The dispersion releaser technology is an effective method for testing drug release from nanosized drug carriers

Accepted Manuscript The dispersion releaser technology is an effective method for testing drug release from nanosized drug carriers Christine Janas, Marc-Philip Mast, Li Kirsamer, Carlo Angioni, Fiona Gao, Werner Mäntele, Jennifer Dressman, Matthias G. Wacker PII: DOI: Reference:

S0939-6411(16)30755-X http://dx.doi.org/10.1016/j.ejpb.2017.02.006 EJPB 12440

To appear in:

European Journal of Pharmaceutics and Biopharmaceutics

Received Date: Revised Date: Accepted Date:

2 November 2016 9 February 2017 12 February 2017

Please cite this article as: C. Janas, M-P. Mast, L. Kirsamer, C. Angioni, F. Gao, W. Mäntele, J. Dressman, M.G. Wacker, The dispersion releaser technology is an effective method for testing drug release from nanosized drug carriers, European Journal of Pharmaceutics and Biopharmaceutics (2017), doi: http://dx.doi.org/10.1016/j.ejpb. 2017.02.006

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The dispersion releaser technology is an effective method for testing drug release from nanosized drug carriers Christine Janasa, Marc-Philip Masta, Li Kirsamerb, Carlo Angionic , Fiona Gaoa, Werner Mänteleb, Jennifer Dressmana, Matthias G. Wackera,d,*

a

Goethe University, Institute of Pharmaceutical Technology, D-60438 Frankfurt, Germany

b

Goethe University, Institute for Biophysics, D-60438 Frankfurt, Germany

c

Goethe University, Pharmazentrum Frankfurt, Institute of Clinical Pharmacology, D-60590 Frankfurt, Germany

d

Fraunhofer-Institute for Molecular Biology and Applied Ecology, Department of Pharmaceutical Technology and Nanosciences, D-60438 Frankfurt, Germany

*Correspondence: Dr. Matthias G. Wacker Fraunhofer-Institute of Molecular Biology and Applied Ecology IME Project group for Translational Research and Pharmacology TMP Department of Pharmaceutical Technology and Nanosciences Max-von-Laue-Str. 9, D-60438 Frankfurt a. M., Germany Phone

+49-69-798-29691

Fax

+49-69-798-29694

E-mail

[email protected]

1

1. Abstract The dispersion releaser (DR) is a dialysis-based setup for the analysis of the drug release from nanosized drug carriers. It is mounted into dissolution apparatus 2 of the United States Pharmacopoeia. The present study evaluated the DR technique investigating the drug release of the model compound flurbiprofen from drug solution and from nanoformulations composed of the drug and the polymer materials poly (lactic acid), poly (lactic-co-glycolic acid) or Eudragit® RS PO. The drug loaded nanocarriers ranged in size between 185.9 and 273.6 nm and were characterized by a monomodal size distribution (PDI < 0.1). The membrane permeability constants of flurbiprofen were calculated and mathematical modeling was applied to obtain the normalized drug release profiles. For comparing the sensitivities of the DR and the dialysis bag technique, the differences in the membrane permeation rates were calculated. Finally, different formulation designs of flurbiprofen were sensitively discriminated using the DR technology. The mechanism of drug release from the nanosized carriers was analyzed by applying two mathematical models described previously, the reciprocal powered time model and the three parameter model.

2. Keywords Drug release, colloidal drug carriers, dialysis, USP apparatus 2, mathematical modeling, flurbiprofen

2

3. Abbreviations API

active pharmaceutical ingredient

BSA

bovine serum albumin

CAR

compartmental accumulation ratio

DB

dialysis bag

DLS

dynamic light scattering

DR

dispersion releaser

FDA

Food and Drug Administration

HPLC

high-performance liquid chromatography

MWCO

molecular weight cut off

LC-MS/MS

liquid chromatography-mass spectrometry/ mass spectrometry

PBS

phosphate buffered saline

PDI

polydispersity index

PEEK

polyether ether ketone

PLA

poly (lactic acid)

PLGA

poly (lactic-co-glycolic acid)

PVA

poly (vinyl alcohol)

PTFE

polytetrafluoroethylene

RC

regenerated cellulose

rcf

relative centrifugal force

rpm

revolutions per minute

rpt

reciprocal powered time

SD

standard deviation

SEC

size exclusion chromatography 3

US-FDA

Food and Drug Administration of the United States of America

USP

United States Pharmacopeia

4

4. Introduction Nanomedicines are becoming increasingly important in global markets. Since a growing number of pharmaceuticals and medical devices are manufactured using nanotechnology [1], there is an emerging need for methods to assure the quality and safety of such products. Further, the availability of suitable tools for predicting the in vivo performance during formulation screening would streamline the development process. In vitro release testing is widely considered to be a ‘gold standard’ in the evaluation of orally administered dosage forms [2]. Additionally, it has been proposed for the quality control of liposomes and parenteral modified-release formulations by the Food and Drug Administration of the United States of America (US-FDA) [3, 4]. To date, only few methods for testing release from microparticles and several types of nanoformulations have been put forward [5, 6]. Often ‘sample and separate’ [7, 8] or dialysisbased techniques [5, 9] are applied. ‘Sample and separate’ methods use filtration [8, 10, 11], solid phase extraction [7] or centrifugation [12] to separate the dissolved active pharmaceutical ingredient (API) from the particulate fraction. In other studies dialysis-based techniques have been applied to measure drug release and in some cases the compendial equipment was modified [5, 9, 13]. For example, the release of ibuprofen from lipid nanocapsules was studied in a dissolution apparatus 1 of the United States Pharmacopoeia (USP) equipped with a glass cylinder holding a dialysis membrane at the bottom. The sample was filled into the system and drug concentration in the acceptor compartment was quantified [9, 14]. Similarly, the release of dexamethasone from polymeric micelles was investigated using a USP apparatus 2 in combination with a dialysis sac [15]. Yet, another study utilized the USP apparatus 2 and a holder for the commercially available Float-A-Lyzer® device to assess the release of naproxen and indomethacin from crystalline nanosuspensions [16]. The USP apparatus 4 (flow-through cell) has also been used for testing release from nanoformulations. A dialysis adapter (‘A4D’) mounted in a 22.6 mm cell 5

was used to investigate the release of dexamethasone-loaded liposome formulations [5, 17]. The release test discriminated between extruded and non-extruded formulations of the API. The authors found the continuous flow and the large membrane surface area as advantageous for this task [5, 17]. This setup was also found to be useful for investigating the release of vitamin E acetate from nanoemulsions [18]. Importantly, most nanoformulations are characterized by an increased dissolution or release rate which is often described using a modification of the Noyes and Whitney equation [19]. Further, as a consequence of Ostwald ripening [20], a temporary supersaturation may occur. Both effects result in fluctuations of the release profile [21]. While for most nanocrystal formulations this rapid release is part of the formulation concept, the ‘burst release’ has been a major shortcoming of other types of nanocarriers [22]. The release from micro- and nanoparticles was investigated for carriers composed of polymethacrylate [11] earlier. Because of their increased surface area a more rapid diffusion from the particle matrix into the surrounding medium may only be compensated by an increased affinity of the compound to the material [11]. These unspecific interactions with binding sites at the carrier matrix are more sensitive to shear forces and competing substances in the release medium. Therefore, many of the existing ‘sample and separate’ techniques are very likely to result in a rapid release of the API from the matrix material [9, 23]. Despite the fact that dialysis has been proposed as the ‘method of choice’ for studying drug release from nanoformulations such as polymeric nanocarriers, micelles, liposomes or even nanocrystals that contain high excipient concentrations [23], the barrier properties of the dialysis membrane do impose a limit on the rate of drug release that can be measured from such formulations [24] - the diffusion rate of an API through the dialysis membrane is often rate limiting to release in these experiments. Further, sink conditions in the donor compartment may not be maintained through the entire experiment [5].

6

The dispersion releaser (DR) is a dialysis-based device composed of a sample holder cell serving as the donor compartment for dispersed dosage forms [25]. A dialysis tubing is mounted around this housing. This donor chamber is agitated by a paddle stirrer that is connected to the USP apparatus 2 and equipped with a permanent magnet. The propulsion is transmitted to a magnetic stirring device in the acceptor compartment (see Figure 1). This design allows control over the shear forces applied to the nanoformulation while facilitating a more rapid membrane transport compared to previous setups. In this study, the utility of the DR setup was illustrated by conducting release experiments with the poorly water soluble weak acid flurbiprofen (pKa = 4.22, logP = 10.417 [26]) from drug solution and drug loaded nanoparticles. For this evaluation a protocol from the USP was applied [27]. The particulate formulations were composed of poly (lactic acid) (PLA), poly lactic-co-glycolic acid (PLGA), and Eudragit® RS PO. Even in the DR setup, the dialysis membrane still limits the sensitivity due to a delay in the membrane transport. Therefore, the release profiles obtained from these experiments were normalized by calculating the rate constants of membrane diffusion (kM). They were used to determine the individual drug release features of each of the nanocarrier formulations by applying the four-step model [2830]. Further, the impact of various experimental conditions (e.g. medium composition, temperature) on the kM values was determined. In this context, also the kM values of two different dialysis-based setups, the DR and the dialysis bag (DB) technique, were compared. Finally, the drug release from polymeric nanoparticles was analyzed using two different mathematical models, the reciprocal powered time (rpt) model [28] and the three-parameter model [30].

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5. Material and Methods

5.1.

Materials

The dispersion releaser was fabricated from polyether ether ketone (PEEK) at the facilities of Goethe University (Frankfurt am Main, Germany). Regenerated cellulose (RC) dialysis tubing with a molecular weight cut-off (MWCO) of 50 kDa and a flat diameter of 28 mm (Spectra/Por®6, Spectrum Labs) was purchased from VWR International GmbH (Darmstadt, Germany). Racemic flurbiprofen was obtained from Cayman Chemical Company (Ann Arbor, Michigan, US). Poly lactide (Resomer® L206S), poly vinyl alcohol (average MW 30-70 kDa, 87-90% hydrolyzed), and penicillin streptomycin solution were purchased from Sigma Aldrich (Steinheim, Germany). Eudragit® RS PO and poly lactic-co-glycolic acid (Resomer 502 H) were kindly provided by Evonik Industries AG (Darmstadt, Germany). Pluronic® F-68 was purchased from AppliChem GmbH (Darmstadt, Germany). Bovine serum albumin (BSA) was purchased at PAA Laboratories GmbH (Egelsbach, Germany). All organic solvents were gradient grade for liquid chromatography.

5.2.

Preparation of nanoparticle formulations

Flurbiprofen-loaded PLA and PLGA nanoparticles were prepared by the emulsion diffusion evaporation method. A protocol similar to the procedure described by Meister et al. [31] for PLA nanoparticles was used. In brief, a total amount of 200 mg of the polymer and 2 mg of the drug were dissolved in 4 mL of dichloromethane. Twelve mL of an aqueous PVA solution 1% [w/w] was added and the mixture was homogenized at 17,000 rpm for 15 minutes on an ice bath using an Ultra Turrax® equipped with a TN25 dispersing tool (IKA, Staufen, Germany). After diluting the emulsion with a volume of 12 mL of the aqueous PVA solution 1% [w/w], the dichloromethane was evaporated overnight at room temperature under

8

constant stirring. Particles were purified by centrifugation at 16,100 rpm for 8 minutes (Centrifuge 5430 R, Eppendorf AG, Hamburg, Germany) and redispersed in half the original volume of purified water. Three particle batches were pooled for further experiments in order to supply sufficient amounts of the nanoparticle formulations. There was no further optimization of the manufacturing process undertaken. The variations in batch quality supported the evaluation of the DR technology. PLA and PLGA nanoparticles were freeze dried in presence of

5% [w/v] of

trehalose

(Epsilon 2-4 LSC,

Martin

Christ

Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) [32]. Eudragit® RS PO nanoparticles were prepared by nanoprecipitation method as described previously [11]. Fifteen mg of the drug and 150 mg of the polymer were dissolved in 1 mL of ethanol 96% [v/v]. An aqueous solution of Pluronic® F-68 0.01% [w/v] was added by using a peristaltic pump under constant stirring. After one hour particles were collected and purification

was

accomplished

as

described

for

PLA

and

PLGA

nanoparticles.

Eudragit® RS PO nanoparticles were stored at 4 °C overnight prior to release testing.

5.3.

Physicochemical characterization of polymeric nanoparticles

The particle size and polydispersity index (PDI) were determined by dynamic light scattering (DLS) in a Malvern Zetasizer Nano (Malvern Instruments, Malvern, UK) equipped with a backscatter detector at an angle of 173 °. Zeta potential was measured by micro electrophoresis with the help of a Malvern Dip cell (Malvern Instruments, Malvern, UK). Prior to the measurement, the drug-loaded nanoparticles and samples from the donor compartment of release experiments were diluted with purified water 1:40 [v/v] and 1:1 [v/v], respectively. The content of solid material in the nanoparticle suspensions was quantified by microgravimetry. For this purpose, samples were dried at 80 °C for 2 hours. The drug content was analyzed by high performance liquid chromatography (HPLC) analysis (see section 5.8).

9

5.4.

Determination of drug solubility in release media

Prior to the release experiment the thermodynamic solubility of flurbiprofen in release media was determined. For this purpose, an excess of the drug was added to 3 mL of each medium and incubated at 37 °C for 24 hours (n=3). Afterwards, the samples were filtered through a 0.45 µm PTFE syringe filter and drug concentration was quantified by HPLC or LC-ESIMS/MS analysis (see section 5.8).

5.5.

In vitro release experiments conducted with the dispersion releaser and dialysis bag

The DR setup was used in combination with a USP apparatus 2 equipped with a mini-vessel configuration [25] (see Figure 1). An RC dialysis membrane with a MWCO of 50 kDa was mounted onto the sample holder cell of the DR and fixed with O-rings. Afterwards the donor chamber was partly filled with release medium. The media in the donor and the acceptor compartments were composed of phosphate buffered saline (PBS) at a pH of 7.4 supplemented with 1% [v/v] of a penicillin streptomycin solution to avoid microbial growth. For de-aeration of the media, the method suggested in the USP was applied [33]. A total weight of 140 g of the medium was filled into each vessel. Prior to the experiment, the dialysis membrane was treated according to the instructions of the manufacturer. The MWCO was more than 100 times higher than the molecular weight of the analyte (flurbiprofen, MW = 244.26 Da) [34]. Furthermore, the volume in the acceptor compartment was ten times higher than the volume of the donor compartment [35] assuring a more rapid diffusion through the dialysis membrane. After 15 minutes, drug formulations (drug in release medium or nanoparticle formulations diluted with release medium, experiment no. 1 to 10), were filled into the donor compartments with a syringe and an injection needle (Sterican®, 0.8 x 22 mm, B.Braun, 10

Melsungen, Germany). The amount of drug was adjusted to 400 µg in each vessel to ensure sink conditions. At predetermined time points (15 and 30 min, 1, 2, 3, 4, 6, 8, 22, and 24 h) samples with a volume of 0.5 mL were collected from the acceptor compartment and replaced with fresh medium. Initial experiments were conducted at a temperature of 37 ± 0.5 °C. For further experiments accelerated conditions (50 ± 0.5 °C) were adjusted. Similar conditions have been used in dialysis experiments earlier [5] in order to accelerate the diffusion process without having a pronounced impact on formulation properties and the dialysis membrane. The stirring rates were set to 75 or 50 rpm which is the range commonly applied in dissolution tests using USP apparatus 2. The results obtained from the DR experiments were compared to the measurements conducted with the DB technique. In this case also, the USP apparatus 2 was fitted with a mini-vessel. All vessels were filled with 248 g of the release medium. A dialysis membrane of the same type was applied. For the dissolved drug (experiment no. 11) and the nanoparticle formulation (experiment no. 12) a total drug amount of 714.3 µg in 2.0 mL of release medium was filled into the dialysis bag. The stirring rate was adjusted to 75 rpm and temperature was kept constant at 37 ± 0.5 °C.

5.6.

Membrane leakage test

The leakage of the dialysis membrane in the DR setup was quantified by determining the diffusion of BSA from the donor into the acceptor compartment (see Table 1, experiment no. 0). The RC dialysis membrane with a MWCO of 50 kDa should retain 90% of the protein over a time period of 17 hours according to the specifications defined by the manufacturer of the tubing [36]. A total amount of 100 mg of BSA was dissolved in 4 mL of PBS at pH 7.4. The solution was filled into the donor compartment. At predetermined time points (1, 2, 4, 6, 8, and 24 h) samples were taken from the acceptor compartment and replaced with fresh medium. The 11

BSA concentration was quantified by size exclusion chromatography (SEC) as reported previously [37]. A Biosep SEC-s3000 (Phenomenex Ltd., Aschaffenburg, Germany) column was used. A total volume of 50 µl of the sample was injected into a Chromaster HPLC system (see section 5.8) equipped with a UV-VIS-detector (no. 5420). The flow rate was adjusted to 1 mL/min and absorbance was monitored at a wavelength of 280 nm. PBS at pH 6.8 with 0.5% [w/v] of sodium azide served as the mobile phase.

5.7.

Intermediate precision and robustness of the dispersion releaser setup

The intermediate precision of the DR technology was evaluated by repeating the release experiment of the pure drug (see Table 1, experiment no. 1) with a second operator and equipment (see Table 1, experiment no. 2). Further, the robustness of the setup was investigated. For this purpose, the drug release test was conducted at two different stirring rates and temperatures. The temperature was adjusted to 37 ± 0.5 °C and 50 ± 0.5 °C (see Table 1, experiment no. 1 and 3). The evaporation from the vessels was monitored over a time period of 24 hours. The stirring rate was adjusted to 50 or 75 rpm, respectively (see Table 1, experiment no. 1 and 4). For liposomal parenterals the US-FDA recommends the use of physiological media such as simulated human plasma [3] to detect differences in the release behavior triggered by protein interaction. The impact of proteins on the release experiment was investigated by using phosphate-albumin buffered saline pH 7.2 R and phosphate-albumin buffered saline pH 7.2 R1 as release media (see Table 1, experiment no. 5 and 6). These compendial media are described by the European Pharmacopeia [38] and contain BSA at a concentration of 10 and1 g/L, respectively. The amounts of albumin do not represent the physiological concentration or protein composition but were identified as a well-accepted standard in the testing of drug formulations.

12

All media were supplemented with 1% [v/v] of penicillin and streptomycin solution to reduce microbial growth. De-aeration was performed with an ultrasonic bath to avoid foaming of the protein components under the conditions of the USP method [33].

5.8.

Quantification of flurbiprofen by HPLC

The drug concentrations were determined by performing a HPLC analysis. All samples were diluted with a mixture of 0.1% [v/m] of trifluoroacetic acid in acetonitrile and centrifuged at 20,800 rcf and 20 °C for 10 minutes (Centrifuge 5430 R with rotor FA-45-30-11, Eppendorf AG, Hamburg, Germany) after appropriate homogenization. Following centrifugation, a volume of 80 µL of the supernatant was injected into the Chromaster HPLC system (Chromaster, VWR Hitachi, Tokyo, Japan). The system was equipped with a diode array detector (DAD, VWR no. 5430), a HPLC pump (VWR no. 5160), an auto sampler (VWR no. 5260), and a column oven (VWR no. 5310). A reversed phase column (Gemini NX-C 18, 250 x 4.60 mm, 110A, Phenomenex Ltd., Aschaffenburg, Germany) and a mobile phase consisting of 42.5% [w/w] of trifluoroacetic acid (0.1% [v/v]) in distilled water and 57.5% [w/w] acetonitrile were used. The column temperature was adjusted to 30 °C and flurbiprofen was detected at a wavelength of 247 nm. The samples of the nanoparticle release experiments were centrifuged at 20 °C and 20,800 rcf (Centrifuge 5430 R, Eppendorf AG, Hamburg, Germany) for 10 minutes prior to the dilution step. After confirming linearity of the HPLC method, the recovery of flurbiprofen from the release medium was investigated over a concentration range of 0.1 to 3.0 µg/mL. For quantification of flurbiprofen from samples containing serum proteins, liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) was used. The drug was separated from the plasma proteins by adding 250 µL of acetonitrile to 50 µl of the sample and 50 µL of an internal standard solution (flurbiprofen-d5 2500 ng/mL in acetonitrile). After mixing and centrifugation (20,100 rcf over 3 min, Centrifuge 5424,

13

Eppendorf AG, Hamburg, Germany) a volume of 20 µL of the supernatant was injected into the LC-MS/MS system. The LC-MS/MS system consisted of a triple quadrupole 5500 QTRAP mass spectrometer (AB Sciex, Darmstadt, Germany) equipped with a Turbo-Vsource operating in negative electro spray ionization (ESI) mode, a binary HPLC pump (Agilent no. 1200), a degassing unit (Agilent, Waldbronn, Germany), and a HTC Pal autosampler (Chromtech, Idstein, Germany). A cooling stack was used to store the samples at 6 °C in the autosampler. High purity nitrogen for the mass spectrometer was produced by a NGM 22-LC/MS nitrogen generator (cmc Instruments, Eschborn, Germany). A Chiralpak AD-RH (150 x 4.6 mm I.D., 5 µm particle size) column (Chiral Technologies, Illkirch Cedex, France) with a pre-column of the same material and a mixture of acetonitrile and water (70:30, [v/v]) with 0.1% [v/v] formic acid was used as mobile phase. The temperature of the column was maintained at 20 °C and the flow rate of the mobile phase was set at 0.6 mL/min. The mass spectrometer was operated in the negative ion mode with an electrospray voltage of -4500 V at 500°C. Multiple reaction monitoring was used for quantification. The mass transitions used were m/z 243.0 to 199.0 for flurbiprofen and m/z 253.0 to 197.0 for the internal standard, all with a dwell time of 100 ms. All quadrupoles were working at unit resolution. Quantitation was performed with Analyst Software V1.6.2 (Applied Biosystems, Darmstadt, Germany) using the internal standard method (isotope-dilution mass spectrometry).

5.9.

Mathematical modeling describing the drug release and membrane permeation kinetics in dialysis-based experiments

The permeation of the drug through the membrane was calculated in order to determine the drug release from the carrier. For this task, mathematical modeling was applied. All calculations were based on a previously described four-step model [29].

14

Initially, the membrane permeability constants (kM) of flurbiprofen were calculated from the release profiles of each experiment performed with the pure compound (see Table 1, experiment no. 1 to 7). The kM values are reported as the mean value (n=6) and standard deviation. For this purpose, the data points were fitted to Fick’s law of diffusion (see equation (1)):



= 

 ∗ ∗ 

 ∗ 
 −  

(1)

where A is the membrane surface area, h is the thickness of the dialysis membrane, Vα is the volume of the acceptor compartment, and Cα and Cd are the concentrations of flurbiprofen within the acceptor and donor compartments, respectively. Defining the initial amount of flurbiprofen as Q0, the concentration of the drug within the donor compartment can be described as follows: 
 =

  ∗  

(2)



Accordingly, equation (1) can be transformed to equation (3) with the analytical solution which is presented in equation (4), where the term Q0/(Vα+Vd) is the concentration of flurbiprofen at the equilibrium.



 ∗

=  ∗ ∗

  = 





  

  ∗ 



 1 −

−  

!∗" ∗#$ %$  &∗$ ∗$

'

(3)

(4)

The rate at which the drug concentration increases within the acceptor compartment and the total amount of the drug released from the nanocarrier can then be calculated according to the remaining steps of the four-step model (step 2: computation of the concentration rate of the acceptor compartment; step 3: computation of the concentration rate of the donor compartment; step 4: calculation of the total amount of released drug) [29]. For each colloidal

15

formulation, plots of the free drug concentration in the donor compartment and the total free drug concentration were constructed using this model. The concentration of the free drug was calculated with the help of equation (5):



≈

∆ ∆

= 

 ∗ ∗ 

 ∗ 
 −  

(5)

The slope was obtained by linear regression of three following data points. The finite differences ∆Cα and ∆t were approximated to calculate the slope dCa/dt. The free drug concentration within the donor compartment was calculated as follows (see equation (6)): ∆

∗ 


 =  ∆  ∗ 

∗

+  

(6)

Finally, the amount of the free drug in both compartments (Qt) was calculated, assuming a constant volume in both chambers, according to equation (7): +  = 
 ∗ ,
+   ∗ ,

(7)

The formulations were additionally compared using the normalized release rate, which can also be obtained from these calculations. The concentration profiles of the free drug determined for each compartment were expressed by the compartmental accumulation ratio (CAR), which is defined as follows:  

 -.
 = /0

(8)

or

 

 -.  = /0

(9)

with the concentrations of free flurbiprofen in each compartment at time t (cd(t) and cα(t)) and the concentration of free flurbiprofen in the equilibrium (c(∞)).

5.10.

Mathematical modeling describing the drug release from nanoparticles

16

Understanding the mechanism of drug release is essential in formulation development and quality control [23]. Two mathematical models were evaluated for the description and interpretation of drug release kinetics from the nanoformulations. In most cases, the release profiles of colloidal drug carriers can be appropriately described by a regression based on a first order kinetics [28, 39-41]. However, in some cases this model was not able to explain the release profile satisfactorily [28]. For our studies, the rpt model [28] and the threeparameter model [30] were both evaluated for their ability to describe the concentration profiles of the total free drug calculated with the four step model. The curve fit achieved with both models was compared. Further, the descriptive parameters obtained from each of the models were taken into account. In the rpt model, the drug release is explained with a combination of diffusion and dissolution mechanisms. The fraction of the drug that is released from the nanoparticle formulation (F) depends on the amount of the drug released in the medium (w) and the total amount of the drug remaining to be released (M). The effective surface area (A), the diffusion coefficient (D) and the diffusion length (h) play a role in this process. A time-dependent variable (X) is used to describe the changes in the release rate which may occur as a result of hydration, swelling or erosion (see equation (10)). 1=

2

23

=

# 5∗!∗6 & #5∗!∗6 7 5∗!∗6 4 & 4# &

4

(10)

The constants and integrals of equation (10) can be condensed into two constants, m and b, as shown in equation (11) [28]: 8

1 =  8 9

(11)

Therefore, the rpt model is more useful in summarizing various release parameters than describing the release mechanism. But a release rate that is the result of dissolution and diffusion mechanisms is more likely to result in an appropriate curve fit.

17

Zeng and coworkers used a three-parameter model to analyze the influence of the molecular weight of polymeric drug carriers, the nanoparticle diameter, and composition of release medium on drug release profiles of various colloidal formulations. In contrast to the limitations of the rpt model, the three-parameter model provides a number of release parameters closely related to the mechanisms of release. During the initial phase, the release rate is strongly affected by the diffusion process while, in the second phase, reversible drug-carrier interactions are more pronounced. The total drug release depends on time (t), the average concentration of the drug in the nanocarrier (cN), the surface area (A) and the nanocarrier volume (VN).
/


= −:; ∗ <=

(12)

The rate constant, kS, is defined as follows :> = - ∗

?

= - ∗

@∗A

(13)

∗

and is a function of the diffusion coefficient (D), the partitioning coefficient (K) and the thickness of the shell (h). In the present study, the release profiles were appropriately described by equation (14) (derived from equation (12)) indicating an initial rapid diffusion of the drug, followed by a slow association/dissociation reaction. During the initial phase, the release is described by the release parameter kS. In the second phase, the rate constants of association (kon) and dissociation (koff) are the primary influences on the release rate. 3#

3

=

BCC

BD BCC

E1 −

F ∗ G

+

BD

BD BCC

E1 −

BCC ∗

G

(14)

From the model the difference in the free energy of the bound and the unbound fraction of the drug was calculated (∆G, see equations (15) and (16)). Essential parameters are the Boltzmann constant (kB), the absolute temperature (T) and the rate constants kon and koff.

18

∆H = −:I ∗ J ∗ ln

BD

BCC

(15)

To obtain ∆G an initial burst release (IniB) was defined in the release profile of every nanoparticle formulation and the difference in free energy was calculated by applying equation (16). IniB =

P

R

(16)

∆S

PQ "T ∗U

Afterwards, the release parameter ks and the rate constant koff were calculated with the help of equations (17) to (19). For this purpose, the relative release rate (reR) of the initial burst (early phase of the release profile, t ≈ 0) was determined by applying linear regression to the early time points of the release profile. Similarly, the slope λ2 was obtained by applying linear regression to the plateau phase. :; = V . ∗ 1 + 3

ln 1 − 3#  = ln E 

YP,Z =

5.11.



∆S "T ∗U



W?∗W? F 

BDBCC G∗W? WX 

(17)  − YZ ∗ 

F BD BCC ±]F BD BCC X^∗F ∗BCC Z

(18)

(19)

Statistical and graphical analysis

All data are expressed as the mean value ± standard deviation (SD). DLS measurements, drug content of nanoparticles, solubility, recovery determination, and release of nanoparticles were performed in triplicate. All other experiments were performed with n=6. The mean value and standard deviation were calculated by using Microsoft Excel (Microsoft, Redmond, USA). The paired t-test for analysis of significance was performed with SigmaPlot 11.0 (Systat Software GmbH, Erkrath, Germany) for DLS data. The release profiles of flurbiprofen were analyzed by using MATHEMATICA® (Wolfram Research, Inc., Champaign, IL, USA). For the 19

three-parameter model, a programmed interface allowed the definition of reR, λ2 and IniB value based on the graphical analysis of the release curve as described above. The R² of the curve fit was calculated with the help of the software by manually defining the threeparameter model with all input parameters as equation (see section 5.10).

20

6. Results and discussion Many efforts have been made to investigate the release of drugs and drug candidates from nanosized drug carriers. In most cases, the inherent barrier properties of the dialysis membrane impose a limit to the sensitivity of dialysis-based techniques. With the DR technology, a more effective membrane transport resulted in a highly sensitive analytical tool for investigating the release rate of flurbiprofen from nanocarriers in various media. A weak acid of the BCS class 2, flurbiprofen, was used to validate this new technology. Further, the mechanisms involved in the release of the drug were investigated.

6.1.

Quantification and equilibrium solubility of flurbiprofen in various media

Initially, the equilibrium solubility of the drug was quantified in order to confirm sink conditions during the time of the release experiments. The observed thermodynamic solubility (24 h) of flurbiprofen in PBS at pH 7.4 at 37 °C was 1.68 ± 0.01 mg/mL. The addition of BSA to the buffer solution improved the solubility of the weak acid even at the slightly lower pH value (7.2). For flurbiprofen, a plasma protein binding of approximately 99% has been reported [42]. The solubility of the API increased to values of 4.10 ± 0.09 mg/mL in phosphate-albumin buffered saline pH 7.2 R1 and to 4.33 ± 0.06 mg/mL in phosphate-albumin buffered saline pH 7.2 R. Accordingly, with a maximum concentration of 2.86 µg/mL of flurbiprofen within the dissolution vessel, sink conditions were maintained during all release experiments. Prior to the release tests, the recovery of flurbiprofen was determined from various release media by HPLC analysis. With an average recovery of 95.30 ± 0.73% the requirements defined by the US-FDA were met for all of the tested concentrations [27].

21

6.2.

Manufacture of polymeric nanoparticles

Two of the three tested polymeric nanocarrier formulations of flurbiprofen were prepared by the emulsion-diffusion evaporation method as described previously [31]. For the two batches of PLA nanoparticles, a particle diameter of 267.3 ± 0.9 nm and 271.9 ± 1.7 nm was determined by DLS. Both batches were comparable in their size and exhibited monodisperse size distribution, as indicated by the PDI (0.029 ± 0.008 for batch 1 and 0.041 ± 0.025 for batch 2). At this point only slight differences in the physicochemical properties were observed. After purification, a total drug load of 4.68 µg/mg for batch 1 and 4.73 µg/mg for batch 2 was measured. Following the same protocol, flurbiprofen-loaded PLGA nanoparticles with a particle size of 273.6 ± 0.9 nm, a PDI of 0.083 ± 0.019, and a drug load of 6.47 µg/mg were prepared. Flurbiprofen-loaded

Eudragit® RS PO

nanoparticles

were

manufactured

by

a

nanoprecipitation method described in section 5.2. This batch exhibited a particle size of 185.9 ± 1.3 nm and a PDI of 0.089 ± 0.017, with a total drug loading of 109.78 µg/mg.

6.3.

Membrane leakage test of the dialysis membrane in the dispersion releaser setup

A number of dialysis-based techniques have been applied to evaluate the release of drugs from nanoformulations [5, 9, 13, 15, 16, 18]. Since the barrier properties of the dialysis membrane are essential for the outcome of the release study, the membrane leakage was evaluated (see Table 1, experiment no. 0). For this purpose, BSA was filled into the donor chamber equipped with a RC membrane with a MWCO of 50 kDa. At an average molecular weight of 66 kDa [43], a retention of approximately 90% of BSA is defined by the manufacturer over a time period of 17 hours [36]. With the DR setup a total release of 22

10.0 ± 3.9% was achieved over a time period of 24 h (see Figure 2). Thus, the retention criteria were met and no further leakage from the donor chamber was observed.

6.4.

Membrane permeation of flurbiprofen in the dispersion releaser

The membrane permeation of flurbiprofen was examined in the DR under various conditions (see Table 1, experiment no. 1 to 6). Firstly, the intermediate precision was investigated by conducting repeated measurements with different operators and equipment. There was no significant effect on the detected release profiles observed. Further, the kM values determined from the release profiles indicate a comparable membrane transport in both experiments (see Table 2, experiment no. 1 and 2). For this reason, only one of these two experiments is presented (see Figure 3). As expected, the release profiles were strongly affected by the composition and the temperature of the release medium (see Figure 3 A and C). The kM values of the membrane transport were calculated from these experiments and have been listed in Table 2. At elevated temperature the kM value was increased by a factor of 1.2 (see experiment no. 3). A stirring rate of 50 rpm in the DR setup was sufficient to maintain a constant and rapid membrane transport (see Figure 3 B). There was no significant difference between the kM values at 50 and 75 rpm observed (see experiment no. 2 and 4). However, the standard deviation of kM was found to be higher at an increased temperature or at a reduced stirring rate. More often, physiological fluids have been recommended by the regulatory authorities for testing the release of non-oral dosage forms. Unfortunately, due to the challenges associated with a higher content of proteins or plasma in dialysis-based setups, most studies are still conducted in buffer media [5, 16, 44].

23

For flurbiprofen a high plasma protein binding (99%) has been reported in literature [42]. Therefore, the effect of BSA on membrane permeability of the compound was studied using two compendial media described by the European Pharmacopeia. They contain BSA concentration of 1.0 g/L and 10.0 g/L, respectively. These concentrations do not correspond to the albumin concentrations found in the human blood stream but represent a wellaccepted standard in the testing of formulations. When adding BSA at a concentration of 1.0 g/L there was only a minor impact on the release profile observed (experiment no. 6, see Figure 3C). At the higher concentration of 10.0 g/L, the membrane permeation was significantly reduced (experiment no. 5, see Figure 3C). An interaction of flurbiprofen with the surface of the membrane or a delayed transport of the compound bound to the protein are the most likely explanations.

6.5.

Discrimination between different formulations with dispersion releaser technology

To discriminate between different particle preparations and particle batches, the drug release of flurbiprofen from PLA (experiment no. 7 and 8), Eudragit® (experiment no. 9), and PLGA (experiment no. 10) nanoparticles was quantified with the help of the DR technology. For PLGA and Eudragit® nanoparticles a burst release of more than 90% within 4 hours has been determined (see Figure 4). The calculation of the concentration of free flurbiprofen within the donor compartment using the four-step model (see section 5.9) confirmed this observation (see Figure 6). Similar findings have been reported for flurbiprofen-loaded PLGA and Eudragit® nanoparticles previously [11, 45]. By contrast, a prolonged release was observed for PLA nanoparticles that exhibited

a

biphasic profile (see Figure 4). Less than 50% of the drug was released within 4 hours and by 48 h only 66.1 ± 0.4% of the flurbiprofen from batch 1 and 63.4 ± 0.7% of the flurbiprofen from batch 2 have been released. Obviously, the poorly standardized small-scale 24

manufacturing process was responsible for the differences between those two release profiles. Importantly, this variation in the performance of both formulations was not indicated by the physicochemical characterization but by the release experiments only. After each release experiment samples were taken from the donor compartment and particle size was measured by DLS in order to detect changes in the particle structure. Even after this

procedure,

there

were

no

pronounced

differences

observed

between

the

physicochemical properties of the two particle batches (279.6 ± 3.1 nm, PDI 0.030 ± 0.010 for batch 1; particle size 263.3 ± 3.4 nm, PDI 0.040 ± 0.028 for batch 2). Conclusively, the release experiments conducted with the DR technology discriminated between different formulations but also between different particle batches manufactured in a poorly standardized manufacturing process.

6.6.

Comparison between dispersion releaser and dialysis bag technique

In addition to the release experiments conducted with the novel DR technology, the DB technique was applied investigating the differences in sensitivity between those methods. The DB setup is frequently used to measure the drug release from colloidal drug carriers such as nanoparticles, liposomes or polymeric micelles [15, 17, 46]. Most experimental parameters including the temperature, the stirring rate, the membrane material, the release medium and MWCO of the dialysis membrane were kept constant. Other parameters had to be changed because of the differences between those setups (e.g. surface area). The DB technique confirmed the results obtained with the DR setup: a modified release profile of flurbiprofen from PLA nanocarriers (experiment no. 12) and an immediate release when an aqueous solution of the drug was added to the donor chamber (experiment no. 11, see Figure 5A and B).

25

Essentially, a disadvantage of the DB technique was revealed by the broader standard deviations when the solution was tested (see Figure 5B). During the early time points of the test, a high amount of the drug is accumulating within the donor chamber. Under such conditions, as they also may occur during the initial burst of a formulation, the DB method is less sensitive than the DR setup. This effect was further investigated by calculating the kM values for DB and DR. The membrane transport was significantly lower when the DB technique was applied. Further, the SD of kM increased by a factor of 10 (see Table 2, experiment no 1 and 11). Although both methods confirm each other in their results, the DR technology is more sensitive to a rapid increase in the drug concentration. Therefore, it is more sensitive to fluctuations in the release profile than the DB technique.

6.7.

Mathematical modeling to describe drug release and membrane permeation kinetics in dialysis-based experiments

For all release experiments conducted with nanoformulations the concentration of the free drug in the donor compartment and the total concentration of free flurbiprofen (see Figure 7) were calculated by applying the four-step model [29]. Further, the CAR was calculated to illustrate the concentration profile in donor compartment (see Figure 6) Whereas the concentration of the free drug in the donor compartment reaches its maximum during the first minutes of the release experiment for Eudragit® and PLGA nanoparticles (t = 0 hours, see Figure 6 C and D ), the concentration of the free drug increases during the first hour for the PLA formulation (see Figure 6 A and B). This confirms the observations made with the non-normalized profiles (see section 6.5). The differences between the formulations are more pronounced when using the normalized drug release rate (see Figure 7).

26

6.8.

Mathematical

modeling

to

describe

the

drug

release

from

nanoparticles

The rpt model and the three-parameter model were applied to investigate the mechanism of drug release for all tested nanoformulations. Good correlations were achieved for all formulations, as indicated by the correlation coefficients (R2, see Table 3 and 4). The release parameters m and b were calculated with the rpt model (see Table 3). They describe the drug release by a combination of diffusion and dissolution processes and do not provide detailed information about the strength of the distinct drug-carrier interactions. For nanocarrier systems involving such mechanisms the profile may not be described appropriately by these two values. However, the model is suitable for nanocarrier systems loaded with flurbiprofen and could be used to detect process changes or batch-to-batch reproducibility. However, since the two parameters are very sensitive to small changes in the profile, applying them in quality control setting would also require careful selection of the rejection criteria. In the three-parameter model the kinetic parameters ks and koff and the amount of the free energy in the drug delivery system, ∆G, are calculated from the release profile (see Table 4). They allow a deeper understanding and interpretation of the release behavior of flurbiprofen from the tested nanoformulations. In particular, ∆G and the dissociation parameter koff are determined by the strength of the drug-polymer interactions. As proposed by the originators of this model, a burst release of the drug is associated with a weak drug-carrier interaction [30] and thus the difference in the free energy ∆G is much higher for Eudragit® and PLGA nanoparticles than observed for nanoparticles composed of PLA (see Table 4). Furthermore, the differences in ∆G among several batches of nanoparticles indicate a change in the encapsulation. Unfortunately, all input parameters in the three-parameter

27

model are obtained by manual graphical analysis of the release profile. This is potentially a weakness of the three-parameter model. The shape of the release profile during the initial phase of the burst release is described by the release parameter kS. For nanoformulations tested, kS values ranged from 2.717 to 5.064 1/h. The higher kS values calculated for the PLGA and Eudragit® nanoparticles indicate that the more rapid release of flurbiprofen from these formulations is due to a weak drugpolymer interaction which was also responsible for higher ∆G values. Since these two parameters (∆G and kS) are closely related to carrier composition and the technical parameters of manufacture, they could be useful tools in formulation development. For those formulations that are very similar in composition as indicated by their ∆G and kS values, the dissociation parameter koff could be applied to further optimization. With 0.0189 and 0.0151 1/h, there were only minor differences in the koff values between the two PLA nanoparticle batches observed. The koff value is related to the dissociation of the drug in the equilibrium and is closely related to the carrier material. However, further sensitivity analysis and the testing of more batches will be needed to evaluate the ranges in which this parameter varies. In summary, an acceptable curve fit was achieved with both models. While the rpt model could be more easily applied in automated processes, the three parameter model describes the different mechanisms involved in the release behavior more appropriately. However, manual graphical analysis still is an important weakness of the three-parameter model.

7. Conclusion The in vitro release of the BCS class 2 weak acid flurbiprofen was investigated by applying the novel DR technology. To study the sensitivity and robustness of this setup at a high release rate and under various experimental conditions, the membrane permeability constants were first calculated for the permeation of flurbiprofen from an aqueous solution. 28

Even at an elevated temperature of 50 °C differences in the membrane transport of flurbiprofen could be sensitively detected in the acceptor compartment (see Table 2). Accelerated testing conditions such as these have previously been applied to the release testing of long acting parenterals [6, 47]. Compared to the DB technique, the DR showed higher reproducibility, as confirmed by smaller standard deviations. The DR technology was able to discriminate effectively among different nanoformulations of flurbiprofen. The normalized release of flurbiprofen from the nanoparticles was calculated by applying the fourstep model. These normalized release profiles were used to study the mechanisms of drug release from different nanoparticle species using the rpt model and the three-parameter model. While these two approaches resulted in optimal fits for all release profiles, they differed with regards to the field of potential applications in research and quality control. Further, for long-acting PLA nanoparticles the DR technology sensitively discriminated between two particle batches exhibiting similar physicochemical properties. In conclusion, the study illustrates the outstanding potential of the DR technology as an analytical tool in pharmaceutical development. A high sensitivity at different release rates and under various experimental conditions will help us gain a better understanding of the mechanisms involved in the drug release from nanoformulations.

8. Acknowledgements The authors acknowledge Dr. Dirk Beilke, Dr. Björn Fähler (Pharma Test Apparatebau AG, Hainburg, Germany) and Susanne Beyer for their support. The authors also acknowledge the LOEWE initiative of the State of Hessen for financial support.

9. Figure captions Figure 1: The Dispersion Releaser: Principle of drug release (A), systematic drawing (B), and the Dispersion Releaser in an USP apparatus 2 in mini vessel configuration (C). 29

Figure 2: Membrane permeation of BSA into the acceptor chamber of the dispersion releaser. Figure 3: Membrane permeability of the pure API. Investigation of the effects of temperature (A), stirring rate (B), and BSA amount within the release medium (C). Experiment no. 1 ( no. 3 (

), no. 4 (

), no. 5 (

), and no. 6 (

).

Figure 4: Cumulative drug release of flurbiprofen from drug solution ( nanoparticles made of Eudragit® (

), PLGA (

), and PLA batch 1 (

) and polymeric

) and batch 2 (

Figure 5: Cumulative drug release of flurbiprofen from drug solution ( nanoparticles batch 2 (

),

).

) and PLA

). Experiments performed with the DR (A) and the DB technique

(B). Figure 6: Compartment accumulation ratio of release experiments performed with nanoparticles composed of PLA (batch 1 (A) and batch 2 (B)), Eudragit® (C), and PLGA (D), CAR of the acceptor compartment (-●-) and donor compartment (-○-). Figure 7: Release profile of the free drug concentration in the acceptor compartment (-●-) and the calculated total free drug concentration (-○-) for nanoparticles composed of PLA (batch 1 (A) and batch 2 (B)), Eudragit® (C), and PLGA (D).

30

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36

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Table 1: Experimental design used for the evaluation of the DR technology.

Experiment

Purpose

Setup

Release

Temperature

Medium

Stirring rate

No. 0

Membrane leakage

DR

PBS pH 7.4

37 °C

75 rpm

No. 1

Drug permeability

DR

PBS pH 7.4

37 °C

75 rpm

No. 2

Intermediate precision

DR

PBS pH 7.4

37 °C

75 rpm

No. 3

Robustness

DR

PBS pH 7.4

50 °C

75 rpm

No. 4

Robustness

DR

PBS pH 7.4

37 °C

50 rpm

No. 5

Robustness

DR

PBS pH 7.2

37 °C

75 rpm

37 °C

75 rpm

+10 g/L BSA No. 6

Robustness

DR

PBS pH 7.2 +1 g/L BSA

No. 7

PLA NP release, batch 1

DR

PBS pH 7.4

37 °C

75 rpm

No. 8

PLA NP release, batch 2

DR

PBS pH 7.4

37 °C

75 rpm

No. 9

Eudragit® NP release

DR

PBS pH 7.4

37 °C

75 rpm

No. 10

PLGA NP release

DR

PBS pH 7.4

37 °C

75 rpm

No. 11

Drug permeability

DB

PBS pH 7.4

37 °C

75 rpm

No. 12

PLA NP release, batch 2

DB

PBS pH 7.4

37 °C

75 rpm

Table 2: Overview of the permeability experiments and calculated kM values of flurbiprofen.

Experiment

Setup

Average kM [cm2 * h-1]

SD of kM [cm2 * h-1]

No. 1

DR, 37 °C, 75 rpm

2.143 x10-3

5.977 x10-5

No. 2

DR, 37 °C, 75 rpm

2.161 x10-3

1.061 x10-4

No. 3

DR, 50 °C, 75 rpm

2.585 x10-3

2.263 x10-4

No. 4

DR, 37 °C, 50 rpm

2.111 x10-3

1.052 x10-4

No. 5

DR, 37 °C, 75 rpm

2.820 x10-4

1.225 x10-5

1.894 x10-3

9.101 x10-5

1.548 x10-3

6.190 x10-4

Medium with 10 g/L BSA No. 6

DR, 37 °C, 75 rpm Medium with 1 g/L BSA

No. 11

DB, 37 °C, 75 rpm

Table 3: Parameters of drug release from flurbiprofen-loaded nanoparticles performed with the DR technique and analyzed with the rpt model.

Experiment

Description

m

b

R2

No. 7

PLA NP, batch 1

1.062

0.242

0.9893

No. 8

PLA NP, batch 2

1.967

0.310

0.9962

No. 9

Eudragit® NP

0.064

0.459

0.9996

No. 10

PLGA NP

0.0003

22.955

0.9961

Table 4: Parameters of drug release from flurbiprofen-loaded nanoparticles performed with the DR technique and analyzed with the three-parameter model.

Experiment

IniB

ΔG [J]

kS [1/h]

koff [1/h]

R2

No. 7

0.47

-4.86 x10-22

3.553

0.0189

0.9942

No. 8

0.46

-6.48 x10-22

2.717

0.0151

0.9987

No. 9

0.97

1.41 x10-20

3.866

0.0462

0.9949

No. 10

0.94

1.11 x10-20

5.064

0.0284

0.9978

Graphical abstract