Self-emulsifying drug delivery systems: Impact of stability of hydrophobic ion pairs on drug release

Accepted Manuscript Self-emulsifying drug delivery systems: Impact of stability of hydrophobic ion pairs on drug release Imran Nazir, Mulazim Hussain Asim, Aida Dizdarević, Andreas BernkopSchnürch PII: DOI: Reference:

S0378-5173(19)30176-0 https://doi.org/10.1016/j.ijpharm.2019.03.001 IJP 18182

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

10 January 2019 28 February 2019 1 March 2019

Please cite this article as: I. Nazir, M.H. Asim, A. Dizdarević, A. Bernkop-Schnürch, Self-emulsifying drug delivery systems: Impact of stability of hydrophobic ion pairs on drug release, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.2019.03.001

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Self-emulsifying drug delivery systems: Impact of stability of hydrophobic ion pairs on drug release Imran Nazir1,2, Mulazim Hussain Asim1,3 , Aida Dizdarević1, Andreas Bernkop-Schnürch1* 1

Center for Chemistry and Biomedicine, Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 80/82, A-6020 Innsbruck, Austria 2

Department of Pharmacy, COMSATS University Islamabad, Abbottabad campus, 22010 Abbottabad, Pakistan 3

Department of Pharmaceutics, Faculty of Pharmacy, University of Sargodha, 40100 Sargodha, Pakistan

*Corresponding author: Andreas Bernkop-Schnürch E-mail address: [email protected] Tel.: +43 512 507 58601; Fax: +43 512 507 58699

1

ABSTRACT The aim of this study was to evaluate the impact of stability of hydrophobic ion pairs (HIPs) in gastrointestinal (GI) fluids on their release from self-emulsifying drug delivery systems (SEDDS). HIPs of leuprolide (LEU), insulin (INS) and bovine serum albumin (BSA) were formed using various mono- and di-carboxylate surfactants (sodium deoxycholate (SDC), sodium dodecanoate (SDD), sodium stearoyl glutamate (SSG) and pamoic acid di-sodium salt (PAM)). HIPs were evaluated regarding precipitation efficiency, log Pn-butanol/water and dissociation behavior at various pH and ionic strength. Solubility studies of these HIPs were accomplished to identify suitable solvents for the formulation of SEDDS. Subsequently, HIPs were incorporated into SEDDS followed by characterization regarding zeta potential, stability and log DSEDDS/release medium. Independent from the type of (poly)peptides, PAM showed most efficient HIP properties among tested surfactants. The highest encapsulation efficiency with PAM was achieved at molar ratios of 1:1 for LEU, 3:1 for INS and 50:1 for BSA and log Pnbutanol/water

of HIPs were increased at least 2.5 units. Dissociation studies showed that LEU-

PAM, INS-PAM, BSA-PAM complexes were dissociated within 6 h up to 25%, 60% and 85% in GI fluids, respectively. These HIPs were successfully incorporated into SEDDS exhibiting negative zeta potential and high stability for 4 h. Log DSEDDS/release medium of LEU-PAM, INSPAM, BSA-PAM complexes were 2.4 ± 0.7, 2.1 ± 0.62 and 1.6 ± 0.45, respectively. Findings of this study showed that stability of HIPs has great impact on log DSEDDS/release medium and consequently on their release from SEDDS. Keywords: Hydrophobic ion pair; Pamoic acid; Self-emulsifying drug delivery system (SEDDS); Dissociation; log DSEDDS/release medium; (poly)peptides.

2

1. INTRODUCTION Self-emulsifying drug delivery systems (SEDDS) are homogeneous, isotropic mixtures of oils, surfactants and co-solvents spontaneously forming oil in water emulsions after dilution with gastrointestinal (GI) fluids (Gursoy and Benita, 2004; Karamanidou et al., 2015). They have been considered as a potential platform for oral delivery of hydrophilic macromolecular drugs such as therapeutic (poly)peptides, DNA- and RNA-based drugs in recent years (AboulFotouh et al., 2018; Rao and Shao, 2008). Incorporating hydrophilic macromolecular drugs into SEDDS protects them towards the enzymatic and sulfhydryl barrier of GI tract. Moreover, SEDDS are able to permeate the mucus gel barrier in a comparatively efficient manner facilitating the transport of incorporated hydrophilic macromolecular drugs to the underlying epithelium (Mahmood and Bernkop-Schnürch, 2018). The incorporation of hydrophilic macromolecular drugs into SEDDS, however, is a great challenge. In recent years, hydrophobic ion pairing (HIP) turned out to be the method of choice to address this challenge (Hetényi et al., 2017; Meyer and Manning, 1998; Powers et al., 1993). HIP is a non-covalent lipidization method, whereby hydrophilic macromolecular drugs form a water insoluble complex with oppositely charged surfactants via electrostatic interactions (Zupančič and Bernkop-Schnürch, 2017). Advantage of SEDDS, however, can only become effective when the incorporated HIPs remain in the oily droplets formed in the intestinal fluid until they have reached the absorption membrane (Li et al., 2017). The release behavior of HIPs from SEDDS seems therefore to play a crucial role in the overall performance of these delivery systems. As equilibrium in drug concentration between the lipophilic phase of SEDDS and the aqueous phase of the intestinal fluid is immediately reached, the distribution coefficient between SEDDS pre-concentrate and release medium –designated log DSEDDS/release medium – can be considered as the key parameter for drug release (Bernkop-Schnürch and Jalil, 2018; Mahmood and Bernkop3

Schnürch, 2018). As due to the self-emulsifying properties of SEDDS the distribution of HIPs between the lipophilic and aqueous phase cannot be evaluated, log DSEDDS/release medium is instead obtained by determining the maximum solubility of HIPs in both phases (Bonengel et al., 2018). This method, however, might be misleading if HIPs dissociate in the aqueous medium over time, as the maximum drug solubility in the aqueous phase strongly increases and log DSEDDS/release medium consequently decreases by this effect. It was therefore the aim of this study to determine the dissociation of HIPs in various aqueous media over time and to evaluate the impact of this dissociation behavior on their release from SEDDS. For comparison short, medium and high molecular weight (poly)peptides were chosen in this study. Various anionic surfactants SDS, SDD, SSG and PAM exhibiting mono- and dicarboxylic moieties were used for HIP with LEU, INS and BSA. Resulting HIPs were incorporated into various SEDDS formulations, which were characterized regarding droplet size, zeta potential and log DSEDDS/release medium. The dissociation of HIPs was investigated under simulated intestinal conditions and the resulting impact on drug release was evaluated. 2.

Materials and methods

2.1.

Materials

Leuprolide acetate was purchased from Chemos GmbH (Regenstauf, Germany). Human recombinant insulin, bovine serum albumin, pamoic acid di sodium salt, sodium dodecanoate, sodium doxycholate, Tween 20 (polyethylene glycol sorbitan monolaurate), DMSO and tetraglycol were purchased from Sigma Aldrich (Vienna, Austria). Sodium stearoyl glutamate was purchased from BASF (Augustin, Germany). Transcutol HP (diethylene glycol monoethyl ether), Labrasol ALF (caprylocaproyl macrogol-8 glycerides), Capryol 90 (propylene glycol monocaprylate type II), Labrafil M2125 CS (linoleoyl macrogol-6 glycerides) and Peceol (glycerol mono-oleates type 40) were a gift from Gattefossé (Lyon, France). All other reagents, chemicals and solvents used were of analytical grade and obtained from commercial sources. 4

2.2.

Methods

2.2.1. HPLC analysis The concentration of LEU, INS and BSA were analyzed by HPLC using a Hitachi Elite LaChrom HPLC- System equipped with L-2130 pump, L-2200 auto sampler and L-2400 UV detector by previously described methods with minor modifications (Griesser et al., 2017; Sarmento et al., 2006; Wong et al., 2018). The samples were quantified by using a reversephase Nucleosil 100-5 C18 5 µm, 4.6 mm x 250 mm column as stationary phase. A binary solvent system was used, where solvent A was 0.1% (v/v) trifluoroacetic acid (TFA) in water and solvent B was acetonitrile. LEU was quantified by a gradient method, where 0-12 min: 75% A/25% B - 50% A/50% B and 12-14 min: 75% A/25% B were used as mobile phase at 40 oC with a flow rate of 1 mL/min. Furthermore, all samples were stored within autosampler at 10 o

C until 20 µL injected and subsequently analyzed at a wavelength of 222 nm. INS was

quantified by a gradient method, where 0-12 min: 70% A/30% B - 45% A/55% B, 12-14 min: 70% A/30% B were used as mobile phase at 40 oC with a flow rate of 1 mL/min. Sample of 20 µL was injected and analyzed at a wavelength of 214 nm. BSA was also quantified by a gradient method, where 0-9 min: 90% A/10% B - 50% A/50% B, 9-15 min: 80% A/20% B and 15-17 min: 90% A/10% B were used as mobile phase at 40 oC with a flow rate of 1 mL/min. The injection volume was 20 µL and samples were analyzed at a wavelength of 222 nm. 2.2.2. Preparations of HIPs Lipophilic complexes of (poly)peptides were prepared by HIP with aqueous solutions of ion pairing agents as described previously with minor modifications (Griesser et al., 2017). Briefly, LEU, INS and BSA were dissolved in 0.01 M HCl in a final concentration of 5 mg/mL as they exhibit net positive charges at this pH as listed in Table 1. Solutions of anionic surfactants were prepared in 1 mL of demineralized water (DW) applying various molar ratios of surfactant to (poly)peptides as listed in Table 1. Thereafter, the solution of each surfactant was added 5

dropwise to each (poly)peptide solution (5 mg/mL) while shaking at 400 rpm for 2 h at 25 oC. A cloudy solution was formed immediately indicating the formation of HIPs of (poly)peptides. The precipitated HIPs formed were isolated by centrifugation at 10,500 g for 10 min using a MiniSpin Centrifuge (Eppendorf, Hamburg, Germany). The HIPs were washed twice with purified water, lyophilized (Christ Gamma 1-16 LSC Freeze dryer) and stored at -20 oC until further use. The precipitation efficiency was calculated by measuring the remaining amount of (poly)peptides in the supernatant by HPLC as described above using the Eq. (1). (𝑝𝑜𝑙𝑦)𝑝𝑒𝑝𝑡𝑖𝑑𝑒𝑠 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑎𝑓𝑡𝑒𝑟 𝐻𝐼𝑃

𝑃𝑟𝑒𝑐𝑖𝑝𝑖𝑡𝑎𝑡𝑖𝑜𝑛 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 [%] = 100 − ((𝑝𝑜𝑙𝑦)𝑝𝑒𝑝𝑡𝑖𝑑𝑒𝑠

𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑏𝑒𝑓𝑜𝑟𝑒 𝐻𝐼𝑃

× 100) (1)

2.2.3. Partitioning coefficient (logPn-butanol/water) determination LogPn-butanol/water of (poly)peptides and their HIPs were determined in n-butanol and in water by using a previously described method with minor modification (Zupančič et al., 2016b). Briefly, 1 mg of (poly)peptides or HIPs was added in 1 mL of n-butanol/water (1:1). The mixture was incubated at 37 oC with shaking at 500 rpm for 24 h. Thereafter, samples were centrifuged at 10,500 g for 10 min. Aliquots of 100 µL were withdrawn from both phases and diluted with 300 µL of methanol containing 0.1% (v/v) TFA. The amount of (poly)peptides in n-butanol and in water was quantified by HPLC as described above. LogPn-butanol/water was calculated according to Eq. (2). 𝐿𝑜𝑔 𝑃𝑛−𝑏𝑢𝑡𝑎𝑛𝑜𝑙/𝑤𝑎𝑡𝑒𝑟 = 𝑙𝑜𝑔

𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 (𝑝𝑜𝑙𝑦)𝑝𝑒𝑝𝑡𝑖𝑑𝑒𝑠 𝑖𝑛 𝑛−𝑏𝑢𝑡𝑎𝑛𝑜𝑙 𝑝ℎ𝑎𝑠𝑒 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 (𝑝𝑜𝑙𝑦)𝑝𝑒𝑝𝑡𝑖𝑑𝑒𝑠 𝑖𝑛 𝑎𝑞𝑢𝑒𝑜𝑢𝑠 𝑝ℎ𝑎𝑠𝑒

(2)

2.2.4. Solubility studies Increasing amounts of HIPs were added to lipids, surfactants and organic solvents. Solubility of HIPs was in particular examined in Transcutol HP, tetraglycol, propylene glycol, Peceol, Capryol 90, Labrasol ALF, Labrafil M 2125 CS, Tween 80 and DMSO. The mixture of HIPs and solvents were sonicated while shaking at 1000 rpm for 24 h at 37 oC. Afterwards, solubility of these HIPs was evaluated visually after centrifugation at 10,500 g for 5 min.

6

2.2.5. SEDDS development and characterization In order to develop SEDDS formulations, different concentrations of oil, surfactants and cosolvents were homogenized as listed in Table 2. The excipients were added to 2 mL reaction tubes and homogenized by vortex mixer (Thermomixer comfort, Eppendorf, Germany) at 40 °C under constant shaking at 1000 rpm, whereby semisolid components were melted before use as described previously (Zupančič et al., 2016b). Based on the results of preliminary solubility studies, SEDDS loaded with HIPs of LEU, INS and BSA were developed. Briefly, HIPs were incorporated into SEDDS in a concentration of 2% via ultrasonification for 10 min at room temperature. As illustrated in Table 2, HIPs of LEU, INS and BSA were incorporated in formulation FI, FII and FIII, respectively. The incorporation of HIPs was evaluated visually after centrifugation at 10,500 g for 5 min. Thereafter, 10 µL of SEDDS were added to 990 µL of 10 mM phosphate buffer (PB) pH 6.8 containing 137 mM NaCl and incubated at 37 ̊ C for 4 h while shaking at 500 rpm. Size and zeta potential of blank as well as HIPs loaded SEDDS formulations were analyzed by photon

correlation

spectroscopy

with

Zetasizer

Nano-ZSP

(Malvern

instruments,

Worcestershire, UK). Aliquots of 10 µL of SEDDS were diluted in 990 µL of 10 mM PB pH 6.8 containing 137 mM NaCl for size measurements and diluted with DW for zeta potential measurement. In order to investigate stability of SEDDS, size and zeta potential of SEDDS were evaluated at 0 and 4 h while shaking at 500 rpm at 37 ̊ C. 2.2.6. Dissociation of HIPs Mechanistic studies were carried out to investigate the influence of pH, ionic strength and time on complex dissociation (Hetényi et al., 2018; Vaishya et al., 2015). Briefly, 2 mg of the HIPs were incubated in 100 µL of DW, 0.01 M HCl containing 137 mM NaCl (pH 2), 10 mM PB containing 137 mM NaCl (pH 6, 6.8 and 7.4) and 10 mM PB pH 7.4 containing various ionic strengths of NaCl (10 mM, 100 mM and 154 mM) while shaking at 500 rpm at predetermined 7

time points (2, 4 and 6 h) at 37 oC. Thereafter, the mixture was centrifuged at 10,500 g for 10 min. Amount of dissociated (poly)peptides in supernatant was quantified using HPLC and the percentage of dissociated peptide was calculated by Eq. (3). 𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 (𝑝𝑜𝑙𝑦)𝑝𝑒𝑝𝑡𝑖𝑑𝑒𝑠 𝑖𝑛 𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡

𝐷𝑖𝑠𝑠𝑜𝑐𝑖𝑎𝑡𝑖𝑜𝑛 [%] = ( 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓(𝑝𝑜𝑙𝑦)𝑝𝑒𝑝𝑡𝑖𝑑𝑒𝑠 2.2.7. Distribution coefficient (log DSEDDS/release

𝑖𝑛 𝐻𝐼𝑃𝑠

medium)

(3)

) × 100

and concentration (Crelease

medium)

determination In order to evaluate release of HIPs from SEDDS, log DSEDDS/release medium was determined as described previously (Shahzadi et al., 2018). Log DSEDDS/release

medium

was evaluated by

measuring solubility of HIPs of (poly)peptides in SEDDS pre-concentrate and in release medium in a separate manner. Increasing concentrations of HIPs were dissolved in respective SEDDS pre-concentrate. For the solubility of HIPs in the release medium, 2 mg of HIPs were dissolved in 100 µL of release medium at 37 oC while shaking at 500 rpm at predetermined time points (2, 4, 6 h). However, DW, 0.01 M HCl containing 137 mM NaCl ( pH 2), 10 mM PB containing 137 mM NaCl (pH 6, 6.8 and 7.4) and 10 mM PB pH 7.4 containing various ionic strengths of NaCl (10 mM, 100 mM and 154 mM) were used as release medium in this study. The solutions were centrifuged at 10,500 g for 10 min and solubility of HIPs in the release medium was quantified using HPLC as described above. Log DSEDDS/release medium was calculated according to Eq. (4). 𝑀𝑎𝑥𝑖𝑚𝑢𝑚 𝑠𝑜𝑙𝑢𝑏𝑖𝑙𝑖𝑡𝑦 𝑜𝑓 𝐻𝐼𝑃𝑠 𝑖𝑛 𝑆𝐸𝐷𝐷𝑆

𝐿𝑜𝑔 𝐷𝑆𝐸𝐷𝐷𝑆/𝑟𝑒𝑙𝑒𝑎𝑠𝑒 𝑚𝑒𝑑𝑖𝑢𝑚 = 𝑙𝑜𝑔 (𝑀𝑎𝑥𝑖𝑚𝑢𝑚 𝑠𝑜𝑙𝑢𝑏𝑖𝑙𝑖𝑡𝑦 𝑜𝑓 𝐻𝐼𝑃𝑠 𝑖𝑛 𝑟𝑒𝑙𝑒𝑎𝑠𝑒 𝑚𝑒𝑑𝑖𝑢𝑚)

(4)

Subsequently, the concentration of HIPs released from SEDDS after emulsification in 10 mM PB containing 137 mM NaCl (pH 6, 6.8 and 7.4) at various time points (2, 4 and 6 h) was calculated using Eq. (5). 𝐶𝑟𝑒𝑙𝑒𝑎𝑠𝑒 𝑚𝑒𝑑𝑖𝑢𝑚 [%] = 100 − (

100 𝑉𝑟𝑒𝑙𝑒𝑎𝑠𝑒 𝑚𝑒𝑑𝑖𝑢𝑚 1+ 𝑉𝑆𝐸𝐷𝐷𝑆 .𝐷𝑆𝐸𝐷𝐷𝑆/𝑟𝑒𝑙𝑒𝑎𝑠𝑒 𝑚𝑒𝑑𝑖𝑢𝑚

)

(5)

8

Where Vrelease medium, VSEDDS and DSEDDS/release medium represents the volume of release medium, volume of the SEDDS pre-concentrate and distribution of the HIPs in SEDDS pre-concentrate and release medium, respectively. 2.2.8. Statistical data analysis Statistical data analysis was performed using GraphPad Prism version 5.02 software. The twoway analysis of variance (ANOVA) and Bonferroni post hoc test were used for multiple comparisons at p < 0.05 for significant (*), p < 0.01 for very significant (**) and p < 0.001 (***) for highly significant. All the data were expressed as the means of at least threeexperiments ± standard deviation (SD). 3.

Results

3.1.

Preparations of HIPs

HIPs of LEU, INS and BSA were formulated by using various anionic surfactants. The impact of these anionic surfactants exhibiting one and two carboxylate moieties were evaluated on ion pair formation as described in Table 3. Water insoluble HIPs were formed via ionic interactions between oppositely charged amines and carboxylate groups. Under acidic conditions (pH ~ 2) basic amino acids i.e. Lysine, Arginine and Histidine of (poly)peptides are ionized (Zupančič and Bernkop-Schnürch, 2017). LEU exhibits two basic amino acids, INS exhibits six basic amino acids and BSA exhibits 103 basic amino acids as shown in Table 1. As (poly)peptides and surfactants precipitated only in combination with each other, ionic interactions between them are responsible for HIPs formation. LEU showed maximum precipitation efficiency at a stoichiometric ratio of 2:1 when using mono-carboxylic acid surfactants while maximum precipitation efficiency was reached at a ratio of 1:1 when di-carboxylic acid surfactants were used. PAM showed highest precipitation efficiency of 90.2% as illustrated in Figure 1(A). In case of INS, a molar ratio of 3:1 with PAM exhibited highest precipitation efficiency of 95%, whereas highest efficiency of approximately 9

84% was obtained for SDC at a ratio of 6:1. Results are shown in Figure 1(B). BSA, a large molecular weight protein demonstrated the highest precipitation efficiency of 98% with PAM at a molar ratio of 50:1 while SDC and SDD showed maximum efficiency at a molar ratio of 100:1 as illustrated in Figure 1(C). According to these results, PAM could be identified as the most efficient for all tested (poly)peptides and was consequently used for further studies. 3.2.

Partitioning coefficient (log Pn-butanol/water) determination

Log Pn-butanol/water was determined in order to evaluate the increase in lipophilicity of the (poly)peptides via HIP as illustrated in Figure 2. Sufficient lipophilicity of HIPs of (poly)peptides is crucial for their incorporation in SEDDS, however, also enhanced their cell membrane permeability (Kahns et al., 1993). For determination of log P, the distribution of (poly)peptides and their corresponding HIPs between n-octanol and aqueous phase was determined. Neither BSA nor its HIP dissolved in n-octanol phase suggesting that n-octanol is a too lipophilic solvent for BSA-partitioning studies. Therefore, log P of HIPs was evaluated in n-butanol as a comparatively more hydrophilic solvent. In previous study, log P was also evaluated using alternative solvents such as Capmul 907 P (Zupančič et al., 2016b). 3.3.

Solubility studies

Solubility studies of HIPs were performed to identify most suitable oils, solvents and surfactants for the development of SEDDS. Visual solubility determination method was selected because it is less complicated and time consuming (Griesser et al., 2017). Results of solubility studies are listed in Table 4. Generally, HIPs are highly soluble in solvents of high dielectric constant. Water insoluble oily SEDDS components such as Peceol having a dielectric constant less than 3.5 showed lower HIPs solubilizing properties. 3.4.

SEDDS development and characterization

Based on solubility studies, three SEDDS formulations (FI, FII and FIII) were developed. Mean droplet size, polydispersity index (PDI) and zeta potential were measured for both blank and 10

loaded formulations. Blank SEDDS formulations demonstrated a mean droplet size below 400 nm with a polydispersity index (PDI) < 0.4 having a constant negative zeta potential over 4 h as shown in Table 5. Results of various in vivo studies of (poly)peptides with increasing molecular weight showed that bigger (poly)peptides are more challenging to form stable ion pairs (Bonengel et al., 2018; Menzel et al., 2018). Therefore, short, medium and high molecular weight (poly)peptides were chosen to evaluate the influence of molecular weight of (poly)peptides on stability of HIPs. Formulations (FI, FII and FIII) were loaded with 2% of LEU, INS and BSA HIPs, respectively. As in previous in vivo studies a payload of 0.5-2% led to encouraging results (Bonengel et al., 2018; Zupančič et al., 2016a), a payload of 2% was chosen. Loaded SEDDS showed an even more negative zeta potential due to the presence of anionic PAM in HIPs. A constant pronounced negative zeta potential is a reliable indicator of emulsion stability. 3.5.

Dissociation of HIPs

Dissociation of HIPs likely directly effects oral bioavailability of (poly)peptides. Therefore, mechanistic studies were carried out to investigate the influence of pH and concentration of ions at various time points on the dissociation of these complexes. HIPs were also incubated in demineralized water serving as negative control. Figure 3(A) illustrates the dissociation of the LEU-PAM in different aqueous media. Less than 5% LEU-PAM complex was dissociated upon incubation in demineralized water. On the one hand, dissociation of LEU-PAM gradually increased by raising pH of the PB. On the other hand, dissociation of LEU-PAM also increased over time by increasing the concentration of ions in the aqueous medium. LEU-PAM dissociated up to 25% at physiological conditions (PB 7.4 137 mM NaCl). INS-PAM exhibited higher complex dissociation as compared to LEU-PAM as illustrated in Figure 3(B). Under physiological conditions, approximately 60% of the complex was dissociated within 6 h. In contrast, a negligible amount of HIPs dissociated in demineralized 11

water as well as at acidic or less basic pH. The dissociation of INS-PAM complex increased at pH 7.4 and further increased by increasing the ionic strength of the medium. These results are in good agreement with previous studies showing that by increasing the concentration of ions up to 150 mM at pH 7.4, a proportional increase in dissociation of the complex was revealed (Hetényi et al., 2018; Vaishya et al., 2015). BSA-PAM exhibited a significantly higher complex dissociation as compared to HIPs of above mentioned (poly)peptides. Under physiological conditions, up to 90% of BSA-PAM was dissociated within 6 h as described in Figure 3(C). The obtained results indicate that dissociation of HIPs depends upon pH, ionic strength and time. 3.6.

Distribution coefficient (log DSEDDS/release

medium)

and concentration (Crelease

medium)

determination The release of HIPs from SEDDS is based on a simple diffusion process from the lipid phase into the aqueous phase. Equilibrium is reached immediately according to the log DSEDDS/release medium

of the HIPs between SEDDS pre-concentrate and release medium. When the HIPs are

absorbed from the membrane further HIPs will move out of the oily droplets until equilibrium is reached again. Accordingly, HIPs release from SEDDS is to a high extent controlled by the absorption rate from the mucosa. Therefore, log DSEDDS/release medium was determined in order to characterize the distribution of HIPs between SEDDS and release medium (Bernkop-Schnürch and Jalil, 2018; Mudie et al., 2010). Figure 3(A) illustrates that log DSEDDS/release medium of LEUPAM decreased by raising pH and ionic strength. Log DSEDDS/release

medium

of INS-PAM

decreased significantly with increasing the pH from 6 to 7.4, whereas remained stable at various ionic strength of the release medium as shown in Figure 3(B). In case of BSA-PAM, log DSEDDS/release medium decreased significantly over time by increasing pH and ionic strength as illustrated in Figure 3(C).

12

Assuming a dilution of SEDDS in release medium of 1:50, Crelease medium (%) of HIPs released from SEDDS was determined as illustrated in Figure 8. Approximately 40% of LEU-PAM was released from the SEDDS after dilution with release medium indicating that the drug will likely remain to a higher extent in the SEDDS as shown in Figure 4(A). A significant Crelease medium (%) of INS-PAM was observed by increasing the pH of the release medium as illustrated in Figure 4(B). Approximately 95% of BSA-PAM was released from the SEDDS after dilution with PB 7.4 containing 137 mM NaCl indicating a faster release as shown in Figure 4(C).

4.

DISCUSSION

Hydrophobic ion pairing is an effective strategy to enhance the lipophilicity of hydrophilic (poly)peptides with charged amino acids by substituting their counter ions with suitable lipophilic surfactants (Lengsfeld et al., 2002). Within present study PAM, a di-carboxylic acid lipophilic ion-pairing agent having a pKa value of 2.7 and log P value of 5.5 was chosen for HIP with (poly)peptides as it exhibited the highest precipitation efficiency among the tested surfactants. As complex formation via ion pairing is based on ionic interactions without any chemical modification, the (poly)peptide functionality is maintained. However, formation of HIPs is predominantly based on Coulombic forces as described previously (Bernkop-Schnürch, 2018). According to Coulomb’s law: 𝐹=𝑘

𝑞1 𝑞2 𝑟 2Ɛ

Where k is the Coulomb’s constant, q1 and q2 are the signed magnitudes of charges, r is the distance between the charges and Ɛ is the dielectric constant of the medium. The magnitude of electrostatic force of attraction between opposite charges (F) is directly proportional to the product of magnitudes of charges and inversely proportional to the square of the distance between them as well as the dielectric constant of the medium. Taking just Coulomb’s forces into account a molar ratio of 1:1 can be assumed between charged (poly)peptides and their 13

corresponding charged surfactant for the net charge of the complexes. However, complexes cannot dissociate completely suggesting the non-Coulombic interactions such as hydrophobic interactions are involved (Koetz et al., 1996). Griesser et al. explained that increase in lipophilicity is not simply dependent on the number of surfactant molecules being attached. Size of (poly)peptides as well as the amount of immobilized surfactants per kDa of (poly)peptide seem to be further key parameters for lipidization (Griesser et al., 2017). However, log P value of HIPs of (poly)peptides has a strong influence on their incorporation in SEDDS. Chamieh et al. reported that HIPs of leuprorelin-docusate having log P ~ 3 remained 100% inside the oily droplets, whereas HIPs of desmopressin-docusate having log P ~ 0.5 remained only to 30% inside the oily droplets (Chamieh et al., 2019). According to these results and considerations, minimum lipophilicity (log P) for incorporation of HIPs should be at least greater than 2. SEDDS formulations are prone to hydrolysis by pancreatic lipase, an endogenous enzyme present in duodenum being responsible for degrading ester structure of SEDDS components resulting in degradation as well as premature release of incorporated (poly)peptides (Karamanidou et al., 2016). Therefore, only SEDDS components that were proven to be stable against enzymatic degradation by lipase were used in this study (Hetényi et al., 2017). Proteolytic enzymes such as pepsin, trypsin, chymotrypsin and elastase are not soluble in the oily SEDDS droplets and (poly)peptides being incorporated in SEDDS are therefore protected against enzymatic degradation. This protection, however, is just as long provided as the (poly)peptides remains in the oily droplet. SEDDS undergo a series of phase changes after dilution with aqueous media. During these phase changes and thereafter ion pairs are partially released from oily droplets according to their distribution coefficient. When the few released ion pairs being poorly soluble in aqueous phase, however, fall apart aqueous drug solubility is strongly improved and consequently much more of the drug is released (Griffin et al., 2014). The stability of HIPs is comparatively low in intestinal fluids due to higher dielectric constant 14

of aqueous medium of around 80. Aprotic and usually water insoluble SEDDS components display a dielectric constant around 3.5, whereas protic components have a dielectric constant in the range of 6.1 to 46.7 (Griesser et al., 2017). Therefore, the stability of HIPs in SEDDS is much higher than in the release medium. In addition, stability of HIPs in the formulation can also be provided by the preparation of solid SEDDS. In case of solid SEDDS where the preconcentrate is adsorbed on mesoporous or microporous silicates, emulsification takes place upon contact with release medium. Hydration, however, can be limited especially in deeper pores hindering drug release (Gumaste and Serajuddin, 2017). Alternatively, solid SEDDS can be prepared by techniques like granulation, spray drying, hot melt extrusion or 3D printing without a solid phase carrier using mixtures of solid or semi-solid excipients (Cho et al., 2013; Mahmood and Bernkop-Schnürch, 2018). The release behavior of HIPs from the SEDDS after dilution with intestinal fluid are predominantly controlled by log DSEDDS/release

medium.

Various methods such as membrane

diffusion methods, sample-and-separate methods and very few in situ methods are used to characterize release kinetics of drugs from SEDDS. The obtained release profiles from SEDDS are often misleading as sink conditions are mostly violated in case of membrane diffusion methods and physical integrity of oily droplets is not preserved in case of sample-and-separate methods (Bernkop-Schnürch and Jalil, 2018). Log DSEDDS/release medium of LEU-PAM was 2.3 after 4 h when SEDDS are diluted 1:50 in intestinal fluid (pH 6.8 137 mM NaCl). Therefore, around 40% of HIPs was released indicating an extended retention in the lipophilic phase due to its high stability. Log DSEDDS/release medium of INS-PAM was 2 after 4 h in water being likely sufficiently high to keep the drug in the oily droplets protecting it towards an enzymatic degradation on the way to the absorption membrane. Under physiological condition (pH 6.8 137 mM NaCl), however, log DSEDDS/release medium was determined to be just 1.5, showing that when SEDDS are diluted 1:50 in the intestinal fluid already around 70% of the drug are 15

released. BSA-PAM, a large molecular weight (poly)peptide was rapidly dissociated under physiological conditions (pH 6.8 137 mM NaCl) over time having a log DSEDDS/release medium of 1 after 4 h. Consequently, around two-thirds of the drug are already released within 2 h. These results showed that highly stable HIPs exhibit sustained drug release whereas less stable HIPs exhibit faster drug release. Bonengel et al. also observed that octreotide-decanoate ion pair having log D of 1.7 exhibits a faster drug release, whereas octreotide-docusate ion pair having log D of 2.7 exhibits a more sustained drug release (Bonengel et al., 2018). Yuan et al. reported that stability of HIPs has inordinate effect on drug release by decreasing the burst release of the drugs from oily vehicles and prolonging the release time (Yuan et al., 2009). Chamieh et al. observed that release of the leuprorelin-docusate and desmopressin-docusate ion pairs from the oily droplets is related to the lipophilicity of the HIPs, ionic strength as well as composition of the release medium (Chamieh et al., 2019). However, in vivo conditions are more complex due to presence of endogenous anions such as phosphatidylserine, phosphatidylinositol, sialic acid or bile acids effecting the stability of HIPs after their release from SEDDS in GI fluids. The effect of these endogenous anions on the stability of HIPs should certainly be subject of further investigations. Improved lipophilicity of hydrophilic drugs was advantageous to entrap drugs on the one hand, whereas less dissociation of these complexes possibly prolongs their release on the other hand. The stability of ion pairs also has a great impact on intestinal permeability after their release from the SEDDS formulation. Neubert described that ion pairing of hydrophilic ionizable drugs has efficiently improved their permeability across membranes (Neubert, 1989). Phan et al. reported that HIPs in SEDDS enhanced the permeation of the horseradish peroxidase through the Caco-2 cell monolayer and freshly excised rat intestine by 4 times and 2.5 times compared to free enzyme, respectively (Phan et al., 2019). On the one hand, lipophilic counter ions accumulated in the membrane facilitate the transport of the cationic drug molecules. On the 16

other hand, the entire complex can also move across the membrane. A sufficient mucosal uptake, however, requires sufficient ion pairs being available at the absorption site that can likely be provided by appropriate SEDDS. 5.

CONCLUSION

Within this study, lipophilicity of various hydrophilic (poly)peptides was enhanced via HIP permitting their incorporation into SEDDS. Lipophilicity of HIPs was estimated by determining their log Pn-butanol/water. The release of HIPs from SEDDS is primarily controlled by log DSEDDS/release medium. Stability of HIPs was assessed by studying dissociation of these complexes in different release media at various pH and ionic strength over time. Results suggest that stability of HIPs is an important controlling factor for improving their affinity towards SEDDS oily droplets and subsequently reducing their premature release upon dilution with intestinal fluids. Hence, the fate of HIPs has a great impact on log DSEDDS/release medium and consequently their release from SEDDS in GI fluids. ACKNOWLEDGEMENT The authors are highly thankful and would like to extend acknowledgement to Higher Education of Pakistan (HEC) and Austrian Agency for International Cooperation in Education and Research, Austria (ÖAD) for their support. CONFLICT OF INTEREST The authors report no conflicts of interest.

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Phan, T.N.Q., Le-Vinh, B., Efiana, N.A., Bernkop-Schnürch, A., 2019. Oral self-emulsifying delivery systems for systemic administration of therapeutic proteins: Science fiction? Journal of Drug Targeting, 1-27. Powers, M.E., Matsuura, J., Brassell, J., Manning, M.C., Shefter, E., 1993. Enhanced solubility of proteins and peptides in nonpolar solvents through hydrophobic ion pairing. Biopolymers: Original Research on Biomolecules 33, 927-932. Rao, S.V.R., Shao, J., 2008. Self-nanoemulsifying drug delivery systems (SNEDDS) for oral delivery of protein drugs: I. Formulation development. International journal of pharmaceutics 362, 2-9. Sarmento, B., Ribeiro, A., Veiga, F., Ferreira, D., 2006. Development and validation of a rapid reversed‐ phase HPLC method for the determination of insulin from nanoparticulate systems. Biomedical Chromatography 20, 898-903. Shahzadi, I., Dizdarević, A., Efiana, N.A., Matuszczak, B., Bernkop-Schnürch, A., 2018. Trypsin decorated self-emulsifying drug delivery systems (SEDDS): Key to enhanced mucus permeation. Journal of colloid and interface science 531, 253-260. Vaishya, R.D., Mandal, A., Gokulgandhi, M., Patel, S., Mitra, A.K., 2015. Reversible hydrophobic ionparing complex strategy to minimize acylation of octreotide during long-term delivery from PLGA microparticles. International journal of pharmaceutics 489, 237-245. Wong, C.Y., Martinez, J., Al-Salami, H., Dass, C.R., 2018. Quantification of BSA-loaded chitosan/oligonucleotide nanoparticles using reverse-phase high-performance liquid chromatography. Analytical and bioanalytical chemistry, 1-16. Yuan, H., Jiang, S.-P., Du, Y.-Z., Miao, J., Zhang, X.-G., Hu, F.-Q., 2009. Strategic approaches for improving entrapment of hydrophilic peptide drugs by lipid nanoparticles. Colloids and surfaces B: Biointerfaces 70, 248-253. Zupančič, O., Bernkop-Schnürch, A., 2017. Lipophilic peptide character–What oral barriers fear the most. Journal of Controlled Release 255, 242-257. Zupančič, O., Grieβinger, J.A., Rohrer, J., de Sousa, I.P., Danninger, L., Partenhauser, A., Sündermann, N.E., Laffleur, F., Bernkop-Schnürch, A., 2016a. Development, in vitro and in vivo evaluation of a selfemulsifying drug delivery system (SEDDS) for oral enoxaparin administration. European Journal of Pharmaceutics and Biopharmaceutics 109, 113-121. Zupančič, O., Leonaviciute, G., Lam, H.T., Partenhauser, A., Podričnik, S., Bernkop-Schnürch, A., 2016b. Development and in vitro evaluation of an oral SEDDS for desmopressin. Drug delivery 23, 2074-2083.

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FIGURE CAPTIONS Figure 1:

Precipitation efficiency of HIPs of (A) LEU (B) INS and (C) BSA with PAM ( ), SSG ( ), SDC ( ), SDD ( ). Precipitated (poly)peptide-surfactant complex was isolated via centrifugation for 10 min and the remaining amount of (poly)peptide in supernatant was quantified by HPLC. Data are shown as mean ± SD (n =3).

Figure 2:

Log Pn-butanol/water of LEU, INS, BSA (gray bars) and their corresponding HIPs of LEU-PAM 1:1, INS-PAM 3:1, BSA-PAM 50:1 (white bars). The amount of LEU, INS and BSA in aqueous and organic phase was determined by HPLC. Data are shown as mean ± SD (n =3).

Figure 3:

(A) Dissociation of LEU-PAM at 2 h (white bars), 4 h (grey bars) and 6 h (black bars) and log DSEDDS/release medium of LEU-PAM at 2 h ( ), 4 h ( ) and 6 h ( ) at indicated pH and ionic strength. (B) Dissociation of INS-PAM at 2 h (white bars), 4 h (grey bars) and 6 h (black bars) and log DSEDDS/release medium of ) at indicated pH and ionic strength. INS-PAM at 2 h ( ), 4 h ( ) and 6 h ( (C) Dissociation of BSA-PAM at 2 h (white bars), 4 h (grey bars) and 6 h (black bars) and log DSEDDS/release medium of BSA-PAM at 2 h ( ), 4 h ( ) and 6 h ( ) at indicated pH and ionic strength. Indicated values are means of at least three experiments ± SD (*p<0.05, **p<0.01, ***p<0.001).

Figure 4:

(A) LEU-PAM concentration in the indicated release medium after 1: 50 dilution of SEDDS pre-concentrate. (B) INS-PAM concentration in the indicated release medium after 1:50 dilution of SEDDS pre-concentrate. (C) BSA-PAM concentration in the indicated release medium after 1: 50 dilution of SEDDS preconcentrate. Indicated values are means of at least three experiments ± SD (*p<0.05, **p<0.01, ***p<0.001).

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Table 1: HIPs of LEU, INS and BSA with tested surfactants. (Poly)peptides

LEU

INS

BSA

Mol.Wt (kDa)

Basic amino acids (AA)

1.2

Arginine Histidine

5.8

66

Arginine Lysine 2 Histidine 2 N-terminal AA 25 Arginine 60 Lysine 17 Histidine N terminal AA

Net positive charge

Tested surfactants

2

PAM SSG SDC SDD

6

PAM SSG SDC SDD

103

PAM SSG SDC SDD

Molar ratios [(poly)peptides : surfactant]

1: 0.5 1: 1 1: 2 1: 3 1: 4 1: 1 1: 3 1: 6 1: 9 1: 12 1: 25 1: 50 1: 100 1: 150 1: 200

Table 2: Composition of SEDDS formulations. Values were indicated in percent (v/v). Formulation

DMSO Transcutol HP Tetraglycol Capryol 90 Labrasol ALF Labrafil M 2125 CS Tween 20 Peceol

FI

– 5 10 10 25 – 30 20

FII

10 – 10 – 20 10 30 20

FIII

10 – 10 – 30 – 30 20

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Table 3: List of anionic surfactants screened and tested within this study. Surfactant

Chemical group

Chemical Formula

Mol. Wt

Dicarboxylic mono sodium salt

C23H42NNaO5

435.58

Monocarboxylic Sodium salt

C24H39NaO4

414.55

Monocarboxylic Sodium salt

C11H23NaO2

222.30

Dicarboxylic di sodium salt

C23H14O6Na2

432.33

PAM

SSG

SDC

SDD

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Solubility study of HIPs of (poly)peptides in certain SEDDS excipients; Table 4: indicated values represents maximum solubility at 37 oC. Excipient Name

Functionality

DMSO Transcutol HP Tetraglycol Capryol 90 Labrasol ALF Labrafil M 2125 CS Tween 20 Peceol

Solvent Sovent and solubilizer Solvent Nonionic water insoluble surfactant Nonionic water dispersible surfactant Nonionic water dispersible surfactant Nonionic surfactant Oily vehicle for lipophilic drugs

HLB value NA NA NA 5 12 9 16.7 1

Dielectric constant 46.7 14.1 15.7 6.1 8.1 3.4 NA 3.5

Solubility of HIP complex [%] LEU-PAM INS-PAM BSA-PAM 10 10 10 4 2 0.25 2 2 3 1 0.5 NA 2 1 0.25 2 1 0.25 1 0.5 0.125 2 1 0.25

Table 5: Evaluation of size distribution and zeta potential of blank SEDDS as well as HIPs loaded SEDDS as function of time using light scattering measurements. Data are shown as mean ± SD (n = 3). BLANK SEDDS

Time [h] HIPs LEUPAM

Formulations FI

INSPAM

FII

BSAPAM

FIII

HIPs loaded SEDDS [2% v/v]

Size[nm] Zeta Potential [mV] PDI 0

Size[nm] Zeta Potential [mV] PDI 4

Size [nm] Zeta Potential [mV] PDI 0

Size [nm] Zeta Potential [mV] PDI 4

346 ± 0.55 0.385 227 ± 0.24 0.455 265 ± 1.02 0.319

366 ± 0.84 0.410 298 ± 0.12 0.504 316 ± 0.51 0.468

382 ± 0.25 0.243 252 ± 0.34 0.265 273 ± 0.62 0.365

392 ± 0.11 0.455 287 ± 0.75 0.304 310 ± 0.96 0.435

-7.75 ± 0.274 -10.19 ± 0.340 -12.86 ± 0.468

-8.75 ± 0.575 -11.20 ± 0.485 -13.27 ± 1.055

-12.56 ± 0.395 -19.40 ± 0.235 -24.65 ± 0.310

-12.82 ± 0.435 -21.52 ± 0.370 -25.44 ± 0.854

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