- Email: [email protected]
Percutaneous penetration and skin metabolism of ethylsalicylate-containing agent, TU-2100: in-vitro and in-vivo evaluation in guinea pigs
Journal of Controlled Release 79 (2002) 113–122 www.elsevier.com / locate / jconrel
Percutaneous penetration and skin metabolism of ethylsalicylate-containing agent, TU-2100: in-vitro and in-vivo evaluation in guinea pigs Amnon C. Sintov a , *, Clara Behar-Canetti a , Yeshayahu Friedman b , Dov Tamarkin b a
Ben Gurion University of the Negev, The Institutes for Applied Research, Beer Sheva 84105, Israel b Tamarkin Pharmaceutical Innovation Ltd., Science Park, Rehovot, Israel Received 30 May 2001; accepted 6 November 2001
Abstract The aim of this study was to investigate the percutaneous penetration and dermal metabolism of a new potential anti-acne prodrug—TU-2100 [bis(o-carboxyphenyl ethyl ester)nonanedioate] in guinea pigs. The fluxes of this agent through excised skin after applications of TU-2100 gels at 3 and 10% concentrations were similar. However, after 24 h from the time of drug application, the total amounts of permeated TU-2100 into the skin compartment and through the skin into the receiver were 271.7 (630.7 S.E.) mg / cm 2 from the 3% gel and 779.4.0 (698.5 S.E.) mg / cm 2 from the 10% gel, demonstrating a relatively high skin accumulation. Higher degradation of TU-2100 to ethylsalicylate occurred after application of drug at 10% concentration than after the application of 3% gel. In contrast, the fraction of permeated drug metabolized was twofold higher after the 3% gel application than after the 10% gel (Fm 520 vs. 10.5 mole %). Since Fm is reversibly related to the total permeating drug, the obtained values actually reflect the significant difference in TU-2100 permeation from the 3% (271.7 mg) and the 10% (779.4 mg) gels. An in vivo–in vitro comparison revealed similar drug accumulations in the skin after application of both 3 and 10% gels, however, skin metabolism was found to be significantly higher in vivo than in vitro. 2002 Elsevier Science B.V. All rights reserved. Keywords: TU-2100; Ethylsalicylate; Skin permeation; Drug metabolism; Topical anti-acne drug
1. Introduction The viable skin serves as a first-pass metabolic organ for many drugs [1]. It is, therefore, important to trace the metabolic pathways of topical drugs and quantify the metabolite formation during percuta*Corresponding author. Institutes for Applied Research, BenGurion University of the Negev, P.O. Box 653, Beer Sheva 84105, Israel. Tel.: 1972-8-6472-709; fax: 1972-8-6472-960. E-mail address: [email protected] (A.C. Sintov).
neous absorption studies, in particular, when active or toxic metabolites are involved. Salicylate esters are a good example for one of the most popular over-the-counter group of topical agents, which undergo skin hydrolysis to an active metabolite. It has been well documented that the skin esterases can rapidly hydrolyze the topically applied salicylate esters and diesters to salicylic and salicyluric acids [2–5]. This cutaneous hydrolysis has been exploited in the design of a new investigational prodrug, TU-
0168-3659 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 01 )00531-4
114
A.C. Sintov et al. / Journal of Controlled Release 79 (2002) 113 – 122
2100 (Azasal-2100—CTFA Dictionary of Cosmetic Ingredients). The drug is composed of two commonly used anti-acne drugs—azelaic acid and salicylate (as ethyl ester) (Fig. 1). Azelaic acid is a C9dicarboxylic acid, which in controlled studies demonstrated (as a 20% cream) comparable anti-acne efficacy to 0.05% topical tretinoin, 5% benzoyl peroxide, and 2% erythromycin [6–9]. As reported [9], approximately 6 mg of the drug was absorbed after 12-h application of 1 g of 20% azelaic acid cream. Salicylic acid is widely used as an over-thecounter topical medication at concentrations up to 2% for a variety of skin diseases, such as acne, psoriasis, ichthyosis and related skin disorders [10]. It is postulated that by the virtue of its in-situ formed active components (at sub-toxic levels) combined with its lipophilicity, TU-2100 can be used as a relatively more effective and safer agent in the treatment of acne and seborrheic dermatitis. In the present study, the percutaneous penetration of TU-2100 was examined in guinea pig skin with two preparations containing 3 and 10% of the parent drug. The metabolism of TU-2100 has been assessed
and the correlation between the in vitro skin diffusion profiles and the obtained in vivo skin accumulation data is discussed.
2. Materials and methods
2.1. Chemicals and drugs TU-2100 was synthesized by Tamarkin Pharmaceutical Innovation (TPI) (Rehovot, Israel). The TU2100 preparations were formulated in ethanol–polyethylene glycol 400 (PEG 400, obtained from Merck, Darmstadt, Germany) (1:3, v / v). TU-2100 was first dissolved in ethanol, and then PEG 400 was added gradually while mixing. The somewhat viscous solutions were considered as gels, although no classical polymeric gelling agents were involved. Similar vehicle preparations with equivalent concentrations (as to 10% TU-2100 on a molar basis) of ethylsalicylate (with and without equimolar concentration of azelaic acid), and salicylic acid were prepared and examined for comparison purposes. Salicylic acid, ethylsalicylate, salicyluric acid and azelaic acid were purchased from Sigma Israel (Rehovot, Israel). HPLC-grade solvents were obtained from Merck.
2.2. Diffusion cells The permeability of topical TU-2100 through guinea pig skin was measured in-vitro with a Franz diffusion cell system (Crown Bioscientific, Clinton, NJ, USA). The diffusion area was 1.767 cm 2 (15 mm diameter orifice), and the receptor compartment volumes varied between 11.1 to 12.1 ml. The solutions on the receiver side were stirred by externally driven, PTFE-coated magnetic bars.
2.3. Skin preparation and permeation study
Fig. 1. The chemical structure of TU-2100.
Male guinea pigs (Dunken Hartley, 600–700 g) were supplied by Harlan Laboratories Breeding Center, Ein Karem, Jerusalem, Israel. The guinea pigs were killed by aspiration of ethyl ether vapors and the abdominal hair was then trimmed off with a hair clipper. Sections of full-thickness abdominal skin were excised from the fresh carcasses of the
A.C. Sintov et al. / Journal of Controlled Release 79 (2002) 113 – 122
animals. Subcutaneous fat was removed with a scalpel, and the skin sections were mounted in the diffusion cells. The skin was placed with the stratum corneum facing up on the receiver chambers, and then the donor chambers were clamped in place. The excess skin was trimmed off and the receiver chamber, defined as the side facing the dermis, was filled with phosphate buffer (4 mM, pH 7.4). After 30 min of skin washing performed at 37 8C, the buffer was removed from the cells. Drug specimens (100 mg) were carefully applied over the skin, and the receiver chambers were filled with phosphate buffer (4 mM, pH 7.4)–ethanol (analytical grade) (1:1). This alcoholic medium was found to be useful by providing a sink condition, while 2% bovine serum albumin in isotonic buffer had failed to solubilize the drug and its metabolites in the receiver compartment. Samples (2 ml) were withdrawn from the receiver solution at predetermined time intervals, and the cells were replenished to their marked volumes with fresh buffer–ethanol solution. Addition of solution into the receiver compartment was performed with great care to avoid trapping air beneath the dermis. The samples were taken into 2-ml amber vials at t50, 3, 5, 7, 9, 12, 20, and 24 h. The sealed vials were kept at 4 8C usually for no more than 2 days until analyzed by high-performance liquid chromatography (HPLC). To validate the data, reproducibility was tested by analyzing the samples immediately after sampling and after 2 days. Since data were found comparable, it has been assumed that enzymatic reactions in the samples were stopped while refrigerated, or alternatively, might not have occurred in the ethanol-containing receiver compartment.
2.4. TU-2100 application on animal skin in vivo Four anesthetized (15 mg / kg pentobarbital sodium i.p.) male guinea pigs (Dunken Hartley, 600–700 g) were placed on their back. The abdominal skin, which its hair had been trimmed off 1 day previously, was washed gently with distilled water. Anesthesia was maintained with 0.1 ml pentobarbital (15 mg / ml) along the experiment. Two small cylinders (15 mm diameter orifice) were attached to the abdomen by silicon glue. One cylinder was located in the lower area of the animal’s abdomen and the
115
other cylinder was located toward the frontal area, approximately 5 cm away. Specimens of 3 and 10% TU-2100 preparations were weighed (100 mg), were put into the open cylinder and were allowed to be spread over the entire area (1.77 cm 2 ). Two animals bearing together four cylinders (n52; four applications) were treated with 3% gel and the other two animals with four more cylinders were treated with 10% gel. After 8 h of in vivo study, the remaining formulations were taken out of the cylinders, the cylinders were removed and the guinea pigs were killed by aspiration of ethyl ether. The drug-exposed skin areas were swabbed 3–4 times with three layers of gauze pads, washed for 30 s with running water, wiped carefully and harvested from the animals.
2.5. Skin extraction After 24 h of in vitro and 8 h of in vivo studies, the drug-exposed skin sections were rinsed with phosphate buffer (pH 7.4), cut to small pieces and inserted in 2-ml vials. The skin pieces in each vial were extracted by 1-ml ethanol. Each extraction was performed by incubation in a 50 8C shaking water bath (150 rpm) for 1 h. The extracts were injected into the HPLC system after 1:1 dilution with phosphate buffer, pH 7.4.
2.6. HPLC analysis of samples from receiver solutions and skin extracts Aliquots of 50 ml from each vial were injected into the HPLC system, equipped with a prepacked C 18 column (LiChrospher 60 RP-select B, 5 mm, 12534 mm). The HPLC system consisted of a Perkin-Elmer Model LC 250 Pump, and a diode array detector Model 235C. TU-2100 and ethylsalicylate were eluted using an isocratic mobile phase consisting of acetonitrile–water (70:30), and salicylic acid was eluted and chromatographed separately using acetonitrile–phosphate buffer (0.02 M, pH 2) (30:70). A flow rate of 1.5 ml / min was used. Calibration curves over the range of 1–120 mg / ml were linear. The detection limit was 0.1 mg / ml. Azelaic acid concentrations in the receiver solutions were analyzed by HPLC with fluorescence detection according to the method published by Gatti et al. [11].
A.C. Sintov et al. / Journal of Controlled Release 79 (2002) 113 – 122
116
2.7. Calculations 2.7.1. Calculations of Fm values from in vitro data The fraction of TU-2100 dose metabolized in mole % was calculated according to the following equation: Fraction metabolized 5 Fm
f M /(2Mw,ES ) g 3 100 5 ]]]]]]] f M /(2Mw,ES ) 1 Pp /Mw,P g
metabolites cleared by the blood circulation. It is just the mole % of detected dermal metabolites as related to the total drug and its metabolites found in the skin: Fm in skin 5
sMES /2Mw,ES 1MSA /2Mw,SA 1MSU /2Mw,SUd 3100 ]]]]]]]]]]]]]] M s ES /2Mw,ES 1MSA /2Mw,SA 1MSU /2Mw,SU 1Pp /Mw,Pd (3)
(1) Total ethylsalicylate formed 5 M 5 MR 1 MS Total unchanged drug permeated 5 Pp 5 PR 1 PS where MR and MS are the quantities of the metabolite found (in mg) in the receiver and the skin, respectively. PR and PS are the quantities of the parent drug (in mg) found in the receiver and the skin, respectively. Mw,ES and Mw,P are the molecular weights of ethyl salicylate (166.17) and TU-2100 (484.56), respectively. The fraction of ethylsalicylate (as a parent drug) dose metabolized in mole % was calculated according to the following equation:
where MES , MSA and MSU are the quantities of the metabolites found (in mg) in the skin. Pp is the quantity of the parent drug (in mg) found in the skin. Mw,ES , Mw,SA , Mw,SU and Mw,P are the molecular weights of ethyl salicylate (166.17), salicylic acid (138.12), salicyluric acid (195.19), and TU-2100 (484.56), respectively.
3. Results
3.1. In vitro skin penetration The cumulative permeation of TU-2100 from alcohol–PEG 400 gels per unit of skin surface area is presented in Fig. 2. The figure illustrating the kinetic
Fraction of ES metabolized 5 Fm
f M /Mw,SA g 3 100 5 ]]]]]]] f M /Mw,SA 1 Pp /Mw,ES g
(2)
Total salicylic acid formed 5 M 5 MR 1 MS Total unchanged drug permeated 5 Pp 5 PR 1 PS where MR and MS are the quantities of the metabolite found (in mg) in the receiver and the skin, respectively. PR and PS are the quantities of the parent drug (in mg) found in the receiver and the skin, respectively. Mw,ES and Mw,SA are the molecular weights of ethyl salicylate (166.17) and salicylic acid (138.12), respectively.
2.7.2. Calculations of Fm values from in vivo data The fraction of TU-2100 dose metabolized in the skin (Fm ) was calculated according to Eq. (3). It should be noted that the fraction metabolized of the in vivo study does not include the TU-2100 and its
Fig. 2. Percutaneous penetration of 3 and 10% TU-2100 in gels after application on guinea pig skin in diffusion cells (n56).
A.C. Sintov et al. / Journal of Controlled Release 79 (2002) 113 – 122
profile of TU-2100 skin penetration, presents a lag period of 5–7 h followed by a pseudo-steady state flux. After 24 h, the total amounts of permeated TU-2100 into the skin compartment and through the skin into the receiver compartment were 271.7 (630.7 S.E.) mg / cm 2 from the 3% gel and 779.4.0 (698.5 S.E.) mg / cm 2 from the 10% gel (Table 1). As shown, most of the TU-2100 is accumulated in the skin, a fact that usually categorizes the drug as a topical agent. While the 10% product exhibited about threefold higher skin levels than the 3% gel (t-test, P,0.05), treatment with the latter product resulted in a comparable percentage of total penetration of TU-2100 from the dosage form into and through the skin (9.05 vs. 7.79%, P.0.05). Ethylsalicylate and azelaic acid formation data as detected in the receiver chambers are presented in Fig. 3. Table 2 shows the metabolite (ethylsalicylate) formation and accumulation in the skin and the receiver after 24 h. No salicylic acid was detected. According to our findings, higher degradation of TU-2100 occurred after administration of 10% gel than after 3% gel was applied on the skin (t-test, P,0.05). In contrast, the fraction of permeated dose metabolized (Fm ) was twofold higher after 3% gel application than after the 10% gel. A total of 44.7 mg ethylsalicylate was found after 24-h application of 3% gel (equal to 20 mole % fraction metabolized, Fm ) comparing to 59.9 mg after application of 10% gel (Fm 510.5 mole %). Since Fm is reversibly related to the total permeating drug (of Table 1), the obtained values (Table 2) actually reflect the significant difference in TU-2100 permeation from the 3% (271.7 mg) and the 10% (779.4 mg) gels. Percutaneous penetration and biotransformation of TU-2100 was compared to its metabolite after application on skin as a drug at the same molar concentrations as in 10% TU-2100, i.e., 6.9% ethylsalicylate in the same gel vehicle. Since the
117
vehicle used (i.e., PEG 400 and ethanol) might influence the skin and play a role as a penetration enhancer, this comparison was only aimed to address differences in formulation-specific permeability values and not to explore molecular-based parameters of skin permeation. An equivalent concentration of free azelaic acid (3.9%) was applied to test its influence on ethylsalicylate penetration. Fig. 4 shows the skin penetration of ethylsalicylate after application of 6.9% solution with and without 3.9% azelaic acid. Fig. 5 presents the concomitant formation of salicylic acid during these applications. Table 3 presents the skin and the receiver contents of salicylic acid and ethylsalicylate after 24-h dermal application of 6.9% ethylsalicylate gel with and without 3.9% azelaic acid, and after application of 5.7% salicylic acid, an equivalent concentration as in the TU-2100 solution. It has been shown that ethylsalicylate penetrated through the skin at higher rate and extent than TU-2100. More than 2 mg / cm 2 skin surface area penetrated after 12 h, while only 10 mg / cm 2 penetrated through the skin at that time. In addition, about 57% of the ethylsalicylate dose was permeated from the vehicle after 24 h, 98% of which was found out of the skin (in the receiver). In contrast, only 7.8% of TU-2100 permeated from the vehicle to the skin and through the skin, from which most of the drug accumulated in the skin (approximately 92%). The metabolism of the permeated TU-2100 to ethylsalicylate was 10.5 % (Table 2), while the biotransformation of permeated ethylsalicylate (applied as drug) to salicylic acid was only 1.28% (Table 3). Another important result was noted, in which the presence of azelaic acid significantly inhibited the penetration of ethylsalicylate through the skin as well as its hydrolysis to salicylic acid. The mechanism by which azelaic acid decreases the rate of ethylsalicylate penetration still remains to be explored.
Table 1 Parent drug accumulation in skin and in the receiver after 24 h [mean (6S.E.)] (n56) Product
Skin In mg / cm skin area
3% 10%
2
128.4 (17.3) 410.4 (50.9)
In mg / g skin weight 1185.8 (161.6) 4292.9 (730.3)
Receiver, in mg / cm 2 skin area
Total drug permeating to and through the skin (mg)
% of applied dose
25.33 (6.28) 30.72 (5.36)
271.67 (30.70) 779.42 (98.51)
9.05 (1.02) 7.79 (0.98)
118
A.C. Sintov et al. / Journal of Controlled Release 79 (2002) 113 – 122
drug content in this in vivo study was similar to the drug content found after 24 h in skin of the in vitro penetration study (Fig. 6a). That is, 456.32 vs. 410.4 mg / cm 2 that permeated to the skin from the 10% gel and 93.45 vs. 128.4 mg / cm 2 from the 3% gel. Although this particular comparison of skin drug contents is related to different time intervals, it may still be a sufficient indicative comparison in cases where a steady state is attained. In contrast to the drug accumulation, the extent of the skin metabolism was found to be significantly higher in the in vivo permeation than in the in vitro (Table 4 and Fig. 6b). Besides, salicylic acid formation in the skin was noted as well as its further conjugation with glycine to salicyluric acid. In addition, an unidentified metabolite was chromatographed as a peak that followed salicylic acid. The quantity of this unidentified peak in the skin was twofold higher after 10% application of TU-2100 than after 3% gel application. In comparison, no salicylic acid or salicyluric acid or any detectable salicylic-related metabolite was found in the excised skin sections used in the in vitro diffusion testing.
4. Discussion
Fig. 3. The in situ formation of (a) ethylsalicylate and (b) azelaic acid after application of 3% and 10% TU-2100 in gels on guinea pig skin in diffusion cells (n56).
3.2. In vivo skin absorption The cutaneous permeation of TU-2100 after 8 h of application of 3 and 10% gels was tested in cylinders placed on the living animal’s abdomen. The results are shown in Table 4. Interestingly, the cutaneous
The fractions of the TU-2100 formed in vivo as metabolites (Fm ) in the skin were calculated and presented in Table 4. As can be seen, these values are found to be dose dependent and about the same as the Fm values calculated from the in vitro data (Table 2), of course, after ignoring the different times of skin sampling by assuming that a steady state has been attained. It should be noted that the Fm values calculated from the in vitro results are apparently not the same as the in vivo skin values, expressing the total fraction of TU-2100 as metabolites (inside and across the skin) rather than those only accumulated in the skin (see explanation in Materials and methods). There is still a question whether this similarity reflects a true comparison and a real correlation between the in vitro and the in vivo conditions. However, it should be noted that this similarity is true only if two assumptions are made: (a) the same permeation flux of the drug (at a steady state) occurs from the vehicle to the skin in both in vitro and in vivo methods, and (b) none or very
A.C. Sintov et al. / Journal of Controlled Release 79 (2002) 113 – 122
119
Table 2 Ethylsalicylate found in the skin and in the receiver after 24 h [mean (6S.E.)] (n56) Product
3% 10%
Skin In mg / cm 2 skin area
In mg / g skin weight
0.37 (0.09) 0.84 (0.14)
3.32 (0.77) 8.17 (0.52)
Receiver, in mg / cm 2 (mg)
Total salicylate found (mole %)*
Fraction of dose metabolized
24.92 (2.75) 33.07 (2.60)
44.68 (4.86) 59.93 (4.42)
20.00 (2.51) 10.50 (1.13)
*Calculated according to Eq. (1) (Materials and methods).
minor drug and metabolites are cleared within 8 h from the skin to the systemic circulation. The first assumption is apparently true (see Fig. 6a), and it can also be assumed according to the results that very minor drug is penetrated in vivo through the skin into the circulation, however, the formed salicylates are probably cleared very rapidly from the skin as reflected in the in vitro results (see receiver in Tables 2 and 3). It is reasonable to postulate that the clearance of ethylsalicylate and other metabolites from the skin would not have been significantly changed from the in vitro to the in vivo method. Therefore, the observed difference in skin accumulation of ethylsalicylate as a metabolite between the in vitro and the in vivo (Fig. 6b) is probably due to the
difference in metabolism and skin viability, but not to the difference in the metabolite clearance. Another finding of this research have shown that the skin accumulation of ethylsalicylate formed after TU2100 application was lower than the ethylsalicylate accumulation after its application as a free drug. This may be explained by the slow degradation of TU2100 in the skin and releasing of ethylsalicylate in a prolonged manner. Thus, while this might be accompanied by relatively lower toxic effects, the free ethylsalicylate application will provide higher drug content for a relatively short time, which may cause adverse side effects. The rapid diffusion and accumulation of lipophilic salicylates have been supported by Megwa et al. [3] who reported on a direct
Fig. 4. Percutaneous penetration of 6.9% ethylsalicylate (ES) solution with or without 3.9% azelaic acid (AZA) [equivalent concentrations as in 10% TU-2100] after application on guinea pig skin in diffusion cells (n54).
Fig. 5. The in situ formation of salicylic acid after application of 6.9% ethylsalicylate (ES) solution with or without 3.9% azelaic acid (AZA) [equivalent concentrations as in 10% TU-2100] on guinea pig skin in diffusion cells (n54).
A.C. Sintov et al. / Journal of Controlled Release 79 (2002) 113 – 122
120
Table 3 Ethylsalicylate as a parent drug and its metabolite found in the skin and in the receiver after 24 h [mean (6S.E.)] (n54) Preparation
Analyte
Skin
Receiver, in mg / cm 2 (mg)
Total salicylate found (mole %)*
439.4 (52.0) Not detected
2189.7 (189.4) 23.90 (0.66)
3964.7 (334.8) 42.25 (1.17)
1.28 (0.10)
22.00 (3.64) Not detected
214.2 (43.1) Not detected
1340.0 (115.1) 6.37 (1.85)
2406.8 (205.2) 11.25 (3.27)
0.56 (0.15)
131.28 (4.42)
1062.5 (67.2)
978.8 (119.4)
1963.3 (203.2)
In mg / cm 2 skin area
In mg / g skin weight
ES SA
54.05 (5.75) Not detected
6.9% ES1 3.9% AZA
ES SA
5.7% SA
SA
6.9% ES
Fraction of dose metabolized
ES5Ethylsalicylate; SA5salicylic acid; AZA5azelaic acid. *Calculated according to Eq. (2) (Materials and methods).
penetration of methyl salicylate accompanying with high levels in deep tissues and blood stream and poor metabolism to salicylic acid. It has also been evidenced by Cross et al. [4] when significant levels of salicylate were detected in the dermis and subcutaneous tissue of volunteers treated with methylsalicylate formulations. The in vitro data presented in this paper support previous indications that direct tissue penetration, and not solely redistribution by the systemic blood circulation, results in skin accumulation and increased deeper tissue concentrations of salicylate [3,4] and salicylic acid [12]. This paper is another evidence of what it has already been confirmed, that the skin is a metabolic
organ capable of hydrolyzing esters [2,4,5,13,14]. Cross et al. [4] performed microdialysis studies with commercial salicylate esters applied on human skin in vitro and on human volunteers. As reported by the researchers, significant amounts of the parent esters, methyl- and glycolsalicylate, penetrated into the receptor fluid, suggesting a relatively low rate of hydrolysis of these substances by skin esterases. The in vivo human microdialysis, however, confirmed the high esterase activity in viable skin as no trace of parent esters were observed. The present paper similarly demonstrates the difference in esterase activity between viable and non-viable skin. Boehnlein et al. [5] detected salicylic acid and salicyluric
Table 4 Accumulation of parent drug and its metabolites in skin after 8 h in vivo [mean (6S.E.)] (n52 animals; total of four applications) Preparation
Analyte
3% TU-2100
TU-2100 ES SA SU
93.45 17.67 0.17 0.79
(618.71) (63.51) (60.04) (60.11)
TU-2100 ES SA SU
456.32 25.78 0.17 0.74
(685.86) (65.70) (60.03) (60.08)
10% TU-2100
Accumulation, in mg / cm 2 skin area
Total metabolites detected (nmole)
Fraction of parent drug as identified metabolites (mole %)*
222.0 (643.5)
22.53 (63.16)
319.2 (668.7)
8.32 (61.89)
ES5Ethylsalicylate; SA5salicylic acid; SU5salicyluric acid. *Similar calculation as in Eq. (3) (Materials and methods). Note that the fraction metabolized is not related to the dose of permeating drug into the body. It is the mole % of detected dermal metabolites as related to the total drug and its metabolites found in the skin.
A.C. Sintov et al. / Journal of Controlled Release 79 (2002) 113 – 122
121
times) than the in vivo metabolism. It was found that 1.88 (62.18 S.E.) mg / cm 2 ethylsalicylate were formed in the skin placed over 3% bovine serum albumin, which is only twofold higher than the value found in skin placed on ethanol–buffer fluid. The apparent difference between the two techniques of in vitro testing was probably due to a non-sink condition for the lipophilic TU-2100 in the bovine serum albumin solution, while the ethanol–buffer provided a sink condition for the drug and its metabolite. To better simulate in vivo conditions when metabolism is measured, further investigations are needed to find out the optimal physiological buffer that keeps the skin viable for long time and also provides a perfect sink condition for in vitro studies.
5. Conclusion
Fig. 6. An in vitro–in vivo comparison of (a) the TU-2100 accumulation in the skin after application of 3 and 10% gels, and (b) the in situ formed ethylsalicylate after application of 3 and 10% TU-2100 gels. Note that TU-2100 and its metabolites were determined in the skin after 8 h of in vivo study vs. 24 h of the in vitro experiments.
acid after methylsalicylate application onto hairless guinea pig skin with 38 and 56% of absorbed dose metabolized in nonviable and viable skin, respectively. Similarly to the cited paper, in which a viable and a nonviable skin were compared, we found salicyluric acid (glycine conjugate) only in vivo, while at the in vitro condition only esterase activity was observed. Interestingly, the viability of the excised skin in the in vitro testing was enough for pertaining esterase activity even with a non-physiological, ethanol-containing fluid in the receptor compartment. In a separate test, we found that even with a physiological fluid, a buffer containing 3% bovine serum albumin, the ethylsalicylate formation from TU-2100 was significantly lower (approximately 14
Our study has provides new data on the percutaneous absorption and dermal metabolism of TU2100. By using abdominal skin of guinea pigs, this prodrug was found to show similar permeability in vitro and in vivo with a high extent of accumulation inside the skin. Although permeability and TU-2100 accumulation were comparable in vitro and in vivo, the skin metabolism of the drug decreased significantly during transport in excised skin that was tested immediately after isolation in diffusion cells. In the in vitro diffusion testing with excised skin sections, no salicylic acid or salicyluric acid or any detectable salicylic-related metabolite besides ethylsalicylate was found, and the extent of biotransformation to ethylsalicylate was found to be significantly lower than in the in vivo findings. This was also found even in skin that was perfused with physiological fluid containing serum albumin. Obviously and once again, it indicates that the common in vitro penetration studies could not be suitable for the evaluation of skin metabolism of topical prodrugs during their permeation, unless a way to preserve skin viability is used. In summary, the evident intracutaneous depot of TU-2100 (with reduced transdermal uptake), and the capability of the skin to hydrolyze its ester groups into active agents in a slow and prolonged manner make this topical agent as a relatively safer and more effective anti-acne drug candidate in daily clinical practice.
122
A.C. Sintov et al. / Journal of Controlled Release 79 (2002) 113 – 122
References [1] J. Kao, The influence of metabolism on percutaneous absorption, in: R.L. Bronaugh, H.I. Maibach (Eds.), Percutaneous Absorption, Mechanisms—Methodology—Drug Delivery, 2nd Edition, Marcel Dekker, New York, Basel, 1989, pp. 259–282. [2] D.B. Guzek, A.H. Kennedy, S.C. McNeill, E. Wakshull, R.O. Potts, Transdermal drug transport and metabolism. 1. Comparison of in vitro and in vivo results, Pharm. Res. 6 (1989) 33–39. [3] S.A. Megwa, H.A.E. Benson, M.S. Roberts, Percutaneous absorption of salicylates from some commercially available topical products containing methyl salicylate or salicylate salts in rats, J. Pharm. Pharmacol. 47 (1995) 891–896. [4] S.E. Cross, C. Anderson, M.S. Roberts, Topical penetration of commercial salicylate esters and salts using human isolated skin and clinical microdialysis studies, Br. J. Clin. Pharmacol. 46 (1998) 29–35. [5] J. Boehnlein, A. Sakr, J.L. Lichtin, R.L. Bronaugh, Characterization of esterase and alcohol dehydrogenase activity in skin. Metabolism of retinyl palmitate to retinol (vitamin A) during percutaneous absorption, Pharm. Res. 11 (1994) 1155–1159. [6] A. Fitton, K.L. Goa, Azelaic acid. A review of its pharmaco-
[7] [8] [9]
[10] [11]
[12]
[13]
[14]
logical properties and therapeutic efficacy in acne and hyperpigmentary skin disorders, Drugs 41 (1991) 780–798. P.S. Mackrides, A.F. Shaughnessy, Azelaic acid therapy for acne, Am. Fam. Physician 54 (1996) 2457–2459. H. Gollnick, M. Schramm, Topical drug treatment in acne, Dermatology 196 (1998) 119–125. Q.H. Nguyen, T.P. Bui, Azelaic acid: pharmacokinetic and pharmacodynamic properties and its therapeutic role in hyperpigmentary disorders and acne, Int. J. Dermatol. 34 (1995) 75–84. L.A. Goldsmith, Salicylic acid, Int. J. Dermatol. 18 (1979) 32–36. R. Gatti, V. Andrisano, A.M. Di Pietra, V. Cavrini, Analysis of aliphatic dicarboxylic acids in pharmaceuticals and cosmetics by liquid chromatography (HPLC) with fluorescence detection, J. Pharm. Biomed. Anal. 13 (1995) 589–595. P. Singh, M.S. Roberts, Dermal and underlying tissue pharmacokinetics of salicylic acid after topical application, J. Pharmacokinet. Biopharm. 21 (1993) 337–373. H. Bundgaard, A. Hoelgaard, B. Mollgaard, Leaching of hydrolytic enzymes from human skin in cutaneous permeation studies as determined with metronidazole and 5-fluorouracil prodrugs, Int. J. Pharm. 15 (1983) 285–292. J. Kao, M.P. Carver, Cutaneous metabolism of xenobiotics, Drug Metab. Rev. 22 (1990) 363–410.
Percutaneous penetration and skin metabolism of ethylsalicylate-containing agent, TU-2100: in-vitro and in-vivo evaluation in guinea pigs Amnon C. Sintov a , *, Clara Behar-Canetti a , Yeshayahu Friedman b , Dov Tamarkin b a
Ben Gurion University of the Negev, The Institutes for Applied Research, Beer Sheva 84105, Israel b Tamarkin Pharmaceutical Innovation Ltd., Science Park, Rehovot, Israel Received 30 May 2001; accepted 6 November 2001
Abstract The aim of this study was to investigate the percutaneous penetration and dermal metabolism of a new potential anti-acne prodrug—TU-2100 [bis(o-carboxyphenyl ethyl ester)nonanedioate] in guinea pigs. The fluxes of this agent through excised skin after applications of TU-2100 gels at 3 and 10% concentrations were similar. However, after 24 h from the time of drug application, the total amounts of permeated TU-2100 into the skin compartment and through the skin into the receiver were 271.7 (630.7 S.E.) mg / cm 2 from the 3% gel and 779.4.0 (698.5 S.E.) mg / cm 2 from the 10% gel, demonstrating a relatively high skin accumulation. Higher degradation of TU-2100 to ethylsalicylate occurred after application of drug at 10% concentration than after the application of 3% gel. In contrast, the fraction of permeated drug metabolized was twofold higher after the 3% gel application than after the 10% gel (Fm 520 vs. 10.5 mole %). Since Fm is reversibly related to the total permeating drug, the obtained values actually reflect the significant difference in TU-2100 permeation from the 3% (271.7 mg) and the 10% (779.4 mg) gels. An in vivo–in vitro comparison revealed similar drug accumulations in the skin after application of both 3 and 10% gels, however, skin metabolism was found to be significantly higher in vivo than in vitro. 2002 Elsevier Science B.V. All rights reserved. Keywords: TU-2100; Ethylsalicylate; Skin permeation; Drug metabolism; Topical anti-acne drug
1. Introduction The viable skin serves as a first-pass metabolic organ for many drugs [1]. It is, therefore, important to trace the metabolic pathways of topical drugs and quantify the metabolite formation during percuta*Corresponding author. Institutes for Applied Research, BenGurion University of the Negev, P.O. Box 653, Beer Sheva 84105, Israel. Tel.: 1972-8-6472-709; fax: 1972-8-6472-960. E-mail address: [email protected] (A.C. Sintov).
neous absorption studies, in particular, when active or toxic metabolites are involved. Salicylate esters are a good example for one of the most popular over-the-counter group of topical agents, which undergo skin hydrolysis to an active metabolite. It has been well documented that the skin esterases can rapidly hydrolyze the topically applied salicylate esters and diesters to salicylic and salicyluric acids [2–5]. This cutaneous hydrolysis has been exploited in the design of a new investigational prodrug, TU-
0168-3659 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 01 )00531-4
114
A.C. Sintov et al. / Journal of Controlled Release 79 (2002) 113 – 122
2100 (Azasal-2100—CTFA Dictionary of Cosmetic Ingredients). The drug is composed of two commonly used anti-acne drugs—azelaic acid and salicylate (as ethyl ester) (Fig. 1). Azelaic acid is a C9dicarboxylic acid, which in controlled studies demonstrated (as a 20% cream) comparable anti-acne efficacy to 0.05% topical tretinoin, 5% benzoyl peroxide, and 2% erythromycin [6–9]. As reported [9], approximately 6 mg of the drug was absorbed after 12-h application of 1 g of 20% azelaic acid cream. Salicylic acid is widely used as an over-thecounter topical medication at concentrations up to 2% for a variety of skin diseases, such as acne, psoriasis, ichthyosis and related skin disorders [10]. It is postulated that by the virtue of its in-situ formed active components (at sub-toxic levels) combined with its lipophilicity, TU-2100 can be used as a relatively more effective and safer agent in the treatment of acne and seborrheic dermatitis. In the present study, the percutaneous penetration of TU-2100 was examined in guinea pig skin with two preparations containing 3 and 10% of the parent drug. The metabolism of TU-2100 has been assessed
and the correlation between the in vitro skin diffusion profiles and the obtained in vivo skin accumulation data is discussed.
2. Materials and methods
2.1. Chemicals and drugs TU-2100 was synthesized by Tamarkin Pharmaceutical Innovation (TPI) (Rehovot, Israel). The TU2100 preparations were formulated in ethanol–polyethylene glycol 400 (PEG 400, obtained from Merck, Darmstadt, Germany) (1:3, v / v). TU-2100 was first dissolved in ethanol, and then PEG 400 was added gradually while mixing. The somewhat viscous solutions were considered as gels, although no classical polymeric gelling agents were involved. Similar vehicle preparations with equivalent concentrations (as to 10% TU-2100 on a molar basis) of ethylsalicylate (with and without equimolar concentration of azelaic acid), and salicylic acid were prepared and examined for comparison purposes. Salicylic acid, ethylsalicylate, salicyluric acid and azelaic acid were purchased from Sigma Israel (Rehovot, Israel). HPLC-grade solvents were obtained from Merck.
2.2. Diffusion cells The permeability of topical TU-2100 through guinea pig skin was measured in-vitro with a Franz diffusion cell system (Crown Bioscientific, Clinton, NJ, USA). The diffusion area was 1.767 cm 2 (15 mm diameter orifice), and the receptor compartment volumes varied between 11.1 to 12.1 ml. The solutions on the receiver side were stirred by externally driven, PTFE-coated magnetic bars.
2.3. Skin preparation and permeation study
Fig. 1. The chemical structure of TU-2100.
Male guinea pigs (Dunken Hartley, 600–700 g) were supplied by Harlan Laboratories Breeding Center, Ein Karem, Jerusalem, Israel. The guinea pigs were killed by aspiration of ethyl ether vapors and the abdominal hair was then trimmed off with a hair clipper. Sections of full-thickness abdominal skin were excised from the fresh carcasses of the
A.C. Sintov et al. / Journal of Controlled Release 79 (2002) 113 – 122
animals. Subcutaneous fat was removed with a scalpel, and the skin sections were mounted in the diffusion cells. The skin was placed with the stratum corneum facing up on the receiver chambers, and then the donor chambers were clamped in place. The excess skin was trimmed off and the receiver chamber, defined as the side facing the dermis, was filled with phosphate buffer (4 mM, pH 7.4). After 30 min of skin washing performed at 37 8C, the buffer was removed from the cells. Drug specimens (100 mg) were carefully applied over the skin, and the receiver chambers were filled with phosphate buffer (4 mM, pH 7.4)–ethanol (analytical grade) (1:1). This alcoholic medium was found to be useful by providing a sink condition, while 2% bovine serum albumin in isotonic buffer had failed to solubilize the drug and its metabolites in the receiver compartment. Samples (2 ml) were withdrawn from the receiver solution at predetermined time intervals, and the cells were replenished to their marked volumes with fresh buffer–ethanol solution. Addition of solution into the receiver compartment was performed with great care to avoid trapping air beneath the dermis. The samples were taken into 2-ml amber vials at t50, 3, 5, 7, 9, 12, 20, and 24 h. The sealed vials were kept at 4 8C usually for no more than 2 days until analyzed by high-performance liquid chromatography (HPLC). To validate the data, reproducibility was tested by analyzing the samples immediately after sampling and after 2 days. Since data were found comparable, it has been assumed that enzymatic reactions in the samples were stopped while refrigerated, or alternatively, might not have occurred in the ethanol-containing receiver compartment.
2.4. TU-2100 application on animal skin in vivo Four anesthetized (15 mg / kg pentobarbital sodium i.p.) male guinea pigs (Dunken Hartley, 600–700 g) were placed on their back. The abdominal skin, which its hair had been trimmed off 1 day previously, was washed gently with distilled water. Anesthesia was maintained with 0.1 ml pentobarbital (15 mg / ml) along the experiment. Two small cylinders (15 mm diameter orifice) were attached to the abdomen by silicon glue. One cylinder was located in the lower area of the animal’s abdomen and the
115
other cylinder was located toward the frontal area, approximately 5 cm away. Specimens of 3 and 10% TU-2100 preparations were weighed (100 mg), were put into the open cylinder and were allowed to be spread over the entire area (1.77 cm 2 ). Two animals bearing together four cylinders (n52; four applications) were treated with 3% gel and the other two animals with four more cylinders were treated with 10% gel. After 8 h of in vivo study, the remaining formulations were taken out of the cylinders, the cylinders were removed and the guinea pigs were killed by aspiration of ethyl ether. The drug-exposed skin areas were swabbed 3–4 times with three layers of gauze pads, washed for 30 s with running water, wiped carefully and harvested from the animals.
2.5. Skin extraction After 24 h of in vitro and 8 h of in vivo studies, the drug-exposed skin sections were rinsed with phosphate buffer (pH 7.4), cut to small pieces and inserted in 2-ml vials. The skin pieces in each vial were extracted by 1-ml ethanol. Each extraction was performed by incubation in a 50 8C shaking water bath (150 rpm) for 1 h. The extracts were injected into the HPLC system after 1:1 dilution with phosphate buffer, pH 7.4.
2.6. HPLC analysis of samples from receiver solutions and skin extracts Aliquots of 50 ml from each vial were injected into the HPLC system, equipped with a prepacked C 18 column (LiChrospher 60 RP-select B, 5 mm, 12534 mm). The HPLC system consisted of a Perkin-Elmer Model LC 250 Pump, and a diode array detector Model 235C. TU-2100 and ethylsalicylate were eluted using an isocratic mobile phase consisting of acetonitrile–water (70:30), and salicylic acid was eluted and chromatographed separately using acetonitrile–phosphate buffer (0.02 M, pH 2) (30:70). A flow rate of 1.5 ml / min was used. Calibration curves over the range of 1–120 mg / ml were linear. The detection limit was 0.1 mg / ml. Azelaic acid concentrations in the receiver solutions were analyzed by HPLC with fluorescence detection according to the method published by Gatti et al. [11].
A.C. Sintov et al. / Journal of Controlled Release 79 (2002) 113 – 122
116
2.7. Calculations 2.7.1. Calculations of Fm values from in vitro data The fraction of TU-2100 dose metabolized in mole % was calculated according to the following equation: Fraction metabolized 5 Fm
f M /(2Mw,ES ) g 3 100 5 ]]]]]]] f M /(2Mw,ES ) 1 Pp /Mw,P g
metabolites cleared by the blood circulation. It is just the mole % of detected dermal metabolites as related to the total drug and its metabolites found in the skin: Fm in skin 5
sMES /2Mw,ES 1MSA /2Mw,SA 1MSU /2Mw,SUd 3100 ]]]]]]]]]]]]]] M s ES /2Mw,ES 1MSA /2Mw,SA 1MSU /2Mw,SU 1Pp /Mw,Pd (3)
(1) Total ethylsalicylate formed 5 M 5 MR 1 MS Total unchanged drug permeated 5 Pp 5 PR 1 PS where MR and MS are the quantities of the metabolite found (in mg) in the receiver and the skin, respectively. PR and PS are the quantities of the parent drug (in mg) found in the receiver and the skin, respectively. Mw,ES and Mw,P are the molecular weights of ethyl salicylate (166.17) and TU-2100 (484.56), respectively. The fraction of ethylsalicylate (as a parent drug) dose metabolized in mole % was calculated according to the following equation:
where MES , MSA and MSU are the quantities of the metabolites found (in mg) in the skin. Pp is the quantity of the parent drug (in mg) found in the skin. Mw,ES , Mw,SA , Mw,SU and Mw,P are the molecular weights of ethyl salicylate (166.17), salicylic acid (138.12), salicyluric acid (195.19), and TU-2100 (484.56), respectively.
3. Results
3.1. In vitro skin penetration The cumulative permeation of TU-2100 from alcohol–PEG 400 gels per unit of skin surface area is presented in Fig. 2. The figure illustrating the kinetic
Fraction of ES metabolized 5 Fm
f M /Mw,SA g 3 100 5 ]]]]]]] f M /Mw,SA 1 Pp /Mw,ES g
(2)
Total salicylic acid formed 5 M 5 MR 1 MS Total unchanged drug permeated 5 Pp 5 PR 1 PS where MR and MS are the quantities of the metabolite found (in mg) in the receiver and the skin, respectively. PR and PS are the quantities of the parent drug (in mg) found in the receiver and the skin, respectively. Mw,ES and Mw,SA are the molecular weights of ethyl salicylate (166.17) and salicylic acid (138.12), respectively.
2.7.2. Calculations of Fm values from in vivo data The fraction of TU-2100 dose metabolized in the skin (Fm ) was calculated according to Eq. (3). It should be noted that the fraction metabolized of the in vivo study does not include the TU-2100 and its
Fig. 2. Percutaneous penetration of 3 and 10% TU-2100 in gels after application on guinea pig skin in diffusion cells (n56).
A.C. Sintov et al. / Journal of Controlled Release 79 (2002) 113 – 122
profile of TU-2100 skin penetration, presents a lag period of 5–7 h followed by a pseudo-steady state flux. After 24 h, the total amounts of permeated TU-2100 into the skin compartment and through the skin into the receiver compartment were 271.7 (630.7 S.E.) mg / cm 2 from the 3% gel and 779.4.0 (698.5 S.E.) mg / cm 2 from the 10% gel (Table 1). As shown, most of the TU-2100 is accumulated in the skin, a fact that usually categorizes the drug as a topical agent. While the 10% product exhibited about threefold higher skin levels than the 3% gel (t-test, P,0.05), treatment with the latter product resulted in a comparable percentage of total penetration of TU-2100 from the dosage form into and through the skin (9.05 vs. 7.79%, P.0.05). Ethylsalicylate and azelaic acid formation data as detected in the receiver chambers are presented in Fig. 3. Table 2 shows the metabolite (ethylsalicylate) formation and accumulation in the skin and the receiver after 24 h. No salicylic acid was detected. According to our findings, higher degradation of TU-2100 occurred after administration of 10% gel than after 3% gel was applied on the skin (t-test, P,0.05). In contrast, the fraction of permeated dose metabolized (Fm ) was twofold higher after 3% gel application than after the 10% gel. A total of 44.7 mg ethylsalicylate was found after 24-h application of 3% gel (equal to 20 mole % fraction metabolized, Fm ) comparing to 59.9 mg after application of 10% gel (Fm 510.5 mole %). Since Fm is reversibly related to the total permeating drug (of Table 1), the obtained values (Table 2) actually reflect the significant difference in TU-2100 permeation from the 3% (271.7 mg) and the 10% (779.4 mg) gels. Percutaneous penetration and biotransformation of TU-2100 was compared to its metabolite after application on skin as a drug at the same molar concentrations as in 10% TU-2100, i.e., 6.9% ethylsalicylate in the same gel vehicle. Since the
117
vehicle used (i.e., PEG 400 and ethanol) might influence the skin and play a role as a penetration enhancer, this comparison was only aimed to address differences in formulation-specific permeability values and not to explore molecular-based parameters of skin permeation. An equivalent concentration of free azelaic acid (3.9%) was applied to test its influence on ethylsalicylate penetration. Fig. 4 shows the skin penetration of ethylsalicylate after application of 6.9% solution with and without 3.9% azelaic acid. Fig. 5 presents the concomitant formation of salicylic acid during these applications. Table 3 presents the skin and the receiver contents of salicylic acid and ethylsalicylate after 24-h dermal application of 6.9% ethylsalicylate gel with and without 3.9% azelaic acid, and after application of 5.7% salicylic acid, an equivalent concentration as in the TU-2100 solution. It has been shown that ethylsalicylate penetrated through the skin at higher rate and extent than TU-2100. More than 2 mg / cm 2 skin surface area penetrated after 12 h, while only 10 mg / cm 2 penetrated through the skin at that time. In addition, about 57% of the ethylsalicylate dose was permeated from the vehicle after 24 h, 98% of which was found out of the skin (in the receiver). In contrast, only 7.8% of TU-2100 permeated from the vehicle to the skin and through the skin, from which most of the drug accumulated in the skin (approximately 92%). The metabolism of the permeated TU-2100 to ethylsalicylate was 10.5 % (Table 2), while the biotransformation of permeated ethylsalicylate (applied as drug) to salicylic acid was only 1.28% (Table 3). Another important result was noted, in which the presence of azelaic acid significantly inhibited the penetration of ethylsalicylate through the skin as well as its hydrolysis to salicylic acid. The mechanism by which azelaic acid decreases the rate of ethylsalicylate penetration still remains to be explored.
Table 1 Parent drug accumulation in skin and in the receiver after 24 h [mean (6S.E.)] (n56) Product
Skin In mg / cm skin area
3% 10%
2
128.4 (17.3) 410.4 (50.9)
In mg / g skin weight 1185.8 (161.6) 4292.9 (730.3)
Receiver, in mg / cm 2 skin area
Total drug permeating to and through the skin (mg)
% of applied dose
25.33 (6.28) 30.72 (5.36)
271.67 (30.70) 779.42 (98.51)
9.05 (1.02) 7.79 (0.98)
118
A.C. Sintov et al. / Journal of Controlled Release 79 (2002) 113 – 122
drug content in this in vivo study was similar to the drug content found after 24 h in skin of the in vitro penetration study (Fig. 6a). That is, 456.32 vs. 410.4 mg / cm 2 that permeated to the skin from the 10% gel and 93.45 vs. 128.4 mg / cm 2 from the 3% gel. Although this particular comparison of skin drug contents is related to different time intervals, it may still be a sufficient indicative comparison in cases where a steady state is attained. In contrast to the drug accumulation, the extent of the skin metabolism was found to be significantly higher in the in vivo permeation than in the in vitro (Table 4 and Fig. 6b). Besides, salicylic acid formation in the skin was noted as well as its further conjugation with glycine to salicyluric acid. In addition, an unidentified metabolite was chromatographed as a peak that followed salicylic acid. The quantity of this unidentified peak in the skin was twofold higher after 10% application of TU-2100 than after 3% gel application. In comparison, no salicylic acid or salicyluric acid or any detectable salicylic-related metabolite was found in the excised skin sections used in the in vitro diffusion testing.
4. Discussion
Fig. 3. The in situ formation of (a) ethylsalicylate and (b) azelaic acid after application of 3% and 10% TU-2100 in gels on guinea pig skin in diffusion cells (n56).
3.2. In vivo skin absorption The cutaneous permeation of TU-2100 after 8 h of application of 3 and 10% gels was tested in cylinders placed on the living animal’s abdomen. The results are shown in Table 4. Interestingly, the cutaneous
The fractions of the TU-2100 formed in vivo as metabolites (Fm ) in the skin were calculated and presented in Table 4. As can be seen, these values are found to be dose dependent and about the same as the Fm values calculated from the in vitro data (Table 2), of course, after ignoring the different times of skin sampling by assuming that a steady state has been attained. It should be noted that the Fm values calculated from the in vitro results are apparently not the same as the in vivo skin values, expressing the total fraction of TU-2100 as metabolites (inside and across the skin) rather than those only accumulated in the skin (see explanation in Materials and methods). There is still a question whether this similarity reflects a true comparison and a real correlation between the in vitro and the in vivo conditions. However, it should be noted that this similarity is true only if two assumptions are made: (a) the same permeation flux of the drug (at a steady state) occurs from the vehicle to the skin in both in vitro and in vivo methods, and (b) none or very
A.C. Sintov et al. / Journal of Controlled Release 79 (2002) 113 – 122
119
Table 2 Ethylsalicylate found in the skin and in the receiver after 24 h [mean (6S.E.)] (n56) Product
3% 10%
Skin In mg / cm 2 skin area
In mg / g skin weight
0.37 (0.09) 0.84 (0.14)
3.32 (0.77) 8.17 (0.52)
Receiver, in mg / cm 2 (mg)
Total salicylate found (mole %)*
Fraction of dose metabolized
24.92 (2.75) 33.07 (2.60)
44.68 (4.86) 59.93 (4.42)
20.00 (2.51) 10.50 (1.13)
*Calculated according to Eq. (1) (Materials and methods).
minor drug and metabolites are cleared within 8 h from the skin to the systemic circulation. The first assumption is apparently true (see Fig. 6a), and it can also be assumed according to the results that very minor drug is penetrated in vivo through the skin into the circulation, however, the formed salicylates are probably cleared very rapidly from the skin as reflected in the in vitro results (see receiver in Tables 2 and 3). It is reasonable to postulate that the clearance of ethylsalicylate and other metabolites from the skin would not have been significantly changed from the in vitro to the in vivo method. Therefore, the observed difference in skin accumulation of ethylsalicylate as a metabolite between the in vitro and the in vivo (Fig. 6b) is probably due to the
difference in metabolism and skin viability, but not to the difference in the metabolite clearance. Another finding of this research have shown that the skin accumulation of ethylsalicylate formed after TU2100 application was lower than the ethylsalicylate accumulation after its application as a free drug. This may be explained by the slow degradation of TU2100 in the skin and releasing of ethylsalicylate in a prolonged manner. Thus, while this might be accompanied by relatively lower toxic effects, the free ethylsalicylate application will provide higher drug content for a relatively short time, which may cause adverse side effects. The rapid diffusion and accumulation of lipophilic salicylates have been supported by Megwa et al. [3] who reported on a direct
Fig. 4. Percutaneous penetration of 6.9% ethylsalicylate (ES) solution with or without 3.9% azelaic acid (AZA) [equivalent concentrations as in 10% TU-2100] after application on guinea pig skin in diffusion cells (n54).
Fig. 5. The in situ formation of salicylic acid after application of 6.9% ethylsalicylate (ES) solution with or without 3.9% azelaic acid (AZA) [equivalent concentrations as in 10% TU-2100] on guinea pig skin in diffusion cells (n54).
A.C. Sintov et al. / Journal of Controlled Release 79 (2002) 113 – 122
120
Table 3 Ethylsalicylate as a parent drug and its metabolite found in the skin and in the receiver after 24 h [mean (6S.E.)] (n54) Preparation
Analyte
Skin
Receiver, in mg / cm 2 (mg)
Total salicylate found (mole %)*
439.4 (52.0) Not detected
2189.7 (189.4) 23.90 (0.66)
3964.7 (334.8) 42.25 (1.17)
1.28 (0.10)
22.00 (3.64) Not detected
214.2 (43.1) Not detected
1340.0 (115.1) 6.37 (1.85)
2406.8 (205.2) 11.25 (3.27)
0.56 (0.15)
131.28 (4.42)
1062.5 (67.2)
978.8 (119.4)
1963.3 (203.2)
In mg / cm 2 skin area
In mg / g skin weight
ES SA
54.05 (5.75) Not detected
6.9% ES1 3.9% AZA
ES SA
5.7% SA
SA
6.9% ES
Fraction of dose metabolized
ES5Ethylsalicylate; SA5salicylic acid; AZA5azelaic acid. *Calculated according to Eq. (2) (Materials and methods).
penetration of methyl salicylate accompanying with high levels in deep tissues and blood stream and poor metabolism to salicylic acid. It has also been evidenced by Cross et al. [4] when significant levels of salicylate were detected in the dermis and subcutaneous tissue of volunteers treated with methylsalicylate formulations. The in vitro data presented in this paper support previous indications that direct tissue penetration, and not solely redistribution by the systemic blood circulation, results in skin accumulation and increased deeper tissue concentrations of salicylate [3,4] and salicylic acid [12]. This paper is another evidence of what it has already been confirmed, that the skin is a metabolic
organ capable of hydrolyzing esters [2,4,5,13,14]. Cross et al. [4] performed microdialysis studies with commercial salicylate esters applied on human skin in vitro and on human volunteers. As reported by the researchers, significant amounts of the parent esters, methyl- and glycolsalicylate, penetrated into the receptor fluid, suggesting a relatively low rate of hydrolysis of these substances by skin esterases. The in vivo human microdialysis, however, confirmed the high esterase activity in viable skin as no trace of parent esters were observed. The present paper similarly demonstrates the difference in esterase activity between viable and non-viable skin. Boehnlein et al. [5] detected salicylic acid and salicyluric
Table 4 Accumulation of parent drug and its metabolites in skin after 8 h in vivo [mean (6S.E.)] (n52 animals; total of four applications) Preparation
Analyte
3% TU-2100
TU-2100 ES SA SU
93.45 17.67 0.17 0.79
(618.71) (63.51) (60.04) (60.11)
TU-2100 ES SA SU
456.32 25.78 0.17 0.74
(685.86) (65.70) (60.03) (60.08)
10% TU-2100
Accumulation, in mg / cm 2 skin area
Total metabolites detected (nmole)
Fraction of parent drug as identified metabolites (mole %)*
222.0 (643.5)
22.53 (63.16)
319.2 (668.7)
8.32 (61.89)
ES5Ethylsalicylate; SA5salicylic acid; SU5salicyluric acid. *Similar calculation as in Eq. (3) (Materials and methods). Note that the fraction metabolized is not related to the dose of permeating drug into the body. It is the mole % of detected dermal metabolites as related to the total drug and its metabolites found in the skin.
A.C. Sintov et al. / Journal of Controlled Release 79 (2002) 113 – 122
121
times) than the in vivo metabolism. It was found that 1.88 (62.18 S.E.) mg / cm 2 ethylsalicylate were formed in the skin placed over 3% bovine serum albumin, which is only twofold higher than the value found in skin placed on ethanol–buffer fluid. The apparent difference between the two techniques of in vitro testing was probably due to a non-sink condition for the lipophilic TU-2100 in the bovine serum albumin solution, while the ethanol–buffer provided a sink condition for the drug and its metabolite. To better simulate in vivo conditions when metabolism is measured, further investigations are needed to find out the optimal physiological buffer that keeps the skin viable for long time and also provides a perfect sink condition for in vitro studies.
5. Conclusion
Fig. 6. An in vitro–in vivo comparison of (a) the TU-2100 accumulation in the skin after application of 3 and 10% gels, and (b) the in situ formed ethylsalicylate after application of 3 and 10% TU-2100 gels. Note that TU-2100 and its metabolites were determined in the skin after 8 h of in vivo study vs. 24 h of the in vitro experiments.
acid after methylsalicylate application onto hairless guinea pig skin with 38 and 56% of absorbed dose metabolized in nonviable and viable skin, respectively. Similarly to the cited paper, in which a viable and a nonviable skin were compared, we found salicyluric acid (glycine conjugate) only in vivo, while at the in vitro condition only esterase activity was observed. Interestingly, the viability of the excised skin in the in vitro testing was enough for pertaining esterase activity even with a non-physiological, ethanol-containing fluid in the receptor compartment. In a separate test, we found that even with a physiological fluid, a buffer containing 3% bovine serum albumin, the ethylsalicylate formation from TU-2100 was significantly lower (approximately 14
Our study has provides new data on the percutaneous absorption and dermal metabolism of TU2100. By using abdominal skin of guinea pigs, this prodrug was found to show similar permeability in vitro and in vivo with a high extent of accumulation inside the skin. Although permeability and TU-2100 accumulation were comparable in vitro and in vivo, the skin metabolism of the drug decreased significantly during transport in excised skin that was tested immediately after isolation in diffusion cells. In the in vitro diffusion testing with excised skin sections, no salicylic acid or salicyluric acid or any detectable salicylic-related metabolite besides ethylsalicylate was found, and the extent of biotransformation to ethylsalicylate was found to be significantly lower than in the in vivo findings. This was also found even in skin that was perfused with physiological fluid containing serum albumin. Obviously and once again, it indicates that the common in vitro penetration studies could not be suitable for the evaluation of skin metabolism of topical prodrugs during their permeation, unless a way to preserve skin viability is used. In summary, the evident intracutaneous depot of TU-2100 (with reduced transdermal uptake), and the capability of the skin to hydrolyze its ester groups into active agents in a slow and prolonged manner make this topical agent as a relatively safer and more effective anti-acne drug candidate in daily clinical practice.
122
A.C. Sintov et al. / Journal of Controlled Release 79 (2002) 113 – 122
References [1] J. Kao, The influence of metabolism on percutaneous absorption, in: R.L. Bronaugh, H.I. Maibach (Eds.), Percutaneous Absorption, Mechanisms—Methodology—Drug Delivery, 2nd Edition, Marcel Dekker, New York, Basel, 1989, pp. 259–282. [2] D.B. Guzek, A.H. Kennedy, S.C. McNeill, E. Wakshull, R.O. Potts, Transdermal drug transport and metabolism. 1. Comparison of in vitro and in vivo results, Pharm. Res. 6 (1989) 33–39. [3] S.A. Megwa, H.A.E. Benson, M.S. Roberts, Percutaneous absorption of salicylates from some commercially available topical products containing methyl salicylate or salicylate salts in rats, J. Pharm. Pharmacol. 47 (1995) 891–896. [4] S.E. Cross, C. Anderson, M.S. Roberts, Topical penetration of commercial salicylate esters and salts using human isolated skin and clinical microdialysis studies, Br. J. Clin. Pharmacol. 46 (1998) 29–35. [5] J. Boehnlein, A. Sakr, J.L. Lichtin, R.L. Bronaugh, Characterization of esterase and alcohol dehydrogenase activity in skin. Metabolism of retinyl palmitate to retinol (vitamin A) during percutaneous absorption, Pharm. Res. 11 (1994) 1155–1159. [6] A. Fitton, K.L. Goa, Azelaic acid. A review of its pharmaco-
[7] [8] [9]
[10] [11]
[12]
[13]
[14]
logical properties and therapeutic efficacy in acne and hyperpigmentary skin disorders, Drugs 41 (1991) 780–798. P.S. Mackrides, A.F. Shaughnessy, Azelaic acid therapy for acne, Am. Fam. Physician 54 (1996) 2457–2459. H. Gollnick, M. Schramm, Topical drug treatment in acne, Dermatology 196 (1998) 119–125. Q.H. Nguyen, T.P. Bui, Azelaic acid: pharmacokinetic and pharmacodynamic properties and its therapeutic role in hyperpigmentary disorders and acne, Int. J. Dermatol. 34 (1995) 75–84. L.A. Goldsmith, Salicylic acid, Int. J. Dermatol. 18 (1979) 32–36. R. Gatti, V. Andrisano, A.M. Di Pietra, V. Cavrini, Analysis of aliphatic dicarboxylic acids in pharmaceuticals and cosmetics by liquid chromatography (HPLC) with fluorescence detection, J. Pharm. Biomed. Anal. 13 (1995) 589–595. P. Singh, M.S. Roberts, Dermal and underlying tissue pharmacokinetics of salicylic acid after topical application, J. Pharmacokinet. Biopharm. 21 (1993) 337–373. H. Bundgaard, A. Hoelgaard, B. Mollgaard, Leaching of hydrolytic enzymes from human skin in cutaneous permeation studies as determined with metronidazole and 5-fluorouracil prodrugs, Int. J. Pharm. 15 (1983) 285–292. J. Kao, M.P. Carver, Cutaneous metabolism of xenobiotics, Drug Metab. Rev. 22 (1990) 363–410.