NANOPARTICLES FOR DERMAL AND SYSTEMIC DELIVERY OF DRUGS
20170065533 ยท 2017-03-09
Assignee
Inventors
- Simon Benita (Tel Aviv, IL)
- Taher Nasser (Tur'an Village, IL)
- Nour KARRA (Jaffa Tel Aviv, IL)
- Amit BADIHI (Jerusalem, IL)
Cpc classification
A61K47/6925
HUMAN NECESSITIES
A61K47/6937
HUMAN NECESSITIES
A61K47/34
HUMAN NECESSITIES
A61K9/06
HUMAN NECESSITIES
International classification
A61K9/00
HUMAN NECESSITIES
Abstract
The present invention relates to a poly(lactic glycolic) acid (PLGA) nanoparticle associated with therapeutic agents for a variety of therapeutic applications.
Claims
1. A poly(lactic glycolic) acid (PLGA) nanoparticle having an average diameter of at most 500 nm, the PLGA having an average molecular weight of between 2,000 and 20,000 Da, wherein said nanoparticle containing cyclosporine.
2. The nanoparticle according to claim 1, wherein the PLGA polymer is a copolymer of polylactic acid (PLA) and polyglycolic acid (PGA).
3. The nanoparticle according to claim 1, wherein the average diameter of the nanoparticle is between about 100 and 200 nm.
4. The nanoparticle according to claim 1, being in the form of a nanocapsule or a nanosphere.
5. The nanoparticle according to claim 1, wherein said nanoparticle being further associated with at least one non-active agent.
6. The nanoparticle according to claim 5, wherein said at least one non-active agent is selected to modulate at least one characteristic of the nanoparticle, said characteristic being selected from size, polarity, hydrophobicity/hydrophilicity, electrical charge, reactivity, chemical stability, clearance rate, distribution and targeting.
7. The nanoparticle according to claim 5, wherein the non-active agent is selected from fatty acids, amino acids, aliphatic or non-aliphatic molecules, aliphatic thiols, and aliphatic amines.
8. The nanoparticle according to claim 7, wherein the non-active agent is a fatty amino acid (alkyl amino acid).
9. The nanoparticle according to claim 1, wherein the cyclosporine is associated with the nanoparticle via one or more linker moieties.
10. The nanoparticle according to claim 9, wherein said one or more linker moieties having a first portion capable of association with the nanoparticle and a second portion capable of association with the therapeutic agent.
11. The nanoparticle according to claim 10, wherein the linker moiety is a fatty amino acid (alkyl amino acid).
12. The nanoparticle according to claim 11, wherein the linker is oleylcysteineamide.
13. A composition comprising at least one nanoparticle according to claim 1.
14. The composition of claim 13 being a pharmaceutical composition.
15. The composition according to claim 14, being adapted for transdermal administration of a therapeutic agent.
16. The composition according to claim 14, for topical administration of a therapeutic across skin layers.
17. The composition according to claim 13, wherein the composition is essentially free of water.
18. A topical formulation comprising at least one nanoparticle according to claim 1.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0158] In order to understand the invention and to see how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
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DETAILED DESCRIPTION OF THE INVENTION
I. Lactic Acid and Glycolic Delivery to the Skin
[0183] Use is made of the clinically well-accepted PLGA polymers as well as PLA particles of a specific molecular weight, to prepare nanoparticles of a certain particle size that are applied onto the skin, penetrate in the upper layers of the dermis and release, in a controlled manner over time, lactic and glycolic acid, or only lactic acid, which are natural moisturizing factors, allowing a prolonged and sustained hydration of the skin without being harmful.
[0184] The PLGA nanoparticles, per se, empty or loaded with appropriate actives are used as the prolonged active hydrating ingredients, as a result of their degradation within the skin leading to the progressive and continuous release of lactic and glycolic acid. Even if the nanoparticles penetrate into the deep layer of the epidermis or even the dermis, they do not induce any damage as previously described since the hydrolysis product lactic and glycolic acids are naturally eliminated or excreted.
[0185] It should be emphasized the PLGA (or PLA), as the active hydrating components of the composition of the invention, are not merely used as carriers for delivery of other components to the skin, although the invention also encompasses the possibility that other beneficial active components are used. Thus, in accordance with the invention the composition is intended for topical application, i.e., contains carriers for topical applications, as well as for other applications.
[0186] The nanoparticles of the invention are typically of a size smaller than 500 nm. Typically, the nanoparticles are of a size range of between 100 and 200 nm, or between 50 and 100 nm.
[0187] In some embodiments, the molecular weight of PLGA and the ratio between PLA and PGA is tailored so that the nanoparticles have the following properties: [0188] (a) Penetrate into the skin to at least the 10 superficial epidermis layers; [0189] (b) Penetrate to a depth of at least 4-20 micrometers into the skin; [0190] (c) Biodegrade in the skin layer into which they penetrate (typically about 15% in the Stratum corneum); [0191] (d) Sustained release of the lactic acid and glycolic acid or only the lactic acid for a period above 24 hours, preferably above 72 hours, more preferably about a week.
[0192] Without wishing to be bound by theory, there seems to be interplay between the size of particle (which influences the penetration rate and the deepness of penetration), the ratio of PLA and PGA and the molecular weight of the PLGA, in such a way that the above properties can be achieved by a number of combinations. Several changes in parameters may neutralize each other.
[0193] In some embodiments, the ratio of PLA:PGA is 85:15; 72:25; or 50:50. In some embodiments, the ratio is 50:50.
[0194] In other embodiments, the molecular weight of the PLGA ranges from 2,000 to 10,000 Da. In some embodiments, the ratio is between 2,000 and 4,000.
[0195] In other embodiments, the PLA particles may be employed per se, in such embodiments the PLA molecular weight is in the range of 4,000 and 20,000.
[0196] The nanoparticles of this disclosure may be comprise or prepared of PLGA having an average molecular weight ranging between 2,000 and 500,000 Da. Namely, the PLGA may have an average molecular weight of between 2,000 and 250,000 Da, between 2,000 and 200,000 Da, between 2,000 and 100,000 Da, between 2,000 and 50,000 Da or between 2,00 and 20,000 Da.
[0197] The therapeutic active associated with the PLGA nanoparticle may be selected from calcitonin, cyclosporine, insulin, dexamethasone, dexamethasone palmitate, cortisone, prednisone and others.
II. Encapsulation Strategies of Insoluble Compounds in Nanoparticlesthe Potential of DHEA Loaded PLGA Nanoparticles
[0198] In the present invention, the nanoparticles may be loaded with active materials such as vitamins, peptides, and others as disclosed hereinabove.
[0199] Humans have adrenals that secrete large amounts of dehydroepiandrosterone (DHEA) and its sulphate derivatives (DHEAS). A remarkable feature of plasma DHEA(S) levels in humans is their great decrease with aging. Researchers have postulated that this age-related decline in DHEA(S) levels may explain some of the degenerative changes associated with aging. Three mechanisms of action of DHEA(S) have been identified. DHEA and DHEA(S) are precursors of testosterone and estradiol. DHEA(S) is a neurosteroid, which modulates neuronal excitability via specific interactions with neurotransmitter receptors, and DHEA is an activator of calcium-gated potassium channels.
[0200] Randomized, placebo-controlled clinical trials which included 280 healthy individuals (140 men and 140 women) aged 60-years and over treated with (near) physiological doses of DHEA (50 mg/day) over one year have yielded very positive results. Impact of DHEA replacement treatment was assessed on mood, well being, cognitive and sexual functions, bone mass, body composition, vascular risk factors, immune functions and skin. Interestingly, an improvement of the skin status was observed, particularly in women, in terms of hydration, epidermal thickness, sebum production, and skin pigmentation. Furthermore, no harmful consequences were observed following this 50 mg/day DHEA administration over one year.
[0201] It is known that DHEA might be related to the process of skin aging through the regulation and degradation of extracellular matrix protein. It was demonstrated that DHEA can increase procollagen synthesis and inhibit collagen degradation by decreasing matrix metalloproteinase (MMP)-1 synthesis and increasing tissue inhibitor of matrix metalloprotease (TIMP-1) production in cultured dermal fibroblasts. DHEA (5%) in ethanol:olive oil (1:2) was topically applied to buttock skin of volunteers 12 times over 4 weeks, and was found to significantly increase the expression of procollagen alpha1 (I) mRNA and protein in both aged and young skin. On the other hand, topical DHEA significantly decreased the basal expression of MMP-1 mRNA and protein, but increased the expression of TIMP-1 protein in aged skin. These recent results suggest the possibility of using DHEA as an anti-skin aging agent.
[0202] Based on the overall reported results, exogenous DHEA, administered topically may promote keratinization of the epidermis, enhance skin hydration by increasing the endogenous production and secretion of sebum subsequently reinforcing the barrier effect of the skin, treat the atrophy of the dermis by inhibiting the loss of collagen and connective tissue and finally can modulate the pigmentation of the skin. These properties render DHEA the active of choice as an anti-aging active ingredient provided DHEA is adequately dissolved in the topical formulation, can diffuse from the formulation towards the skin and be fully bioavailable for skin penetration following dermal application. Indeed, DHEA exhibits complex solubility limitations in common cosmetic and pharmaceutical solvents such as water, polar oils and vegetable oils. DHEA is practically insoluble in water (0.02 mg/ml) and is known for its tendency to precipitate rapidly within topical regular formulations even at concentrations lower than 0.5%, yielding several polymorphic crystal forms which are difficult to control and exhibit very slow dissolution rate. Furthermore, DHEA shows low solubility in lipophilic phases with a maximum solubility of 1.77% in mid chain triglycerides. The most accepted topical dosage form is the o/w emulsion in which the DHEA should be dissolved in the lipophilic phase. However, this solution is very difficult to accomplish since very high concentrations of oil phase (more than 70%) are needed to achieve a DHEA concentration eliciting an adequate efficacy activity (approximately 0.5% w/v). Topical products with such high oil phase concentrations will be unpleasant and unappealing, ruling out their usefulness as cosmetic products.
[0203] There is no doubt that the recrystallization process of DHEA should be prevented since it can potentially cause significant variations in therapeutic bioavailability and efficacy. The drug crystals need first to re-dissolve in the skin prior to diffusing and penetrating the superficial skin layers. Such a process is unlikely to occur easily and will significantly affect the activity of the product. Moreover, the recrystallization process can affect the stability and the physical appearance of the formulation. Thus, there is clearly a need to prepare pleasant and convenient o/w topical formulations where DHEA loaded nanoparticles can be dispersed at an adequate concentration precipitate out of the formulation. Furthermore, the DHEA embedded nanocarrier should be incorporated in a topical formulation, which can promote penetration of the active ingredient within the epidermis and dermis layers where its action is most needed.
III. Delivery of Surface Bound Macromolecules and Minerals into the Skin Using Thiol Activated Nanoparticles
[0204] Commercially available products utilizing transdermal delivery have been mainly limited to low molecular weight lipophilic drugs (MW<500 Da) [16], with larger molecular weights (MW>500 Da) facing penetration difficulties [17]. Due to the impervious nature of the Stratum corneum towards macromolecules, a suitable penetration enhancer should substantially improve transport of macromolecules through the skin. Various technologies have been developed for this purpose, including the use of microneedles, electroporation, laser generated pressure waves, hyperthermia, low-frequency sonophoresis, iontophoresis, penetration enhancers, or a combination of these methods. Many penetration enhancement techniques face inherent challenges, such as scale-up and safety concerns [17]. The present invention proposes the delivery of macromolecules, mostly hydrophilic, by a non invasive method, using a surface binding technique of macromolecules to thiolated nanoparticles.
Thiolated NPsState of the Art
[0205] Nanoparticles were functionalized with a maleimide moiety, which were then conjugated to a thiolated protein. Alternatively, nanoparticles can be functionalized with a thiol group then conjugated to a maleimidic residue on the protein. Traditionally, such delivery systems have been mostly used for the targeted delivery of drug loaded nanoparticles, principally to malignant tumors, where the surface conjugated protein is used simply as a targeting moiety recognizing disease specific epitopes.
IV. Experimental
1. DiD Loaded PLGA NPs and NCs and/or Rhodamine B PLGA Conjugated NPs or NCS Preparation
[0206] PLGA was dissolved in acetone containing 0.2% w/v Tween 80, at a concentration of 0.6% w/v. In case were NCs were prepared, octanoic acid or MCT at a concentration of 0.13% w/v was also added to the organic phase. If DiD loaded NPs were prepared then, an aliquot of acetone DiD solution at a concentration of 1 mg/ml was also added to the organic phase, resulting in a final concentration of 15-30 g/ml. If rhodamine B PLGA conjugated NPs or NCs were prepared, 0.03% w/v rhodamine B tagged PLGA were dissolved in acetone together with 0.57% w/v non labeled PLGA. The organic phase was added to the aqueous phase containing 0.1% w/v Solutol HS 15. The suspension was stirred at 900 rpm over 15 minutes and then concentrated by evaporation to a final polymer concentration of 30 mg/ml. The aqueous and oil control compositions were identical to the formulation described above, only without the polymer presence.
2. [.SUP.3.H]DHEA and [.SUP.3.H]COE PLGA Solid Nanoparticle Encapsulation and Evaluation
DHEA NPs Preparation
[0207] DHEA loaded PLGA nanocapsules were prepared using the interfacial deposition method [18]. DHEA was solubilized in octanoic acid/MCT/oleic acid and in acetone. If positively charged DHEA NCs were prepared, the cationic lipid, DOTAP [1,2-dioleoyl-3-trimethylammonium-propane], at a concentration of 0.1% w/v was added to the organic phase. In case were radioactive DHEA NCs were prepared, 15 Ci of tritiated DHEA were inserted into the oil core of the NCs during the preparation of the NCs together with 1 mg of cold DHEA. In case [.sup.3H]Cholesteryl oleyl ether ([.sup.3H]COE) were prepared, 80 and 127 Ci [.sup.3H]COE were either dissolved in MCT to form NCs or simply added to the organic phase for NPs formation, respectively. The organic phase was added drop wise to the aqueous phase under stirring at 900 rpm, and the formulation was concentrated by evaporation to a polymer concentration of 8 mg/ml. The formulations were filtered through 0.8 m membrane and then 3 ml from the different [.sup.3H]DHEA NCs were dia-filtrated with 30 ml PBS (pH 7.4) (Vivaspin 300,000 MWCO, Vivascience, Stonehouse, UK) and filtered through 1.2 m filter (w/0.8 m Supor Membrane, Pall corporation, Ann Arbor, USA). The radioactivity intensity for the overall formulations and their respective controls was set so a finite dose applied will be in the range of a total of 0.63-1.08 Ci/ml. The compositions of the organic phase and the aqueous phase are presented in Table 1.
TABLE-US-00001 TABLE 1 compositions of organic phase and aqueous phase Organic phase Aqueous phase PLGA 4500 MW - 150 mg Solutol HS 15 - 50 mg Octanoic acid - 75 l Water - 100 ml DHEA - 10 mg TWEEN 80 - 50 mg Acetone - 50 ml
[0208] Particle Size Analysis:
[0209] mean diameter and particle size distribution measurements were carried out utilizing an ALV Noninvasive Back Scattering High Performance Particle Sizer (ALV-NIBS HPPS, Langen, Germany) at 25 C. and using water as diluent.
[0210] Zeta Potential Measurements:
[0211] the zeta potential of the NPs was measured using the Malvern zetasizer (Malvern, UK) diluted in HPLC grade water.
[0212] Scanning (SEM) and Transmission Electron Microscopy (TEM):
[0213] morphological evaluation was performed by means of scanning and transmission TEM (Philips Technai F20 100 KV). Specimens for TEM visualization are prepared by mixing the sample with phosphotungstic acid 2% (w/v) pH 6.4 for negative staining.
[0214] Cryo-Transmission Electron Microscopy (Cryo-TEM):
[0215] A drop of the aqueous phase was placed on a carbon-coated holey polymer film supported on a 300 mesh Cu grid (Ted Pella Ltd), the excess liquid was blotted and the specimen was vitrified via a fast quench in liquid ethane to 170 C. The procedure was performed automatically in the Vitrobot (FEI). The vitrified specimens were transferred into liquid nitrogen for storage. The fast cooling is known to preserve the structures present at the bulk solution and therefore provides direct information on the morphology and aggregation state of the objects in the bulk solution without drying. The samples were studied using a FEI Tecnai 12 G2 TEM, at 120 kV with a Gatan cryo-holder maintained at 180 C., and images were recorded on a slow scan cooled charge-coupled device CCD Gatan manufactured camera. Images were recorded with the Digital Micrograph software package, at low dose conditions, to minimize electron beam radiation damage.
3. Diffusion Experiments
[0216] Franz diffusion cells (Crown Glass, Sommerville, N.J., USA) with an effective diffusion area of 1/0.2 cm.sup.2 and an acceptor compartment of 8 ml were used. The receptor fluid was a phosphate buffer, pH 7.4.
[0217] Throughout the experiment, the receptor chamber content was continuously agitated by a small magnetic stirrer. The temperature of the skin was maintained at 32 C. by water circulating system regulated at 37 C. Finite doses of the vehicle and formulations (10-50 mg polymer per cell) were applied on the horny layer of the skin or cellulose membrane. The donor chamber was opened to the atmosphere. The exact time of application was noted and considered as time zero for each cell. At 4, 8, 12 and 24 h or 26 h, the complete receptor fluid was collected and replaced with fresh temperature equilibrated receptor medium. The determination of the diffused active ingredient concentration was determined from aliquots. At the end of the 24- or 26-h period, the skin surface was washed 5 times with 100 ml of distilled water or ethanol. The washing fluids were pooled and an aliquot part (1 ml) was assayed for the active ingredient concentration.
[0218] The cells were then dismantled and the dermis separated from the epidermis by means of elevated temperature as described above. The active ingredient content was determined by means of HPLC or other validated analytical techniques. Furthermore, the presence of lactic or glycolic acid in the receptor medium was examined.
4. DiD Loaded PLGA NPs and NCs and/or Rhodamine PLGA Conjugated NPs or NCS Site Localization
[0219] Excised human skin or porcine ear skin samples were placed on Franz diffusion cells (PermeGear, Inc., Hellertown, Pa.), with an orifice diameter of 5/11.28 mm, 5/8 mL receptor volume and an effective diffusion area of 0.2/1.0 cm.sup.2. The receptor fluid was phosphate buffer, pH 7.4. Throughout the experiment, the receptor chamber content was continuously agitated by a small magnetic stirrer. The temperature of the skin was maintained at 32 C. by water circulating system regulated at 37 C. The solutions and different NP and NCs formulations either loaded with entrapped DiD fluorescent probe with free PLGA or PLGA covalently bound to rhodamine B were applied on the skin as detailed below. This protocol was adopted to follow the skin localization of both the entrapped DiD probe and of the conjugated rhodamine B polymer. The various formulations were prepared as described in the experimental section above. The dose applied for each formulation on the excised skin samples was 125 l of a 30 mg/ml PLGA polymer concentration with an initial entrapped fluorescent content of DiD 30 g/ml.
[0220] After single incubation period or at different time intervals, some of the skin samples were dissected to identify the localization site of the nanocarrier in the various skin layers by confocal microscope. The procedure was as follows using histological sectioning. The skin specimens were fixated using formaldehyde 4% for 30 minutes. The fixated tissues were placed in an adequate plastic cubic embedding in tissue freezing medium (OCT, Tissue-Tek). Skin samples were then deeply frozen at 80 C. and vertically cut into 10 m thick sections, utilizing Cryostat at 20 C. Then, the treated specimens were stored in a refrigerator until to the confocal microscopic analysis.
[0221] In addition, some whole mount skin specimens were kept intact after Franz cells incubation at selected time interval of 2 h and immediately observed by confocal microscope and further reconstructed using 3D imaging from z-stacks pictures. The fluorescence intensity versus skin depth for nanocarriers and respective controls using line profile (calculated intensity for each section and whole specimen accumulative intensity are reported). Samples data is provided in Table 2.
TABLE-US-00002 TABLE 2 Description of the composition of each formulation topically applied with specific equivalent dose PLGA- DiD eq. PLGA, rhodamine B Oil core Volume dose Formulation mg/cm.sup.2 conjugated % type in applied Applied Composition (MW, kDa) w/w from NPs NCs (l) (l) (g) DiD NPs 4.5 (4) 150 1.125 DiD NPs 4.5 (50) 150 1.125 DiD NCs 4.5 (4) Octanoic 150 1.125 acid (75) DiD micellar 150 1.125 solution DiD incorporated 3.75 (4) 5 125 3.75 rhodamine B conjugated PLGA NPs DiD incorporated 3.75 (4) 5 MCT (113) 125 3.75 rhodamine B conjugated PLGA NCs Rhodamine B 3.75 (NA) 125 incorporated Latex NPs DiD and rhodamine 5 125 3.75 B conjugated PLGA aqueous dispersion DiD and rhodamine 5 MCT (113) 125 3.75 B conjugated PLGA oil containing aqueous dispersion
5. [.SUP.3.H]DHEA NCs Site Localization and Deep Skin Layer Localization
[0222] [.sup.3H]DHEA NCs Formulations were Applied on the Skin Using the Franz Cell diffusion system. [.sup.3H]DHEA localization in the various skin layers was determined by skin compartment dissection technique. Dermatome pig skin (600-800 m thick) was mounted on Franz diffusion cells (Crown Glass, Sommerville, N.J., USA) with an effective diffusion area of 1 cm.sup.2 and an acceptor compartment of 8 ml (PBS, pH 7.4). At different time intervals, skin compartment dissection was carried out to identify the localization site of the nanocarriers in the skin surface, upper corneocytes layers, epidermis, dermis and receptor cell. First, the remainder of the formulation was collected following serial washings to allow adequate recovery. Then, the skin surface was removed by adequate sequential tapes stripping, contributing the first strip to the donor compartment. The rest of the viable epidermis was separated from the dermis by means of heat elevated temperature, and then chemically dissolved by solvable digestion liquid. Finally the receptor fluids were also collected and further analyzed.
[0223] In addition, in an attempt to reveal quantitatively the biofate of the NCs and NPs in the various layers of the skin, 80 and 127 Ci [.sup.3H]Cholesteryl oleyl ether ([.sup.3H]COE) were either dissolved in the oil core of the NCs or entrapped in the nanomatrices of the NPs respectively. The radioactive tracer, [.sup.3H]Cholesteryl oleyl ether ([.sup.3H]COE) is highly lipophilic with a log P above 15 (>15) and its localization within skin layers reflects the localization of either the oil core of the NC or the nanomatrix of the NP since the probe cannot be released from the nanocarriers in view of its extremely high lipophilicity.
6. Oleylcysteineamide Synthesis and Characterization
Oleylcysteineamide Synthesis
[0224] Under a flow of nitrogen the flask was charged via syringe with oleic acid (OA) (2.0 g, 7.1 mmol), 60 ml of dry tetrahydrofuran, and triethylamine (0.5 ml, 7.1 mmol). Stirring was commenced, and the solution was cooled to an internal temperature of 15 C. using a dry ice-isopropyl alcohol bath at 5 to 10 C. Ethyl chloroformate (0.87 ml, 6.1 mmol) was added and the solution was stirred for 5 min. The addition of ethyl chloroformate resulted in an internal temperature rise to +8 to +10 C. and the precipitation of a white solid. Following the precipitation the continuously stirred mixture, still in the dry-isopropyl alcohol bath, was allowed to reach an internal temperature of 14 C. Cysteine (1.0 g, 8.26 mmol) dissolved in 5% Na.sub.2CO.sub.3 solution (10 ml) introduced into the flask via a syringe needle, was vigorously bubbled through the solution for 10 min with manual stirring: the internal temperature rises abruptly to 25 C. With the flask still in the cooling bath, stirring was continued for an additional 30 min, and the reaction mixture was stored in the freezer at 15 C. overnight. The slurry was stirred with tetrahydrofuran (100 ml) at room temperature for 5 min and ammonium salts were removed by suction filtration through a Bchner funnel. After the solids were rinsed with tetrahydrofuran (20 ml), the filtrate was passed through a plug of silica gel (25 g Merck 60 230-400 mesh) in a coarse porosity sintered-glass filter funnel with aspirator suction. The funnel was further washed with acetonitrile (100 ml) and the combined filtrates were evaporated (rotary evaporator) to give a viscous liquid.
[0225] Formation of oleylcysteineamide was confirmed by H-NMR (Mercury VX 300, Varian, Inc., CA, USA) and LC-MS (Finnigan LCQDuo, ThermoQuest, NY, USA).
Oleylcysteineamide Characterization
[0226] .sup.1H-NMR (CDCl.sub.3, ): 0.818, 0.848, 0.868, 0.871, 0.889, 1.247, 1.255, 1.297, 1.391, 1.423, 1.452, 1.621, 1.642, 1.968, 1.989, 2.008, 2.174, 2.177, 2.268, 2.2932.320, 2.348, 3.005, 3.054, 4.881, 5.316, 5.325, 5.335, 5.343, 5.353, 5.369, 6.516, 6.540, 7.259 ppm.
[0227] LC-MS: Peak at: 384.42.
[0228] The NMR analysis confirms the formation of the linker oleylcysteineamide, while the LC-MS spectrum clearly corroborates the molecular weight of the product which is 385.6 g/mol
7. Preparation and Characterization of Surface Activated Nanoparticles and Macromolecules Conjugation
[0229] Nanoparticles were prepared using the well established interfacial deposition method [18]. The oleylcysteineamide linker molecule was dissolved in the organic phase containing the polymer dissolved in water soluble organic solvent. The organic phase was then added drop wise to the aqueous phase which contains a surfactant. The suspension was evaporated at 37 C. under reduced pressure to a final nanoparticulate suspension volume of 10 ml. A maleimide bearing spacer molecule (LC-SMCC) was reacted with the desired macromolecule at pH 8 for subsequent conjugation to the thiol moiety. The thiol activated NPs and the relevant maleimide bearing molecule were then mixed and allowed to react overnight under a nitrogen atmosphere. The following day, free unbound molecules were separated from the conjugated NPs using a dia-filtration method.
TABLE-US-00003 TABLE 3 Formulation composition Organic phase Aqueous phase Polymer Solutol HS 15 300 mg 100 mg Oleyl cysteine Water 20 mg 100 ml Tween 80 100 mg Acetone 50 ml
Size and Zeta Potential Characterization:
[0230] The size and zeta potential of the various NPs were measured in water using a DTS zetasizer (Malvern, UK).
Determination of the Conjugation Efficiency of the Various Macromolecules to NPs:
[0231] The conjugation efficiency of the macromolecules such as MAbs was determined using the calorimetric Bicinchoninic acid assay (BCA) for protein quantification (Pierce, Ill., USA).
[0232] It should be noted, that the same procedure disclosed herein has been used to link hyaluronic acid to the nanoparticles.
8. Incorporation of Nanoparticles into Anhydrous Cream
[0233] The advantages of dispersing the final product in anhydrous cream are enormous. Increasing amounts (0.1-10%) of freeze-dried powders of the NPs and the NPs prepared are incorporated into a novel cream comprising no water. The relative amounts of the ingredients of this cream are detailed in Table 4.
TABLE-US-00004 TABLE 4 relative amounts of ingredients Ingredient Relative amont/100 Dow corning 9040 - 40.0-50.0 Cyclopentasiloxane (and) Dimethicone crosspolymer Dimethicone 5.0-7.0 Cyclopentasiloxane 10.0-15.0 Shin etsu KSG-16 20.0-35.0 Dimethicone (and) Dimethicone/Vinyl dimethicone Crosspolymer Boron Nitride 0.3-0.70 lauroyl Lysine Ajinomoto 0.2-0.70 hyaluronic acid MP 50000 0.1-0.40 Palmitoyloligopeptide - 0.05-0.3 Biopeptide CL Sederma Palmitoyl tetrapeptide - N- 0.05-0.3 Palmitoyl-Rigin
IV. Preliminary Results
Nanoparticle Formulation and Characterization
[0234] Fluorescent nanoparticles were prepared to facilitate visual detection of the nanoparticles. PLA was conjugated to the fluorescent Rhodamine B probe. The nanoparticles were then prepared as described in the experimental section above.
[0235] The results demonstrate a homogenous nanoparticle formulation. It was possible to see the nanoparticles owing to the fluorescence labeling with Rhodamine fluorophore at excitation/emission 560/580 nm. The nanoparticles exhibited a mean diameter of 52 nm and a Zeta potential value of 37.3 mV.
[0236] This technique was used to detect and identify the localization of the nanoparticles with time in the various layers of the skin following topical application.
Cryo-TEM Visualization of PLGA Biodegradable NPs One Month Following Preparation
[0237] The Cryo-TEM images of blank PLGA.sub.4500 nanoparticles at various areas of the carbon grid are depicted in
DHEA Loaded PLGA Nanoparticles
[0238] DHEA was encapsulated within the oil core of PLGA nanocapules. The Cryo-TEM images at various areas of the carbon grid are depicted in
[0239] For encapsulation efficiency and active substance content determination, [.sup.3H] DHEA was incorporated within MCT NCs. The initial theoretical DHEA content for the cationic and anionic NCs, following diafiltration with PBS (pH 7.4), were 0.49 and 0.52%, while the observed contents were 0.18 and 0.15% respectively. The encapsulation efficiency was therefore 36.5 and 30.4% for the positively and negatively charged NCs, respectively (as shown in Table 5).
TABLE-US-00005 TABLE 5 DHEA content and loading efficiency within MCT NCs Theoretical Observed conc. conc. Yield Formulation (%, w/v) (%, w/v) (%) Positively charged [.sup.3H]DHEA 0.013 0.006 36.53 loaded MCT NCs Negatively charged [.sup.3H]DHEA 0.013 0.005 30.40 loaded MCT NCs
Skin Penetration of Fluorescent Labeled Nanospheres
[0240] To evaluate skin penetration of NPs, nanospheres comprising of PLGA were prepared, while a quantity of the polymer was covalently labeled with the infra-red dye NIR-783. Fluorescent formulations were topically administered on abdominal human skin of 60 years old male, using Franz cells (2.25 mg/cm.sup.2). After 3 h, skin specimens were washed and scanned using ODYSSEY Infra Red Imaging System (LI-COR Biosciences, NE, USA). Fluorescent images of various consecutive tape stripping following topical administration are presented in
Skin Penetration of Fluorescent Labeled Nanocapsules
[0241] To evaluate skin penetration of nanocapsules (NCs), as compared to nanospheres (NSs), formulations were incorporated with the fluorescent probe DiD. In order to define the bio-fate of PLGA nanocarrier, DiD fluorescent-probe-loaded-MCT NCs coated with PLGA covalently bound to rhodamine B were prepared. In the absence of MCT, NPs were formed. Non-degradable commercially available rhodamine B loaded Latex nanospheres were also investigated.
[0242] The fluorescent formulations were topically administered on abdominal human skin of 40 years old female, using Franz cells (4.5 mg/cm.sup.2). After 2 h, skin specimens were washed and scanned using Zeiss LSM710 confocal laser scanning microscope. Reconstructed fluorescent images of whole skin specimens are depicted in
[0243] The dually labeled nanocarriers formulations and their respective controls were applied for 2 hours on abdominal human skin of 50 years old female. Reconstructed fluorescent images of whole skin specimens are depicted in
[0244] Finally, poor rhodamine B intensity was recorded following 2 hours incubation of non-degradable rhodamine B latex NSs on abdominal human skin of 30 years female. This result suggests that non-degradable based carrier has a major limit to release its cargo when compared to degradable systems (
[.SUP.3.H]DHEA NCs Site Localization and Deep Skin Layer Localization
[0245] The results reported in
TABLE-US-00006 TABLE 6 [.sup.3H]DHEA distribution over time in the different SC layers of porcine skin following incubation with different nanocapsule formulations. Values are mean SD. N = 4 Incubation periods Stratum corneum layers (strips number) Formulation (hours) A (1-2) B (3-4) C (5-6) D (7-8) E (9-10) Positively 1 0.2% 0.0 0.2% 0.1 0.1% 0.0 0.1% 0.1 0.1% 0.1 charged 3 0.3% 0.2 0.2% 0.1 0.1% 0.1 0.1% 0.1 0.1% 0.1 [.sup.3H]DHEA 6 0.3% 0.1 0.1% 0.1 0.1% 0.1 0.1% 0.1 0.1% 0.1 loaded MCT NCs 8 0.7% 0.6 0.3% 0.2 0.2% 0.1 0.1% 0.1 0.1% 0.1 12 2.0% 1.8 0.8% 0.7 0.3% 0.2 0.3% 0.2 0.2% 0.1 24 1.9% 0.9 0.8% 0.1 0.6% 0.2 0.4% 0.1 0.3% 0.1 Negatively 1 1.3% 0.1 0.4% 0.1 0.2% 0.1 0.1% 0.1 0.1% 0.0 charged 3 0.3% 0.0 0.2% 0.0 0.1% 0.0 0.1% 0.0 0.1% 0.0 [.sup.3H]DHEA 6 0.2% 0.1 0.2% 0.0 0.1% 0.0 0.1% 0.0 0.1% 0.0 loaded MCT NCs 8 0.8% 0.8 0.3% 0.3 0.3% 0.1 0.2% 0.1 0.2% 0.1 12 1.9% 1.3 0.8% 0.3 0.4% 0.1 0.3% 0.2 0.3% 0.2 24 2.9% 1.8 1.4% 0.5 0.7% 0.3 0.5% 0.3 0.4% 0.2 Positively 1 1.5% 0.9 1.1% 1.1 0.4% 0.5 0.2% 0.1 0.2% 0.1 charged oil 3 3.4% 1.4 1.4% 0.7 0.5% 0.3 0.2% 0.1 0.2% 0.1 control 6 2.4% 0.8 0.8% 0.3 0.3% 0.1 0.3% 0.1 0.2% 0.1 8 1.5% 0.5 0.7% 0.3 0.3% 0.1 0.2% 0.1 0.1% 0.1 12 4.6% 1.7 1.4% 0.6 0.5% 0.2 0.3% 0.1 0.2% 0.1 24 2.7% 0.8 0.9% 0.3 0.5% 0.2 0.3% 0.2 0.2% 0.2 Negatively 1 2.2% 2.4 0.8% 0.7 0.2% 0.2 0.1% 0.0 0.1% 0.1 charged oil 3 1.7% 0.7 0.5% 0.2 0.2% 0.1 0.1% 0.1 0.1% 0.0 control 6 1.1% 0.3 0.3% 0.0 0.1% 0.1 0.1% 0.1 0.1% 0.0 8 1.3% 0.1 0.4% 0.1 0.2% 0.1 0.1% 0.1 0.1% 0.0 12 1.0% 0.5 0.2% 0.1 0.1% 0.0 0.1% 0.0 0.0% 0.0 24 2.0% 0.6 0.7% 0.2 0.3% 0.1 0.2% 0.1 0.1% 0.1
[0246] Increasing levels of the radioactive DHEA were found over time in the receptor compartment fluids when both positively and negatively DHEA loaded NCs were incubated, reaching 0.5%, 2.5% and 14% from the initial dose applied following 1 hour, 8 and 24 hours, respectively. On the other hand, the respective oil controls exhibited constant [.sup.3H]DHEA levels lower than 1% radioactivity at most time intervals. Although lag time of 3 hours was observed for the different formulations, [.sup.3H]DHEA appearance in the receptor fluids following positively and negatively NCs application was significantly higher than from the respective oil controls. The total amount of DHEA in the receptor fluids (g/cm.sup.2), released from the different treatments, is plotted against the square root of time (
[0247] The highly lipophilic radioactive compound, [.sup.3H]COE, was incorporated into PLGA NSs and MCT containing NCs, in an attempt to identify the fate of the empty nanocarrier when topically applied. Following diafiltration with PBS (pH=7.4) the encapsulation efficiency was 45% and 70% for the NSs and the NCs, respectively. Aqueous and oil controls of [.sup.3H]COE, without polymer, were prepared for the ex-vivo experiments. Again, over 90% from the initial amount of the tritiated COE were collected from the donor compartment following each incubation period, irrespective of the formulation type (data not shown). Table 7 exhibits [.sup.3H]COE dermatobiodistribution as a function of the SC layers following the different treatments, as was previously described for [.sup.3H]DHEA. Up to 8 hours incubation of [.sup.3H]COE loaded NSs and NCs, less than 1% from the applied dose were extracted from the upper skin layers. Interestingly, a notable increase in layers A and B of was observed following 12 hours incubation of the NSs and NCs, similar to the previous observation reported when DHEA NCs were applied. Although no notable differences, associate to the incubation periods, in the levels of [.sup.3H]COE were recorded when the different controls were topically applied, the constant distribution of the [.sup.3H]COE in MCT was higher in comparison to the [.sup.3H]COE surfactant solution (Table 7). Finally, less than 0.5% of radioactivity was counted in the viable compartments (epidermis, dermis and receptor fluids) during the incubation periods, when both nanocarriers formulations and their respective control were applied (
TABLE-US-00007 TABLE 7 [.sup.3H]COE distribution over time in the different SC layers of porcine skin following incubation with different nanocapsules formulations. Values are mean SD. N = 3 Incubation periods Stratum corneum layers (strips number) Formulation (hours) A (1-2) B (3-4) C (5-6) D (7-8) E (9-10) [.sup.3H]Cholesteryl 1 0.7% 0.8 0.2% 0.2 0.1% 0.1 0.1% 0.1 0.1% 0.1 oleyl ether 3 0.2% 0.1 0.2% 0.1 0.1% 0.1 0.1% 0.0 0.1% 0.0 loaded PLGA NSs 6 0.3% 0.2 0.2% 0.2 0.1% 0.1 0.1% 0.1 0.1% 0.1 8 0.9% 1.0 0.3% 0.4 0.1% 0.1 0.1% 0.1 0.1% 0.1 12 0.9% 1.3 0.4% 0.5 0.4% 0.5 0.2% 0.2 0.1% 0.2 24 3.6% 0.7 1.5% 0.8 0.9% 0.5 0.7% 0.4 0.5% 0.4 [.sup.3H]Cholesteryl 1 0.2% 0.2 0.1% 0.0 0.1% 0.0 0.0% 0.0 0.0% 0.0 oleyl ether 3 0.4% 0.5 0.1% 0.1 0.1% 0.0 0.0% 0.0 0.0% 0.0 loaded PLGA NCs 6 0.4% 0.6 0.1% 0.1 0.2% 0.3 0.1% 0.1 0.0% 0.0 8 0.6% 0.7 0.2% 0.2 0.1% 0.2 0.1% 0.1 0.1% 0.1 12 1.2% 0.8 0.4% 0.4 0.2% 0.1 0.2% 0.2 0.1% 0.1 24 2.4% 1.8 0.8% 0.6 0.4% 0.3 0.3% 0.2 0.2% 0.2 [.sup.3H]Cholesteryl 1 0.4% 0.8 0.2% 0.3 0.2% 0.3 0.1% 0.2 0.3% 0.6 oleyl ether 3 0.1% 0.1 0.1% 0.1 0.1% 0.0 0.0% 0.0 0.0% 0.0 surfactant 6 0.2% 0.1 0.1% 0.1 0.1% 0.0 0.1% 0.0 0.1% 0.0 solution 8 0.5% 0.5 0.3% 0.3 0.3% 0.3 0.1% 0.2 0.1% 0.1 12 0.5% 0.4 0.3% 0.2 0.2% 0.2 0.1% 0.1 0.1% 0.1 24 0.5% 0.1 0.3% 0.2 0.2% 0.1 0.2% 0.1 0.1% 0.1 [.sup.3H]Cholesteryl 1 1.8% 0.5 0.6% 0.4 0.3% 0.2 0.1% 0.1 0.1% 0.0 oleyl ether oil 3 1.0% 0.7 0.4% 0.3 0.1% 0.1 0.1% 0.0 0.0% 0.0 control 6 1.2% 0.3 0.4% 0.2 0.1% 0.0 0.1% 0.0 0.1% 0.0 8 1.7% 0.5 0.8% 0.5 0.3% 0.1 0.1% 0.1 0.1% 0.0 12 1.2% 0.5 0.7% 0.2 0.2% 0.1 0.1% 0.1 0.1% 0.1 24 1.6% 0.3 0.5% 0.3 0.3% 0.1 0.1% 0.1 0.1% 0.0
Thiol Surface Activated NPs and MAbs Conjugated NPs
[0248] Thiol surface activated NPs were prepared from the following polymers:
[0249] PLGA of a MW of approximately 48,000 Da,
[0250] PEG-PLGA.sub.50,000 and PLGA.sub.4500,
[0251] PEG-PLA.sub.100,000
Preparation of immunoNPs Conjugated to Various MAbs
[0252] The following MAbs were successfully conjugated to the surface of the thiolated NPs with high conjugation efficiency (see Table 8):
[0253] Cetuximab
[0254] Rituximab
[0255] Herceptin
[0256] Avastin
TABLE-US-00008 TABLE 8 Properties of INPs conjugated to relevant MAbs Zeta Size potential Conjugation Polymer MAb (nm) (mV) (%) PEG- Cetuximab 75 46 93 PLGA.sub.50,000/ PLGA.sub.48,000 PEG- Rituximab 73.75 N.A 86.7% PLGA.sub.50,000/ PLGA.sub.48,000
Morphological Evaluation Using TEM
[0257] The coupling of cetuximab MAb to INPs was qualitatively confirmed by TEM observations, using 12 nm gold labeled goat anti-human IgG (Jackson ImmunoResearch Laboratories, PA, USA). Each gold black spot observed in
Binding Capacity Determination In Vitro in A549 Cell Line by Flow Cytometry
[0258] For evaluation of the binding properties evaluation using flow cytometry, cells were detached using a 0.05% solution of EDTA. Cells were re-suspended in FACS medium (1% BSA, 0.02% Sodium Azide in PBS). 200,000 cells in 200 l were used for each treatment. Cells were centrifuged at 1200 rpm at 4 degrees. Then, cells were incubated with either native cetuximab antibody or equivalent concentrations of cetuximab immunonanoparticles over ice, for 1 h. 0.005 g/ml, 0.01 g/ml, 0.025 g/ml, 0.05 g/ml, 0.1 g/ml and 0.5 g/ml cetuximab antibody or INPs equivalents were used. The anti-CD-20 antibody, rituximab (Mabthera) was used as an isotype matched irrelevant nonbinding control. Cells were also incubated with equivalent concentrations of surface activated NPs and rituximab INPs as negative controls, to exclude non specific binding of INPs. Following 1 h incubation, cells were centrifuged and washed twice with FACS medium. Cells were then incubated for forty minutes at 4 C. in the dark with FITC-conjugated AffiniPure F(ab).sub.2 Fragment goat anti-human IgG (Jackson Immunoresearch). Cells were then centrifuged and washed twice with FACS medium. Cells were re-suspended in FACS medium and fluorescence was determined by flow cytometry. The results are depicted in
Skin Penetration of Immunonanoparticles
[0259] To evaluate the ability of nanoparticles to enhance the penetration of macromolecules into the skin, INPs covalently conjugated to cetuximab MAb were prepared. 6 mg/cm.sup.2 equivalent to 0.12 mg MAb/cm.sup.2 were topically administered to 44 years old female abdominal human skin, over 3 h, against relevant controls. Then, skin specimens were washed and immunostained with Cy5 labeled goat anti-human secondary IgG (Jackson ImmunoResearch Laboratories, PA, USA). Reconstructed fluorescent images were performed using an Olympus confocal Microscope (
[0260]
V. Nanoparticles Containing Cyclosporine
Materials
[0261] Cyclosporine A was purchased from TEVA (Opava, Komrov, Czech Republic); Polysorbate 80 (Tween 80), Ovalbimun (OVA), 1-Fluoro-2,4-dinitrobenzene (DNFB) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) from Sigma-Aldrich (Rehovot, Israel); [.sup.3H]-Cyclosporine A, Ultima-Gold liquid scintillation cocktail and Solvable were from Perkin-Elmer (Boston, Mass., USA). PLGA 100K (MW: 100000 Da) was from SurModics Pharmaceuticals (Birmingham, Ala., USA), Macrogol 15-hydroxystearate (Solutol HS 15) from BASF (Ludwigshafen, Germany) and oleic acid from Fisher Chemicals. Labrafil 1944 was provided by Gattefoss (Lyon, France). 0.05% clobetasol 17-propionate ointment (Dermovate, Glaxo operations, UK), 0.1% tacrolimus ointment (Protopic, Lot: 036160, Expiry: 07/2016, Astellas, The Netherlands) and 0.1% mometasone furoate (M.F.) ointment (Elocom, Lot: 4UHKA12001, Expiry: 01/2017, Schering-Plough Labo N.V. Belgium) were purchased from local pharmacy. Semi-solid silicone elastomer blend Dow Corning 9040, Dimethicone PMX-200 (Dow Corning 350) and Cyclohexasiloxane PMX-246 (Dow Corning 246) were from Dow Corning (Midland, Mich., USA). (2-Hydroxypropyl)--cyclodextrin (HPCD) was from Crabosynth (Compton, UK), Dimethyl sulfoxide (DMSO) was from Fluka (Steinheim, Switzerland), all organic solvents were HPLC grade and purchased from J.T. Baker (Deventer, Holland). All tissue culture media were from Biological Industries Ltd (Beit Ha Emek, Israel). ELISA kits for cytokines determination (mouse interleukin (IL)-2, -4, -5, interferon- (INF-), human IL-2, -6, -8), cytokines for T-cell activation (anti-mouse CD3, anti-human CD3, -28), anti-mouse antibodies (CD8 PerCP.Cy7, CD25 Pacific Blue and CD69 APC) used in FACS analysis and Carboxyfluorescein succinimidyl ester (CFSE) Cell Division Tracker Kit were from Biolegend Inc. (San Diego, Calif., USA). Anti-mouse IgG (H+L) was from Jackson ImmunoResearch (West Grove, Pa., USA), and anti-mouse IgE:HRP from BioRad (Hercules, Calif., USA).
Preparation and Characterization of Empty or Drug-Loaded NCs
Preparation of Empty or Drug-Loaded NCs
[0262] PLGA nanocarriers were prepared according to the well-established solvent displacement method. Briefly, the polymer poly(lactic-co-glycolic) acid (PLGA) 100K (50:50 blend of lactic:glycolic acid; Lactel), was dissolved in acetone containing 0.2% w/v Tween 80 and 0.8% w/v blend of different oils at different compositions, at a concentration of 0.6% w/v. CsA was added at various concentrations into the organic phase, that was added to the aqueous phase containing 0.1% w/v Solutol HS15, resulting in the formation of NCs. The suspension was stirred at 900 rpm for 15 min and then concentrated 5 (from the initial aqueous medium) by reduced pressure evaporation. The NCs dispersed in aqueous media were diluted with 10% HPCD solution, at a volume ratio of 1:1, prior to lyophilization in epsilon 2-6 LSC Pilot Freeze Dryer (Martin Christ, Germany). Finally, semi-solid anhydrous preparation consisting of semi-solid silicone elastomer blend, cyclohexasiloxane (and) cyclopentasiloxane, polydimethylsiloxane polymer and lyophilized CsA-loaded NCs was prepared at weight ratios 80:15:3:2 respectively.
Physicochemical Characterization of NCs
[0263] The mean diameter and the zeta potential of NCs were characterized using Malvern's Zetasizer (Nano series, Nanos-ZS, UK) at 25 C. and using water as diluent. Morphological evaluation was performed using transmission electron microscopy (TEM, Philips Technai F20 100 KV) following negative staining with phosphotungstic acid and by cryo-scanning electron microscopy (Cryo-SEM; Ultra 55 SEM, Zeiss, Germany). In the cryo-SEM method, the sample was sandwiched between two flat aluminum platelets with a 200 mesh TEM grid used as a spacer between them. The sample was then high-pressure frozen in a HPM010 high-pressure freezing machine (Bal-Tec, Liechtenstein). The frozen samples were mounted on a holder and transferred to a BAF 60 freeze fracture device (Bal-Tec) using a VCT 100 Vacuum Cryo Transfer device (Bal-Tec). After fracturing at a temperature of 120 C. samples were transferred to SEM using a VCT 100 and were observed using a secondary back-scattered and in-lens electrons detector at 1 kV at a temperature of 120 C. X-ray diffraction (XRD) measurements were performed on the D8 Advance diffractometer (Bruker AXS, Karlsruhe, Germany) with a secondary Graphite monochromator, 2 Sollers slits and 0.2 mm receiving slit. XRD patterns within the range 2 to 55 20 were recorded at room temperature using CuK radiation (=1.5418 ) with following measurement conditions: tube voltage of 40 kV, tube current of 40 mA, step-scan mode with a step size of 0.02 20 and counting time of 1 s/step. EVA 3.0 software (Bruker AXS) was using for all calculations. The equation for calculation of the degree of crystallinity is as follows: DC=100%.Math.Ac/(Ac+Aa) where DC is the degree of crystallinity, Ac and Aa are the crystalline and amorphous areas on the X-ray diffractogram.
Drug Loading
[0264] The loading efficiency of CsA in NCs was determined by HPLC. 10 l of aqueous dispersion of CsA-loaded NCs were injected into an HPLC system equipped with UV detector (Dionex ultimate 300, Thermo Fisher Scientific). Using a 5 m XTerra MS C8 column (3.9150 mm) (Waters corporation, Mildfold, Mass., USA) and a 60:40 v/v mixture of Acetonitrile:water as mobile phase, CsA was detected at the wavelength of 215 nm, with a retention time of 6.6 min. CsA content in the lyophilized powder was determined as described in equation (1).
[0265] Various nanoparticulate formulations were prepare, and their physical characteristics are summarized in Table 9.
TABLE-US-00009 TABLE 9 Composition and properties of the different nanocarrier formulations Zeta Oil CsA Mean potential Formulation (%, w/w) (%, w/w) diameter (nm) PDI (mV) CsA loaded NCs 36% 5% 153.8 1.8 0.152 40.6 Oleic acid CsA loaded NCs 13% 5% 162.0 0.8 0.06 36.0 Oleic acid:Labrafil (1:1) CsA loaded NCs 36% 5% 192.8 3.1 0.174 35.2 MCT CsA loaded NCs 36% 5% 122.1 2.7 0.166 35.6 Glyceryl tributirate CsA loaded NSs 0 5% 106.7 0.2 0.082 34.5
[0266] The mean diameter of the various nanocarriers varied from 100 to 200 nm with a relatively narrow distribution range as reflected by the low PDI values obtained. MCT-containing CsA NCs mean diameter was two-fold higher than that of the CsA NSs, while the rest of the oils had a lesser effect on the particle size distribution of NCs. The incorporation of the active agent CsA, with or without oil presence, did not alter the negatively-charged nature of the smooth and spherical PLGA-based NPs surfaces. High drug encapsulation efficiency (92-100% recovery) lead to the drug content of 5-7% (w/w). According to the X-ray diffraction (XRD) patterns shown in
[0267] NPs of PLGA are highly sensitive in aqueous medium and exhibit poor stability within water-based topical formulations. Therefore, they needed to be stored as freeze-dried powders following lyophilization and incorporated within a water-free topical formulation. The oleic:labrafil (Ol:La)-CsA-loaded NCs formulation were chosen for further investigation in view of the satisfactory results achieved following the lyophilization process. The NCs remained stable and intact following lyophilization as confirmed by freeze fracture cryo-SEM depictions. As shown in
Effect of Lyophilization
[0268] 100K PLGA (50:50 blend of lactic:glycolic acid, Lactel) NCs loaded with various amounts of CsA were prepared with different oils (i.e. oleic acid or castor oil), according to the solvent evaporation method described hereinabove. The NCs dispersed in aqueous media were diluted with a 10% HPCD aqueous solution (as cryoprotectant), at volume ratio of 1:1, prior to lyophilization in Epsilon 2-6 LSC Pilot Freeze Dryer (Martin Christ, Germany). The lyophilization process of 150 ml batches of dispersion is described in Table 10.
TABLE-US-00010 TABLE 10 Process parameters of lyophilization of the lab batch (total time: ~17 hr) Process phase Time (h:min) Temp. ( C.) Vacuum (mbar) 1 Loading 00:00 20 2 Freezing 01:00 35 3 Freezing 01:00 35 4 Sublimation 00:15 35 1.03 5 Sublimation 00:15 20 1.03 6 Sublimation 00:10 10 0.94 7 Sublimation 04:00 0 0.94 8 Sublimation 05:00 20 0.94 9 Second drying 05:00 20 0.001 Total time 16.40
Physicochemical Evaluation of the Anhydrous Semi-Solid Preparation
[0269] Mean diameter and zeta potential of the NCs were characterized using Malvern's Zetasizer (Nano ZSP) at 25 C. For the sample preparation, 200 mg of the anhydrous semi-solid preparation were dissolved in 2 mL HPLC water. The sample was vortexed and further centrifuged (4000 rpm, 10 min). Then, 1.2 mL of the supernatant was collected and centrifuged again (14000 rpm, 10 min). Finally, 1 mL of the obtained supernatant was collected and evaluated.
[0270] CsA content in the formulation was determined by HPLC. 200 mg of the anhydrous semi-solid preparation were dissolved in 2 mL DMSO in a 4 mL vial. The sample was shacked 30 min at 37 C. and then centrifuged (4000 rpm, 10 min). 1 mL of the supernatant was centrifuge (14000 rpm, 10 min). Finally, 10 L of the supernatant was diluted into 990 L Acetonitrile (factor dilution 200). The amount of CsA was quantified by HPLC.
[0271] The free CsA was evaluated using an extraction procedure. Approximately 500 mg of the anhydrous semi-solid preparation were weighted in a 4 mL vial and then 2.5 mL Tributyrin were added. The solutions was vortexed and further centrifuged (14000 rpm, 10 min). Then, 100 L of the supernatant was diluted in 1900 L Acetonitrile, then the solution was vortexed and centrifuged (14000 rpm, 10 min). Finally, 800 L of the supernatant was collected and evaluated by HPLC (factor dilution 50).
[0272] HPLC protocol for CsA: 10 l of samples were injected into an HPLC system consisting of a pump, autosampler, column oven and UV detector (Dionex ultimate 300, Thermo Fisher Scientific). With 5 m XTerra MS C8 column (3.9150 mm) (Waters corporation, Mildfold, Mass., USA), identification of CsA was obtained at the wavelength of 215 nm. The column was thermostated at 60 C. CsA determination was achieved using a mobile phase consisted of a mixture of Acetonitrile: water (60:40 v/v) which elicited a retention time of 6.6 min. CsA stock solution (200 g/mL) was prepared weighting 2 mg CsA in a 20 mL scintillation vial and adding 10 mL acetonitrile. The stock was vortexed and calibration curve was prepared at concentration ranging from 1 to 100 g/mL. The results for various formulations tested are provided in Tables 11-1 to 11-3.
TABLE-US-00011 TABLE 11-1 Composition and properties for 100K PLGA nanocapsules, before lyophilization with various oil core formulations % CsA Mean CsA content CsA observed Oil core (w/w) diameter (nm) PDI (%, w/w) (%) Oleic acid:Labrafil 5% 165.7 6.6 0.11 0.01 13.7 0.4 96.10 2.9 7% 169.8 7.1 0.10 0.01 18.6 3.7 98.3 19.2 8% 162.0 0.121 19.8 94.2 9% 172 2.0 0.1 0.02 22.1 0.7 95.4 2.9 Castor oil:Labrafil 5% 117.4 12.9 0.09 0.01 14.3 1.3 100.4 9.0 7% 120.9 16.3 0.1 0.01 18.2 1.3 96.2 7.0 8% 114.5 0.125 19.8 94.5 9% 118.9 5.8 0.09 0 20.8 1.3 90.3 5.8
TABLE-US-00012 TABLE 11-2 Composition and properties for 100K PLGA nanocapsules, after lyophilization and extraction from the cream Mean CsA CsA % CsA Yield diameter content observed Free CsA Oil core (w/w) (%) (nm) PDI (%, w/w) (%) (% w/w) Oleic acid: 5% 91.2 1.1 268.0 16.8 0.16 0.05 4.8 0.4 96 8 15.4 3.9 Labrafil 7% 90.4 1.1 265.3 18.1 0.20 0.02 6.8 0.3 97.1 4.3 16.4 2.3 8% 90.3 240.3 0.205 7.77 97.1 12.44 9% 91.0 0.8 272.5 21.5 0.18 0.01 8.5 1.2 94.4 13.3 18.4 6.0 Castor oil: 5% 89.4 0.7 200.2 5.8 0.12 0.01 .sup.5 0.1 100 2 7.7 0.9 Labrafil 7% 86.8 3.2 209.2 17.9 0.13 0.02 6.9 0.3 98.6 4.3 7.96 0.4 8% 87.8 201.9 0.159 7.8 97.6 6.33 9% .sup.89 1.1 199.9 7.8 0.12 0 7.8 0.4 86.7 4.4 9.2 0.4
TABLE-US-00013 TABLE 11-3 Summary of NCs formulation suspension and lyophilized powder following reconstitution, castor oil core NCs suspension NCs diameter (nm) 117.4 12.9 PDI (nm) 0.09 0.01 CsA content (% w/w) 14.3 1.3 CsA (%) from initial content 100.4 9.0 NCs Lyo NCs diameter (nm) 200.2 5.8 powder PDI (nm) 0.12 0.01 following CsA content (% w/w) 5 0.1 dispersion CsA (%) from initial content 100 2 reconstitution Water content (%) 3.1 0.9 Free CsA (%) 7.7 0.9 Yield (%) 89.4 0.7
[0273] Following lyophilization and reconstitution of the powder, the mean diameter of the NCs increased by about 100 nm, irrespective of the formulation composition. This was attributed to the presence of the Kleptose cryoprotectant which surround every NC and protects it from the lyophilization process stress. The PDI value is lower than 0.15-0.2, indicating homogeneity of the NC populations before and after lyophilization and reconstitution.
[0274] The total concentration of CsA in the formulation was maintained to be between 5-9% (w/w), depending on the formulation used, and the leakage of CsA from the NCs following lyophilization and reconstitution was 7.70.9%.
Anhydrous Topical Formulation
Preparation of Anhydrous Cream
[0275] An anhydrous semi-solid base consisting of 80% silicone-based Elastomer 10 (cyclopentasiloxane and dimethicone crosspolymer in DMF, Dow Corning), 16% ST-cyclomethicone 56-NF (cyclopentaxiloane and cyclohexasiloxane, Dow Corning), and 4% Q7-9120 Silicone 350 (polydimethylsiloxane) was prepared. Then, 2% lyophilized NCs was dispersed in the base (CysA-loaded PLGA NCs, 100 kDa (Poly-D,L-lactide-co-glycolide at 50:50 blend of LA:GA); CysA was solubilized in either oleic acid or castor oil). The mixture was stirred using head stirrer set to 1800 rpm. For large scale preparation up to 1 kg, IKA LR 1000 basic reactor was used (100 rpm, at temperature controlled conditions).
[0276] The NCs remained stable within the formulation, with a maximal leakage of cyclosporine of no more than about 7%.
Dermato-Biodistribution (DBD) of CsA NCs Using Fresh Pig Skin in an Ex Vivo Model
Porcine Tissue Treatment
[0277] About 750 m thick, trimmed, porcine ear skin was purchased from Lahav Animal Research Institute (Kibbutz Lahav, Israel). The skin was cleaned and the dermatomed skin was either treated or stored frozen at 20 C. for up to one month before use. Skin integrity was measured by transepidermal water loss (TEWL) using a VapoMeter device (Delfin technologies, Finland). Skin samples with TEWL values 15 g h.sup.1m.sup.2 were used in the experiments.
Ex Vivo DBD Experiments
[0278] The excised pig skin was placed on Franz diffusion cells with the acceptor compartment containing 10% ethanol in PBS (pH 7.4). Various doses of radioactivity, equivalent to 937.5 g of CsA, in NC formulations and respective controls were applied to the mounted skin. At different time intervals, the distribution of radioactively-labeled CsA was determined in several skin compartments. First, the remaining formulation on the skin surface was collected by serial washings, and combined with the first strip collected by D-SQUAME skin sampling discs (CuDERM corporation, Dallas, USA), made up the donor compartment. The subsequent 10 strips, consisting of 5 sequential tape stripping couples, were pooled as upper SC. Viable epidermis, containing also the lower SC, was heat-separated (1 min in PBS at 56 C.) from the dermis. Then, the various separated layers were chemically dissolved with Solvable. It is emphasized that the remaining skin residuals were also digested in Solvable and the residual radioactivity found was negligible. Aliquots of the receptor fluid were also collected. All the radioactive compounds were determined in Ultima-Gold scintillation liquid in a Tri-CARB 2900TR beta counter.
[0279] The results shown in
[0280] Notably, CsA scarcely penetrated to the viable epidermis layers when administered in respective oil controls at any time point. In contrast, when CsA was encapsulated within NCs, higher concentrations of CsA were observed at 6 and 24 hours following application. Between 300 and 500 ng CsA per mg tissue weight were recovered at each time point. Although a similar pattern was observed in the dermis compartment (
CsA NCs Anti-Inflammatory In Vitro Activity
[0281] The encapsulated CsA was biologically active, as shown in vitro on mouse splenocytes (
Ex-Vivo Activity in Human Skin Organ Culture
Human Skin Organ Culture
[0282] Human skin was obtained from patients undergoing elective cosmetic surgery (abdominoplasty), approved by the Hadassah University Hospital Ethics Committee. Full-thickness human skin organ culture was prepared as described elsewhere [23]. The skin was kept at 37 C. in an atmosphere containing 5% CO.sub.2 in serum-free DMEM containing 2 mM L-glutamine, 100 IU/mL penicillin and 100 g/mL streptomycin. Lipopolysaccharide (LPS) (10 g/ml) was added to the growth medium to induce inflammation and skin explants were treated topically for 48 h. Cytokines IL-8 and IL-6 secreted to the medium were determined by specific ELISA kits.
Ex Vivo Proliferation and Activation Assay
[0283] Activation of mouse T cells: Splenocytes in single cell suspension were isolated from BALB/c mice. Briefly, excised spleens were minced in ice-cold PBS with a 5-ml syringe plunger tip and cells were centrifuged at 1250 rpm at 4 C. for 7 min. Hypotonic red blood cells lysis was performed by RBC Lysis Solution (Biological Industries Ltd., Bet HaEmek, Israel) for 3 min. Lysis was neutralized by five volumes of PBS and centrifuged, cells were resuspended in PBS and passed through a 70-m cell strainer. Cells were then resuspended in serum-free RPMI (2010.sup.6/ml) and labeled for cell proliferation using a Carboxyfluorescein succinimidyl ester (CFSE) Cell Division Tracker Kit, according to slightly modified manufacturer's procedure (1 M CFSE, 5 min at room temperature). CFSE-labeled cells were seeded in anti-mouse CD3 antibody (1 g/ml)-coated 96-well plate (0.410.sup.6 cells/well). For the experiments, cells were incubated in RPMI supplemented with 10% heat-inactivated FCS with different treatments of 10 nM CsA formulations. After three days, cells were analyzed for cell proliferation (CFSE staining) and activation (expression of CD8, CD25 and CD69 antigens) by fluorescence-activated cell sorter analysis (FACS). Briefly, cells were labeled with a mixture of fluorescence-labeled anti-mouse CD8 (PE-Cy7), anti-mouse CD25 (Pacific Blue) and anti-mouse CD69 (APC) antibodies for 30 min on ice and analyzed on a BD LSRII flow cytometer (Becton Dickinson, Franklin Lakes, N.J.). Live cells were gated based on forward- and side-scatter properties and data analyzed using FCS Express V3 analysis software (De Novo, Glendale, Calif.). Secreted cytokine IL-2 was determined in cell culture media by a mouse IL-2 specific ELISA kit, according to manufacturer's instructions.
[0284] It is known that skin activation by LPS strongly correlates with IL-6 and IL-8 secretion in the growth medium; this was confirmed for the untreated specimens (
OVA-Induced AD Efficacy Studies
[0285] Strict animal care procedures set forth by The Authority for Biological and Biomedical Models of the Hebrew University of Jerusalem based on guidelines from the NIH guide for the Care and Use of Laboratory Animals were followed (approval No. MD-13-13918-4). Treatment effects were compared using one-way ANOVA followed by a post-ANOVA test at each time point using IBM SPSS Statistics Software version. Differences were considered significant if P<0.05.
[0286] Localized AD in mice was established by repeated epicutaneous (EC) sensitization with OVA. Briefly, 6-7 week-old BALB/c mice (Harlan Laboratories, Jerusalem, Israel) were anesthetized with isoflurane (Fluotec 3: anesthesia unit, Ohmeda (GE Healthcare)) and the back of the mice was shaved with an electronic razor followed by tape-stripping by adhesive tape to mimic a standardized skin injury. 100 g of either OVA in 100 L PBS or PBS alone was placed on 11 cm sterile gauze that was secured to the skin by Tegaderm transparent adhesive film (3M, MN, USA) (day 1). The patch was kept for a 1-week period and then removed. Two weeks later an identical EC sensitization was reapplied at the same skin site. Overall, mice received a total of three 1-week OVA exposures, separated from each other by 2-week intervals, totaling seven weeks. Mice were treated topically on days 38 and 41 (week 6), before the third sensitization period, by either 0.1% CsA topical preparations, 0.1% tacrolimus ointment (Protopic) or 0.1% mometasone furoate (M.F.) ointment (Elocom) (about 20 mg of topical preparation equivalent to 20 g/cm.sup.2 drug). At the end of the experiment (day 49), the mice were euthanized and the treated skin-site integrity was evaluated by TEWL measurements. Skin specimens from patched areas were fixed in 10% buffered formalin. For histological analysis, paraffin-embedded 4 m sections were stained with H&E. Dermal thickness was quantitatively assessed by ImageJ software. Blood was withdrawn and serum anti-OVA IgE titer was determined by ELISA. Splenocytes were isolated as a single cell suspension as described above in section 2.8, sensitized in vitro by OVA for 48 h and cytokines levels (IL-4, IL-5 and INF-) secreted in cell culture media were determined by ELISA.
[0287] Skin lesions and systemic AD markers were evaluated after the third sensitization week (week 7 from the beginning). Untreated AD mice had increased trans-epidermal water loss (TEWL) related to the level of skin disruption, increased skin thickness, elevated levels of serum anti-OVA-IgE, and high cytokines levels (INF-, IL-4, and IL-5) secreted in cell culture media from isolated splenocytes following OVA activation in vitro.
[0288] The six above-mentioned parameters were evaluated following topical application of OVA and various treatments. Skin of saline instead of OVA-treated mice exhibited a TEWL of 7.92.1 g/h/m.sup.2 whereas the TEWL following OVA EC sensitization (untreated group) was significantly elevated to 13.33.3 g/h/m.sup.2. 0.1% CsA NCs in topical preparation and 0.1% tacrolimus commercial product yielded a TEWL of 9.32.1 and 9.61.8 g/h/m.sup.2, respectively, suggesting an effective skin-barrier preserving activity (
[0289] Following EC sensitization, either with saline or OVA, the average dermal thickness increased by 100 and 120% respectively, compared to the naive tissue (
[0290] EC OVA sensitization significantly increased serum OVA-IgE titer of the untreated, blank NCs vehicle and M.F. mice groups compared to the saline-treated group, but not for the free CsA, CsA NCs and tacrolimus treatments applied (
[0291] To investigate allergen-induced production of major Th1 and Th2 cytokines, cells from mice spleen were harvested and stimulated with OVA in vitro. Cells from saline-treated mice did not respond to OVA stimulation. However, in vitro stimulation of splenocytes from OVA-sensitized mice elicited a strong production of IL-4 and IL-5 cytokines. In addition, a marked elevation in INF- secretion was observed upon in vitro OVA challenge of cells from both saline and OVA-treated mice. Significantly, a low amount of INF- was secreted when splenocytes from CsA NCs- and free CsA-treated mice only were challenged in vitro, but not in any other treatment (
Contact Hypersensitivity (CHS) Mice Model Efficacy Study
[0292] Strict animal care procedures set forth by The Authority for Biological and Biomedical Models of the Hebrew University of Jerusalem based on guidelines from the NIH guide for the Care and Use of Laboratory Animals were followed (approval No. MD-13-13918-4). Treatment effects were compared using one-way ANOVA followed by a post-ANOVA test at each time point using IBM SPSS Statistics Software version. Differences were considered significant if P<0.05.
[0293] Induction of CHS was performed as described by Wang et al. [24]. Four days before CHS sensitization the mice abdomens were carefully shaved and allowed to rest for recovery. On the day of sensitization, various topical CsA formulations were applied to the shaved skin (20 mg of CsA NC semisolid anhydrous preparation or CsA in carbohydrate ointment, both equivalent to 20 g/cm.sup.2 CsA). Four hours after topical treatments, to elicit CHS, mice were sensitized with 50 l 0.5% DNFB in acetone/olive oil (AOO) 4:1 on the shaved abdomen. They were challenged five days later with 25 l 0.25% DNFB in AOO on the back of the right ear only. The left ear was untreated and swelling responses were measured by micrometer (Mytutoyo, USA), recording the difference between left and right ears at 24, 48, 72, 96 and 168 h after challenge. The average swelling of 150 m was considered an allergic reaction.
[0294] The anti-inflammatory effect of the different CsA preparations, either encapsulated in NCs and dispersed in an anhydrous preparation or freely dissolved in a carbohydrate base was evaluated in a slightly modified CHS mice model. In this model, the topical treatments were applied 4 hours before the initial challenge with 0.5% DNFB as hapten, upon the shaved abdomen on undamaged skin of mice. Five days later, the mice were stimulated with 0.25% DNFB on the right ear lobe only and the degree of inflammation was quantified by the amount of ear swelling which developed. Upon re-exposure to the hapten in the elicitation phase, the untreated control group developed an intense skin inflammation on the right ear for about one week, manifested by a reaction peak two days post-elicitation followed by a gradual decrease over seven days (
[0295] In recent years CsA has been widely investigated in the design of topical formulations for the treatment of AD. Many of the researches concentrated on the enhanced dermal delivery through the ex vivo animal or human skin samples, whereas only a few studies reported on the potentially improved efficacy in various animal models. Some of the approaches for enhancing skin penetration of the poorly absorbed drug included the addition of penetrating enhancers or peptide penetrating moieties, whereas other researchers attempted to enhance the penetration of poorly absorbed drugs via nanocarriers such as liposomes, micelles, microemulsion and polymeric NPs.
[0296] To date, many topical formulations of CsA-loaded nanocarriers have not reached the dermatology market because of the limited stability of the nanocarriers in the topical formulation, and the subsequent leakage of the active ingredient from the nanocarriers towards the external phase of the topical formulation, resulting in significant damage to the transport efficiency of the active ingredient through the skin. The results provided herein present an original design of CsA NCs dispersed in a topical anhydrous formulation ensuring long term stability of CsA in the NCs and minimum leakage from the NCs towards the silicone-based formulation. CsA was successfully incorporated up to 14% (w/w) in a non-crystalline state, within a smooth and spherical oil core of PLGA-NCs, with high encapsulation efficiency (above 92%).
[0297] The topical delivery of CsA using PLGA NCs as drug carriers significantly enhanced its penetration into the viable skin layers where it is expected to be biologically active (as seen in
[0298] At 6- and 24-h post topical application of the NCs with oleic:labrafil oil core, the concentrations of CsA in the viable epidermis and dermis, were 215 and 260; 11 and 21 ng/mg, respectively. It is of note that no significant correlation could be established between the average blood concentrations of CsA and efficacy in psoriasis and AD treatment following oral administration. The lack of correlation was tentatively explained by the day to day variation of blood concentrations and a threshold limit which needs to be attained for efficacy. Farlanut et al. [25] reported that in human patients with psoriasis, a CsA concentration higher than 100 ng/ml, at a 12-h trough is associated with good clinical response. Apparently, the threshold effect is a plausible explanation for the lack of correlation. Indeed, CsA appeared to be concentrated in the skin at levels estimated to be near the peak values in blood and about 10-fold higher than the levels in trough blood samples of patients suffering from plaque-type psoriasis who responded to the treatment.
[0299] In AD, CsA target cells include Langerhans cells, mast cells eosinophils and keratinocytes located in the skin. Without wishing to be bound by theory, skin levels of 1000 ng/g equivalent to 1 ng/mg reported to be active for psoriasis are sufficient to inhibit the activation of these cells involved in the inflammatory process. As shown herein, the levels of CsA in the epidermis and dermis were well above such threshold levels since 24-h post application, 260 and 21 ng/mg were observed respectively. Furthermore, no detectable radioactivity permeation in the receptor fluids through the porcine ear skin could be measured over time, suggesting that very low, if any, radioactivity could traverse the whole skin barrier. Thus, marked systemic exposure of CsA following topical application is not likely to occur.
[0300] Moreover, it was found that the intrinsic immunosuppressive activity of CsA which blocks the T cell activation was not compromised following its encapsulation into the NCs, but in fact was improved as shown in vitro in primary mice cells (as shown in
[0301] The potential anti-inflammatory topical effect of CsA NCs ex vivo in human skin organ culture model was demonstrated since IL-6 and IL-8 concentrations in the medium diminished markedly following LPS activation, and were close to the activity of the marketed highly potent steroid, clobetasol propionate, following topical application of these formulations on the skin samples (
[0302] Without wishing to be bound by theory, the presence of the NCs may markedly increase the penetration of poorly-absorbed drug CsA in the viable epidermis and dermis following topical application due to accumulation of the drug-loaded NCs within hair follicles, triggering enhanced passage of the drug through the deep skin layers.
[0303] A delivery system in the form of an anhydrous topical preparation containing the CsA-loaded NCs was tested in vivo in different animal models. For this purpose, two well-known animal models were selected, OVA-induced AD and CHS elicited by DNFB, as described herein. These models allowed the assessment of the potential effect of CsA NCs in a topical application either in a well-established chronic AD induced by repetitive EC OVA challenges, or in the acute phase of CHS allergic response to DNFB.
[0304] It was observed that following EC exposure to OVA Balb/c, mice developed cutaneous inflammatory reactions, as measured by elevated TEWL, and allergen specific immune responses of serum OVA-specific IgE, TH.sub.2 cytokines, including IL-4 and IL-5, and the TH.sub.1 cytokine INF- (
[0305] CsA NCs preserved skin integrity, prevented skin thickening, reduced INF-, OVA-IgE serum levels, IL-4 levels but not IL-5 levels whereas tacrolimus improved only three of the six parameters (skin integrity, OVA-IgE serum levels and IL-4) and M.F. only improved two of the six parameters (IL-4 and IL-5 levels). It is interesting to note that free CsA in a topical formulation improved two of the six parameters (INF-, OVA-IgE serum levels) probably owing to the lack of skin integrity which allowed the standard topical formulation of CsA to reach sufficient therapeutic levels in the skin to be able to inhibit the immune reaction to OVA and reduce INF- levels.
[0306] It can be deduced that CsA NCs reached sufficient therapeutic CsA levels in the animal skins to be able to inhibit T-cell proliferation which is associated with pro-inflammatory cytokines secretion, including INF-. For the purpose of confirmation of these findings, a second CHS efficacy animal study was carried out as described herein, where the free CsA, blank NCs and CsA NCs respective topical formulations were applied on healthy intact skin allowing possible skin penetration discrimination between the formulations. Again, it was noted that at day two the CsA NC formulation was able to elicit a significant anti-inflammatory response reducing the ear swelling by 30% as assessed by ear thickness measurement compared to the different groups. CsA NCs treatment prior to hapten sensitization led to a significant suppression of skin inflammation. Moreover, rapid recovery of the inflammatory response was observed for the CsA NCs group (
[0307] The overall results presented herein show that topical application of CsA-loaded NCs dispersed in an anhydrous formulation can promote the penetration of the potent drug at therapeutic levels in the epidermis and dermis of mice, eliciting a therapeutic action resulting in a significant repression of immunological and inflammatory reactions.