1401x + 0.0808

y = 3.2346x + 0.0359

y = 27.097x - 0.8234

y = 61.674x + 1.5721

0

1

2

3

4

5

6

7

0 2 4

DXR

Sus

p. C

onc.

ent

rapp

ed (µ

g/m

L)

Time (h)

37 C42 C47 C52 C57 C

Arrhenius plot – uptake rate vs. 1/T

Ea = 65.6 kcal/mol

(consistent with chain ordering effect on bilayer barrier properties & size selectivity)

Xiang & Anderson, Biophys. J., 72, 223 (1997)

10° ↑ => 24-fold ↑ in k

Historical Model for Structure-Transport Relationships

• “Bulk” Solubility-Diffusion theory – Graham (1866); Overton’s rule (1896, 1899, 1902)

Permeability: Po = KmemDmem/h incorrect!

Kmem = PCmembrane/water ~ PCoctanol/water

Dmem = const/MW1/2

log (Po) = const + 1.0 log PCoctanol/water/MW1/2

Not for bilayer transport! Xiang & Anderson, Adv. Drug Delivery Revs., 58, 1357 (2006)

Bulk Solubility Diffusion Theory fails to account for phase transitions

Data plotted from Xiang and Anderson, Biophys. J. 1998.

-10

-9

-8

-7

-6

-5

-4

-3

-2

3 3.1 3.2 3.3 3.4

1/T x 1000 (K)

ln P

erm

(obs

erve

d an

d pr

edic

ted)

DPPC phase transition

Formic Acid across DPPC

“Bulk” permeability

Bulk solubility-diffusion model fails to account for the amplified size

selectivity

20 140 260 380 500

V (A 3 )

10 -11

10 -10

10 -9

10 -8

10 -7

10 -6

10 -5

10 -4

P m

δ / K

or D

Decane diffusion (n=0.8)

Egg PC - liquid crystalline bilayers (n=1.48)

DPPC - gel phase bilayers (n=6.2)

log Pmδ /K = const - n log V

Permeability theory - barrier domain model

PK D

hf Pm

barrier water barrier

barriero= = ∗/

Po = from solubility-diffusion theory f = reflects chain ordering factor

f = foexp(-λas/af)

Permeant cross-sectional area

Bilayer free-surface area – decreases with increase in chain-order

Xiang & Anderson, Adv. Drug Delivery Revs., 58, 1357 (2006)

Joguparthi et al., J Pharm Sci 97:400–420, 2008

Lactone t1/2 = 3 hr

Translation from in vitro to in vivo • Release methods in blood or plasma

– Pitfalls of dynamic dialysis • see Modi & Anderson, Mol. Pharmaceutics, 10, 3076 (2013)

– Non-sink release methods • see Fugit & Anderson, Mol. Pharmaceutics, 11, 1314 (2014)

– Real-time spectroscopic methods • see Fugit et al., J. Control. Release, 197, 10 (2015)

Topotecan (TPT)

Liposomal TPT release in human plasma monitored by fluorescence excitation

spectra Dependence of TPT release t1/2 on

extravesicular ammonia concentration

Environmental factors in vivo may influence liposomal release rates

• Exchange of plasma lipid components with vesicle lipids • Disruption of pH gradients by permeable (small

molecule) plasma components – see Joguparthi & Anderson, J. Pharm. Sci., 97, 433 (2008) – accelerated

silatecan release due to plasma CO2

– see Fugit et al., J. Control. Release, 197, 10 (2015)

Liu et al., Anticancer Drugs, 13, 709 (2002)

Liposomal TPT retention in PBS vs. human plasma

NH3 in human plasma (8 donors) as rec’d from supplier

Increase in NH3 in human plasma vs storage time at various temps.

Acknowledgments Eva Csuhai Sogol Kangarlou Tian-Xiang Xiang Andre Ponta

Kyle Fugit Duhyung Choi Paul Bummer Amar Jyoti

Portions of the work presented were supported by Grant Number R25CA153954 from the National Cancer Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Instittute or the National Institutes of Health.

drug release from liposomes: role of mechanism based...

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