International Journal of Pharmaceutics 472 (2014) 20–26
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Pharmaceutical Nanotechnology
In vivo evaluation of an oral self-microemulsifying drug delivery system (SMEDDS) for leuprorelin Fabian Hintzen, Glen Perera, Sabine Hauptstein, Christiane Müller, Flavia Laffleur, Andreas Bernkop-Schnürch * Department of Pharmaceutical Technology, Institute of Pharmacy, Center for Molecular Biosciences, Leopold-Franzens-University Innsbruck, Innrain 80/82, Center for Chemistry and Biomedicine, Innsbruck 6020, Austria
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 March 2014 Received in revised form 22 May 2014 Accepted 26 May 2014 Available online 29 May 2014
The objective of this study was to develop a self-microemulsifying drug delivery system (SMEDDS) for the model peptide drug leuprorelin to prove a protective effect against luminal enzymatic metabolism. In order to incorporate leuprorelin into microemulsion droplets (o/w), the commercially available hydrophilic leuprolide acetate was modified by hydrophobic ion paring with sodium oleate. The obtained hydrophobic leuprolide oleate was dissolved in the SMEDDS formulation (30% (m/m) Cremophor EL, 30% (m/m) Capmul MCM, 10% (m/m) propylene glycol and 30% (m/m) Captex 355) in a concentration of 4 mg/g showing a mean droplet size of 50.1 nm when dispersed in a concentration of 1% (m/v) in phosphate buffer pH 6.8. The microemulsion was able to shield leuprolide oleate from enzymatic degradation by trypsin and a-chymotrypsin, so that after 120 min 52.9% and 58.4%, respectively, of leuprolide oleate were still intact. Leuprolide acetate dissolved in an aqueous control solution was completely metabolized by trypsin within 60 min and by a-chymotrypsin within 5 min. Moreover, an in vivo study in rats showed a 17.2-fold improved oral bioavailability of leuprolide oleate SMEDDS compared to a leuprolide acetate control solution. This is the first time, to our knowledge, that hydrophobic ion pairing is utilized in order to incorporate a peptide drug in SMEDDS and evidence of a protective effect of oil-in-water (o/w) microemulsion droplets against enzymatic degradation of a peptide drug was provided. According to these results, the system could be likely a novel platform technology to improve the oral bioavailability of peptide drugs. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Self-microemulsifying drug delivery system Protection against enzymatic degradation Leuprorelin Hydrophobic ion pairing
1. Introduction The oral administration of peptide and protein drugs remains a significant challenge for pharmaceutical researcher due to several physiological barriers limiting gastrointestinal absorption. The rapid degradation by luminal enzymes is one of the main problems that needs to be overcome to enhance systemic uptake (Guo et al., 2004). An increasingly popular approach to improve the bioavailability of peptide drugs via the oral route are self-microemulsifying drug delivery systems (SMEDDS). SMEDDS are isotropic mixtures of oil(s), one or more surfactants and a co-surfactant (or co-solubilizer) (Gursoy and Benita, 2004). Dispersion of these mixtures in an aqueous environment leads to transparent or slightly bluish,
* Corresponding author. Tel.: +43 512 507 58601; fax: +43 512 507 58699. E-mail address: [email protected] (A. Bernkop-Schnürch). http://dx.doi.org/10.1016/j.ijpharm.2014.05.047 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
thermodynamically stable oil-in-water (o/w) microemulsions with a droplet size range from 10 to 300 nm (Sarciaux et al., 1995; Gursoy and Benita, 2004; Anton and Vandamme, 2011). As just a gentle agitation is required to emulsify these formulations, the digestive motility of stomach and intestine is sufficient after oral application (Charman et al., 1992; Shah et al., 1994; Constantinides, 1995). Among many factors contributing to the improved oral bioavailability are the large surface area, permeation enhancement and protection against luminal enzymatic degradation. Although the latter point is often mentioned in the literature, to our knowledge, it is not yet demonstrated. In addition, it was rarely successful to incorporate peptides into o/w microemulsion droplets due to their generally hydrophilic nature. Leuprorelin (leuprolide acetate) is a synthetic gonadotropinreleasing hormone (GnRH) analogue used in the treatment of sex hormone-related disorders such as advanced prostate cancer, endometriosis and precocious puberty (Plosker and Brogden, 1994; Kutscher et al., 1997). The highly water-soluble nonapeptide has
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two ionizable basic side chains, imidazole group of histidine (pKa 6.0) and guanidine group of arginine (pKa 13.0) (Choi and Park, 2000). As most peptide drugs, its bioavailability is low, and it is usually given intramuscularly as depot injection (e.g., Lupron1). The Transport studies already showed that inhibition of proteolytic enzymes could improve the intestinal absorption of leuprorelin (Guo et al., 2004). Therefore, it was the aim of the study to prove a protective effect of a SMEDDS for the model peptide drug leuprorelin against metabolism by intestinal enzymes. In order to incorporate leuprorelin into the lipophilic core of the SMEDDS droplets, the commercially available hydrophilic leuprolide acetate was modified by hydrophobic ion paring with sodium oleate to obtain the hydrophobic leuprolide oleate. As the peptide drugs in general show poor permeability across intestinal membranes, a formulation with permeation enhancing properties seems appropriate for the preparation of leuprorelin SMEDDS. Therefore, leuprolide oleate SMEDDS were prepared employing a formulation with a permeation enhancing effect. Increased permeation for the hydrophilic macromolecular compound fluorescein isothiocyanate–dextran 4 (FD4) via tight junction opening was shown in a previous study (Hintzen et al., 2013); as leuprolide acetate is also mainly absorbed by the paracellular route (Guo et al., 2004) this formulation seems suitable.
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phosphate buffer pH 6.8 in a concentration of 1% (m/v) under gentle stirring (200 rpm) resulting in a leuprolide loaded microemulsion (0.04 mg/mL). The mean droplet size was measured by dynamic light scattering using a PSS NICOMP TM 380 DLS (Santa Barbara, CA, USA). A microemulsion without leuprolide served as control. Additionally, the lyophilized leuprolide oleate (molar ratio 3:1) was dispersed in phosphate buffer pH 6.8 in a concentration of 0.04 mg/mL. The leuprolide oleate suspension and leuprolide oleate microemulsion were analyzed by HPLC with respect to the solubility of leuprolide oleate in both media and the purity of the lyophilized product. 2.4. Quantitation of leuprorelin via RP-HPLC HPLC analysis was performed according to a method previously described by our research group (Iqbal et al., 2011). In brief, samples containing leuprorelin were analyzed on a Nucleosil 100-5 C18 column (250 4 mm) at 40 C with gradient elution (1 mL/min): 0–10 min; linear gradient; from 25% A/75% B to 40% A/60% B (eluent A: acetonitrile; eluent B: 0.1% trifluoroacetic acid) at 278 nm. The calibration curve was established with leuprolide acetate in a range from 1.5 to 200 mg/mL. The detection and quantitation limit was investigated in the validation study, and can be specified with 0.48 mg/mL and 1.44 mg/mL (Iqbal et al., 2011).
2. Materials and methods 2.5. Drug release and payload 2.1. Materials Capmul MCM (mono/diglycerides of caprylic acid, HLB = 5–6) and Captex 355 (caprylic/capric triglyceride) was supplied by Abitec Corporation, USA. Cremophor EL (non-ionic emulsifier obtained by causing ethylene oxide to react with castor oil in a molar ratio of 35 to 1, HLB = 12–14) was purchased from BASF, Germany. Propylene glycol was obtained from Gatt-Koller, Absam, Austria. Acetonitrile and water for HPLC analysis were purchased from Avantor Performance Materials, Netherlands. Trifluoroacetic acid was obtained from Carl Roth, Germany. Leuprolide acetate (Mr 1209.4 Da (free peptide)) was supplied by Chemos, Germany. All other chemicals were purchased from Sigma–Aldrich, Austria. All chemicals were of analytical grade. 2.2. Hydrophobic ion pairing of leuprorelin In order to increase hydrophobicity of leuprolide acetate to improve the lipid-solubility, hydrophobic ion pairing with sodium oleate was performed as described previously (Choi and Park, 2000). Therefore, sodium oleate dissolved in demineralized water was added to leuprolide acetated solutions 1 mg/mL in a molar ratio of 1:1, 2:1, 3:1 and 4:1 under continuous stirring. The obtained solutions with precipitated ion pairs were centrifuged at 5000 rpm. The aqueous supernatant was analyzed for remaining dissolved leuprorelin via HPLC as described below. The precipitated pellet was freeze-dried at 30 C and 0.01 mbar (Christ Gamma 1-16 LSC Freeze dryer) and stored at 24 C. 2.3. Preparation and characterization of SMEDDS A previously developed SMEDDS formulation showing a permeation enhancing effect via tight junction opening was employed. The self-emulsifying mixtures was composed of 30% (m/m) Cremophor EL, 30% (m/m) Capmul MCM, 10% (m/m) propylene glycol and 30% (m/m) Captex 355 (Hintzen et al., 2013). The lyophilized leuprolide oleate was dissolved in a concentration of 4 mg/g in the SMEDDS by using a thermomixer at 37 C for 12 h. Subsequently, the SMEDDS were emulsified in
Determining the content of incorporated leuprorelin in droplets, microemulsions were filled into dialysis tubes (MWCO 10,000 Da) and placed in a beaker with phosphate buffer pH 6.8. This system was incubated under stirring, and the amount of leuprorelin in the outer water phase was quantified. The mixture resulting from sodium oleate: leuprolide acetate in a ratio of 3:1 showed the lowest amount of residual leuprolide acetate in the water phase during hydrophobic ion pairing. Therefore, this precipitate was used for further release studies. Briefly, a thoroughly mixed formulation containing leuprolide oleate in SMEDDS (4 mg/g) was emulsified in phosphate buffer pH 6.8 (1% m/v). Afterwards, 50 mL of the microemulsion was filled into the dialysis tube and placed in a beaker with 50 mL phosphate buffer pH 6.8. The release of leuprorelin to the outer phase was analyzed via HPLC over 30 h. An aqueous leuprolide acetate solution (0.04 mg/mL) served as control. 2.6. Enzymatic degradation by intestinal enzymes Enzymatic degradation studies were performed according to a modified method, as described previously by our research group (Werle et al., 2006; Perera et al., 2009). Leuprolide acetate and leuprolide oleate SMEDDS were dissolved in a buffer containing 1.36 g/L of Na2HPO4, 0.22 g/L of KH2PO4 and 8.5 g/L of NaCl to obtain a final concentration of 0.04 mg/mL. Enzyme solutions were prepared in a final activity of 9.35 IU/mL for trypsin, 7.16 BTEE U/mL for a-chymotrypsin and 0.29 IU/mL for elastase. The enzyme activities were in accordance with physiological conditions (Bernkop-Schnurch, 1998). The degradation studies were performed by adding 100 mL of each enzyme solution to 100 mL of leuprolide acetate solution (0.04 mg/mL) and leuprolide oleate microemulsion (0.04 mg/mL leuprolide oleate/1% m/v microemulsion), respectively, and incubated in a thermomixer at 37 C. The enzymatic reaction was stopped at predetermined time points by adding 100 mL of 2% trifluoroacetic acid (TFA) to the reaction mixture. The samples were analyzed by HPLC. The observed retention time for leuprolide acetate was 5.7 min and for leuprolide oleate 9.7 min.
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Table 1 Formulations used for in vivo studies in male Sprague Dawley rats. Formulation
Route of administration
Dose
Dosage form
Volume
Leuprolide Leuprolide Leuprolide Leuprolide Leuprolide
Iv Oral Oral Oral Oral
0.25 mg 1 mg 1 mg 1 mg 1 mg
Solution Solution Suspension SMEDDS SMEDDS
250 mL 250 mL 250 mL 250 mL 250 mL
acetate loaded aqueous solution acetate loaded aqueous solution oleate loaded aqueous suspension acetate loaded SMEDDS oleate loaded SMEDDS
2.7. In vivo evaluation of leuprorelin SMEDDS in rats The protocol for the in vivo study on animals was approved by the Animal Ethical Committee of Vienna, Austria and adheres to the Principles of Laboratory Animal Care. The in vivo study was performed on 25 Sprague Dawley rats weighing 200–250 g, which were divided into 5 cohorts of 5 animals each (Table 1). The rats were housed in polycarbonate cages (46 cm 25 cm 21 cm) at room temperature and fasted for 2 h before administration of all dosage forms, but with free access to water. Aliquots (250 mL) of each oral formulation were administered through a flexible plastic stomach tube with a round tip in order to minimize trauma, followed by administration of 250 mL of water. Intravenous injections were applied into tail veins. The blood samples were taken at predetermined time points from the tail vein. The blood samples (approximately 120 mL) were spiked with 20 mL of a 3.8% (m/v) sodium citrate solution in order to prevent blood clotting. The samples were immediately centrifuged at 10,000 rpm to obtain the plasma.
ISCID energy, 0 eV; hexapole RF, 500 Vpp; ion energy, 6.0 eV; collision energy, 10.0 eV; collision RF, 600 Vpp; transfer time, 60 ms; prepulse storage, 12.0 ms; mass range, 200–2000 m/z. 2.10. Pharmacokinetic and statistical data analysis Pharmacokinetic parameters of leuprorelin were calculated by applying a non-compartmental pharmacokinetic analysis to the plasma concentration–time data using the software GraphPad Prism 5 version 5.01. The area under the concentration versus time curve (AUC0–last) was calculated in accordance to the linear trapezoidal rule, using kinetic data collected from individual values. The absolute bioavailability was calculated from the dose corrected AUCs for oral versus intravenous administration. Statistical data analysis was performed using one-way ANOVA and the Kruskal–Wallis test with 95% confident interval (p-value < 0.05) as the minimal level of significance followed by a multiple-comparison Bonferroni and Dunns post hoc test (p < 0.05). The results were expressed as the mean of at least 3 experiments SD.
2.8. Standard and sample preparation 3. Results and discussion The stock solution of leuprolide acetate (1 mg/mL) was prepared in 50% acetonitrile and 50% water with 0.1% formic acid. Calibration curves (1–1000 ng/mL) were established by adding 20 mL of leuprolide standards to 100 mL of rat plasma. Afterwards, standards and samples were treated 2 times with 200 mL of icecold acetonitrile in order to precipitate plasma proteins. Subsequently, the samples were centrifuged at 13,400 rpm for 10 min, and the supernatant was transferred into glass vials, followed by evaporating to dryness with a Univapo 100 ECH (Uniequip, Germany) for 60 min at 40 C. The residue was dissolved with 125 mL of mobile phase (80% A and 20% B) before analyzing via LC– MS (Sofianos et al., 2008; Iqbal et al., 2012).
3.1. Hydrophobic ion pairing of leuprorelin
2.9. Quantitation of leuprorelin via LC–MS
The hydrophobic ion pair exchange of leuprolide acetate with sodium oleate led to precipitation in aqueous media. In the following, this precipitate could be separated from the water soluble fraction by centrifugation. The extent of ion pairing was determined by measuring residual leuprolide in the water phase. As shown in Fig. 1, the leuprolide water solubility decreased up to a molar ratio of 3:1 and increased with further increasing the concentration of oleate. Although leuprolide has two basic amino acid groups that should be able to bind two molecules of oleate, the aqueous solubility decreased by adding more oleate. Choi and Park explained this effect with the formation of a hydrophobic complex when reaching a molar
Leuprorelin was quantified in blood samples of the in vivo study via LC–MS by a slightly modified method described previously by our research group (Sofianos et al., 2008; Iqbal et al., 2012). In brief, the analysis was carried out by LC on an Agilent 1200 Series system (Agilent Technologies, Waldbronn, Germany) equipped with a G1312B SL binary pump, G1329B autosampler, vacuum degasser and G1316B column oven. The mobile phase consisted of solvents A: 10% acetonitrile, 90% water, 2 mM ammonium acetate, 0.1% formic acid and B: 90% acetonitrile, 10% water, 2 mM ammonium acetate, 0.1% formic acid. The plasma samples and the standards were separated on a YMC-Pack C4 column (250 4.6 mm, 5 mm, 30 nm pore size) using isocratic elution (80% A: 20% B) for 15 min at a flow rate of 1 mL/min, an oven temperature of 60 C and injection volume of 40 mL. Mass spectrometry was performed on a Bruker MicrOTOF-Q II system operated in positive ion mode under the following conditions: end plate offset, 500 V; capillary voltage, 4500 V; nebulizer pressure, 29 psi; dry gas (nitrogen) flow rate, 6.0 L/min; dry temperature, 200 C; funnel 1 RF, 200 Vpp; funnel 2 RF, 300 Vpp;
Fig. 1. Comparison of residual leuprolide acetate in the water phase depending on the ratio between leuprolide acetate and sodium oleate during hydrophobic ion pairing process. Indicated values are means (SD, n = 3).
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ratio of 3:1. However, further addition of sodium oleate (molar ratio 4:1) led to an efficient micellar solubilization of leuprorelin by sodium oleate (Choi and Park, 2000). Further investigation of the lyophilized leuprolide oleate pellet (molar ratio 3:1) as aqueous dispersion and microemulsion by HPLC indicated that the hydrophobic ion pairing was not complete. The leuprolide oleate pellet was dispersed in water and centrifuged at 10,000 rpm for 5 min. The chromatogram of the aqueous supernatant after centrifugation showed a single peak at the retention time of leuprolide acetate, an undissolved pellet remained. However, when the leuprolide oleate pellet was dissolved in SMEDDS, no precipitation in the generated microemulsion after centrifugation could be observed, and the chromatogram showed two peaks: one for leuprolide acetate and one for leuprolide oleate (data not shown). Accordingly, the microemulsion as well as the lyophilized pellets contained both ion pairs. The results demonstrated that leuprolide oleate is only soluble in the microemulsion, whereas leuprolide acetate could be dissolved in the aqueous control solution and in the aqueous phase of the o/w microemulsion. The ratio of leuprolide acetate to oleate in the microemulsion was 59:41. 3.2. Preparation and characterization of SMEDDS The lyophilized leuprolide oleate could be dissolved in the SMEDDS formulation in a concentration of 4 mg/g. All four products of hydrophobic ion pairing (molar ratio 1:1, 2:1, 3:1 and 4:1) led to clear, homogenous mixtures that were able to selfemulsify in phosphate buffer pH 6.8 within a minute. The obtained microemulsions were slightly bluish. Moreover, as shown in Fig. 2, the droplet size of control microemulsion without leuprorelin and all other leuprorelin microemulsions did not differ significantly. The fact that the droplet size does not increase when the leuprolide oleate is incorporated seems beneficial from the drug delivery point of view providing a large surface area dispersion to improve drug absorption. PDI values are 0.060 0.002 for unloaded SMEDDS and 0.103 0.005 for loaded SMEDDS. 3.3. Drug release and payload The drug release of leuprorelin across a dialysis membrane (MWCO 10,000 Da) in an aqueous environment over time is shown in Fig. 3. As already mentioned, the microemulsion contained
Fig. 2. Comparison of the droplet sizes of Leu-SMEDDS emulsified in phosphate buffer pH 6.8 with respect to the ratio between leuprolide acetate and sodium oleate during hydrophobic ion pairing process. Indicated values are means (SD, n = 3).
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leuprolide acetate and oleate; therefore two release profiles are shown. The release of leuprolide acetate is slower, but similar to the control solution indicating that leuprolide acetate is incorporated into the microemulsion droplet to a very low extent. However, the profile of leuprolide oleate is significantly sustained and after 30 h approximately 50% were released. As the plateau phase was reached, the remaining leuprolide oleate was likely still incorporated in SMEDDS droplets. Consequently, the drug release studies confirm the assumption that leuprolide oleate was at least partially incorporated into the microemulsion droplets. The sustained release of leuprorelin by embedding it into the droplets is advantageous as for oral delivery of peptide drugs, a drug release as close as possible to the site of absorption is favored. The microemulsion avoids an immediate release and could shield the peptide drug from rapid degradation until the absorption site is reached. Although the peptide might be degraded to a certain extent, the probability of the peptide drug reaching the apportion side unaltered is increased. Furthermore, the permeation enhancing effect of the microemulsion may support the absorption to increase bioavailability (Hintzen et al., 2013). A drawback of the developed microemulsion might be the incomplete release of leuprolide oleate under the chosen conditions. However, in vivo the excipients are likely digested by lipases or mixed micelles with bile acids and phospholipids could be formed, which leads to an enhanced release of the embedded leuprorelin. 3.4. Enzymatic degradation by intestinal enzymes One of the main obstacles to improve the oral bioavailability of leuprorelin is the enzymatic barrier of the gastrointestinal tract due to an intensively degradation by proteolytic enzymes (Woodley, 1994; Imanidis et al., 1995; Guo et al., 2004). Therefore, a selfemulsifying drug delivery system was developed to protect leuprorelin from enzymatic degradation in the intestine. Trypsin, a-chymotrypsin and elastase were separately investigated to measure a specific protective effect and to evaluate the extent of degradation by each enzyme. Fig. 4 shows the degradation profiles of leuprorelin in a microemulsion prepared with the lyophilized product of hydrophobic ion paring (molar ratio 3:1) and an aqueous control solution containing leuprolide acetate by trypsin as a function of time. The microemulsion contained leuprolide acetate as well as leuprolide oleate as described in Section 3.1. The leuprolide acetate control was completely degraded within 60 min, whereas leuprolide acetate in the microemulsions was metabolized rapidly within
Fig. 3. Leuprorelin release profiles from an aqueous leuprolide acetate solution ( ) and Leu-SMEDDS (1:3) emulsified in phosphate buffer pH 6.8 ( ) (&). As the leuprorelin precipitated incorporated into the SMEDDS contained leuprolide acetate and leuprolide oleate, release profile for both ion pairs are indicated serparately: leuprolide acetate ( ) and leuprolide oleate (&) from Leu-SMEDDS microemulsion. Indicated values are means (SD, n = 3).
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examined. This lack of information is a obstacle regarding the development of self-emulsifying drug delivery systems. Furthermore, the incorporation of peptides into the lipophilic droplets is a great challenge due to their hydrophilic nature and was rarely successful so far (Rao and Shao, 2008; Rao et al., 2008a,b). All other studies in which emulsions were investigated as peptide carriers, the drug is assumed to be in the aqueous phase (Swenson and Curatolo, 1992; Ho et al., 1996; Rao and Shao, 2008). Within the present study it could be demonstrated that the protective effect against enzymatic metabolism is improved, when the peptide is embedded in the lipophilic phase. 3.5. In vivo evaluation of leuprorelin SMEDDS Fig. 4. Degradation profile of leuprolide acetate by trypsin in an aqueous leuprolide acetate solution ( ) and as Leu-SMEDDS (1:3) emulsified in phosphate buffer pH 6.8 ( ) (&). As the leuprorelin precipitated incorporated into the SMEDDS contained leuprolide acetate and leuprolide oleate, degradation profiles of both ion pairs are indicated serparately: leuprolide acetate ( ) and leuprolide oleate (&) in the microemulsion. Fig. 5B highlights the first 5 min of the degradation process due to the rapid metabolism by a-chymotrypsin. Indicated values are means (SD, n = 3).
the first 60 min; it remained stable thereafter (Fig. 4A). Leuprolide oleate seemed to be protected by the microemulsion droplets right from the beginning, resulting in a comparatively lower extent of degradation (Fig. 4B). Accordingly, the leuprolide oleate is likely embedded into the lipid phase due to the higher hydrophobicity and therefore protected against degradation by trypsin. The degradation of leuprolide acetate control and of leuprorelin SMEDDS (3:1) by a-chymotrypsin was similar but more rapid (Fig. 5). Therefore, the reaction was stopped at different time points, and the first 5 min are separately presented in Fig. 5B. After 5 min no remaining leuprolide acetate was to be detected in the solution, whereas in the microemulsion a small amount remained stable. In contrast, leuprolide oleate was metabolized very slowly more than 50% remained unaltered within 120 min due to the protective effect of SMEDDS droplets. However, leuprorelin was not metabolized by elastase at all as the whole amount of leuprolide acetate and oleate in solution, and microemulsion could be found after 180 min of incubation (data not shown). Consequently, a protective effect was only shown for trypsin and a-chymotrypsin. The degradation studies with trypsin and a-chymotrypsin showed that leuprorelin SMEDDS had a limited protective effect for the hydrophilic leuprolide acetate. This might be caused by an insufficient–if at all–incorporation of leuprolide acetate into SMEDDS droplets. The nevertheless lower degradation might be a result of enzyme inhibitory properties of microemulsion components. However, ion pairing of leuprolide acetate with sodium oleate led to a hydrophobic product that was likely incorporated into the microemulsion droplets. As leuprolide oleate was shielded from degradation right from the start of the experiment, the peptide seems to be embedded into the microemulsion droplets. As mentioned in Section 3.1, both ion pairs (leuprolide acetate and oleate) were present in the microemulsion that allows good comparability in the same formulation in respect to the investigated protective effect. Self-microemulsifying drug delivery systems were extensively investigated in the last decade to improve the oral bioavailability of poorly water soluble drugs. Despite of the promising concept of improving oral bioavailability due to increased solubility (Patel and Vavia, 2007; Woo et al., 2007; Mezghrani et al., 2011), with a few exceptions (e.g., Neoral1) SMEDDS play a minor role on the pharmaceutical market. One reason therefore could be that most studies focus on a higher bioavailability by increasing watersolubility. Other promising properties of SMEDDS, e.g., protection of peptide drugs against enzymatic degradation are rarely
The detail information about administered formulations and the calculated pharmacokinetic parameters of the in vivo study are shown in Tables 1 and 2, respectively. The plasma–concentration profile of intravenous injections of leuprolide acetate to male Sprague Dawley rats indicate a rapid metabolism as more than 99% were metabolized within 90 min (Fig. 6A). Moreover, an orally administered leuprolide acetate solution proved that the peptide drug cannot be easily absorbed from the gastrointestinal tract (Cmax 5.98 ng/mL; absolute bioavailability 0.074%), which might be caused by severe degradation by luminal enzymes and a low permeability across intestinal membranes (Fig. 6B). However, leuprolide acetate given orally as SMEDDS showed a 6.5fold improved absolute bioavailability and reached a Cmax of
Fig. 5. (A and B) Degradation of leuprorelin by a-chymotrypsin in an aqueous leuprolide acetate solution ( ) and as Leu-SMEDDS (1:3) emulsified in phosphate buffer pH 6.8 ( ) (&). As the leuprorelin precipitated incorporated into the SMEDDS contained leuprolide acetate and leuprolide oleate, degradation profiles of both salts are indicated separately: leuprolide acetate ( ) and leuprolide oleate (&) in the microemulsion. B highlights the first 5 min of the degradation process due to the rapid metabolism by a-chymotrypsin. Indicated values are means (SD, n = 3).
F. Hintzen et al. / International Journal of Pharmaceutics 472 (2014) 20–26
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Table 2 Pharmacokinetic parameters calculated after iv and oral administration of investigated formulations in rats. Absolute bioavailability (BA) was calculated with reference to iv injection and indicated values are means of five rats. Delivery system
AUC0–last (min ng/mL)
Cmax (ng/mL)
Tmax (min)
Absolute BA (%)
Iv solution Leuprolide Leuprolide Leuprolide Leuprolide
144,653 426 0 2796ns 7385*
16,226.7 5.98 – 15.66ns 51.68*
– 30 – 30 30
– 0.074 – 0.483 1.276
acetate solution oleate suspension acetate SMEDDS oleate SMEDDS
ns: not significant compared to leuprolide acetate solution. * p < 0.05 compared to leuprolide acetate solution.
15.66 ng/mL after 30 min (Table 2). Moreover, the absorption was sustained as compared to the solution (Fig. 6B). This is likely caused by an enhanced permeability across intestinal membranes as the system was able to open tight junctions in vitro (Hintzen et al., 2013) representing the predominant way of uptake for hydrophilic macromolecular drugs like leuprorelin (Guo et al., 2004). Furthermore, the SMEDDS showed a slight protection of leuprolide acetate against degradation by intestinal serine proteases in vitro that might support the gastrointestinal uptake. If the freeze-dried pellet, obtained in the hydrophobic ion pairing process, was dissolved in the self-emulsifying system and orally administered to rats, the uptake was even further enhanced. The results showed a significant improvement of the area under the plasma concentration–time curve of leuprorelin (Fig. 6B), resulting in a 17.2-fold increased absolute bioavailability and a 8.6-fold increase of Cmax as compared to the control solution (Table 2). Moreover, the control
leuprolide acetate was no more detectable after 2 h when given as solution, whereas in case of leuprolide oleate SMEDDS plasma levels of around 8 ng/mL were found after 6 h. The enhanced and sustained uptake is likely caused by the combination of an improved transport across the intestinal gut wall, the protective effect against proteases and a sustained release of the peptide drug from the SMEDDS droplets. Up to now, most peptide and protein drugs are given intravenously or subcutaneously by injections due to several physiological barriers limiting the oral bioavailability of hydrophilic macromolecular drugs. In order to overcome the userunfriendly route of administration, especially for long-term disease treatment, it was the aim to develop a system that enables oral administration of peptides. The main obstacles are a low intestinal permeability and a rapid degradation by luminal enzymes. The developed self-emulsifying drug delivery system addresses these problems as the system can increase the permeability, particularly by tight junction opening, and shield leuprorelin against proteases. 4. Conclusion This is the first time, to our knowledge, that hydrophobic ion pairing of a peptide drug was used in order to embed a peptide drug successful into microemulsion droplets. Moreover, the selfmicroemulsifying drug delivery system for leuprorelin proved that SMEDDS can shield peptides from degradation by intestinal proteases. Furthermore, a sustained release of leuprorelin could be demonstrated that avoids an initial rapid degradation of the drug. These considerations could be confirmed by an in vivo study in rats and led to significant enhanced plasma profiles of leuprorelin via oral delivery. According to these results, the novel system could likely be a novel platform technology to improve the oral bioavailability of peptide drugs. Acknowledgement The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/ 2007–2013) under grant agreement n 280761. References
Fig. 6. (A) Plasma–concentration curve of leuprorelin after intravenous injection of leuprolide acetate to rats (dose = 1 mg/kg). (B) Plasma concentration curves of leuprorelin after oral administration of a leuprolide acetate solution ( ), a leuprolide oleate suspension (&) a leuprolide acetate SMEDDS ( ) and a leuprolide oleate SMEDDS ( ) to rats (dose = 4 mg/kg). Indicated values are means (SD) of five rats. Leuprolide oleate SMEDDS ( ) are significantly different compared to all the others (p < 0.05); leuprolide acetate SMEDDS ( ) are not significantly different compared to leuprolide acetate solution.
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Guo, J., Ping, Q., Jiang, G., Dong, J., Qi, S., Feng, L., Li, Z., Li, C., 2004. Transport of leuprolide across rat intestine, rabbit intestine and Caco-2 cell monolayer. Int. J. Pharm. 278, 415–422. Gursoy, R.N., Benita, S., 2004. Self-emulsifying drug delivery systems (SEDDS) for improved oral delivery of lipophilic drugs. Biomed. Pharmacother. 58, 173–182. Hintzen, F., Laffleur, F., Sarti, F., Müller, C., Bernkop-Schnürch, A., 2013. In vitro and ex vivo evaluation of an intestinal permeation enhancing self-microemulsifying drug delivery system (SMEDDS). J. Drug Deliv. Sci. Tech. 23, 261–267. Ho, H.O., Hsiao, C.C., Sheu, M.T., 1996. Preparation of microemulsions using polyglycerol fatty acid esters as surfactant for the delivery of protein drugs. J. Pharm. Sci. 85, 138–143. Imanidis, G., Hartner, K.C., Mazer, N.A., 1995. Intestinal permeation and metabolism of a model peptide (leuprolide) and mechanisms of permeation enhancement by nonionic surfactants. Int. J. Pharm. 120, 41–50. Iqbal, J., Shahnaz, G., Perera, G., Hintzen, F., Sarti, F., Bernkop-Schnurch, A., 2012. Thiolated chitosan: development and in vivo evaluation of an oral delivery system for leuprolide. Eur. J. Pharm. Biopharm. 80, 95–102. Iqbal, J., Vigl, C., Moser, G., Gasteiger, M., Perera, G., Bernkop-Schnurch, A., 2011. Development and in vivo evaluation of a new oral nanoparticulate dosage form for leuprolide based on polyacrylic acid. Drug Deliv. 18, 432–440. Kutscher, B., Bernd, M., Beckers, T., Polymeropoulos, E.E., Engel, J., 1997. Chemistry and molecular biology in the search for new LHRH antagonists. Angew. Chem. Int. Edit. 36, 2149–2162. Mezghrani, O., Ke, X., Bourkaib, N., Xu, B.H., 2011. Optimized self-microemulsifying drug delivery systems (SMEDDS) for enhanced oral bioavailability of astilbin. Pharmazie 66, 754–760. Patel, A.R., Vavia, P.R., 2007. Preparation and in vivo evaluation of SMEDDS (selfmicroemulsifying drug delivery system) containing fenofibrate. AAPS J. 9, E344–E352. Perera, G., Greindl, M., Palmberger, T.F., Bernkop-Schnurch, A., 2009. Insulin-loaded poly(acrylic acid)-cysteine nanoparticles: stability studies towards digestive enzymes of the intestine. Drug Deliv. 16, 254–260.
Plosker, G.L., Brogden, R.N., 1994. Leuprorelin – a review of its pharmacology and therapeutic use in prostatic-cancer, endometriosis and other sex hormonerelated disorders. Drugs 48, 930–967. Rao, S.V., Shao, J., 2008. Self-nanoemulsifying drug delivery systems (SNEDDS) for oral delivery of protein drugs: I. Formulation development. Int. J. Pharm. 362, 2– 9. Rao, S.V., Agarwal, P., Shao, J., 2008a. Self-nanoemulsifying drug delivery systems (SNEDDS) for oral delivery of protein drugs: II. In vitro transport study. Int. J. Pharm. 362, 10–15. Rao, S.V., Yajurvedi, K., Shao, J., 2008b. Self-nanoemulsifying drug delivery system (SNEDDS) for oral delivery of protein drugs: III. In vivo oral absorption study. Int. J. Pharm. 362, 16–19. Sarciaux, J.M., Acar, L., Sado, P.A., 1995. Using microemulsion formulations for oraldrug delivery of therapeutic peptides. Int. J. Pharm. 120, 127–136. Shah, N.H., Carvajal, M.T., Patel, C.I., Infeld, M.H., Malick, A.W., 1994. Self-emulsifying drug-delivery systems (Sedds) with polyglycolyzed glycerides for improving invitro dissolution and oral absorption of lipophilic drugs. Int. J. Pharm. 106, 15– 23. Sofianos, Z.D., Katsila, T., Kostomitsopoulos, N., Balafas, V., Matsoukas, J., Tselios, T., Tamvakopoulos, C., 2008. In vivo evaluation and in vitro metabolism of leuprolide in mice – mass spectrometry-based biomarker measurement for efficacy and toxicity. J. Mass Spectrom. 43, 1381–1392. Swenson, E.S., Curatolo, W.J., 1992. Intestinal permeability enhancement for proteins, peptides and other polar drugs – mechanisms and potential toxicity. 2. Adv. Drug Deliv. Rev. 8, 39–92. Werle, M., Samhaber, A., Bernkop-Schnurch, A., 2006. Degradation of teriparatide by gastro-intestinal proteolytic enzymes. J. Drug Target. 14, 109–115. Woo, J.S., Kim, T.S., Park, J.H., Chi, S.C., 2007. Formulation and biopharmaceutical evaluation of silymarin using SMEDDS. Arch. Pharm. Res. 30, 82–89. Woodley, J.F., 1994. Enzymatic barriers for Gi peptide and protein delivery. Crit. Rev. Ther. Drug Carrier Syst. 11, 61–95.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical Nanotechnology
In vivo evaluation of an oral self-microemulsifying drug delivery system (SMEDDS) for leuprorelin Fabian Hintzen, Glen Perera, Sabine Hauptstein, Christiane Müller, Flavia Laffleur, Andreas Bernkop-Schnürch * Department of Pharmaceutical Technology, Institute of Pharmacy, Center for Molecular Biosciences, Leopold-Franzens-University Innsbruck, Innrain 80/82, Center for Chemistry and Biomedicine, Innsbruck 6020, Austria
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 March 2014 Received in revised form 22 May 2014 Accepted 26 May 2014 Available online 29 May 2014
The objective of this study was to develop a self-microemulsifying drug delivery system (SMEDDS) for the model peptide drug leuprorelin to prove a protective effect against luminal enzymatic metabolism. In order to incorporate leuprorelin into microemulsion droplets (o/w), the commercially available hydrophilic leuprolide acetate was modified by hydrophobic ion paring with sodium oleate. The obtained hydrophobic leuprolide oleate was dissolved in the SMEDDS formulation (30% (m/m) Cremophor EL, 30% (m/m) Capmul MCM, 10% (m/m) propylene glycol and 30% (m/m) Captex 355) in a concentration of 4 mg/g showing a mean droplet size of 50.1 nm when dispersed in a concentration of 1% (m/v) in phosphate buffer pH 6.8. The microemulsion was able to shield leuprolide oleate from enzymatic degradation by trypsin and a-chymotrypsin, so that after 120 min 52.9% and 58.4%, respectively, of leuprolide oleate were still intact. Leuprolide acetate dissolved in an aqueous control solution was completely metabolized by trypsin within 60 min and by a-chymotrypsin within 5 min. Moreover, an in vivo study in rats showed a 17.2-fold improved oral bioavailability of leuprolide oleate SMEDDS compared to a leuprolide acetate control solution. This is the first time, to our knowledge, that hydrophobic ion pairing is utilized in order to incorporate a peptide drug in SMEDDS and evidence of a protective effect of oil-in-water (o/w) microemulsion droplets against enzymatic degradation of a peptide drug was provided. According to these results, the system could be likely a novel platform technology to improve the oral bioavailability of peptide drugs. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Self-microemulsifying drug delivery system Protection against enzymatic degradation Leuprorelin Hydrophobic ion pairing
1. Introduction The oral administration of peptide and protein drugs remains a significant challenge for pharmaceutical researcher due to several physiological barriers limiting gastrointestinal absorption. The rapid degradation by luminal enzymes is one of the main problems that needs to be overcome to enhance systemic uptake (Guo et al., 2004). An increasingly popular approach to improve the bioavailability of peptide drugs via the oral route are self-microemulsifying drug delivery systems (SMEDDS). SMEDDS are isotropic mixtures of oil(s), one or more surfactants and a co-surfactant (or co-solubilizer) (Gursoy and Benita, 2004). Dispersion of these mixtures in an aqueous environment leads to transparent or slightly bluish,
* Corresponding author. Tel.: +43 512 507 58601; fax: +43 512 507 58699. E-mail address: [email protected] (A. Bernkop-Schnürch). http://dx.doi.org/10.1016/j.ijpharm.2014.05.047 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
thermodynamically stable oil-in-water (o/w) microemulsions with a droplet size range from 10 to 300 nm (Sarciaux et al., 1995; Gursoy and Benita, 2004; Anton and Vandamme, 2011). As just a gentle agitation is required to emulsify these formulations, the digestive motility of stomach and intestine is sufficient after oral application (Charman et al., 1992; Shah et al., 1994; Constantinides, 1995). Among many factors contributing to the improved oral bioavailability are the large surface area, permeation enhancement and protection against luminal enzymatic degradation. Although the latter point is often mentioned in the literature, to our knowledge, it is not yet demonstrated. In addition, it was rarely successful to incorporate peptides into o/w microemulsion droplets due to their generally hydrophilic nature. Leuprorelin (leuprolide acetate) is a synthetic gonadotropinreleasing hormone (GnRH) analogue used in the treatment of sex hormone-related disorders such as advanced prostate cancer, endometriosis and precocious puberty (Plosker and Brogden, 1994; Kutscher et al., 1997). The highly water-soluble nonapeptide has
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two ionizable basic side chains, imidazole group of histidine (pKa 6.0) and guanidine group of arginine (pKa 13.0) (Choi and Park, 2000). As most peptide drugs, its bioavailability is low, and it is usually given intramuscularly as depot injection (e.g., Lupron1). The Transport studies already showed that inhibition of proteolytic enzymes could improve the intestinal absorption of leuprorelin (Guo et al., 2004). Therefore, it was the aim of the study to prove a protective effect of a SMEDDS for the model peptide drug leuprorelin against metabolism by intestinal enzymes. In order to incorporate leuprorelin into the lipophilic core of the SMEDDS droplets, the commercially available hydrophilic leuprolide acetate was modified by hydrophobic ion paring with sodium oleate to obtain the hydrophobic leuprolide oleate. As the peptide drugs in general show poor permeability across intestinal membranes, a formulation with permeation enhancing properties seems appropriate for the preparation of leuprorelin SMEDDS. Therefore, leuprolide oleate SMEDDS were prepared employing a formulation with a permeation enhancing effect. Increased permeation for the hydrophilic macromolecular compound fluorescein isothiocyanate–dextran 4 (FD4) via tight junction opening was shown in a previous study (Hintzen et al., 2013); as leuprolide acetate is also mainly absorbed by the paracellular route (Guo et al., 2004) this formulation seems suitable.
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phosphate buffer pH 6.8 in a concentration of 1% (m/v) under gentle stirring (200 rpm) resulting in a leuprolide loaded microemulsion (0.04 mg/mL). The mean droplet size was measured by dynamic light scattering using a PSS NICOMP TM 380 DLS (Santa Barbara, CA, USA). A microemulsion without leuprolide served as control. Additionally, the lyophilized leuprolide oleate (molar ratio 3:1) was dispersed in phosphate buffer pH 6.8 in a concentration of 0.04 mg/mL. The leuprolide oleate suspension and leuprolide oleate microemulsion were analyzed by HPLC with respect to the solubility of leuprolide oleate in both media and the purity of the lyophilized product. 2.4. Quantitation of leuprorelin via RP-HPLC HPLC analysis was performed according to a method previously described by our research group (Iqbal et al., 2011). In brief, samples containing leuprorelin were analyzed on a Nucleosil 100-5 C18 column (250 4 mm) at 40 C with gradient elution (1 mL/min): 0–10 min; linear gradient; from 25% A/75% B to 40% A/60% B (eluent A: acetonitrile; eluent B: 0.1% trifluoroacetic acid) at 278 nm. The calibration curve was established with leuprolide acetate in a range from 1.5 to 200 mg/mL. The detection and quantitation limit was investigated in the validation study, and can be specified with 0.48 mg/mL and 1.44 mg/mL (Iqbal et al., 2011).
2. Materials and methods 2.5. Drug release and payload 2.1. Materials Capmul MCM (mono/diglycerides of caprylic acid, HLB = 5–6) and Captex 355 (caprylic/capric triglyceride) was supplied by Abitec Corporation, USA. Cremophor EL (non-ionic emulsifier obtained by causing ethylene oxide to react with castor oil in a molar ratio of 35 to 1, HLB = 12–14) was purchased from BASF, Germany. Propylene glycol was obtained from Gatt-Koller, Absam, Austria. Acetonitrile and water for HPLC analysis were purchased from Avantor Performance Materials, Netherlands. Trifluoroacetic acid was obtained from Carl Roth, Germany. Leuprolide acetate (Mr 1209.4 Da (free peptide)) was supplied by Chemos, Germany. All other chemicals were purchased from Sigma–Aldrich, Austria. All chemicals were of analytical grade. 2.2. Hydrophobic ion pairing of leuprorelin In order to increase hydrophobicity of leuprolide acetate to improve the lipid-solubility, hydrophobic ion pairing with sodium oleate was performed as described previously (Choi and Park, 2000). Therefore, sodium oleate dissolved in demineralized water was added to leuprolide acetated solutions 1 mg/mL in a molar ratio of 1:1, 2:1, 3:1 and 4:1 under continuous stirring. The obtained solutions with precipitated ion pairs were centrifuged at 5000 rpm. The aqueous supernatant was analyzed for remaining dissolved leuprorelin via HPLC as described below. The precipitated pellet was freeze-dried at 30 C and 0.01 mbar (Christ Gamma 1-16 LSC Freeze dryer) and stored at 24 C. 2.3. Preparation and characterization of SMEDDS A previously developed SMEDDS formulation showing a permeation enhancing effect via tight junction opening was employed. The self-emulsifying mixtures was composed of 30% (m/m) Cremophor EL, 30% (m/m) Capmul MCM, 10% (m/m) propylene glycol and 30% (m/m) Captex 355 (Hintzen et al., 2013). The lyophilized leuprolide oleate was dissolved in a concentration of 4 mg/g in the SMEDDS by using a thermomixer at 37 C for 12 h. Subsequently, the SMEDDS were emulsified in
Determining the content of incorporated leuprorelin in droplets, microemulsions were filled into dialysis tubes (MWCO 10,000 Da) and placed in a beaker with phosphate buffer pH 6.8. This system was incubated under stirring, and the amount of leuprorelin in the outer water phase was quantified. The mixture resulting from sodium oleate: leuprolide acetate in a ratio of 3:1 showed the lowest amount of residual leuprolide acetate in the water phase during hydrophobic ion pairing. Therefore, this precipitate was used for further release studies. Briefly, a thoroughly mixed formulation containing leuprolide oleate in SMEDDS (4 mg/g) was emulsified in phosphate buffer pH 6.8 (1% m/v). Afterwards, 50 mL of the microemulsion was filled into the dialysis tube and placed in a beaker with 50 mL phosphate buffer pH 6.8. The release of leuprorelin to the outer phase was analyzed via HPLC over 30 h. An aqueous leuprolide acetate solution (0.04 mg/mL) served as control. 2.6. Enzymatic degradation by intestinal enzymes Enzymatic degradation studies were performed according to a modified method, as described previously by our research group (Werle et al., 2006; Perera et al., 2009). Leuprolide acetate and leuprolide oleate SMEDDS were dissolved in a buffer containing 1.36 g/L of Na2HPO4, 0.22 g/L of KH2PO4 and 8.5 g/L of NaCl to obtain a final concentration of 0.04 mg/mL. Enzyme solutions were prepared in a final activity of 9.35 IU/mL for trypsin, 7.16 BTEE U/mL for a-chymotrypsin and 0.29 IU/mL for elastase. The enzyme activities were in accordance with physiological conditions (Bernkop-Schnurch, 1998). The degradation studies were performed by adding 100 mL of each enzyme solution to 100 mL of leuprolide acetate solution (0.04 mg/mL) and leuprolide oleate microemulsion (0.04 mg/mL leuprolide oleate/1% m/v microemulsion), respectively, and incubated in a thermomixer at 37 C. The enzymatic reaction was stopped at predetermined time points by adding 100 mL of 2% trifluoroacetic acid (TFA) to the reaction mixture. The samples were analyzed by HPLC. The observed retention time for leuprolide acetate was 5.7 min and for leuprolide oleate 9.7 min.
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Table 1 Formulations used for in vivo studies in male Sprague Dawley rats. Formulation
Route of administration
Dose
Dosage form
Volume
Leuprolide Leuprolide Leuprolide Leuprolide Leuprolide
Iv Oral Oral Oral Oral
0.25 mg 1 mg 1 mg 1 mg 1 mg
Solution Solution Suspension SMEDDS SMEDDS
250 mL 250 mL 250 mL 250 mL 250 mL
acetate loaded aqueous solution acetate loaded aqueous solution oleate loaded aqueous suspension acetate loaded SMEDDS oleate loaded SMEDDS
2.7. In vivo evaluation of leuprorelin SMEDDS in rats The protocol for the in vivo study on animals was approved by the Animal Ethical Committee of Vienna, Austria and adheres to the Principles of Laboratory Animal Care. The in vivo study was performed on 25 Sprague Dawley rats weighing 200–250 g, which were divided into 5 cohorts of 5 animals each (Table 1). The rats were housed in polycarbonate cages (46 cm 25 cm 21 cm) at room temperature and fasted for 2 h before administration of all dosage forms, but with free access to water. Aliquots (250 mL) of each oral formulation were administered through a flexible plastic stomach tube with a round tip in order to minimize trauma, followed by administration of 250 mL of water. Intravenous injections were applied into tail veins. The blood samples were taken at predetermined time points from the tail vein. The blood samples (approximately 120 mL) were spiked with 20 mL of a 3.8% (m/v) sodium citrate solution in order to prevent blood clotting. The samples were immediately centrifuged at 10,000 rpm to obtain the plasma.
ISCID energy, 0 eV; hexapole RF, 500 Vpp; ion energy, 6.0 eV; collision energy, 10.0 eV; collision RF, 600 Vpp; transfer time, 60 ms; prepulse storage, 12.0 ms; mass range, 200–2000 m/z. 2.10. Pharmacokinetic and statistical data analysis Pharmacokinetic parameters of leuprorelin were calculated by applying a non-compartmental pharmacokinetic analysis to the plasma concentration–time data using the software GraphPad Prism 5 version 5.01. The area under the concentration versus time curve (AUC0–last) was calculated in accordance to the linear trapezoidal rule, using kinetic data collected from individual values. The absolute bioavailability was calculated from the dose corrected AUCs for oral versus intravenous administration. Statistical data analysis was performed using one-way ANOVA and the Kruskal–Wallis test with 95% confident interval (p-value < 0.05) as the minimal level of significance followed by a multiple-comparison Bonferroni and Dunns post hoc test (p < 0.05). The results were expressed as the mean of at least 3 experiments SD.
2.8. Standard and sample preparation 3. Results and discussion The stock solution of leuprolide acetate (1 mg/mL) was prepared in 50% acetonitrile and 50% water with 0.1% formic acid. Calibration curves (1–1000 ng/mL) were established by adding 20 mL of leuprolide standards to 100 mL of rat plasma. Afterwards, standards and samples were treated 2 times with 200 mL of icecold acetonitrile in order to precipitate plasma proteins. Subsequently, the samples were centrifuged at 13,400 rpm for 10 min, and the supernatant was transferred into glass vials, followed by evaporating to dryness with a Univapo 100 ECH (Uniequip, Germany) for 60 min at 40 C. The residue was dissolved with 125 mL of mobile phase (80% A and 20% B) before analyzing via LC– MS (Sofianos et al., 2008; Iqbal et al., 2012).
3.1. Hydrophobic ion pairing of leuprorelin
2.9. Quantitation of leuprorelin via LC–MS
The hydrophobic ion pair exchange of leuprolide acetate with sodium oleate led to precipitation in aqueous media. In the following, this precipitate could be separated from the water soluble fraction by centrifugation. The extent of ion pairing was determined by measuring residual leuprolide in the water phase. As shown in Fig. 1, the leuprolide water solubility decreased up to a molar ratio of 3:1 and increased with further increasing the concentration of oleate. Although leuprolide has two basic amino acid groups that should be able to bind two molecules of oleate, the aqueous solubility decreased by adding more oleate. Choi and Park explained this effect with the formation of a hydrophobic complex when reaching a molar
Leuprorelin was quantified in blood samples of the in vivo study via LC–MS by a slightly modified method described previously by our research group (Sofianos et al., 2008; Iqbal et al., 2012). In brief, the analysis was carried out by LC on an Agilent 1200 Series system (Agilent Technologies, Waldbronn, Germany) equipped with a G1312B SL binary pump, G1329B autosampler, vacuum degasser and G1316B column oven. The mobile phase consisted of solvents A: 10% acetonitrile, 90% water, 2 mM ammonium acetate, 0.1% formic acid and B: 90% acetonitrile, 10% water, 2 mM ammonium acetate, 0.1% formic acid. The plasma samples and the standards were separated on a YMC-Pack C4 column (250 4.6 mm, 5 mm, 30 nm pore size) using isocratic elution (80% A: 20% B) for 15 min at a flow rate of 1 mL/min, an oven temperature of 60 C and injection volume of 40 mL. Mass spectrometry was performed on a Bruker MicrOTOF-Q II system operated in positive ion mode under the following conditions: end plate offset, 500 V; capillary voltage, 4500 V; nebulizer pressure, 29 psi; dry gas (nitrogen) flow rate, 6.0 L/min; dry temperature, 200 C; funnel 1 RF, 200 Vpp; funnel 2 RF, 300 Vpp;
Fig. 1. Comparison of residual leuprolide acetate in the water phase depending on the ratio between leuprolide acetate and sodium oleate during hydrophobic ion pairing process. Indicated values are means (SD, n = 3).
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ratio of 3:1. However, further addition of sodium oleate (molar ratio 4:1) led to an efficient micellar solubilization of leuprorelin by sodium oleate (Choi and Park, 2000). Further investigation of the lyophilized leuprolide oleate pellet (molar ratio 3:1) as aqueous dispersion and microemulsion by HPLC indicated that the hydrophobic ion pairing was not complete. The leuprolide oleate pellet was dispersed in water and centrifuged at 10,000 rpm for 5 min. The chromatogram of the aqueous supernatant after centrifugation showed a single peak at the retention time of leuprolide acetate, an undissolved pellet remained. However, when the leuprolide oleate pellet was dissolved in SMEDDS, no precipitation in the generated microemulsion after centrifugation could be observed, and the chromatogram showed two peaks: one for leuprolide acetate and one for leuprolide oleate (data not shown). Accordingly, the microemulsion as well as the lyophilized pellets contained both ion pairs. The results demonstrated that leuprolide oleate is only soluble in the microemulsion, whereas leuprolide acetate could be dissolved in the aqueous control solution and in the aqueous phase of the o/w microemulsion. The ratio of leuprolide acetate to oleate in the microemulsion was 59:41. 3.2. Preparation and characterization of SMEDDS The lyophilized leuprolide oleate could be dissolved in the SMEDDS formulation in a concentration of 4 mg/g. All four products of hydrophobic ion pairing (molar ratio 1:1, 2:1, 3:1 and 4:1) led to clear, homogenous mixtures that were able to selfemulsify in phosphate buffer pH 6.8 within a minute. The obtained microemulsions were slightly bluish. Moreover, as shown in Fig. 2, the droplet size of control microemulsion without leuprorelin and all other leuprorelin microemulsions did not differ significantly. The fact that the droplet size does not increase when the leuprolide oleate is incorporated seems beneficial from the drug delivery point of view providing a large surface area dispersion to improve drug absorption. PDI values are 0.060 0.002 for unloaded SMEDDS and 0.103 0.005 for loaded SMEDDS. 3.3. Drug release and payload The drug release of leuprorelin across a dialysis membrane (MWCO 10,000 Da) in an aqueous environment over time is shown in Fig. 3. As already mentioned, the microemulsion contained
Fig. 2. Comparison of the droplet sizes of Leu-SMEDDS emulsified in phosphate buffer pH 6.8 with respect to the ratio between leuprolide acetate and sodium oleate during hydrophobic ion pairing process. Indicated values are means (SD, n = 3).
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leuprolide acetate and oleate; therefore two release profiles are shown. The release of leuprolide acetate is slower, but similar to the control solution indicating that leuprolide acetate is incorporated into the microemulsion droplet to a very low extent. However, the profile of leuprolide oleate is significantly sustained and after 30 h approximately 50% were released. As the plateau phase was reached, the remaining leuprolide oleate was likely still incorporated in SMEDDS droplets. Consequently, the drug release studies confirm the assumption that leuprolide oleate was at least partially incorporated into the microemulsion droplets. The sustained release of leuprorelin by embedding it into the droplets is advantageous as for oral delivery of peptide drugs, a drug release as close as possible to the site of absorption is favored. The microemulsion avoids an immediate release and could shield the peptide drug from rapid degradation until the absorption site is reached. Although the peptide might be degraded to a certain extent, the probability of the peptide drug reaching the apportion side unaltered is increased. Furthermore, the permeation enhancing effect of the microemulsion may support the absorption to increase bioavailability (Hintzen et al., 2013). A drawback of the developed microemulsion might be the incomplete release of leuprolide oleate under the chosen conditions. However, in vivo the excipients are likely digested by lipases or mixed micelles with bile acids and phospholipids could be formed, which leads to an enhanced release of the embedded leuprorelin. 3.4. Enzymatic degradation by intestinal enzymes One of the main obstacles to improve the oral bioavailability of leuprorelin is the enzymatic barrier of the gastrointestinal tract due to an intensively degradation by proteolytic enzymes (Woodley, 1994; Imanidis et al., 1995; Guo et al., 2004). Therefore, a selfemulsifying drug delivery system was developed to protect leuprorelin from enzymatic degradation in the intestine. Trypsin, a-chymotrypsin and elastase were separately investigated to measure a specific protective effect and to evaluate the extent of degradation by each enzyme. Fig. 4 shows the degradation profiles of leuprorelin in a microemulsion prepared with the lyophilized product of hydrophobic ion paring (molar ratio 3:1) and an aqueous control solution containing leuprolide acetate by trypsin as a function of time. The microemulsion contained leuprolide acetate as well as leuprolide oleate as described in Section 3.1. The leuprolide acetate control was completely degraded within 60 min, whereas leuprolide acetate in the microemulsions was metabolized rapidly within
Fig. 3. Leuprorelin release profiles from an aqueous leuprolide acetate solution ( ) and Leu-SMEDDS (1:3) emulsified in phosphate buffer pH 6.8 ( ) (&). As the leuprorelin precipitated incorporated into the SMEDDS contained leuprolide acetate and leuprolide oleate, release profile for both ion pairs are indicated serparately: leuprolide acetate ( ) and leuprolide oleate (&) from Leu-SMEDDS microemulsion. Indicated values are means (SD, n = 3).
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examined. This lack of information is a obstacle regarding the development of self-emulsifying drug delivery systems. Furthermore, the incorporation of peptides into the lipophilic droplets is a great challenge due to their hydrophilic nature and was rarely successful so far (Rao and Shao, 2008; Rao et al., 2008a,b). All other studies in which emulsions were investigated as peptide carriers, the drug is assumed to be in the aqueous phase (Swenson and Curatolo, 1992; Ho et al., 1996; Rao and Shao, 2008). Within the present study it could be demonstrated that the protective effect against enzymatic metabolism is improved, when the peptide is embedded in the lipophilic phase. 3.5. In vivo evaluation of leuprorelin SMEDDS Fig. 4. Degradation profile of leuprolide acetate by trypsin in an aqueous leuprolide acetate solution ( ) and as Leu-SMEDDS (1:3) emulsified in phosphate buffer pH 6.8 ( ) (&). As the leuprorelin precipitated incorporated into the SMEDDS contained leuprolide acetate and leuprolide oleate, degradation profiles of both ion pairs are indicated serparately: leuprolide acetate ( ) and leuprolide oleate (&) in the microemulsion. Fig. 5B highlights the first 5 min of the degradation process due to the rapid metabolism by a-chymotrypsin. Indicated values are means (SD, n = 3).
the first 60 min; it remained stable thereafter (Fig. 4A). Leuprolide oleate seemed to be protected by the microemulsion droplets right from the beginning, resulting in a comparatively lower extent of degradation (Fig. 4B). Accordingly, the leuprolide oleate is likely embedded into the lipid phase due to the higher hydrophobicity and therefore protected against degradation by trypsin. The degradation of leuprolide acetate control and of leuprorelin SMEDDS (3:1) by a-chymotrypsin was similar but more rapid (Fig. 5). Therefore, the reaction was stopped at different time points, and the first 5 min are separately presented in Fig. 5B. After 5 min no remaining leuprolide acetate was to be detected in the solution, whereas in the microemulsion a small amount remained stable. In contrast, leuprolide oleate was metabolized very slowly more than 50% remained unaltered within 120 min due to the protective effect of SMEDDS droplets. However, leuprorelin was not metabolized by elastase at all as the whole amount of leuprolide acetate and oleate in solution, and microemulsion could be found after 180 min of incubation (data not shown). Consequently, a protective effect was only shown for trypsin and a-chymotrypsin. The degradation studies with trypsin and a-chymotrypsin showed that leuprorelin SMEDDS had a limited protective effect for the hydrophilic leuprolide acetate. This might be caused by an insufficient–if at all–incorporation of leuprolide acetate into SMEDDS droplets. The nevertheless lower degradation might be a result of enzyme inhibitory properties of microemulsion components. However, ion pairing of leuprolide acetate with sodium oleate led to a hydrophobic product that was likely incorporated into the microemulsion droplets. As leuprolide oleate was shielded from degradation right from the start of the experiment, the peptide seems to be embedded into the microemulsion droplets. As mentioned in Section 3.1, both ion pairs (leuprolide acetate and oleate) were present in the microemulsion that allows good comparability in the same formulation in respect to the investigated protective effect. Self-microemulsifying drug delivery systems were extensively investigated in the last decade to improve the oral bioavailability of poorly water soluble drugs. Despite of the promising concept of improving oral bioavailability due to increased solubility (Patel and Vavia, 2007; Woo et al., 2007; Mezghrani et al., 2011), with a few exceptions (e.g., Neoral1) SMEDDS play a minor role on the pharmaceutical market. One reason therefore could be that most studies focus on a higher bioavailability by increasing watersolubility. Other promising properties of SMEDDS, e.g., protection of peptide drugs against enzymatic degradation are rarely
The detail information about administered formulations and the calculated pharmacokinetic parameters of the in vivo study are shown in Tables 1 and 2, respectively. The plasma–concentration profile of intravenous injections of leuprolide acetate to male Sprague Dawley rats indicate a rapid metabolism as more than 99% were metabolized within 90 min (Fig. 6A). Moreover, an orally administered leuprolide acetate solution proved that the peptide drug cannot be easily absorbed from the gastrointestinal tract (Cmax 5.98 ng/mL; absolute bioavailability 0.074%), which might be caused by severe degradation by luminal enzymes and a low permeability across intestinal membranes (Fig. 6B). However, leuprolide acetate given orally as SMEDDS showed a 6.5fold improved absolute bioavailability and reached a Cmax of
Fig. 5. (A and B) Degradation of leuprorelin by a-chymotrypsin in an aqueous leuprolide acetate solution ( ) and as Leu-SMEDDS (1:3) emulsified in phosphate buffer pH 6.8 ( ) (&). As the leuprorelin precipitated incorporated into the SMEDDS contained leuprolide acetate and leuprolide oleate, degradation profiles of both salts are indicated separately: leuprolide acetate ( ) and leuprolide oleate (&) in the microemulsion. B highlights the first 5 min of the degradation process due to the rapid metabolism by a-chymotrypsin. Indicated values are means (SD, n = 3).
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Table 2 Pharmacokinetic parameters calculated after iv and oral administration of investigated formulations in rats. Absolute bioavailability (BA) was calculated with reference to iv injection and indicated values are means of five rats. Delivery system
AUC0–last (min ng/mL)
Cmax (ng/mL)
Tmax (min)
Absolute BA (%)
Iv solution Leuprolide Leuprolide Leuprolide Leuprolide
144,653 426 0 2796ns 7385*
16,226.7 5.98 – 15.66ns 51.68*
– 30 – 30 30
– 0.074 – 0.483 1.276
acetate solution oleate suspension acetate SMEDDS oleate SMEDDS
ns: not significant compared to leuprolide acetate solution. * p < 0.05 compared to leuprolide acetate solution.
15.66 ng/mL after 30 min (Table 2). Moreover, the absorption was sustained as compared to the solution (Fig. 6B). This is likely caused by an enhanced permeability across intestinal membranes as the system was able to open tight junctions in vitro (Hintzen et al., 2013) representing the predominant way of uptake for hydrophilic macromolecular drugs like leuprorelin (Guo et al., 2004). Furthermore, the SMEDDS showed a slight protection of leuprolide acetate against degradation by intestinal serine proteases in vitro that might support the gastrointestinal uptake. If the freeze-dried pellet, obtained in the hydrophobic ion pairing process, was dissolved in the self-emulsifying system and orally administered to rats, the uptake was even further enhanced. The results showed a significant improvement of the area under the plasma concentration–time curve of leuprorelin (Fig. 6B), resulting in a 17.2-fold increased absolute bioavailability and a 8.6-fold increase of Cmax as compared to the control solution (Table 2). Moreover, the control
leuprolide acetate was no more detectable after 2 h when given as solution, whereas in case of leuprolide oleate SMEDDS plasma levels of around 8 ng/mL were found after 6 h. The enhanced and sustained uptake is likely caused by the combination of an improved transport across the intestinal gut wall, the protective effect against proteases and a sustained release of the peptide drug from the SMEDDS droplets. Up to now, most peptide and protein drugs are given intravenously or subcutaneously by injections due to several physiological barriers limiting the oral bioavailability of hydrophilic macromolecular drugs. In order to overcome the userunfriendly route of administration, especially for long-term disease treatment, it was the aim to develop a system that enables oral administration of peptides. The main obstacles are a low intestinal permeability and a rapid degradation by luminal enzymes. The developed self-emulsifying drug delivery system addresses these problems as the system can increase the permeability, particularly by tight junction opening, and shield leuprorelin against proteases. 4. Conclusion This is the first time, to our knowledge, that hydrophobic ion pairing of a peptide drug was used in order to embed a peptide drug successful into microemulsion droplets. Moreover, the selfmicroemulsifying drug delivery system for leuprorelin proved that SMEDDS can shield peptides from degradation by intestinal proteases. Furthermore, a sustained release of leuprorelin could be demonstrated that avoids an initial rapid degradation of the drug. These considerations could be confirmed by an in vivo study in rats and led to significant enhanced plasma profiles of leuprorelin via oral delivery. According to these results, the novel system could likely be a novel platform technology to improve the oral bioavailability of peptide drugs. Acknowledgement The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/ 2007–2013) under grant agreement n 280761. References
Fig. 6. (A) Plasma–concentration curve of leuprorelin after intravenous injection of leuprolide acetate to rats (dose = 1 mg/kg). (B) Plasma concentration curves of leuprorelin after oral administration of a leuprolide acetate solution ( ), a leuprolide oleate suspension (&) a leuprolide acetate SMEDDS ( ) and a leuprolide oleate SMEDDS ( ) to rats (dose = 4 mg/kg). Indicated values are means (SD) of five rats. Leuprolide oleate SMEDDS ( ) are significantly different compared to all the others (p < 0.05); leuprolide acetate SMEDDS ( ) are not significantly different compared to leuprolide acetate solution.
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