manuscript


doi: 10.5599/admet.2.4.155 248 

ADMET & DMPK 2(4) (2014) 248-253; doi: 10.5599/admet.2.4.155 

 
Open Access : ISSN : 1848-7718  

http://www.pub.iapchem.org/ojs/index.php/admet/index   

Original scientific paper 

Rapid Screening of Permeation of Rutin through Skin Using 
Alaptide Enantiomers 

Aneta Cernikova, Radka Opatrilova, Pavel Bobal and Josef Jampilek 

Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, 
Palackeho 1/3, 61242 Brno, Czech Republic 

Corresponding Author:  E-mail: josef.jampilek@gmail.com; Tel.: +420-541-562-926 

Received: December 21, 2014; Revised: December 22, 2014; Published: January 09, 2015  

 

Abstract 
The investigation deals with the influence of permeation of rutin from carboxymethyl cellulose gel through 

full-thickness pig ear skin by (S)- and (R)-alaptide as potential excipients. Alaptide, 8-methyl-6,9-

diazaspiro[4.5]decan-7,10-dione, is the original Czech compound. By means of this rapid screening it was 

found out that the permeation of rutin through the skin increased linearly with time and was enhanced by 

both enantiomers of alaptide: approx. 1.2-fold by (R)-alaptide and approx. 1.5-fold by (S)-alaptide. 

Keywords 
(S)-Alaptide; (R)-alaptide, rutin; carboxymethyl cellulose gel; skin permeation modifiers. 

 

Introduction 

Permeation modifiers (enhancers and retardants) affect the barrier properties of the skin. Chemical 

permeation enhancers (CPEs) are employed to increase the transdermal penetration of therapeutic agents 

[1−6 and refs. therein]. Alaptide, (S)-8-methyl-6,9-diazaspiro[4.5]decan-7,10-dione (see Fig. 1) was designed 

as an analogue of melanocyte-stimulating hormone release-inhibiting factor (MIF-1) [7−10] and thus is able 

to influence the creation and function of keratinocytes [11−13]. Based on these facts and structural analogy 

with other CPEs [4], it was suggested to investigate alaptide as a potential CPE of many anti-inflammatory 

drugs, antimicrobial chemotherapeutics, sex hormones/genital system modulators or drugs of 

central/vegetative nervous system [14−16]. Permeation through the skin of micronized and nanonized  

(S)-alaptide from semisolid formulations was investigated recently [17], and the transdermal modifying 

effect of micronized and nanonized (S)-alaptide was also described [14−16,18,19]. It was found that 

enhancers can become retardants in different formulation environments or when different particle sizes 

were used and vice versa. Since modifiers influence drug delivery, further exploration of these compounds 

is required to understand their modifying action on the properties of the skin. 

Today more than 50 % of marketed drugs are chiral. It has been well recognized that the 

stereochemistry of chiral drugs has a major influence on their pharmacological, pharmacokinetic and 

toxicological actions. Studies on enantiomeric differences in the percutaneous permeation of chiral 

compounds have been actively pursued in recent years. Stratum corneum, the rate-limiting barrier in 

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ADMET & DMPK 2(4) (2014) 248-253 Rutin permeation enhancement by alaptide enantiomers 

doi: 10.5599/admet.2.4.155 249 

transdermal permeation, is made up mainly of keratin and ceramides, which could potentially provide 

chiral environment. Transdermal delivery is facilitated by the presence of penetration enhancers, which act 

primarily by altering the diffusion by disrupting the highly ordered membrane structure or by affecting the 

partitioning behaviour of the diffusant molecules. Enantioselective permeation was observed with some 

chiral excipients, including, for example, terpene enhancers [20−22]. 

Based on the above mentioned facts and the excellent enhancement effect of (S)-alaptide [14−16],  

(R)-alaptide was prepared, and both enantiomers were tested by means of rapid screening for their ability 

to modify the skin barrier properties. Bioflavonoid glycoside rutin, 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-

oxo-4H-chromen-3-yl 6-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranoside, see Fig. 1, was chosen as an 

model compound, since rutin is one of the most often used drugs for decreasing capillary permeability and 

for treatment of haemorrhoids, varicosis and microangiopathy [23]. Enhanced permeation through the skin 

and higher availability of the compound would be favourable for the treatment of vascular wall fragility. 

Within this study the permeation of rutin from carboxymethyl cellulose gel and subsequently the 

permeation of rutin with the addition of the individual enantiomers of alaptide as modifiers of transdermal 

permeation from the same formulation are investigated. 

Flavonoids (bioflavonoids, Vitamin P) are a class of plant secondary phenolic metabolites. Today more 

than 4000 flavonoid-based compounds were described [24,25]. Flavonoids are glycosides containing 

aglycone derived from benzopyran (chromene) with lateral phenyl in the positions C(2), C(3) or C(4) of the 

benzopyran scaffold. The phenyl rings are marked A and B, and the pyran ring C is ortho-condensed with 

the ring A. In nature flavonoids are substituted (especially in position 3 of the benzopyran scaffold) by 

various sugars and generate flavonoid glycoside. Aglycone or the glycosyl group can be substituted by a 

hydroxyacid [25,26]. Both aglycone and glycosides have biological effects. Bioflavonoids normalize 

permeability of capillaries and remove their fragility; they have antihaemorrhagic and anti-edematous 

effects [25−28]. Also antibacterial, antioxidant and anti-inflammatory activities are typical for them [25−28]. 

Bioflavonoids are used as venopharmaceuticals, i.e. drugs for the treatment of chronic venous insufficiency, 

superficial thrombophlebitis and haemorrhoids. They can be applied locally for the treatment of 

dermatomicroangiopathy, periphlebitis, haematomas and traumatic oedema [28]. 

 
Figure 1. Structures of investigated compounds. 

Experimental 

Preparation of alaptide 

Both alaptide enantiomers were synthesized by the standard process [29] and characterized [30]. The 

specific optical rotation of 0.2 mol/L methanolic solution is [α]D
20

 = +21.2° for the (R)-enantiomer and 

[α]D
20

 = -21.2° for the (S)-enantiomer. The melting point of (S)-alaptide is 232-234 °C, while that of  



Jampilek et al.  ADMET & DMPK 2(4) (2014) 248-253 

250  

(R)-alaptide is 230-232 °C. Rutin was purchased from Sigma, all other reagents were purchased from Aldrich 

and Lach-Ner. 

Investigated Semisolid Formulations 

The composition of gel with 10% (w/w) rutin was the following: rutin 10 g and carboxymethyl cellulose 

gel (carboxymethyl cellulose sodium 5 g, macrogol 300 10 g, propylene glycol 2.5 g, methylparaben 0.2 g, 

propylparaben 0.2 g and purified water 87.3 g) up to 100 g. The composition of gel with the addition of 

alaptide 1% (w/w; in relation to rutin amount) was the following: rutin 10 g, alaptide 0.1 g and 

carboxymethyl cellulose gel up to 100 g. 

In vitro transdermal permeation experiments 

Skin samples were obtained from porcine ear. Full thickness skin was cut in fragments and stored at 

−20 °C until utilized. Skin samples were slowly thawed (at 4 °C overnight and then at ambient temperature) 

before each experiment. The permeation enhancing effect of both alaptide enantiomers was evaluated in 

vitro, using a vertical Franz diffusion cell (SES – Analytical Systems, Germany) with a donor surface area of 

0.6359 cm
2
 and a receptor volume of 5.2 mL. The skin was mounted between the donor and receptor 

compartments of the Franz diffusion cell with the epidermal side up. The receptor compartment was filled 

with phosphate buffered saline (pH 7.4) and maintained at 34.0±0.5 °C, using a circulating water bath. The 

receptor compartment content was continuously stirred using a magnetic stirring bar. The skin was kept in 

contact with the receptor phase for 0.5 h prior to the experiment. The investigated gel formulations were 

applied to the skin surface as a donor sample, and the donor compartment of the cell was covered by 

Parafilm®. Samples (0.5 mL) of the receptor phase were withdrawn at five pre-determined time intervals 

(30, 60, 90, 120 and 180 min), and the cell was refilled with an equivalent amount of fresh buffer solution. A 

minimum of five determinations was performed using skin fragments from a minimum of 2 animals for 

each compound, and the data was expressed as means ± SD. The samples were immediately analysed by 

HPLC. 

The analysis of the samples was performed using an Agilent 1200 series HPLC system, equipped with a 

diode array detection (DAD) system, a quaternary model pump and an automatic injector (Agilent 

Technologies, Germany). Data acquisition was performed using ChemStation chromatography software. A 

Zorbax Eclipse XDB C18 5 μm, 4.6×150 mm (Agilent, USA) chromatographic column was used. The total flow 

of the column was 0.5 mL/min; injection was 10 μL; column temperature was 30 °C; and sample 

temperature was 10 °C. The detection wavelength of 254 nm was chosen; the time of analysis was 10 min. 

A mixture of MeOH (HPLC grade, 85.0 %) and H2O (HPLC – Mili-Q Grade, 15.0%) was used as a mobile 

phase. The retention time (tR) of rutin was 3.70±0.05 min; the limit of detection (LOD) was 0.99 ng/mL; and 

the limit of quantification (LOQ) was 3.3 ng/mL. 

Results and Discussion 

Rutin was chosen as a frequently applied venopharmaceutical for the topical treatment of the fragility of 

vascular wall that can be associated with various injuries of the skin, because alaptide itself shows 

significant curative effect on the skin [8,31]. The rapid in vitro skin permeation screening experiments were 

performed using static Franz diffusion cells [32] within 3 hours only. Full-thickness pig ear skin was selected 

for in vitro evaluation of permeation. This tissue is a suitable in vitro model of human skin [33], because 

porcine skin has shown to be histologically and biochemically similar to human skin, and it has been used in 

numerous studies [4]. The permeation of rutin through full-thickness pig ear skin without and with 1 % 



ADMET & DMPK 2(4) (2014) 248-253 Rutin permeation enhancement by alaptide enantiomers 

doi: 10.5599/admet.2.4.155 251 

(w/w related to rutin) (S)- or (R)-enantiomer of alaptide was tested from carboxymethyl cellulose gel as a 

donor vehicle. All the results are provided in Table 1 and illustrated in Figure 2. 

Table 1. Cumulative permeated amounts Qt per unit area [μg/cm
2
] of rutin from carboxymethyl cellulose gel without 

and with (S)-alaptide or (R)-alaptide as modifiers of transdermal permeation achieved in in vitro transdermal 
permeation experiments using Franz diffusion cell. Qt are expressed as mean ± SD (n = 5 experiments). 

Time [h] 
Cumulative permeated amounts Qt per unit area[µg/cm

2
] 

rutin rutin+(S)-alaptide rutin+(R)-alaptide 

0.5 3.7±0.6 5.5±0.7 4.6±0.6 

1.0 6.5±0.7 9.7±0.9 7.7±0.8 

1.5 9.0±0.5 15.4±1.0 11.9±1.0 

2.0 13.2±1.1 19.4±1.2 15.1±0.8 

3.0 16.7±1.3 25.9±1.5 20.0±1.1 

0

5

10

15

20

25

30

0.5 1 1.5 2 3

Q
t 
[

g
/c

m
2
]

Time [h]

rut rut+SALA rut+RALA  
Figure 2. In vitro profile of cumulative permeated amounts Qt per unit area [μg/cm

2
] in time of rutin 

(rut) alone and after addition of 1 % w/w of (S)-alaptide (SALA) or (R)-alaptide (RALA) from 
carboxymethyl cellulose gel through skin. Qt values are expressed as mean ± SD (n = 5 experiments). 

As the presented study represents a preliminary rapid in vitro screening, all the experiments were 

performed only in the time range from 0.5 to 3.0 h. The values obtained from the permeation experiments 

were expressed as the cumulative permeated amount of the drug (Qt [μg]) per unit of skin surface area 

(0.6359 cm
2
), see Table 1. Due to the short time interval of experiments steady-state permeation flux and 

lag time cannot be calculated, but this was not the aim of the experiments. The dependences of the 

cumulative permeated amount of the drug per unit of skin surface area in time (Qt versus t) are shown in 

Figure 2. Based on the results it can be stated that permeation of rutin through the skin increased linearly 

with time; for rutin alone correlation coefficient was r = 0.9891 and slope 5.95 /cm
2
/h; for rutin with  

(R)-alaptide r = 0.9896 and slope 7.16 /cm
2
/h; and for rutin with (S)-alaptide r = 0.9915 and slope 

9.21 /cm
2
/h. Based on the ratios slopes of the linear dependences Qt versus t permeation enhancement 

within the studied time span (up to 3 h) can be evaluated. The permeation of rutin with (R)-alaptide was 

1.22-fold higher than without the addition of (R)-alaptide. The addition of (S)-alaptide to 

carboxymethyl cellulose gel caused 1.54-fold higher permeation of rutin through the skin than from the 



Jampilek et al.  ADMET & DMPK 2(4) (2014) 248-253 

252  

formulation without (S)-alaptide. On the other hand, rutin permeated from the formulation containing  

(S)-alaptide only 1.27-fold more compared with (R)-alaptide. 

As mentioned above the components of stratum corneum form a chiral environment, therefore there is 

potential for stereoselective interactions of an enhancer with skin components. Stereoselective interactions 

of individual drug enantiomers (e.g., propranolol, metoprolol, timolol, ketorolac, ketoprofen, flurbiprofen, 

ephedrine, oxybutynin, amlodipine) with the skin components involving different permeation rates of 

individual enantiomers through the skin were tested. Some authors found different permeation rates for 

enantiomers, while other studies showed no enantiomeric differences in the permeation [20−22,34−36]. 

Similarly, investigations of interactions of individual CPE enantiomers with the skin provided opposite 

results [20,22,37,38]; therefore additional studies are needed to be performed to answer whether the skin 

is an enantioselective membrane and given its mechanism of action, enhancer conformation can influence 

its enhancing properties or the differences in the permeation rates between the individual enantiomers are 

not related to any stereoselective interactions, but they can be explained only by different physicochemical 

properties. 

Conclusions 

The ability of (S)- and (R)-alaptide to modify the permeation of rutin through the porcine ear skin was 

examined using the Franz cell in the rapid in vitro screening. Both enantiomers of alaptide applied in 

concentration 1 % w/w related to the amount of rutin influenced the permeation of the drug from the 

carboxymethyl cellulose gel through the skin. According to the calculated cumulative permeated amount of 

the drug per unit of skin surface area in time, rutin with addition of the (R)-enantiomer of alaptide 

permeated 1.22-fold more than from the formulation without the alaptide enantiomers, and with addition 

of the (S)-enantiomer of alaptide permeated 1.54-fold more than without the alaptide enantiomers. The 

permeation of rutin through the skin increased approximately linearly with time. It can be concluded that 

the presence of alaptide facilitated permeation of rutin. 

Acknowledgements: This study was supported by the centralized development project “Improving 
qualification of students of Ph.D. educational programs & postdoctorates of agricultural and veterinary 
branches through engagement in multidisciplinary interuniversity teams”. (Studie byla finančně podpořena 
centralizovaným rozvojovým projektem “Zvyšování kvalifikace studentů doktorských studijních programů 
a post-doktorandů zemědělských a veterinárních oborů zapojením do multidisciplinárních meziuniverzitních 
týmů”.) 

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