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Oleodendrimers: A novel class of multicephalous heterolipids as chemical penetration enhancers for transdermal drug delivery
International Journal of Pharmaceutics 454 (2013) 158–166
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Oleodendrimers: A novel class of multicephalous heterolipids as chemical penetration enhancers for transdermal drug delivery Rahul S. Kalhapure, Krishnacharya G. Akamanchi ∗ Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Matunga (E), Mumbai 400019, India
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Article history: Received 29 May 2013 Received in revised form 4 July 2013 Accepted 7 July 2013 Available online xxx Keywords: Dendrimers Transdermal delivery Penetration enhancers Diclofenac sodium Oleic acid Anti-inflammatory Oleodenrimers
a b s t r a c t This paper reports synthesis and evaluation of Janus type generation G-1 and G-2 dendrimers. The dendrimers have been constructed by linking two building blocks, dendrons and oleic acid, through ester and amide bonds and were well characterized by Fourier-transform infrared (FT-IR), 1 H NMR, 13 C NMR and electrospray ionization mass spectrometry (ESI-MS). The dendrimers have been evaluated for in vitro cytotoxicity using sulforhodamine B assay (SRB assay) and in vivo skin irritation potential. The ester linked dendrimers did not exhibit any cytotoxicity even up to 80 g/ml while G-1 and G-2 generations dendrimers with amide linkage exhibited toxicity above 70 g/ml and 21 g/ml, respectively, none of the dendrimers showed any skin irritation. All the dendrimers, tested for their skin permeation enhancement potential using diclofenac sodium (DS) as a model drug at a concentration of 1% in gels, showed significant increase in steady-state flux (ERflux ) of the drug as compared to control (without enhancer), and oleic acid. Amongst the dendrimers, the ester linked G-1 and G-2 dendrimers showed highest ERflux , 3.33 ± 0.31 and 3.39 ± 0.21, respectively. © 2013 Elsevier B.V. All rights reserved.
1. Introduction For the last 30 years dendrimer chemistry has been the center of attraction for scientists from all over the fields and in particular in drug delivery technology and disease diagnosis. With their unique size and tree like shape with large number of peripheral functionalities, dendrimers, in addition to drug entrapment, provide scope for property modifications and alternative sites for drug conjugation. Some of their applications include vehicles for drug delivery (Esfand and Tomalia, 2001; Jain et al., 2006; Yang and Lopina, 2007), as component in transdermal drug delivery (Chauhan et al., 2003), preparation of prodrugs by drug conjugation (Emanuele et al., 2004; Najlah et al., 2007), tissue targeting of the drugs (Bhadra et al., 2005; Kono et al., 2008; Agarwal et al., 2008), enhancement of solubility of poorly soluble or practically insoluble drugs (Devarakonda et al., 2005), as drugs (Klajnert et al., 2006; Chauhan et al., 2009; Refat et al., 2009) and HIV prophylactics (Jiang et al., 2005). A class of dendrimers, popularly referred as Janus type dendrimers, have two sides, polar at one end and non-polar at another end are amphiphilic molecules and attract both lipophilic as well hydrophilic compounds (Lenoble et al., 2007; Feng et al., 2007;
∗ Corresponding author. Tel.: +91 22 33611111; fax: +91 22 33611020. E-mail addresses: [email protected], [email protected] (K.G. Akamanchi). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.07.028
Fuchs et al., 2008; Tuuttila et al., 2008). Recently, libraries of Janus dendrimers have been synthesized which can self-assemble into uniform dendrimersomes and other complex architectures. These dendrimersomes have been used to encapsulate doxorubicin (Percec et al., 2010). Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in treatment of variety of local and systemic ailments; however there are some problems in their usage, like gastrointestinal adverse effects and nephrotoxicity. These problems have been addressed by adopting different approaches, including structural modifications to make new analogs, making prodrug, alternative route of administration, alternative delivery strategies, amongst others. Delivery of these drugs via alternate routes, like transdermal (Cordero et al., 1997), offers many advantages such as easy accessibility and painless delivery, protecting the active compound from gastric enzymes, avoidance of hepatic first pass effect and termination of therapy by simply removing medication from the site of application. Stratum corneum (SC), the uppermost layer of the skin, is the major barrier in the systemic transport of drug through the skin. “Brick” and “mortar” structure of SC is analogous to a wall with corneocytes of hydrated keratin comprising bricks and multiple lipid gel and liquid domains forming the mortar. Numerous methods are used to modify barrier properties of SC to facilitate drug transport across skin, amongst them, use of chemical penetration enhancers (CPEs), as excipients in the delivery systems, is widely considered (Barry, 2001; Williams and Barry, 2004). After initial rise in the number of CPEs
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in 1980s very few CPEs have been synthesized and the active pool of CPEs has reached to a constant level. Only 1 in 100,000 molecules represent a CPE because of their slow rate of synthesis and development as compared to bioactive molecules (Karande et al., 2005). Diclofenac sodium (DS) is a potent NSAID used in management of acute conditions of inflammation and pain, musculoskeletal disorders, rheumatism and arthritis. Clinical use of DS has been often limited due to drawbacks of irritation and ulceration of gastrointestinal mucosa after oral administration (McCarthy, 1999) therefore; transdermal delivery systems have been attempted. However major drawback is, it cannot reach in effective concentration at the site of application because, it does not penetrate well through the skin (Prasaee et al., 2002). Permeation enhancers have been employed to improve skin penetration of DS (Özgüney et al., 2006; Nokhodchi et al., 2007; Kisak and Singh, 2008) and oleic acid (OA), when solubilized in propylene glycol, has been found to be the best (Kim et al., 2008). Some of the oils and amphipaths have potential to improve transdermal drug delivery, or can even serve as drug carriers. In polar solvents (including water) many oils and amphipaths spontaneously form a plethora of structures including micelles, cubic phases, microemulsions, hexagonal phases, vesicles, etc. and can affect molecular transport across skin barrier, more directly, by acting as skin permeation enhancers (Cevc and Vierl, 2010). Fatty acid-alcohol esters have been used in enhancement of percutaneous penetration of DS (Takahashi et al., 2001). Fatty acids and their derivatives including OA and its derivatives, also find wide applications as CPEs in transdermal delivery of number of both lipophilic and hydrophilic drugs (Aungst, 1989; Yamamoto et al., 2003; Williams and Barry, 2004). Fatty acids are preferred because they have the advantage of being endogenous components of human skin and are known to enter the hydrophobic tails of the stratum corneum lipid bilayer, where they disturb lipid bilayer packing, increase fluidity and subsequently decrease the diffusional resistance to permeants (Golden et al., 1987). We are engaged in synthesis of OA based dendritic heterolipids and have recently reported synthesis of one such heterolipids and its application as an oil for development of self-microemulsifying drug delivery system of furosemide (Kalhapure and Akamanchi, 2012). Encouraged by the success we continued our efforts toward synthesis and evaluation of many such heterolipids and herein report our recent results on synthesis of OA based G-1 and G-2 Janus type poly (propyl ether imine) dendrimers and their evaluation as potential CPEs.
2. Materials and methods 2.1. Materials DS was obtained as a generous gift sample from Glenmark Pharmaceuticals, Nashik, India. OA was obtained from Sigma, USA. 3-Amino-1-propanol and tert-butyl acrylate were obtained from Alfa-Aesar, USA. Thionyl chloride, p-dimethylaminopyridine (DMAP), triethylamine (TEA), methyl acrylate, benzyl amine, acrylonitrile, lithium aluminum hydride (LAH) and benzyl alcohol were purchased from s d fine Chemicals, India. Other materials were procured as follows: 2,2,2-trichloroethyl chloroformate from Spectrochem, India, Raney Nickel and Pd/C (10%) from Monarch catalysts, India, and Carbopol 974 P NF from Lubrizol, USA. All the solvents used were of analytical grade and obtained from Merck. For thin layer chromatography, Merck precoated Silica-gel 60F254 plates were used. Methyl acrylate and acrylonitrile were purified by distillation before use. TEA was purified by distillation over ptoluenesulfonic acid.
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2.2. Instrumentation FT-IR spectra were recorded using Perkin Elmer spectrophotometer. 1 H NMR and 13 C NMR spectra were recorded on Jeol nuclear magnetic resonance spectrometer at frequencies 300 MHz and 75 MHz, respectively. Electrospray ionization-mass spectra (ESI-MS) were recorded on Varian mass spectrometer and Agilent 6524 Q-TOF accurate Mass LC MS/MS system, Agilent (USA). UV spectra were recorded on Shimadzu UV-1650 PC, UV-visible spectrophotometer. 2.3. Dendron synthesis Dendrons with primary alcohol as a focal functionality were synthesized following, with some modification, literature procedure (Krishna and Jayaraman, 2003). Dendrons with secondary amine as a focal functionality were synthesized starting with benzyl amine followed by iterative Michael addition and reduction reactions and at the end debenzylated by catalytic hydrogenolysis. 2.3.1. G-1 dendron (compound I) with aliphatic alcohol as a focal functionality (Scheme 1) To a solution of tert-butyl acrylate (1.92 g, 15 mmol) in MeOH (50 ml) was added, drop wise, a solution of 3-amino-1-propanol (5 mmol, 0.37 g) in MeOH (100 ml) maintaining the temperature below 30 ◦ C. The reaction mixture was stirred at room temperature for 8 h and allowed to stand overnight. MeOH and excess tert-butyl acrylate were removed under reduced pressure to get compound I as pure colorless liquid (1.64 g, 99%). 2.3.2. G-2 dendron (compound VII) with aliphatic alcohol as a focal functionality (Scheme 1) 2.3.2.1. Compound II. Acrylonitrile (6.36 g, 120 mmol) was added, drop wise, to a stirred mixture of benzyl alcohol (10.8 g, 100 mmol) and aqueous NaOH (40%) (1 ml) over a period of 1 h maintaining the temperature below 35 ◦ C. The reaction mixture was stirred further for 10 h, diluted with water and acidified with conc. HCl to pH 2 and extracted with CHCl3 , solvent was evaporated under reduced pressure to get compound II as colorless liquid (15.6 g, 97%). 2.3.2.2. Compound III. To a solution of compound II (10 g) in MeOH (200 ml) and 20% aqueous NaOH (10 ml) was added Raney Ni (1 g) and hydrogenated in Parr hydrogenator (H2 , 100 psi) for 12 h. The reaction mixture was filtered through a bed of celite, evaporated under vacuum to remove MeOH and water. To the residue dry methanol was added and solid separated out was removed by filtration. The filtrate was concentrated under vacuum to obtain the amine (9.95 g, 97%) which was used in the next step, without purification, as follows. The amine (8.25 g, 50 mmol) was dissolved in MeOH (100 ml) and added drop wise to a solution of methyl acrylate (12.9 g, 150 mmol) in MeOH (150 ml) over a period of 2 h. The reaction mixture was stirred further for 8 h and allowed to stand at room temperature for 24 h, solvent evaporated under reduced pressure to afford, after purification by chromatography (Silica gel #60-120 and hexane:EtOAc = 7:3) compound III as a colorless liquid (11.96 g, 71%). 2.3.2.3. Compound IV. To a suspension of LAH (8.36 g, 220 mmol) in dry THF (300 ml) at 0 ◦ C was added, drop wise, compound III (33.7 g, 100 mmol) in THF (150 ml) maintaining the temperature at 0 ◦ C. The suspension was stirred at 0 ◦ C for 1 h and brought to room temperature; and stirring was further continued for 2 h. The reaction mixture was quenched by careful addition of solid Na2 SO4 ·8H2 O followed by ice (16 g), and left until all of the unreacted LAH was destroyed and free white suspension settled at the bottom. Resulting suspension was filtered through a bed of celite and the filtrate
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Scheme 1. Synthesis of G-1 and G-2 dendrons with primary alcohol as a focal functionality.
was concentrated under reduced pressure to obtain compound IV as colorless oil (16.8 g, 60%). 2.3.2.4. Compound V. Acrylonitrile (160 mmol, 8.48 g) was added to a mixture of compound IV (11.25 g, 40 mmol) and aqueous NaOH (40%) (4 ml) and stirred for 18 h. The reaction mixture was diluted with water and acidified with concentrated HCl to pH 2 and extracted with CHCl3 , solvent was evaporated under vacuum and the residue obtained was purified by column chromatography using SiO2 (60-120 mesh) and hexane/EtOAc, 6:4 as eluent to get compound V as colorless liquid (8.96 g, 58%). 2.3.2.5. Compound VI. Compound V (7.74 g, 10 mmol) was dissolved in a mixture of MeOH (100 ml) and 20% aqueous NaOH (20 ml) and to this solution Raney Ni (1.32 g) was added and hydrogenated (H2 , 100 psi) at room temperature for 12 h, and worked up as described in Section 2.3.2.2 above to obtain amine as liquid residue (5.92 g, 75%). This amine was taken to next step without further purification as follows. The amine (3.95 g, 10 mmol) was added drop wise to a solution of tert-butyl acrylate (15.36 g, 120 mmol) in MeOH (100 ml) over a period of 1 h, and stirring was continued further for 8 h at room temperature. Excess tert-butyl acrylate and MeOH were evaporated under reduced pressure, crude product obtained as residue was purified by column chromatography (Silica gel #60-120 and hexane:EtOAc = 6:4) to get compound VI as a colorless liquid (4.08 g, 45%). 2.3.2.6. Compound VII. To a solution of compound VI (4 g) in MeOH (50 ml) and water (5 ml) was added 0.6 g of Pd/C (10%) and hydrogenated (H2 , 100 psi) at room temperature for 5 days to obtain
compound VII, after purification (SiO2 ) (hexane/EtOAc, 7:3), as a colorless to yellow oil (1.47 g, 41%; after recovery of 2.3 g of unreacted compound VI). 2.3.3. G-1 dendron (compound IX) with secondary amine as a focal functionality (Scheme 2) To a solution of methyl acrylate (172 g, 2000 mmol) in MeOH (500 ml) was added drop wise solution of benzylamine (53.5 g, 500 mmol) in MeOH (100 ml) at room temperature. Reaction mixture was stirred for 6 h. Excess methyl acrylate and MeOH was removed under vacuum to get clear yellow liquid (VIII) (136.71 g, 98%). Compound VIII (20 g) in MeOH (100 ml) was added with 0.1 g Pd/C (10%) and hydrogenated (H2 , 100 psi) at room temperature for 12 h, filtered through a celite bed and concentrated in vacuo to get colorless oil (12.87 g, 95%). 2.3.4. G-2 dendron (compound XIII) with secondary amine as a focal functionality (Scheme 2) 2.3.4.1. Compound X. To a suspension of LAH (6.69 g, 176 mmol) in dry THF (300 ml) at 0 ◦ C was added drop wise compound VIII (22.34 g, 80 mmol) in THF (100 ml) maintaining the temperature at 0 ◦ C and worked up as described under Section 2.3.2.3 to afford a colorless liquid (14.4 g, 81%). 2.3.4.2. Compound XI. Acrylonitrile (7.95 g, 150 mmol) was added drop wise to a mixture of X (11.15 g, 50 mmol) and aqueous NaOH (40%) (5 ml). Reaction was continued and worked up as described under Section 2.3.2.4 to afford a colorless to yellow liquid (12.35 g, 75%).
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Scheme 2. Synthesis of G-1 and G-2 dendrons with secondary amine as a focal functionality.
2.3.4.3. Compound XII. Compound XI (6.58 g, 20 mmol) was dissolved in a mixture of 100 ml MeOH and 20% aqueous NaOH (20 ml). Raney Ni (1.316 g) was added to the solution and hydrogenated (H2 , 100 psi) at room temperature for 12 h and worked up as described in Section 2.3.2.2 to obtain liquid amine (6 g, 89%). Amine (3.37 g, 10 mmol) was added drop wise to a solution of tert-butyl acrylate (15.36 g, 120 mmol) in 100 ml of MeOH over a period of 1 h. Reaction was continued and worked as described under Section 2.3.2.5 to obtain a colorless liquid (6.62 g, 78%).
2.4.2. General procedure for preparation of oleodendrimers E1E and E2E To a two necked flask fitted with Dean-stark water trap and reflux condenser was charged toluene, dendron (1 equiv.), and DMAP (1 equiv.) and refluxed for 3 h, and cooled to room temperature. To this cooled solution oleoyl chloride (1 equiv.) was added and refluxed for 8 h followed by removal of solvent under reduced pressure. Residue obtained was chromatographed (Silica gel #60120 and hexane:EtOAc = 9:1) to obtain pure product.
2.3.4.4. Compound XIII. 2,2,2-trichloro ethyl chloroformate (0.211 g, 1 mmol) was added to a solution of compound XII (0.850 g, 1 mmol) in acetonitrile (10 ml). Reaction mixture was stirred at room temperature for 30 min. Acetonitrile was removed under vacuum to get intermediate. Powdered zinc (0.0091 g) was added to intermediate in acetic acid (5 ml) in portions over a period of 1 h. Reaction was continued further for 2 h, following which the reaction mixture was added to 40 ml CHCl3 and mixture was neutralized with 5% aqueous NaHCO3 ·CHCl3 was removed under vacuum to obtain XIII as a colorless liquid (0.532 g, 70%).
2.4.3. General procedure for preparation of oleodendrimers A1E and A2E Dendron (1 equiv.) and TEA (2 equiv.) and CHCl3 were taken in two necked flask fitted with a guard tube, oleoyl chloride (1 equiv.) in CHCl3 was added drop wise with stirring maintaining the temperature below 35 ◦ C, stirred for further 2 h. Reaction mixture was diluted with water and organic layer separated, washed with brine, dried over Na2 SO4 and concentrated in vacuo. Residue obtained was purified by chromatography (Silica gel #60-120 and hexane:EtOAc = 7:3).
2.4. Dendrimer synthesis
2.4.4. Oleodendrimer E1E Prepared by following the general procedure using compound I (3.31 g, 100 mmol), and 50 ml of toluene to get E1E as light yellow liquid (5.35 g, 90%).
OA was converted to oleoyl chloride using thionyl chloride and was condensed with dendrons to afford after purification by chromatography (Silica gel #60-120 and hexane:EtOAc = 9:1), G-1 and G-2 Janus type dendrimers, hereafter, referred as oleodendrimer E1E, E2E, A1E and A2E (Scheme 3). 2.4.1. Oleoyl chloride To a solution of OA (0.1 mol, 28.25 g) in CHCl3 (200 ml) contained in 1 L round bottom flask, fitted with a reflux condenser and guard tube, was added SOCl2 (0.15 mol, 17.85 g) drop wise over a period of 2 h under continuous magnetic stirring, and heated at 60 ◦ C for additional 6 h. CHCl3 and excess of SOCl2 were removed under reduced pressure to get oleoyl chloride as a yellow colored oil (29.5 g, 98%).
2.4.5. Oleodendrimer E2E Prepared by following the general procedure using compound VII (0.818 g, 1 mmol) and 30 ml of toluene to get E2E as light yellow liquid (0.96 g, 75%). 2.4.6. Oleodendrimer A1E Oleoyl chloride (3.01 g, 10 mmol) in CHCl3 (50 ml) was added drop wise to a solution of IX (1.89 g, 10 mmol) and TEA (2.02 g, 20 mmol) in CHCl3 (50 ml) over a period of 1 h and worked up as described in Section 2.4.3 to obtain a colorless to yellow liquid (2.72 g, 60%).
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Scheme 3. Synthesis of oleodendrimers.
2.4.7. Oleodendrimer A2E Oleoyl chloride (0.301 g, 1 mmol) in CHCl3 (10 ml) was added drop wise to a solution of XIII (0.760 g, 1 mmol) and TEA (0.202 g, 2 mmol) in CHCl3 (20 ml) over a period of 1 h and worked up as described in Section 2.4.3 to obtain a colorless to yellow liquid (0.71 g, 69%). 2.5. In vitro cytotoxicity study Cytotoxicity of all the synthesized oleodendrimers was determined in Human Cervix Cancer Cell Line (HeLa) by sulforhodamine B (SRB) colorimetric assay at concentrations 10, 20, 40 and 80 g/ml (Skehn et al., 1990; Vichai and Kirtikara, 2006) using adriamycin as positive control. The percentage growth was calculated at each of the concentration levels using the six absorbance measurements. The dose response parameters were calculated for each test article. Values were calculated for three parameters; growth inhibition of 50% (GI50 ), the concentration of test compound resulting in total growth inhibition (TGI), and the net loss of cells following treatment (LC50 ). 2.6. In vivo skin irritation study New Zealand white male rabbits, of body weight in the range of 2–3 kg, were used for in vivo skin irritation test and the test was performed as per OECD guideline 404. The study protocol was approved by the Institutional Animal Ethics Committee (Approval No. ICT/IAEC/0910/13). Initially test was carried out using only one animal. Lactic acid was used as standard skin irritant (Schliemann-Willers et al., 2005) and was applied on left side area of body part and untreated right side was kept as control. Suitable semi-occlusive dressing patch was loosely held in contact with the skin for the duration of the exposure period. Neck collar was used to prevent the ingestion of the test substance, from the application site, by the animal. At the end of 4 h exposure period, the residual test substance was washed off using distilled water taking care that the existing response or the integrity of the epidermis is not altered. No dermal reactions were observed at 1 h and 24 h after removal of patch. As the animal, in the initial test, did not exhibit any dermal reaction the test was repeated with two additional animals to confirm the initial test findings. Sequential testing strategy was followed for other oleodendrimers. The dermal responses viz. erythema, eschar formation and edema were recorded at 1, 24, 48 and 72 h after removal of patch and the dermal reactions were classified into five grades point (0–4) on following basis; point
0, without erythema or edema; point 1, very slight erythema or edema; point 2, obvious erythema or edema; point 3, medium erythema or edema; point 4, strong erythema or edema with slight incrustation (Draize, 1959). From these grades point primary irritation index (PII) was determined for all the oleodendrimers and graded as follows: 0.00, no irritation; 0.04–0.99, irritation barely perceptible, 1.00–1.99, slight irritation; 2.00–2.99, mild irritation; 3.00–5.99, moderate irritation; 6.00–8.00, severe irritation. 2.7. Solubility study of DS To determine the solubility of DS, an excess amount of DS was added to distilled water and stirred at room temperature for 24 h with a magnetic stirrer. The sample was then filtered through a 0.45-m cellulose acetate filter (Millipore, India). The concentration of DS in the filtrate was determined spectrophotometrically at 277 nm (Obata et al., 1993; Kantarci et al., 2005). 2.8. Preparation of topical formulation DS gels (Table 1) were prepared using carbopol 974P NF, TEA, water and oleodendrimer E1E, E2E, A1E, and A2E as penetration enhancers in 1% (w/w) concentration. OA was used as a standard and a control formulation was prepared without any penetration enhancer. 2.9. Assay of DS A spectrophotometric method was used to quantify the amount of DS penetrating through rat skin in vitro. First, a stock solution containing 10 mg/10 ml of DS was prepared with phosphate buffer saline (PBS) of pH 6.8. Solutions of 0.5, 1, 1.5, 2 and 2.5 g/ml were prepared by the stock solution using PBS. The absorbances of the solutions were determined against a blank at 277 nm. Absorbance against concentration were plotted to get calibration Table 1 Formula for diclofenac sodium topical gel. Ingredients
Quantity (g)
Diclofenac sodium Carbopol 974P NF Oleodendrimer (E1E/E2E/A1E/A2E) Triethylamine Distilled water
0.3 0.5 0.1 q.s. 10
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(y = 0.346x + 0.003), which was linear over the concentration range of 0.5–2.5 g/ml with a R2 of 0.999. 2.10. In vitro skin permeation studies Permeation study was carried out using rat skin. The study protocol was approved by the Institutional Animal Ethics Committee (Approval No. ICT/IAEC/0910/12). Female Wistar rats (weight 150–200 g) were sacrificed by CO2 euthanasia. Full thickness abdominal skin was removed, after shaving off the hair, and subcutaneous fat was scratch up, examined microscopically to ensure the integrity (Durrheim et al., 1980) and stored in a freezer at −20 ◦ C until further use. Just before starting the experiment the skin was taken out and thawed until it reached the room temperature and equilibrated for an hour in pH 6.8 buffer and examined microscopically for its integrity. The skin piece was mounted between the compartments of Keshary-Chien diffusion cell (Panigrahi et al., 2005) with epidermis facing upward in the donor compartment. The active diffusion area was 1 cm2 . The receptor compartment was filled with 10 ml PBS of pH 6.8. Temperature of the cells was maintained at 37 ◦ C and 0.5 g of gel was applied on the skin surface of donor compartment. Solution in the receptor compartment was stirred magnetically at 500 rpm throughout the experiment. Samples (2 ml) were withdrawn at regular intervals through the sampling port, and fresh receptor fluid solution of the same volume was added to make up. Aliquots from the receptor solution were withdrawn periodically (up to 8 h) and measured spectrophotometrically at 277 nm against blank (PBS pH 6.8). For each release point experiment were carried in quadruplet, for each preparation, and average release was calculated. The steady state flux (Jss ) was obtained from the linear portion of the cumulative drug permeated versus time graph. Permeability coefficient (Kp ) was calculated using the formula: Kp =
Jss , Cdonor
where Cdonor is the concentration of drug in the donor compartment (Prasaee et al., 2002). The permeation-enhancing activities were expressed as enhancement ratios of flux, ERflux (El-Kattan et al., 2000); ERflux = DS flux with permeation enhancer/DS flux without permeation enhancer. 2.11. Calculation of log Poctanol/water and hydrophilic–lipophilic balance (HLB) values Molecular structures of the synthesized oleodendrimers were constructed using ChemSW software and relaxed to represent 3D structures in lowest energy conformation. For molecular energy minimization MM2 minimizer was used and then surfactant properties (log Poctanol/water and hydrophilic-lipophilic balance) were determined keeping other default settings. These properties were calculated in order to understand the effect of surfactant properties of oleodendrimers on penetration enhancement efficacy. 2.12. Statistical analysis Results were expressed as mean ± SD and statistical analysis was done by ANOVA. A probability level of P < 0.05 was considered significant.
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Synthetic sequence involved changes in functional group therefore it was easy to monitor reaction progress by FT-IR. Thus nitrile formation (C N : 2251 cm−1 ), reduction of nitrile to amine (C NH2 : 3363 cm−1 ), formation of ester (C O : 1728 cm−1 ) and conversion of ester to alcohol ( OH : 3350 cm−1 ) could be recognized by FTIR. Condensation of oleoyl chloride with dendrons to form ester bond could be confirmed by disappearance of OH ( = 3350 cm−1 ) and apperence of ester (C O : 1728 cm−1 ). Similarly, attachment of oleoyl chloride with dendrons to form amide bond could be identified by disappearance of NH2 ( = 3383 cm−1 ) and apppearance of C O of amide ( = 1651 cm−1 ). Characteristics of 1 H NMR spectra were changes in chemical shifts of CH2 groups at peripheries depending on the functional group present. Thus, disappearance of triplet at ∼2.46 ppm of CH2 C O group and appearance of triplet at ∼1.86 ppm corresponding to CH2 CH2 OH, and disappearance of triplet ∼2.74 ppm of the CH2 OH and appearance ∼2.52 and 3.56 ppm corresponding to CH2 O CH2 and CH2 CN protons, respectively, could be used comfortably to monitor the progress of the reaction. NMR data of indivisual compounds are given in the supporting information. For dendrimers and higher generation dendrons 1 H NMR was not very useful for as a decisive means for structure characterization because of increase in number of H atoms in the molecule sharply. Therefore they were analyzed by 13 C NMR for confirmation of structure. 3.1.1. Oleodendrimer E1E FT-IR (neat) : 1731, 1455, 1367, 1255, 1166 cm−1 . 1 H NMR (300 MHz, CDCl3 ) ␦: 0.88 (t, 3 H), 1.29 (m, 20 H), 1.44 (s, 18 H), 1.63 (q, 2 H), 1.75 (q, 2 H), 2.01 (m, 4 H), 2.33 (m, 6 H), 2.47 (t, 2 H), 2.71 (m, 4 H), 4.08 (t, 2 H), 5.34 (t, 2 H). 13 C NMR (75 MHz, CDCl3 ) ␦: 14.12, 22.68, 24.99, 26.69, 27.21, 28.10, 29.18, 29.33, 29.52, 29.71, 29.77, 31.92, 33.81, 34.36, 49.42, 50.15, 62.45, 80.30, 129.7, 129.99, 171.97, 173.85. ESI-MS m/z: 596 [M+ ]. 3.1.2. Oleodendrimer E2E FT-IR (neat) : 1731, 1462, 1247, 1161 cm−1 . 1 H NMR (300 MHz, CDCl3 ) ␦: 0.88 (t, 3 H), 1.29 (m, 20 H), 1.44 (s, 36 H), 1.63 (m, 8 H), 1.75 (m, 4 H), 2.21 (m, 4 H), 2.33 (t, 2 H), 2.47 (m, 10 H), 2.71 (m, 8 H), 3.34 (m, 8 H), 3.84 (m, 8 H), 4.08 (t, 2 H), 5.34 9t, 2 H). 13 C NMR (75 MHz, CDCl3 ) ␦: 14.08, 21.37, 22.66, 24.65, 27.19, 29.04, 29.12, 29.30, 29.50, 29.66, 29.74, 31.89, 33.99, 34.35, 48.09, 51.44, 62.17, 67.18, 67.98, 70.39, 72.29, 81.57, 129.7, 130, 170.91, 173.56. ESI-MS m/z: 1082.91 [M+ ]. 3.1.3. Oleodendrimer A1E FT-IR (neat) : 2926, 2855, 1738, 1651, 1440, 1378, 1176, 1070 cm−1 . 1 H NMR (300 MHz, CDCl3 ) ␦: 0.88 (t, 3H), 1.27 (m, 18H), 1.30 (q, 2H), 1.61 (m, 2 H), 2.01 (t, 4 H), 2.31 (t, 2 H), 2.60 (m, 4 H), 3.58 (t, 4 H), 3.68 (s, 6 H), 5.34 (t, 2 H). 13 C NMR (75 MHz, CDCl3 ) ␦: 13.77, 22.34, 24.95, 26.87, 28.83, 28.97, 29.17, 29.42, 31.56, 32.26, 32.74, 33.45, 43.95, 51.61, 129.40, 129.62, 172.23, 173.01. ESI-MS m/z: 454.2 [M+ ]. 3.1.4. Oleodendrimer A2E FT-IR (neat) : 1728, 1651, 1462, 1367, 1255, 1158 cm−1 . 1 H NMR (60 MHz, CDCl3 ) ␦: 0.89 (t, 3 H), 1.28 (m, 18 H), 1.45 (s, 36 H), 1.71–3.51 (m, 50 H), 5.44 (t, 2 H). 13 C NMR (75 MHz, CDCl3 ) ␦: 14.26, 21.99, 22.67, 24.67, 27.19, 29.04, 29.17, 29.30, 29.57, 29.66, 29.74, 31.84, 33.96, 34.36, 60.87, 64.01, 67.00, 67.77, 82.29, 129.74, 130.00, 172.01, 173.56. ESI-MS m/z: 1024.89 [M+ ].
3. Results and discussion 3.2. In vitro cytotoxicity study 3.1. Characterization Techniques used for characterization of synthesized dendrons and dendrimers were FT-IR, 1 H NMR, 13 C NMR and ESI-MS.
The relative cell viability for HeLa cell line in the presence of oleodendrimers at different concentrations is presented in Fig. 1. In case of oleodendrimers E1E and E2E, the cell survival rate was
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Fig. 2. Cumulative drug permeated through rat skin vs. time profile in the absence and presence of permeation enhancer; (a) control, oleic acid, oleodendrimer E1E, E2E, A1E, and A2E. Each point represents mean ± SD (n = 4). Fig. 1. Growth curve: human cervix cancer cell line HeLa.
3.3. In vivo skin irritation studies
Table 2 LC50 , TGI and GI50 values derived from Fig. 1. Oleodendrimer
Value (g/ml)
E1E E2E A1E A2E
LC50
TGI
GI50
>80 >80 >80 >80
>80 >80 >80 44.6
>80 >80 69.7 20.9
The acute skin irritation evaluation was carried out for oleodendrimer E1E, E2E, A1E and A2E by Draize test using the dorsal skin of rabbits, the results are shown in Table 3. At the end of 72 h, after removal of the patch, all oleodendrimers produced PII of 0, thereby indicating that they were safe for use in transdermal delivery. However the rabbit skin, to which the patch containing lactic acid (positive control) was applied, showed signs of skin abrasion, erythema, and eschar at all the observed time points. 3.4. In vitro skin permeation studies
more than 95% whereas; the rate had reduced to 70% and 14% for oleodendrimers A1E and A2E, respectively. SRB study revealed that oleodendrimer E1E and E2E did not exhibit any cytotoxicity even up to 80 g/ml concentration, whereas, 50% inhibition in cell growth was observed for oleodendrimer A1E and A2E at 69.7 and 20.9 g/ml concentrations, respectively (P < 0.05). LC50 values of >80 g/ml for oleodendrimers revealed their non-cytotoxicity. Fig. 1 illustrates the growth curves for HeLa cell lines, and LC50 , TGI and GI50 values, derived from the growth curves are given in Table 2.
Dendrimers were used in 1% concentration to study the effect of dendrimer generation and type of dendron linkage to OA moiety on the permeation flux of DS. All formulations with oleodendrimers showed significant increase in flux, as compared to formulation with OA, as penetration enhancer and control, respectively (Fig. 2). Increase in generation of dendrimer from 1 to 2 resulted in increases in flux of DS (Table 4). Oleodendrimer E2E showed the highest permeation flux, Jss (E2E > E1E > A2E > A1E) (P < 0.05). Dendrimers with amide linkage between OA moiety and
Table 3 Primary skin irritation on rabbit dorsal skin after treating with 0.5 g of the enhancers in a 6-cm2 patch. Code
Type of response
E1E
Erythema Edema Erythema Edema Erythema Edema Erythema Edema
E2E A1E A2E a
PIIa
Time after removal of dressing 1h
24 h
48 h
72 h
6 days
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
– – – – – – – –
0 0 0 0 0 0 0 0
PII (primary irritation index), calculated by averaging the erythema values and the edema values at 1, 24, 48, 72 h, respectively, then combining the averages.
Table 4 In vitro permeability parameters for diclofenac sodium across the rat skin (n = 4). Parameter
Jss Kp ERflux
Permeation enhancer Oleic acid
E1E
E2E
A1E
A2E
Control
1.63 ± 0.21 1.09 ± 0.14 × 10−4 1.87 ± 0.074
2.90 ± 0.53 1.94 ± 0.35 × 10−4 3.33 ± 0.31
2.95 ± 0.51 1.98 ± 0.34 × 10−4 3.39 ± 0.28
1.76 ± 0.23 1.17 ± 0.15 × 10−4 2.02 ± 0.08
2.24 ± 0.32 1.50 ± 0.21 × 10−4 2.58 ± 0.13
0.87 ± 0.08 0.58 ±0.05 × 10−4 –
Jss (g/cm2 /h), steady-state flux; Kp (cm/h), permeability coefficient; ERflux , enhancement ratio of flux.
R.S. Kalhapure, K.G. Akamanchi / International Journal of Pharmaceutics 454 (2013) 158–166 Table 5 Log Poctanol/water , HLB and ERflux values of oleodendrimers. Oleodendrimer
Log Poctanol/water
HLB
ERflux
E2E E1E
10.25 9.40
7.60 4.79
3.39 3.33
A2E A1E
9.79 5.84
7.02 4.51
2.58 2.02
dendron (A2E > A1E) showed low permeation flux (Jss ) compared to dendrimers with ester linkage between OA moiety and dendron (E2E > E1E) (P < 0.05). The results obtained in this study are consistent with diclofenac permeation studies using monounsaturated fatty acids, where, increase in permeation was observed with increase in carbon chain length, and change in carboxylic acid moiety of OA to amide resulted in decrease in permeation rate (Kim et al., 2008). Enhancement ratios of oleodendrimers E2E, E1E, A2E and A1E were 3.39, 3.33, 2.58, and 2.02, respectively, whereas for OA it was 1.87 (P < 0.05). Interestingly, oleodendrimer A1E showed low flux and enhancement ratio as compared to oleodendrimer E1E (P < 0.05). This may be because of presence of amide linkage in oleodendrimer A1E and methyl ester at the periphery, which makes it less lipophilic as compared to that of oleodendrimer E1E having ester linkage between OA and dendron and presence of t-butyl ester at the periphery. Our results were in good agreement with the previous study, using the same model drug DS and aliphatic diesters as permeation enhancers. It has been observed that increasing lipophilicity of enhancers resulted in increased permeation of DS and, within the same homologous series, branched diesters showed more enhancements compared to linear diesters, as evidenced by higher ER value of DS for diisopropyl adipate than diethyl adipate (Takahashi et al., 2002). Log Poctanol/water and HLB values calculated using ChemSW software (Table 5) revealed that order for lipophilicity and HLB values for oleodendrimers was E2E > A2E > E1E > A1E. The result of in vitro penetration studies indicated linear relationship between lipophilicity, HLB and ER of DS (Fig. 3a and b). Increase in log Poctanol/water and HLB resulted in increase in ERflux (E2E > E1E
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and A2E > A1E) (Table 5). Higher enhancement ratio was shown by E1E than A2E, though A2E was found to be bit more lipophilic from log Poctanol/water values. This higher ER by E1E than A2E might be attributed to dominance of type of linkage in between oleic acid moiety and dendron over lipophilicity of oleodendrimers in facilitation of transdermal penetration of drug. According to HLB classification system, E1E, E2E, A2E would be classified as wetting agents and A1E as water-in-oil surfactant, but the mechanism of penetration enhancement by wetting agents and water-in-oil surfactants still remains unclear (Cornwell et al., 1998). Thus percutaneous absorption of the drug depends on the physicochemical properties of the drug, enhancer, and skin, changes in any of these parameters would affect drug penetration across the epidermis of the skin. 4. Conclusion New G-1 and G-2 Janus type dendrimers with OA moiety as a lipophilic side chain were successfully synthesized, characterized and evaluated for their penetration enhancement efficacy in transdermal formulations using DS as a model drug. All the dendrimers showed significant increase in permeation flux of the drug as compared to that of OA. The enhancement effect was influenced by the dendrimer generation, type of linkage between OA moiety and dendron, log Poctanol/water and HLB. From the present study, it can be concluded that these oleodendrimers have great potential for use in transdermal drug delivery systems, as penetration enhancers for hydrophilic drugs such as DS, and can be further evaluated for transdermal permeation enhancement of hydrophobic drugs as well. Additionally, these oleodendrimers can be investigated as potential excipients for different applications such as oil phase, a wetting agent and surfactant for other types of drug delivery systems and formulation development. Conversion of peripheral functionality to carboxylic acid and further neutralization with alkali earth metals may result in new OA based amphiphilic dendritic anionic surfactants. These amphiphilic anionic surfactants can be evaluated for development of drug delivery systems where surfactants form a major component. Thus attaching dendritic head groups to OA could be potential approach toward structural modifications to get new lipids with desired properties. Acknowledgements The authors are thankful to Glenmark Pharmaceuticals, Nashik, India for gift sample of Diclofenac sodium. Rahul S. Kalhapure is thankful to University Grants Commission, Government of India for financial support under UGC-SAP. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijpharm. 2013.07.028. References
Fig. 3. Graph of ERflux versus (a) log P and (b) HLB of oleodendrimers.
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Oleodendrimers: A novel class of multicephalous heterolipids as chemical penetration enhancers for transdermal drug delivery Rahul S. Kalhapure, Krishnacharya G. Akamanchi ∗ Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Matunga (E), Mumbai 400019, India
a r t i c l e
i n f o
Article history: Received 29 May 2013 Received in revised form 4 July 2013 Accepted 7 July 2013 Available online xxx Keywords: Dendrimers Transdermal delivery Penetration enhancers Diclofenac sodium Oleic acid Anti-inflammatory Oleodenrimers
a b s t r a c t This paper reports synthesis and evaluation of Janus type generation G-1 and G-2 dendrimers. The dendrimers have been constructed by linking two building blocks, dendrons and oleic acid, through ester and amide bonds and were well characterized by Fourier-transform infrared (FT-IR), 1 H NMR, 13 C NMR and electrospray ionization mass spectrometry (ESI-MS). The dendrimers have been evaluated for in vitro cytotoxicity using sulforhodamine B assay (SRB assay) and in vivo skin irritation potential. The ester linked dendrimers did not exhibit any cytotoxicity even up to 80 g/ml while G-1 and G-2 generations dendrimers with amide linkage exhibited toxicity above 70 g/ml and 21 g/ml, respectively, none of the dendrimers showed any skin irritation. All the dendrimers, tested for their skin permeation enhancement potential using diclofenac sodium (DS) as a model drug at a concentration of 1% in gels, showed significant increase in steady-state flux (ERflux ) of the drug as compared to control (without enhancer), and oleic acid. Amongst the dendrimers, the ester linked G-1 and G-2 dendrimers showed highest ERflux , 3.33 ± 0.31 and 3.39 ± 0.21, respectively. © 2013 Elsevier B.V. All rights reserved.
1. Introduction For the last 30 years dendrimer chemistry has been the center of attraction for scientists from all over the fields and in particular in drug delivery technology and disease diagnosis. With their unique size and tree like shape with large number of peripheral functionalities, dendrimers, in addition to drug entrapment, provide scope for property modifications and alternative sites for drug conjugation. Some of their applications include vehicles for drug delivery (Esfand and Tomalia, 2001; Jain et al., 2006; Yang and Lopina, 2007), as component in transdermal drug delivery (Chauhan et al., 2003), preparation of prodrugs by drug conjugation (Emanuele et al., 2004; Najlah et al., 2007), tissue targeting of the drugs (Bhadra et al., 2005; Kono et al., 2008; Agarwal et al., 2008), enhancement of solubility of poorly soluble or practically insoluble drugs (Devarakonda et al., 2005), as drugs (Klajnert et al., 2006; Chauhan et al., 2009; Refat et al., 2009) and HIV prophylactics (Jiang et al., 2005). A class of dendrimers, popularly referred as Janus type dendrimers, have two sides, polar at one end and non-polar at another end are amphiphilic molecules and attract both lipophilic as well hydrophilic compounds (Lenoble et al., 2007; Feng et al., 2007;
∗ Corresponding author. Tel.: +91 22 33611111; fax: +91 22 33611020. E-mail addresses: [email protected], [email protected] (K.G. Akamanchi). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.07.028
Fuchs et al., 2008; Tuuttila et al., 2008). Recently, libraries of Janus dendrimers have been synthesized which can self-assemble into uniform dendrimersomes and other complex architectures. These dendrimersomes have been used to encapsulate doxorubicin (Percec et al., 2010). Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in treatment of variety of local and systemic ailments; however there are some problems in their usage, like gastrointestinal adverse effects and nephrotoxicity. These problems have been addressed by adopting different approaches, including structural modifications to make new analogs, making prodrug, alternative route of administration, alternative delivery strategies, amongst others. Delivery of these drugs via alternate routes, like transdermal (Cordero et al., 1997), offers many advantages such as easy accessibility and painless delivery, protecting the active compound from gastric enzymes, avoidance of hepatic first pass effect and termination of therapy by simply removing medication from the site of application. Stratum corneum (SC), the uppermost layer of the skin, is the major barrier in the systemic transport of drug through the skin. “Brick” and “mortar” structure of SC is analogous to a wall with corneocytes of hydrated keratin comprising bricks and multiple lipid gel and liquid domains forming the mortar. Numerous methods are used to modify barrier properties of SC to facilitate drug transport across skin, amongst them, use of chemical penetration enhancers (CPEs), as excipients in the delivery systems, is widely considered (Barry, 2001; Williams and Barry, 2004). After initial rise in the number of CPEs
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in 1980s very few CPEs have been synthesized and the active pool of CPEs has reached to a constant level. Only 1 in 100,000 molecules represent a CPE because of their slow rate of synthesis and development as compared to bioactive molecules (Karande et al., 2005). Diclofenac sodium (DS) is a potent NSAID used in management of acute conditions of inflammation and pain, musculoskeletal disorders, rheumatism and arthritis. Clinical use of DS has been often limited due to drawbacks of irritation and ulceration of gastrointestinal mucosa after oral administration (McCarthy, 1999) therefore; transdermal delivery systems have been attempted. However major drawback is, it cannot reach in effective concentration at the site of application because, it does not penetrate well through the skin (Prasaee et al., 2002). Permeation enhancers have been employed to improve skin penetration of DS (Özgüney et al., 2006; Nokhodchi et al., 2007; Kisak and Singh, 2008) and oleic acid (OA), when solubilized in propylene glycol, has been found to be the best (Kim et al., 2008). Some of the oils and amphipaths have potential to improve transdermal drug delivery, or can even serve as drug carriers. In polar solvents (including water) many oils and amphipaths spontaneously form a plethora of structures including micelles, cubic phases, microemulsions, hexagonal phases, vesicles, etc. and can affect molecular transport across skin barrier, more directly, by acting as skin permeation enhancers (Cevc and Vierl, 2010). Fatty acid-alcohol esters have been used in enhancement of percutaneous penetration of DS (Takahashi et al., 2001). Fatty acids and their derivatives including OA and its derivatives, also find wide applications as CPEs in transdermal delivery of number of both lipophilic and hydrophilic drugs (Aungst, 1989; Yamamoto et al., 2003; Williams and Barry, 2004). Fatty acids are preferred because they have the advantage of being endogenous components of human skin and are known to enter the hydrophobic tails of the stratum corneum lipid bilayer, where they disturb lipid bilayer packing, increase fluidity and subsequently decrease the diffusional resistance to permeants (Golden et al., 1987). We are engaged in synthesis of OA based dendritic heterolipids and have recently reported synthesis of one such heterolipids and its application as an oil for development of self-microemulsifying drug delivery system of furosemide (Kalhapure and Akamanchi, 2012). Encouraged by the success we continued our efforts toward synthesis and evaluation of many such heterolipids and herein report our recent results on synthesis of OA based G-1 and G-2 Janus type poly (propyl ether imine) dendrimers and their evaluation as potential CPEs.
2. Materials and methods 2.1. Materials DS was obtained as a generous gift sample from Glenmark Pharmaceuticals, Nashik, India. OA was obtained from Sigma, USA. 3-Amino-1-propanol and tert-butyl acrylate were obtained from Alfa-Aesar, USA. Thionyl chloride, p-dimethylaminopyridine (DMAP), triethylamine (TEA), methyl acrylate, benzyl amine, acrylonitrile, lithium aluminum hydride (LAH) and benzyl alcohol were purchased from s d fine Chemicals, India. Other materials were procured as follows: 2,2,2-trichloroethyl chloroformate from Spectrochem, India, Raney Nickel and Pd/C (10%) from Monarch catalysts, India, and Carbopol 974 P NF from Lubrizol, USA. All the solvents used were of analytical grade and obtained from Merck. For thin layer chromatography, Merck precoated Silica-gel 60F254 plates were used. Methyl acrylate and acrylonitrile were purified by distillation before use. TEA was purified by distillation over ptoluenesulfonic acid.
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2.2. Instrumentation FT-IR spectra were recorded using Perkin Elmer spectrophotometer. 1 H NMR and 13 C NMR spectra were recorded on Jeol nuclear magnetic resonance spectrometer at frequencies 300 MHz and 75 MHz, respectively. Electrospray ionization-mass spectra (ESI-MS) were recorded on Varian mass spectrometer and Agilent 6524 Q-TOF accurate Mass LC MS/MS system, Agilent (USA). UV spectra were recorded on Shimadzu UV-1650 PC, UV-visible spectrophotometer. 2.3. Dendron synthesis Dendrons with primary alcohol as a focal functionality were synthesized following, with some modification, literature procedure (Krishna and Jayaraman, 2003). Dendrons with secondary amine as a focal functionality were synthesized starting with benzyl amine followed by iterative Michael addition and reduction reactions and at the end debenzylated by catalytic hydrogenolysis. 2.3.1. G-1 dendron (compound I) with aliphatic alcohol as a focal functionality (Scheme 1) To a solution of tert-butyl acrylate (1.92 g, 15 mmol) in MeOH (50 ml) was added, drop wise, a solution of 3-amino-1-propanol (5 mmol, 0.37 g) in MeOH (100 ml) maintaining the temperature below 30 ◦ C. The reaction mixture was stirred at room temperature for 8 h and allowed to stand overnight. MeOH and excess tert-butyl acrylate were removed under reduced pressure to get compound I as pure colorless liquid (1.64 g, 99%). 2.3.2. G-2 dendron (compound VII) with aliphatic alcohol as a focal functionality (Scheme 1) 2.3.2.1. Compound II. Acrylonitrile (6.36 g, 120 mmol) was added, drop wise, to a stirred mixture of benzyl alcohol (10.8 g, 100 mmol) and aqueous NaOH (40%) (1 ml) over a period of 1 h maintaining the temperature below 35 ◦ C. The reaction mixture was stirred further for 10 h, diluted with water and acidified with conc. HCl to pH 2 and extracted with CHCl3 , solvent was evaporated under reduced pressure to get compound II as colorless liquid (15.6 g, 97%). 2.3.2.2. Compound III. To a solution of compound II (10 g) in MeOH (200 ml) and 20% aqueous NaOH (10 ml) was added Raney Ni (1 g) and hydrogenated in Parr hydrogenator (H2 , 100 psi) for 12 h. The reaction mixture was filtered through a bed of celite, evaporated under vacuum to remove MeOH and water. To the residue dry methanol was added and solid separated out was removed by filtration. The filtrate was concentrated under vacuum to obtain the amine (9.95 g, 97%) which was used in the next step, without purification, as follows. The amine (8.25 g, 50 mmol) was dissolved in MeOH (100 ml) and added drop wise to a solution of methyl acrylate (12.9 g, 150 mmol) in MeOH (150 ml) over a period of 2 h. The reaction mixture was stirred further for 8 h and allowed to stand at room temperature for 24 h, solvent evaporated under reduced pressure to afford, after purification by chromatography (Silica gel #60-120 and hexane:EtOAc = 7:3) compound III as a colorless liquid (11.96 g, 71%). 2.3.2.3. Compound IV. To a suspension of LAH (8.36 g, 220 mmol) in dry THF (300 ml) at 0 ◦ C was added, drop wise, compound III (33.7 g, 100 mmol) in THF (150 ml) maintaining the temperature at 0 ◦ C. The suspension was stirred at 0 ◦ C for 1 h and brought to room temperature; and stirring was further continued for 2 h. The reaction mixture was quenched by careful addition of solid Na2 SO4 ·8H2 O followed by ice (16 g), and left until all of the unreacted LAH was destroyed and free white suspension settled at the bottom. Resulting suspension was filtered through a bed of celite and the filtrate
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Scheme 1. Synthesis of G-1 and G-2 dendrons with primary alcohol as a focal functionality.
was concentrated under reduced pressure to obtain compound IV as colorless oil (16.8 g, 60%). 2.3.2.4. Compound V. Acrylonitrile (160 mmol, 8.48 g) was added to a mixture of compound IV (11.25 g, 40 mmol) and aqueous NaOH (40%) (4 ml) and stirred for 18 h. The reaction mixture was diluted with water and acidified with concentrated HCl to pH 2 and extracted with CHCl3 , solvent was evaporated under vacuum and the residue obtained was purified by column chromatography using SiO2 (60-120 mesh) and hexane/EtOAc, 6:4 as eluent to get compound V as colorless liquid (8.96 g, 58%). 2.3.2.5. Compound VI. Compound V (7.74 g, 10 mmol) was dissolved in a mixture of MeOH (100 ml) and 20% aqueous NaOH (20 ml) and to this solution Raney Ni (1.32 g) was added and hydrogenated (H2 , 100 psi) at room temperature for 12 h, and worked up as described in Section 2.3.2.2 above to obtain amine as liquid residue (5.92 g, 75%). This amine was taken to next step without further purification as follows. The amine (3.95 g, 10 mmol) was added drop wise to a solution of tert-butyl acrylate (15.36 g, 120 mmol) in MeOH (100 ml) over a period of 1 h, and stirring was continued further for 8 h at room temperature. Excess tert-butyl acrylate and MeOH were evaporated under reduced pressure, crude product obtained as residue was purified by column chromatography (Silica gel #60-120 and hexane:EtOAc = 6:4) to get compound VI as a colorless liquid (4.08 g, 45%). 2.3.2.6. Compound VII. To a solution of compound VI (4 g) in MeOH (50 ml) and water (5 ml) was added 0.6 g of Pd/C (10%) and hydrogenated (H2 , 100 psi) at room temperature for 5 days to obtain
compound VII, after purification (SiO2 ) (hexane/EtOAc, 7:3), as a colorless to yellow oil (1.47 g, 41%; after recovery of 2.3 g of unreacted compound VI). 2.3.3. G-1 dendron (compound IX) with secondary amine as a focal functionality (Scheme 2) To a solution of methyl acrylate (172 g, 2000 mmol) in MeOH (500 ml) was added drop wise solution of benzylamine (53.5 g, 500 mmol) in MeOH (100 ml) at room temperature. Reaction mixture was stirred for 6 h. Excess methyl acrylate and MeOH was removed under vacuum to get clear yellow liquid (VIII) (136.71 g, 98%). Compound VIII (20 g) in MeOH (100 ml) was added with 0.1 g Pd/C (10%) and hydrogenated (H2 , 100 psi) at room temperature for 12 h, filtered through a celite bed and concentrated in vacuo to get colorless oil (12.87 g, 95%). 2.3.4. G-2 dendron (compound XIII) with secondary amine as a focal functionality (Scheme 2) 2.3.4.1. Compound X. To a suspension of LAH (6.69 g, 176 mmol) in dry THF (300 ml) at 0 ◦ C was added drop wise compound VIII (22.34 g, 80 mmol) in THF (100 ml) maintaining the temperature at 0 ◦ C and worked up as described under Section 2.3.2.3 to afford a colorless liquid (14.4 g, 81%). 2.3.4.2. Compound XI. Acrylonitrile (7.95 g, 150 mmol) was added drop wise to a mixture of X (11.15 g, 50 mmol) and aqueous NaOH (40%) (5 ml). Reaction was continued and worked up as described under Section 2.3.2.4 to afford a colorless to yellow liquid (12.35 g, 75%).
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Scheme 2. Synthesis of G-1 and G-2 dendrons with secondary amine as a focal functionality.
2.3.4.3. Compound XII. Compound XI (6.58 g, 20 mmol) was dissolved in a mixture of 100 ml MeOH and 20% aqueous NaOH (20 ml). Raney Ni (1.316 g) was added to the solution and hydrogenated (H2 , 100 psi) at room temperature for 12 h and worked up as described in Section 2.3.2.2 to obtain liquid amine (6 g, 89%). Amine (3.37 g, 10 mmol) was added drop wise to a solution of tert-butyl acrylate (15.36 g, 120 mmol) in 100 ml of MeOH over a period of 1 h. Reaction was continued and worked as described under Section 2.3.2.5 to obtain a colorless liquid (6.62 g, 78%).
2.4.2. General procedure for preparation of oleodendrimers E1E and E2E To a two necked flask fitted with Dean-stark water trap and reflux condenser was charged toluene, dendron (1 equiv.), and DMAP (1 equiv.) and refluxed for 3 h, and cooled to room temperature. To this cooled solution oleoyl chloride (1 equiv.) was added and refluxed for 8 h followed by removal of solvent under reduced pressure. Residue obtained was chromatographed (Silica gel #60120 and hexane:EtOAc = 9:1) to obtain pure product.
2.3.4.4. Compound XIII. 2,2,2-trichloro ethyl chloroformate (0.211 g, 1 mmol) was added to a solution of compound XII (0.850 g, 1 mmol) in acetonitrile (10 ml). Reaction mixture was stirred at room temperature for 30 min. Acetonitrile was removed under vacuum to get intermediate. Powdered zinc (0.0091 g) was added to intermediate in acetic acid (5 ml) in portions over a period of 1 h. Reaction was continued further for 2 h, following which the reaction mixture was added to 40 ml CHCl3 and mixture was neutralized with 5% aqueous NaHCO3 ·CHCl3 was removed under vacuum to obtain XIII as a colorless liquid (0.532 g, 70%).
2.4.3. General procedure for preparation of oleodendrimers A1E and A2E Dendron (1 equiv.) and TEA (2 equiv.) and CHCl3 were taken in two necked flask fitted with a guard tube, oleoyl chloride (1 equiv.) in CHCl3 was added drop wise with stirring maintaining the temperature below 35 ◦ C, stirred for further 2 h. Reaction mixture was diluted with water and organic layer separated, washed with brine, dried over Na2 SO4 and concentrated in vacuo. Residue obtained was purified by chromatography (Silica gel #60-120 and hexane:EtOAc = 7:3).
2.4. Dendrimer synthesis
2.4.4. Oleodendrimer E1E Prepared by following the general procedure using compound I (3.31 g, 100 mmol), and 50 ml of toluene to get E1E as light yellow liquid (5.35 g, 90%).
OA was converted to oleoyl chloride using thionyl chloride and was condensed with dendrons to afford after purification by chromatography (Silica gel #60-120 and hexane:EtOAc = 9:1), G-1 and G-2 Janus type dendrimers, hereafter, referred as oleodendrimer E1E, E2E, A1E and A2E (Scheme 3). 2.4.1. Oleoyl chloride To a solution of OA (0.1 mol, 28.25 g) in CHCl3 (200 ml) contained in 1 L round bottom flask, fitted with a reflux condenser and guard tube, was added SOCl2 (0.15 mol, 17.85 g) drop wise over a period of 2 h under continuous magnetic stirring, and heated at 60 ◦ C for additional 6 h. CHCl3 and excess of SOCl2 were removed under reduced pressure to get oleoyl chloride as a yellow colored oil (29.5 g, 98%).
2.4.5. Oleodendrimer E2E Prepared by following the general procedure using compound VII (0.818 g, 1 mmol) and 30 ml of toluene to get E2E as light yellow liquid (0.96 g, 75%). 2.4.6. Oleodendrimer A1E Oleoyl chloride (3.01 g, 10 mmol) in CHCl3 (50 ml) was added drop wise to a solution of IX (1.89 g, 10 mmol) and TEA (2.02 g, 20 mmol) in CHCl3 (50 ml) over a period of 1 h and worked up as described in Section 2.4.3 to obtain a colorless to yellow liquid (2.72 g, 60%).
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Scheme 3. Synthesis of oleodendrimers.
2.4.7. Oleodendrimer A2E Oleoyl chloride (0.301 g, 1 mmol) in CHCl3 (10 ml) was added drop wise to a solution of XIII (0.760 g, 1 mmol) and TEA (0.202 g, 2 mmol) in CHCl3 (20 ml) over a period of 1 h and worked up as described in Section 2.4.3 to obtain a colorless to yellow liquid (0.71 g, 69%). 2.5. In vitro cytotoxicity study Cytotoxicity of all the synthesized oleodendrimers was determined in Human Cervix Cancer Cell Line (HeLa) by sulforhodamine B (SRB) colorimetric assay at concentrations 10, 20, 40 and 80 g/ml (Skehn et al., 1990; Vichai and Kirtikara, 2006) using adriamycin as positive control. The percentage growth was calculated at each of the concentration levels using the six absorbance measurements. The dose response parameters were calculated for each test article. Values were calculated for three parameters; growth inhibition of 50% (GI50 ), the concentration of test compound resulting in total growth inhibition (TGI), and the net loss of cells following treatment (LC50 ). 2.6. In vivo skin irritation study New Zealand white male rabbits, of body weight in the range of 2–3 kg, were used for in vivo skin irritation test and the test was performed as per OECD guideline 404. The study protocol was approved by the Institutional Animal Ethics Committee (Approval No. ICT/IAEC/0910/13). Initially test was carried out using only one animal. Lactic acid was used as standard skin irritant (Schliemann-Willers et al., 2005) and was applied on left side area of body part and untreated right side was kept as control. Suitable semi-occlusive dressing patch was loosely held in contact with the skin for the duration of the exposure period. Neck collar was used to prevent the ingestion of the test substance, from the application site, by the animal. At the end of 4 h exposure period, the residual test substance was washed off using distilled water taking care that the existing response or the integrity of the epidermis is not altered. No dermal reactions were observed at 1 h and 24 h after removal of patch. As the animal, in the initial test, did not exhibit any dermal reaction the test was repeated with two additional animals to confirm the initial test findings. Sequential testing strategy was followed for other oleodendrimers. The dermal responses viz. erythema, eschar formation and edema were recorded at 1, 24, 48 and 72 h after removal of patch and the dermal reactions were classified into five grades point (0–4) on following basis; point
0, without erythema or edema; point 1, very slight erythema or edema; point 2, obvious erythema or edema; point 3, medium erythema or edema; point 4, strong erythema or edema with slight incrustation (Draize, 1959). From these grades point primary irritation index (PII) was determined for all the oleodendrimers and graded as follows: 0.00, no irritation; 0.04–0.99, irritation barely perceptible, 1.00–1.99, slight irritation; 2.00–2.99, mild irritation; 3.00–5.99, moderate irritation; 6.00–8.00, severe irritation. 2.7. Solubility study of DS To determine the solubility of DS, an excess amount of DS was added to distilled water and stirred at room temperature for 24 h with a magnetic stirrer. The sample was then filtered through a 0.45-m cellulose acetate filter (Millipore, India). The concentration of DS in the filtrate was determined spectrophotometrically at 277 nm (Obata et al., 1993; Kantarci et al., 2005). 2.8. Preparation of topical formulation DS gels (Table 1) were prepared using carbopol 974P NF, TEA, water and oleodendrimer E1E, E2E, A1E, and A2E as penetration enhancers in 1% (w/w) concentration. OA was used as a standard and a control formulation was prepared without any penetration enhancer. 2.9. Assay of DS A spectrophotometric method was used to quantify the amount of DS penetrating through rat skin in vitro. First, a stock solution containing 10 mg/10 ml of DS was prepared with phosphate buffer saline (PBS) of pH 6.8. Solutions of 0.5, 1, 1.5, 2 and 2.5 g/ml were prepared by the stock solution using PBS. The absorbances of the solutions were determined against a blank at 277 nm. Absorbance against concentration were plotted to get calibration Table 1 Formula for diclofenac sodium topical gel. Ingredients
Quantity (g)
Diclofenac sodium Carbopol 974P NF Oleodendrimer (E1E/E2E/A1E/A2E) Triethylamine Distilled water
0.3 0.5 0.1 q.s. 10
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(y = 0.346x + 0.003), which was linear over the concentration range of 0.5–2.5 g/ml with a R2 of 0.999. 2.10. In vitro skin permeation studies Permeation study was carried out using rat skin. The study protocol was approved by the Institutional Animal Ethics Committee (Approval No. ICT/IAEC/0910/12). Female Wistar rats (weight 150–200 g) were sacrificed by CO2 euthanasia. Full thickness abdominal skin was removed, after shaving off the hair, and subcutaneous fat was scratch up, examined microscopically to ensure the integrity (Durrheim et al., 1980) and stored in a freezer at −20 ◦ C until further use. Just before starting the experiment the skin was taken out and thawed until it reached the room temperature and equilibrated for an hour in pH 6.8 buffer and examined microscopically for its integrity. The skin piece was mounted between the compartments of Keshary-Chien diffusion cell (Panigrahi et al., 2005) with epidermis facing upward in the donor compartment. The active diffusion area was 1 cm2 . The receptor compartment was filled with 10 ml PBS of pH 6.8. Temperature of the cells was maintained at 37 ◦ C and 0.5 g of gel was applied on the skin surface of donor compartment. Solution in the receptor compartment was stirred magnetically at 500 rpm throughout the experiment. Samples (2 ml) were withdrawn at regular intervals through the sampling port, and fresh receptor fluid solution of the same volume was added to make up. Aliquots from the receptor solution were withdrawn periodically (up to 8 h) and measured spectrophotometrically at 277 nm against blank (PBS pH 6.8). For each release point experiment were carried in quadruplet, for each preparation, and average release was calculated. The steady state flux (Jss ) was obtained from the linear portion of the cumulative drug permeated versus time graph. Permeability coefficient (Kp ) was calculated using the formula: Kp =
Jss , Cdonor
where Cdonor is the concentration of drug in the donor compartment (Prasaee et al., 2002). The permeation-enhancing activities were expressed as enhancement ratios of flux, ERflux (El-Kattan et al., 2000); ERflux = DS flux with permeation enhancer/DS flux without permeation enhancer. 2.11. Calculation of log Poctanol/water and hydrophilic–lipophilic balance (HLB) values Molecular structures of the synthesized oleodendrimers were constructed using ChemSW software and relaxed to represent 3D structures in lowest energy conformation. For molecular energy minimization MM2 minimizer was used and then surfactant properties (log Poctanol/water and hydrophilic-lipophilic balance) were determined keeping other default settings. These properties were calculated in order to understand the effect of surfactant properties of oleodendrimers on penetration enhancement efficacy. 2.12. Statistical analysis Results were expressed as mean ± SD and statistical analysis was done by ANOVA. A probability level of P < 0.05 was considered significant.
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Synthetic sequence involved changes in functional group therefore it was easy to monitor reaction progress by FT-IR. Thus nitrile formation (C N : 2251 cm−1 ), reduction of nitrile to amine (C NH2 : 3363 cm−1 ), formation of ester (C O : 1728 cm−1 ) and conversion of ester to alcohol ( OH : 3350 cm−1 ) could be recognized by FTIR. Condensation of oleoyl chloride with dendrons to form ester bond could be confirmed by disappearance of OH ( = 3350 cm−1 ) and apperence of ester (C O : 1728 cm−1 ). Similarly, attachment of oleoyl chloride with dendrons to form amide bond could be identified by disappearance of NH2 ( = 3383 cm−1 ) and apppearance of C O of amide ( = 1651 cm−1 ). Characteristics of 1 H NMR spectra were changes in chemical shifts of CH2 groups at peripheries depending on the functional group present. Thus, disappearance of triplet at ∼2.46 ppm of CH2 C O group and appearance of triplet at ∼1.86 ppm corresponding to CH2 CH2 OH, and disappearance of triplet ∼2.74 ppm of the CH2 OH and appearance ∼2.52 and 3.56 ppm corresponding to CH2 O CH2 and CH2 CN protons, respectively, could be used comfortably to monitor the progress of the reaction. NMR data of indivisual compounds are given in the supporting information. For dendrimers and higher generation dendrons 1 H NMR was not very useful for as a decisive means for structure characterization because of increase in number of H atoms in the molecule sharply. Therefore they were analyzed by 13 C NMR for confirmation of structure. 3.1.1. Oleodendrimer E1E FT-IR (neat) : 1731, 1455, 1367, 1255, 1166 cm−1 . 1 H NMR (300 MHz, CDCl3 ) ␦: 0.88 (t, 3 H), 1.29 (m, 20 H), 1.44 (s, 18 H), 1.63 (q, 2 H), 1.75 (q, 2 H), 2.01 (m, 4 H), 2.33 (m, 6 H), 2.47 (t, 2 H), 2.71 (m, 4 H), 4.08 (t, 2 H), 5.34 (t, 2 H). 13 C NMR (75 MHz, CDCl3 ) ␦: 14.12, 22.68, 24.99, 26.69, 27.21, 28.10, 29.18, 29.33, 29.52, 29.71, 29.77, 31.92, 33.81, 34.36, 49.42, 50.15, 62.45, 80.30, 129.7, 129.99, 171.97, 173.85. ESI-MS m/z: 596 [M+ ]. 3.1.2. Oleodendrimer E2E FT-IR (neat) : 1731, 1462, 1247, 1161 cm−1 . 1 H NMR (300 MHz, CDCl3 ) ␦: 0.88 (t, 3 H), 1.29 (m, 20 H), 1.44 (s, 36 H), 1.63 (m, 8 H), 1.75 (m, 4 H), 2.21 (m, 4 H), 2.33 (t, 2 H), 2.47 (m, 10 H), 2.71 (m, 8 H), 3.34 (m, 8 H), 3.84 (m, 8 H), 4.08 (t, 2 H), 5.34 9t, 2 H). 13 C NMR (75 MHz, CDCl3 ) ␦: 14.08, 21.37, 22.66, 24.65, 27.19, 29.04, 29.12, 29.30, 29.50, 29.66, 29.74, 31.89, 33.99, 34.35, 48.09, 51.44, 62.17, 67.18, 67.98, 70.39, 72.29, 81.57, 129.7, 130, 170.91, 173.56. ESI-MS m/z: 1082.91 [M+ ]. 3.1.3. Oleodendrimer A1E FT-IR (neat) : 2926, 2855, 1738, 1651, 1440, 1378, 1176, 1070 cm−1 . 1 H NMR (300 MHz, CDCl3 ) ␦: 0.88 (t, 3H), 1.27 (m, 18H), 1.30 (q, 2H), 1.61 (m, 2 H), 2.01 (t, 4 H), 2.31 (t, 2 H), 2.60 (m, 4 H), 3.58 (t, 4 H), 3.68 (s, 6 H), 5.34 (t, 2 H). 13 C NMR (75 MHz, CDCl3 ) ␦: 13.77, 22.34, 24.95, 26.87, 28.83, 28.97, 29.17, 29.42, 31.56, 32.26, 32.74, 33.45, 43.95, 51.61, 129.40, 129.62, 172.23, 173.01. ESI-MS m/z: 454.2 [M+ ]. 3.1.4. Oleodendrimer A2E FT-IR (neat) : 1728, 1651, 1462, 1367, 1255, 1158 cm−1 . 1 H NMR (60 MHz, CDCl3 ) ␦: 0.89 (t, 3 H), 1.28 (m, 18 H), 1.45 (s, 36 H), 1.71–3.51 (m, 50 H), 5.44 (t, 2 H). 13 C NMR (75 MHz, CDCl3 ) ␦: 14.26, 21.99, 22.67, 24.67, 27.19, 29.04, 29.17, 29.30, 29.57, 29.66, 29.74, 31.84, 33.96, 34.36, 60.87, 64.01, 67.00, 67.77, 82.29, 129.74, 130.00, 172.01, 173.56. ESI-MS m/z: 1024.89 [M+ ].
3. Results and discussion 3.2. In vitro cytotoxicity study 3.1. Characterization Techniques used for characterization of synthesized dendrons and dendrimers were FT-IR, 1 H NMR, 13 C NMR and ESI-MS.
The relative cell viability for HeLa cell line in the presence of oleodendrimers at different concentrations is presented in Fig. 1. In case of oleodendrimers E1E and E2E, the cell survival rate was
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Fig. 2. Cumulative drug permeated through rat skin vs. time profile in the absence and presence of permeation enhancer; (a) control, oleic acid, oleodendrimer E1E, E2E, A1E, and A2E. Each point represents mean ± SD (n = 4). Fig. 1. Growth curve: human cervix cancer cell line HeLa.
3.3. In vivo skin irritation studies
Table 2 LC50 , TGI and GI50 values derived from Fig. 1. Oleodendrimer
Value (g/ml)
E1E E2E A1E A2E
LC50
TGI
GI50
>80 >80 >80 >80
>80 >80 >80 44.6
>80 >80 69.7 20.9
The acute skin irritation evaluation was carried out for oleodendrimer E1E, E2E, A1E and A2E by Draize test using the dorsal skin of rabbits, the results are shown in Table 3. At the end of 72 h, after removal of the patch, all oleodendrimers produced PII of 0, thereby indicating that they were safe for use in transdermal delivery. However the rabbit skin, to which the patch containing lactic acid (positive control) was applied, showed signs of skin abrasion, erythema, and eschar at all the observed time points. 3.4. In vitro skin permeation studies
more than 95% whereas; the rate had reduced to 70% and 14% for oleodendrimers A1E and A2E, respectively. SRB study revealed that oleodendrimer E1E and E2E did not exhibit any cytotoxicity even up to 80 g/ml concentration, whereas, 50% inhibition in cell growth was observed for oleodendrimer A1E and A2E at 69.7 and 20.9 g/ml concentrations, respectively (P < 0.05). LC50 values of >80 g/ml for oleodendrimers revealed their non-cytotoxicity. Fig. 1 illustrates the growth curves for HeLa cell lines, and LC50 , TGI and GI50 values, derived from the growth curves are given in Table 2.
Dendrimers were used in 1% concentration to study the effect of dendrimer generation and type of dendron linkage to OA moiety on the permeation flux of DS. All formulations with oleodendrimers showed significant increase in flux, as compared to formulation with OA, as penetration enhancer and control, respectively (Fig. 2). Increase in generation of dendrimer from 1 to 2 resulted in increases in flux of DS (Table 4). Oleodendrimer E2E showed the highest permeation flux, Jss (E2E > E1E > A2E > A1E) (P < 0.05). Dendrimers with amide linkage between OA moiety and
Table 3 Primary skin irritation on rabbit dorsal skin after treating with 0.5 g of the enhancers in a 6-cm2 patch. Code
Type of response
E1E
Erythema Edema Erythema Edema Erythema Edema Erythema Edema
E2E A1E A2E a
PIIa
Time after removal of dressing 1h
24 h
48 h
72 h
6 days
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
– – – – – – – –
0 0 0 0 0 0 0 0
PII (primary irritation index), calculated by averaging the erythema values and the edema values at 1, 24, 48, 72 h, respectively, then combining the averages.
Table 4 In vitro permeability parameters for diclofenac sodium across the rat skin (n = 4). Parameter
Jss Kp ERflux
Permeation enhancer Oleic acid
E1E
E2E
A1E
A2E
Control
1.63 ± 0.21 1.09 ± 0.14 × 10−4 1.87 ± 0.074
2.90 ± 0.53 1.94 ± 0.35 × 10−4 3.33 ± 0.31
2.95 ± 0.51 1.98 ± 0.34 × 10−4 3.39 ± 0.28
1.76 ± 0.23 1.17 ± 0.15 × 10−4 2.02 ± 0.08
2.24 ± 0.32 1.50 ± 0.21 × 10−4 2.58 ± 0.13
0.87 ± 0.08 0.58 ±0.05 × 10−4 –
Jss (g/cm2 /h), steady-state flux; Kp (cm/h), permeability coefficient; ERflux , enhancement ratio of flux.
R.S. Kalhapure, K.G. Akamanchi / International Journal of Pharmaceutics 454 (2013) 158–166 Table 5 Log Poctanol/water , HLB and ERflux values of oleodendrimers. Oleodendrimer
Log Poctanol/water
HLB
ERflux
E2E E1E
10.25 9.40
7.60 4.79
3.39 3.33
A2E A1E
9.79 5.84
7.02 4.51
2.58 2.02
dendron (A2E > A1E) showed low permeation flux (Jss ) compared to dendrimers with ester linkage between OA moiety and dendron (E2E > E1E) (P < 0.05). The results obtained in this study are consistent with diclofenac permeation studies using monounsaturated fatty acids, where, increase in permeation was observed with increase in carbon chain length, and change in carboxylic acid moiety of OA to amide resulted in decrease in permeation rate (Kim et al., 2008). Enhancement ratios of oleodendrimers E2E, E1E, A2E and A1E were 3.39, 3.33, 2.58, and 2.02, respectively, whereas for OA it was 1.87 (P < 0.05). Interestingly, oleodendrimer A1E showed low flux and enhancement ratio as compared to oleodendrimer E1E (P < 0.05). This may be because of presence of amide linkage in oleodendrimer A1E and methyl ester at the periphery, which makes it less lipophilic as compared to that of oleodendrimer E1E having ester linkage between OA and dendron and presence of t-butyl ester at the periphery. Our results were in good agreement with the previous study, using the same model drug DS and aliphatic diesters as permeation enhancers. It has been observed that increasing lipophilicity of enhancers resulted in increased permeation of DS and, within the same homologous series, branched diesters showed more enhancements compared to linear diesters, as evidenced by higher ER value of DS for diisopropyl adipate than diethyl adipate (Takahashi et al., 2002). Log Poctanol/water and HLB values calculated using ChemSW software (Table 5) revealed that order for lipophilicity and HLB values for oleodendrimers was E2E > A2E > E1E > A1E. The result of in vitro penetration studies indicated linear relationship between lipophilicity, HLB and ER of DS (Fig. 3a and b). Increase in log Poctanol/water and HLB resulted in increase in ERflux (E2E > E1E
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and A2E > A1E) (Table 5). Higher enhancement ratio was shown by E1E than A2E, though A2E was found to be bit more lipophilic from log Poctanol/water values. This higher ER by E1E than A2E might be attributed to dominance of type of linkage in between oleic acid moiety and dendron over lipophilicity of oleodendrimers in facilitation of transdermal penetration of drug. According to HLB classification system, E1E, E2E, A2E would be classified as wetting agents and A1E as water-in-oil surfactant, but the mechanism of penetration enhancement by wetting agents and water-in-oil surfactants still remains unclear (Cornwell et al., 1998). Thus percutaneous absorption of the drug depends on the physicochemical properties of the drug, enhancer, and skin, changes in any of these parameters would affect drug penetration across the epidermis of the skin. 4. Conclusion New G-1 and G-2 Janus type dendrimers with OA moiety as a lipophilic side chain were successfully synthesized, characterized and evaluated for their penetration enhancement efficacy in transdermal formulations using DS as a model drug. All the dendrimers showed significant increase in permeation flux of the drug as compared to that of OA. The enhancement effect was influenced by the dendrimer generation, type of linkage between OA moiety and dendron, log Poctanol/water and HLB. From the present study, it can be concluded that these oleodendrimers have great potential for use in transdermal drug delivery systems, as penetration enhancers for hydrophilic drugs such as DS, and can be further evaluated for transdermal permeation enhancement of hydrophobic drugs as well. Additionally, these oleodendrimers can be investigated as potential excipients for different applications such as oil phase, a wetting agent and surfactant for other types of drug delivery systems and formulation development. Conversion of peripheral functionality to carboxylic acid and further neutralization with alkali earth metals may result in new OA based amphiphilic dendritic anionic surfactants. These amphiphilic anionic surfactants can be evaluated for development of drug delivery systems where surfactants form a major component. Thus attaching dendritic head groups to OA could be potential approach toward structural modifications to get new lipids with desired properties. Acknowledgements The authors are thankful to Glenmark Pharmaceuticals, Nashik, India for gift sample of Diclofenac sodium. Rahul S. Kalhapure is thankful to University Grants Commission, Government of India for financial support under UGC-SAP. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijpharm. 2013.07.028. References
Fig. 3. Graph of ERflux versus (a) log P and (b) HLB of oleodendrimers.
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