Drug Deliv. and Transl. Res. (2014) 4:416–428 DOI 10.1007/s13346-014-0204-0
RESEARCH ARTICLE
Pouch drug delivery systems for dermal and transdermal administration Jana Zailer & Elka Touitou
Published online: 18 October 2014 # Controlled Release Society 2014
Abstract In this work, we have designed and investigated a new carrier for dermal and transdermal drug delivery. The delivery system is composed of high (>60 %) ethanol concentration, phospholipid, polymer, and water. The system forms a structured matrix following non-occluded application on the skin. We call these structured carriers as pouch drug delivery systems (PDDS). The pouch-structured matrix was characterized by electron microscopy, 31P-NMR and FTIR. The new delivery system exhibits a number of properties adequate for the design of improved dermal and transdermal drug administration for various treatments. Lidocaine PDDS dry faster and has an enhanced dermal drug delivery when compared to a clinical-used product. These proprieties are important for the prevention of premature ejaculation. Results obtained in pharmacodynamics test carried out with brotizolam PDDS in a mice-sleeping model and with ibuprofen PDDS in fevered rats indicated a prolonged hypnotic and antipyretic effect, respectively. The carrier was found nonirritant in tests carried out on EpiDermTM skin model.
Ethosomal carriers contain phospholipid vesicles formed in the presence of 20–50 % ethanol. At these alcohol concentrations, the phospholipid generates vesicles with fluid bilayers, that are able to penetrate the skin and deliver actives to the skin layers. Here, we present a new skin delivery carrier composed of phospholipid, a high alcohol concentration (>60 %), and polymer, which we call pouch drug delivery systems (PDDS). Applied on the skin, the carrier forms a pouch-structured matrix having enhanced skin delivery properties. The present study was carried out to characterize the new delivery system, to learn its delivery proprieties to skin and pharmacodynamics efficacy. For this purpose, we studied PDDS containing molecules with various physicochemical and pharmacological properties and using appropriate animal models.
Keywords Dermal . Transdermal . Ibuprofen . Lidocaine . Brotizolam . Antipyretic . Hypnotic . Phospholipid . Ethanol
Materials
Introduction More than one decade ago, we have introduced ethosomes for dermal and transdermal delivery [1]. These carriers were then worldwide investigated by many research groups and were used in pharmaceutical and cosmetic products [2–9]. J. Zailer : E. Touitou (*) The Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, PO box 12065, Jerusalem 91120, Israel e-mail:
[email protected]
Materials and methods
Ibuprofen, fluorescein D HPE (N-(fluorescein-5thiocarbamoyl)-1,2-dihexadecanoyl-sn + glycerol-3phosphoethanolamine, triethylammonium salt), fluorescein isothiocyanate (FITC), Rhodamine B, phosphotungstic acid (PTA), brewer’s yeast, MTT ((3-4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide), sodium dodecyl sulfate (SDS) were purchased from Sigma (USA). Phospholipid (Phospholipon® 90G) was purchased from Lipoid GmbH (Germany). Hydroxypropylcellulose (Klucel® HF) was from Hercules (USA). Lidocaine (base) was a kind gift from Trima (Israel). Ethanol 96 EP was purchased from Gadot (Israel). Brotizolam was a gift from ANL (Israel). Pentobarbitone sodium (Pental veterinary, CTS Israel) and STUD 100® (Pound International Ltd., UK) were acquired from a private
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pharmacy. EpiDermTM tissues were purchased from MatTek Corporation (USA). Dulbecco’s phosphate buffered saline (DPBS) was obtained from Biological Industries (Israel). All other materials used were of analytical grade.
dose mode by a Gatan US1000 high-resolution cooled CCD camera with the Digital Micrograph software package.
Composition and preparation of the new drug delivery systems
In these tests, several systems were analyzed: PDDS, control (no phospholipid), phospholipid reverse micelles solution composed of 5 % PL in 95 % ethanol and an ethosomal system composed of 5 % Phospholipon® 90G, 30 % ethanol, 0.7 % Klucel® HF, and water to 100 % w/w. Proton decoupled 31P-NMR spectra of the systems were recorded on a Varian 300 spectrometer (Varian Inc., Palo Alto, CA, USA) at a frequency of 121.6 MHz. The number of transients varied from 3000 up to 10,000. The acquisition times were 1 s with a microsecond excitation pulse. Chemical shifts were measured relative to external phosphoric acid in D2O. All samples contained 10 % D2O for locking. Line broadening between 10 and 100 Hz was applied [1].
PDDS are mainly composed of phospholipid (PL), ethanol (>60 %), polymer, water, and active. They may also contain glycols. In this first publication on PDDS, we present results of experiments carried out using a representative formulation of the system having the main components (% w/w): 65 % ethanol, 5 % Phospholipon® 90G, 0.7 % Klucel® HF, water and drug, or fluorescent probe. For system preparation, the phospholipid is dissolved in ethanol in a covered container, the drug or fluorescent probe is added to this solution. Water is then added slowly in a fine stream with constant mixing with an overhead stirrer (Heidolph digital 2000 RZR-2000, Germany). Klucel is dispersed in the solution while mixing at the same speed. The system is left to repose and then remixed. Control systems, without phospholipid or without ethanol, were prepared following a similar procedure. Physical characterization of PDDS Visualization of pouch structures by light microscopy, confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and cryo-TEM PDDS samples and control (no phospholipid) were visualized by light microscopy using the Zeiss, Axioskop (Germany), with Achroplan ×40/0.65 objective. CLSM visualization was carried out using the Olympus fluoview 300 microscope (Japan) with air plan ×20 objective lens and the 488-nm laser line excitation. For this test, PDDS were prepared using PL and Fluorescein DHPE (a fluorescent phospholipid) and the control systems were prepared using FITC. A further analysis was done by TEM using the Philips TECHNAI CM 120 electron microscope (The Netherlands) at 9.7 K magnification following a negative staining with 1 % aqueous solution of phosphotungstic acid [1, 10]. Five microliters of PDDS were applied on a microscope carbon-coated grid (after glow discharge) and left for 30 s before staining. The excess solution was removed by blotting. After 20 min of drying, the specimen was viewed under the microscope. Specimens for cryo-TEM were prepared in a controlled environment vitrification chamber (CEVS) and quenched into liquid ethane at its freezing point. Specimens, kept below −178 °C, were examined in a FEI T12 G2 transmission electron microscope, operated at 120 kV, using a Gatan 626 cryo-holder system. Images were recorded digitally in the minimal electron-
Determination of phospholipids configuration by 31P-NMR
Fourier transform infrared spectroscopy (FTIR) analysis Infrared spectra of PDDS and control (no phospholipid) were recorded. Samples were casted on KBr plates on Nicolet iS10 equipped with Smart iTR software (Thermo Scientific), at 15,798.7 cm-1 laser frequency. Each sample was scanned 32 times. Measurement of skin penetration of various molecules incorporated in PDDS Assessment of Rhodamine B and FITC skin penetration by CLSM: an in vivo experiment All animal experiments were carried out in accordance with institutional guidelines for animal care, by protocols approved by the Animal Ethical Care Committee of the Hebrew University of Jerusalem. In this experiment, the skin penetration of two probes, Rhodamine B and FITC, an ionic and a lipophilic molecule, respectively, was evaluated in rats. PDDS contained either 0.1 % FITC or 0.5 % Rhodamine B. Control systems were prepared from the same ingredients as PDDS but without phospholipid. The experiments were carried out on adult Sprague–Dawley (SD) male rats, weighting ∼350 g (Harlan, Israel). Briefly, the dorsal skin of the animals was shaved using electric clipper (Oster, USA) a day prior to the experiment. Twenty-five microliters of PDDS or control system were applied on rat’s dorsal skin nonocclusive on an area of 1 cm2. After the application, each rat was kept in an individual cage at dark. Animals were sacrificed 1 h after the application and the treated skin area was carefully cleaned from the residual non-absorbed fluorescent material and removed. The skin samples were then immersed in O.C.T. and frozen
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gradually to −70 °C. The frozen blocks were cryosectioned into 14 μm slices with a cryo-microtome (Leica CM3000). The skin slices were then examined with a CLS microscope (Olympus Fluoview FV10i, Japan). For excitation of FITC and Rhodamine B, 488- and 546-nm laser lines were used respectively. Skin samples from untreated skin animals similarly processed were used for the evaluation of skin auto fluorescence [5, 10].
30 °C, with a mobile phase composed from acetonitrile to phosphate buffer 0.01 M pH 6 (35:65 v/v) at a flow rate of 1.2 ml/min.
Evaluation of Rhodamine B skin penetration by CLSM: an in vitro experiment
To evaluate the efficacy of the transdermal administration of Brotizolam PDDS, we used the pentobarbitone-induced sleeping model [12, 13]. In brief, 15 female C57/B1 mice, 8–9 weeks (Harlan, Israel), were randomly divided into three equal groups, five animals in each of two treatment groups and five mice in untreated positive control group. On the day before the experiment, the dorsal skin area of animal was clipped (Oster, USA). The animals in the treatment groups received a single dose of 1 mg/kg brotizolam from the tested systems, PDDS, and control (without PL and ethanol), each containing 0.02 % brotizolam. Thirty minutes after drug administration, by system application on the skin, pentobarbitone sodium 35 mg/kg was IP injected to each animal. For each mouse, the sleep onset time was recorded. A mouse losing its righting reflex was considered asleep. The time elapsed between the pentobarbitone injection and the loss of righting reflex is the sleep latency time. The time elapsed between the loss and the recovery of the righting reflex was recorded as sleep duration.
The skin penetration profile of Rhodamine B, from PDDS and control system (without phospholipid), was evaluated using Franz diffusion cells and porcine ear skin (Lahav, Israel). Full thickness clipped skin was mounted on the diffusion cells with a receiver volume of 5 ml and effective diffusion area of 0.64 cm2. Twenty-five microliters of the system containing 0.1 % Rhodamine B was applied on the stratum corneum side of the skin. The receiver medium was constantly stirred and consisted of 1:1 ethanol to water solution. The water bath of the diffusion cells was kept at 37±0.5 °C. At the end of 1 h experiment, the skins were removed, and their surface was carefully washed and wiped. The treated skin area was then optically scanned at 10-μm increments through the z-axis using a confocal laser-scanning microscope, Zeiss LSM 410, with an air plan ×10 objective lens. For excitation of the label, the 561nm laser line was used, with an electron gain of 320. The fluorescence intensity of the probe (arbitrary units) in the skin was further assessed using the Image Pro-Plus software [1, 11]. Measurement of Lidocaine skin penetration The evaluation of skin penetration of lidocaine from the systems containing 9.6 % w/w lidocaine was carried out in Franz diffusion cells. It was interesting to measure the drug amount delivered in 10-min application time, by the new delivery carrier as compared with a clinical used formulation containing the same drug concentration, STUD 100® (Pound International Ltd., UK). Twenty-five microliters of each formulation was applied on the stratum corneum side of full thickness clipped porcine ear skin. At the end of the 10 min experiment, the skin was removed from the diffusion cells and cleaned for surface material and extracted for 48 h with 7:3 ethanol to water solution [6]. The amount of lidocaine in the extraction solution was quantified by reverse phase HPLC method using a MerckHitachi D-7000 apparatus equipped with an L-7400 variable UV detector, L-7300 column oven, L-7200 auto-sampler, L7100 pump, and an HSM computerized analysis program. The assay was carried out at 210 nm, using a Zorbax Eclipse XDB C18, 5 μm 4.6×150 mm (Agilent, USA) column kept at
Measurement of pharmacodynamic effect of drug loaded PDDS in animal models Hypnotic effect of Brotizolam PDDS in mice sleeping model
Antipyretic effect of Ibuprofen PDDS in fevered rats The antipyretic effect of ibuprofen incorporated in the system was measured in rats fevered by injection of brewer’s yeast [14, 15]. Briefly, 20 ml/kg of 20 % w/v aqueous suspension of brewer’s yeast was injected subcutaneously below the nape of the animal’s neck to 15 adult male SD rats (∼300 mg each). Two rats, that did not receive the brewer’s yeast injection, were considered the normal temperature untreated control animals. The body temperature was measured by a digital thermometer (Vega, China), lubricated and inserted 3 cm into the animal’s rectum for 10 s. All animals were habituated to the thermometer before the experiment in order to minimize the stress response induced by the measurement procedure. Brewer’s yeast injected rats were randomly divided into three groups of five rats each, treated as follows: about 750 μl PDDS applied (200 mg/kg ibuprofen) on 6-cm2 preclipped skin of each rat, 62.5 mg/kg ibuprofen in suspension administrated orally, and five fevered rats served as control. Ibuprofen PDDS and ibuprofen suspension contained 10 mg drug/100 mg formulation. The drug treatment was done 18 h following the yeast injection. Animal body temperature was recorded at the following time intervals: 0, 18, 19, 20, 21, 22, 23, 24, 25, and 30 h post fever induction.
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Evaluation of drug plasma concentration following ibuprofen PDDS application on rat skin
Pharmacokinetic parameters calculated from the ibuprofen blood concentration curve
In this experiment, the type of animals and pre-experiment conditions were similar to those described in the section of antipyretic effect of ibuprofen PDDS in fevered rats. Fifteen animals were randomly divided into three equal groups of five. On the day before the experiment, the back area of the animals was clipped. One hour before the experiment, the rats were restricted from food, having free access to water during all experimental period. The compositions and mode of drug administration were similar to the pharmacodynamics experiment described in the previous part of this paper. Five hundred microliters of blood samples were collected from the animal tail at 0.5, 1, 2, 4, 8, 12, and 24 h post drug administration. For ibuprofen extraction, animal blood samples were centrifuged at 4000 rpm for 10 min at room temperature (RT). Hundred microliters of supernatant was transferred into 1.5 ml tubes, and then 50 μl buffer acetate 0.03 M pH 4 was added to each sample and vigorously mixed for 0.5 min by Vortex Genie®-2 (Scientific industries, USA). Finally, 450 μl of acetonitrile was added to each sample and vigorously mixed for 3 min. All samples were centrifuged again for 5 min at 14,000 rpm at RT (Hermle Z 160 M centrifuge). Fifty microliters of filtered sample was injected into HPLC. Ibuprofen was quantified, using a Merck-Hitachi D-7000 apparatus equipped with an L-7400 variable UV detector, L7300 column oven, L-7200 auto-sampler, L-7100 pump, and an HSM computerized analysis program. The assay was carried out at 225 nm, using a LiChospher® 100 C18 5 μm, 4× 250 mm (MERCK, USA) column kept at 25 °C, with a mobile phase composed of methanol to acetate buffer 0.03 M pH 4 (80:20 v/v) at a flow rate of 1 ml/min.
The pharmacokinetic parameters were calculated from the values plotted in the blood levels versus time curve by using the NCOMP version 3.1 [16]. AUC values were obtained from the mean of the area under the plasma drug concentration curve of individual rats. The relative bioavailability (Ftransdermal relative to oral) of ibuprofen was calculated using the relationship: Ftransdermal relative to oral
. ¼ ðAUCtransdermal Doseoral Þ ðAUCoral Dosetransdermal Þ
Skin irritation test following PDDS application The evaluation of the carrier possible irritation effect was performed by Harlan (Israel) in in vitro experiment using reconstructed human epidermis (RhE), EpiDermTM tissues. The EpiDermTM skin model closely parallels human skin, thus providing a useful in vitro means to assess dermal irritancy and toxicology [17]. In this assay, EpiDermTM tissues were exposed for 60 min to the tested system and the controls. Dulbecco’s phosphate-buffered saline (DPBS) and 5 % sodium dodecyl sulfate (SDS) solution were used as a negative and positive control, respectively. At the end of the exposure time, the tissue was rinsed 15 times with sterile DPBS and incubated with a fresh assay medium for 24 h in humidified atmosphere at 37±1 °C in the presence of 5 % CO2. Then, the medium was changed and the tissue was incubated at the same conditions for another 18 h. At the end of the incubation period, MTT assay was carried out: 0.3 ml of 1 mg/ml MTT
Fig. 1 Structures present in PDDS. Representative micrographs of (a) PDDS matrix versus (b) control (system with the same composition without phospholipid) visualized by light microscopy (Zeiss, Axioskop, Germany), using an Achroplan ×40/0.65 objective, bar=1000 nm
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Fig. 2 Pouches in PDDS. Representative CLSM micrographs of (a) PDDS matrix containing fluorescent phospholipid versus controls: (b) system without phospholipid and (c) system without both phospholipid
and ethanol, visualized by Olympus fluoview 300, Japan with an air plan ×20 objective lens, excited at 488-nm laser line, bar=10 μm
in PBS medium was added, and the plates were incubated for 3 h at the same conditions as above. The tissue was then transferred to DPBS and washed three times by aspiration of the liquid and refilling the wells by fresh DPBS. Then, the media was discarded. The absorbance signal was measured at 570 nm in a microplate spectrophotometer (Multiskan FC, Thermo Scientific). The relative cell viability was calculated as a percent of the mean of the negative control tissues [18].
Results
Data analysis The results are expressed as mean values±standard deviations (SD). Statistical analysis was performed using unpaired twotailed t test or ANOVA with posttest at a significance level set at P<0.05 or less.
Physical characteristics of PDDS Existence of pouch structures visualized by microscopy PDDS micrographs obtained by various microscopic techniques show the existence of a structured matrix containing pouches. Light microscopy micrographs (Fig. 1) of PDDS matrix (Fig. 1a) show the presence of structures. A control system with the same composition but without phospholipid does not have any structure (Fig. 1b). The existence of pouch structures in PDDS containing fluorescent phospholipid was further confirmed by confocal microscopy. From the micrographs in Fig. 2, it is evident that
Fig. 3 Pouches in PDDS. Representative TEM micrographs of (a) PDDS matrix versus (b) control (system without phospholipid), visualized by Philips TECHNAI CM 120, magnification ×9.7 K, following negative staining with 1 % phosphotungstic acid, bar=1000 nm
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TEM micrographs of negatively stained PDDS confirmed that the new matrix system consists of pouches (Fig. 3a). On the other hand, from Fig. 3b, it is clear that the TE micrograph of the control system, system with the same composition but without phospholipid, shows no pouch structures. Further, in Fig. 4, we present a cryo-TE micrograph of PDDS where the pouch is clearly apparent. The micrographs presented above evidence the presence of structures in the matrix that we investigate here. Noteworthy, the pouches are absent from the different control systems.
31
P-NMR spectra for phospholipids
the phospholipid is responsible for the formation of structures in the carrier (Fig. 2a). The two control systems, one without phospholipid and the second without both phospholipid and ethanol, do not present any structures (Fig. 2b, c).
In order to investigate the configuration of phospholipids in the pouch forming system prior to solvent evaporation, 31P NMR studies were carried out on PDDS compared to various control compositions. 31P-NMR spectra of PDDS prepared as above (Fig. 5a) exhibit a sharp line shape at about −28 ppm, which is usually observed for phospholipids in high concentration of ethanol [1]. Such a sharp line shape is similar to the
Fig. 5 31P-NMR spectra of (a) PDDS system containing 5 % phospholipid, 65 % ethanol, 0.7 % hydroxypropylcellulose, and water. (b) System with the same composition but without phospholipid, (c) phospholipid
reverse micelles, and (d) ethosomal system [1] (5 % phospholipid, 30 % ethanol, 0.7 % hydroxypropylcellulose, and water). Recorded on Varian 300 spectrometer
Fig. 4 Pouches in PDDS. Representative cryo-TEM micrograph of PDDS matrix, visualized by FEI T12 G2, bar=200 nm
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line shape in 31P-NMR spectra of phospholipids in a reverse micelle configuration, presented in Fig. 5c. At 30 % ethanol concentration in the ethosome sample, exhibited a broader asymmetrical 31P-NMR spectra at about 0 ppm, as presented in Fig. 5d. The total line width of the 31P-NMR resonance also decreased with the increasing ethanol concentrations. At an ethanol concentration higher than 50 %, the signal becomes isotropic and narrow, showing that the phospholipids exist in the form of fast-tumbling micelles [1]. The 31P-NMR spectra suggest that the pouch structures are associated with reverse micelles of phospholipid in polymer matrix following ethanol evaporation and not a vesicular system. Control system, system with the same composition as PDDS but without phospholipid (Fig. 5b) does not present any peak because of the absence of phosphorous group in the system.
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indicating stretching of CH3 and of C-H, respectively, than in the control system. The increase in isoprene groups in the PDDS spectrum is a sign of interaction and creation of new bonds between the lipophilic chains of the phospholipids and the polymer. In addition, this spectrum exhibits a sharp peak at 1736 cm-1 according to the O-C=O vibration and two peaks at 1243 and 1174 cm-1. This may be due to both PO2 and P-O-C vibrations [19]. This can suggest some interaction and new bonds formation between the hydrophilic head of the phospholipids and oxygen and carbon in the polymer molecules. There is only a low (24 %) similarity between spectra recorded for PDDS and the control system, sustaining the existence of structures only in the presence of the phospholipid. Enhanced dermal delivery from PDDS Delivery to deep skin layers: In vivo experiment in rat
FTIR spectra Spectra of PDDS versus a system with the same composition but without phospholipid are presented in Fig. 6. As we can see, the pouch forming system provided much greater isoprene functional groups at 2923 and 2853 cm-1,
The ability of PDDS to deliver ionic and non-ionic molecules into full thickness rat skin following in vivo application was tested by CLSM using two fluorescent probes, the ionic Rhodamine B and the lipophilic FITC. The apparatus parameters were kept constant for each probe during all
Fig. 6 FTIR spectra (% transmittance) of PDDS versus control (system with the same composition but without phospholipid). The samples were casted on KBr plates on Nicolet iS10 equipped with Smart iTR software (Thermo Scientific), at 15,798.7 cm-1 laser frequency
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measurements in order to allow a comparison of the micrographs. CLSM images of skin cross-sections (Fig. 7) show the penetration depth and fluorescence intensity obtained following 1-h nonocclusive application of systems on the back skin of the animal. It can be seen that both the depth and fluorescence intensity in different skin layers were the highest when the probes were applied in PDDS. The pouch systems were able to deliver both molecule through stratum corneum and viable epidermis into the deep dermis at more than 450-μm depth. We see a much weaker fluorescence and only up to the viable epidermis (∼65 μm) for these probes delivered from the control system.
Skin penetration profile of a hydrophilic molecule: In vitro experiment in Franz diffusion cells
Fig. 7 Skin penetration of Rhodamine B from PDDS (a) and from control (b) and of FITC from PDDS (c) and from control (d). CLS micrographs of cross-section of rat back skin following 1h nonocclusive
application in vivo. The stratum corneum at the right side of the images. Control system with the same composition as PDDS without phospholipid
In this experiment, we have measured the penetration profile into porcine ear skin of the ionic molecule, Rhodamine B incorporated in PDDS and compared to the control system. The systems were applied nonocclusive and left 1 h on the skin in Franz diffusion cells. Figure 8 presents the average fluorescence intensity (arbitrary units) at various skin depths. The delivery from PDDS resulted in an increase in both depth of penetration (120 vs. 70 μm) and fluorescence intensity in the skin.
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Fig. 8 Skin penetration profiles of Rhodamine B delivered from PDDS and control (system with the same composition without phospholipid) into porcine ear skin in 1h nonocclusive application. Depth of skin penetration and fluorescence intensity were determined by CLSM (Zeiss LSM 410) and analyzed by Image Pro-Plus software
A two times higher cumulative fluorescence intensity was measured in the skin following probe application in PDDS relative to control (AUC 1395 vs. 701). Lidocaine delivered to skin from PDDS relative to a spray in clinical use Lidocaine is a lipophilic drug used to prevent immature ejaculation. STUD 100® containing 9.6 % lidocaine in volatile solvents is one pharmaceutical product in clinical use for this indication. In this experiment, we were interested to measure the amount of lidocaine delivered to skin from the new delivery system as compared to STUD 100®, both containing the same drug concentration, in 10 min non-occluded application on the skin. We carried out the experiment in Franz diffusion cells and porcine ear skin. The amounts of lidocaine measured in the skin at the end of the experiment, as presented in Fig. 9, were 79.2 ±16.2 and 39.6±4.6 μg/cm2 for PDDS formulation and STUD 100®, respectively. The PDDS composition was able to deliver into the skin 100 % more drug in a short
period. The statistical significance of the difference between the systems clearly designates that the PDDS are much more effective in skin penetration enhancer for lidocaine than in the commercial volatile solvent system. Another advantage of the lidocaine PDDS over STUD 100® is the shorter drying time for the formulation applied on the site, 3 versus 6 min, respectively. This is a critical parameter in the mode of use of this product. Pharmacodynamic effect of drugs incorporated in PDDS Hypnotic effect of brotizolam PDDS in mice Brotizolam is a drug prescribed to induce and maintain sleep. Chemically, it is a triazolothienodiazepine that shortens the time needed to fall asleep, reduces the frequency of awakenings, and prolongs total sleep time. The hypnotic effect of PDDS containing brotizolam was tested in mice model of pentobarbitone-induced sleeping. Systems each containing 0.02 % brotizolam were applied on the skin 30 min prior to pentobarbitone sodium administration. The loss of righting reflex and the duration of the loss of the righting reflex were recorded as latency time and sleep duration, respectively. Results show that the drug significantly shortened the sleep latency in both treated groups. Sleep latency decreased from 6 ±3.8 min in the untreated control group to 1.3±0.6 and 2.1± 0.2 min in the PDDS- and control carrier-treated groups, respectively (Fig. 10a). Noteworthy, only in the PDDStreated group the sleeping duration was prolonged for a significant longer time relative to the treated control group and the untreated positive control. Sleep duration increased from 29.5±5.1 to 196.9±22.2 min in the PDDS-treated group and only to 98.6±17.8 min in the treated control group (Fig. 10b). Antipyretic effect of ibuprofen PDDS in fevered rats
Fig. 9 Amount extracted from skin following 10 min application of lidocaine PDDS and STUD 100®, each containing 9.6 % drug. The results are expressed as mean±SD; n=4; *P<0.05
Ibuprofen is an effective antipyretic agent currently administrated per os. One drawback of the current mode of use is the need to be administrated every 4 to 6 h.
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injection induces an increase in the animal body temperature with a peak effect at 18 h post-injection. Figure 11 presents the time course of fever and changes in the body temperature relative to the normal values (36.2±0.1 °C) before brewer’s yeast injection (ΔT). The temperature-time profiles presented in Fig. 11 show that following ibuprofen PDDS application on the skin, the body temperature of the fevered rats was gradually reduced achieving normal values in 2 h. The body temperature remained low (36.4±0.2 °C) until the end of the tested time, at least 12 h. By oral administration, rat’s body temperature returned to baseline after 1 h but was maintained for only 3 h when a rise in temperature was seen again. The body temperature of the untreated fevered animals remained high during the whole experiment period, 37.7±0.2 °C.
Plasma drug concentration in rats treated with Ibuprofen PDDS
We have tested here the effect of the drug incorporated in the new carrier and compared to the oral administration. The antipyretic effect of PDDS containing ibuprofen was measured in rats following fever induction. Brewer’s yeast
Plasma drug concentration versus time profiles obtained in rats that were treated with ibuprofen PDDS single application or per os with aqueous ibuprofen suspension are given in Fig. 12 and Table 1. It can be seen that a peak plasma concentration of 70.7±26.9 μg/ml was achieved 1 h following the PDDS application. For oral administration, the Cmax and Tmax values were 86.6±21.2 μg/ml and 0.5 h, respectively. A relative bioavailability (F) value of 0.51 was calculated for PDDS versus ibuprofen oral administration. Results of this pharmacokinetic study show that, when administered transdermal from PDDS, the drug was present in rat plasma for a much longer period as compared to the oral administration, 24 versus 12 h, respectively.
Fig. 11 Effect of ibuprofen applied on the skin from PDDS versus ibuprofen liquid preparation administrated orally on fevered rats. Brewer’s yeast was injected 18 h prior to drug administration. Mean± SD (n=5 in each fevered rats group, n=2 in the normal rats group). ΔT
change of the body temperature from the initial normal value in each group. P<0.01 transdermal versus fevered untreated groups; P<0.05 transdermal versus oral groups at 25 h post yeast injection; P<0.01 transdermal versus oral groups at 30 h post yeast injection
Fig. 10 The sleep latency (a) and the sleep duration (b) following skin application of brotizolam PDDS versus brotizolam control system. All data are presented as mean±SD; n=5 for each group. *P<0.05 and ***P<0.001 versus the brotizolam control group and the untreated animals group (one way ANOVA with posttest)
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Fig. 12 Plasma concentrations of ibuprofen in rats following transdermal and oral administration. Transdermal PDDS administrated at a single dose of 200 mg/kg drug and oral drug suspension administrated at dose of 62.5 mg/kg. Data is presented as mean±SD; n=5 in each group
It was further interesting to compare the PK and PD profiles obtained in the experiments presented above. It is noteworthy that the animals had a similar weight and age. Figure 13 shows the existence of a good correlation between the decrease in body temperature of animals and the plasma drug concentration following ibuprofen PDDS application.
Results of safety test for PDDS The skin safety of PDDS was tested in vitro by the EpiDerm™ (reconstructed human epidermal model) skin irritation test. The results of the controls confirmed the validity of the test. In accordance to Table 2, the mean OD570 of the negative control tissues exposed to DPBS was 1.674 (within the acceptance criteria, ≥1.0 and ≤2.5). The positive control tissues exposed to 5 % SDS had only 11.0 % mean viability (within the acceptance criteria, ≤20 %) [20]. Measurement of the effect of topical exposure of the EpiDerm™ by the MTT test showed that the new delivery system, PDDS, has no effect on Table 1 Pharmacokinetic parameters obtained with a single dose ibuprofen administrated in PDDS applied on the skin or as oral suspension (Ibuprofen, 200 and 62.5 mg/kg, respectively)
Cmax (μg/ml) Tmax (h) AUC0→24 (μg×h/ml) AUC0→∞ (μg×h/ml) F (relative bioavailability) transdermal versus oral
Transdermal PDDS
Oral suspension
70.74±26.94 1 388.09±67.41 423.29±59.51 0.51
86.57±21.18 0.5 236.99±34.74 257.33±31.03
Fig. 13 The correlation between the pharmacokinetic profile and pharmacodynamic effect after transdermal administration of ibuprofen PDDS in rats. Mean±SD; n=5 in each group
cell viability, 146 versus 100 % in the negative control (DPBS) untreated cells. When the positive control solution (SDS) causes severe skin irritation, the cell viability was only 11.0 % (Fig. 14 and Table 2). These results suggest that PDDS had no irritating effect on skin model used here. The test material is considered skin irritating if the remaining relative cell viability is below 50 %.
Discussion The PDDS representative composition investigated here exhibits a matrix containing pouch looking structures. 31 P-NMR spectra suggest that the phospholipid molecules in the initial system solution are organized as micelles. This is in contrast to phospholipids spectra of dispersions at lower ethanol concentration, up to 45 %, or alcohol free phospholipid dispersion. The last two systems present bilayered vesicles (ethosomes and liposomes) [1, 21]. This data together with the micrographs suggest that the pouch structures could be the result of association of phospholipid reverse micelles in the polymer matrix following solvent evaporation. We found a deeper and more intense penetration into the skin layers, by both lipophilic and hydrophilic probes when the delivery system applied to the skin was PDDS. Table 2 Results from the irritation test of PDDS in EpiDerm™ in vitro experiment. The test uses MTT signals, viability, and classification of positive (5% SDS) and negative (DPBS) controls and PDDS after exposure to reconstructed human epidermis (RhE) EpiDermTM model Treatment
O.D. values (570 nm) Viability (%) Classification mean±SD mean±SD
Negative control 1.674±0.323 Positive control 0.184±0.066 PDDS 2.444±0.022
100.0±19.32 Non-irritant 11.0±3.92 Irritant 146.0±1.32 Non-irritant
Drug Deliv. and Transl. Res. (2014) 4:416–428
427
Fig. 14 Results of EpiDerm™ test carried out for PDDS: Cell viability after exposure to PDDS and positive control (SDS) relative to control untreated tissue. Mean±SD percent of cell viability
Confocal laser scanning micrographs demonstrated that PDDS is able to enhance skin penetration of ionic and nonionic molecules into the deep skin layers after relatively short application time. While systems with the same composition just without phospholipid resulted in only a small reservoir in the upper layers of the skin. These findings indicate that PDDS is able to efficiently enhance the delivery of the drugs into the deep skin layers and thus can possibly improve the efficiency of topical skin therapy. PDDS is a good penetration enhancer for lidocaine. We have seen that after a 10 min application of lidocaine in the pouch system the drug quantity in the skin was twice to that from STUD 100®. Brotizolam and ibuprofen, each incorporated in the new carrier, have shown a significant sustained action in vivo. The hypnotic effect of brotizolam PDDS in sleeping mice model significantly prolonged the sleep duration as compared to both animal groups, treated with the control solution or to the untreated group. Ibuprofen PDDS in fevered rats gradually reduced the animal’s body temperature to normal values. These values Scheme 1 A cartoon describing ways for enhanced skin penetration by PDDS
were maintained for at least 12 h. In contrast, the oral ibuprofen administration was able to maintain the antipyretic effect for only 3 h. Interestingly, ibuprofen blood levels increased rapidly and reached a peak in the first hour was followed by a plateau for 24 h. This shows a good PK-PD correlation for ibuprofen PDDS. Overall, the data indicate that PDDS is effective at delivering molecules deeply into and through the skin and promote a sustained action of drugs. It is suggested that the enhanced delivery by PDDS is contributed by the system’s components and its pouchstructure. A number of concomitant processes may take place when compositions containing reverse phospholipid micelles in a polymer solution of high ethanol concentration (65 %) are applied on the skin. Scheme 1 illustrates a hypothetical model of PDDS affecting penetration through the stratum corneum lipids. It is known that ethanol interdigitates stratum corneum lipid bilayers enhancing their fluidity and disturbing their organization [1, 22, 23]. This disorganization may facilitate penetration of other molecules. The drug molecules and the phospholipid micelles may penetrate the disturbed bilayers. Micelles association in pouches may be responsible for the drug-sustained effect observed with two drug molecules tested here.
Conclusions The results obtained in this work indicate that the system investigated has enhanced skin penetration, promotes sustained drug effect, and is safe. This first publication opens the doors for further research on PDDS as carriers for dermal and transdermal delivery of various drugs
PDDS Stratum corneum lipid mullayers Viable epidermis Blood vessels in the dermis
Reverse micelle
Drug
Ethanol
428 Conflicts of interest None.
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All institutional and national guidelines for the care and use of laboratory animals were followed.