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Life Sciences, Vol. 57, No. 13, pp.

1293-1297, 1995
Pergamon Copyright 0 1995 Ekwier Scie.nce Ltd
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D. Fleisherl, S. M. Niemiecl, C. K. Oh2 , Z. Hut, C. Ramachandranl and N. Weinerl*

1College of Pharmacy, University of Michigan , Ann Arbor, MI 48109.

2 Smith Kline & French Company, King of Prussia, PA .

(Received in final fom July 11, 1995)


The results of this study clearly demonstrates the utility of novel non-ionic liposomal
systems in facilitating transfer of GHRP-6 into and across deeper strata of skin following topical
application. These findings indicate that it may be possible to deliver therapeutic doses of a
wide variety of peptides to local skin tissue via topical application. The results also suggest the
possibility of controlled enhancement of skin penetration or metered polypeptide deposition
through appropriate choice of liposomal lipid components. The pronounced enhancement of
GHRP-6 and mannitol transport from emulsions containing the nonionic lipids suggests a
promising delivery system for hydrophilic drugs in general.

Key Words: nonionic liposomes, topical delivery, growth hormone

At present, treatment of disease states affecting the skin and underlying tissue with
peptide drugs require systemic administration of the drug. This mode of treatment is clearly
inefficient and we have recently demonstrated that pharmacologically effective doses of large
hydrophilic peptides such as alpha-interferon (IX-IFN) or of large hydrophobic peptides such as
cyclosporin-A (CsA) can be delivered to living skin strata via topical administration of
nonionic liposomal formulations (1). In this study we describe the disposition of a model
dicationic oligopeptide, GHRP-6, in hairless mouse skin after topical application from several
liposomal formulations using in vitro diffusion experiments. GHRP-6, His-D-Trp-Ala-Trp-D-
Phe-Lys-NH2 (mol. wt. 873.03) is an oligopeptide with a non-polar core and a basic amino
acid at each extremity (2).

The synthetic nonionic lipids, glyceryl distearate (GDS), glyceryl dilaurate (GDL) and
polyoxyethylene-lo-stearyl ether (POE-lo) as well as cholesterol (CH) were supplied by IGI
Inc., Little Falls, NJ. Bovine brain ceramides Type III (CM), stearic acid (SA), cholesteryl
sulfate (CS), isopropyl myristate (IPM) and HEPES free acid were obtained from Sigma, St.
Louis, MO. Egg phosphatidylcholine (PC) and egg phosphatidylserine (PS) were obtained
from Lipoid KG, Ludwigstrasse, Germany. cL-Tocopherol was obtained from Eastman Kodak,
Rochester, NY. GHRP-6 (SK&F 110679) and 3H-GHRP-6 were kindly supplied by Smith
Kline & French, King of Prussia, PA. Sephadex G-75 was obtained from Pharmacia Inc.,
Piscataway, NJ. All other chemicals were of analytical grade. The water used was double-
distilled and deionized using a Millipore Milli-Q@ system.

To whom all correspondence should be addressed.

1294 Topical Delivery of Liposomal Polypeptides Vol. 57, No. 13, 199.5

Preuaration of GHRP formulations

Previous studies with two non-ionic liposomal cyclosporin-A formulations suggested that
the two formulations exhibited widely differing transport abilities following topical application.
It was of interest to determine if these differences were drug-related or were due to differences
in the physico-chemical nature of the liposomes and their lipid components. Thus, the two non-
ionic liposomal formulations, one containing GDL:CH:POE-10 (Non-l) and the other
GDS:CH:POE-10 (Non-2) at a weight percent ratio of 57:15:28 were prepared as follows:
Appropriate amounts of the lipids were mixed in a beaker and melted at 75°C. The melt was
then drawn into a syringe preheated in a water-bath at 75°C. A second syringe containing
0.05M isotonic HEPES buffer, pH 7.4, was preheated to 70°C. The two syringes were then
connected via a 3-way Teflon or metal stopcock. The aqueous buffer was then injected into
the lipid phase syringe. The mixture was mixed back and forth between the two syringes
rapidly several times while being cooled under cold tap water. This process was continued until
the mixture was at room temperature. The resulting liposomal suspensions were then examined
using a Nikon Diaphot light microscope to assure integrity and quality of the liposomal
preparations. If lipid particulates were present or if the liposomes were not uniform and
spherical, the suspension was discarded and a fresh batch prepared. Typically, one out of ten
batches were unsatisfactory. The total lipid concentration in the suspensions was 50 mg/ml . An
equal volume of 2 mg/ml GHRP-6 solution in isotonic HEPES buffer, pH 7.4, containing trace
amount of 3H-GHRP was then mixed with the empty liposomes to give a suspension containing
1 mg/ml GHRP-6 and a total lipid concentration of 25 mg/ml. The non-ionic liposomal
formulations were stored at 4°C overnight before use in diffusion experiments.

Phospholipid-based and ceramide-based formulations were prepared as follows:

PC:CH:PS (1:0.5:0.1, mole ratio) and CM:CH:SA:CS (42:28: 18: 12, weight ratio) dehydration-
rehydration liposomes (DRV) were prepared by a modification of the method reported by Kirby
and Gregoriadis (3). Briefly, appropriate amounts of the various lipids and a-tocopherol (1
percent by weight of the total lipids) were dissolved in chloroform or chloroform-methanol (2: 1
v/v) mixtures in a round-bottomed flask. The solvents were then removed using a
rotoevaporator under vacuum and the flask containing the film was dried overnight in a
desiccator to remove any residual solvents. An appropriate amount of isotonic 0.05M HEPES
buffer, pH 7.4, was then added to the film in the flask and the film was hydrated at 40°C for egg
PC systems and 55°C for skin lipid systems, for 30 minutes with intermittent vortexing. The
resultant suspensions were then dehydrated at 50°C under vacuum using the rotoevaporator.
When the suspensions became very viscous, an amount of water equivalent to that removed
(determined by weighing the flask and its contents before and after dehydration) was added
back to the suspensions and rehydrated at 50°C for 45 minutes. The suspensions were then
annealed at 40°C for 15 minutes and stored at 4°C overnight before use in the diffusion
experiments. The total lipid concentration in the suspensions was 50 mg/ml. Equal volumes of 2
mg/ml GHRP-6 solution in isotonic HEPES, pH 7.4 buffer, containing trace amount of 3H-
GHRP were then mixed with the empty liposomes to yield suspensions containing 1 mg/ml
GHRP-6 and a total lipid concentration of 25 mg/ml. The liposomal formulations were stored at
4°C overnight before use in diffusion experiments.

The possibility that the drug may associate to some extent with the liposomes cannot be
ruled out. However, no attempts were made to separate unassociated drug. Thus, all liposomal
peptide formulations used in deposition studies contained unassociated and liposome-associated
peptide. The liposomal preparations were stable for over a test period of two months as judged
visually for phase separation, sedimentation and flocculation and also when examined for
particle size variations using a NiComp 370 submicron laser light scattering particle sizer.

The emulsion formulations were prepared using emulsifier blends of the same
composition used to prepare the Non-l and Non-2 liposomes. Oil-in-water emulsions
containing GHRP-6 were prepared by mixing the respective liposomal GHRP-6 preparations
with isopropyl myristate at a 80:20 ratio by weight followed by sonication for 2 minutes. Oil-
in-water emulsions containing only POE-IO-stearyl ether as the emulsifier were prepared as
follows: Appropriate amounts of POE- lo-stearyl ether were weighed in a scintillation vial and
Vol. 57, No. l3, 1995 Topical Delivery of Liposomal Polypeptides 1295

dissolved in isopropyl myristate. An aqueous solution of GHRP-6 in isotonic 0.05M HEPES

buffer, pH 7.4, containing trace amount of 3H-GHRP-6, was then mixed with the POE-10
solution and sonicated for 2 minutes. The aqueous to oil phase ratio was 80:20 by weight. All
emulsions were physically stable over the test period of 2 weeks as judged visually for phase

In Vitro Diffusion Exueriments

Hairless mice were sacrificed and full thickness dorsal skin was excised. Subcutaneous
fat was carefully removed using a dull scalpel. Appropriate-sized pieces of skin were then
mounted on Franz diffusion cells with a surface area of 1.77 sq. cm and a receiver capacity of 7
ml (Crown Glass, Somerville, NJ). The epidermal side of the skin was exposed to ambient
conditions while the dermal side was bathed by a 0.05M isotonic HEPES, pH 7.4, buffer. The
receiver solution was stirred continuously using a small Teflon-covered magnet. Care was
exercised to remove any air bubbles between the underside of the skin and the receiver solution.
The temperature of the receiver solution was maintained at 37°C. Following mounting of the
skin, 200 u.1 of the test formulation were applied to the epidermal surface of the hairless mouse
skin and carefully spread evenly to achieve complete surface coverage. A minimum of three
cells using skin from at least three different animals were used . All experiments were carried
out under non-occluded conditions. At 24 hr the diffusion set-up was dismantled. Upon
dismantling, the donor cap was rinsed in 10 ml of buffer followed by a 20 ml methanol rinse.
The methanol rinse was allowed to dry in a hood at which time scintillation cocktail was added.
The buffer rinse along with the methanol rinse were then assayed for radiolabeled drug. The
skin piece was then mounted on a board and stripped as follows: A piece of adhesive tape
(Scotch Magic Tape, 810, 3M Commercial Office Supply Division, St. Paul, MN), 1.9 cm wide
and about 6 cm long was used to strip the skin. The tape was of sufficient size to cover the area
of skin that was in contact with the test formulation. At least nine strippings were carried out
and each strip was analyzed separately for radiolabeled drug. If at the end of nine strippings the
skin did not appear shiny and glossy, additional strippings were carried out to ensure complete
removal of the stratum corneum. The remaining skin and the receiver solution were then
assayed for radiolabeled drug. Assay of the donor rinses, strips, remaining skin and receiver
solution was carried out using a scintillation counter after addition of 15 ml of Ecolite+ (ICN
Biomedicals Inc., Irvine, CA) to each system .

Results and Discussion

Table I shows the disposition of GHRP-6 in various strata of hairless mouse skin 24
hours after topical in vitro application of various formulations. The recovery of total
radioactivity was better than 90 percent for all systems. The results indicate that the ceramide-
based liposomal formulation is not significantly different from the aqueous control when the
amounts in the deeper skin strata and in the receiver compartments are compared (p>O. 1, two-
tailed t-test). The phospholipid-based formulation was not significantly different from the
aqueous or skin-lipid systems in facilitating deposition of GHRP-6 into the deeper skin strata
and across the skin (p > 0.2). This is not surprising since transport of the cationic drug would
be severely retarded via complexation with the negatively charged liposomal system. The Non-
1 liposomal formulation was not significantly different from the Non-2 liposomal formulation in
transporting GHRP-6 into and through the skin. However, of the two nonionic liposomal
formulations tested, Non-l was significantly better than all the other systems (p < 0.02) in
enhancing transport into and across skin. The combined uptake of GHRP-6 in the deeper skin
strata and the receiver compartment for Non-l was roughly 3-fold to 4-fold higher than the
aqueous control and about two to three times better than the phospholipid-based liposomal
formulation. The differences in the combined uptake between Non-2 and the aqueous or PC or
CM formulation were found to be statistically insignificant (p > 0.1).

Non-l formulations thus appear to be superior to the other systems in facilitating transfer
of water-soluble drugs such as GHRP-6 and of large hydrophobic drugs such as cyclosporin-A
(1). The general enhancement in drug transfer efficiency, irrespective of the nature of the drug,
suggests strongly that Non-l may be exerting enhancer effects on the stratum corneum
permeability. The effects of the consequences of the release of Non-l components upon partial
12% Topical Delivery of Liposomal Polypeptides Vol. 57, No. l3, 1995

or complete melting of GDL following topical application to skin has been discussed in detail
elsewhere (4). The release of POE-10 -stearyl ether, a known skin penetration enhancer (5,6),
from the bilayer configuration was suggested as a plausible explanation for Non-l behavior.

Distribution of Growth Hormone Releasing Peptide (GHRP-6) in Various Strata of Hairless
Mouse Skin 24 Hours after Topical Zi V&o Application bf Various Formulations.

Formulation Total Living Skin Receiver

Strips Strata

Aqueous solution 69.0 k 5.0 0.1 kO.0 0.5+0.1

25 mg/ml Non-l liposome 48.0 f 13.6 l.Of0.4 1.4kO.3

25 mg/ml Non-2 liposome 85.7 f 2.1 0.5 + 0.2 l.OkO.3

25 mg/ml PL-based liposome 83.5 k 2.6 0.2 k 0.2 0.7 + 0.3

25 mg/ml Ceramide-based liposome 82.4 + 17.3 0.0 * 0.0 0.6kO.l

20 mg/ml Non- 1 I IPM emulsion 29.7 k 9.4 2.3 f 0.5 6.4 + 0.9

20 mg/ml Non-2 / IF’Memulsion 93.3 + 2.1 2.6 f 1.1 7.0* 1.7

50 mg/ml POE-IO / IPM emulsion 54.2 k 7.1 2.7 zk1.4 7.0 + 4.0

^ ..I. . 1 . . .. - 1 r 1. .. . .
_ as^_-
percent or applied dose + stanclarci aeviauon.
. ._ 1 otal recovery or raaioacuvity was
greater than Y5% m all cases.

We had previously shown that Non-l systems were significantly better than
phospholipid-based or ceramide-based liposomes for deposition of cyclosporin-A and CI-
interferon (1) into skin. It was also shown previously that deposition of hydrophilic markers
such as carboxyfluorescein from phospholipid-based oil-in-water emulsions was markedly
lower than deposition from phospholipid-based liposomal formulations (7). This finding
suggested that phospholipids need to be in a bilayer configuration for maximum transfer
efficiency of hydrophilic drugs. If indeed nonionics exert their effects via two separate
mechanisms, 1) deposition of drugs from liposomal bilayers and 2) enhancer effects of the
individual lipids upon skin permeability, then freeing up of part of the nonionic lipids
(enhancers) should improve deposition and not retard it as it did with the phospholipid systems.
The mode of preparation of the nonionic emulsions and the evaporation of water following
topical application of the emulsions could result in the co-existence of nonionic lipids that are
either free or are incorporated in a liposomal (bilayer) configuration. The results of the
disposition of GHRP-6 following application of Non-l and Non-2 emulsions are shown in
Table 1. The nearly 4 to 6-fold enhancement of GHRP amounts in the living skin strata and in
the receiver strongly supports our hypothesis of dual action for nonionic liposomes. That this
enhancement is greater for Non-2 emulsions further corroborates the hypothesis since the
relative increase in free lipids would be much higher for Non-2 emulsions than Non-l
emulsions compared to the respective liposomal systems (m.p CDL =3O”C; m.p. GDS = 55°C).
Finally, the enhanced amounts of GHRP-6 found in the dermis and receiver compartment
(Table 1) with an emulsion containing only POE-10 underlines the potent enhancer action of the
surfactant. Although enhancement of drug absorption from emulsions and microemulsions
containing medium-chain glycerides following oral, nasal, rectal or intraduodenal
administration has been reported (8-13), this is the first report on enhancement of peptide
deposition/absorption following topical delivery from nonionic emulsions. It has also been
Vol. 57, No. 13, 1995 Topical Delivery of Liposomal Polypeptides 1297

suggested that isopropyl myristate may facilitate fluidization of the stratum corneum lipid
domains allowing a greater degree of permeation of drugs. In addition to these enhancer effects,
an increase in the thermodynamic activity of the drug in the emulsion system due to a less
favorable environment being provided for the charged drug or an increase in partitioning into
skin arising out of ion-pairing effects in a low dielectric medium may also contribute to the
overall increase in GHRP-6 amounts transported into and across skin (14). However, in a recent
study it was observed that the absorption of mannitol, an uncharged polar hydrophilic marker,
into and across hairless mouse skin was lo-fold higher following topical in vivo application of
Non-l emulsions compared to that from Non-l liposomes. It was also found that such
enhancement effects were not observed with phospholipid-based emulsions (unpublished
results). These findings appear to rule out major contributions to enhancement from isopropyl
myristate effects on the stratum corneum or from ion-pairing.


Supported by NIH Grants # AI 22303 and AI 30876.



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