ethosome seminar final
TRANSCRIPT
ETHOSOMES AS DRUG CARRIERS
1. Introduction
Transdermal drug delivery offers many advantages as compared to traditional drug
delivery systems, including oral and parenteral drug delivery system. Transdermal
route is therefore, a better alternative to achieve constant plasma levels for prolonged
periods of time, which additionally could be advantageous because of less frequent
dosing regimens. Advantages claimed are increased patient acceptability (non
invasiveness), avoidance of gastrointestinal disturbances and first pass metabolism of
the drug1. The major advances in vesicle research was the finding a vesicle derivatives
known as an “Ethosomes2”.
Ethosomes are non-invasive delivery carriers that enable drugs to reach the deep skin
layers and/or the systemic circulation. Ethosomes are soft, malleable vesicles
composed mainly of phospholipids (phosphatidylcholine, phosphatidylserine, and
phosphatitidic acid), ethanol (relatively high concentration) and water3. These “soft
vesicles” represents novel vesicular carrier for enhanced delivery through skin. The
soft, malleable vesicles tailored for enhanced delivery of active agents. The size of
ethosomes can be modulated to range anywhere from 30nm to a few microns.
Although ethosomal systems are conceptually sophisticated, they are characterized by
simplicity in their preparation, safety and efficacy that can highly expand their
application. Ethosomal systems were much more efficient at delivering a fluorescent
probe to the skin in terms of quantity and depth than either liposomes or
hydroalcoholic solution. Ethosomes provides a number of important benefits
including improving the drug's efficacy, enhancing patient compliance, comfort and
reducing the total cost of treatment. Ethosomes were found to be suitable for various
applications within the pharmaceuticals, biotechnology, veterinary, cosmetic and
nutraceutical markets. Enhanced delivery of bioactive molecules through the skin and
cellular membranes by means of an ethosomal carrier opens numerous challenges and
opportunities for the research and future development of novel improved therapies.
Ethosomes are lipid vesicles containing phospholipids, alcohol (ethanol and isopropyl
alcohol) in relatively high concentration and water4. Ethosomes are soft vesicles made
of phospholipids and ethanol (in higher quantity) and water. Ethosomes permeate
1
through the skin layers more rapidly and possess significantly higher transdermal flux
in comparison to conventional liposomes5, 6. Visualization of ethosomes is shown in
Figure 1. Although, the exact mechanism for better permeation into deeper skin layers
from ethosomes is still not clear. The synergistic effects of combination of
phospholipids and high concentration of ethanol in vesicular formulations have been
suggested to be responsible for deeper distribution and penetration in the skin lipid bi-
layers.
1.1 Definition:
Ethosomes are vesicular carrier comprise of hydroalcoholic or hydro/ alcoholic
/glycolic phospholipids in which the concentration of alcohols or their combination is
relatively high.
1.2 Composition of ethosomes:
Typically, ethosomes may contain phospholipids with various chemical structures like
phosphatidyl choline (PC), hydrogenated PC, phosphatidic acid (PA), phosphatidyl
serine (PS), phosphatidyl ethanolamine (PE), phosphatidyl glycerol (PPG),
phosphatidyl inositol (PI), hydrogenated PC, alcohol (ethanol or isopropyl alcohol),
water and propylene glycol (or other glycols) 7, 8. Such a composition enables delivery
of high concentration of active ingredients through skin. Drug delivery can be
modulated by altering alcohol: water or alcohol-polyol: water ratio. Some preferred
phospholipids are soya phospholipids such as Phospholipon 90 (PL-90). It is usually
employed in a range of 0.5-10% w/w. Cholesterol at concentrations ranging between
0.1-1 % can also be added to the preparation9. Examples of alcohols, which can be
used, include ethanol and isopropyl alcohol. Among glycols, propylene glycol and
transcutol are generally used. In addition, non-ionic surfactants (PEG-alkyl ethers)
2
VISUALIAZATION OF ETHOSOME(TEM MAGNIFICATION: 31500)
VISUALIAZATION OF ETHOSOME (SEM X 100, 000)
can be combined with the phospholipids in these preparations. Cationic lipids like
cocoamide, alkyl amines, dodecylamine, cetrimide etc. can be added too. The
concentration of alcohol in the final product may range from 20 to 50%. The
concentration of the non-aqueous phase (alcohol and glycol combination) may range
between 22 to 70%10, 11. Different additives used in the ethosomal formulation are
presented in the Table 1.
TABLE 1: Tabular form represents different additives used in the ethosomal formulations:
Class Examples Uses
Phospholopids
Soya phosphatidyl choline; Egg phosphatidyl choline; Diestearyl phopshatidyl choline
Vesicle forming components
Polyglcerol Propylene glycol; Transcutol RTM
As a skin penetration enhancer
Alcohol Ethanol; Isopropyl alcohol For providing the softness for vesicle membrane; As a skin penetration enhancer
Cholesterol Cholesterol For providing the stability for vesicle membrane
Dyes Rhodamine 123; Rhodamine red
For characterization studies
Vehicles Carbopol D934 As a gel former
2. Advantages 9, 10:
Ethosomes have enhanced permeation of drug through skin for transdermal drug
delivery.
The delivery of large molecules (peptides, protein molecule) is possible. It contains
non-toxic raw material in formulation.
High patient compliance- The ethosomal drug is administrated in semisolid form (gel
or cream) hence producing high patient compliance.
Ethosomal system is passive, non-invasive and is available for immediate
commercialization.
Simple method for drug delivery in comparison to Iontophoresis and Phonophoresis
and other complicated methods.
Ethosomes are platform for the delivery of large and diverse group of drugs.
(peptides, protein molecules)
3
Ethosome composition is safe and the components are approved for pharmaceutical
and cosmetic use.
Low risk profile- The technology has no large-scale drug development risk.
High market attractiveness for products with proprietary technology. Relatively
simple to manufacture with no complicated technical investments required for
production of Ethosomes.
Various application in Pharmaceutical, Veterinary, Cosmetic field.
3. Disadvantages:
Drugs that require high blood levels cannot be administered – limited only to potent
molecules, those requiring a daily dose of 10mg or less.
Ethosomal administration is not a means to achieve rapid bolus type drug input,
rather it is usually designed to offer slow, sustained drug delivery.
Adequate solubility of the drug in both lipophilic and aqueous environments, to reach
dermal microcirculation and gain access to the systemic circulation.
The molecular size of the drug should be reasonable that it should be absorbed
percutaneously.
Adhesive may not adhere well to all types of skin.
Uncomfortable.
May not be economical.
Skin irritation or dermatitis due to excipients and enhancers of drug delivery system.
4. Mechanism Of Drug Penetration 12:
The main advantage of ethosomes over liposomes is the increase permeation of the
drug. The mechanism of the drug absorption from ethosomes is not clear. The drug
absorption probably occurs in following two phases. 13, 14 (Figure 2):
1. Ethanol effect: Ethanol acts as a penetration enhancer through the skin. The
mechanism of its penetration enhancing effect is well known. Ethanol
penetrates into intercellular lipids and increases the fluidity of cell membrane
lipids and decrease the density of lipid multilayer of cell membrane.
2. Ethosomal effect: Increased cell membrane lipid fluidity caused by the
ethanol of ethosomes results increased skin permeability. So the ethosomes
4
permeates very easily inside the deep skin layers, where it gets fused with skin
lipids and releases the drugs into deep layer of skin.
FIGURE 2: Mechanism of action of ethosomes
5. Method of preparation15 :
There are two methods which can be used for the formulation and preparation of
ethosomes. Both of the methods are very simple and convenient and do not involve
any sophisticated instrument or complicated process. The formulation of ethosomes
involves hot and cold method (table 2).
1. Hot method: In this method disperse phospholipids in water by heating in a water
bath at 40°C until a colloidal solution is obtained. In a separate vessel properly
mix ethanol and propylene glycol and heat up to 40°C. Add the organic phase into
the aqueous phase. Dissolve the drug in water or ethanol depending on its
solubility. The vesicle size of ethosomal formulation can be decreased to the
desire extent using probe sonication or extrusion method.
2. Cold method: This is the most common and widely used method for the
ethosomal preparation. Dissolve phospholipids, drug and other lipid materials in
ethanol in a covered vessel at room temperature with vigorous stirring. Add
propylene glycol or other polyglycol during stirring. Heat the mixture up to 30°C
5
in a water bath. Heat the water up to 30°C in a separate vessel and add to the
mixture and then stir it for 5 min in a covered vessel. The vesicle size of
ethosomal formulation can be decreased to desire extend using sonication. or
extrusion method. Finally, the formulation should be properly stored under
refrigeration.
TABLE 3: Flow chart representation for method of preparation:
6. Various methods for characterization of ethosomes:
Visualization: Visualization of ethosomes can be done using transmission
electron microscopy (TEM) and by scanning electron microscopy (SEM) 16.
Vesicle size and zeta potential: Particle size and zeta potential can be
determined by dynamic light scattering (DLS) using a computerized inspection
system and photon correlation spectroscopy (PCS) 17.
Entrapment efficiency: The entrapment efficiency of drug by ethosomes can be
measured by the ultracentrifugation technique 18.
Transition temperature: The transition temperature of the vesicular lipid
systems can be determined by using differential scanning calorimetry 19.
Surface tension activity measurement: The surface tension activity of drug in
aqueous solution can be measured by the ring method in a Du Nouy ring
tensiometer 20.
6
Vesicle stability: The stability of vesicles can be determined by assessing the
size and structure of the vesicles over time. Mean size is measured by DLS and
structure changes are observed by TEM 21.
Drug content: Drug can be quantified by a modified high performance liquid
chromatographic method 22.
Penetration and permeation studies: Depth of penetration from ethosomes can
be visualized by confocal laser scanning microscopy (CLSM) 23.
7. Applications:
a. Therapeutic application of ethosomes:
5% acyclovir ethosomal preparation compared to the 5% acyclovir cream
showed significant improvements in treatment of herpetic infections 24.
New approach to treat deep skin and soft tissue bacterial infections by dermal
application of erythromycin in an ethosomal carrier.
The effect of an ethosomal insulin formulation that was applied to the skin and
there is a significant decrease in blood glucose level.
b. Ethosomes as a drug carrier:
Ethosomes can be used for many purposes in drug delivery. Ethosomes are
mainly used as replacement of liposomes. Ethosomes can be used for transdermal
delivery of hydrophilic and impermeable drugs through the skin. Following drugs
have been used with ethosomal carrier.
7
TABLE 4: Drug incorporated in ethosomal carrier
NAME OF DRUG
DRUG INCORPORATED IN ETHOSOMAL CARRIER
USES
Acyclovir25 Improved skin permeation. Improved in pharmacodynamics profile. Improved in biological activity two to three times.
Treatment of Herpes labialis
Anti-HIV agents26
(Zidovudine, Lamivudine)
Reduced drug toxicity. Prolonging drug action. Improved transdermal flux. Affected the normal histology of skin. Improved in biological activity two to three times.
Anti-HIV
Azelaic acid27 Ammonium glycyrrhizinate28
Prolong drug release. Improved in biological anti-inflammatory activity. Improved dermal deposition exhibiting sustained release.
Treatment of various inflammatory based skin diseases
Bacitracin29 Increased bioavailability. Improved dermal deposition. Improved intracellular delivery.
Anti-bacterial
Cannabidol30 Improve bioavailability. Increased skin permeation. Improved GIT degradation.
Treatment of Rheumatoid arthritis
a. Delivery of Anti-Parkinsonism agent:
Ethosomal formulation of psychoactive drug Trihexyphenidyl hydrochloride (THP)
was compared with that from classical liposomal formulation.THP is a M1 muscarinic
receptors antagonist and used in the treatment of Parkinson’s disease 31.
b. Transdermal delivery of hormones:
Oral administration of hormones is associated with problems like high first pass
metabolism, low oral bioavailability and several dose dependent side effects. The risk
of failure of treatment is known to increase with each pill missed.
c. Targeting pilosebaceous:
Hair follicles and sebaceous glands are increasingly being recognized as potentially
significant elements in the percutaneous drug delivery. Furthermore, considerable
attention has also been focused on exploiting the follicles as transport shunts for
systemic drug delivery. With the purpose of pilosebaceous targeting, maiden et al32.
prepared and evaluated minodixil ethosomal formulation.
8
d. Topical delivery of DNA:
Many environmental pathogens attempt to enter the body through the skin. Skin
therefore has evolved into excellent protective barrier, which is also immunologically
active and able to express the gene 33 .On the basis of above facts another important
application of ethosomes is to use them for topical delivery of DNA molecules to
express gene in skin cells.
e. Delivery of Antibiotics:
Topical delivery of antibiotics is a better choice for increasing the therapeutic efficacy
of these agents. Conventional oral therapy causes several allergic reactions along with
several side effects. Conventional external preparations possess low permeability to
deep skin layers and subdermal tissues34. Ethosomes can overcome this problem by
delivering sufficient quantity of antibiotic into deeper layers of skin. Ethosomes
penetrate rapidly through the epidermis and bring appreciable amount of drugs into
the deeper layer of skin and suppress infection at their root.
f. Delivery of problematic drug molecules:
The oral delivery of large biogenic molecules such as peptides or proteins is difficult
because they are completely degraded in the GI tract. Non-invasive delivery of
proteins is a better option for overcoming the problems associated with oral delivery.
8. Commercial use of ethosomes:
1.) Preparation of a ligustrazine ethosome patch.
2.) Ethosomes for antifungal drugs in the treatment of tropical fungal diseases.
3.) Cannabinol in ethosomal formulation was used as an anti-arthritis drug.
4.) Zidovudine an anti-viral drug is given in ethosomal formulation.
9. Evaluation:
a. Filter membrane-vesicle interaction study by scanning electron microscopy
Vesicle suspension (0.2 mL) was applied to filter membrane having a pore size of
50nm and placed in diffusion cells. The upper side of the filter was exposed to the air,
whereas the lower side was in contact with PBS (phosphate buffer saline solution),
(pH 6.5). The filters were removed after 1 hour and prepared for SEM studies by
9
fixation at 4°C in Karnovsky’s fixative overnight followed by dehydration with
graded ethanol solutions (30%, 50%, 70%, 90%, 95%, and 100% vol/vol in water).
Finally, filters were coated with gold and examined in SEM (Leica, Bensheim,
Germany) 35.
b. Skin permeation studies
The hair of test animals (rats) were carefully trimmed short (<2 mm) with a pair of
scissors and the abdominal skin was separated from the underlying connective tissue
with a scalpel. The excised skin was placed on aluminium foil and the dermal side of
the skin was gently teased off for any adhering fat and/or subcutaneous tissue36.The
effective permeation area of the diffusion cell and receptor cell volume was 1.0 cm2
and 10 ml respectively. The temperature was maintained at 32°C ± 1°C. The receptor
compartment contained PBS (10ml of pH 6.5). Excised skin was mounted between
the donor and the receptor compartment. Ethosomal formulation (1.0 ml) was applied
to the epidermal surface of skin Samples (0.5 ml) were withdrawn through the
sampling port of the diffusion cell at 1, 2, 4, 8, 12, 16, 20, and 24 hour time intervals
and analyzed by high performance liquid chromatography (HPLC) assay.
c. Stability study
Stability of the vesicles was determined by storing the vesicles at 4°C ± 0.5°C.
Vesicle size, zeta potential, and entrapment efficiency of the vesicles was measured
after 180 days.
d. Vesicle-skin interaction study by TEM and SEM
From animals ultra thin sections were cut (Ultra cut, Vienna, Austria), collected on
formvar-coated grids and examined under transmission electron microscope. For SEM
analysis, the sections of skin after dehydration were mounted on stubs using an
adhesive tape and were coated with gold palladium alloy37 using a fine coat ion
sputter coater. The sections were examined under scanning electron microscope.
e. Vesicle-skin interaction study by fluorescence microscopy
Fluorescence microscopy was carried according to the protocol used for TEM and
SEM study. Paraffin blocks are used were made, 5-μm thick sections were cut using
microtome and examined under a fluorescence absorbance at 540 nm.
10
f. Drug uptake studies
The uptake of drug into MT-2 cells (1×106 cells/mL) was performed in 24 well plates
in which 100 μl RPMI medium was added. Cells were incubated with 100 μl of the
drug solution in PBS (pH 7.4), ethosomal formulation, or marketed formulation, and
then drug uptake was determined by analyzing the drug content by HPLC assay.
g. HPLC assay
The amount of drug permeated in the receptor compartment during in vitro skin
permeation experiments was determined by HPLC assay using methanol: distilled-
water: acetonitrile (70:20:10 vol/vol) mixture as mobile phase delivered at 1 mL/min..
The column eluent was monitored at 271 nm using UV detector. 37
TABLE 5: Evaluation parameters and instrument/methods used in Ethosomes
PARAMETERS INSTRUMENTS/METHODS USED
IMPORTANCE
Vesicle Shape Transmission Electron Microscopy (TEM) Scanning Electron Microscopy (SEM)
Determines skin penetration
Vesicle Size and Zeta Potential
Dynamic Light Scattering (DLS), Photon Correlation Spectroscopy (PCS) and Zeta Meter
Determines skin penetration and stability of vesicles
Transition Temperature
Differential Scanning Calorimetry (DSC)
Determines transition temperature of lipid vesicles
Drug Entrapment Ultracentrifugation Technique Suitability of method Drug Content UV Spectrophotometer, High
Performance Liquid Chromatographic Method (HPLC)
Important in deciding the amount of vesicle preparation to be used
Surface Tension Measurement
Ring Method in a Du Nouy ring tensiometer
Determines surface tension activity of drug in aqueous solution
Stability Studies Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM)
To determine the shelf-life of vesicle formulation
Skin Permeation Studies
Confocal Laser Scanning Microscopy (CLSM)
Determines rate of drug transport through skin
In-vitro dissolution Franz diffusion cell Determines the drug release rate from vesicle
11
10. Future prospects
Introduction of ethosomes has initiated a new era in vesicular research for transdermal
drug delivery. Different reports show a promising future of ethosomes in making
transdermal delivery of various agents more effective. Further, research in this area
will allow better control over drug release in vivo, allowing physician to make the
therapy more effective. Ethosomes offer a good opportunity for the non-invasive
delivery of small, medium and large sized drug molecules .The results of the first
clinical study of acyclovir-ethosomal formulations support this conclusion. Multiliter
quantities of ethosomal formulations can be prepared very easily. Thus, it can be a
logical conclusion that ethosomal formulations possess promising future in effective
dermal/transdermal delivery of bio active agents.
11. CONCLUSION:
It can be easily concluded that ethosomes can provide better skin permeation than
liposomes. The main limiting factor of transdermal drug delivery system i.e.
epidermal barrier can be overcome by ethosomes to significant extent. Application of
ethosomes provides the advantages such as improved permeation through skin and
targeting to deeper skin layers for various skin diseases. Ethosomes have been tested
to encapsulate hydrophilic drugs, cationic drugs, proteins and peptides. Ethosomal
carrier opens new challenges and opportunities for the development of novel
improved therapies.
12
12. REFERENCES:
1. Br. J. Obstet. Gynaecol., The metabolic consequences of treating postmenopausal
women with normal hormone replacement therapy, 1997; 104:4-13.
2. Touitou E, Drug delivery across the skin, Expert Opin. Biol. Ther., 2002; 2:723-733.
3. Merdan VM, Alhaique F, and Touitou E, Vesicular carriers for topical delivery. Acta
Techno. Legis Medicament., 1998; 12:1-6.
4. Patel S: Ethosomes: A promising tool for transdermal delivery of drug, Pharma. Info.
Net. 2007; 5(3): 47-53.
5. Touitou E, Dayan N, Bergelon L., Goldin B., Eliaz M: Ethosomes novel vesicular
carriers for enhanced delivery, characterization and skin permeation properties,
Control Release 2000; 65: 403-418.
6. Bendas E.R, Tadros M.I: Enhanced transdermal delivery of salbutamol sulfate via
ethosomes. AAPS Pharm. Sci. Tech. 2007; 8(4): 1-15.
7. Verma D.D, Fahr A: Synergistic penetration effect of ethanol and phospholipids on
the topical delivery of cyclosporine A. J Control Release 2004; 97: 55-66.
8. Bhalaria M.K, Nail S, Miera A.N: Ethosomes: - A novel delivery system for
antifungal drugs in the treatment of topical fungal diseases. Indian Journal of
Experimental Biology 2009; 47: 368-375.
9. Ijeoma F, Uchegbu, and Vyas S.P: Non-ionic surfactant based vesicles in drug
delivery. Int. J. Pharm., 1998; 172: 33-70.
10. Touitou E, Godin B, Dayan N, Piliponsky A, Levi Schaffer F, Weiss C: Intracellular
delivery mediated by an ethosomal carrier and biomaterials 2001; 22: 3053-3059.
11. Gangwar S, Singh S, Garg G: Ethosomes: A novel tool for drug delivery through the
skin. Journal of Pharmacy Research 2010; 3(4): 688-691.
12. Pandey N: Proniosomes and Ethosomes: New prospect in transdermal drug delivery
system. International Journal of Pharmaceutical sciences and research 2011; 2(8):
1988-1996.
13. Anitha P, Ramkanth S, Umasankari K, Alagusundaram M, Devaki Devi P, and Indira
Prasanna: Ethosomes: A non-invasive vesicular carrier for transdermal drug
delivery. IJRLS 2011; 1: 23-30.
13
14. Jain S, Mishra D, Kuksal A, Tiwary A.K, Jain N.K: Vesicular approach for drug
delivery into or across the skin:- current status and future prospectus.
www.pharmainfo.net.
15. Toll R, Jacobi U, Richter H, Lademann J, Schaefer H, Blume U: Penetration profile
of microsphere in follicular targeting of terminal hair follicles. J. Invest Dermatol
2004; 123: 168-176.
16. New RRC, Preparation and size determination, In: Liposomes A practical approach,
New RRC (Ed.), Oxford University Press, Oxford, 1990: 36-39.
17. Maghraby G.M.M, Williams A.C, and Barry B.W: Oestradiol skin delivery from
ultradeformable liposomes: refinement of surfactant concentration. Int. J. Pharm.
2000; 196(1): 63-74.
18. Guo J, Ping Q, Sun G, and Jiao C, Lecithin vesicular carriers for transdermal
delivery of cyclosporine A. Int. J. Pharm., 2000;194(2):201-207.
19. El Maghraby GMM, Williams AC, and Barry BW, Oestradiol skin delivery from
ultradeformable liposomes: refinement of surfactant concentration. Int. J. Pharm.,
2000; 196(1):63-74.
20. Fry DW, White JC, and Goldman ID, Rapid secretion of low molecular weight
solutes from liposomes without dilution. Anal. Biochem, 1978; 90:809-815.
21. New RRC, Preparation of liposomes and size determination, In: Liposomes A
Practical Approach, New RRC (Ed.), Oxford University Press, Oxford, 1990:36-39.
22. Cevc G, Schatzlein A, and Blume G, Transdermal drug carriers: Basic properties,
optimization and transfer efficiency in case of epicutaneously applied peptides, J.
Control. Release, 1995; 36:3-16.
23. Vanden Berge BAI, Swartzendruber VAB, and Geest J, Development of an optimal
protocol for the ultra structural examination of skin by transmission electron
microscopy. J. Microsc., 1997; 187(2):125-133.
24. Dayan N, and Touitou E, Carrier for skin delivery of trihexyphenidyl HCl:
Ethosomes vs. liposomes. Biomaterials, 2002; 21:1879-1885.
25. Toll R, Jacobi U, Richter H, Lademann J, Schaefer H, and Blume U, Penetration
profile of microspheres in follicular targeting of terminal hair follicles, J. Invest.
Dermatol. 2004; 123:168-176.
26. New RRC, Preparation and size determination, In: Liposomes A practical approach,
New RRC (Ed.), Oxford University Press, Oxford, 1990: 36-39.
14
27. Horwitz E, Pisanty S, Czerninski R, Helser M, Eliav E, Touitou E: A clinical
evaluation of a novel liposomal carrier for acyclovir in the topical treatment of
recurrent herpes labialis. Oral Surg. Oral Med. Oral Pathol. Oral Radial. Endod.
1999; 88: 700-705.
28. Jain S, Umamaheshwari R.B, Bhadra D, Jain N.K, Ethosomes- A novel vesicular
carrier for enhanced transdermal delivery of an Anti-HIV agents. Indian J. Pharm.
Sci. 2004; 66(1): 72-81.
29. Esposito E, Menegatti E, Cortesi R: Ethosomes and liposomes as topical vehicles for
azelaic acid: a preformulation study. International Journal of Cosmetic Science
2004: 26(5): 270.
30. Lauer AC, Ramachandran C, Lieb L.M, Niemiec S, Weiner ND. Targeted delivery to
the pilosebaceous unit via liposomes. Adv. Drug Delivery 1996; 18: 311-324.
31. Johnsen SG, Bennett EP, Jensen, VG Lance, Therapeutic effectiveness of oral
testosterone. 1974; 2:1473-1475.
32. Fang J, Hong C, Chiu W, Wang Y. Effect of liposomes and niosomes on skin
permeation of enoxacin. Int. J. Pharm. 2001; 219: 61-72.
33. Donatella P, Giuseppe L, Domenico M, Franco A, Massimo F: Ethosomes for skin
delivery of ammonium glycyrrhizinate: - In vitro percutaneous permeation through
human skin and in-vivo anti-inflammatory activity on human volutrees. Journal of
Controlled Release 2005.
34. Goldin B, Touitou E: Mechanism of Bacitracin permeation enhancement through the
skin and cellular membrane from an ethosomal carrier. J. Control. Release 2004; 94:
365-379.
35. Lodzki M, Godin B, Rakou L, Mechoulam R, Gallily R, Touitou E: Cannabidol
transdermal delivery and anti-inflammatory effect in a marine model. J. Control.
Release 2003; 93: 79-389.
36. Lopez-Pinto JM, Gonzalez-Rodriguez ML, Rabasco AM. Effect of cholesterol and
ethanol on dermal delivery from DPPC liposomes. Int J Pharm. 2005; 298:1-12.
37. Kelly HW, Murphy S.Beta-Adrenergic agonists for acute, severe asthma. Ann
pharmacotherapy 1992; 26:81-91
15
16