J. Biochem. 2010;147(4):463–470

doi:10.1093/jb/mvp185

Transdermal delivery of a readthrough-inducing drug: a new
approach of gentamicin administration for the treatment ofnonsense mutation-mediated disorders
Received July 13, 2009; accepted November 4, 2009; published online November 11, 2009

Masataka Shiozuka1, Akira Wagatsuma1,
Tadafumi Kawamoto2, Hiroyuki Sasaki3,
Kenichi Shimada1, Yoshikazu Takahashi4,
Yoshiaki Nonomura4 and Ryoichi Matsuda1,**Ryoichi Matsuda, Department of Life Sciences, Graduate schoolof Arts and Sciences, The University of Tokyo, 3-8-1 Komaba,
Meguro-ku, Tokyo 153-8902, Japan. Tel.: þ81-3-5454-6637;
Fax: þ81-3-5454-4306; E-mail: cmatsuda@mail.ecc.u-tokyo.ac.jp

To induce the readthrough of premature termination
codons, aminoglycoside antibiotics such as gentamicin
have attracted interest as potential therapeutic agentsfor diseases caused by nonsense mutations. The transdermal delivery of gentamicin is considered unfeasible
because of its low permeability through the dermis.
However, if the skin permeability of gentamicin could
be improved, it would allow topical application without
the need for systemic delivery. In this report, wedemonstrated that the skin permeability of gentamicin
increased with the use of a thioglycolate-based depilatory agent. After transdermal administration, the readthrough activity in skeletal muscle, as determined using
a lacZ/luc reporter system, was found to be equivalent
to systemic administration when measured in transgenic
mice. Transdermally applied gentamicin was detectedby liquid chromatography-tandem mass spectrometry
in the muscles and sera of mice only after depilatory
agent-treatment. In addition, expansion of the intercellular gaps in the basal and prickle-cell layers wasobserved by electron microscopy only in the depilatory
agent-treated mice. Depilatory agent-treatment may be
useful for the topical delivery of readthough-inducing
drugs for the rescue of nonsense mutation-mediatedgenetic disorders. This ﬁnding may also be applicablefor the transdermal delivery of other pharmacologically
active molecules.
Keywords: Gentamicin/nonsense mutation/readthrough/thioglycolates/transdermal drug delivery.
Abbreviations: TEM, transmission electron microscopy; TCA, trichloroacetic acid.
More than 1800 distinctly inherited human diseases
are caused by a single gene that carries nonsenseß The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

463

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1
Department of Life Sciences, Graduate school of Arts and Sciences,
The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo
153-8902; 2Radioisotope Research Institute, Tsurumi University,
Yokohama 230-8501; 3Institute of DNA Medicine, The Jikei
University School of Medicine, Tokyo 105-8461; and 4Microbial
Chemistry Research Foundation, Tokyo 141-0021, Japan

mutations (1). In Duchenne muscular dystrophy(DMD), up to 20% of patients carry nonsense mutations (2). Similarly, up to 10% of patients (450% in
Israel) with cystic ﬁbrosis (CF) have nonsense mutations in the CF transmembrane regulator gene (3).
Nonsense mutations in tumour-suppressor genes arealso common during the development and progressionof cancer (4). Despite advances in gene therapy, clinicalsuccess is pending. One limitation involves the inducing genes used in the therapy, which are often targetedby the patient’s autoimmune system in response to theviral proteins encoded in the vector. An alternative,
pharmacologic approach to induce translational readthrough involves blocking the nonsense mutations
using antibiotics. It has been reported that aminoglycoside antibiotics can interfere with the ﬁdelity of thetranslation machinery. Aminoglycosides cause extensive miscoding of the mRNA in vitro (5) and allow
readthrough of premature termination codons, as
demonstrated in Escherichia coli (6), tetrahymena (7),wheat embryos (8), yeast (9), cultured mouse cells (10)and human cells (1114).
The mdx mouse, which is a naturally occurring
animal model for DMD, carries a point mutation(from CAA to TAA) at position 3185 in exon 23 of
the dystrophin gene. Barton-Davis et al. (1999)
reported that gentamicin restored functional dystrophin in the mdx mouse (15). Moreover, clinical trials
involving patients with DMD or CF caused by nonsense mutations have shown that aminoglycosides
suppress premature termination mutations in somecases (16, 17).
Gentamicin is one of the most commonly used aminoglycoside antibiotics, and it is usually administeredby intramuscular injection. However, it cannot be
administered either orally or transdermally as it is apolarized water-soluble compound with very poor
intestinal and dermal permeability. Less painful andsimpler methods of gentamicin administration areneeded to improve the care of patients with genetic
disorders caused by nonsense mutations. To this end,transdermal drug delivery has several advantages: it:(i) bypasses gastrointestinal incompatibility and thehepatic ‘ﬁrst-pass’ effect; (ii) reduces side-effects
due to the optimization of the blood concentrationtime
proﬁle;(iii) involves
patient-activated/patient-modulated delivery which enhances patientcompliance; and (iv) enhances target speciﬁcity (18).
However, a major limitation of transdermal administration is the difﬁculty associated with delivering
gentamicin through the skin barrier.

M. Shiozuka et al.

Materials and Methods
Chemicals
Gentamicin solution (Gentacin injection) and cream (0.1% Gentacincream) were purchased from Schering-Plough K. K. (Osaka, Japan).
Hair removal gel mousse was obtained from Reckitt Benckiser Co.,
Ltd. (Tokyo, Japan). All other chemicals were purchased from
Sigma Chemical Co. (St Louis, MO, USA), Wako Pure Chemical
Industries (Osaka, Japan) and Promega (Madison, WI, USA).
Animals
A dual-reporter transgenic mouse strain on a C57/BL6 backgroundwas generated by the Bioindustry Division of Oriental Yeast Co.,Ltd (Tokyo, Japan). The dual-reporter gene consisted of the genes
encoding b-galactosidase and luciferase connected with the premature termination codon ‘Opal (TGA)’ region (a 27-mer that contains
the sequence surrounding the premature termination codon of exon
23 of the mdx gene for mouse dystrophin; TTGAAAGAG
CAATAAAATGGCTTCAAC), and was driven by a cytomegalovirus/b-actin hybrid promoter (Fig. 1). Transgene DNA was injected
into the male pronuclei of fertilized eggs which were then incubatedat 37 C and transferred into the uteri of pseudopregnant ICR recipient female mice. Founder mice bearing the transgene were identiﬁedby PCR analysis of the DNA isolated from partially excised tails.
Homozygous transgenic mice were obtained after crossing heterozygous littermates.
Male hairless (HR1) and normal (C57/BL6) mice (5 weeks old;
25 g body weight) were obtained from Japan SLC, Inc. The mice
were housed individually under controlled conditions of temperatureand humidity and had free access to water and food. The mice were
procured after approval for the present study from the University ofTokyo Animal Ethics Committee.
Skin treatment and readthrough analysis
A thioglycolate-based depilatory cream was applied for 1 min to themouse skin, which was then rinsed with warm water to remove thecream. Gentamicin cream (1 mg gentamicin/day) was then applied
and rubbed gently onto the skin daily for 7 days. At the completion

lacZ

luc

beta-globin poly A

Premature Termination Codon

Fig. 1 Schematic structure of the dual-reporter gene construct. The
dual reporter was composed of the lacZ and luc genes connectedwith a premature termination codon region derived from exon 23 of
the mdx mouse dystrophin gene and driven by a cytomegalovirus/
b-actin hybrid promoter. Although the premature termination
codon was originally TAA, we used TGA in the present study.

464

Liquid chromatography-tandem mass spectrometry
To trace the gentamicin introduced into the mice, LC-MS/MS analysis was performed using the 1100 Quaternary HPLC System(Agilent Technologies, USA) coupled to the API 5000 (Applied
Biosystems, CA, USA). Gentamicin solution (10 ng/ml in 5%
TCA), 5% TCA and blank mouse serum or muscle tissue extract
in reporter lysis buffer were added in equal quantities to serve as the
gentamicin standard samples. To prepare the samples for LC-MS/
MS, the supernatant of the serum or muscle tissue extract was mixed
with 5% TCA (twice the volume of the sample). Chromatographic
separation was performed on an XTerra column (3.5 mm,
2.1 mm  50 mm, Waters, MA, USA) maintained at 30 C in acolumn oven. Five millimolar hepataﬂuorobutyric acid (solvent A)
and acetonitrile (solvent B) were used as the mobile phase under thecondition of 10% B (02 min), 1030% B (24 min, linear gradient),
30% B (410 min), 1030% B (1012 min, linear gradient), and 10%
B (1215 min) at a ﬂow rate of 0.2 ml/min with an injection volumeof 10 ml. The parent to product ion transitions for gentamicin (m/z478.39!322.30 as gentamicin C1) was monitored on a triple quadrupe mass spectrometer, operating in the multiple reaction monitoring (MRM) with a positive ion mode of electrospray ionization (ESI)
and a data collection time of 15 min. The relative quantitative value
was calculated using the single-level, calibration-curve method.
Histochemical and electron microscopic analyses
Serial fresh-frozen sections (10 mm thickness) from the whole bodyof a mouse were prepared according to Kawamoto’s ﬁlm method(19). The whole-body sections were freeze-dried and then incubatedin X-Gal solution (Nakarai, Japan) overnight at 37 C, and thenplaced in PBS. The adjacent sections were stained with
Hematoxylin and Eosin.
For TEM analysis, small pieces (10 mm  10 mm) of the dorsalside of the skin were excised from the hairless mice with a single-edge
razor blade. To prepare ultra-thin sections for electron microscopy,
tissues were doubly ﬁxed with 2% glutaraldehyde in 0.1 M phosphate buffer and 1% osmium tetroxide (in 0.1 M phosphatebuffer), and then dehydrated with a graded series of ethanol. The
tissues were then embedded in epoxy resin. Ultra-thin sections were
stained with uranyl acetate and lead citrate and observed under
an electron microscope (H-7500; Hitachi, Tokyo, Japan) at anacceleration voltage of 100 kV. The images were optimized for contrast and brightness using Photoshop CS3 software (Adobe Inc.,San Jose, USA).

ResultspCAGGS
CMV/beta-actin
promoterof the treatment, the mice were euthanized with an overdose of ether.
Tissue samples were collected from the latissimus dorsi, gluteus
major, femoris lateralis, biceps femoris and quadriceps femoris.
Dissected tissues were minced with scissors and homogenized inthree volumes of the reporter lysis buffer (Promega) with thinglass fragments using a tissue grinder (Physcotron, Niti-on,
Japan). Tissue homogenates were subjected to one round of
freeze-thawing. For the readthrough assay, the lysate supernatants
were collected after centrifugation at 17710g for 10 min, and thenanalyzed using the Beta-Glo and Bright-Glo luciferase assay systems(Promega).
The b-galactosidase and luciferase activities were measured
according to the manufacturers’ instructions using a luminometer(Luminescencer-JNRII, AB-2300; Atto, Japan). The readthroughefﬁciency was determined as the ratio of luciferase activity tob-galactosidase activity.

Establishment of a transgenic mouse fortranscriptional readthrough evaluation

In order to measure transcriptional readthrough activity, we established a novel transgenic mouse strain,
named READ, which expressed a dual-reporter gene.
The dual reporter construct was composed of the lacZand luc genes connected with a premature termination
codon region (Fig. 1). Although the premature termination codon of the mdx mouse was originally TAA,
we adopted TGA-centered sequences because

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The objective of the present study was to evaluate
the in vivo, chemically enhanced transdermal deliveryof gentamicin as a readthrough-inducing drug. To thisend, we established a novel transgenic mouse strain,
named READ (Readthrough Evaluation andAssessment by Dual-reporter), that carries a dualreporter gene composed of the lacZ and luc genes connected with a premature termination codon. In thissystem, only b-galactosidase can be translated withouttranscriptional readthrough, however, both enzymes
would be translated when readthrough occurs. In addition, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to validate the effect of a
depilatory agent on the in vivo permeation of gentamicin. Finally, ultrastructural studies using electron
microscopy were performed to provide insight into
the potential mechanism of barrier alterations in depilatory agent-treated skin.

Transdermal delivery of readthrough drug

Transgenic

Wild

A

B

C

D

H&E

X-gal

aminoglycoside antibiotics tend to exhibit the highest
readthrough activity for TGA (20). When a test substance without readthrough activity was administered
to READ mice, only b-galactosidase was translated.
We conﬁrmed b-galactosidase expression in striatedmuscles, including the diaphragm and heart, by X-gal
staining of whole-body sections (Fig. 2C). The readthrough efﬁciency was determined as the ratio of
luciferase activity to b-galactosidase activity.
Readthrough activity of gentamicin in READ mice

The readthrough activities of gentamicin following
single, daily subcutaneous injections (1 mg in 0.1 ml,n ¼ 6) and transdermal administration (1 mg in 1 g,
n ¼ 5) after depilatory treatment were compared after
7 days. The mice used for transdermal administration
were treated with depilatory cream on day zero. A
commercially available topical cream containing
0.1% gentamicin was then applied daily to the skinof the back, hip and thigh of each transgenic mouse.
Gentamicin applied to the depilatory agent-treatedskin induced readthrough in the muscle tissues at thesame level as that observed after subcutaneous injection (Fig. 3). Transdermally administered gentamicin
resulted in readthrough activity only in depilatory
agent-treated READ mice, but not in untreated ones.
Detection of gentamicin in muscle and serum ofsubcutaneously and transdermally treated mice

To study the permeability of gentamicin into themuscle tissues, LC-MS/MS analysis was performed.
The presence of gentamicin C1 was conﬁrmed inboth the subcutaneously and transdermally treatedgroups (Fig. 4). The gentamicin components C1a andC2 were also detected in the same manner (data notshown). To evaluate whether pretreatment with thethioglycolate-based depilatory agent enhances gentamicin permeation through the skin, the detection ofFig. 3 Effects of topical and subcutaneous administration of
gentamicin on readthrough activity in READ mice. The readthroughactivity of gentamicin was compared between mice that were treatedby daily topical administration (1 mg in 1 g, n ¼ 5) onto
depilatory-treated skin and those treated by subcutaneous injection(1 mg in 100 ml, n ¼ 6) for 7 days. The data for gentamicin creamadministration showed the same increase in readthrough efﬁciency
as that observed for subcutaneous injection (gentamicin solution).
Error bars indicate SDs.

gentamicin in the sera and muscle tissue extractsof hairless mice was carried out using LC-MS/MS.
We conﬁrmed that the LC-MS/MS peak observed
for the depilatory-treated group was in the identical
position as that of the reference material, whereas thepeak associated with gentamicin C1 was undetectablein the untreated group (Fig. 5). The results indicate that the depilatory agent signiﬁcantly increased
the absorption of gentamicin by the skin.
Ultrastructure of depilatory agent-treated skin

To examine whether ultrastructural changes in the skinwere caused by treatment with the depilatory agent,
465

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Fig. 2 Whole-body X-gal staining of a transgenic mouse. Whole-body fresh sections of 5-week-old transgenic (A and C) and control (B and D)
mice were stained with Hematoxylin-Eosin (A and B) and X-gal (C and D). The transgenic mouse expressed b-galactosidase in the skeletal andcardiac muscles. The contents of the digestive tracts stained positive with X-gal due to the native bacterial b-galactosidase of ﬂora (D).
Bar ¼ 1 cm.

M. Shiozuka et al.

TEM analysis was performed with control, 1 min- and24 h-treated samples. In the non-treated control
sample, the cells were closely packed together andintercellular spaces were not observed (Fig. 6A).
However, even after just 1 min of treatment with the
depilatory agent, the formation of gaps could beobserved when the control (Fig. 6A) and experimental
sample (Fig. 6B) were compared. In the sample treated
24 h with the depilatory agent, a large expansion of theintercellular gaps and extraordinary spaces in the basal
and prickle-cell layers were clearly evident (Fig. 6C).

system used in this study makes it possible not only
to screen new molecules which induce readthrough,but also to examine the pharmacokinetics and side
effects associated with such molecules. This system
can also be used for the optimization of various
routes of drug administration. Our READ mouse provides a powerful and valuable tool for the developmentof novel readthrough therapeutics.

Discussion

Transdermal drug delivery systems offer many advantages over conventional dosage forms, such as
improved patient compliance, reduced side effects, nohepatic ﬁrst pass effects, and the possibility to easilyinterrupt or terminate treatment (2224). Moreover,compared with oral administration, such a noninvasive drug delivery route signiﬁcantly reduces drug
degradation due to the lower metabolic activity at thesite of administration. It also bypasses hepatic circulation which is a major site of potential drug metabolism(25). However, given the low permeability of external
molecules, such as gentamicin, the skin remains aminor portal of entry for drugs (26). Therefore, various
approaches aimed at decreasing the resistance ofskin to drug penetration have been investigated (27).
The effects of a depilatory agent on the percutaneousabsorption of testosterone and theophylline in Guinea
pigs (28) and rats (29), respectively, and on the

Establishment of a transgenic mouse for thein vivo assay of readthrough activity

We established and demonstrated the efﬁcacy of adual-reporter transgenic mouse strain, named READ,
which can be used for the detection of readthroughactivity in vivo for the ﬁrst time. It is difﬁcult to measure the amount of dystrophin quantitatively because
it is relatively large (molecular weight of 427 kDa) andis only present in small amounts in striated muscles.
Therefore, the reporter assay with READ mouse isquantitative and efﬁcient in comparison to the detection of dystrophin using a mdx mouse. In rare cases,
some luciferase inhibitors, a compound such as
PTC124, acting through post-translational luc reporter
stabilization, appear to activate gene expression (21).
We consider the importance of implementing theappropriate control assays. The READ mouse assay
466

Depilatory agent-treatment enhances dermal
permeability and is useful for transdermal
drug delivery

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Fig. 4 LC-MS/MS detection of gentamicin in the muscle tissue extracts from gentamicin-treated mice. The mass chromatograms of gentamicin
C1 in muscle tissue extracts from C57/BL6 mice: gentamicin standard sample (A); no administration (B); subcutaneous injection of gentamicin
daily for 7 days (C); transdermal administration of gentamicin cream daily for 7 days after one-time depilatory treatment (D). The major(black-painted) peak obtained for both administration groups was identical to the gentamicin standard sample.

Transdermal delivery of readthrough drug

ionotophoretic delivery of insulin in diabetic rats (30,31), porcine epidermis (32, 33), and on the human stratum corneum (34) have been reported. As shown in
Fig. 3, topically applied gentamicin cream induced thereadthrough of a premature termination codon and
was as effective as subcutaneous injection.
Presence of transdermally delivered gentamicin
in muscle and serum

To determine whether a depilatory agent could inﬂuence the permeability of skin, we next investigated thepermeation of gentamicin following transdermal
administration using a validated LC-MS/MS system(Fig. 4). The quantity of gentamicin was calculated
from the peak area and when appropriate, wenormalized the measured levels to a peak area ofknown concentration in a gentamicin standard
sample. In the muscle tissue extracts, the concentrationof gentamicin was 2-fold higher on average (n ¼ 3)
for transdermal administration when compared to subcutaneous injections. It is likely that transdermal
administration can extend the duration of treatment
with gentamicin, which has a short half-life.
The administered gentamicin was present in both thesera and muscle tissue extracts of depilatory
agent-treated mice (Fig. 5). Furthermore, LC-MS/MS
results indicated that the amount of gentamicin was
signiﬁcantly higher (7000-fold) in the muscle tissue
than in the serum. In vivo transdermal absorption
experiments demonstrated that the depilatory agent
drastically reduced the barrier function of the skin467

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Fig. 5 LC-MS/MS detection of gentamicin in the sera and muscle tissue extracts of hairless mice with or without depilatory treatment. Mass
chromatograms of gentamicin C1 in the sera (A, C and E) and muscle tissue extracts (B, D and F) from hairless mice: non-depilatory-treated andgentamicin-administrated daily for 7 days (A and B); one-time depilatory-treated 24 h prior to administration and gentamicin-administrated
daily for 7 days (C and D); part of (C) at a higher magniﬁcation (E); gentamicin standard sample (F). The black-painted peak showed theexistence of gentamicin. Gentamicin was not detected in the non-depilatory-treated group, whereas gentamicin was present in the
depilatory-treated-group, as assessed by comparison with the reference material in LC-MS/MS.

M. Shiozuka et al.

A

Control (No treatment)B

1 min treatment

C

24 h treatment

and exerted a direct penetration-enhancing effect on
the transdermal route. However, these may be basedon the difference in hairless mouse and normal hairy
mouse. The hairless mice have been widely and frequently used to predict the effects of penetration
enhancers in human skin. Nevertheless, the information on the suitability of hairless mice in percutaneous
penetration is not uniform and sometimes contradictory (35). The skin of hairless mice is obviously thin incomparison with normal mice and has many folds.
Moreover, because the skin penetration is a complex process, a judgement about this penetrationenhancing effect on hairless mouse skin must be
cautious. In addition, we can conclude that differencesin the delivery vehicles affect the penetration and bioavailability of the drug. Speciﬁcally, the effectiveness ofgentamicin in an emulsion base (i.e. a cream) was
higher than in oils and fats as determined by the readthrough activity and the LC-MS/MS analysis (data notshown).
Depilatory agent-treatment causes
ultrastructural changes in the skin

To gain further insight into the mechanisms by which
depilatory agents affect skin permeability, we evaluated ultrastructural changes in the skin caused by athioglycolate-based depilatory agent (Fig. 6). As compared to the non-treated control, both the 1 min- and24 h-treatments disrupted the structural integrity of thebasal and prickle-cell layers. Jin-Ning Lee and colleagues (34) have shown that depilatory agentsenhance transepidermal drug delivery by reducing theresistance of both the transcellular and intercellularroutes of the stratum corneum. Our present results
indicate that the alteration and expansion of the intracellular spaces in the basal and prickle-cell layers could
468

be due to the shrinkage of cells in those layers, which inturn leads to reduced resistance.
Readthrough for rescuing muscular dystrophy

In this study, we selected gentamicin to evaluate the
efﬁcacy of topical drug delivery. Aminoglycosides haveemerged as vanguard pharmacogenetic agents for thetreatment of human genetic disorders due to their ability to suppress translation termination caused by nonsense mutations. Gentamicin injections into mdx mice,a model for DMD, restored dystrophin in up to 20%of the muscle ﬁbres and ameliorated the clinical symptoms of diseases (15). As gentamicin can cause serious
side effects, such as inner ear and kidney toxicities, andmay generate resistant bacteria, non-aminoglycoside,
readthrough-inducing molecules are being actively
sought. Previously, we reported that the dipeptide antibiotic negamycin has the ability to suppress nonsense
mutations with lower toxicity than gentamicin (3638).
Thus, negamycin and its derivatives represent therapeutic candidates for genetic disorders, and are a
topic for future studies. The small-molecular agent
PTC124 (also known as Ataluren) has also beenreported as a promising readthrough drug for DMD
patients with nonsense mutations (39). Our transgenic
mouse would be applicable to investigate the potential
in vivo readthrough activity of these and other new
molecules.
In the present study, we demonstrated that a transdermally delivered readthrough drug promotes the
bypassing of a premature termination codon, which
represents a novel approach for the treatment ofgenetic diseases caused by nonsense mutations. The
transdermal delivery of drugs involves the continuous
administration of therapeutic molecules through theskin, and has many advantages including the maintenance of low plasma drug levels and improving patient

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Fig. 6 Electron micrographs of skin samples from hairless mice treated with a depilatory cream. Dorsal skin samples from 5-week-old hairless
mice; control (non-depilatory-treated) (A); treated with the depilatory cream for 1min (B); and treated with depilatory cream for 24 h during thestudy (C). Expansion of the intercellular gaps in the basal and prickle-cell layers caused by depilatory processing was evident (B and C, arrows).
Bar ¼ 5 mm.

Transdermal delivery of readthrough drug

compliance. We also showed the effectiveness of athioglycolate-based depilatory agent to enhance thetopical delivery of gentamicin. This ﬁnding may also
be applicable for the transdermal delivery of otherpharmacologically active molecules.

Acknowledgements
The authors would like to thank Dr Ryuichi Sawa for his helpfuladvice regarding the LC-MS/MS method.

Conflict of interest
None declared.

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the Ministry of Health, Labour and Welfare, Japan.

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