Synthesis and Evaluation of Transdermal Permeation, Pharmacological Activity of Bornyl NSAID Esters

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Send Orders for Reprints to [email protected] 72 Letters in Drug Design & Discovery, 2015, 11, 72-77 Synthesis and Evaluation of Transdermal Permeation, Pharmacological Activity of Bornyl NSAID Esters

Jiufeng Lina, Kefeng Nia, Yang Zhangb, Dongzhi Weia and Yuhong Rena,* aState Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Sci- ence and Technology, Shanghai 200237, China bInstitute of Chemical Biology and Pharmacy, East China University of Science and Technology, Shanghai 200237, P.R. China

Abstract: The bornyl ester derivatives of non-steroidal anti-inflammatory drugs (NSAIDs) including naproxen, ketoprofen and flufenamic acid, were synthesized and their transdermal permeation efficiency and pharmacological activity were determined. The bornyl NSAID esters exhibited more than 10-fold increase of permeation efficiency across mice skin than parent NSAIDs. The preliminary biological and pharmacological evaluations suggested that the bornyl NSAID esters retain most of their inhibition activity toward cyclooxygenase (COX) enzymes and show significant analgesic effect against acetic acid-induced writhing in mice.

Keywords: Anti-nociceptive, borneol, cyclooxygenase, esters, non-steroidal anti-inflammatory drugs, transdermal permeation.

1. INTRODUCTION improve skin permeation of the drug. Gels which were composed of a mixture of solution of polyethylene glycol Over decades, NSAIDs have been used extensively as (PEG400), Tween 80 and (2-hydroxypropyl)--cyclodextrin, anti-inflammatory, anti-pyretic and antinociceptive agents and oleic acid showed to significantly promote the percuta- in many conditions, including osteoarthritis and rheumatoid neous adsorption of zaltoprofen [16]. Therefore, topical for- arthritis [1-3]. NSAIDs exert their effects by inhibiting the mulation consisting of chemical penetration enhancers is the activity of COX for the synthesis of prostaglandins [4]. most widely implemented approach to facilitate the drugs However, it also underlies some of their common side ef- delivery through the skin. fects, particularly their gastrointestinal toxicity [5]. Gener- ally, oral administration of NSAIDs is very effective, but the Borneol, a Chinese traditional medicine, has refreshing, problem of side effects after long-term administration, such anti-pyretic and analgesic effects and has been used for more as irritation and ulceration of the gastrointestinal mucosa has than thousand years in China. Recent studies have shown arisen in clinical trials [6,7]. These gastroenteropathies are that borneol can be used as a permeation enhancer to help believed to be resulted from the direct contact effect between some drugs permeate through various physiological barriers, the NSAIDs and gastrointestinal tract [8, 9]. In contrast, per- including brain, skin, gastric mucosa and cornea [17-20]. cutaneous administration of NSAIDs can avoid these disad- Moreover, some nature bornyl ester compounds extracted vantages and provide better patient compliance than oral from Piper methysticum roots demonstrated substantial administration. Various transdermal delivery methods have COX-I and moderate COX-II inhibitory activities [21]. been designed in order to improve the skin permeation of However, the percutaneous adsorption of these compounds NSAIDs, including using appropriate vehicles [10, 11], per- was not investigated in the report. meation enhancers [12] or utilization of pro-drugs [13]. The Inspired by the structures of these borynl esters com- common approach is the use of excipients. Microemulsions pounds [21], we designed the NSAIDs bornyl ester to expect were composed of triglycerides, a mixture of lecithin and n- enhanced the permeation of NSAIDs moiety. We presumed butanol, and an aqueous solution which displayed an excel- that the prevalent hydrolases, especially the lipases in body lent enhanced permeation of ketoprofen with respect to con- could hydrolyze the bornyl esters into borneol and NSAIDs, ventional formulation [14]. Microemulsion system using the released NSAIDs then operate the therapeutic effect. Our oleic acid as the oil phase also showed a good solubilizing previous study of in-vitro enzymatic hydrolysis proved that capacity and skin permeation rate of ketoprofen [15]. In these NSAIDs bornyl ester could be quantitatively recon- these cases, additional permeation enhancers (terpenes [14] verted into NSAIDs at a very fast rate (data not shown). and oleic acid [15]) were added to the microemulsions to These NSAIDs bornyl ester may work as pro-drugs. As new compounds, the preliminary study of toxicity was investigated by the acute toxicity test in our lab, which was carried *Address correspondence to this author at the State Key Laboratory of out in mice via hypodermic injection. LD50 values for Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, China; naproxen and its bornyl ester is 475 mg/kg and 680 mg/kg, Tel: +86-2164252163; Fax: +86-2164250068; E-mail: [email protected] respectively. Similar results were obtained with ketoprofen

1875-628X/15 $58.00+.00 ©2015 Bentham Science Publishers Synthesis and Evaluation of Transdermal Permeation Letters in Drug Design & Discovery, 2015, Vol. 12, No. 1 73 and flufenamic acids, which indicated that the toxicity is n-hexane = 5:1). The bornyl NSAID esters were analyzed by lower after borneol esterification. We deduce that the side 1H-NMR and 13C NMR on a Brücker AM 500 spectrometer effects of these esters would mainly come from the NSAID in CDCl3. Mass spectrometry (MS) was performed on an moiety. Certainly, as potential drugs, they need to go through Agilent 5975E mass spectrometer. very complicated regulatory approval process before their application in the future. 1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl 2-(6-methoxynaph- In this study, the bornyl esters of naproxen, ketoprofen thalen-2-yl)propanoate 1b 1 and flufenamic acid were successfully synthesized in one Yield 61%; H NMR (500MHz, CDCl3/TMS, , ppm): step by directly coupling carboxyl group of NSAIDs to hy- 1.1 (m,3H), 1.5 (m, 1H), 1.6 (m, 5H), 1.8 (m, 5H), 2.3 (m, droxyl group of borneol. The transdermal permeation effi- 1H), 3.8 (m, 1H), 3.9 (s, 3H), 4.9 (t, J 7.1 Hz, 1H), 7.1 (d, J ciency of the NSAIDs bornyl esters was investigated com- 8.1 Hz, 2H), 7.4 (t, J 7.9 Hz, 1H), 7.7 (t, J 7.9 Hz, 3H); 13C pared to the NSAIDs and physical mixture of NSAIDs and NMR (126 MHz, CDCl3/TMS, , ppm): 173.6, 156.1, 132.9, borneol. Pharmacological activity of bornyl NSAID esters, 132.7, 129.4, 128.5/129, 126.1/126.7, 118.5, 105.6, 82.4, including their inhibition activity toward COX enzymes and 55.8, 49.3, 48.0, 45.3, 41.1, 38.1, 27.0/27.1, 19.5/19.5, 13.7, analgesic effect were evaluated. Moreover, in-vivo research 13.5; MS: m/z = 367.4 [M + H]+. of the pharmacokinetic (such as elimination half-life in blood), pharmacological action and toxicology properties of 1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl 2-(3-benzoylphen- these NSAIDs esters will be investigated in the next study. yl)propanoate 2b Yield 58%; 1H NMR (500MHz, CDCl /TMS, , ppm): 2. MATERIALS AND METHOD 3 1.1 (m,3H), 1.5 (m, 1H), 1.6 (m, 5H), 1.8 (m, 5H), 2.3 (m, 2.1. Experimental Animals 1H), 3.8 (m, 1H), 3.9 (s, 3H), 4.9 (t, J 7.1 Hz, 1H), 7.4 (m, 3H), 7.5 (m,2H), 7.7 (d, J 7.9 Hz, 1H), 7.8 (t, J 7.7 Hz, 3H); 13 Male nude mice (6 weeks old; BALB/cA-nu) and KM C NMR (126 MHz, CDCl3/TMS, , ppm): 194.2, 173.6, mice in CL grade (4-6 weeks old) used in this study were 140.1, 138.4, 135.1, 133.4, 132.3, 131.2, 130.4/130.3, 129.1, purchased from Shanghai Experimental Animal Center (Chi- 128.8, 128.4/128.3, 82.4, 49.5, 48.0, 45.3, 40.7, 38.1, nese Academy of Sciences, Shanghai, China). Mice were 27.0/27.1, 19.5/19.5, 13.7, 13.5; MS: m/z = 391.5 [M + H]+ housed in plastic cages at 20-25 °C and maintained on a standard pellet diet with free access to water. 1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl 2-((3-(trifluorome- thyl)phenyl)amino)benzoate 3b 2.2. Materials 1 Yield 65%; H NMR (500MHz, CDCl3/TMS, , ppm): Naproxen, ketoprofen, flufenamic acid and borneol were 1.1 (m,3H), 1.5 (m, 1H), 1.6 (m, 5H), 1.8 (m, 5H), 2.3 (m, purchased from Hengyuan chemical company (Wuhan, 1H), 3.6 (s, 1H), 3.9 (d, J 6.9 Hz, 2H), 6.0 (s, 1H), 6.8 (t, J China). 1,3-Dicyclohexylcarbodiimide (DCC) and 4- 7.6 Hz, 1H), 7.0 (m,1H), 7.2 (s, 1H), 7.3 (s, 1H), 7.4 (t, J 7.9 Dimethylaminopyridine (DMAP) were obtained from 13 Hz, 2H); C NMR (126 MHz, CDCl3/TMS, , ppm): 168.3, Rongxuan Biotechnology Co., Ltd. (Shanghai, China). COX 147.7, 142.1, 134.0, 131.8, 130.7, 129.9, 124.1, 123.8, 120.5, inhibitor screen assay kit was purchased from Cayman 118.8, 118.2, 114.6, 113.3, 81.7, 49.4, 48.1, 45.3, 38.0, Chemical Company (USA). All other chemicals were pur- 27.0/27.1, 19.5/19.5, 13.5; MS: m/z = 418.2 [M + H]+ chased from other commercial source and of reagent grade

2.3. Synthesis of NSAID Bornyl Esters 2.4. Measurement of Skin Permeation Rate of NSAIDs and their Bornyl Eaters The synthsis of the NSAIDs bornyl esters was shown in Fig. (1). Briefly, 43.5 mmol (10 g) of naproxen or 39.3 mmol Skins used in the transdermal permeation tests were ob- (10g) of ketoprofen, or 35.6 mmol (10 g) of flufenamic acid tained from the dorsa of male nude mice (6 weeks old; and 0.3 g (2.5 mmol) of DMAP were dissolved in 200 mL of BALB/cA-nu). After the subcutaneous fat and other extrane- anhydrous toluene in a 250 mL round bottomed flask fitted ous tissues were carefully removed, the excised skins were with a reflux condenser and an anhydrous CaCl2 guard tube. stored at -20 °C and used within one week. 65.3 mmol (10.1 g) borneol (or 59 mmol (9.1 g) for ketopro- Vertical Franz diffusion cells with 6.5 mL receptor com- fen, or 53.4mmol (8.2 g) for flufenamic acid) and 87mmol partments and 3.46 cm2 diffusion areas were used during the (17.9 g) DCC (or 78.6 mmol (16.2 g) for ketoprofen, or 71.2 permeation tests. The receptor compartment was filled with mmol (14.7 g) for flufenamic acid) were added to the above 6.5 mL receptor buffer (ethanol: saline =7:3) and its tem- solution according to the molar ratio of 1:1.5:2 (NSAID: perature was maintained at 37 °C and stirred at 200 rpm dur- borneol: DCC). The mixture was refluxed for 12 hours. ing the experiment. After equilibrated for 1 hour, 5 mL of When cooled to room temperature, the mixture was filtrated and washed with ethyl acetate (200 mL). The organic layer ethanol containing 5.2 mM NSAIDs or the mixture of NSAIDs and borneol (5.2 mM each) or 5.2 mM of the esters was washed with 20% Na2CO3 (150 mL), water (200 mL) and saturated NaCl solution (150 mL), respectively, and then was added to each donor compartment on the epidermal sur- face of the skin. At predetermined intervals (2, 4, 8, 12 and was dried over anhydrous NaSO4 and evaporated in vacuo to remove the solvent. The crude product was further purified 24 h), 0.65mL of the receptor medium was withdrawn and by column chromatography (silica gel, ethyl acetate: replaced with an equal volume of fresh receptor buffer 74 Letters in Drug Design & Discovery, 2015, Vol. 12, No. 1 Lin et al.

R O O

OH O

DCC,DMAP R OH

a b

O O O OH O F HO H F N F HO O

1a 2a 3a

O O O O O F H F N O O O F

1b 2b 3b

Fig. (1). Synthesis of bornyl NSAID esters. equilibrated at 37 °C immediately. The withdrawn samples ing test according to reported method [13] with some modi- were analyzed by HPLC to determine the concentrations of fications. The KM mice were divided into three groups with the compounds which had permeated through the skin. seven animals in each group and 2 mL of 45 mM NSAIDs ethanol solution or the physical mixture of NSAIDs and bor- 2.5. HPLC Procedure for Analysis of NSAIDs and their neol (45 mM each) or 45 mM the bornyl esters was smeared Bornyl Eaters on the skin within 20 min and smeared again after 12 hours after the hair on abdominal region of mice was removed The NASIDs and bornyl NSAID esters were quantita- carefully with 2% Na2S. The sole ethanol was used as con- tively analyzed by HPLC using a Zorbax SB-C18 column trol group. 24 hours later, 0.2-0.3 mL 0.6% acetic acid solu- (Agilent, USA) in Agilent 1100 series (USA). Each injected tion was intraperitoneally administered. Immediately after sample (20 L) was eluted at 30 °C with isocratic elution of administration of acetic acid, each mouse was placed in an 80% methanol and 20% water at a flow rate of 1 mL/min, individual clear plastic observational chamber, and the num- and the products were detected at 220 nm. Calibration curves ber of writhing per mouse was recorded within 15 min. The were constructed ranging from concentrations of 0.01 2 decrease in number of writhing was expressed as percentage mg/mL to 1 mg/mL. Excellent linearity (0.95r 0.99) was protection by test compounds with reference to control obtained for the NSAIDs and their bornyl esters. group.

2.6. Pharmacological Activity of NSAIDs and their 2.6.3. Statistical Analyses Bornyl Esters Data are expressed as mean ± SD. For all tests, difference 2.6.1. COX Inhibitor Screening Assay with a probability of P<0.05 was considered to be significant. The COX enzymatic activity inhibition assay was performed using COX inhibitor screen assay kit (Cayman Chemical Company, USA) according to the assay protocol 3. RESULTS AND DISCUSSIONS instructions. Generally, NSAIDs (10 M) and bornyl NSAID esters (10 M) were incubated with COX-1 (ovine) or COX- 3.1. Skin Permeation Study 2 (human recombinant) enzyme for 10 min. The remained The transdermal permeation efficiency of NSAIDs and enzyme activity of COX was then determined according to their bornyl esters were determined and the results were the assay protocol instruction. showen in Fig. (2). A mixture of ethanol and saline (7:3) was used for acceptor buffer during skin permeation tests due to 2.6.2. Acetic Acid-Induced Writhing Test the tested compounds which have very low aqueous solubil- The analgesic effect of NSAIDs and their bornyl esters ity. Compared to the parent NSAIDs, which were transported was determined in KM mice using acetic acid-induced writh- across mice skin slightly, the equimolar physically mixed Synthesis and Evaluation of Transdermal Permeation Letters in Drug Design & Discovery, 2015, Vol. 12, No. 1 75

their inhibition activity toward COX (COX-1, COX-2). The COX inhibition activity of NSAIDs and their bornyl esters were investigated with COX inhibitor screen assay kit (Cayman Chemical Company, USA) according to the assay protocol instructions. As shown in Table 1, all parent NSAIDs under tests were found to be active against both COX-1 (ovine) and COX-2 (human recombinant) enzymes exhibiting about 41%-52% inhibition and showed non- selectivity for COX enzymes. These results are found to be in agreement with the reports [24, 25]. Similarly, the bornyl NSAIDs esters also displayed non-selectivity toward COX-1 and COX-2, but showed a slight decrease of inhibitiory activity than parent NSAIDs (31%-44%). It can thus be con- cluded that the presence of bornyl at NSAIDs did not change their inhibitiory activity and selectivity toward COX enzymes to a great extent. Fig. (2). Permeation efficiency of NSAIDs, the physical mixture of equimolar NSAIDs and borneol and the NSAIDs bornyl esters 3.3. Analgesic Effect through excised mice skins. The concentration of each test compounds was 5.2 mM. The analgesic effect of NSAIDs, the physical mixture and the bornyl esters were assessed by their ability to relieve NSADIs and borneol displayed slightly enhanced transder- acetic acid-induced writhing in mice. Because the bornyl mal permeation efficiency although almost half of borneol NSAID esters can permeate across the skin rapidly in 12 was permeated. Strikingly, the permeation of bornyl NSADI hours (Fig. 2), twice-percutaneous administration was car- esters increased rapidly within 12 hours and then began to ried out in 24 hours. As shown in Fig. (3), intraperitoneal flatten in 24 hours. The permeation efficiency of NSAIDs in injection of acetic acid produced average 21 writhes in the 12 hours calculated to be less than 8 g/cm2/h, which is in control group for 15 min. The parent NSAIDs group did not agreement with previous reports [22]. By comparison, the show any significant reduction in the number of writhes, permeation efficiency of bornyl NSAID esters was increased which may be because they cannot penetrate the skin enough about 13-17 times over parent NSAIDs and about 10-13 to reach therapeutic level. While the equimolar physical mix- times over the physical mixture of NSAIDs and borneol. The ture of NSAIDs and borneol displayed a certain amount of results suggested that the introduction of bornyl group to decrease in writhes number, which may be caused by the NSAIDs can promote their permeation efficiency signifi- combined effects of the permeated borneol and the slight cantly. A possible explanation is that the bornyl group can NSAIDs. However, percutaneous administration of bornyl loosen the tight junctions between epithelial cells, which in NSAIDs esters produced a significant reduction in writhes turn, accelerate the permeation of bornyl NSAID esters compared to other test groups. However, no significant dif- across the epithelium via the paracellular route. Meanwhile, ference could be observed among three esters with respect to the bornyl group can also increase the orderliness of lipid their analgesic activity. The percentage of pain inhibition was calculated from the decrease in number of writhing due molecular chains on the skin epithelial cell membrane [18, to analgesic effect of the tested compounds with reference to 23], which decreases the collision between the lipid molecu- the control group. Compared to parent NSAIDs, which only lar and bornyl NSAID esters, resulting in the increased per- brought less than 6% analgesic effect, the physical mixture meation of NSAIDs bornyl esters through the membrane. group showed less than 18% analgesic effect and the bornyl NSAIDs esters showed more than 31% analgesic effect. This 3.2. COX Inhibition Assay is due to the enhanced permeation ability of bornyl NSAID It is known that NSAIDs are widely used as the anti- esters, as well as the most of inhibition activity of bornyl inflammatory, anti-nociceptive pharmaceuticals because of NSAID esters toward COX was retained. Although treatment

Table 1. COX inhibition activity of NSAIDs and their esters (n=3, mean ± S.D.).

Compound Concentration (M) COX-1 Activity (% Inhibition) COX-2 Activity (% Inhibition)

1a 10 52.2±3.8 46.3±2.4

1b 10 44.6±2.9 41.1±3.3

2a 10 47.3±2.6 45.5±2.1

2b 10 42.8±3.5 31.7±2.2

3a 10 42.4±2.3 45.2±3.0

3b 10 37.3±3.4 34.7±2.7 76 Letters in Drug Design & Discovery, 2015, Vol. 12, No. 1 Lin et al.

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Received: January 22, 2014 Revised: June 18, 2014 Accepted: June 26, 2014