Mayank Singhala, Grégoire Schwachb, Torsten Weissc, Yogeshvar N. Kaliaa
aSchool of Pharmaceutical Sciences, University of Geneva & University of Lausanne, CMU, 1 Rue Michel Servet, 1211 Geneva, Switzerland.
bEarly Stage Development, Ferring Pharmaceuticals A/S, Kay Fiskers Plads 11, 2300 Copenhagen, Denmark; Currently F. Hoffman-La Roche Ltd., Pharmaceuticals Division, Grenzacherstr. 124, CH-4070 Basel, Switzerland.
cEarly Stage Development, Ferring Pharmaceuticals A/S, Kay Fiskers Plads 11, 2300 Copenhagen, Denmark.
Abstract
The ability of electric current to improve the delivery of gonadotropin releasing hormone antagonist, degarelix, was studied in vitro. Initially, degarelix electrotransport at fixed current density (0.5 mA/cm2, 6 h) was measured as a function of concentration (0.5 mM and 2 mM) across porcine skin. The results indicated that increasing the concentration did not increase degarelix delivery. This was attributed to its lipophilicity – as a lipophilic cation, degarelix is able to bind to negative charge sites in the skin and inhibit – determined from co-iontophoresis of acetaminophen and using heat separated dermis skin. Electron microscopy revealed the time and concentration dependent aggregation behavior of degarelix that further impaired its electrotransport. In an approach to trigger the release of degarelix from a subcutaneous depot a new model was developed. Degarelix was found to form aggregates in the presence of negatively charged plasma proteins, thereby neutralizing its positive charge and thus shutting off its response to external electric current stimuli. Overall, this research highlights the complicated interaction between peptide structure, skin membranes and plasma proteins that must be judiciously considered in order to improve delivery.
Keywords: degarelix, fibrils, iontophoresis, peptide delivery, subcutaneous depot, protein binding
104 Transit of the water-soluble, polar or charged molecules across the stratum corneum (SC) is the rate limiting step for the transdermal delivery of therapeutic agents [1,2]. The application of electric field facilitates the cutaneous delivery by reversibly impairing the skin barrier function [3-7]. Constant current iontophoresis is an active delivery technique that involves the application of a current density (< 0.5 mA/cm2) to control the transport of polar and ionized molecules during the current application period [8,9].
The oral route is not suitable for peptide delivery and therefore, most of the peptides are delivered by parenteral route. Several reports have successfully demonstrated the application of transdermal iontophoresis to deliver several peptides and proteins [10-16]. Degarelix, a gonadotropin-releasing hormone antagonist, is administered by subcutaneous injection for the treatment of advanced prostate cancer. Its starting dose is 240 mg which is given as two subcutaneous (s.c.) injections of 120 mg (40 mg/ml) followed by maintenance dose of 80 mg given as one s.c. injection (20 mg/ml) every 28 days [17]. Degarelix is a synthetic linear decapeptide amide with a molecular weight of 1632.26 Da and isoelectric point of 10.4. At a physiological pH 7.4, it acquires about +1 net charge, which is similar to other peptides such as triptorelin [14], nafarelin [18] and leuprolide [19]. Triptorelin, nafarelin and leuprolide are GnRH analogues and differ from GnRH (H-Pyr-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) only in the amino acid at position 6 (a glycine in GnRH, tryptophan in triptorelin, D-naphthylalanine in nafarelin, and D-leucine in leuprolide). In contrast to these GnRH agonists, degarelix contains five D-amino acids out of which seven are unnatural amino acids (Figure 1) [20]. Degarelix has different physicochemical properties with more hydrophobic regions then triptorelin, nafarelin and leuprolide and it forms a gel depot after s.c. administration that enables sustained drug release for over a month.
There has been a considerable interest in finding an alternative to deliver peptides in non-invasive manner [10,21-23]. The iontophoretic delivery of triptorelin has been successfully achieved via transdermal route where the input rates were sufficient to achieve therapeutic plasma level [14,24,25] An investigation of the effect of current density revealed corresponding increase in the amount of peptide transported across the epidermis. However, reduction in the delivery was observed at two fold higher concentration, which was attributed to an inhibition of electroosmosis (EO) and also suggested possible involvement of peptide aggregation. Essentially identical behavior was also observed for nafarelin and leuprolide
105 membrane and peptide’s lipophilicity was responsible for impaired electroosmotic flow [26].
Evaluation of iontophoretic delivery of degarelix across the skin would enable us to assess its suitability for non-invasive delivery and co-iontophoresis of acetaminophen aids in determining relative contribution of electromigration (EM) and EO to report on the impact of degarelix iontophoresis on skin permselectivity.
Figure 1. Chemical structure of degarelix (MW: 1632.26 Da, pKa 10.4). The drug substance is commonly designated as D-Alaninamide, N-acetyl-3-(2-naphthalenyl)-D-alanyl-4-chloro-
D-phenylalanyl-3-(3-pyridinyl)-D-alanyl-L-seryl-4-[[[(4S)-hexahydro-2,6-dioxo-4- pyrimidinyl]carbonyl]amino]-L-phenylalanyl-4-[(aminocarbonyl)amino]-D-phenylalanyl-L-leucyl-N6-(1-methylethyl)-L-lysyl-L-prolyl.
In an attempt to avoid s.c. administration, the specific objectives of this study were (i) to investigate the feasibility of iontophoresis to deliver degarelix across porcine skin, (ii) to determine the effect of concentration on degarelix delivery and (iii) to study the effect of degarelix on electroosmotic solvent flow. However, after the initial surprising results, they were expanded in order (iv) to study degarelix delivery across heat separated dermis to find out the affinity of degarelix with the skin membranes – epidermis and dermis, and (v) to study the degarelix aggregation behavior and its impact on the iontophoretic delivery. The secondary objectives of the project were (vi) to study the ability of electric current to trigger the release of degarelix from the in situ formed depot in the human s.c. tissue and finally, (vii) to explore degarelix depot formation and visualization under confocal laser scanning microscopy (CLSM).
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106 2.1. Materials
Degarelix was provided by Ferring Pharmaceuticals SA, Copenhagen. Human plasma was kindly obtained from the Laboratory of Toxicology, Service of Laboratory Medicine at the Geneva University Hospital, Geneva, CH. Acetaminophen (ACM), 2-(N-morpholino)ethanesulfonic acid (MES), sodium citrate, silver wire and silver chloride for fabricating electrodes, agarose, tween 80, bovine serum albumin (BSA), fluorescein isothiocyanate-albumin conjugate (FITC-BSA), sodium chloride, potassium chloride, calcium chloride, sodium bicarbonate, sodium hydroxide and trifluoroacetic acid were all purchased from Sigma-Aldrich (Buchs, Switzerland). Citric acid and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were purchased from Acros Organics (Chemie Brunschwig; Basel, Switzerland). Acetonitrile and PVC tubing (ID 3.17 mm; OD 4.97 mm) were purchased from VWR International (Nyon, Switzerland). Dialysis membrane (MWCO 6-8 kDa) was purchased from Spectrum Laboratories Inc., Switzerland. All solutions were prepared using deionised reverse osmosis filtered water (resistivity ≥ 18 MΩ·cm). All other chemicals were at least of analytical grade.
2.2. HPLC-UV analysis 2.2.1. Degarelix
The analytical method provided by Ferring Pharmaceuticals SA was transferred to the Dionex HPLC system – ASI-100 auto-sampler equipped with a P680A LPG-4 pump and UVD 170U detector controlled by Chromeleon® Chromatography Management Software. The mobile phase (MP) comprised 35 % ACN with 0.1% (v/v) trifluoroacetic acid (TFA) and 65 % Milli-Q water with 0.1% (v/v) TFA. A LiChrospher 100 RP8-endc. (250 x 4.0 mm and 5 μm particle size) column was used. The flow rate was 0.8 ml/min, column temperature was 30 °C and UV wavelength of 226 nm was used for detection. The LOD and LOQ were 0.26 and 0.7 µg/ml, respectively.
2.2.2. Acetaminophen
The acetaminophen was analyzed using Dionex HPLC system – ASI-100 auto-sampler equipped with a P680A LPG-4 pump and UVD 170U detector controlled by Chromeleon® Chromatography Management Software. The mobile phase was comprised of 50 % methanol and 50 % Milli-Q water with 0.1% (v/v) TFA. A LiChrospher 100 RP8-endc. (250 x 4.0 mm
107 was 30 °C and UV wavelength of 243 nm was used for detection. The LOD and LOQ were 0.17 and 0.5 µg/ml, respectively.
2.3. Skin preparation 2.3.1. Porcine skin
Porcine ears were acquired soon after sacrifice from a local abattoir (CARRE; Rolle, Switzerland).
Ears were cleaned with cold running water and the outer region was carefully excised from the underlying cartilage. Then, 32 mm circular disks were punched out from the excised full thickness skin samples followed by slicing to the thickness of 1.0 ± 0.1 mm before storing at −20 °C. Heat-separated dermis was prepared by separating epidermis from the dermis by immersing porcine skin in water at 60 °C for 45s; [27] the epidermis was then removed carefully with the aid of forceps. Transport experiments were also performed with heat-separated dermis.
2.3.2. Human skin
Given that degarelix is indicated to be injected into the s.c. tissue in the abdominal region – excised human abdominal tissue (skin with s.c. fat) was chosen as the best model for in vitro electroactive release experiments from degarelix depot. Human abdominal tissue samples were collected immediately after surgery from the Department of Plastic, Aesthetic and Reconstructive Surgery, Geneva University Hospital (Geneva, Switzerland), The study was approved by the Central Committee for Ethics in Research (CER: 08-150 (NAC08-051);
Geneva University Hospital). The skin was supplied in large triangular pieces (typically with sides of length 10 cm x 15 cm x 15 cm) and these were processed on receipt and stored as small circular disks with a diameter of either 16 mm at -20ºC until use.
2.4. Stability studies
2.4.1. Degarelix stability in the presence of porcine skin and human skin with s.c. tissue Degarelix standard solutions of 50 µg/ml were prepared separately in MP and 10 mM citrate buffer with 133 mM NaCl, pH 5.5 (CB). Stability in porcine and human skin with s.c. tissue was determined by placing 2 cm2 of porcine skin (1 mm thickness) or s.c. fat (1 cm thick) with human skin (2 cm2) in contact with the degarelix standard solution (5 ml) in both solvents for 24 h. The samples after 24 h were centrifuged (Eppendorf Centrifuge 5804;
Schönenbuch, Switzerland) and supernatant was taken and analyzed for degarelix content
108 the solvents.
2.4.2. Degarelix stability in the presence of current
This was evaluated by subjecting a 50 µg/ml degarelix solution in CB to 1 mA current for a period of 7 h. In the first study, current was delivered directly using Ag/AgCl electrodes and in the second via salt bridges. The control was carried out using the same set-up but in the absence of current. The sample after 7 h was analyzed using HPLC-UV analytical method.
The study was performed in triplicate.
2.5. Iontophoretic delivery of degarelix from solution 2.5.1. Iontophoretic set-up and Protocol
Modified vertical diffusion cells of cross-sectional area 2 cm2 were used to clamp the skin (32 cm2) where the receiver compartment was equipped with two side arms (one for housing cathode and second to take samples). Skin was equilibrated for 30 min by filling both compartments with CB solution. The donor compartment was connected to the anodal compartment (containing PBS buffer at pH 7.4) by a salt bridge (3% agarose) to minimize the effect of competing ions. After equilibration, the donor compartment was emptied completely and filled with 1 mL of drug solution (either 0.5, or 2 mM degarelix and 15 mM acetaminophen (ACM) in 10 mM MES, pH 5.5). Ag/AgCl electrodes connected to a power supply (Kepco APH 1000M; Flushing, NY) were used to deliver constant current of 0.5 mA/cm2 for 6 h. One mL of solution was sampled hourly from the receiver compartment and replaced with the same volume of fresh buffer solution. Samples were centrifuged at 10621g for 10 min and supernatants were analyzed for degarelix content using HPLC. Experiments were performed with at least four replicates.
2.5.2. Skin biodistribution
After completion of the iontophoresis permeation study, skin biodistribution of degarelix was investigated as a function of depth for permeation area in the following order from top to bottom: 20, 40, 40, 40, 40, 100, 100, and > 620 µm and laterally as a function of distance from the permeation area of 2 cm2 that is defined as (1) Lateral diffusion area 1 (LD1): 1 cm2 surrounding the permeation area and (2) Lateral diffusion area 2 (LD2): the remaining surrounding area (Figure 2). Degarelix was extracted from each lamella of permeation area
109 samples were centrifuged and supernatant was injected in HPLC for degarelix quantification.
Figure 2. Schematic representation of the parts of skin used to determine degarelix biodistribution. (a) Biodistribution of permeation area to a total depth of 1 mm in the following order: 20, 40, 40, 40, 40, 100, 100, 100, 100, and > 580 µm. (b) Lateral diffusion area 1, i.e., 1 cm2 surrounding the permeation area. (c) Lateral diffusion area 2, i.e., 5 cm2 of the remaining no permeated area.
2.5.3. Quantification of electroosmotic solvent flow
ACM is an uncharged hydrophilic compound and therefore, is primarily transported through the skin by EO. Electroosmotic flux (JEO) is the product of electroosmotic solvent flow (Vw) and concentration (CACM). ACM is included in the donor compartment formulation as a marker for the magnitude of convective solvent flow. Once the solvent flow is known, the electromigration flux (JEM) and electroosmotic flux (JEO) for degarelix electrotransport can be calculated (assuming that there is no passive delivery of degarelix).[14] For each experiment, an inhibition factor (IF) was calculated according to the following equation:
IF = [QACM-6h;control]/[QACM-6h;DEG] (1)
where QACM-6h;control and QACM-6h;DEG are the amount of acetaminophen transported in the receiving compartment during 6 h of iontophoresis in the absence and in the presence of degarelix in the donor solution, respectively.
110 A previously reported method of transmission electron microscopy (TEM) (FEI Tecnai G2 Sphera, Eindhoven, Netherlands) was adapted to visualize the aggregation of degarelix peptide in the solution form.[28] Degarelix solution of different concentrations, used in iontophoresis experiments, were visualized after 6 h and after 3 weeks of solution preparation.
TEM images were processed using ImageJ software (ImageJ 1.45s).
2.6. Electrotransport from degarelix depot 2.6.1. In vitro depot formation
Degarelix is subcutaneously injected to form a depot and achieve 1 month of sustained therapeutic effect. Therefore, to test the response of degarelix release from a depot in response to electric field, a depot was formed under in vitro conditions. Square pieces (2.5 x 2.5 cm2) of washed dialysis membrane (regenerated cellulose membrane, MWCO: 6-8 KDa), were mounted on Franz diffusion cells. Over the dialysis membrane hydrophilic polypropylene membrane (diameter: 30 mm, pore size: 0.2 µm) (Pall Life Sciences, GH Polypro, Mexico) was placed and both were clamped in between the two compartments of Franz cells. Then 1 ml of 20% plasma solution (20% human plasma in Ringer’s solution pH 7 ± 0.2) was placed in the donor compartment over the dialysis membrane. The donor cells were then sealed with parafilm. Franz diffusion cells containing 20% plasma solution were then maintained in an oven at 51.5 °C for at least 1 h. Parafilm was removed from the donor cells and 150 µl of reconstituted degarelix drug product solution (clinical dose concentration, 40 mg/ml in MilliQ) was slowly injected onto the surface of the dialysis membranes by placing the tip perpendicular to the membrane. Donor cells were sealed again using parafilm and diffusion cells were placed in the heating chamber for 22 ± 1 h at 51.5 ± 1.5 °C to permit maturation of degarelix depot.
2.6.2. Electrically assisted release from depot: set-up and protocol
To permit release of degarelix from the depot site in response to current, the current path had to cross the entire skin and s.c. tissue to reach depot (Figure 3). After maturation of degarelix depot, cellulose dialysis membrane (MWCO 6000KD) was removed and propylene membrane (pore size 0.22 µm) with the matured depot was clamped between two compartments of a two-arm vertical Franz diffusion cell. In a separate study, it was confirmed that propylene membrane was not limiting the diffusion of degarelix once released from depot into the receiver media. To mimic in vivo conditions, human skin with s.c. tissue
111 silicone gel and 1 mL of PBS was placed over skin. The donor cell was connected with the anode via a salt bridge and cathode electrode was placed in the receiver compartment through one of the two arms. A current of 0.5 mA/cm2 was applied for 3 h and samples were taken hourly from the second arm of the receiver compartment.
Figure 3. Schematic representation of the model used to study degarelix release from s.c.
depot preparation in response to external electric stimuli.
2.6.3. Determining factors contributing to the depot formation
Degarelix depot was prepared using 20% plasma solution in Ringer solution. To understand the factors responsible for degarelix aggregation and depot formation different media were prepared and tested (Table 1).
Receiver medium Sampling
arm
Power supply PBS
Donor compartment
Receptor
compartment Cathode
Anode (via salt bridge) Subcutaneous tissue
Human skin Depot Supporting
membrane
112 selecting them.
Condition Medium Rational
1 Ringer’s solution Contribution of ions
2 20% plasma in Ringer’s solution Contribution of plasma components 3 20% fetal calf serum in Ringer’s solution Contribution of serum components
(without fibrinogen)
(equivalent BSA representing 20% total plasma proteins)
For confocal laser scanning microscopy
2.7. Confocal laser scanning microscopy 2.7.1. Fluorescent labelling
Degarelix (10 mg) was dissolved in a borate buffer system (0.05 M; pH 8.5) and rhodamine isothiocyanate (dissolved in DMSO) was added such that the molar ratio of peptide:
rhodamine remained at 1:2. The tagging reaction was carried out with constant stirring for 2 h in the dark. After completion, the reaction mixture was dialyzed through a 3.5 kDa membrane for 4 h in 0.1 % tween 80 solution in order to remove unreacted rhodamine isothiocyanate.
Rhodamine conjugated degarelix (rhodamine-degarelix; labeling procedure explained below), 0.5 mM, was used to visualize the biodistribution of degarelix in the normal and heat separated dermis skin samples. After 6h of iontophoresis, the diffusion cells were disassembled and the skin samples were cleaned three times using 1 mL PBS and gently dried with a paper towel. Skin slices of 40 µm were obtained using a cryotome (Microm HM 560 Cryostat, Walldorf, Germany) and visualized under confocal microscope (LSM 700, Zeiss;
Germany). The images were analyzed using Zen software (Carl Zeiss, Germany) and further processed using ImageJ 1.45s software.
113 protein present in 20% plasma solution) was observed under a CLSM. The confocal images were analyzed using Zen software and processed using ImageJ 1.45s software. Non fluorescent depot formed using BSA was visualized under an optical microscope.
2.7.2. Fluorescence resonance energy transfer
Steady-state fluorescence was measured at room temperature using a BioTek Synergy Mx Multi-Mode Reader. For measurements of fluorescence, emission spectra from 500 nm to 700 nm were collected (λex 480 nm). 96 well plates were loaded with 200 µl of samples. FITC-BSA, rhodamine-degarelix, and BSA were used to compare the fluorescence of sample mixture of FITC-BSA and rhodamine-degarelix conjugate in a 100:1 molar ratio.
Fluorescence of sample mixture of BSA and rhodamine-degarelix conjugate in a same molar ratio was also recorded as a control. All samples were prepared fresh for spectral acquisition.
Fluorescence energy transfer from FITC-BSA to rhodamine-degarelix was also observed using CLSM. On two separate glass slides a drop of a mixture of BSA/rhodamine-degarelix solution and FITC-BSA/ rhodamine-degarelix (100:1 molar ratio) was placed and the images were taken at excitation wavelength of 488 nm and 555 nm.
2.8. Statistical analysis
Data were expressed as mean ± SD. Outliers determined using the Grubbs test were discarded. Results were evaluated statistically using analysis of variance (ANOVA followed by Student–Newman–Keuls test) or Student’s t-test. The level of significance was fixed at α=0.05.
3. Results
3.1. Demonstrating degarelix stability
The solution concentrations of degarelix in MP following exposure to porcine skin and s.c. fat with human skin for 24 h were 95.65% ± 6.85% and 97.20% ± 1.04%, respectively of the initial values. From the study using CB as solvent, no degarelix was detected after 24 h.
However, same skin samples after being exposed to CB resulted in the recovery of 93.68% ± 4.41% of degarelix when placed in MP under continuous stirring for another 24 h.
After current application for 7 h via salt bridges, the solution concentration of degarelix was 94.4 ± 5.7% of its initial value. A slight decrease of peptide in solution was observed when
114 concentration was 84.63 ± 4.6% of its initial value.
3.2. Iontophoretic delivery of degarelix from solution
3.2.1. Degarelix electrotransport and the effect of concentration
No degarelix was permeated across porcine skin during in vitro permeation studies performed with 0.5 mM and 2 mM solution in the donor compartments from 6 h of anodal iontophoresis at 0.5 mA/cm2 of current density. The amounts deposited after 6 h for 0.5 and 2 mM solution was 12.1 ± 2.8 µg/cm2 and 11.7 ± 2.1 µg/cm2, respectively. Control experiments showed that passive delivery of degarelix from aqueous solution of 2 mM concentration after 6 h was 4.6
± 1.2 µg/cm2 (Figure 4a). A small but statistically significant difference was observed over the control, with 7.2 ± 2.2 μg/cm2 of peptide was iontophoresed. In contrast, only 2.1 ± 0.2 µg/cm2 of degarelix, following iontophoresis, was found deposited in the heat separated dermis when 0.5 mM of degarelix solution was placed in the donor compartment.
Figure 4. (a) Comparison of degarelix delivery in the permeation area of the intact skin after application of 2 mM and 0.5 mM of degarelix donor solution for 6 h at a current density of 0.5 mA/cm2. (b) Comparison of degarelix biodistribution in skin to a total depth of 280 µm (expressed as a percentage of the total amount deposited). (Mean ± SD; n ≥ 4).
3.2.2. Effect of degarelix on acetaminophen EO delivery
Co-iontophoresis of acetaminophen reported on the effect of degarelix transport on electroosmotic solvent flow (Table 2). A significant decrease in ACM permeation was observed in the presence of degarelix at 2 mM concentration in comparison to control (ACM
0
115 electroosmotic inhibition of ACM was noted; 0.95 inhibition factor. No ACM inhibition was recorded when delivered through heat separated dermis with 19 fold higher ACM permeated in comparison to intact skin with epidermis.
Table 2. Effect of degarelix concentration and skin barrier on degarelix electrotransport and its impact on electroosmotic solvent flow after 6 h of transdermal iontophoresis, 0.5 mA/cm2. (Mean ± SD; n ≥ 4)
a Cumulative permeation and skin deposition of degarelix.
a Cumulative permeation and skin deposition of degarelix.