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In vitro and in vivo evaluation of the delivery of topical formulations containing glycoalkaloids of Solanum lycocarpum fruits
European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx
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European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb
Research paper
In vitro and in vivo evaluation of the delivery of topical formulations containing glycoalkaloids of Solanum lycocarpum fruits Renata F.J. Tiossi a,1, Juliana C. Da Costa a,1, Mariza A. Miranda a, Fabíola S.G. Praça a, James D. McChesney b, Maria Vitória L.B. Bentley a, Jairo K. Bastos a,⇑ a b
Faculty of Pharmaceutical Science of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil Arbor Therapeutics, LLC, Etta, MS, USA
a r t i c l e
i n f o
Article history: Received 19 September 2013 Accepted in revised form 31 January 2014 Available online xxxx Keywords: Solanum lycocarpum Glycoalkaloids Solasonine Solamargine Topical delivery Skin cancer
a b s t r a c t The glycoalkaloids solasonine (SN) and solamargine (SM) have been studied for their antiparasitic, antifungal, and anticancer properties, especially in vitro and in vivo against non-melanoma skin cancer. Thus, the alkaloidic extract of Solanum lycocarpum, which contains approximately 45% each of SN and SM, was used to define the best experimental conditions for in vitro and in vivo assays. The in vitro assays were performed with the Franz cell diffusion porcine skin model to evaluate the effects of different pHs and the presence of monoolein, ethoxydiglycol or ethanol penetration enhancers on the skin penetration and retention of SN and SM after 3, 6, 9 and 12 h of exposure. The in vivo assay was performed on hairless mice with the formulation selected in the in vitro assays. The results showed that pH 6.5 was optimal for SM penetration. The formulation containing 5% alkaloidic extract, 5% propylene glycol, 5% monoolein and a hydroxyethyl cellulose gel base (NatrosolÒ) (pH 6.5) was optimal for the delivery of SN and SM into the skin, and this formulation is potentially useful for the topical therapy of several skin disorders. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Solanum lycocarpum A. St.-Hil. (Solanaceae) is a native species of Brazilian Cerrado belonging to the genus Solanum that is known for the biosynthesis of glycoalkaloids. The glycoalkaloids solasonine (SN) and solamargine (SM) (Fig. 1) are found in the fruits of more than a hundred species of Solanum [1], but S. lycocarpum stands out for its production of these compounds. SN and SM can be obtained by selectively extracting them from the fruits of this species [2]. These compounds are structurally similar because they contain the same steroidal moiety, solasodine, differing only in their sugar chain moieties: solatriose for SN and chacotriose for SM [3]. SN and SM have been studied as antiparasitics [4,5], antidiabetics [3], anti-virals against herpes [6], antifungals [7–9], immunomodulators [10], and anticancer agents in several cell lines [11–15] and in vivo against human skin cancer [16,17]. Studies have shown that SN and SM are selective for cancerous cells due to the sugar chain moiety, mainly rhamnose, of which SN ⇑ Corresponding author. Faculty of Pharmaceutical Science of Ribeirão Preto, University of São Paulo, Av. do Café, s/n, Ribeirão Preto, SP 14040-903, Brazil. Tel.: +55 16 36024230; fax: +55 16 36024230, 16 36024879. E-mail address: [email protected] (J.K. Bastos). 1 These authors contributed equally to this work.
and SM contain one and two units, respectively [18,19]. These compounds have been studied to treat non-melanoma skin cancer by employing a mixture of glycoalkaloids extracted from Solanum species [18]. However, the efficacy of these compounds not only depends on the quantity used but also on the composition of the topical formulation. For instance, cetomacrogol-based cream with 10% glycoalkaloids and 10% dimethylsulfoxide (DMSO) effected an 83% skin carcinoma cure rate [16], while another formulation with 0.005% glycoalkaloids and keratolytic agents was 100% curative [20]. In contrast, a 66% cure rate was achieved with 0.005% glycoalkaloids and keratolytic agents in emulsifying wax and white soft paraffin base [17]. It is important to note that these studies were performed with an alkaloidic mixture from another Solanum species, which is composed of 33% SN, 33% SM and 34% di- and mono-glycosides of solasodine [18], while the alkaloidic extract from S. lycocarpum studied in this work contained approximately 45% SN and 45% SM [21]. It is desirable that topical formulations adequately allow for skin penetration and retention with minimal systemic absorption [22]. In this context, penetration enhancers have been widely used in formulations to improve the skin penetration of several compounds [23]. Moreover, it is known that glycoalkaloids have a basic character, which plays an important role in their skin permeability, depending on their degree of ionization, solubility in the applied
http://dx.doi.org/10.1016/j.ejpb.2014.01.010 0939-6411/Ó 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: R.F.J. Tiossi et al., In vitro and in vivo evaluation of the delivery of topical formulations containing glycoalkaloids of Solanum lycocarpum fruits, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.01.010
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Fig. 1. Structures of solasodine and their glycosides: solasonine (SN) and solamargine (SM).
phase and partitioning into the skin [24]. Taking into consideration that penetration and the consequent effects of glycoalkaloids in the skin could be greatly influenced by the formulation pH [25], it is mandatory to understand the physicochemical properties of these compounds and the skin penetration of SN and SM in the development and optimization of new skin anticancer therapies. Therefore, considering the remarkable activity of glycoalkaloids on cancer cells and the lack of data on their topical delivery, as well as their high content in the alkaloidic extract of S. lycocarpum fruits, we have performed studies on the development of a topical formulation with the ability to deliver these compounds into deeper skin layers so that they can be used in the treatment of non-melanoma skin cancer. 2. Material and methods
described in our previous work [21]. Briefly, a Zorbax SB-C18 analytical reverse phase column was used as stationary phase, and the binary gradient consisted of sodium phosphate buffer (pH 7.2, 0.01 M) (pump A) and MeCN (pump B). Thus, the calibration curves were constructed for both glycoalkaloids, and linearity was achieved for concentrations in the range of 0.77–990.00 lg/mL for SN and 0.78–1000.00 lg/mL for SM, with correlation coefficients P 0.999. The retention times of SN and SM were 10.35 and 12.35 min, respectively. The intra-assay and inter-assay variations were less than 9.18% for SN and 8.74% for SM. The method recoveries were evaluated for samples of stratum corneum (SC) and epidermis plus dermis (EP + D), determined at three concentrations, and the values were higher than 88.94% for SN and 94.75% for SM. The limits of detection and quantification were 0.29 lg/mL and 0.86 lg/mL for SN, and 0.57 lg/mL and 1.74 for SM, respectively [21].
2.1. Chemicals and reagents High performance liquid chromatography (HPLC)-grade acetonitrile (MeCN) and methanol (MeOH) were obtained from Mallinckrodt Co. (Xalostoc, Mexico). Anhydrous disodium hydrogen phosphate was acquired from Carlo Erba Reagents (Brazil). Deionized water was purified by Milli-Q-plus filter systems (Millipore, USA). The analytical grade cetylpyridinium chloride, ethanol (EtOH), propylene glycol, hydrochloric acid, sodium hydroxide, sodium phosphate, methylparaben (NipaginÒ) and propylparaben (NipasolÒ) were purchased from Synth (Brazil). Hydroxyethyl cellulose gel (NatrosolÒ) was purchased from Galena (Netherlands). Ethoxydiglycol (Transcutol CGÒ) was obtained from Gatefosse (France). Monoolein 18-99K (Myverol™) was obtained from Quest (Netherlands). The standard compounds, SN and SM, with 96% purity, were kindly provided by Dr. James D. McChesney from Cypress Creek Pharma. 2.2. Glycoalkaloid extraction The alkaloidic extract incorporated into the topical formulation, containing 45.09% ± 1.14 SN and 44.37% ± 0.60 SM, was obtained using a selective acid–base extraction method and was quantified by analytical HPLC with ultraviolet (UV) detection, as described previously [2]. 2.3. Analytical HPLC conditions The analysis with SN and SM standards was performed using a validated reverse-phase HPLC-UV method with gradient elution, as
2.4. Evaluation of physicochemical properties of SN and SM at different pHs To evaluate the influence of pH on the solubility and partition coefficient of these compounds, a buffer was prepared by mixing different volumes of sodium citrate (0.1 M) and sodium phosphate (0.2 M) solutions, obtaining pHs of 3.0, 4.0, 5.0, 5.5, 6.0, 6.5, 7.0 and 8.0, according to Macilvaine [26]. To determine the saturation concentration (Cs), excess of alkaloidic extract were mixed in these solutions, filtered, and the amounts of SN and SM were quantified by HPLC, as described in Section 2.3. The partition coefficient between octanol and water (Koctanol/w) was determined using a shake flash method (n = 3) [27]. Both buffer solutions at different pHs were saturated with octanol, and octanol was saturated with buffer solutions. The buffer phases were then separated, and an excess of extract was added. After achieving equilibrium, the dispersions were filtered, and the alkaloids were quantified before partition (A1). To each remaining buffer solution sample (1.5 mL), 1.5 mL of buffer-saturated octanol was added. This system was shaken for 24 h at room temperature. After this period, an aliquot of the aqueous phase was removed for analysis and quantification (A2). The Koctanol/w values were determined for glycoalkaloids at each pH and were obtained by the following equation:
K octanol=w ¼ ðConc: A1 Conc: A2Þ=Conc: A2
ð1Þ
where Conc. is the concentration of each glycoalkaloid [27].
Please cite this article in press as: R.F.J. Tiossi et al., In vitro and in vivo evaluation of the delivery of topical formulations containing glycoalkaloids of Solanum lycocarpum fruits, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.01.010
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2.5. In vitro skin permeation and retention studies 2.5.1. Experimental conditions The topical delivery of glycoalkaloids was assessed in vitro with porcine ear skin as a biological membrane model, as described by Lopes et al. [23] and Tiossi et al. [21]. The skin of the outer portion of the porcine ear was dissected, dermatomized to a thickness of 500 lm, stored in aluminum foil at 20 °C and used within 30 days. The diffusion experiments were conducted in static vertical Franz diffusion cells (Hanson Instruments, Chatsworth, CA), in which the porcine ear skin was positioned with the SC exposed to the donor compartment (0.78 cm2) and the dermis (D) in contact with an acceptor compartment filled with 3 mL of receptor solution (RS). This solution was composed of sodium phosphate buffer (pH 7.2, 0.1 M) and cetylpyridinium chloride (1%), in which SN and SM displayed solubilities of 6.09 and 6.24 mg/mL, respectively. Approximately 400 mg of each formulation was applied to the SC, and the temperature of the diffusion cells was kept at 32 ± 0.5 °C by a water jacket. The RS was mixed with a magnetic stirring bar (300 rpm) throughout the experiment, as recommended by the Scientific Committee on Consumer Products [28]. The experiment lasted 12 h (n = 5). After the predetermined time for each experiment, the RS of each diffusion cell was collected for the quantification of glycoalkaloid content. The skin was collected, gently washed with purified water to remove the excess formulation and was submitted to the extraction procedure. For the quantification of glycoalkaloids skin retention, the SC was removed by a tape-stripping technique using 13 pieces of tape, as described previously with modifications [21,23]. The first tape was discarded, and others were combined after extraction. The remaining skin, considered EP + D, was cut in small pieces and submitted to extraction. The samples obtained from SC and EP + D were extracted using 3 mL of methanol by vortex stirring for 1 min, followed by an ultrasound bath for 30 min. The resulting homogenates were centrifuged at 3125g for 10 min, filtered (0.45 lm pore size, MilliporeÒ), and an aliquot of 20 lL was analyzed by HPLC, as previously described. The amounts of glycoalkaloids in the skin and in the RS after the penetration studies were quantified using the calibration curves obtained for SN and SM and the penetration area (0.78 cm2). The amounts of glycoalkaloids in the SC, EP + D and RS were expressed in lg/cm2.
2.5.2. Formulations preparation The gel base used was composed of 2% hydroxyethyl cellulose, 5% propylene glycol, 0.18% methylparaben, 0.02% propylparaben and phosphate buffer (pH 6.5, 0.1 M). To this gel base, the alkaloidic extract (5%) dissolved in propylene glycol was added in combination with different types and quantities of the following penetration enhancers, as described in Table 1. Table 1 Formulations and penetration enhancers evaluated. Formulations
Penetration enhancer
A B C D E F Control formulationa
Monoolein (1%) Monoolein (5%) Monoolein (1%) + ethanol (10%) Monoolein (5%) + ethanol (10%) Ethanol (10%) Ethoxydiglycol (5%) –
a The control formulation contains propylene glycol (5%), alkaloidic extract (5%) and hydroxyethyl cellulose gel base at pH 6.5.
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The pH was measured (Digimed, DM20) in triplicate in a distilled water solution containing 10% of each formulation. The viscosity of the different formulations was carried out using a rheometer (DHR-2 TA Instruments, New Castec, USA). Approximately 5 g of each formulation was transferred at 25 ± 1 °C to the rheometer and viscosity of the formulations was performed at a rotational speed 0–500 s1 and 500–0 s1 in 120 s. Under the same conditions, the viscosity of the control formulation was examined. The average of three readings was used to calculate the viscosity. 2.6. In vivo penetration study in animal model Animal experiments were performed in male hairless mice, weighing 31.69 ± 3.27 g, provided by the Pharmacy School, University of São Paulo, Ribeirão Preto – SP, Brazil. The mice were housed in a controlled ambient temperature (23 ± 2 °C) under a 12 h:12 h light:dark cycle with access to water and food ad libitum. The experiment was conducted in accordance with the rules of the Ethics Committee on Animal Use of USP – Campus Ribeirão Preto (protocol number 07.1.1108.53.9). Formulations containing the penetration enhancers 5% monoolein (B) and 5% monoolein + 10% ethanol (D) or with the control formulation (CF) were evaluated. Aliquots of each formulation (200 mg) were applied to a limited area (1.77 cm2) of the dorsal skin of hairless mice with five replicates animals for each formulation. After a period of 12 h, animals were euthanized by carbon dioxide. The skin area where the formulations were applied was then carefully dissected and submitted to the processes of extraction and quantification of glycoalkaloids in both SC and EP + D, according to Sections 2.3 and 2.5. 2.7. Statistical analysis The results are reported as the means ± S.E.M. (standard error of the mean). The data were statistically analyzed, and the differences between groups were determined using one-way ANOVA, followed by Tukey’s multiple comparison test. Differences of P < 0.05 were considered significant for both analyses. 3. Results and discussion 3.1. Evaluation of the physicochemical properties of SN and SM at different pHs Physicochemical properties, such as the solubility and partition coefficients, play important roles in the capacity of drugs to penetrate biological barriers [24]. Determining the solubility of a drug is important because it is known that when the solubility of a compound is high, there is a greater tendency for it to be absorbed [29]. The distribution coefficient can predict the ability of a compound to passively penetrate through the skin. This coefficient gives information about the interaction of compounds with octanol, which has similar characteristics to the SC [30]. For ionizable species such as glycoalkaloids, properties such as solubility and the partition coefficient can be affected considerably by the pH. For this reason, both properties were evaluated in the pH range 3–8. Koctanol/w was determined at different pHs, except for pH 8.0, at which pH the glycoalkaloids totally partitioned to the octanol phase. The solubility and Koctanol/w were pH dependent; at acidic pH, the solubility of glycoalkaloids increased, while the partition coefficients decreased (Fig. 2). The observed effect of pH on solubility and the partition coefficient can be explained by the ability of the amine group of glycoalkaloids to be ionized; at alkaline pH, these compounds are free
Please cite this article in press as: R.F.J. Tiossi et al., In vitro and in vivo evaluation of the delivery of topical formulations containing glycoalkaloids of Solanum lycocarpum fruits, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.01.010
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the drug is the use of penetration enhancers in topical formulations [33,34]. Preliminary studies conducted by our research group showed the importance of pH control of formulations, once it was observed that at pH 6.5 occurred higher penetration of glycoalkaloids into the skin layers. To evaluate the role of known penetration enhancers on the glycoalkaloids skin retention, monoolein [23] was used with or without ethanol or ethoxydiglycol (Fig. 3). There was no significant variation of the pHs among the formulations, because it had used the same Natrosol gel base containing both sodium citrate and sodium phosphate buffers. As the formulations behaved as a pseudoplastic non-Newtonian fluid, it was possible to measure the apparent viscosity. At the loop apex (487.67 s1) the formulations presented a viscosity of 0.63 ± 0.04 Pa s, showing that the different penetration enhancers used did not modify the viscosity value significantly. The results showed that the penetration enhancers allowed the glycoalkaloids to reach SC and EP + D. However, detectable amounts of SN and SM in the RS were not found. Ethoxydiglycol [35,36] and ethanol [37,38] are solvents with penetration enhancer effects for some drugs, in the present work these solvents were not able to significantly affect the permeation into the skin; permeation into deep layers (EP + D) was not significantly different from the control formulation (CF). According to Fig. 3, the total retention, SC + [EP + D], of glycoalkaloids in the skin from formulations containing 5% monoolein (B), 1% monoolein + 10% ethanol (C), 5% monoolein + 10% ethanol (D) and 10% ethanol (E) was statistically significantly different
Fig. 2. Effect of pH on the solubility (a) and partition coefficient (Koctanol/w) (b) of solasonine (SN) and solamargine (SM) in a mixture of buffer solutions of sodium citrate (0.1 M) and sodium phosphate (0.2 M) at different pHs.
bases, reducing their aqueous solubility and increasing their affinity for the organic phase. Considering the partition coefficient, it is expected that at alkaline pH, the glycoalkaloids will have increased skin affinity. However, it is known that higher values of Koctanol/w can result in a greater affinity to SC, which can hinder the permeation of compounds from the SC into more hydrated layers, such as the dermis [30]. Furthermore, increased pH may negatively affect the skin barrier, and the long-term application of an alkaline formulation to the skin is not suitable for clinical use [31]. However, glycoalkaloids at acidic pH have increased solubility. Considering the physicochemical properties of drugs that permeate through the skin, it is necessary not only to achieve the highest solubility possible but also to have a partition coefficient that allows the interaction of drugs with skin layers. Therefore, for the successful penetration of compounds into the skin, these properties must be combined. In the present work, the results indicated that pHs between 5.5 and 7.0 are optimal for both properties, so this range was chosen for subsequent studies. 3.2. Effect of penetration enhancers on the penetration There are many factors related to skin penetration that should be taken into consideration for good passive penetration into the skin, such as molecular size (300–500 Da), log P (1–3.5) and solubility (greater than 100 lg/mL) [32]. SN and SM have a very similar molecular size of 884.50 and 868.08 Da, respectively, one strategy to overcome the skin barrier and to have an effective permeation of
Fig. 3. Effect of absorption enhancers on the in vitro penetration of solasonine (SN) (a) and solamargine (SM) (b) by Franz cell diffusion for 12 h (n = 5) into SC and EP + D, along with the total retention, SC + [EP + D]. Absorption enhancers evaluated were: 1% monoolein (A), 5% monoolein (B), 1% monoolein + 10% ethanol (C), 5% monoolein + 10% ethanol (D), 10% ethanol (E), 5% ethoxydiglycol (F) and control formulation (CF) (, # and d indicate P < 0.05 difference from retention with CF in SC, EP + D and SC + [EP + D], respectively).
Please cite this article in press as: R.F.J. Tiossi et al., In vitro and in vivo evaluation of the delivery of topical formulations containing glycoalkaloids of Solanum lycocarpum fruits, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.01.010
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(P < 0.05) than with control formulation (CF) or other formulations assayed. Considering the skin layers, the SC retention of SN only revealed a significant difference (P < 0.05) when the formulation containing 1% monoolein + 10% ethanol (C) was compared to the control formulation (CF) (P < 0.05). However, the SC retention of SM was significantly different with formulations containing 5% monoolein (B), 1% monoolein + 10% ethanol (C) and 10% ethanol (D) in comparison with the control formulation (CF) (P < 0.05). These data suggest that SM has a greater capacity than SN to penetrate the SC. It is interesting to highlight that the penetration event begins with the retention of compounds in the SC before they can consequently reach the deeper skin layers. Thus, by comparing the different formulations against the control formulation (CF) (P < 0.05), it could be observed that penetration enhancers, such as monoolein and ethanol, in combination or alone, were able to enhance SC retention significantly, but only formulations containing 5% monoolein (B) or 5% monoolein + 10% ethanol (D) were effective in promoting the retention in EP + D, which represents the skin layers where non-melanoma cancers commonly occur. The in vitro penetration results allowed us to select formulations containing 5% monoolein (B) and 5% monoolein + 10% ethanol (D) as the most promising for the in vivo permeation assays because they were significantly different (P < 0.05) than the control formulation (CF) but were not significantly different from each other. Moreover, preliminary kinetic studies with formulation B allowed to observed that 12 h was the best time for glycoalkaloids permeation. 3.3. In vivo penetration study in animal model The penetration of glycoalkaloids from formulations containing 5% monoolein (B) and 5% monoolein + 10% ethanol (D) was evaluated in comparison with the control formulation (CF) in an in vivo penetration assay, after 12 h of application (Fig. 4). In this study it was not used any formulation as a positive control, despite of the availability of a commercial formulation with the same glycoalkaloids, but in a concentration 1000 times lower, which could not be quantified with the analytical developed method. The SC and EP + D penetration for the glycoalkaloids was significantly different for the formulations containing 5% monoolein (B) and 5% monoolein + 10% ethanol (D) compared to the control formulation (CF). The mean values of the SC retention of the formulations assayed were four times higher than the control formulation (CF) for both glycoalkaloids, while the EP + D retention was about ten times greater than control formulation (CF). However, as seen in the in vitro tests, the in vivo experiments revealed no significant differences (P < 0.05) between formulations containing 5% monoolein (B) and 5% monoolein + 10% ethanol (D). It is known that porcine ear skin is a suitable substitute for human skin in dermatological research as it has similar permeability [39,40], whereas in hairless mice, the percutaneous absorption is relatively greater [41,42]. Thus, the retention of glycoalkaloids in the in vivo assay was about two fold greater than in the in vitro assay. Considering the presence of ethanol in formulation D, it could be observed that this co-solvent did not promote any absorption enhancing activity. Therefore, the formulation containing only 5% monoolein (B) as an penetration enhancer is more suitable for further topical in vivo anticancer assays (Fig. 4). The developed formulation could be employed in association with conventional therapies, such as epirubicin and cisplatin, to increase the susceptibility of cancer cells, including those with multidrug resistance [43,44]. It is important to note that the developed formulation could be employed for future studies not only on skin cancers but also on other dermatological diseases because these glycoalkaloids display other biological properties, such as the following: antiparasitic against Leishmania amazonensis [5], anti-viral
Fig. 4. Effect of absorption enhancers on the in vivo penetration of solasonine (SN) (a) and solamargine (SM) (b) in SC, EP + D and SC + [EP + D] in different hydroxyethyl cellulose gel formulations with absorption enhancers 5% monoolein (B), 5% monoolein + 10% ethanol (D) or control formulation (CF) at pH 6.5 (n = 5 animals per formulation) for 12 h. (, # and d indicate P < 0.05 compared to CF for SC, EP + D and SC + [EP + D], respectively).
against Herpes genitalis, Herpes zoster and Herpes simplex [6], and antifungal against dermatophytes and yeasts, including Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis and Microsporum gypseum, which have high recurrence rates, despite the broad spectrum of available antifungal agents [7]. 4. Conclusion In this paper, we report a promising topical formulation containing glycoalkaloids from S. lycocarpum that allows the penetration of SN and SM into the skin layers where cancerous lesions commonly take place. Moreover, the developed formulation containing 5% monoolein in a hydroxyethyl cellulose gel base presented a higher permeation of the glycoalkaloids SN and SM into the target site EP + D in both in vitro and in vivo tests. This finding has great relevance because the glycoalkaloids, especially SM, are primarily responsible for the anticancer properties of S. lycocarpum. Moreover, the developed formulation could be employed in association with conventional anticancer drugs. Additionally, the developed formulation could be employed for future studies, not only to treat skin cancer but also other dermatological diseases, such as leishmaniasis, herpes and dermatophytosis. In conclusion, this study may contribute to the use of new anticancer therapies, but further studies are needed to evaluate the pharmacological and toxicological potential of the developed formulation in the treatment of skin cancer and other dermatological diseases. Acknowledgments The authors are grateful to Mário Ogasawara, Valter Lopes, José Orestes Del Ciampo and Henrique Diniz for technical support. We
Please cite this article in press as: R.F.J. Tiossi et al., In vitro and in vivo evaluation of the delivery of topical formulations containing glycoalkaloids of Solanum lycocarpum fruits, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.01.010
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also thank the São Paulo Research Foundation (FAPESP), Brazil (Grant 2007/57538-1) for financial support.
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Research paper
In vitro and in vivo evaluation of the delivery of topical formulations containing glycoalkaloids of Solanum lycocarpum fruits Renata F.J. Tiossi a,1, Juliana C. Da Costa a,1, Mariza A. Miranda a, Fabíola S.G. Praça a, James D. McChesney b, Maria Vitória L.B. Bentley a, Jairo K. Bastos a,⇑ a b
Faculty of Pharmaceutical Science of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil Arbor Therapeutics, LLC, Etta, MS, USA
a r t i c l e
i n f o
Article history: Received 19 September 2013 Accepted in revised form 31 January 2014 Available online xxxx Keywords: Solanum lycocarpum Glycoalkaloids Solasonine Solamargine Topical delivery Skin cancer
a b s t r a c t The glycoalkaloids solasonine (SN) and solamargine (SM) have been studied for their antiparasitic, antifungal, and anticancer properties, especially in vitro and in vivo against non-melanoma skin cancer. Thus, the alkaloidic extract of Solanum lycocarpum, which contains approximately 45% each of SN and SM, was used to define the best experimental conditions for in vitro and in vivo assays. The in vitro assays were performed with the Franz cell diffusion porcine skin model to evaluate the effects of different pHs and the presence of monoolein, ethoxydiglycol or ethanol penetration enhancers on the skin penetration and retention of SN and SM after 3, 6, 9 and 12 h of exposure. The in vivo assay was performed on hairless mice with the formulation selected in the in vitro assays. The results showed that pH 6.5 was optimal for SM penetration. The formulation containing 5% alkaloidic extract, 5% propylene glycol, 5% monoolein and a hydroxyethyl cellulose gel base (NatrosolÒ) (pH 6.5) was optimal for the delivery of SN and SM into the skin, and this formulation is potentially useful for the topical therapy of several skin disorders. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Solanum lycocarpum A. St.-Hil. (Solanaceae) is a native species of Brazilian Cerrado belonging to the genus Solanum that is known for the biosynthesis of glycoalkaloids. The glycoalkaloids solasonine (SN) and solamargine (SM) (Fig. 1) are found in the fruits of more than a hundred species of Solanum [1], but S. lycocarpum stands out for its production of these compounds. SN and SM can be obtained by selectively extracting them from the fruits of this species [2]. These compounds are structurally similar because they contain the same steroidal moiety, solasodine, differing only in their sugar chain moieties: solatriose for SN and chacotriose for SM [3]. SN and SM have been studied as antiparasitics [4,5], antidiabetics [3], anti-virals against herpes [6], antifungals [7–9], immunomodulators [10], and anticancer agents in several cell lines [11–15] and in vivo against human skin cancer [16,17]. Studies have shown that SN and SM are selective for cancerous cells due to the sugar chain moiety, mainly rhamnose, of which SN ⇑ Corresponding author. Faculty of Pharmaceutical Science of Ribeirão Preto, University of São Paulo, Av. do Café, s/n, Ribeirão Preto, SP 14040-903, Brazil. Tel.: +55 16 36024230; fax: +55 16 36024230, 16 36024879. E-mail address: [email protected] (J.K. Bastos). 1 These authors contributed equally to this work.
and SM contain one and two units, respectively [18,19]. These compounds have been studied to treat non-melanoma skin cancer by employing a mixture of glycoalkaloids extracted from Solanum species [18]. However, the efficacy of these compounds not only depends on the quantity used but also on the composition of the topical formulation. For instance, cetomacrogol-based cream with 10% glycoalkaloids and 10% dimethylsulfoxide (DMSO) effected an 83% skin carcinoma cure rate [16], while another formulation with 0.005% glycoalkaloids and keratolytic agents was 100% curative [20]. In contrast, a 66% cure rate was achieved with 0.005% glycoalkaloids and keratolytic agents in emulsifying wax and white soft paraffin base [17]. It is important to note that these studies were performed with an alkaloidic mixture from another Solanum species, which is composed of 33% SN, 33% SM and 34% di- and mono-glycosides of solasodine [18], while the alkaloidic extract from S. lycocarpum studied in this work contained approximately 45% SN and 45% SM [21]. It is desirable that topical formulations adequately allow for skin penetration and retention with minimal systemic absorption [22]. In this context, penetration enhancers have been widely used in formulations to improve the skin penetration of several compounds [23]. Moreover, it is known that glycoalkaloids have a basic character, which plays an important role in their skin permeability, depending on their degree of ionization, solubility in the applied
http://dx.doi.org/10.1016/j.ejpb.2014.01.010 0939-6411/Ó 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: R.F.J. Tiossi et al., In vitro and in vivo evaluation of the delivery of topical formulations containing glycoalkaloids of Solanum lycocarpum fruits, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.01.010
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Fig. 1. Structures of solasodine and their glycosides: solasonine (SN) and solamargine (SM).
phase and partitioning into the skin [24]. Taking into consideration that penetration and the consequent effects of glycoalkaloids in the skin could be greatly influenced by the formulation pH [25], it is mandatory to understand the physicochemical properties of these compounds and the skin penetration of SN and SM in the development and optimization of new skin anticancer therapies. Therefore, considering the remarkable activity of glycoalkaloids on cancer cells and the lack of data on their topical delivery, as well as their high content in the alkaloidic extract of S. lycocarpum fruits, we have performed studies on the development of a topical formulation with the ability to deliver these compounds into deeper skin layers so that they can be used in the treatment of non-melanoma skin cancer. 2. Material and methods
described in our previous work [21]. Briefly, a Zorbax SB-C18 analytical reverse phase column was used as stationary phase, and the binary gradient consisted of sodium phosphate buffer (pH 7.2, 0.01 M) (pump A) and MeCN (pump B). Thus, the calibration curves were constructed for both glycoalkaloids, and linearity was achieved for concentrations in the range of 0.77–990.00 lg/mL for SN and 0.78–1000.00 lg/mL for SM, with correlation coefficients P 0.999. The retention times of SN and SM were 10.35 and 12.35 min, respectively. The intra-assay and inter-assay variations were less than 9.18% for SN and 8.74% for SM. The method recoveries were evaluated for samples of stratum corneum (SC) and epidermis plus dermis (EP + D), determined at three concentrations, and the values were higher than 88.94% for SN and 94.75% for SM. The limits of detection and quantification were 0.29 lg/mL and 0.86 lg/mL for SN, and 0.57 lg/mL and 1.74 for SM, respectively [21].
2.1. Chemicals and reagents High performance liquid chromatography (HPLC)-grade acetonitrile (MeCN) and methanol (MeOH) were obtained from Mallinckrodt Co. (Xalostoc, Mexico). Anhydrous disodium hydrogen phosphate was acquired from Carlo Erba Reagents (Brazil). Deionized water was purified by Milli-Q-plus filter systems (Millipore, USA). The analytical grade cetylpyridinium chloride, ethanol (EtOH), propylene glycol, hydrochloric acid, sodium hydroxide, sodium phosphate, methylparaben (NipaginÒ) and propylparaben (NipasolÒ) were purchased from Synth (Brazil). Hydroxyethyl cellulose gel (NatrosolÒ) was purchased from Galena (Netherlands). Ethoxydiglycol (Transcutol CGÒ) was obtained from Gatefosse (France). Monoolein 18-99K (Myverol™) was obtained from Quest (Netherlands). The standard compounds, SN and SM, with 96% purity, were kindly provided by Dr. James D. McChesney from Cypress Creek Pharma. 2.2. Glycoalkaloid extraction The alkaloidic extract incorporated into the topical formulation, containing 45.09% ± 1.14 SN and 44.37% ± 0.60 SM, was obtained using a selective acid–base extraction method and was quantified by analytical HPLC with ultraviolet (UV) detection, as described previously [2]. 2.3. Analytical HPLC conditions The analysis with SN and SM standards was performed using a validated reverse-phase HPLC-UV method with gradient elution, as
2.4. Evaluation of physicochemical properties of SN and SM at different pHs To evaluate the influence of pH on the solubility and partition coefficient of these compounds, a buffer was prepared by mixing different volumes of sodium citrate (0.1 M) and sodium phosphate (0.2 M) solutions, obtaining pHs of 3.0, 4.0, 5.0, 5.5, 6.0, 6.5, 7.0 and 8.0, according to Macilvaine [26]. To determine the saturation concentration (Cs), excess of alkaloidic extract were mixed in these solutions, filtered, and the amounts of SN and SM were quantified by HPLC, as described in Section 2.3. The partition coefficient between octanol and water (Koctanol/w) was determined using a shake flash method (n = 3) [27]. Both buffer solutions at different pHs were saturated with octanol, and octanol was saturated with buffer solutions. The buffer phases were then separated, and an excess of extract was added. After achieving equilibrium, the dispersions were filtered, and the alkaloids were quantified before partition (A1). To each remaining buffer solution sample (1.5 mL), 1.5 mL of buffer-saturated octanol was added. This system was shaken for 24 h at room temperature. After this period, an aliquot of the aqueous phase was removed for analysis and quantification (A2). The Koctanol/w values were determined for glycoalkaloids at each pH and were obtained by the following equation:
K octanol=w ¼ ðConc: A1 Conc: A2Þ=Conc: A2
ð1Þ
where Conc. is the concentration of each glycoalkaloid [27].
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2.5. In vitro skin permeation and retention studies 2.5.1. Experimental conditions The topical delivery of glycoalkaloids was assessed in vitro with porcine ear skin as a biological membrane model, as described by Lopes et al. [23] and Tiossi et al. [21]. The skin of the outer portion of the porcine ear was dissected, dermatomized to a thickness of 500 lm, stored in aluminum foil at 20 °C and used within 30 days. The diffusion experiments were conducted in static vertical Franz diffusion cells (Hanson Instruments, Chatsworth, CA), in which the porcine ear skin was positioned with the SC exposed to the donor compartment (0.78 cm2) and the dermis (D) in contact with an acceptor compartment filled with 3 mL of receptor solution (RS). This solution was composed of sodium phosphate buffer (pH 7.2, 0.1 M) and cetylpyridinium chloride (1%), in which SN and SM displayed solubilities of 6.09 and 6.24 mg/mL, respectively. Approximately 400 mg of each formulation was applied to the SC, and the temperature of the diffusion cells was kept at 32 ± 0.5 °C by a water jacket. The RS was mixed with a magnetic stirring bar (300 rpm) throughout the experiment, as recommended by the Scientific Committee on Consumer Products [28]. The experiment lasted 12 h (n = 5). After the predetermined time for each experiment, the RS of each diffusion cell was collected for the quantification of glycoalkaloid content. The skin was collected, gently washed with purified water to remove the excess formulation and was submitted to the extraction procedure. For the quantification of glycoalkaloids skin retention, the SC was removed by a tape-stripping technique using 13 pieces of tape, as described previously with modifications [21,23]. The first tape was discarded, and others were combined after extraction. The remaining skin, considered EP + D, was cut in small pieces and submitted to extraction. The samples obtained from SC and EP + D were extracted using 3 mL of methanol by vortex stirring for 1 min, followed by an ultrasound bath for 30 min. The resulting homogenates were centrifuged at 3125g for 10 min, filtered (0.45 lm pore size, MilliporeÒ), and an aliquot of 20 lL was analyzed by HPLC, as previously described. The amounts of glycoalkaloids in the skin and in the RS after the penetration studies were quantified using the calibration curves obtained for SN and SM and the penetration area (0.78 cm2). The amounts of glycoalkaloids in the SC, EP + D and RS were expressed in lg/cm2.
2.5.2. Formulations preparation The gel base used was composed of 2% hydroxyethyl cellulose, 5% propylene glycol, 0.18% methylparaben, 0.02% propylparaben and phosphate buffer (pH 6.5, 0.1 M). To this gel base, the alkaloidic extract (5%) dissolved in propylene glycol was added in combination with different types and quantities of the following penetration enhancers, as described in Table 1. Table 1 Formulations and penetration enhancers evaluated. Formulations
Penetration enhancer
A B C D E F Control formulationa
Monoolein (1%) Monoolein (5%) Monoolein (1%) + ethanol (10%) Monoolein (5%) + ethanol (10%) Ethanol (10%) Ethoxydiglycol (5%) –
a The control formulation contains propylene glycol (5%), alkaloidic extract (5%) and hydroxyethyl cellulose gel base at pH 6.5.
3
The pH was measured (Digimed, DM20) in triplicate in a distilled water solution containing 10% of each formulation. The viscosity of the different formulations was carried out using a rheometer (DHR-2 TA Instruments, New Castec, USA). Approximately 5 g of each formulation was transferred at 25 ± 1 °C to the rheometer and viscosity of the formulations was performed at a rotational speed 0–500 s1 and 500–0 s1 in 120 s. Under the same conditions, the viscosity of the control formulation was examined. The average of three readings was used to calculate the viscosity. 2.6. In vivo penetration study in animal model Animal experiments were performed in male hairless mice, weighing 31.69 ± 3.27 g, provided by the Pharmacy School, University of São Paulo, Ribeirão Preto – SP, Brazil. The mice were housed in a controlled ambient temperature (23 ± 2 °C) under a 12 h:12 h light:dark cycle with access to water and food ad libitum. The experiment was conducted in accordance with the rules of the Ethics Committee on Animal Use of USP – Campus Ribeirão Preto (protocol number 07.1.1108.53.9). Formulations containing the penetration enhancers 5% monoolein (B) and 5% monoolein + 10% ethanol (D) or with the control formulation (CF) were evaluated. Aliquots of each formulation (200 mg) were applied to a limited area (1.77 cm2) of the dorsal skin of hairless mice with five replicates animals for each formulation. After a period of 12 h, animals were euthanized by carbon dioxide. The skin area where the formulations were applied was then carefully dissected and submitted to the processes of extraction and quantification of glycoalkaloids in both SC and EP + D, according to Sections 2.3 and 2.5. 2.7. Statistical analysis The results are reported as the means ± S.E.M. (standard error of the mean). The data were statistically analyzed, and the differences between groups were determined using one-way ANOVA, followed by Tukey’s multiple comparison test. Differences of P < 0.05 were considered significant for both analyses. 3. Results and discussion 3.1. Evaluation of the physicochemical properties of SN and SM at different pHs Physicochemical properties, such as the solubility and partition coefficients, play important roles in the capacity of drugs to penetrate biological barriers [24]. Determining the solubility of a drug is important because it is known that when the solubility of a compound is high, there is a greater tendency for it to be absorbed [29]. The distribution coefficient can predict the ability of a compound to passively penetrate through the skin. This coefficient gives information about the interaction of compounds with octanol, which has similar characteristics to the SC [30]. For ionizable species such as glycoalkaloids, properties such as solubility and the partition coefficient can be affected considerably by the pH. For this reason, both properties were evaluated in the pH range 3–8. Koctanol/w was determined at different pHs, except for pH 8.0, at which pH the glycoalkaloids totally partitioned to the octanol phase. The solubility and Koctanol/w were pH dependent; at acidic pH, the solubility of glycoalkaloids increased, while the partition coefficients decreased (Fig. 2). The observed effect of pH on solubility and the partition coefficient can be explained by the ability of the amine group of glycoalkaloids to be ionized; at alkaline pH, these compounds are free
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the drug is the use of penetration enhancers in topical formulations [33,34]. Preliminary studies conducted by our research group showed the importance of pH control of formulations, once it was observed that at pH 6.5 occurred higher penetration of glycoalkaloids into the skin layers. To evaluate the role of known penetration enhancers on the glycoalkaloids skin retention, monoolein [23] was used with or without ethanol or ethoxydiglycol (Fig. 3). There was no significant variation of the pHs among the formulations, because it had used the same Natrosol gel base containing both sodium citrate and sodium phosphate buffers. As the formulations behaved as a pseudoplastic non-Newtonian fluid, it was possible to measure the apparent viscosity. At the loop apex (487.67 s1) the formulations presented a viscosity of 0.63 ± 0.04 Pa s, showing that the different penetration enhancers used did not modify the viscosity value significantly. The results showed that the penetration enhancers allowed the glycoalkaloids to reach SC and EP + D. However, detectable amounts of SN and SM in the RS were not found. Ethoxydiglycol [35,36] and ethanol [37,38] are solvents with penetration enhancer effects for some drugs, in the present work these solvents were not able to significantly affect the permeation into the skin; permeation into deep layers (EP + D) was not significantly different from the control formulation (CF). According to Fig. 3, the total retention, SC + [EP + D], of glycoalkaloids in the skin from formulations containing 5% monoolein (B), 1% monoolein + 10% ethanol (C), 5% monoolein + 10% ethanol (D) and 10% ethanol (E) was statistically significantly different
Fig. 2. Effect of pH on the solubility (a) and partition coefficient (Koctanol/w) (b) of solasonine (SN) and solamargine (SM) in a mixture of buffer solutions of sodium citrate (0.1 M) and sodium phosphate (0.2 M) at different pHs.
bases, reducing their aqueous solubility and increasing their affinity for the organic phase. Considering the partition coefficient, it is expected that at alkaline pH, the glycoalkaloids will have increased skin affinity. However, it is known that higher values of Koctanol/w can result in a greater affinity to SC, which can hinder the permeation of compounds from the SC into more hydrated layers, such as the dermis [30]. Furthermore, increased pH may negatively affect the skin barrier, and the long-term application of an alkaline formulation to the skin is not suitable for clinical use [31]. However, glycoalkaloids at acidic pH have increased solubility. Considering the physicochemical properties of drugs that permeate through the skin, it is necessary not only to achieve the highest solubility possible but also to have a partition coefficient that allows the interaction of drugs with skin layers. Therefore, for the successful penetration of compounds into the skin, these properties must be combined. In the present work, the results indicated that pHs between 5.5 and 7.0 are optimal for both properties, so this range was chosen for subsequent studies. 3.2. Effect of penetration enhancers on the penetration There are many factors related to skin penetration that should be taken into consideration for good passive penetration into the skin, such as molecular size (300–500 Da), log P (1–3.5) and solubility (greater than 100 lg/mL) [32]. SN and SM have a very similar molecular size of 884.50 and 868.08 Da, respectively, one strategy to overcome the skin barrier and to have an effective permeation of
Fig. 3. Effect of absorption enhancers on the in vitro penetration of solasonine (SN) (a) and solamargine (SM) (b) by Franz cell diffusion for 12 h (n = 5) into SC and EP + D, along with the total retention, SC + [EP + D]. Absorption enhancers evaluated were: 1% monoolein (A), 5% monoolein (B), 1% monoolein + 10% ethanol (C), 5% monoolein + 10% ethanol (D), 10% ethanol (E), 5% ethoxydiglycol (F) and control formulation (CF) (, # and d indicate P < 0.05 difference from retention with CF in SC, EP + D and SC + [EP + D], respectively).
Please cite this article in press as: R.F.J. Tiossi et al., In vitro and in vivo evaluation of the delivery of topical formulations containing glycoalkaloids of Solanum lycocarpum fruits, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.01.010
R.F.J. Tiossi et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx
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(P < 0.05) than with control formulation (CF) or other formulations assayed. Considering the skin layers, the SC retention of SN only revealed a significant difference (P < 0.05) when the formulation containing 1% monoolein + 10% ethanol (C) was compared to the control formulation (CF) (P < 0.05). However, the SC retention of SM was significantly different with formulations containing 5% monoolein (B), 1% monoolein + 10% ethanol (C) and 10% ethanol (D) in comparison with the control formulation (CF) (P < 0.05). These data suggest that SM has a greater capacity than SN to penetrate the SC. It is interesting to highlight that the penetration event begins with the retention of compounds in the SC before they can consequently reach the deeper skin layers. Thus, by comparing the different formulations against the control formulation (CF) (P < 0.05), it could be observed that penetration enhancers, such as monoolein and ethanol, in combination or alone, were able to enhance SC retention significantly, but only formulations containing 5% monoolein (B) or 5% monoolein + 10% ethanol (D) were effective in promoting the retention in EP + D, which represents the skin layers where non-melanoma cancers commonly occur. The in vitro penetration results allowed us to select formulations containing 5% monoolein (B) and 5% monoolein + 10% ethanol (D) as the most promising for the in vivo permeation assays because they were significantly different (P < 0.05) than the control formulation (CF) but were not significantly different from each other. Moreover, preliminary kinetic studies with formulation B allowed to observed that 12 h was the best time for glycoalkaloids permeation. 3.3. In vivo penetration study in animal model The penetration of glycoalkaloids from formulations containing 5% monoolein (B) and 5% monoolein + 10% ethanol (D) was evaluated in comparison with the control formulation (CF) in an in vivo penetration assay, after 12 h of application (Fig. 4). In this study it was not used any formulation as a positive control, despite of the availability of a commercial formulation with the same glycoalkaloids, but in a concentration 1000 times lower, which could not be quantified with the analytical developed method. The SC and EP + D penetration for the glycoalkaloids was significantly different for the formulations containing 5% monoolein (B) and 5% monoolein + 10% ethanol (D) compared to the control formulation (CF). The mean values of the SC retention of the formulations assayed were four times higher than the control formulation (CF) for both glycoalkaloids, while the EP + D retention was about ten times greater than control formulation (CF). However, as seen in the in vitro tests, the in vivo experiments revealed no significant differences (P < 0.05) between formulations containing 5% monoolein (B) and 5% monoolein + 10% ethanol (D). It is known that porcine ear skin is a suitable substitute for human skin in dermatological research as it has similar permeability [39,40], whereas in hairless mice, the percutaneous absorption is relatively greater [41,42]. Thus, the retention of glycoalkaloids in the in vivo assay was about two fold greater than in the in vitro assay. Considering the presence of ethanol in formulation D, it could be observed that this co-solvent did not promote any absorption enhancing activity. Therefore, the formulation containing only 5% monoolein (B) as an penetration enhancer is more suitable for further topical in vivo anticancer assays (Fig. 4). The developed formulation could be employed in association with conventional therapies, such as epirubicin and cisplatin, to increase the susceptibility of cancer cells, including those with multidrug resistance [43,44]. It is important to note that the developed formulation could be employed for future studies not only on skin cancers but also on other dermatological diseases because these glycoalkaloids display other biological properties, such as the following: antiparasitic against Leishmania amazonensis [5], anti-viral
Fig. 4. Effect of absorption enhancers on the in vivo penetration of solasonine (SN) (a) and solamargine (SM) (b) in SC, EP + D and SC + [EP + D] in different hydroxyethyl cellulose gel formulations with absorption enhancers 5% monoolein (B), 5% monoolein + 10% ethanol (D) or control formulation (CF) at pH 6.5 (n = 5 animals per formulation) for 12 h. (, # and d indicate P < 0.05 compared to CF for SC, EP + D and SC + [EP + D], respectively).
against Herpes genitalis, Herpes zoster and Herpes simplex [6], and antifungal against dermatophytes and yeasts, including Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis and Microsporum gypseum, which have high recurrence rates, despite the broad spectrum of available antifungal agents [7]. 4. Conclusion In this paper, we report a promising topical formulation containing glycoalkaloids from S. lycocarpum that allows the penetration of SN and SM into the skin layers where cancerous lesions commonly take place. Moreover, the developed formulation containing 5% monoolein in a hydroxyethyl cellulose gel base presented a higher permeation of the glycoalkaloids SN and SM into the target site EP + D in both in vitro and in vivo tests. This finding has great relevance because the glycoalkaloids, especially SM, are primarily responsible for the anticancer properties of S. lycocarpum. Moreover, the developed formulation could be employed in association with conventional anticancer drugs. Additionally, the developed formulation could be employed for future studies, not only to treat skin cancer but also other dermatological diseases, such as leishmaniasis, herpes and dermatophytosis. In conclusion, this study may contribute to the use of new anticancer therapies, but further studies are needed to evaluate the pharmacological and toxicological potential of the developed formulation in the treatment of skin cancer and other dermatological diseases. Acknowledgments The authors are grateful to Mário Ogasawara, Valter Lopes, José Orestes Del Ciampo and Henrique Diniz for technical support. We
Please cite this article in press as: R.F.J. Tiossi et al., In vitro and in vivo evaluation of the delivery of topical formulations containing glycoalkaloids of Solanum lycocarpum fruits, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.01.010
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also thank the São Paulo Research Foundation (FAPESP), Brazil (Grant 2007/57538-1) for financial support.
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Please cite this article in press as: R.F.J. Tiossi et al., In vitro and in vivo evaluation of the delivery of topical formulations containing glycoalkaloids of Solanum lycocarpum fruits, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.01.010