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Hydrophobic ion pairing: Key to highly payloaded self-emulsifying peptide drug delivery systems
Accepted Manuscript Title: Hydrophobic ion pairing: Key to highly payloaded self-emulsifying peptide drug delivery systems Authors: Janine Griesser, Gergely Het´enyi, Michael Moser, Fr´ed´eric Demarne, Vincent Jannin, Andreas Bernkop-Schnurch ¨ PII: DOI: Reference:
S0378-5173(17)30100-X http://dx.doi.org/doi:10.1016/j.ijpharm.2017.02.019 IJP 16423
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
11-1-2017 2-2-2017 6-2-2017
Please cite this article as: Griesser, Janine, Het´enyi, Gergely, Moser, Michael, Demarne, Fr´ed´eric, Jannin, Vincent, Bernkop-Schnurch, ¨ Andreas, Hydrophobic ion pairing: Key to highly payloaded self-emulsifying peptide drug delivery systems.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2017.02.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Hydrophobic ion pairing: Key to highly payloaded self-emulsifying peptide drug delivery systems
Janine Griesser1, Gergely Hetényi1, Michael Moser1, Frédéric Demarne3, Vincent Jannin3, Andreas Bernkop-Schnürch1,2 *
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Thiomatrix Forschungs-und Beratungs GmbH, Trientlgasse 65, Innsbruck, Austria
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Center for Chemistry and Biomedicine, Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria
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Gattefossé SAS, 36 chemin de Genas, 69804 Saint-Priest Cedex, France
*Corresponding Author: Andreas Bernkop-Schnürch Center for Chemistry and Biomedicine, Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria Tel.: +43-512-507 58601 Fax: +43-512-507 58699 e-mail: [email protected]
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Graphical abstract
Highlights
Independent from the type of peptide, docusate showed the most efficient hydrophobic ion pairing properties and was revealed as universal counter ion.
Novel SEDDS containing Capryol 90, Labrafil M 2125 CS, Labrasol ALF, Peceol, propylene glycol, tetraglycol, Transcutol HP and Tween 20 were developed.
The complexes were incorporated into SEDDS achieving a payload over 10%.
SEDDS exhibited high stability and constant negative zeta potential over a 4 h incubation time.
Correlation between the type of lipid excipients and the solubility was detected.
Abstract Aim: The aim of this study was the formation and characterization of various ion pairs of therapeutic peptides with different surfactants in order to reach a high payload in selfemulsifying drug delivering systems (SEDDS).
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Methods: Hydrophobic ion pairs (HIP) were formed between the anionic surfactants sodium docusate, dodecylsulfate and oleate and the peptides leuprorelin (LEU), insulin (INS) and desmopressin (DES). The efficiency of HIP formation was evaluated by quantifying the amount of formed complexes, log P value determination in n-octanol/water via HPLC and zeta potential measurements. Solvents and surfactants were screened regarding their complex solubilizing properties. Subsequently, peptide complexes were incorporated into SEDDS followed by payload and stability determination. Results: Independent from the type of peptide, docusate showed the most efficient HIP properties followed by dodecylsulfate and oleate. Ratios of 2:1 for LEU, 6:1 for INS and 1.5:1 for DES led to the highest quantity of formed complexes with docusate and log P increased at least by 3 units. The more docusate was added to each peptide, the more negative became the zeta potential of the resulting complex. Incorporating these optimized complexes into novel SEDDS containing Capryol 90, Labrafil M 2125 CS, Labrasol ALF, Peceol, propylene glycol, tetraglycol, Transcutol HP and Tween 20 allowed payloads of the LEU, DES and INS complexes above 10%. Moreover, SEDDS exhibited high stability and constant negative zeta potential over a 4 h incubation time. Conclusion: Following the procedure described herein payloads > 10 % can be achieved for peptide drugs in SEDDS.
Keywords: Leuprorelin, Insulin, Desmopressin, Self-emulsifying drug delivery systems, Hydrophobic ion paring, Peptide delivery
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1. Introduction Self-emulsifying drug delivery systems (SEDDS) representing isotropic mixtures of oils, solvents and emulsifiers are likely opening the door for the oral administration of as challenging drugs as therapeutic peptides and proteins (Leonaviciute and Bernkop-Schnürch, 2015). Incorporating peptide drugs in lipid droplets protects them towards thiol/disulfide exchange reactions with food and endogenous glutathione as well as towards an enzymatic degradation in the gastrointestinal tract (Dahm and Jones, 1994; Ijaz et al., 2016; Schmitz et al., 2006). Moreover, these lipid droplets can be designed in a way that they are able to permeate the mucus gel barrier in a comparatively efficient manner (Friedl et al., 2013). Once having reached the underlying absorption membrane, SEDDS were shown to exhibit even permeation enhancing properties for peptide drugs (Leonaviciute and Bernkop-Schnürch, 2015). Up to date, however, this promising strategy has by far not reached its full potential, as various hurdles still need to be overcome. One of these hurdles is certainly the poor solubility of peptide drugs in the lipophilic phase of SEDDS. Because of their mainly hydrophilic nature peptides and proteins can likely only be incorporated in lipids via hydrophobic ion pairing (HIP). But even by utilizing this technique only comparatively low payloads were achieved. Hintzen et al. (Hintzen et al., 2014), for instance, reached a payload of just 0.4% for leuprorelin, Karamanidou et al. achieved 1.13% for insulin (Karamanidou et al., 2015) and Zupančič et al. (Zupančič et al., 2016) obtained 0.25% for desmopressin in SEDDS. According to these results, the low peptide payload of SEDDS had to be identified as worrying bottleneck for this otherwise promising strategy. In order to address this problem, it was the aim of this study to focus just on this issue and to provide a broader understanding on parameters influencing the efficiency of HIP and the subsequent incorporation of peptide complexes in SEDDS. For comparison reasons three
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peptides including cyclic or non-cyclic as well as high molecular weight and low molecular weight peptides were chosen. As leuprorelin (LEU), insulin (INS) and desmopressin (DES) are cationic peptides, the anionic surfactants sodium docusate, sodium dodecylsulfate and sodium oleate exhibiting different acidic groups, namely a sulfonate, sulfate and carboxylic moiety, were tested for HIP. In order to achieve comparatively high payloads, the solubility of the most lipophilic complexes was investigated in various solvents. Those solvents in which complexes could be dissolved most efficiently were in the following utilized to form SEDDS.
2. Materials and methods 2.1.
Materials
Leuprorelin acetate and desmopressin acetate were purchased from Chemos GmbH, Germany. Insulin from porcine pancreas was bought from Prospec Protein, Israel. Lipids utilized within this study were provided by Gattefossé, France. Tween 20 was a gift from Croda, Germany. All other reagents were purchased from Sigma-Aldrich, Austria. 2.2.
Methods
2.2.1. HPLC analysis Peptides were analyzed on a Hitachi Elite LaChrom HPLC-System equipped with L-2130 pump, L-2200 autosampler and L-2400 UV detector. As mobile phase a binary solvent system of solvent A – water with 0.1% (v/v) trifluoroacetic acid (TFA) and solvent B – acetonitrile with 0.1% (v/v) TFA was used. Leuprorelin was quantified utilizing XBridge BEH300 C18 3.5 µm 4.6 mm x 150 mm as stationary phase. An isocratic method (70% mobile phase A and 30% mobile phase B) over 12 minutes at 40°C with a flow rate of 0.8 ml/min was applied. Moreover, all samples were stored at 10°C within the auto sampler until injection of 20 µl and thereafter
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analyzed at a wavelength of 222 nm. Insulin was quantified with LiChrosorb RP-18 LiChroCART 5 µm, 100 Å, 125 x 4 as stationary phase. An isocratic method (65% mobile phase A and 35% mobile phase B) over 7 minutes at 40°C with a flow rate of 1 ml/min was used. Aliquots of 50 µl were injected and analyzed at a wavelength of 214 nm. For desmopressin Nucleosil 100-5 C18 5 µm 4 mm x 250 mm as stationary phase was used. An isocratic method (75% mobile phase A and 25% mobile phase B) over 10 minutes at 40°C with a flow rate of 1 ml/min was applied. The injection volume was 20 µl and samples were analyzed at a wavelength of 222 nm. 2.2.2. Hydrophobic ion pairing (HIP) First, 1 ml of each peptide solution was prepared in a concentration of 10 mg/ml utilizing 0.01 M HCl as solvent. As leuprorelin, desmopressin and insulin exhibit different net positive charges at this pH , several ratios of peptide to surfactant were analyzed. Thereafter, surfactants as listed in Table 1 were dissolved in 1 ml of demineralized water applying ratios as indicated in Table 1. The solution of each surfactant was added drop wisely to the peptide solution under vigorous stirring (400 rpm). The mixtures were stirred for two hours and subsequently centrifuged at 12,500 rpm for 10 min with High-Speed Mini Centrifuge (Fisher Scientific, Illinois, USA). During the reaction, an immediate white precipitation indicated the formation of the hydrophobic peptide complex. The supernatant was separated from the precipitate and washed with 0.01 M HCl. Thereafter, the water-soluble fraction of the peptide remaining in the supernatant was determined by measuring its concentration via HPLC as described above. Finally, the pellets were frozen, lyophilized and stored at -30°C. As blank reference, the surfactant solutions were added to 0.01 M HCl without peptide. Precipitation efficiency was defined utilizing the following equation: Precipitation efficiency [%] = 100 − (
𝑃𝑒𝑝𝑡𝑖𝑑𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑎𝑓𝑡𝑒𝑟 𝐻𝐼𝑃 × 100) 𝑃𝑒𝑝𝑡𝑖𝑑𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑏𝑒𝑓𝑜𝑟𝑒 𝐻𝐼𝑃 6
2.2.3. Log P determination To 1 ml of n-octanol/water (1:1), 1 mg of peptide or 1 mg of complex was added and incubated at 37°C while shaking at 300 rpm for 24 hours. Samples were centrifuged for 10 min at 10,000 rpm with High-Speed Mini Centrifuge (Fisher Scientific, Illinois, USA). Thereafter, 100 µl aliquots were withdrawn from both of the aqueous and the n-octanol phase and diluted with 300 µl of methanol containing 0.1% (v/v) TFA. The concentration of peptides and complexes in aqueous as well as n-octanol phase was analyzed via HPLC as described above. The partition coefficient was calculated using the following equation: log P = log
c peptide in octanol phase c peptide in aqueous phase
2.2.4. Evaluation of zeta potential during HIP First, 0.5 ml of 1% (m/v) peptide solutions were prepared in 0.01 M HCl and surfactants were dissolved in 0.5 ml of demineralized water in concentrations corresponding to molar ratios as indicated in Table 1. The solution of each surfactant was added dropwisely to the peptide solution under vigorous stirring (400 rpm). Thereafter, zeta potential of the resulting precipitate was measured utilizing Zetasizer Nano-ZSP (Malvern Instruments, Worcestershire, UK). 2.2.5. Solubility studies Peptide-surfactant complexes applying four concentrations, namely 0.625%, 1.25%, 2.5% and 5% (m/v) were added to organic solvents, lipids and surfactants. In particular, solubility of complexes in Capryol 90, Labrafac Lipophile WL 1349, Labrafil M 1944 CS, Labrafil M 2125 CS, Labrasol ALF, Lauroglycol 90, Maisine CC, Peceol, Plurol Oleique CC 497, tetraglycol, propylene glycol, Tween 20 and Transcutol HP was examined. Mixtures of complexes and solvents were sonicated and optionally heated to 37°C while shaking. Afterwards, the mixtures
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were incubated for 12 hours at room temperature. The dissolution was evaluated visually after centrifugation at 12500 rpm for 5 min. 2.2.6. Preparation and characterization of SEDDS Four novel SEDDS containing excipients as listed in Table 2 were developed and peptide complexes were incorporated in an increasing concentration via ultrasonification for 15 min at room temperature and examined for precipitation and homogeneity. The incorporation was evaluated visually after centrifugation at 12500 rpm for 5 min and the highest achievable payload was additionally confirmed via HPLC analysis. Therefore, 100 µl SEDDS were diluted with 6 ml of methanol containing 0.1% trifluoroacetic acid followed by HPLC analysis as described above utilizing on a six-point-calibration curve. Size and zeta potential of SEDDS were determined by photon correlation spectroscopy with Zetasizer Nano-ZSP (Malvern Instruments, Worcestershire, UK) at 37°C. As control, SEDDS were prepared and investigated without incorporating peptide complexes. The formulations were diluted 2% (v/v) in 50 mM phosphate buffer saline (PBS) pH 6.8 for droplet size measurements and with demineralized water for zeta potential measurements. Furthermore, SEDDS were examined by measuring droplet size after a time period of 120 and 240 minutes at 37°C while shaking at 300 rpm in order to investigate their stability. The payload of complexes and peptides in SEDDS was calculated as follows: Payload complex [%] = (
Payload peptide [%] = (
𝑚 𝑑𝑖𝑠𝑠𝑜𝑙𝑣𝑒𝑑 𝑐𝑜𝑚𝑝𝑙𝑒𝑥 × 100) 𝑉𝑆𝐸𝐷𝐷𝑆 𝑏𝑙𝑎𝑛𝑘
𝑚 𝑝𝑒𝑝𝑡𝑖𝑑𝑒 × 𝑝𝑎𝑦𝑙𝑜𝑎𝑑 𝑐𝑜𝑚𝑝𝑙𝑒𝑥) 𝑚 𝑝𝑒𝑝𝑡𝑖𝑑𝑒 + 𝑚 𝑠𝑢𝑟𝑓𝑎𝑐𝑡𝑎𝑛𝑡
2.2.7. Statistical data analyses
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Statistical data analyses were performed using Student’s t-test to analyze the significant difference between two mean values. Level of p≤ 0.05 was set for significant, p≤ 0.01 for very significant and p≤ 0.001 for highly significant. The results were expressed as the mean of at least three experiments ± standard deviation (SD).
3. Results 3.1.
Hydrophobic Ion Pairing
The impact of anionic surfactants exhibiting different functional groups and number of carbon atoms as illustrated in Table 3 was evaluated. The more of these surfactants were added to each peptide, to a higher extend lipophilic complexes were formed until a maximum was reached. Increasing the surfactant concentration beyond that maximum did not lead to a plateau phase. In contrast, the amount of formed complex decreased when the amount of surfactants was further raised. This observation can be explained by the formation of micelles re-dissolving the complex (Choi and Park, 2000). Dai e Dong (Dai and Dong, 2007), for instance, demonstrated for INS that an excessive amount of sodium dodecyl sulfate above the ratio 6:1 leads to a decrease in turbidity of the HIP dispersion and a lower amount of precipitated peptide. LEU containing the two basic amino acids Arginine and Histidine, showed the highest precipitation efficiency of 99.1% at a molar ratio of 2:1 applying sodium docusate, as illustrated in Figure 1. In case of INS with six basic amino acids (2 Histidine, 1 Arginine, 1 Lysine and 2 N-terminal amino acids), a molar ratio of 6:1 exhibited the highest efficiency utilizing sodium docusate and sodium dodecyl sulfate (Figure 2). Moreover, DES as cyclic peptide with one basic amino acid (Arginine) demonstrated the highest precipitation efficiency of 93.4% applying sodium docusate at a molar ratio of 1.5:1 (Figure 3) compared to sodium dodecyl sulfate and sodium oleate. According to these results at least to a certain extent a correlation between the number of cationic
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substructures on the peptide and the most efficient molar ratio of peptide to surfactant could be found. Comparing the complex formation efficacy determined by the degree of precipitation in aqueous medium, sodium docusate ranked independently from the number of cationic substructures, form and size of peptides in all cases first. Whether the superior properties of docusate are mainly related to its branched structure, its dipole moment or the sulfonate group could not be determined. Nevertheless, these results point out the crucial role of the structure of surfactants for efficient HIP. 3.2.
Evaluation of zeta potential during HIP
Apart from the complex formation efficiency the shift in zeta potential due to HIP was investigated utilizing sodium docusate as representative surfactant. Results showed for all peptides an increase in negative charge the more docusate was bound to their surface. Nevertheless, a direct correlation between the number of attached surfactant molecules and the resulting zeta potential could not be observed. Generally, the impact of docusate on the zeta potential was high. Leuprorelin, for instance, demonstrated a change in zeta potential from +21 mV to -13 mV when comparing a surfactant to leuprorelin ratio of 0.5:1 with 4:1 as illustrated in Figure 4A. In case of the surfactant to insulin ratios from 1:1 to 12:1, the zeta potential shifted even from +40 mV to -72 mV. These results as shown in Figure 4B are in good agreement with a study performed by Sun et al. demonstrating that an excess of anionic deoxycholate counter ion results in an increasing negative zeta potential of insulin - deoxycholate complexes (Sun et al., 2011). As pictured in Figure 4C, the surfactant to desmopressin ratio of 0.5:1 showed a zeta potential of +3 mV and for 3:1 a zeta potential of -52 mV was measured. Results showed also that the surfactant is comparatively tightly bound to each peptide, as it would unless not have such a strong impact on the zeta potential. Furthermore, the lipophilic substructures of the surfactant seem to have a greater impact on lipidization than their charge neutralizing effect, as the surfactant to peptide ratio resulting according to Figure 1 – Figure 3 in the most 10
hydrophobic complexes does in no case correlate with the ratio leading to an almost uncharged complex. 3.3.
Log P determination
In order to evaluate the increase in lipophilicity of the chosen model peptides via HIP, log P was determined for comparison. Results of this study are illustrated in Figure 5. Leuprorelin with the highest increase in log P showed an improvement of 5 units. Taking the number of surfactants per peptide with 2 for LEU, 6 for INS and 1.5 for DES into account, the increase in lipophilicity is not simply dependent on the number of surfactant molecules being attached to each peptide. When also the size of these peptides is considered, however, a correlation with obtained data can be seen leading to the following rank order: LEU with 1.7 surfactant molecules per kDa (s/kDa) > DES with 1.4 s/kDa > INS with 1.0 s/kDa. According to this, the number of immobilized surfactants per kDa peptide seems to be one of the key parameters for lipidization. 3.4.
Solubility studies
Solubility studies of the complexes were performed with solvents being most appropriate for the development of SEDDS. Results of these studies are illustrated in Table 4. The LEU – docusate complex exhibiting the comparatively highest log P of all tested complexes showed also the highest solubility dissolving in all tested excipients expect Labrafac Lipophile WL 1349 and Labrafil M 2125 CS. INS – docusate complex was highly soluble in Labrasol ALF, Transcutol HP and Peceol, whereas the desmopressin complex demonstrated high solubility in Labrasol ALF, Maisine CC, Capryol 90, Peceol and Transcutol HP. Generally, all complexes were highly soluble in Transcutol HP followed by Labrasol ALF. Although the dielectric constant of Maisine CC is comparatively very low and no complex could be dissolved in solvents of a dielectric constant in the same range – namely Labrafac Lipophile WL 1349 and Labrafil M 2125 CS – all complexes could be dissolved in this solvent in comparatively high concentrations. Generally,
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aprotic solvents such as Labrafac Lipophile WL 1349, Labrafil M 1944 CS, Labrafil M 2125 CS, Maisine CC and Peceol showed lower complex solubilizing properties compared to the protic solvents Transcutol HP, tetraglycol, propylene glycol and Labrasol ALF. As water possesses a dielectric constant of 80, a convergence to 80 is essential for increasing solubility and payload. Low solubilizing properties were also confirmed for aprotic and usually water insoluble SEDDS components having a dielectric constant from 2.9 to 3.7, whereas protic components of a dielectric constant between 8.1 and 32.0 showed comparatively higher solubilizing properties. Monoesters such as monoglycerides (Peceol, Maisine CC) or monoesters of propylene glycol (Lauroglycol, Capryol) showed higher complex solubilizing properties compared to triesters (triglycerides - Labrafac Lipophile WL 1349), as monoesters exhibit higher hydrophilic and lipophilic balance (HLB) values and higher polarity than triesters. 3.5.
Preparation and characterization of SEDDS
SEDDS were developed based on the results of the solubility studies as described above and initially the unloaded SEDDS as listed in Table 5 were characterized. Thereafter, LEU and DES docusate complexes were incorporated into formulations A and B whereas INS docusate complexes were loaded into formulations C and D. As listed in Table 6, 7 different payloads were tested for all formulations and the payloads were additionally confirmed via HPLC. In all cases a payload of 10.67% representing the highest tested concentration could be achieved. For LEU this result represents a 25-fold increase in payload compared to the payload obtained in former studies by Hintzen et al. (Hintzen et al., 2014; Leonaviciute et al., 2016) and for INS the improvement was 6-fold compared to that achieved by Karamanidou et al. (Karamanidou et al., 2015). In case of DES the improvement in payload was even 40-fold compared to that reported in a recent study by Zupančič et al. (Zupančič et al., 2016). As listed in Table 5 and Table 7, all formulations showed stability over 4 hours. Formulations A and B demonstrated a mean droplet size of 0.3 µm with a polydispersity index (PDI) < 0.750, a constant negative zeta potential and 12
no change in droplet size over 4 hours. Formulations C and D exhibited a mean droplet size of 6 µm with a PDI < 0.850. SEDDS loaded with complexes showed an even higher value of negative zeta potential due to the presence of anionic docusate in the complexes. A constant negative zeta potential guarantees the stability of the droplets and it is a reliable indicator concerning emulsion stability. As the zeta potential of the emulsions did not change over the incubation time, these SEDDS even at comparatively high complex payload can be assessed as highly stable delivery systems.
4. Discussion Although most peptides and protein drugs are very potent and highly active resulting in comparatively low doses being often in the microgram range, a high payload in SEDDS is nevertheless substantial. On the one hand the comparatively much lower oral bioavailability of therapeutic peptides and proteins being in most cases still below 1% has to be taken into consideration, as a consequently 10- up to 200-fold higher amount of drug is needed to achieve the same effect as via parenteral administration. On the other hand, the amount of SEDDS containing surfactants and solvents, that must – although having GRAS status – from the toxicological point of view in particular when being used for treatment of chronic diseases such as diabetes type 2 be addressed very carefully, needs to be kept at a feasible minimum. Furthermore, for certain peptides and protein drugs even considerable high amounts need to be administered. Based on the results obtained in this study, however, it seems likely that reaching sufficient high payloads cannot be regarded anymore as bottle neck for oral peptide administration via SEDDS. The generation of highly lipophilic complexes of therapeutic peptides and proteins will furthermore have also a great impact on other key parameters regarding their oral administration via SEDDS. On the one hand the release rate of peptides from SEDDS is mainly controlled by the distribution coefficient of the lipophilic complex between the 13
lipid phase of SEDDS and the intestinal fluid. Accordingly, the more lipophilic the complex are the more sustained is the release out of the oily droplets. Such a more sustained release can certainly be regarded as advantages, as it was shown by Hintzen et al. and Karamanidou et al. (Hintzen et al., 2014; Karamanidou et al., 2015; Zupančič et al., 2016) that due to the incorporation of peptides into SEDDS they are protected in the gastrointestinal tract towards a presystemic metabolism by peptidases and proteases. On the other hand, the lipophilic complexes described herein will likely exhibit also improved membrane permeability. The more lipophilic a drug is, the more easily it can permeate the phospholipid bilayer of cell membranes. As shown in this study, surfactants form in an efficient manner lipophilic complexes with peptides. According to this, it is our opinion that most surfactants exhibiting a permeation enhancing effect for peptides act rather via a lipidization of peptides than making the cell membrane more leaky or binding calcium or magnesium ions on the membrane loosing thereby tight junctions. The stability of these peptides – surfactant complexes in the body, however, remains unclear. At least, orientating studies of our own research group heading in this direction failed due to the high complexity of the analytic of such complexes in human full blood (data not shown). Although forming considerable stable lipophilic complexes peptide drugs seem to maintain their therapeutic efficacy. An oral insulin formulation based on SEDDS, for instance was even already tested in phase II clinical trial (OramedPharmaceuticals, 2016). Apart from their comparatively high payload, SEDDS having been generated within this study exhibit also other favorable properties. As the cell membrane is covered with negatively charged mucus gel layer, SEDDS with negative zeta potential are necessary to avoid interactions with mucus and biogenic substances (He et al., 2010; Ishiwata et al., 2000; Zhou and Huang, 1994) and to permeate the mucus more easily compared to positively charged SEDDS already sticking on the surface without reaching the underlying absorption membrane (Loo et al., 2012). 5. Conclusion 14
Comparing the formulation of lipophilic complexes of different peptides with different surfactants provided a fundamental overview on key parameters having to be addressed in order to incorporate these hydrophilic macromolecular drugs in SEDDS. Firstly, the choice of surfactant plays a crucial role representing the likely most important parameter. Secondly, the number of surfactant molecules being directly attached per kDa peptide is essential. Increasing the number of surfactant molecules just via the formation of likely mixed micelles does not improve the lipophilic character of the peptide. In contrast, this approach leads even to an increase in hydrophilicity of the complex. Thirdly, the right choice of solvents for the complex in SEDDS can strongly contribute to improved payloads. In particular protic solvents of a dielectric constant around 10 seem to be favorable. Taking these points into account, payload in the even double digit percentage range can be achieved for self-emulsifying peptide drug delivery systems.
6. Conflict of interest Vincent Jannin and Frédéric Demarne were employed by Gattefossé SAS manufacturing and selling lipid-based excipients at the time this study was performed.
7. Acknowledgement The authors would like to thank Dr Joachim Quadflieg from Gattefossé GmbH, Germany for supply excipients for the development of SEDDS and for the technical support.
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References Choi, S.H., Park, T.G., 2000. Hydrophobic ion pair formation between leuprolide and sodium oleate for sustained release from biodegradable polymeric microspheres. Int J Pharm 203, 193202. Dahm, L.J., Jones, D.P., 1994. Secretion of cysteine and glutathione from mucosa to lumen in rat small intestine. Am J Physiol 267, G292-300. Dai, W.G., Dong, L.C., 2007. Characterization of physiochemical and biological properties of an insulin/lauryl sulfate complex formed by hydrophobic ion pairing. Int J Pharm 336, 58-66. Friedl, H., Dünnhaupt, S., Hintzen, F., Waldner, C., Parikh, S., Pearson, J.P., Wilcox, M.D., Bernkop-Schnürch, A., 2013. Development and evaluation of a novel mucus diffusion test system approved by self-nanoemulsifying drug delivery systems. J Pharm Sci 102, 4406-4413. He, C., Hu, Y., Yin, L., Tang, C., Yin, C., 2010. Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials 31, 3657-3666. Hintzen, F., Perera, G., Hauptstein, S., Müller, C., Laffleur, F., Bernkop-Schnürch, A., 2014. In vivo evaluation of an oral self-microemulsifying drug delivery system (SMEDDS) for leuprorelin. Int J Pharm 472, 20-26. Ijaz, M., Bonengel, S., Zupančič, O., Yaqoob, M., Hartl, M., Hussain, S., Huck, C.W., BernkopSchnürch, A., 2016. Development of oral self nano-emulsifying delivery system(s) of lanreotide with improved stability against presystemic thiol-disulfide exchange reactions. Expert Opin Drug Deliv 13, 923-929. Ishiwata, H., Suzuki, N., Ando, S., Kikuchi, H., Kitagawa, T., 2000. Characteristics and biodistribution of cationic liposomes and their DNA complexes. J Control Release 69, 139-148. Karamanidou, T., Karidi, K., Bourganis, V., Kontonikola, K., Kammona, O., Kiparissides, C., 2015. Effective incorporation of insulin in mucus permeating self-nanoemulsifying drug delivery systems. Eur J Pharm Biopharm 97, 223-229. Leonaviciute, G., Bernkop-Schnürch, A., 2015. Self-emulsifying drug delivery systems in oral (poly)peptide drug delivery. Expert Opin Drug Deliv 12, 1703-1716. Leonaviciute, G., Zupančič, O., Prüfert, F., Rohrer, J., Bernkop-Schnürch, A., 2016. Impact of lipases on the protective effect of SEDDS for incorporated peptide drugs towards intestinal peptidases. Int J Pharm 508, 102-108. Loo, Y., Grigsby, C.L., Yamanaka, Y.J., Chellappan, M.K., Jiang, X., Mao, H.Q., Leong, K.W., 2012. Comparative study of nanoparticle-mediated transfection in different GI epithelium coculture models. J Control Release 160, 48-56. OramedPharmaceuticals, 2016. http://www.oramed.com/pipeline/ormd-0801-type-2/ (accessed in December 2016). Schmitz, T., Huck, C.W., Bernkop-Schnürch, A., 2006. Characterisation of the thiol-disulphide chemistry of desmopressin by LC, mu-LC, LC-ESI-MS and Maldi-Tof. Amino Acids 30, 35-42. Sun, S., Liang, N., Kawashima, Y., Xia, D., Cui, F., 2011. Hydrophobic ion pairing of an insulin-sodium deoxycholate complex for oral delivery of insulin. Int J Nanomedicine 6, 30493056. Zhou, X., Huang, L., 1994. DNA transfection mediated by cationic liposomes containing lipopolylysine: characterization and mechanism of action. Biochim Biophys Acta 1189, 195-203. Zupančič, O., Leonaviciute, G., Lam, H.T., Partenhauser, A., Podričnik, S., Bernkop-Schnürch, A., 2016. Development and in vitro evaluation of an oral SEDDS for desmopressin. Drug Deliv 23, 2074-2083.
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Figure Captions Figure 1: Hydrophobic ion pairing of LEU with sodium docusate (■), sodium oleate (∆) and sodium dodecyl sulfate (x). After the addition of surfactants, the precipitated LEU complex was centrifuged and the remaining peptide in supernatant was analyzed by HPLC. Indicated values are means (n ≥ 3) ± SD.
Figure 2: Hydrophobic ion pairing of INS with sodium docusate (■), sodium oleate (∆) and sodium dodecyl sulfate (x). After the addition of surfactants, the precipitated INS complex was centrifuged and the remaining peptide in supernatant was analyzed by HPLC. Indicated values are means (n ≥ 3) ± SD.
17
Figure 3: Hydrophobic ion pairing of DES with sodium docusate (■), sodium oleate (∆) and sodium dodecyl sulfate (x). After the addition of surfactants, the precipitated DES complex was centrifuged and the remaining peptide in supernatant was analyzed by HPLC. Indicated values are means (n ≥ 3) ± SD.
Figure 4: Zeta potential [mV] measurement during HIP of (A) LEU, (B) INS and (C) DES with sodium docusate. Indicated values are means (n ≥ 3) ± SD.
18
Figure 5: Log P of indicated peptide drugs (LEU, INS and DES) (gray bars) and the corresponding HIP complexes LEU-docusate 2:1, INS-docusate 6:1 and DES-docusate 1.5:1 (white bars). Indicated values are means (n ≥ 3) ± SD.
19
Tables Table 1: Complexes of leuprorelin, insulin and desmopressin with indicated surfactants.
Peptide
Net positive charges
Basic amino acids (AA)
Tested surfactants
Molar ratios [surfactant: peptide] 0.5:1
Sodium docusate
1:1
Sodium dodecyl sulfate
2:1
Sodium oleate
3:1
Arginine Leuprorelin
2 Histidine
4:1 1:1 Arginine Sodium docusate
3:1
Sodium dodecyl sulfate
6:1
Sodium oleate
9:1
Histidine Insulin
6 2 Lysine 2 N-terminal AA
12:1 0.5:1
Desmopressin
1
Arginine
Sodium docusate
1:1
Sodium dodecyl sulfate
1.5:1
Sodium oleate
2:1 3:1
20
Table 2: Composition of novel SEDDS containing the three peptide complexes. Values are indicated in percent (v/v). Formulation
A
B
C
D
Transcutol HP
5
5
-
-
Tetraglycol
-
10
10
30
Propyleneglycol
-
-
10
-
Peceol
20
20
10
30
Capryol 90
10
10
-
-
Labrasol ALF
35
25
60
40
Labrafil M 2125 CS
-
-
10
-
Tween 20
30
30
-
-
Table 3: List and chemical structure of tested surfactants. Surfactant
Chemical group
Number of carbon atoms
sulfonate
20
sulfate
12
carboxylic
18
Sodium docusate
Sodium dodecyl sulfate
Sodium oleate
21
Table 4: Classification of lipid based excipients utilized within this study; (HLB) hydrophilic and lipophilic balance; (NA) not applicable. Solubility of peptide docusate complexes in four concentrations 0.625%, 1.25%, 2.5% and 5.0% (m/v). (-) not dissolved or lower than 0.625%. Excipient
Definition
HLB
Dielectric constant
Maximum concentration of dissolved complexes [%] LEU INS DES docusate docusate docusate
Capryol 90
Propylene glycol monocaprylate (type II)
5
6.1
5.0
1.25
2.5
Labrafac Lipophile WL 1349
Triglycerides medium chain
1
2.9
-
-
-
Labrafil M 1944 CS
Oleoyl macrogol-6 glycerides
9
3.7
0.625
-
-
Labrafil M 2125 CS
Linoleoyl macrogol-6 glycerides
9
3.4
-
-
-
Labrasol ALF
Caprylocaproyl macrogol-8 glycerides
12
8.1
5.0
2.5
5.0
Lauroglycol 90
Propylene glycol monolaurate (type II)
3
4.7
5.0
-
-
Maisine CC
Glycerol monolinoleate
1
3.3
5.0
0.625
2.5
Peceol
Glycerol monooleate (type 40)
1
3.5
2.5
2.5
2.5
Plurol Oleique CC 497
Polyglyceryl-3 dioleate
3
3.0
0.625
0.625
-
Propylene glycol
1,2-Propanediol
NA
32.0
5.0
5.0
5.0
Tetraglycol
Glycofurol
NA
15.7
5.0
5.0
5.0
Transcutol HP
Diethylene glycol monoethyl ether
NA
14.1
5.0
5.0
5.0
Tween 20
Sorbitan monolaurate
17
NA
2.5
-
2.5
22
Table 5: Characterization of unloaded SEDDS diluted 2% (v/v) in 50 mM PBS pH 6.8 regarding droplet size [µm] and zeta potential [mV] as a function of time. Indicated values are means (n ≥ 3) ± SD. Mean droplet size [µm]
Zeta potential [mV]
PDI Time [min]
Mean droplet size [µm]
Zeta potential [mV]
Mean droplet size [µm]
PDI
Zeta potential [mV]
PDI
0
120
240
SEDDS 0.295±0.001
A
0.238±0.010
0.321±0.003
-8.33±0.22
-7.87±0.25
0.470
0.328±0.006
B
0.547
0.439±0.052 -9.31±0.49
0.307±0.002 -10.73±0.35
0.518
0.521
2.866±0.459
2.980±1.049
-5.51±0.19
-7.09±0.86
0.242
-6.48±0.21
0.427
3.977±0.286
D
-9.68±0.22
0.735
5.618±1.090
C
-7.19±0.14
0.432
0.737
6.859±0.742 -13.73±0.76
5.554±0.907 -10.93±0.78
0.307
-7.94±0.25
0.237
0.579
Table 6: Payload of peptide complexes incorporated into novel SEDDS; dissolved. Payload Payload Complex Formulation complex peptide [%] [%] LEU
Tested payload [%] 0.17
0.33
0.67
1.33
2.67
5.33
10.67
A&B
10.67
5.93
C&D
10.67
7.31
A&B
10.67
6.67
Docusate INS Docusate DES Docusate
23
Table 7: Characterization of peptide complexes loaded SEDDS diluted 2% (v/v) in 50 mM PBS pH 6.8 regarding droplet size [µm] and zeta potential [mV] as a function of time. Indicated values are means (n ≥ 3) ± SD. Mean droplet size [µm]
Zeta potential [mV]
Mean droplet size [µm]
PDI Time [min] Complex
Zeta potential [mV]
PDI 0
Mean droplet size [µm]
Zeta potential [mV]
PDI 120
240
SEDDS 0.221±0.016
A
0.249 ±0.002 -13.93±0.21
0.529
LEU Docusate
0.527
0.287±0.009
B
0.554
Docusate
DES
0.439
0.278±0.009 -26.63±0.15
0.514
0.368±0.013
B
-17.90±0.20 0.533
0.214±0.003 -24.90±0.26
0.583
Docusate
5.591±0.263 -18.27±0.31
0.396 0.271±0.009
-24.03±0.67 0.849
4.706±0.325 -19.73±0.38
A
4.713±1.016 -25.87±1.25
0.415
4.946±1.317
D
-21.33±0.33 0.547
3.188±0.362 -25.80±0.70
0.754
INS
0.256±0.003 -20.93±0.15
0.467 4.838±1.257
-16.53±0.47 0.453
0.277±0.008 -21.13±0.72
C
0.205±0.001 -16.40±0.30
0.355±0.002 -27.97±0.75
-26.70±0.92 0.547 0.383±0.004
-29.27±1.00
0.576
0.573
24
-28.73±0.21 0.557
S0378-5173(17)30100-X http://dx.doi.org/doi:10.1016/j.ijpharm.2017.02.019 IJP 16423
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
11-1-2017 2-2-2017 6-2-2017
Please cite this article as: Griesser, Janine, Het´enyi, Gergely, Moser, Michael, Demarne, Fr´ed´eric, Jannin, Vincent, Bernkop-Schnurch, ¨ Andreas, Hydrophobic ion pairing: Key to highly payloaded self-emulsifying peptide drug delivery systems.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2017.02.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Hydrophobic ion pairing: Key to highly payloaded self-emulsifying peptide drug delivery systems
Janine Griesser1, Gergely Hetényi1, Michael Moser1, Frédéric Demarne3, Vincent Jannin3, Andreas Bernkop-Schnürch1,2 *
1
Thiomatrix Forschungs-und Beratungs GmbH, Trientlgasse 65, Innsbruck, Austria
2
Center for Chemistry and Biomedicine, Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria
3
Gattefossé SAS, 36 chemin de Genas, 69804 Saint-Priest Cedex, France
*Corresponding Author: Andreas Bernkop-Schnürch Center for Chemistry and Biomedicine, Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria Tel.: +43-512-507 58601 Fax: +43-512-507 58699 e-mail: [email protected]
1
Graphical abstract
Highlights
Independent from the type of peptide, docusate showed the most efficient hydrophobic ion pairing properties and was revealed as universal counter ion.
Novel SEDDS containing Capryol 90, Labrafil M 2125 CS, Labrasol ALF, Peceol, propylene glycol, tetraglycol, Transcutol HP and Tween 20 were developed.
The complexes were incorporated into SEDDS achieving a payload over 10%.
SEDDS exhibited high stability and constant negative zeta potential over a 4 h incubation time.
Correlation between the type of lipid excipients and the solubility was detected.
Abstract Aim: The aim of this study was the formation and characterization of various ion pairs of therapeutic peptides with different surfactants in order to reach a high payload in selfemulsifying drug delivering systems (SEDDS).
2
Methods: Hydrophobic ion pairs (HIP) were formed between the anionic surfactants sodium docusate, dodecylsulfate and oleate and the peptides leuprorelin (LEU), insulin (INS) and desmopressin (DES). The efficiency of HIP formation was evaluated by quantifying the amount of formed complexes, log P value determination in n-octanol/water via HPLC and zeta potential measurements. Solvents and surfactants were screened regarding their complex solubilizing properties. Subsequently, peptide complexes were incorporated into SEDDS followed by payload and stability determination. Results: Independent from the type of peptide, docusate showed the most efficient HIP properties followed by dodecylsulfate and oleate. Ratios of 2:1 for LEU, 6:1 for INS and 1.5:1 for DES led to the highest quantity of formed complexes with docusate and log P increased at least by 3 units. The more docusate was added to each peptide, the more negative became the zeta potential of the resulting complex. Incorporating these optimized complexes into novel SEDDS containing Capryol 90, Labrafil M 2125 CS, Labrasol ALF, Peceol, propylene glycol, tetraglycol, Transcutol HP and Tween 20 allowed payloads of the LEU, DES and INS complexes above 10%. Moreover, SEDDS exhibited high stability and constant negative zeta potential over a 4 h incubation time. Conclusion: Following the procedure described herein payloads > 10 % can be achieved for peptide drugs in SEDDS.
Keywords: Leuprorelin, Insulin, Desmopressin, Self-emulsifying drug delivery systems, Hydrophobic ion paring, Peptide delivery
3
1. Introduction Self-emulsifying drug delivery systems (SEDDS) representing isotropic mixtures of oils, solvents and emulsifiers are likely opening the door for the oral administration of as challenging drugs as therapeutic peptides and proteins (Leonaviciute and Bernkop-Schnürch, 2015). Incorporating peptide drugs in lipid droplets protects them towards thiol/disulfide exchange reactions with food and endogenous glutathione as well as towards an enzymatic degradation in the gastrointestinal tract (Dahm and Jones, 1994; Ijaz et al., 2016; Schmitz et al., 2006). Moreover, these lipid droplets can be designed in a way that they are able to permeate the mucus gel barrier in a comparatively efficient manner (Friedl et al., 2013). Once having reached the underlying absorption membrane, SEDDS were shown to exhibit even permeation enhancing properties for peptide drugs (Leonaviciute and Bernkop-Schnürch, 2015). Up to date, however, this promising strategy has by far not reached its full potential, as various hurdles still need to be overcome. One of these hurdles is certainly the poor solubility of peptide drugs in the lipophilic phase of SEDDS. Because of their mainly hydrophilic nature peptides and proteins can likely only be incorporated in lipids via hydrophobic ion pairing (HIP). But even by utilizing this technique only comparatively low payloads were achieved. Hintzen et al. (Hintzen et al., 2014), for instance, reached a payload of just 0.4% for leuprorelin, Karamanidou et al. achieved 1.13% for insulin (Karamanidou et al., 2015) and Zupančič et al. (Zupančič et al., 2016) obtained 0.25% for desmopressin in SEDDS. According to these results, the low peptide payload of SEDDS had to be identified as worrying bottleneck for this otherwise promising strategy. In order to address this problem, it was the aim of this study to focus just on this issue and to provide a broader understanding on parameters influencing the efficiency of HIP and the subsequent incorporation of peptide complexes in SEDDS. For comparison reasons three
4
peptides including cyclic or non-cyclic as well as high molecular weight and low molecular weight peptides were chosen. As leuprorelin (LEU), insulin (INS) and desmopressin (DES) are cationic peptides, the anionic surfactants sodium docusate, sodium dodecylsulfate and sodium oleate exhibiting different acidic groups, namely a sulfonate, sulfate and carboxylic moiety, were tested for HIP. In order to achieve comparatively high payloads, the solubility of the most lipophilic complexes was investigated in various solvents. Those solvents in which complexes could be dissolved most efficiently were in the following utilized to form SEDDS.
2. Materials and methods 2.1.
Materials
Leuprorelin acetate and desmopressin acetate were purchased from Chemos GmbH, Germany. Insulin from porcine pancreas was bought from Prospec Protein, Israel. Lipids utilized within this study were provided by Gattefossé, France. Tween 20 was a gift from Croda, Germany. All other reagents were purchased from Sigma-Aldrich, Austria. 2.2.
Methods
2.2.1. HPLC analysis Peptides were analyzed on a Hitachi Elite LaChrom HPLC-System equipped with L-2130 pump, L-2200 autosampler and L-2400 UV detector. As mobile phase a binary solvent system of solvent A – water with 0.1% (v/v) trifluoroacetic acid (TFA) and solvent B – acetonitrile with 0.1% (v/v) TFA was used. Leuprorelin was quantified utilizing XBridge BEH300 C18 3.5 µm 4.6 mm x 150 mm as stationary phase. An isocratic method (70% mobile phase A and 30% mobile phase B) over 12 minutes at 40°C with a flow rate of 0.8 ml/min was applied. Moreover, all samples were stored at 10°C within the auto sampler until injection of 20 µl and thereafter
5
analyzed at a wavelength of 222 nm. Insulin was quantified with LiChrosorb RP-18 LiChroCART 5 µm, 100 Å, 125 x 4 as stationary phase. An isocratic method (65% mobile phase A and 35% mobile phase B) over 7 minutes at 40°C with a flow rate of 1 ml/min was used. Aliquots of 50 µl were injected and analyzed at a wavelength of 214 nm. For desmopressin Nucleosil 100-5 C18 5 µm 4 mm x 250 mm as stationary phase was used. An isocratic method (75% mobile phase A and 25% mobile phase B) over 10 minutes at 40°C with a flow rate of 1 ml/min was applied. The injection volume was 20 µl and samples were analyzed at a wavelength of 222 nm. 2.2.2. Hydrophobic ion pairing (HIP) First, 1 ml of each peptide solution was prepared in a concentration of 10 mg/ml utilizing 0.01 M HCl as solvent. As leuprorelin, desmopressin and insulin exhibit different net positive charges at this pH , several ratios of peptide to surfactant were analyzed. Thereafter, surfactants as listed in Table 1 were dissolved in 1 ml of demineralized water applying ratios as indicated in Table 1. The solution of each surfactant was added drop wisely to the peptide solution under vigorous stirring (400 rpm). The mixtures were stirred for two hours and subsequently centrifuged at 12,500 rpm for 10 min with High-Speed Mini Centrifuge (Fisher Scientific, Illinois, USA). During the reaction, an immediate white precipitation indicated the formation of the hydrophobic peptide complex. The supernatant was separated from the precipitate and washed with 0.01 M HCl. Thereafter, the water-soluble fraction of the peptide remaining in the supernatant was determined by measuring its concentration via HPLC as described above. Finally, the pellets were frozen, lyophilized and stored at -30°C. As blank reference, the surfactant solutions were added to 0.01 M HCl without peptide. Precipitation efficiency was defined utilizing the following equation: Precipitation efficiency [%] = 100 − (
𝑃𝑒𝑝𝑡𝑖𝑑𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑎𝑓𝑡𝑒𝑟 𝐻𝐼𝑃 × 100) 𝑃𝑒𝑝𝑡𝑖𝑑𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑏𝑒𝑓𝑜𝑟𝑒 𝐻𝐼𝑃 6
2.2.3. Log P determination To 1 ml of n-octanol/water (1:1), 1 mg of peptide or 1 mg of complex was added and incubated at 37°C while shaking at 300 rpm for 24 hours. Samples were centrifuged for 10 min at 10,000 rpm with High-Speed Mini Centrifuge (Fisher Scientific, Illinois, USA). Thereafter, 100 µl aliquots were withdrawn from both of the aqueous and the n-octanol phase and diluted with 300 µl of methanol containing 0.1% (v/v) TFA. The concentration of peptides and complexes in aqueous as well as n-octanol phase was analyzed via HPLC as described above. The partition coefficient was calculated using the following equation: log P = log
c peptide in octanol phase c peptide in aqueous phase
2.2.4. Evaluation of zeta potential during HIP First, 0.5 ml of 1% (m/v) peptide solutions were prepared in 0.01 M HCl and surfactants were dissolved in 0.5 ml of demineralized water in concentrations corresponding to molar ratios as indicated in Table 1. The solution of each surfactant was added dropwisely to the peptide solution under vigorous stirring (400 rpm). Thereafter, zeta potential of the resulting precipitate was measured utilizing Zetasizer Nano-ZSP (Malvern Instruments, Worcestershire, UK). 2.2.5. Solubility studies Peptide-surfactant complexes applying four concentrations, namely 0.625%, 1.25%, 2.5% and 5% (m/v) were added to organic solvents, lipids and surfactants. In particular, solubility of complexes in Capryol 90, Labrafac Lipophile WL 1349, Labrafil M 1944 CS, Labrafil M 2125 CS, Labrasol ALF, Lauroglycol 90, Maisine CC, Peceol, Plurol Oleique CC 497, tetraglycol, propylene glycol, Tween 20 and Transcutol HP was examined. Mixtures of complexes and solvents were sonicated and optionally heated to 37°C while shaking. Afterwards, the mixtures
7
were incubated for 12 hours at room temperature. The dissolution was evaluated visually after centrifugation at 12500 rpm for 5 min. 2.2.6. Preparation and characterization of SEDDS Four novel SEDDS containing excipients as listed in Table 2 were developed and peptide complexes were incorporated in an increasing concentration via ultrasonification for 15 min at room temperature and examined for precipitation and homogeneity. The incorporation was evaluated visually after centrifugation at 12500 rpm for 5 min and the highest achievable payload was additionally confirmed via HPLC analysis. Therefore, 100 µl SEDDS were diluted with 6 ml of methanol containing 0.1% trifluoroacetic acid followed by HPLC analysis as described above utilizing on a six-point-calibration curve. Size and zeta potential of SEDDS were determined by photon correlation spectroscopy with Zetasizer Nano-ZSP (Malvern Instruments, Worcestershire, UK) at 37°C. As control, SEDDS were prepared and investigated without incorporating peptide complexes. The formulations were diluted 2% (v/v) in 50 mM phosphate buffer saline (PBS) pH 6.8 for droplet size measurements and with demineralized water for zeta potential measurements. Furthermore, SEDDS were examined by measuring droplet size after a time period of 120 and 240 minutes at 37°C while shaking at 300 rpm in order to investigate their stability. The payload of complexes and peptides in SEDDS was calculated as follows: Payload complex [%] = (
Payload peptide [%] = (
𝑚 𝑑𝑖𝑠𝑠𝑜𝑙𝑣𝑒𝑑 𝑐𝑜𝑚𝑝𝑙𝑒𝑥 × 100) 𝑉𝑆𝐸𝐷𝐷𝑆 𝑏𝑙𝑎𝑛𝑘
𝑚 𝑝𝑒𝑝𝑡𝑖𝑑𝑒 × 𝑝𝑎𝑦𝑙𝑜𝑎𝑑 𝑐𝑜𝑚𝑝𝑙𝑒𝑥) 𝑚 𝑝𝑒𝑝𝑡𝑖𝑑𝑒 + 𝑚 𝑠𝑢𝑟𝑓𝑎𝑐𝑡𝑎𝑛𝑡
2.2.7. Statistical data analyses
8
Statistical data analyses were performed using Student’s t-test to analyze the significant difference between two mean values. Level of p≤ 0.05 was set for significant, p≤ 0.01 for very significant and p≤ 0.001 for highly significant. The results were expressed as the mean of at least three experiments ± standard deviation (SD).
3. Results 3.1.
Hydrophobic Ion Pairing
The impact of anionic surfactants exhibiting different functional groups and number of carbon atoms as illustrated in Table 3 was evaluated. The more of these surfactants were added to each peptide, to a higher extend lipophilic complexes were formed until a maximum was reached. Increasing the surfactant concentration beyond that maximum did not lead to a plateau phase. In contrast, the amount of formed complex decreased when the amount of surfactants was further raised. This observation can be explained by the formation of micelles re-dissolving the complex (Choi and Park, 2000). Dai e Dong (Dai and Dong, 2007), for instance, demonstrated for INS that an excessive amount of sodium dodecyl sulfate above the ratio 6:1 leads to a decrease in turbidity of the HIP dispersion and a lower amount of precipitated peptide. LEU containing the two basic amino acids Arginine and Histidine, showed the highest precipitation efficiency of 99.1% at a molar ratio of 2:1 applying sodium docusate, as illustrated in Figure 1. In case of INS with six basic amino acids (2 Histidine, 1 Arginine, 1 Lysine and 2 N-terminal amino acids), a molar ratio of 6:1 exhibited the highest efficiency utilizing sodium docusate and sodium dodecyl sulfate (Figure 2). Moreover, DES as cyclic peptide with one basic amino acid (Arginine) demonstrated the highest precipitation efficiency of 93.4% applying sodium docusate at a molar ratio of 1.5:1 (Figure 3) compared to sodium dodecyl sulfate and sodium oleate. According to these results at least to a certain extent a correlation between the number of cationic
9
substructures on the peptide and the most efficient molar ratio of peptide to surfactant could be found. Comparing the complex formation efficacy determined by the degree of precipitation in aqueous medium, sodium docusate ranked independently from the number of cationic substructures, form and size of peptides in all cases first. Whether the superior properties of docusate are mainly related to its branched structure, its dipole moment or the sulfonate group could not be determined. Nevertheless, these results point out the crucial role of the structure of surfactants for efficient HIP. 3.2.
Evaluation of zeta potential during HIP
Apart from the complex formation efficiency the shift in zeta potential due to HIP was investigated utilizing sodium docusate as representative surfactant. Results showed for all peptides an increase in negative charge the more docusate was bound to their surface. Nevertheless, a direct correlation between the number of attached surfactant molecules and the resulting zeta potential could not be observed. Generally, the impact of docusate on the zeta potential was high. Leuprorelin, for instance, demonstrated a change in zeta potential from +21 mV to -13 mV when comparing a surfactant to leuprorelin ratio of 0.5:1 with 4:1 as illustrated in Figure 4A. In case of the surfactant to insulin ratios from 1:1 to 12:1, the zeta potential shifted even from +40 mV to -72 mV. These results as shown in Figure 4B are in good agreement with a study performed by Sun et al. demonstrating that an excess of anionic deoxycholate counter ion results in an increasing negative zeta potential of insulin - deoxycholate complexes (Sun et al., 2011). As pictured in Figure 4C, the surfactant to desmopressin ratio of 0.5:1 showed a zeta potential of +3 mV and for 3:1 a zeta potential of -52 mV was measured. Results showed also that the surfactant is comparatively tightly bound to each peptide, as it would unless not have such a strong impact on the zeta potential. Furthermore, the lipophilic substructures of the surfactant seem to have a greater impact on lipidization than their charge neutralizing effect, as the surfactant to peptide ratio resulting according to Figure 1 – Figure 3 in the most 10
hydrophobic complexes does in no case correlate with the ratio leading to an almost uncharged complex. 3.3.
Log P determination
In order to evaluate the increase in lipophilicity of the chosen model peptides via HIP, log P was determined for comparison. Results of this study are illustrated in Figure 5. Leuprorelin with the highest increase in log P showed an improvement of 5 units. Taking the number of surfactants per peptide with 2 for LEU, 6 for INS and 1.5 for DES into account, the increase in lipophilicity is not simply dependent on the number of surfactant molecules being attached to each peptide. When also the size of these peptides is considered, however, a correlation with obtained data can be seen leading to the following rank order: LEU with 1.7 surfactant molecules per kDa (s/kDa) > DES with 1.4 s/kDa > INS with 1.0 s/kDa. According to this, the number of immobilized surfactants per kDa peptide seems to be one of the key parameters for lipidization. 3.4.
Solubility studies
Solubility studies of the complexes were performed with solvents being most appropriate for the development of SEDDS. Results of these studies are illustrated in Table 4. The LEU – docusate complex exhibiting the comparatively highest log P of all tested complexes showed also the highest solubility dissolving in all tested excipients expect Labrafac Lipophile WL 1349 and Labrafil M 2125 CS. INS – docusate complex was highly soluble in Labrasol ALF, Transcutol HP and Peceol, whereas the desmopressin complex demonstrated high solubility in Labrasol ALF, Maisine CC, Capryol 90, Peceol and Transcutol HP. Generally, all complexes were highly soluble in Transcutol HP followed by Labrasol ALF. Although the dielectric constant of Maisine CC is comparatively very low and no complex could be dissolved in solvents of a dielectric constant in the same range – namely Labrafac Lipophile WL 1349 and Labrafil M 2125 CS – all complexes could be dissolved in this solvent in comparatively high concentrations. Generally,
11
aprotic solvents such as Labrafac Lipophile WL 1349, Labrafil M 1944 CS, Labrafil M 2125 CS, Maisine CC and Peceol showed lower complex solubilizing properties compared to the protic solvents Transcutol HP, tetraglycol, propylene glycol and Labrasol ALF. As water possesses a dielectric constant of 80, a convergence to 80 is essential for increasing solubility and payload. Low solubilizing properties were also confirmed for aprotic and usually water insoluble SEDDS components having a dielectric constant from 2.9 to 3.7, whereas protic components of a dielectric constant between 8.1 and 32.0 showed comparatively higher solubilizing properties. Monoesters such as monoglycerides (Peceol, Maisine CC) or monoesters of propylene glycol (Lauroglycol, Capryol) showed higher complex solubilizing properties compared to triesters (triglycerides - Labrafac Lipophile WL 1349), as monoesters exhibit higher hydrophilic and lipophilic balance (HLB) values and higher polarity than triesters. 3.5.
Preparation and characterization of SEDDS
SEDDS were developed based on the results of the solubility studies as described above and initially the unloaded SEDDS as listed in Table 5 were characterized. Thereafter, LEU and DES docusate complexes were incorporated into formulations A and B whereas INS docusate complexes were loaded into formulations C and D. As listed in Table 6, 7 different payloads were tested for all formulations and the payloads were additionally confirmed via HPLC. In all cases a payload of 10.67% representing the highest tested concentration could be achieved. For LEU this result represents a 25-fold increase in payload compared to the payload obtained in former studies by Hintzen et al. (Hintzen et al., 2014; Leonaviciute et al., 2016) and for INS the improvement was 6-fold compared to that achieved by Karamanidou et al. (Karamanidou et al., 2015). In case of DES the improvement in payload was even 40-fold compared to that reported in a recent study by Zupančič et al. (Zupančič et al., 2016). As listed in Table 5 and Table 7, all formulations showed stability over 4 hours. Formulations A and B demonstrated a mean droplet size of 0.3 µm with a polydispersity index (PDI) < 0.750, a constant negative zeta potential and 12
no change in droplet size over 4 hours. Formulations C and D exhibited a mean droplet size of 6 µm with a PDI < 0.850. SEDDS loaded with complexes showed an even higher value of negative zeta potential due to the presence of anionic docusate in the complexes. A constant negative zeta potential guarantees the stability of the droplets and it is a reliable indicator concerning emulsion stability. As the zeta potential of the emulsions did not change over the incubation time, these SEDDS even at comparatively high complex payload can be assessed as highly stable delivery systems.
4. Discussion Although most peptides and protein drugs are very potent and highly active resulting in comparatively low doses being often in the microgram range, a high payload in SEDDS is nevertheless substantial. On the one hand the comparatively much lower oral bioavailability of therapeutic peptides and proteins being in most cases still below 1% has to be taken into consideration, as a consequently 10- up to 200-fold higher amount of drug is needed to achieve the same effect as via parenteral administration. On the other hand, the amount of SEDDS containing surfactants and solvents, that must – although having GRAS status – from the toxicological point of view in particular when being used for treatment of chronic diseases such as diabetes type 2 be addressed very carefully, needs to be kept at a feasible minimum. Furthermore, for certain peptides and protein drugs even considerable high amounts need to be administered. Based on the results obtained in this study, however, it seems likely that reaching sufficient high payloads cannot be regarded anymore as bottle neck for oral peptide administration via SEDDS. The generation of highly lipophilic complexes of therapeutic peptides and proteins will furthermore have also a great impact on other key parameters regarding their oral administration via SEDDS. On the one hand the release rate of peptides from SEDDS is mainly controlled by the distribution coefficient of the lipophilic complex between the 13
lipid phase of SEDDS and the intestinal fluid. Accordingly, the more lipophilic the complex are the more sustained is the release out of the oily droplets. Such a more sustained release can certainly be regarded as advantages, as it was shown by Hintzen et al. and Karamanidou et al. (Hintzen et al., 2014; Karamanidou et al., 2015; Zupančič et al., 2016) that due to the incorporation of peptides into SEDDS they are protected in the gastrointestinal tract towards a presystemic metabolism by peptidases and proteases. On the other hand, the lipophilic complexes described herein will likely exhibit also improved membrane permeability. The more lipophilic a drug is, the more easily it can permeate the phospholipid bilayer of cell membranes. As shown in this study, surfactants form in an efficient manner lipophilic complexes with peptides. According to this, it is our opinion that most surfactants exhibiting a permeation enhancing effect for peptides act rather via a lipidization of peptides than making the cell membrane more leaky or binding calcium or magnesium ions on the membrane loosing thereby tight junctions. The stability of these peptides – surfactant complexes in the body, however, remains unclear. At least, orientating studies of our own research group heading in this direction failed due to the high complexity of the analytic of such complexes in human full blood (data not shown). Although forming considerable stable lipophilic complexes peptide drugs seem to maintain their therapeutic efficacy. An oral insulin formulation based on SEDDS, for instance was even already tested in phase II clinical trial (OramedPharmaceuticals, 2016). Apart from their comparatively high payload, SEDDS having been generated within this study exhibit also other favorable properties. As the cell membrane is covered with negatively charged mucus gel layer, SEDDS with negative zeta potential are necessary to avoid interactions with mucus and biogenic substances (He et al., 2010; Ishiwata et al., 2000; Zhou and Huang, 1994) and to permeate the mucus more easily compared to positively charged SEDDS already sticking on the surface without reaching the underlying absorption membrane (Loo et al., 2012). 5. Conclusion 14
Comparing the formulation of lipophilic complexes of different peptides with different surfactants provided a fundamental overview on key parameters having to be addressed in order to incorporate these hydrophilic macromolecular drugs in SEDDS. Firstly, the choice of surfactant plays a crucial role representing the likely most important parameter. Secondly, the number of surfactant molecules being directly attached per kDa peptide is essential. Increasing the number of surfactant molecules just via the formation of likely mixed micelles does not improve the lipophilic character of the peptide. In contrast, this approach leads even to an increase in hydrophilicity of the complex. Thirdly, the right choice of solvents for the complex in SEDDS can strongly contribute to improved payloads. In particular protic solvents of a dielectric constant around 10 seem to be favorable. Taking these points into account, payload in the even double digit percentage range can be achieved for self-emulsifying peptide drug delivery systems.
6. Conflict of interest Vincent Jannin and Frédéric Demarne were employed by Gattefossé SAS manufacturing and selling lipid-based excipients at the time this study was performed.
7. Acknowledgement The authors would like to thank Dr Joachim Quadflieg from Gattefossé GmbH, Germany for supply excipients for the development of SEDDS and for the technical support.
15
References Choi, S.H., Park, T.G., 2000. Hydrophobic ion pair formation between leuprolide and sodium oleate for sustained release from biodegradable polymeric microspheres. Int J Pharm 203, 193202. Dahm, L.J., Jones, D.P., 1994. Secretion of cysteine and glutathione from mucosa to lumen in rat small intestine. Am J Physiol 267, G292-300. Dai, W.G., Dong, L.C., 2007. Characterization of physiochemical and biological properties of an insulin/lauryl sulfate complex formed by hydrophobic ion pairing. Int J Pharm 336, 58-66. Friedl, H., Dünnhaupt, S., Hintzen, F., Waldner, C., Parikh, S., Pearson, J.P., Wilcox, M.D., Bernkop-Schnürch, A., 2013. Development and evaluation of a novel mucus diffusion test system approved by self-nanoemulsifying drug delivery systems. J Pharm Sci 102, 4406-4413. He, C., Hu, Y., Yin, L., Tang, C., Yin, C., 2010. Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials 31, 3657-3666. Hintzen, F., Perera, G., Hauptstein, S., Müller, C., Laffleur, F., Bernkop-Schnürch, A., 2014. In vivo evaluation of an oral self-microemulsifying drug delivery system (SMEDDS) for leuprorelin. Int J Pharm 472, 20-26. Ijaz, M., Bonengel, S., Zupančič, O., Yaqoob, M., Hartl, M., Hussain, S., Huck, C.W., BernkopSchnürch, A., 2016. Development of oral self nano-emulsifying delivery system(s) of lanreotide with improved stability against presystemic thiol-disulfide exchange reactions. Expert Opin Drug Deliv 13, 923-929. Ishiwata, H., Suzuki, N., Ando, S., Kikuchi, H., Kitagawa, T., 2000. Characteristics and biodistribution of cationic liposomes and their DNA complexes. J Control Release 69, 139-148. Karamanidou, T., Karidi, K., Bourganis, V., Kontonikola, K., Kammona, O., Kiparissides, C., 2015. Effective incorporation of insulin in mucus permeating self-nanoemulsifying drug delivery systems. Eur J Pharm Biopharm 97, 223-229. Leonaviciute, G., Bernkop-Schnürch, A., 2015. Self-emulsifying drug delivery systems in oral (poly)peptide drug delivery. Expert Opin Drug Deliv 12, 1703-1716. Leonaviciute, G., Zupančič, O., Prüfert, F., Rohrer, J., Bernkop-Schnürch, A., 2016. Impact of lipases on the protective effect of SEDDS for incorporated peptide drugs towards intestinal peptidases. Int J Pharm 508, 102-108. Loo, Y., Grigsby, C.L., Yamanaka, Y.J., Chellappan, M.K., Jiang, X., Mao, H.Q., Leong, K.W., 2012. Comparative study of nanoparticle-mediated transfection in different GI epithelium coculture models. J Control Release 160, 48-56. OramedPharmaceuticals, 2016. http://www.oramed.com/pipeline/ormd-0801-type-2/ (accessed in December 2016). Schmitz, T., Huck, C.W., Bernkop-Schnürch, A., 2006. Characterisation of the thiol-disulphide chemistry of desmopressin by LC, mu-LC, LC-ESI-MS and Maldi-Tof. Amino Acids 30, 35-42. Sun, S., Liang, N., Kawashima, Y., Xia, D., Cui, F., 2011. Hydrophobic ion pairing of an insulin-sodium deoxycholate complex for oral delivery of insulin. Int J Nanomedicine 6, 30493056. Zhou, X., Huang, L., 1994. DNA transfection mediated by cationic liposomes containing lipopolylysine: characterization and mechanism of action. Biochim Biophys Acta 1189, 195-203. Zupančič, O., Leonaviciute, G., Lam, H.T., Partenhauser, A., Podričnik, S., Bernkop-Schnürch, A., 2016. Development and in vitro evaluation of an oral SEDDS for desmopressin. Drug Deliv 23, 2074-2083.
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Figure Captions Figure 1: Hydrophobic ion pairing of LEU with sodium docusate (■), sodium oleate (∆) and sodium dodecyl sulfate (x). After the addition of surfactants, the precipitated LEU complex was centrifuged and the remaining peptide in supernatant was analyzed by HPLC. Indicated values are means (n ≥ 3) ± SD.
Figure 2: Hydrophobic ion pairing of INS with sodium docusate (■), sodium oleate (∆) and sodium dodecyl sulfate (x). After the addition of surfactants, the precipitated INS complex was centrifuged and the remaining peptide in supernatant was analyzed by HPLC. Indicated values are means (n ≥ 3) ± SD.
17
Figure 3: Hydrophobic ion pairing of DES with sodium docusate (■), sodium oleate (∆) and sodium dodecyl sulfate (x). After the addition of surfactants, the precipitated DES complex was centrifuged and the remaining peptide in supernatant was analyzed by HPLC. Indicated values are means (n ≥ 3) ± SD.
Figure 4: Zeta potential [mV] measurement during HIP of (A) LEU, (B) INS and (C) DES with sodium docusate. Indicated values are means (n ≥ 3) ± SD.
18
Figure 5: Log P of indicated peptide drugs (LEU, INS and DES) (gray bars) and the corresponding HIP complexes LEU-docusate 2:1, INS-docusate 6:1 and DES-docusate 1.5:1 (white bars). Indicated values are means (n ≥ 3) ± SD.
19
Tables Table 1: Complexes of leuprorelin, insulin and desmopressin with indicated surfactants.
Peptide
Net positive charges
Basic amino acids (AA)
Tested surfactants
Molar ratios [surfactant: peptide] 0.5:1
Sodium docusate
1:1
Sodium dodecyl sulfate
2:1
Sodium oleate
3:1
Arginine Leuprorelin
2 Histidine
4:1 1:1 Arginine Sodium docusate
3:1
Sodium dodecyl sulfate
6:1
Sodium oleate
9:1
Histidine Insulin
6 2 Lysine 2 N-terminal AA
12:1 0.5:1
Desmopressin
1
Arginine
Sodium docusate
1:1
Sodium dodecyl sulfate
1.5:1
Sodium oleate
2:1 3:1
20
Table 2: Composition of novel SEDDS containing the three peptide complexes. Values are indicated in percent (v/v). Formulation
A
B
C
D
Transcutol HP
5
5
-
-
Tetraglycol
-
10
10
30
Propyleneglycol
-
-
10
-
Peceol
20
20
10
30
Capryol 90
10
10
-
-
Labrasol ALF
35
25
60
40
Labrafil M 2125 CS
-
-
10
-
Tween 20
30
30
-
-
Table 3: List and chemical structure of tested surfactants. Surfactant
Chemical group
Number of carbon atoms
sulfonate
20
sulfate
12
carboxylic
18
Sodium docusate
Sodium dodecyl sulfate
Sodium oleate
21
Table 4: Classification of lipid based excipients utilized within this study; (HLB) hydrophilic and lipophilic balance; (NA) not applicable. Solubility of peptide docusate complexes in four concentrations 0.625%, 1.25%, 2.5% and 5.0% (m/v). (-) not dissolved or lower than 0.625%. Excipient
Definition
HLB
Dielectric constant
Maximum concentration of dissolved complexes [%] LEU INS DES docusate docusate docusate
Capryol 90
Propylene glycol monocaprylate (type II)
5
6.1
5.0
1.25
2.5
Labrafac Lipophile WL 1349
Triglycerides medium chain
1
2.9
-
-
-
Labrafil M 1944 CS
Oleoyl macrogol-6 glycerides
9
3.7
0.625
-
-
Labrafil M 2125 CS
Linoleoyl macrogol-6 glycerides
9
3.4
-
-
-
Labrasol ALF
Caprylocaproyl macrogol-8 glycerides
12
8.1
5.0
2.5
5.0
Lauroglycol 90
Propylene glycol monolaurate (type II)
3
4.7
5.0
-
-
Maisine CC
Glycerol monolinoleate
1
3.3
5.0
0.625
2.5
Peceol
Glycerol monooleate (type 40)
1
3.5
2.5
2.5
2.5
Plurol Oleique CC 497
Polyglyceryl-3 dioleate
3
3.0
0.625
0.625
-
Propylene glycol
1,2-Propanediol
NA
32.0
5.0
5.0
5.0
Tetraglycol
Glycofurol
NA
15.7
5.0
5.0
5.0
Transcutol HP
Diethylene glycol monoethyl ether
NA
14.1
5.0
5.0
5.0
Tween 20
Sorbitan monolaurate
17
NA
2.5
-
2.5
22
Table 5: Characterization of unloaded SEDDS diluted 2% (v/v) in 50 mM PBS pH 6.8 regarding droplet size [µm] and zeta potential [mV] as a function of time. Indicated values are means (n ≥ 3) ± SD. Mean droplet size [µm]
Zeta potential [mV]
PDI Time [min]
Mean droplet size [µm]
Zeta potential [mV]
Mean droplet size [µm]
PDI
Zeta potential [mV]
PDI
0
120
240
SEDDS 0.295±0.001
A
0.238±0.010
0.321±0.003
-8.33±0.22
-7.87±0.25
0.470
0.328±0.006
B
0.547
0.439±0.052 -9.31±0.49
0.307±0.002 -10.73±0.35
0.518
0.521
2.866±0.459
2.980±1.049
-5.51±0.19
-7.09±0.86
0.242
-6.48±0.21
0.427
3.977±0.286
D
-9.68±0.22
0.735
5.618±1.090
C
-7.19±0.14
0.432
0.737
6.859±0.742 -13.73±0.76
5.554±0.907 -10.93±0.78
0.307
-7.94±0.25
0.237
0.579
Table 6: Payload of peptide complexes incorporated into novel SEDDS; dissolved. Payload Payload Complex Formulation complex peptide [%] [%] LEU
Tested payload [%] 0.17
0.33
0.67
1.33
2.67
5.33
10.67
A&B
10.67
5.93
C&D
10.67
7.31
A&B
10.67
6.67
Docusate INS Docusate DES Docusate
23
Table 7: Characterization of peptide complexes loaded SEDDS diluted 2% (v/v) in 50 mM PBS pH 6.8 regarding droplet size [µm] and zeta potential [mV] as a function of time. Indicated values are means (n ≥ 3) ± SD. Mean droplet size [µm]
Zeta potential [mV]
Mean droplet size [µm]
PDI Time [min] Complex
Zeta potential [mV]
PDI 0
Mean droplet size [µm]
Zeta potential [mV]
PDI 120
240
SEDDS 0.221±0.016
A
0.249 ±0.002 -13.93±0.21
0.529
LEU Docusate
0.527
0.287±0.009
B
0.554
Docusate
DES
0.439
0.278±0.009 -26.63±0.15
0.514
0.368±0.013
B
-17.90±0.20 0.533
0.214±0.003 -24.90±0.26
0.583
Docusate
5.591±0.263 -18.27±0.31
0.396 0.271±0.009
-24.03±0.67 0.849
4.706±0.325 -19.73±0.38
A
4.713±1.016 -25.87±1.25
0.415
4.946±1.317
D
-21.33±0.33 0.547
3.188±0.362 -25.80±0.70
0.754
INS
0.256±0.003 -20.93±0.15
0.467 4.838±1.257
-16.53±0.47 0.453
0.277±0.008 -21.13±0.72
C
0.205±0.001 -16.40±0.30
0.355±0.002 -27.97±0.75
-26.70±0.92 0.547 0.383±0.004
-29.27±1.00
0.576
0.573
24
-28.73±0.21 0.557