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Journal ArticleDOI

Permeation of hyaluronan tetrasaccharides through hairless mouse skin: an in vitro and in vivo study.

01 Jan 2013-Archives of Dermatological Research (Springer-Verlag)-Vol. 305, Iss: 1, pp 69-77
TL;DR: Results suggest that treatment with HA4 improved skin functional recovery after UVA irradiation by skin penetration of HA4.
Abstract: Hyaluronan (HA) is a well-known active ingredient for cosmetic and drug applications. However, based on its varying molecular size, HA may have limited skin permeation. Therefore, the aim of the present study was to investigate the in vitro skin permeability of HA tetrasaccharide (HA4). In addition, the effects of HA4 on in vivo skin barrier function were examined. The cumulative amounts of HA4 through stratum corneum (SC)-stripped skin and full-thickness skin after 8 h were 2,109.6 and 0.8 μg/cm2, respectively. Furthermore, the cumulative amounts of HA4 permeated after 8 h were 784.4 ng/cm2 for a HA4 solution with a pH 4 and 70.0 ng/cm2 with a pH 7 on full-thickness skin. Next, the in vivo effects of HA4 on the water content of the SC and transepidermal water loss (TEWL) were investigated. The dorsal skins of hairless mice were irradiated to a UVA dose of 22.3 J/cm2/d, 5 times a week. In the control group, the water content of the SC was decreased and TEWL and epidermal thickness were increased with UVA irradiation than the normal group. However, the water content of the SC was increased in the HA4 group than that of the control group in the non-UVA irradiation groups. In addition, the water content of the SC was increased and TEWL and epidermal thickness were decreased in the HA4 group than those of the control and HA groups. These results suggest that treatment with HA4 improved skin functional recovery after UVA irradiation by skin penetration of HA4.

Summary (1 min read)

Introduction

  • The stratum corneum (SC), the uppermost layer of the skin, functions as a primary barrier against the penetration of compounds.
  • Generally, skin permeability of lipophilic compounds is higher than that of hydrophilic compounds.
  • In contrast, low molecular weight HA has been implicated in several biological processes including angiogenesis, cell proliferation, maturation, migration, activation of protein tyrosine kinase cascades, and inflammatory gene expression [5, 6, 9, 17, 18, 21, 22].
  • UVB irradiation to the skin induces a variety of responses including erythema, hyperproliferation of keratinocytes, and skin barrier function alterations.
  • Due to its strong water binding potential, HA is a well-known active ingredient for cosmetic and 4 drug applications.

Materials and methods

  • At appropriate intervals, 300 µL aliquots of the receiver medium were withdrawn and immediately r placed by an equal volume of fresh distilled water.
  • Determination of HA4 Determination of HA4 was performed using a LC/MS system.
  • Hairless mice were exposed to a UVA dose of 22.3 J/cm2/day five times a week for three weeks.
  • All measurements were performed in triplicate for each skin, and the mean values were obtained.

Results

  • Passive skin permeation of HA4 across stripped skin and full-thickness skin Figure 1 shows the permeation profile of the cumulative amount of HA4 that permeated through SC-stripped skin and full-thickness skin, and Table 1 summarizes the calculated permeation parameters.
  • Figure 2a and b show permeation profiles of the cumulative amounts of various concentrations of HA4 solutions that perm ated through SC-stripped skin and full-thickness skin, and Table 2 summarizes the calcul ted permeation parameters.
  • Flux and P showed similar increases (Table 2).
  • In addition, the water content of the SC was significantly increased in the HA4/UVA(-) group than that of the Control/UVA(-) group (Fig. 4).
  • In addition, epidermal thickness was significantly decreased in the HA4/UVA(+) group than that of the Control/UVA(+) group (Fig. 6b).

Discussion

  • The barrier function of the skin is principally attributed to the SC.
  • The in vitro skin permeability of HA4 was initially investigated.
  • Various investigations have already been conducted to improve skin permeation of drugs into or through the SC using techniques such as application of chemical enhancers [13, 19, 28], sonophoresis [12], iontophoresis [25], and electrophoration [16].
  • Chronic UVA irradiation significantly increased epidermal thickness [2].
  • If the skin differentiation happens by HA4, it may be involved in the skin functional recovery after UVA irradiation.

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1
Permeation of hyaluronan tetrasaccharides through hairless mouse skin: an in vitro and in vivo study
Madoka Kage, Yoshihiro Tokudome, Fumie Hashimoto*
Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama, 350-0295,
Japan
*Corresponding Author:
Fumie Hashimoto, Ph. D. (
hasimoto@josai.ac.jp)
Faculty of Pharmaceutical Sciences, Josai University

2
Abstract
AbstractAbstract
Abstract Hyaluronan (HA) is a well-known active ingredient for cosmetic and drug applications.
However, based on its varying molecular size, HA may have limited skin permeation. Therefore,
the aim of the present study was to investigate the in vitro skin permeability of HA tetrasaccharide
(HA4). In addition, the effects of HA4 on in vivo skin barrier function were examined. The
cumulative amounts of HA4 through stratum corneum (SC)-stripped skin and full-thickness skin
after 8 h were 2109.6 µg/cm
2
and 0.8 µg/cm
2
, respectively. Furthermore, the cumulative amounts
of HA4 permeated after 8 h were 784.4 ng/cm
2
for a HA4 solution with a pH 4 and 70.0 ng/cm
2
with
a pH 7 on full-thickness skin. Next, the in vivo effects of HA4 on the water content of the SC and
transepidermal water loss (TEWL) were investigated. The dorsal skins of hairless mice were
irradiated to a UVA dose of 22.3 J/cm
2
/d, 5 times a week. In the control group, the water content of
the SC was decreased and TEWL and epidermal thickness were increased with UVA irradiation than
the normal group. However, the water content of the SC was increased in the HA4 group than that
of the control group in the non-UVA irradiation groups. In addition, the water content of the SC
was increased and TEWL and epidermal thickness were decreased in the HA4 group than those of
the control and HA groups. These results suggest that treatment with HA4 improved skin
functional recovery after UVA irradiation by skin penetration of HA4.
Keywords: Hyaluronan oligosaccharide Skin permeation Barrier function UVA
Transepidermal water loss Water content of stratum corneum

3
Introduction
The stratum corneum (SC), the uppermost layer of the skin, functions as a primary barrier against the
penetration of compounds. When formulations are applied on the skin surface, they are generally
permeated through the skin barrier by passive diffusion. Generally, skin permeability of lipophilic
compounds is higher than that of hydrophilic compounds. Molecular weight is also an index which
affects the skin permeation profiles of compounds. Only low molecules, usually less than 500 Da,
can penetrate the skin membrane [8]. However, tacrolimus (804.02 Da), used in the treatment of
atopic eczema, is reported to have a low skin permeation [1].
Hyaluronan (HA), which is composed of repeated β-1,4-glucuronic
acid-β-1,3-N-acetylglucosamine disaccharide units, is a non-sulfated glycosaminoglycan with a
molecular weight of over 1,000 kDa. HA is abundant in the extracellular matrix of the skin. HA
exists freely in extracellular matrix spaces, but is also involved in many biological processes such as
tissue homeostasis, cell proliferation, cell migration, cell differentiation, angiogenesis, tumor biology,
and repair processes by the interface of each protein [23]. The amount of HA in the skin is
equivalent to 50% or more of the total amount of HA in the body.
HA plays different biological roles, depending on its molecular weight. High molecular weight
HA elicits anti-inflammatory and anti-angiogenic responses [3, 20]. In contrast, low molecular
weight HA has been implicated in several biological processes including angiogenesis, cell
proliferation, maturation, migration, activation of protein tyrosine kinase cascades, and inflammatory
gene expression [5, 6, 9, 17, 18, 21, 22]. HA oligosaccharides, which are smaller than low
molecular weight HA, up-regulate heat shock protein 72 expression [27].
The amount of ultraviolet (UV) A radiation reaching the earth’s surface is approximately 20
times greater than that of UVB radiation, and solar UVA radiation contributes to photoaging and
photocarcinogenesis. UVB irradiation to the skin induces a variety of responses including
erythema, hyperproliferation of keratinocytes, and skin barrier function alterations. UVB
irradiation also induces disruptions in epidermal permeability barrier function. Diminished
permeability barrier function has been reported in response to UVB; combined UVA and UVB; or
UVC. UV irradiation of either human or rat skin results in increased percutaneous absorption of
xenobiotics [7].
Due to its strong water binding potential, HA is a well-known active ingredient for cosmetic and

4
drug applications. Cosmetics combined with HA impede transepidermal water loss from skin by
forming a coated skin surface. Additionally, injectable HA fillers are well known for improving
skin contour defects related to aging (wrinkles and lines), depressed acne scars, and other traumatic
or congenital conditions. However, based on its varying molecular size, skin penetration of HA
may be limited.
Therefore, we focused our attention on a tetrasaccharide (HA4) containing two units, with a
single unit being -β-1,4-glucuronic acid- β-1,3-N-acetylglucosamine, and the in vitro skin
permeability of HA4 across hairless mice skins was assessed. In addition, the in vivo effect of HA4
on skin function was investigated.

5
Materials and methods
Materials
HA4 (99.14%) (776.3 Da) were provided by Glycoscience Laboratories Inc. (Tokyo, Japan).
High-molecular weight hyaluronan (HA) (>1200 kDa) was used. All other chemicals and solvents
used were analytical grade.
Animals
Seven-week-old male hairless mice (HR-1) were purchased from Hoshino Experiment Animal
Center (Ibaraki, Japan). All experiment animals had free access to food and water, and were
housed in rooms where the lighting was automatically regulated on a 12 hour light and dark cycle.
All animal experiments and maintenance were performed under conditions approved by the animal
research committee of Josai University.
HA4 sample preparation
A test solution was prepared with 1 mL of distilled water containing HA4 at three different
concentrations: 0.02, 0.1, and 0.5%. Additionally, pH 4 and pH 7 adjustment of 0.5% HA4 solution
was performed using formic acid and 1.5 M ammonium acetate.
Skin membrane preparations
Permeation studies were conducted with excised intact hairless mouse skin (8-10 weeks old) as a
diffusion membrane. To obtain SC-stripped skin, adhesive tape was applied to hairless mouse skin
with uniform pressure. The procedure was repeated about 20 times, until the SC was entirely
removed from the skin.
In vitro permeation experiments

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Abstract: Permeation enhancers are defined as substances that are capable of promoting penetration of drugs into skin and transdermal therapeutic systems offers a more reliable mean of administering drug through the skin. Skin is a natural barrier so it is necessary to employ enhancement strategies to improve topical bioavailability. This review explores that natural products have got potential to enhance the permeation of the drug through skin by reversibly reducing the skin barrier resistance. The use of natural products is the most reliable means of permeation enhancement of transdermally administered drugs and permits the delivery of broader classes of drugs through the stratum corneum. They are safe, non-toxic, pharmacologically inert, non-irritating, and non-allergenic to use as permeation enhancers. The present review initially highlights the current status of natural products on the basis of SAR studies which have shown significant enhancer activities.

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Abstract: Purpose Hyaluronic acid (HA) is an imperative biomaterial with desirable rheological properties to alleviate symptoms of osteoarthritis. Nevertheless, scantly percutaeous permeation of this macromolecule handicaps its effective use for orthopedics and triggers intra-articular injection as the only surrogate. This study presents novel self-assembeld HA-based gel core elastic nanovesicles, (hyaluosomes; GC-HS), for non-invasive transdermal delivery of HA.

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  • ...(25) reported low penetration of HA through intact skin when examining the permeation of HA tetrasaccharides through hairless mouse skin....

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TL;DR: The results of the study suggest that orally administered HA is degraded to oligosaccharides by intestinal bacteria, and oligOSaccharide HA is absorbed in the large intestine and is subsequently distributed throughout the tissues, including the skin.
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References
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Journal ArticleDOI
TL;DR: For pharmaceutical development purposes, it seems logical to restrict the development of new innovative compounds to a MW of under 500 Dalton, when topical dermatological therapy or percutaneous systemic therapy or vaccination is the objective.
Abstract: Human skin has unique properties of which functioning as a physicochemical barrier is one of the most apparent. The human integument is able to resist the penetration of many molecules. However, especially smaller molecules can surpass transcutaneously. They are able to go by the corneal layer, which is thought to form the main deterrent. We argue that the molecular weight (MW) of a compound must be under 500 Dalton to allow skin absorption. Larger molecules cannot pass the corneal layer. Arguments for this "500 Dalton rule" are; 1) virtually all common contact allergens are under 500 Dalton, larger molecules are not known as contact sensitizers. They cannot penetrate and thus cannot act as allergens in man; 2) the most commonly used pharmacological agents applied in topical dermatotherapy are all under 500 Dalton; 3) all known topical drugs used in transdermal drug-delivery systems are under 500 Dalton. In addition, clinical experience with topical agents such as cyclosporine, tacrolimus and ascomycins gives further arguments for the reality of the 500 Dalton rule. For pharmaceutical development purposes, it seems logical to restrict the development of new innovative compounds to a MW of under 500 Dalton, when topical dermatological therapy or percutaneous systemic therapy or vaccination is the objective.

1,132 citations

Journal ArticleDOI
TL;DR: The general physicochemical and biological properties of hyaluronan, and how these properties may be utilized in the various processes of wound healing: inflammation, granulation and reepithelization, are presented.
Abstract: Hyaluronan is a major carbohydrate component of the extracellular matrix and can be found in skin, joints, eyes and most other organs and tissues. It has a simple, repeated disaccharide linear copolymer structure that is completely conserved throughout a large span of the evolutionary tree, indicating a fundamental biological importance. Amongst extracellular matrix molecules, it has unique hygroscopic, rheological and viscoelastic properties. Hyaluronan binds to many other extracellular matrix molecules, binds specifically to cell bodies through cell surface receptors, and has a unique mode of synthesis in which the molecule is extruded immediately into the extracellular space upon formation. Through its complex interactions with matrix components and cells, hyaluronan has multifaceted roles in biology utilizing both its physicochemical and biological properties. These biological roles range from a purely structural function in the extracellular matrix to developmental regulation through effects of cellular behavior via control of the tissue macro- and microenvironments, as well as through direct receptor mediated effects on gene expression. Hyaluronan is also thought to have important biological roles in skin wound healing, by virtue of its presence in high amounts in skin. Hyaluronan content in skin is further elevated transiently in granulation tissue during the wound healing process. In this review, the general physicochemical and biological properties of hyaluronan, and how these properties may be utilized in the various processes of wound healing: inflammation, granulation and reepithelization, are presented.

1,041 citations


"Permeation of hyaluronan tetrasacch..." refers background in this paper

  • ...proliferation, maturation, migration, activation of protein tyrosine kinase cascades, and inflammatory gene expression [5, 6, 9, 17, 18, 21, 22]....

    [...]

Journal ArticleDOI
11 Aug 1995-Science
TL;DR: Low-frequency ultrasound was shown to increase the permeability of human skin to many drugs, including high molecular weight proteins, by several orders of magnitude, thus making transdermal administration of these molecules potentially feasible.
Abstract: Transdermal drug delivery offers a potential method of drug administration. However, its application has been limited to a few low molecular weight compounds because of the extremely low permeability of human skin. Low-frequency ultrasound was shown to increase the permeability of human skin to many drugs, including high molecular weight proteins, by several orders of magnitude, thus making transdermal administration of these molecules potentially feasible. It was possible to deliver and control therapeutic doses of proteins such as insulin, interferon gamma, and erythropoeitin across human skin. Low-frequency ultrasound is thus a potential noninvasive substitute for traditional methods of drug delivery, such as injections.

803 citations


"Permeation of hyaluronan tetrasacch..." refers methods in this paper

  • ...Various investigations have already been conducted to improve skin permeation of drugs into or through the SC using techniques such as application of chemical enhancers [13, 19, 28], sonophoresis [12], iontophoresis [25], and electroporation [16]....

    [...]

Journal ArticleDOI
TL;DR: The measurements suggest that electroporation occurs in the intercellular lipid bilayers of the stratum corneum by a mechanism involving transient structural changes, which may have significance for drug delivery and other medical applications.
Abstract: Mammalian skin owes its remarkable barrier function to its outermost and dead layer, the stratum corneum. Transdermal transport through this region occurs predominantly through intercellular lipids, organized largely in bilayers. Electroporation is the creation of aqueous pores in lipid bilayers by the application of a short (microseconds to milliseconds) electric pulse. Our measurements suggest that electroporation occurs in the intercellular lipid bilayers of the stratum corneum by a mechanism involving transient structural changes. Flux increases up to 4 orders of magnitude were observed with human skin in vitro for three polar molecules having charges between -1 and -4 and molecular weights up to slightly more than 1000. Similar flux increases were observed in vivo with animal skin. These results may have significance for drug delivery and other medical applications.

642 citations


"Permeation of hyaluronan tetrasacch..." refers methods in this paper

  • ...Various investigations have already been conducted to improve skin permeation of drugs into or through the SC using techniques such as application of chemical enhancers [13, 19, 28], sonophoresis [12], iontophoresis [25], and electroporation [16]....

    [...]

Frequently Asked Questions (1)
Q1. What have the authors contributed in "Permeation of hyaluronan tetrasaccharides through hairless mouse skin: an in vitro and in vivo study" ?

In this paper, the authors focused on a tetrasaccharide ( HA4 ) containing two units, with a single unit being -β-1,4-glucuronic acid- β- 1,3-N-acetylglucosamine, and the in vitro skin permeability of HA4 across hairless mice skins was assessed.