TOPICAL DELIVERY SYSTEMS FOR ACTIVE COMPOUNDS

Abstract

Provided concerns viscous or gelled delivery systems based on oily nano-domains dispersed in a viscosified/gelled continuous aqueous phase, and suitable for prolonged and/or sustained topical delivery of various active compounds.

Claims

1. A topical formulation comprising an oily phase integrated into a gelled aqueous continuous phase, the oily phase being in the form of oily nano-domains dispersed in the continuous phase, wherein the oily phase comprises an active agent, at least one oil, at least two hydrophilic surfactants, at least two polar solvents, and at least two penetrating promotors, and wherein the gelled aqueous continuous phase comprises an aqueous diluent and at least one gellant.

2. The topical formulation of claim 1, wherein the oily phase further comprises at least one lipophilic co-surfactant, optionally wherein the lipophilic co-surfactant is a phospholipid.

3. A topical formulation of claim 1, wherein the oily domains have an average domain size of between 5 and 150 nm.

4. The formulation of claim 1, wherein the oily domains have an aspect ratio of between about 1.1 and 1.5.

5. The formulation of claim 1, wherein the oily domains have an elongated shape.

6. The formulation of claim 1, wherein the active agent may be selected from compounds having a main aromatic ring substituted by a secondary amino group.

7. The formulation of claim 1, wherein the active agent may be selected from diclofenac, lidocaine, clonidine, fentanyl, trebenifine, alprostadil, sulfamethoxazole, cephalexin, vancomycin, daptomycin, oritavancin, tazabactam, benzocaine, minocycline, doxycycline, or any pharmaceutically acceptable salt, derivative or analogue thereof.

8. The formulation of claim 1, wherein the active agent is selected from diclofenac, diclofenac sodium (DCF-Na), diclofenac potassium (DCF-K), DCF-ammonium, diclofenac diethylamine (DCF-DEA) and mixtures thereof.

9. The formulation of claim 8, wherein said active agent is present in the formulation at an amount of between about 1 wt % and about 6 wt %.

10.-13. (canceled)

14. The formulation of claim 1, wherein said oil is selected from isopropyl-myristate (IPM), ethyl oleate, methyl oleate, lauryl lactate, oleyl lactate, oleic acid, linoleic acid, monoglyceride oleate and monoglyceride linoleate, coco caprylocaprate, hexyl laurate, oleyl amine, oleyl alcohol, hexane, heptanes, nonane, decane, dodecane, short chain paraffinic compounds, terpenes, D-limonene, L-limonene, DL-limonene, olive oil, soybean oil, canola oil, cotton oil, palmolein, sunflower oil, corn oil, essential oils, such as peppermint oil, pine oil, tangerine oil, lemon oil, lime oil, orange oil, citrus oil, neem oil, lavender oil, anise oil, pomegranate seed oil, grapeseed oils, pumpkin oil, rose oil, clove oil, sage oil, eucalyptol oil, jasmine oil, oregano oil, capsaicin and similar essential oils, triglycerides (e.g. unsaturated and polyunsaturated tocopherols), medium-chain triglycerides (MCT), avocado oil, punicic (omega 5 fatty acids) and CLA fatty acids, omega 3-, 6-, 9-fatty acids and ethylesters of omega fatty acids and mixtures thereof.

15. The formulation of claim 14, wherein the oil is selected from isopropyl-myristate (IPM), oleic acid, oleyl alcohol, vegetable oils, terpenes, peppermint oil, eucalyptol oil, and mixtures thereof.

16.-17. (canceled)

18. The formulation of claim 1, wherein said two hydrophilic surfactants are selected from polyoxyethylene sorbitan monolaurate (polysorbate 20 or T20), polyoxyethylene sorbitan monopalmitate (T40), polyoxyethylene sorbitan monooleate (T80), polyoxyethylene sorbitan monostearate (T60) and polyoxyethylene esters of saturated (hydrogenated) and unsaturated castor oil, ethoxylated monoglycerol esters, hydroxystearate, ethoxylated fatty acids and ethoxylated fatty alcohols of short and medium and long chain fatty acids, sugar esters of saturated and unsaturated fatty acids, mono- and polyesters of sucrose, polyglycerol esters (3, 6, 8, 10 glycerols) of fatty acids, ethoxylated mono glycerides (8, 10, 12, 20, 40 EO) and ethoxylated diglycerides, ethoxylated fatty acids and ethoxylated fatty alcohols.

19. The formulation of claim 18, wherein said first and second hydrophilic surfactants are each being independently selected from polyoxyethylenes, ethoxylated (20EO) sorbitan monolaurate (T20), ethoxylated (20EO) sorbitan monostearate/palmitate (T60), ethoxylated (20EO) sorbitan mono oleate/linoleate (T80), ethoxylated (20EO) sorbitan trioleate (T85), castor oil ethoxylated (20EO to 60EO); hydrogenated castor oil ethoxylated (20 to 60EO), ethoxylated (5-40 EO) monoglyceride stearate/palmitate, polyoxyl 35 and 40 EOs castor oil, polyoxyl 35 castor oil, polysorbate 20 (Tween 20), polysorbate 40 (Tween 40), polysorbate 60 (Tween 60), polysorbate 80 (Tween 80), Mirj S40, Mirj S20, oleoyl macrogolglycerides, polyglyceryl-3 dioleate, ethoxylated hydroxyl stearic acid (Solutol HS15), sugar esters such as sucrose mono oleate, sucrose mono laurate, sucrose mono stearate, polyglycerol esters such as deca glycerol mono oleate or monolaurate, hexa glycerol monolaurate or mono oleate.

20. (canceled)

21. The formulation of claim 1, wherein said at least two polar solvents comprise at least a first solvent and a second solvent, and the first solvent being selected from short chain alcohols and/or the second solvents being selected from polyols.

22.-30. (canceled)

31. The formulation of claim 1, wherein said at least two penetrating promotors are independently selected from dimethyl sulfoxide (DMSO), dimethyl isosorbide (DMI), isopropyl myristate (IPM), 2-(2-ethoxyethoxy)ethanol (transcutol), phosphatidylcholine (PC), ethanol, isopropyl alcohol (IPA), ethyl acetate, oleyl alcohol, oleic acid, oleyl esters, beta-cyclodextrines, urea and its derivatives such as dimethyl or diphenyl urea, glycerol and propyleneglycol (PG), pyrrolidone and derivatives, peppermint oil, terpene and terpenoids (essential oils) oils, and combinations thereof.

32. (canceled)

33. The formulation of claim 1, wherein said gellant is present in the formulation in an amount of between about 0.75 and 3.5 wt %.

34. The formulation of claim 1, wherein said gellant is selected from cellulose ethers (e.g., hydroxyethyl cellulose, methyl cellulose, hydroxypropylmethyl cellulose), polyvinylalcohol, polyquaternium-10, guar gum, hydroxypropyl guar gum, xanthan gum, gellan, Aloe vera gel, amla, carrageenan, oat flour, starch (from corn, rice, or other plants), gelatin (porcine skin), ghatty gum, gum Arabic, inulin (from chicory), Konjac gum, locust bean gum, marshmallow root, pectin (high and low methoxy), quinoa extract, red alga, solagum, tragacanth gum (TG), Carbopol resins, and mixtures thereof.

35.-37. (canceled)

38. The formulation of claim 1, wherein said diluent is selected from water, purified water, distilled (DW), double distilled (DDW) and triple distilled water (TDW), deionized water, water for injection, saline, dextrose solution, or a buffer having a pH between 4 and 8.

39. The formulation of claim 1, wherein said aqueous diluent that is viscosified/gelled by the gellant is any suitable aqueous liquid.

40.-60. (canceled)

61. An active-loaded oily composition for preparation of a formulation according to claim 1, the active-loaded oily composition comprising at least one active agent, at least one oil, at least two hydrophilic surfactants, at least two polar solvents, and at least two penetrating promotors, and optionally at least one co-surfactant, said active-loaded oily composition being substantially devoid of water.

62.-79. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0176] In order to better understand the subject matter that is disclosed herein and to exemplify 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:

[0177] FIG. 1 shows LUMiFuge™ test results of commercial emulsion for 17 hr at 3000 rpm, showing that a commercial emulsion does not maintain transparency nor stability for long periods of time.

[0178] FIG. 2 shows the effect of changing the phospholipid component on the transparency of 2 wt % DCF-Na loaded gelled formulation.

[0179] FIG. 3 shows the effect of increasing the gellant content on the transparency of 2 wt % DCF-Na loaded gelled formulation.

[0180] FIG. 4 shows the effect of changing the perfuming agent on the transparency of 2 wt % DCF-Na loaded gelled formulation.

[0181] FIGS. 5A-5B show the effect of dilution on unloaded and 2 wt % DCF-Na loaded formulation as measured by electrical conductivity tests, respectively.

[0182] FIGS. 6A-6B show non-gelled formulation un-loaded and loaded with DCF-Na, respectively at different water dilutions, respectively.

[0183] FIG. 7 shows non-gelled formulation loaded with lidocaine at different water dilutions.

[0184] FIGS. 8A-8D are cryo-TEM micrographs of non-gelled formulation A of Table 2 (×650K magnification): 80 wt % water, unloaded with DCF-Na (FIG. 8A); 80 wt % water, 2 wt % DCF-Na (FIG. 8B); 90 wt % water, unloaded with DCF-Na (FIG. 8C); and 90 wt % water, 2 wt % DCF-Na (FIG. 8D).

[0185] FIG. 9 is a cryo-TEM micrograph of a gelled 2 wt % DCF-Na loaded formulation.

[0186] FIGS. 10A-10D are SAXS measurements of Formulation A in Table 2 at various storage temperatures and duration: freshly made (FIG. 10A); stored at 5° C. for 2 weeks (FIG. 10B); stored at 5° C. for 6 months (FIG. 10C); and stored at 25° C. for 6 months (FIG. 10D).

[0187] FIG. 11A shows the diffusion coefficients (Dx, m.sup.2/sec) of the main components for un-loaded and 2 wt % DCF-Na loaded formulation, 80 wt % water dilution, in non-gelled and gelled systems (0.75 wt % gellant). FIG. 11B shows the diffusion coefficients of the main components of a 2 wt % DCF-Na at various water dilutions.

[0188] FIG. 12 shows a comparison of visual appearance of a 2 wt % DCF-Na gelled formulation, 80 wt % water (named NDS 506(A)) (right) and Voltaren Emulgel® (left).

[0189] FIGS. 13A-13B show polarized light microscopic images of Voltaren Emulgel® (FIG. 13A) and NDS 506(A) (FIG. 13B), magnification ×10.

[0190] FIGS. 14A-14B show oily domains size distribution of gelled DCF-Na formulation, as measured by DLS (Dynamic Light Scattering) analysis; water concentration being 80 wt % (FIG. 14A) and 90 wt % (FIG. 14B), as measured without the addition of a gelling agent.

[0191] FIGS. 15A-15B show rheological behavior tests of stress r (Pa) as a function of shear rate y (l/s) of Voltaren Emulgel® (FIG. 15A) and NDS 506(A) (FIG. 15B).

[0192] FIG. 16 shows viscosity measurements at constant sheer rate at 50 Hz, against time (sec) for gelled aqueous phase (without an oily phase) and for gelled DCF-Na loaded formulations for various xanthan contents (0.75%, 0.85% and 1.0%).

[0193] FIG. 17A shows the dynamic complex viscosity of the flow u of the gelled aqueous phase (without an oily phase) against the shear rate (l/s) and gelled 2 wt % DCF-Na loaded formulation; FIG. 17B shows the viscosity of aqueous phase (without an oily phase), an un-loaded gelled formulation and 2 wt % DCF-Na loaded gelled formulation over time at a constant shear rate.

[0194] FIG. 18A shows the storage and loss moduli (G′, G″) for gelled aqueous phase (without an oily phase) and gelled 2 wt % DCF-Na loaded formulation; and FIG. 18B shows the storage and loss moduli (G′, G″) for un-loaded and 2 wt % DCF-Na loaded gelled formulations.

[0195] FIG. 19 shows complex viscosity measurements for NDS 506(A) formulation with various xanthan concentrations (ranging from 0.75 wt % to 2.85 wt %) compared to Voltaren Emulgel®.

[0196] FIGS. 20A-20D show spreadability test results for Voltaren Emulgel® Forte (FIGS. 20A-20B) and formulation NDS 506(A) (FIGS. 20C-20D).

[0197] FIG. 21 show ex vivo penetration and permeation after 24 hours (% Na-DCF form applied dose) Franz cell diffusion tests results carried out on pig skin samples, comparing between NDS 506(A) and Voltaren Emulgel® Forte.

[0198] FIG. 22 shows penetration profiles of DCF-Na concentration (m/cm.sup.2) for NDS 506(A), viscosified with 0.75 wt % or 2.85 wt % of xanthan gum.

[0199] FIGS. 23A-23B show LUMiFuge™ test results for NDS 506(A) (FIG. 23A) and typical commercial emulsion (FIG. 23B).

DETAILED DESCRIPTION OF EMBODIMENTS

Preparation of an Active-Loaded Gelled Formulation

[0200] Step 1: Preparation of Concentrate or Oily Phase

[0201] An excipient mixture was prepared by mixing phosphatidylcholine phospholipid (PC) (preheated to 45° C. until full melting), hydrogenated castor oil (40EO), Tween 60, propylene glycol (PG), isopropyl myristate (IPM), transcutol, dimethyl isosorbide (DMI), fragrance, ethanol (EtOH), and isopropyl alcohol (IPA). The mixture was thoroughly mixed at 300-600 RPM at 25° C. The mixture resulted in a clear, transparent yellowish liquid.

[0202] The active compound was added in powdered form to the mixture and mixed for 10-30 minutes to obtain full entrapment of the active agent.

[0203] Step 2: Preparation of Active-Loaded Gelled Formulation

[0204] The active-loaded oily composition may be diluted with any desired amount of water in order to obtain a desired concentration of the active. Typically, the concentrate is diluted by adding between 70 to 90 wt % of water.

[0205] In order to obtain the gelled formulation, xanthan gum was dissolved into purified water that was buffered to pH of 7.2-7.4 by gentle mixing to obtain homogeneity without lumps of xanthan gel.

[0206] The xanthan gel was added to the loaded oily composition under mixing conditions at room temperature, with gentle mixing until uniform, almost clear gel is formed. The formulation is placed under vacuum or centrifugation to remove any bubbles that may have been entrapped in the final product having spontaneously formed oily-phase domains having a size of <20 nm within the gelled aqueous phase.

[0207] In another sequence of preparation, Heco40 and Tween 60 are heated to 45° C. and allowed to fully melt. The temperature is lowered and PG, IPA, ethanol, IPM, transcutol, DMI, fragrance, and optionally antioxidant are added and mixed to obtain a clear solution. The PC is then added to the oily mixture, and optionally heated to 45° C. to allow full integration of the PC into the oily phase. The system is cooled to room temperature and then powdered Na-DCF is added stepwise into the oily phase to form a concentrate.

[0208] The gelled aqueous phase is prepared by dissolving the xanthan gum in purified buffered aqueous solution or purified water in which pH was adjusted to the desired pH. The concentrate is then added to the aqueous phase at room temperature, under mixing until uniform homogeneous almost clear gel is formed. The formulation is placed under vacuum or centrifugation to remove any bubbles that may have been entrapped in the final product.

[0209] The resulting system in a diluted gelled formulation with the spontaneously formed oily-phase domains having a size of <20 nm dispersed within the gelled aqueous phase.

[0210] The composition of the active-loaded gelled formulation is provided in Table 1.

TABLE-US-00001 TABLE 1 Diluted gelled active-loaded formulation Component Function Amount (wt %) Lecithin (PC) Phospholipid, 0.5 to 1.5 lipophilic co- surfactant Tween 60 First hydrophilic 3.0 to 5.0 surfactant Hydrogenated castor oil Second hydrophilic 0.6 to 1.5 (40EO) surfactant Propylene glycol (PG) Co-surfactant/solvent 2.0 to 6.0 Isopropyl myristate (IPM) Oil 1.0 to 4.0 Transcutol Solvents and/or 1.5 to 3.5 penetrating promoters Dimethyl isosorbide (DMI) 0.9 to 3.0 Peppermint oil Fragrance/Oil/ 0.2 to 0.6 Penetrating promoter Ethanol (EtOH) Polar solvent 1.5 to 2.5 Isopropyl alcohol (IPA) 1.5 to 2.5 Xanthan gum Viscosifier/gellant 0.75 to 3.0  Water — 60-90 Active agent API 1.0-5.0

Variance in Formulation

[0211] Table 2 shows some additional exemplary formulations according to this disclosure, including variations of the formulations that include, inter alia, antioxidants (for example BHT).

TABLE-US-00002 TABLE 2 Exemplary formulations (all amounts are given in wt % out of the formulation) Component A B C D E F G Lecithin (PC) 0.90 0.90 0.90 0.90 — — 0.90 Ethoxylated castor oil (HECO-40) 0.90 0.90 0.90 0.90 0.90 0.90 0.90 Propylene glycol (PG) 3.50 3.50 3.50 3.50 3.50 3.50 3.50 Tween 60 (Tw60) 4.50 4.50 4.50 4.50 5.40 4.50 4.48 Iso propyl mirystate (IPM) 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Dimethyl isosorbide (DMI) 1.60 1.60 1.60 1.60 1.60 1.60 1.60 Diethylene glycol 2.40 2.40 2.40 2.40 2.40 2.40 2.40 monoethyl ether (TC) Perfume 0.60 — 0.60 — 0.60 0.60 0.60 Ethanol (EtOH) 1.30 1.30 1.30 1.30 1.30 1.30 1.30 Isopropyl alcohol (IPA) 1.30 1.90 1.30 1.30 1.30 2.20 1.30 Diclofenac sodium (API) 2.00 2.00 2.00 2.00 2.00 2.00 2.00 Water 79.25 79.25 79.25 79.25 79.25 79.25 79.25 Xanthan gum 0.75 0.75 0.75 0.75 0.75 0.75 0.75 Butylated hydroxytoluene (BHT) — — — — — — 0.02 Component H I J K L M N Lecithin (PC) 0.90 0.90 0.90 0.90 0.90 0.90 0.90 Ethoxylated castor oil (HECO-40) 0.90 0.90 0.90 0.90 0.90 0.90 0.90 Propylene glycol (PG) 3.50 3.50 3.50 3.50 3.50 3.50 3.50 Tween 60 (Tw60) 11.63 4.50 4.50 4.50 4.90 4.50 4.50 Iso propyl mirystate (IPM) 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Dimethyl isosorbide (DMI) 1.60 1.60 1.60 1.60 1.60 1.60 1.60 Diethylene glycol 2.40 2.40 2.40 2.40 2.40 2.40 2.40 monoethyl ether (TC) Perfume 0.60 0.60 0.60 0.60 0.20 0.60 0.60 Ethanol (EtOH) 1.30 1.30 1.30 — 1.30 1.30 1.30 Isopropyl alcohol (IPA) 8.43 15.55 15.55 2.60 1.30 1.30 1.30 Diclofenac sodium (API) 2.00 2.00 2.00 2.00 2.00 2.00 2.00 Water 65.00 65.00 65.00 79.25 79.25 79.00 78.50 Xanthan gum 0.75 0.75 0.75 0.75 0.75 1.00 1.50 Butylated hydroxytoluene (BHT) — — — — — — — Component O P Q R S Lecithin (PC) 0.90 0.90 0.90 1.35 0.80 Ethoxylated castor oil (HECO-40) 0.90 0.90 0.90 1.35 0.80 Propylene glycol (PG) 3.50 3.50 3.50 5.25 3.40 Tween 60 (Tw60) 4.50 4.50 3.50 6.25 4.40 Iso propyl mirystate (IPM) 1.00 1.00 1.00 1.50 0.90 Dimethyl isosorbide (DMI) 1.60 1.60 1.60 2.40 1.50 Diethylene glycol 2.40 2.40 2.40 3.60 2.30 monoethyl ether (TC) Perfume — — — — — Peppermint oil 0.60 0.60 0.60 0.90 0.50 Ethanol (EtOH) 1.30 1.30 1.30 1.95 1.20 Isopropyl alcohol (IPA) 1.30 1.30 1.30 1.95 1.20 Diclofenac sodium (API) 2.00 2.00 3.00 3.00 3.00 Water 78.75 77.15 77.15 69.75 79.25 Xanthan gum 2.00 2.85 2.85 0.75 0.75 Butylated hydroxytoluene — — — — — (BHT)

[0212] All the formulations in Table 2 were obtained by mixing the ingredients according to the processes described herein. The resulting formulations were clear and transparent, without any evidence of phase separation or droplets coalescence.

[0213] Incorporation of various perfuming agents, antioxidants and/or pH adjusting agents (buffers) did not change the nanostructure of the formulation.

[0214] Variance in Type of Phospholipid

[0215] The influence of changing the phospholipid components on the formulations of the invention was tested for Formulation A of Table 2. Various sources of phosphatidyl choline (PC) from various lecithin derivatives and PC levels ranging from 70% to 94% were tested: [0216] Lipoid-S75 (70% PC), Lipoid-S100 (94% PC), Phospholipon 90G (94% PC), Epicorn 200 (94% PC) are soy-based; [0217] Lipoid-P100 GMO-free, 90% PC from soybean; [0218] Lipoid-H100 GMO-free, 90% PC from sunflower seed; and [0219] Lipoid-R100 GMO-free, 90% PC from rapeseed.

[0220] As seen in FIG. 2, all of the phospholipid tested resulted in clear and transparent formulations, without evidence of phase separation or droplets coalescence.

[0221] Variance in Type and Amount of Gellant

[0222] The influence of changing the type of gellant on the formulations of the invention was tested for Formulation A of Table 2. Various types of xanthans were tested, at 2 concentrations: 1 wt % and 0.75 wt % out of the formulation. Table 3 presents characterization of the gelled formulations tested with three different xanthans (Xantural® 75, 180 and 11K, all provided by PC Kelco).

TABLE-US-00003 TABLE 3 Characterization of Formulation A gelled with different gellants Viscosity (mPas) Micros- Turbidity 0.75 1 # Xanthan type Appearance copy (NTU) pH wt % wt % LUMiFuge* 1 Xantural 75 Transparent Clear 45 7.25 109.6 165 Good 2 Xantural 180 Transparent Clear 45 7.16 119.2 172.1 Good 3 Xantural 11K Transparent Clear 25 7.12 116.1 169.1 Good *see explanation about the LUMiFuge ™ test further below.

[0223] As can be seen, the formulations maintain their properties when varying the type of xanthan used as a gellant.

[0224] The influence of the amount of gellant was also assessed. Based on Formulation A in Table 2, the amount of xanthan (Xantural 11K) was varied between 0.75 wt % and 2.85 wt %. The pH, turbidity and long-term stability were measured for these formulations as shown in Table 4 (and FIG. 3).

TABLE-US-00004 TABLE 4 Characterization of formulation A with varying amount of xanthan Xanthan (wt %) 0.75 1.5 2.0 2.5 2.85 pH 6.85 6.85 6.78 6.73 6.77 Turbidity 20 24 33 37 75 (NTU) LUMiFuge Good Good Good Good Good

[0225] Although increasing the amount of xanthan, all formulations remained transparent, without any significant change in pH or turbidity. No changes in transparency of the formulations was detected in LUMiFuge™ tests, indicating that increasing the amount of xanthan does not damage the long-term stability of the formulation.

[0226] Variance in Perfuming Agent

[0227] As perfuming agents are typically oil-based and oil-soluble, the effect of the presence or absence of perfume on the nano-structure and the stability of the formulation was testes, as well as the effect of variance in the type of perfume. Table 5 details the compositions of the tested formulations, all based on Formulation A in Table 2, from which 0.6 wt % is a varying perfume.

TABLE-US-00005 TABLE 5 Formulation A with various perfuming agents Composition DLS** Formu- Completing Size Volume Turbidity lation Perfume component* (nm) (%) PDI (NTU) *** A 0.6 wt % Perfume 1 — 6.395 100 0.197 30 AB — 0.6 wt % PG 7.649 100 0.567 40 AC — 0.6 wt % water 6.930 100 0.554 32 AD 0.2 wt % Perfume 1 0.4 wt % PG 6.993 100 0.295 50 AE 0.1 wt % Perfume 2 0.5 wt % IPA 6.545 100 0.312 60 *Formulation A contained 0.6 wt % of perfume 1; the completing component refers to the component added to the formulation when reducing or eliminating the perfume. **Tested by DLS Zeta sizer by Malvern, Model ZEN1600; due to the nature of the test, DLS measurements were carrying out on non-gelled formulations. *** Tested by Turbidity HANNA Instrument, model HI183414 (230VAC/50 Hz/10VA- Fuse 400 mA).

[0228] As evident by Table 5 and FIG. 4, replacing the perfume agent and/or eliminating the perfume agent from the formulation does not affect its transparency. Optical microscopy and DLS measurements revealed that no change in nanostructure was visible: the samples remain clear (transparent), without any visible change in turbidity. In all samples, the nanodomain size measured with non-gelled system was maintained below 10 nm (monodispersed), without any evidence of phase separation or coalescence of the nanodomains, indicating good compatibility of the nanodomains and different fragrances. This suggest that the perfumes, which are oil-soluble, are solubilized in the core of the droplets and well integrated into the interphase. Stability and transparency was gained with the gelled systems as well.

[0229] This is also supported by the SD-NMR measurements carried out for the examples that are shown in Table 6. No significant changes in diffusion coefficients were measured, meaning that the active agent (DCF-Na) is maintained at the interphase although replacing or eliminating the oil-soluble perfume agent.

TABLE-US-00006 TABLE 6 SD-NMR* results for formulations with different perfumes Diffusion coefficient × 10.sup.−9 (m.sup.2s.sup.−1) Component A AB AC AD AE Surfactants 0.01 0.01 0.01 0.01 0.01 Co-surfactant 0.50 0.56 0.59 0.55 0.59 Water 1.50 1.55 1.52 1.48 1.48 DCF-Na 0.1 0.1 0.1 0.1 0.1 *see detailed explanation about the SD-NMR measurement technique further below.

[0230] As an indicator to the long term stability of the formulations, LUMiFuge™ measurements were carried out for 17 hr at 3000 rpm, and full transparency of the samples was maintained over the entire duration of the test. These conditions are comparable to 3 years of storage, indicating that changing the perfume agent or eliminating it from the formulation is will not influence the long term stability of the formulations.

[0231] Diclofenac Sodium (DCF-Na) Loaded Gelled Formulation

[0232] Effect of Dilution on the Oily Phase Structure

[0233] 2 wt % DCF-Na loaded oily composition which is substantially devoid of water (i.e. a concentrate) was prepared according to the process described above. The un-diluted oily composition was constituted by self-assembled oil-solvated clusters or short domains of surfactants, which differ from the classical reverse micelles. These concentrates are dilutable by any suitable diluent, for example by purified water, to form a diluted delivery system.

[0234] The effect of water dilution on the oily domains structure was investigated by using electrical conductivity tests. Electrical conductivity measurements were performed at 25±2° C. using a conductivity meter, type CDM 730 (Mettler Toledo GmbH, Greifensee, Switzerland). Measurements were made on empty and DCF-Na loaded samples upon dilution with water up to 90 wt %. No electrolytes were added to the samples. The conductivity allowed the identification of the continuous phase and the inner phase. The results are shown in FIGS. 5A-5B.

[0235] The oily domains undergo phase transitions upon increasing the amount of diluent (e.g. water). When in the concentrated form, the oily composition is in the form of oil solved clusters (short surfactant domains), such that DCF-Na resides within the oil domains. When mixed with increasing amounts of water, hydrated domains are formed; upon further dilution with water, structure progressively and continuously transforms into oily domains dispersed in water, such that the DCF-Na molecules are located and entrapped by the tails of the surfactants at the interface of the oily domains with the water phase. It is of note that the absolute values of the conductivity of the empty system are significantly lower than those of the loaded system due to the ionic nature of DCF-Na.

[0236] It was noted that the oily carrier, i.e. the oily composition without DCF-Na, could not be fully diluted. Only upon addition of the DCF-Na, stable oily domains were obtained, as seen in FIGS. 6A and 6B. In FIG. 6A, un-loaded oily phase was diluted to various water concentrations; as can be seen, above 50 wt % water, the system phase separates. When the oily phase was loaded with 2 wt % of DCF-Na, the system was fully dilutable up to 90 wt %, resulting in a clear and transparent formulation, as seen in FIG. 6B.

[0237] As seen in FIG. 7, similar results were obtained when the oily phase was loaded with lidocaine, a structure builder similar in function to that of Na-DCF.

[0238] This attests to the function of the active agent in stabilizing the oily domains interface; the active agent functions as a structurant, contributing and facilitating the final structure of the oily domains. This behavior differs from classic carrier systems, in which the active agent is merely loaded into the formulation, without taking part of the actual structure of the system. Thus, throughout the phase transformations occurring upon dilution, DCF-Na stabilized the structure of the delivery system and is entrapped within the interface, (as will be further explained below in connection with SD-NMR analysis).

[0239] Additional formulations with various dilution levels are shown in Tables 7-1 and 7-2.

TABLE-US-00007 TABLE 7-1 Formulations with various water-dilution levels (between 1 and 4 wt % DCF) Dilution factor 1.00 10.00 5.00 4.00 3.33 2.86 2.50 Lecithin (PC) 4.5 0.45 0.9 1.13 1.35 1.58 1.8 Ethoxylated castor oil (HECO-40) 4.5 0.45 0.9 1.13 1.35 1.58 1.8 Propylene glycol (PG) 22.5 2.25 4.5 5.63 6.75 7.88 9.0 Tween 60 (Tw60) 17.5 1.75 3.5 4.38 5.25 6.13 7.0 Iso propyl mirystate (IPM) 5 0.5 1.0 1.25 1.5 1.75 2 Dimethyl isosorbide (DMI) 1.60 0.8 1.6 2 2.4 2.8 3.2 Diethylene glycol 12 1.2 2.4 3. 3.6 4.2 4.8 monoethyl ether (TC) Perfume 0.5 0.05 0.1 0.13 0.15 0.18 0.2 Ethanol (EtOH) 6.5 0.65 1.3 1.63 1.95 2.28 2.6 Isopropyl alcohol (IPA) 9 0.9 1.8 2.25 2.7 3.15 3.6 Diclofenac sodium (API) 10 1 2 2.5 3 3.5 4 Water 0 87.15 77.15 72.15 67.15 62.15 57.15 Xanthan gum 0 2.85 2.85 2.85 2.85 2.85 2.85

TABLE-US-00008 TABLE 7-2 Formulations with various water-dilution levels (for 2 wt % DCF) Lecithin 4.5 0.45 0.9 1.13 1.35 1.58 1.8 1.8 1.8 HECO-40 4.5 0.45 0.9 1.13 1.35 2.58 2.8 1.8 3.8 PG 22.5 2.5 5 6.25 7.5 8.25 10 11 5 Tw60 17.5 1.75 3.5 4.38 3.4 6.13 7 7 8 IPM 5 0.5 1.0 1.25 1.5 1.75 2 2 2 DMI 1.60 0.8 1.6 2 2.4 2.8 3.2 3.2 3.2 TC 12 1.2 2.4 3 3.6 4.2 6.8 4.8 4.8 Perfume 0.5 0.05 0.1 0.13 0.15 0.18 0.2 0.2 0.2 Ethanol 6.5 0.65 1.3 1.63 1.95 2.28 2.75 2.6 2.6 IPA 9 0.65 1.3 2.13 1.95 2.43 2.6 3.6 3.6 DCF-Na 10 2 2 2 2 2 2 2 2 Water 0 86.15 77.15 72.15 70 63 56 57.15 60.15 Xanthan 0 2.85 2.85 2.85 2.85 2.85 2.85 2.85 2.85

[0240] Structure of Nanodomains

[0241] Photomicrographs of diluted formulations (×650K magnification, FIGS. 8A-8D) indicate that the domains are almost mono dispersed in size. The domains are not necessarily spherical and consist of an oily core and an interface comprising surfactants and co-surfactants. The domains are dispersed in aqueous continuous phase. While the empty droplets (FIGS. 8A and 8C) are more spherical, the loaded systems (FIGS. 8B and 8D) have droplets with substantially elongated shape with an aspect ratio of 1.1 to 1.5. Upon further dilution (i.e. increasing the dilution from 80 wt % water to 90 wt % water) the droplets become less packed and smaller in number per volume.

[0242] As seen in FIG. 9, although the formulation is gelled, the nanodomains remain structured, meaning that the solubilization capacity, stability and release profiles are not affected by the formation of a viscoelastic network in the aqueous phase. In other words, the gelling process of the aqueous phase does not affect the structure and stability of the nanodomains.

[0243] Small-Angle X-ray Scattering (SAXS) measurements suggest that the domains are well structured with almost constant size and distance between droplets (lattice parameters), which do not change over time or temperature (FIGS. 10A-10D). All samples measured have shown similar domains sizes, ranging from 7.1 nm to 8.6 nm with a distance of ca. 1.6 nm between droplets.

[0244] When comparing the unloaded system with the DCF-loaded system, it seems that the presence of DCF-Na allows to obtain smaller oily domains; namely, when DCF-Na was loaded into the system, smaller and more uniform domains were spontaneously obtained (16 nm vs. 6-10 nm for un-loaded and loaded oily phases, respectively). This also attests to the function of the DCF-Na as a structurant (functioning as a cosmotropic agent), as shown in Table 8.

TABLE-US-00009 TABLE 8 Oily domains average size values Domain size (nm) Water content (wt %) Unloaded DCF-Na loaded 80 16.3 (±1.3) 5.8 (±0.6) 90 15.9 (±0.4) 6.7 (±0.3)

[0245] Upon adding the gellant to the formulation, the structure is further modified and larger oily domains are formed. These domains are not spherical and their average size increased (via estimated measurements) to about 10-15 nm, as detailed in Table 9.

TABLE-US-00010 TABLE 9 Oily domains average size values in DCF-Na loaded gelled formulations Domain size (nm) Water content (wt %) Non-gelled Gelled 80 5.8 (±0.6) 14.5 (±1.2) 90 6.7 (±0.3) 10.1 (±1.6)

[0246] Thus, it is suggested that the gellant itself also has an influence on the structure of the delivery system, as once the gellant is added, the domains slightly grow in size and transform to an elongated shape, rather than assembling into globular droplets.

[0247] In order to characterize the structure of the oily domains, self-diffusion NMR (SD-NMR) analysis was carried out. SD-NMR analysis provides an indication on the location of each component within the structure, by calculating the diffusion coefficient of each component in the system. Rapid diffusion (>100×10.sup.−11 m.sup.2s.sup.−1) is characteristic of small or free molecules in solution, while slow diffusion coefficients (<0.1×10.sup.−11 m.sup.2s.sup.−1) suggest low mobility of macromolecules or bound/aggregated molecules.

[0248] SD-NMR measurements were performed with a Bruker AVII 500 spectrometer equipped with GREAT 1/10 gradients, a 5 mm BBO and a 5 mm BBI probe, both with a z-gradient coil and with a maximum gradient strength of 0.509 and 0.544 T m.sup.−1, respectively. Diffusion was measured using an asymmetric bipolar longitudinal eddy-current delay (bpLED) experiment, or and asymmetric bipolar stimulated echo (known as one-shot) experiment with convection compensation and an asymmetry factor of 20%, ramping the strongest gradient from 2% to 95% of maximum strength in 32 steps. The spectrum was processed with the Bruker TOPSPIN software. NMR spectra were recorded at 25±0.2° C. The components were identified by their chemical shift in 1H NMR.

[0249] FIG. 11A shows the diffusion coefficients (Dx, m.sup.2/sec) of the main components for 2 wt % DCF-Na un-loaded and loaded formulation, at 80 wt % water, in non-viscosified (non-gelled) and viscosified (gelled) systems. FIG. 11B shows the effect of dilution on the diffusion coefficient of the loaded and gelled formulation.

[0250] As can be seen from FIGS. 11A-11B, the diffusion coefficient of DCF-Na is similar to that of the hydrophilic surfactants compared to the other components in the system. The Na-DCF diffuses slightly faster than the tails of the surfactants indicating that the Na-DCF is located at the interface and not within the oil core of the oily domains (as the formulation is very poor in oil). Further, the results indicate that the polar solvents are mostly located in the layer, far from the surfactants' heads, however still interact with the heads and are not entirely free (for surfactant tails Dx=0.02×10.sup.−11 and for DCF-Na Dx=0.1×10.sup.−11).

[0251] This suggests that binding occurs between DCF-Na and the surfactants' heads, suggesting that the DCF-Na molecules are interlocked by the surfactant's tails at the interface of the oily domains, and the DCF-Na molecules may also function as a co-surfactant.

[0252] It is also noted that the diffusion coefficient of DCF-Na is lower in the gelled formulation than in the non-viscosified system. Such reduction also contributes to the increased stability of DCF-Na in the viscosified/gelled system and provides for better control over the release of DCF-Na from the oily domains once applied onto the skin.

[0253] From the SD-NMR results, the so-called “obstruction factor (OF)” can be calculated. This factor is derived from the diffusivity of each component in the structure at each certain dilution point normalized to diffusion coefficient of the component itself in a liquid form or in a reference solution [OF=D/D.sub.0]. The obstruction factor is suggestive of the resistance of the components to be released from the structure at a given solubilizate concentration of DCF-Na (2 wt %). It can be seen that due to their close behavior and diffusion coefficients correlation, the components that are hindering the release of Na-DCF from the interface are the set of the surfactants. Low OF values of 0.1 to 0.2 are indicating of significant binding effects of the DCF-Na to the surfactants and, hence, slower release and the formation of a depot effect. The solvents and the water are not obstructing the drug molecule (OF values of 0.5 and 0.6).

Gelled DCF-Na Formulation Compared to Commercial Product

[0254] Gelled DCF-Na formulations were prepared as described above. Their various properties were compared to Voltaren Emulgel® Forte, which is currently the leading commercial product for topical delivery of diclofenac. Voltaren Emulgel® Forte contains 2.32 wt % diclofenac diethylamine (DCF-DEA, which is comparable to 2 wt % DCF-Na) in a gelled emulsion formulation that primarily comprises inactive ingredients (excipients) such as butylhydroxytoluene, carbomers, cocoyl caprylocaprate, diethylamine, isopropyl alcohol, liquid paraffin, macrogol cetostearyl ether, oleyl alcohol, propylene glycol, and purified water.

[0255] Visual Appearance

[0256] The physical properties of 2 wt % gelled DCF-Na formulation, at 80 wt % water dilution (named for ease of reference NDS 506(A)) in comparison to Voltaren Emulgel® Forte, are provided in Table 10.

TABLE-US-00011 TABLE 10 Comparison of physical properties Parameter NDS 506(A) Voltaren Emulgel ® Transparency Transparent Opaque Color Clear to White opaque slightly yellow Texture Gel Gel Microscopy .sup.a Uniform Uniform Turbidity (NTU) .sup.b  80-100 1900-2500 pH .sup.c 7.1-7.5 7.9 Droplet size (nm) .sup.d 6.2 N/A Poly Dispersion 0.4 N/A Index (PDI) .sup.d .sup.a Microscopy analysis: Nikon Eclipse 80i, magnification ×10, polarized light .sup.b Turbidity evaluation: HI 83414 Turbidity and free/Total Chlorine Meter by HANNA instruments (using calibration curve samples). All samples were diluted ×11 with distilled water, shaking at 300 RPM for 1 hour at room temperature .sup.c pH measurements: SevenEasy Metller Toledo .sup.d Drop size examination: Zeta sizer, nano sizer (nano-s), MALVERN instrument

[0257] The differences in appearance between NDS 506(A) and Voltaren Emulgel® Forte are shown in FIG. 12, while microscopic images are provided in FIGS. 13A-13B.

[0258] Commercial products which are based on emulsions, such as Voltaren Emulgel® or Voltaren Emulgel® Forte, are typically a dispersion of two immiscible liquids, formed in the presence of emulsifiers/surfactants, which reduce the interfacial tension between the two phases and cover the dispersed droplets to retard aggregation, flocculation, coalescence and phase separation. Since the emulsifiers do not reduce the interfacial tension to zero and the coverage is not complete, emulsions require application of relatively high shear forces of multistage homogenizer to reduce the droplets size upon preparation of the emulsion. The resulting non-uniform droplets have a strong tendency to coalesce and/or result in phase separation, thereby stabilizing the system energetically. Thus, commercial product show a relatively non-uniform dispersity of the droplets together with large droplet size, far from being homogenous, resulting in a milky, white-opaque appearance.

[0259] In comparison, the NDS 506(A) formulation are spontaneously formed as energetically balanced systems due to their substantially zero interfacial tension. Such formulations are characterized by a small and uniform oily domains size, as seen in FIGS. 14A-14B, resulting in transparent and stable systems.

[0260] Viscosity and Rheology

[0261] Rheological properties of Voltaren Emulgel® and NDS 506(A) was measured by ThermoHaake (Thermo Electron GmbH, Karlsruhe, Germany) using a cone (60 mm diameter) and glass plate, at 25±1° C., shear rates were 0-100 s.sup.−1, as shown in FIGS. 15A-15B, respectively.

[0262] As evident from the viscosity measurements, the viscosity of Voltaren Emulgel® Forte is significantly higher compared to that of NDS 506(A). As explained above, Voltaren Emulgel® Forte is a thermodynamically unstable emulsion, and hence requires relatively strong gelation and high viscosities in order to stabilize the emulsion. Further, such high viscosities often lower the absorbance of the formulation into the skin after application, and may also reduce the penetration and release of diclofenac into the skin and relevant tissues.

[0263] The viscosity of the gelled systems measured at 50 hz against time, demonstrated in FIG. 16 remains constant over time, and is generally dependent on the xanthan gum (or other viscosifying agent) concentration in the formulation.

[0264] As noted, the structures of empty systems are different than those formed by gelled systems loaded with DCF-Na. Since these differences were found to have significant effects on the release of DCF-Na from the delivery system, and hence on the formation of a depot effect, the rheological properties of each system was characterized.

[0265] Thus, the rheological properties of xanthan gel (i.e. the gelled aqueous phase, without the addition of the oily phase), the un-loaded gelled formulation and the DCF-Na loaded gelled formulation were measured and compared. The comparison provided data on the dynamic complex viscosity (η*), as well as the storage modulus (G′) and loss modulus (G″), which reflect the visco-elastic behavior of the systems.

[0266] As seen in FIG. 17A, for both the gelled aqueous phase (without an oily phase) and gelled 2 wt % DCF-Na loaded formulation the complex viscosity drops significantly with the increase of shear rate, where at high shear rates the complex viscosity increases, indicating destruction of the gel structure (a gel-sol transition). However, it is important to note that the loaded gelled formulation shows higher complex viscosities throughout the shear rate sweep compared to the pure xanthan gel, indicating high stability of the formulation. As seen in FIG. 17B, the loading of DCF-Na into the gelled formulation has no significant effect on the viscosity, and its complex viscosity is similar to that of the un-loaded gelled system.

[0267] As seen in FIG. 18A, the storage and loss moduli (G′ and G″) of loaded gelled formulation are higher than that of the pure xanthan gel, meaning that the loaded gelled formulations have a higher energy storage. However, the loss of energy is smaller in the loaded gelled formulation compared to the pure xanthan gel, indicating that the loaded gelled formulation behaves in a viscoelastic manner, and is expected to form a viscoelastic film onto the skin once applied. From FIG. 18B it can be seen that the loading of DCF-Na into the gelled system has no effect on the storage and loss moduli.

[0268] Further insight into the rheological characteristics of the formulations was investigated by measuring the complex viscosity at very low shear rates of the loaded systems with varying amounts of xanthan (0.75 wt % to 2.85 wt %) in comparison to Voltaren Emulgel® Forte (FIG. 19). Under these low shear rates, mimicking the rubbing of the gel onto the surface of the skin, the measured viscosity is lower compared to Voltaren Emulgel® Forte. However, with 2.85 wt % gellant, the formulation loss of viscosity against increasing shear rate drops slower and eventually is similar to the viscosity of the commercial emulsions (at 0.99 l/s). Without wishing to be bound by theory, the commercial emulsion has relatively large droplets and is highly anisotropic. Hence overcoming the interactions between the oil droplets in order to induce flow requires larger input of energy into the system (i.e. higher shear rates). The NDS 506(A) formulation, on the contrary, have smaller and homogenous nanodomains, resulting in a relatively isotropic system; these systems do not demonstrate significant interactions between the nanodomain, hence flow can be induced and maintained at very low shear rates.

[0269] As the formulations are designed for topical application, the viscosity of the formulations have an impact on their spreadability. This is demonstrated by utilizing a spreadability test.

[0270] Spreadability is assessed by placing 350 mg of a tested formulation in the middle of a clean, dry and uniform glass surface. The sample is covered by another glass surface having a weight of 180 g. After 60 second, the diameter of the spread sample is measured and compared to its initial diameter (before the weight was applied). The spreading value S is calculated by the following formula: S=m.Math.A/t, in which m is the weight (g) placed on the sample, A is the spreading area (cm.sup.2) and t (sec) is the time the sample was exposed to the weight. Each formulations was tested 3 times.

[0271] FIGS. 20A-20B show spreadability test for Voltaren Emulgel® Forte, while FIGS. 20C-20D show test results for NDS 506(A). As also seen from Table 11, formulation NDS 506(A) shows improved spreading compared to Voltaren Emulgel® Forte, indicating that NDS 506(A) can cover a larger skin surface using the given amount of formulation.

TABLE-US-00012 TABLE 11 Spreadability test results Mean Quantity diameter Mean area Mean Sample (g) (cm) (cm.sup.2) spreading NDS 506(A) 0.35 6.3 ± 0.1 31.17 ± 0.98 93.51 ± 2.96 Voltaren 0.35 4.1 ± 0.2 13.72 ± 1.28 39.67 ± 3.86 Emulgel ®Forte

[0272] Sensorial Testing

[0273] NDS 506(A) was compared to Voltaren Emulgel® Forte in a series of sensorial tests. 20 human volunteers were asked to wash their hands thoroughly and completely dry them from any residues of water. A predefined weight amount of the formulation (350 mg of either NDS 506(A) or Voltaren Emulgel® Forte) material was placed on the back of their hand. The volunteers were asked to score the immediate contact feel of the gel in regards to its texture, consistency and creaminess using a scale of 1 to 6. Next, the volunteers were asked to rub-in the gel and score again from a scale of 1-6, the tackiness, greasiness and softness feel. In the last stage, the volunteers were asked to score the after-feel effect including softness, greasy, tackiness—residue and the possible performance of a film using the same scoring system as before.

[0274] As shown in Tables 12-1 and 12-2, various parameters were assessed before, during and after application onto the skin.

TABLE-US-00013 TABLE 12-1 Sensorial and textural test results for NDS 506(A) (score 1-6) Immediate contact Rub-in After feel Parameter score Parameter score Parameter score Texture 6 Tackiness 0 Soft 6 Consistency 6 Greasiness 1 Greasy 0.5 Creaminess 4 Softness 6 Tacky 0.5 — — Spreadability 5 Film residue 0 — — — — Absorbency 6

TABLE-US-00014 TABLE 12-2 Sensorial and textural test results for Voltaren Emulgel ®Forte (score 1-6) Immediate contact Rub-in After feel Parameter score Parameter score Parameter score Texture 5 Tackiness 1 Soft 5 Consistency 3 Greasiness 1 Greasy 3 Creaminess 6 Softness 5 Tacky 2 — — Spreadability 5 Film residue 0.5 — — — — Absorbency 5

[0275] As evident from the sensory results, the NDS 506(A) formulation showed better sensorial and textural parameters, suggesting that such formulations are better absorbed into the skin. This may also contribute to improvement in user's compliance to treatment.

[0276] Ex Vivo Permeation and Penetration of DCF-Na

[0277] Ex vivo permeability and penetration of NDS 506(A) was measured compared to Voltaren Emulgel® Forte using Franz cell diffusion (FC) system (PermeGear, Inc., Hellertown, Pa.), using freshly dermatome pig's ear skin. Comparison was carried out between NDS 506(A) and Voltaren Emulgel® Forte (2.32 wt % DCF-DEA). It is noted that 2.32 wt % DCF-DEA is comparable to 2.0 wt % DCF-Na.

[0278] Permeation Procedure Protocol: Five replicates of FC permeation studies were performed for each formulation sample. Skin samples selected showed no wounds, warts or hematomas. The skin's integrity was measured by Trans-epidermal water loss (TEWL) (Dermalab Cortex Technology instrument, Hadsund, Denmark). Only pieces showing TWEL levels less than 10±2.5 g/m.sup.2 h were further used.

[0279] Skin was mounted on receiver chamber with stratum corneum (SC) facing upwards and the donor compartments were clamped in place. The receiver compartment was filled with freshly prepared phosphate buffer PBS (pH 7.4) with constant stirring using a Teflon-coated magnetic stirrer, while heated to 34±2° C. (depending on the RT) to produce 32° C. at the receptor cell. Before initializing the experiment the skin was left to acclimatize with pre-warmed (32° C.) 0.5 ml PBS placed in the donor cell.

[0280] After 30 minutes, PBS was removed and a defined infinite dosage (5 mg/cm.sup.2) of NDS 506(A) and Voltaren Emulgel® Forte were applied onto the skin by spreading the formulations homogeneously. The donor compartment was left open for 30 minutes to enable gel to adhere to the membrane properly and result in a fine film on the surface of the skin. Next, the donor cells and sampling port opening were sealed with Parafilm to avoid further evaporation.

[0281] 0.5 ml samples were withdrawn from the receiver cell at predetermined intervals using a long glass Pasteur pipette to reach near the string area and achieve maximum homogeneity. Cells were replenished to their marked volume with fresh heated (32° C.) buffer solution. The addition of the solution to the receiver compartment was carried out with great care to avoid the entrapment of air bubbles beneath the dermis. Samples were taken to 1.5 ml amber vial and stored at −20° C. until analysis was completed.

[0282] All samples were measured using HPLC (Waters, Milford, Mass./autosampler Waters 717 plus equipped with a photodiode array detector—Waters 996), according to the procedure described further herein. Diclofenac concentration of samples was evaluated from an eight point standards calibration curve, with a R.sup.2 value not less than 0.999. Cumulative drug permeation (mcg/sq. cm) was calculated and plotted against time.

[0283] HPLC Waters 600 series; Autosampler Waters 717 plus; photodiode array detector Waters 996. Mobile phase: 65% acetonitrile/35% water/0.1% trifluoric-acetic acid or formic-acid. Column type: aqua 5 μm, C18, 250 mm×4.6 mm (Phenomenex). Guard column: SecurityGuard cartridge, C18, 4×3.0 mm ID (Phenomenex). Flow rate: 1 ml/min; 30° C.; injection volume 5 μl.

[0284] Penetration Procedure Protocol (Tape Striping) [9]: The procedure was followed as listed above. However, sampling from receptor cell was carried only after 24 hours. Remaining formulations were carefully removed from the donor cell using a spatula. The formulations were placed in a vial containing 10 ml methanol, and donor compartment was thoroughly washed, using the same methanol volume, to ensure that all residual formulation left on the glass was also removed.

[0285] An adhesive film was applied onto the skin surface and pressed using a constant weight roller to avoid the formation of furrows and wrinkles and enable a uniform adhesion of the tape. An additional strip was taken from the skin (total 3). Strips 1-3 were placed into the same vial together with the formulation. This vial content represents the diclofenac remaining in the donor sample (the formulation) after 24 hours together with that found on the surface of the skin termed “Formulation+Upper” (data not shown).

[0286] Seven additional strips (4-10#) were pulled and placed together in a separate 10 ml methanol vial for the analysis of diclofenac depot skin effect termed “Deep skin”.

[0287] The remaining striped skin was place in a third 10 ml methanol vial for analysis of diclofenac content in the this skin layer, termed “Stripped Skin”). As a positive control, and to determined diclofenac content, the same amount of fresh formulation was dissolved in 10 ml methanol vial and recovery of all collected diclofenac concentration (from all steps) were combined form all layer and permeation to show ca. 90-100%.

[0288] All vials (except the samples taken from the receptor cell) were shaken at room temperature for 1.5 hr at 200 rpm and sonicated for 15 min. Samples were filtered using a 0.45 μm cone and transferred into a clean new amber glass vials. All samples were measured using HPLC (same as above). Quantification of diclofenac was calculated from an eight standards calibration curve having a R.sup.2 not less than 0.999.

[0289] Comparative results of ex vivo tests are provided in FIG. 21. Measurements of the levels of DCF-Na within the skin layers (‘Deep’ and ‘Stripped Skin’) demonstrated an increased concentrations of the DCF-Na when testing formulation NDS506(A) compared to commercial Voltaren Emulgel® Forte (2.32 wt % diethylamine diclofenac). However, the permeation levels of the drug as measured after 24 hours in the receiver cell were similar within all three tested formulations. This demonstrates that the permeation of DCF-Na using NDS506(A) reaches deeper skin levels in higher concentrations and then to the desired tissue compared to the reference product, with limited systemic exposure. Without wishing to be bound to theory, the Franz cell analysis results demonstrates the skin depot effect of NDS506(A) and its permeation to the applied joint treated tissue with limited systemic exposure.

[0290] Increasing the xanthan content from 0.75 wt % to 2.85 wt % did not have an effect on the permeation of DCF-Na in a Franz cell test, as seen in FIG. 22. Namely, although the viscosity of the formulation was increased and a denser or stronger network was formed in the aqueous phase, this did not hinder the release of DCF-Na from the formulation.

[0291] Stability

[0292] The stability of NDS 506(A) with antioxidants was evaluated for a period of 3 months, at four different temperatures and relative humidity (% RH) conditions (4° C., 25° C./60% RH and 40° C./75% RH).

[0293] Appearance, pH, % DCF-Na (by HPLC) were measured at each time point for triplicate samples, and compared to the initial measurements taken immediately after preparation of the formulations. The results are presented in Table 13.

[0294] NDS506(A) was also found to maintain stability through 72 hours of freezing and thawing back to room temperature (data not shown), namely the formulation's structure was maintained, without any phase separation or changes in the appearance of the formulation.

TABLE-US-00015 TABLE 13 3 months stability 25.sup.°C. 40.sup.°C. Test conditions Initial 4° C. 60% RHA 75% RHA Incubation time — 3 months 3 months 3 months Appearance Homogenous, Homogenous, Homogenous, Homogenous, transparent transparent transparent transparent slightly- slightly- slightly- slightly- yellow weak yellow weak yellow weak yellow weak gel gel gel gel pH 7.25 7.32 7.33 7.21 Assay (avg. label 101.21 ± 0.91 98.35 ± 1.35 99.35 ± 1.35 100.04 ± 1.77 claim % ± % RDS)

[0295] As can be observed, the DCF-Na loaded gelled formulations maintain their clarity, pH and active concentration values over prolonged periods of time, i.e. at least up to 3 months, when stored at various storage conditions. Thus, these formulations may be stored for prolonged periods of time without adversely affecting their properties.

[0296] To determine long term stability of formulations, a rapid measurement was carried out using LUMiFuge™ analytical centrifugation. LUMiFuge™ analysis enables to predict the shelf-life of a formulation in its original concentration, even in cases of slow destabilization processes like creaming, sedimentation, flocculation, coalescence and fractionation. During LUMiFuge™ measurements, parallel light illuminates the entire sample cell in a centrifugal field; the transmitted light is detected by sensors arranged linearly along the total length of the sample-cell. Local alterations of particles or droplets are detected due to changes in light transmission over time. The results are presented in a graph plotting the percentage of transmitted light (Transmission %) as a function of local position (mm), revealing the corresponding transmission profile over time.

[0297] LUMiFuge™ test results for NDS 506(A) and typical commercial emulsion are shown in FIGS. 23A-23B, respectively, over a time period of 24 hours.

[0298] The analysis of emulsion (having white milky appearance) scattered and absorbed the light resulting in significant decrease in light transmission over time, as the gelled emulsion's stability was impaired. In contrast, the NDS 506(A) formulations, (having a clear and transparent appearance) enabled light to be transmitted (100%) throughout the whole measured cell length. The transmitted light, reflecting the transparency of the sample, was even obtained over 24 hours of centrifugal forces of 3000 rpm tested during analysis. These results support expectation for long shelf life stability properties of the NDS 506(A) formulation after long storage conditions.

[0299] Stability to Freezing and Thawing

[0300] Stability to freezing and thawing was assessed by placing a sample of formulation NDS506(A) at −20° C. for 72 hours and then thawing at room temperature for 2 hours. The formulations remained clear and homogenous after freezing and thawing, with no apparent change.