Intra-articular drug delivery from an optimized topical patch containing teriflunomide and lornoxicam for rheumatoid arthritis treatment: Does the topical patch really enhance a local treatment?

Journal of Controlled Release 169 (2013) 73–81

Contents lists available at SciVerse ScienceDirect

Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel

Intra-articular drug delivery from an optimized topical patch containing teriflunomide and lornoxicam for rheumatoid arthritis treatment: Does the topical patch really enhance a local treatment? Honglei Xi a, Dongmei Cun a, Rongwu Xiang b,⁎⁎, Yanli Guan a, Yuxiu Zhang a, Yuanru Li a, Liang Fang a,⁎ a b

Department of Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China School of Medical Instrument, Shenyang Pharmaceutical University, Shenyang 110016, China

a r t i c l e

i n f o

Article history: Received 19 July 2012 Accepted 17 March 2013 Available online 6 April 2013 Keywords: Teriflunomide Lornoxicam Amine salt Compound transdermal patch Central composite design Intra-articular drug delivery

a b s t r a c t Patients with rheumatoid arthritis (RA) often bear joint destruction and symptomatic pain. The aim of this work is to develop a compound transdermal patch containing teriflunomide (TEF) and lornoxicam (LOX) to transport these drugs across the skin with the isochronous permeation rates for RA therapy and investigate intra-articular delivery of TEF and LOX following transdermal patches applied topically. The salts of TEF and LOX with organic amines diethylamine (DEtA), triethylamine (TEtA), diethanolamine (DEA), triethanolamine (TEA) and N-(2′-hydroxy-ethanol)-piperdine (NP) were prepared to improve the skin permeation of the parent drug. The optimized patch formulation is obtained from a 3-factor, 2-level central composite design. After topical application of the optimized compound patch to only one knee joint in rabbit, intra-articular delivery of TEF and LOX on the application site was compared with that on the non-application site. Anti-inflammatory and analgesic effects of the optimized compound patch were evaluated using the adjuvant arthritis model and the pain model induced by acetic acid, respectively. The in vitro experiment results showed that the amine salts of TEF and LOX, especially TEF-TEtA and LOX-TEtA, enhanced the skin permeation of TEF and LOX from the transdermal patch system. The optimal formulation successfully displayed isochronous permeation rates for TEF and LOX across rabbit skin, and was defined with 5% of TEF-TEtA, 10% of LOX-TEtA and 15% of azone. The in vivo study showed that TEF and LOX from transdermal patches were transferred into skin, ligament and fat pad on the application site by direct diffusion and on the non-application site by the redistribution of systemic blood supply, while local absorption of TEF and LOX in synovial fluid originated from the systemic blood supply rather than direct diffusion. In the RA rat model, the results of swelling inhibition on primary arthritis of bilateral hind paws further confirmed the above-mentioned point. The optimal formulation displayed a double response on joint inflammation and symptomatic pain. In conclusion, although transdermal administration applied topically can provide a local enhanced drug delivery for the superficial joint tissues by direct diffusion, it seemed unlikely to do that for the deeper tissue synovial fluid. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Rheumatoid arthritis (RA) is a chronic inflammatory disease occurring frequently in older individuals, which is characterized by polyarticular inflammation and gradual joint destruction [1]. In current therapeutic strategies, DMARDs are prescribed for slowing the progression of RA and actually modifying the disease but do not directly relieve pain; NSAIDs are prescribed for the relief of RA-related pain and inflammation but have not been shown to slow the progression ⁎ Correspondence to: L. Fang, Department of Pharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning 110016, China. Tel./fax: +86 24 23986330. ⁎⁎ Correspondence to: R. Xiang, School of medical instrument, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, Liaoning, 110016, China. Tel.: +86 24 23986529. E-mail addresses: [email protected] (L. Fang), [email protected] (R. Xiang). 0168-3659/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jconrel.2013.03.028

of the disease [2,3]. A combination of disease-modifying anti-rheumatic drugs (DMARDs), non-steroidal anti-inflammatory drugs (NSAIDs), and steroids has been used to manage RA patients against the complex RA process since the early 1950s [4]. However, to our knowledge, there are no reports or products on compound agents containing DMARDs and NSAIDs. Leflunomide (LEF) is an oral nonbiologic DMARD recommended by the American College of Rheumatology for RA treatment. In the liver, LEF can be rapidly converted to its active metabolite teriflunomide (TEF, Table 1) which is responsible for therapeutic effects of LEF [5,6]. Lornoxicam (LOX, Table 1) is an effective derivative of the oxicam class of NSAIDs, used in the treatment of various types of pain, especially resulting from inflammatory diseases of the joints [7]. It has been reported that LEF can be effectively used in combination with NSAIDs [8]. However, the gastrointestinal side effects of both LEF and LOX after oral administration are the major therapeutic

74

H. Xi et al. / Journal of Controlled Release 169 (2013) 73–81

limitations to successful RA long-term management [9,10]. Recently, LEF has been added to the boxed warning statement by the FDA for RA patients who are having pre-existing liver disease and taking other drugs that can cause liver injury [11]. As in some cases RA is restricted to only one or a few larger joints; transdermal drug delivery provides an opportunity for drug localization at the target site, which often transports drug to target tissues directly below the application site to produce high local levels of drug despite appreciably reduced blood concentrations [12]. Transdermal delivery of TEF and LOX is an ideal alternative to avoid LEF hepatic metabolism and their systemic side effects including gastrointestinal irritation. Moreover, a compound transdermal patch containing TEF and LOX will provide double responses of controlling RA activity and relieving symptomatic pain after one-dose medication. Generally, drugs are thought to permeate across the skin by a passive diffusion process governed by Fick's law of diffusion: J ¼ −D

∂C ∂x

ð1Þ

where J is the “diffusion flux” which indicates the amount of substance that will flow through a small area during a small time interval, D is the diffusion coefficient or diffusivity, C is the concentration of the diffusive substance and x is the space coordinates. Based on Fick's law of diffusion, drug diffusive flux is related to the concentration under the assumption of steady state. When a drug is applied to the skin surface, based on concentration gradient, drug molecules must first partition and diffuse through the skin, and then pass into the dermal blood microcirculation or penetrate into the subjacent tissues. As the synovium is the primary target of the RA process [1], delivering drug into the synovial tissues is quite crucial for RA therapy. It is well accepted that transdermal products applied topically can achieve an excellent therapeutic effect on the inflammatory joints [13,14]. However, how to transport drug into the deeper synovial tissues after transdermal application on the joints is still controversial. Shinkai et al. [15] claimed that local enhanced topical delivery of ketoprofen (M.W. 254.28, log P = 2.91, pKa = 4.23, obtained from the SciFinder database) was attributed to direct drug diffusion in the joint tissues rather than the systemic circulation by a microdialysis technique in pigs and rats. However, Radermacher et al. [16] showed an opposite result that, after transdermal application to a single knee in patients, the small difference between diclofenac (M.W. 296.15, log P = 4.55, pKa = 4.18, obtained from the SciFinder database) concentrations in synovial fluids of bilateral knees indicated that local accumulation of diclofenac by direct transport or diffusion into the knee joint seemed unlikely. This point is contradicted by the study of Shinkai et al. about the topical delivery of ketoprofen. Two main reasons lead to the contradiction. On the one hand, the relative synovial permeability of these drugs is probably different [17]. The study of Niu et al. [18] shows that ketoprofen has an excellent permeability across the skin membrane. On the other hand, in their

work, only free ketoprofen was measured using a microdialysis system with aqueous perfusate and a molecular mass cutoff of 50 kDa on account of the high protein-binding ratio of ketoprofen (99%) and the larger molecular mass (~ 100 kDa) of protein in synovial fluid [19]. Thus the question of whether the topical patches really enhance a local treatment for RA is brought out. In this work, we systemically investigated intra-articular delivery of TEF and LOX for the above question after topical application of an optimized compound patch to only one knee joint in animals. To date, there are no research papers available to date on topical delivery of TEF and LOX in animals or patients. 2. Material and methods 2.1. Chemicals TEF was synthesized in our laboratory by a method described in the patent US5532259 (the purity of obtained TEF is up to 99%; 1 H NMR: (300 MHz, DMSO-d6, major (enol) tautomer), d: 13.43 (s, 1H, OH), 10.74 (s, 1H, NH), 7.78 (d, J = 8.61 Hz, 2H, ArH), 7.66 (d, J = 8.67 Hz, 2H, ArH), 2.27 (s, 3H, CH3); MS (EI) m/z: 269.1 (M–H)). LOX was purchased from Zhuhai Yuancheng Chemical Co., Ltd. (Guangdong, China). DURO-TAK® 87-4098 pressure sensitive adhesive (PSA) was purchased from Henkel Corp. (New Jersey, USA). Complete Freund's adjuvant (CFA) was obtained from Sigma-Aldrich Co. LLC. (Missouri, USA). Ketotop® ketoprofen patch (30 mg/70 cm2) was obtained from Pacific Pharmaceutical Co. Ltd. (Seoul, Korea). Diethylamine (DEtA), triethylamine (TEtA), diethanolamine (DEA), triethanolamine (TEA), methyl paraben and oleic acid (OA) were supplied by Bodi Chemical Holding Co. Ltd. (Tianjin, China). N-(2′hydroxy-ethanol)-piperdine (NP), N-methyl pyrrolidone (NMP), azone (AZ), isopropyl myristate (IPM), Transcutol® P (TP) and L-menthol (MT) were supplied by Alfa Aesar (Massachusetts, USA), International Specialty Products Inc. (New Jersey, USA), Tianmen Kejie Pharmacy Co. Ltd. (Hubei China), Leasun Chemical Co. Ltd. (Shanghai, China), Gattefossé (Lyon, France) and Suzhou Healthytech Bio-Pharmaceutical Co. Ltd. (Jiangsu, China), respectively. All other chemicals were of the highest reagent grade available. 2.2. Animals Rabbits (male, 2.0–2.2 kg), Wistar rats (male, 200–210 g) and KM mice (male, 18–22 g) were obtained from the Experimental Animal Center of Shenyang Pharmaceutical University (Shenyang, China). The procedures followed were in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals and also in accordance with the guidelines for animal use published by the Life Science Research Center of Shenyang Pharmaceutical University. All efforts were made to minimize animal suffering and to limit the number of animals used.

Table 1 Physicochemical properties and pharmacokinetics of TEF and LOX. M.W.a

log P

pKa

Teriflunomide (TEF)

270.21

2.51a

5.20

Lornoxicam (LOX)

371.82

1.8 [7]

4.7 [7]

Chemical structure

a b c

Data were obtained from the SciFinder database. Data from clinical trials of LEF in RA. Data from clinical trials of LOX in RA.

a

t1/2

Bioavailability

Oral dose/day

2 weeks [6]

100% [6]

20 mgb

3–5 h [7]

100% [11]

12–16 mgc

H. Xi et al. / Journal of Controlled Release 169 (2013) 73–81

75

and mixed thoroughly with a mechanical stirrer to obtain a homogeneous coating formulation. A laboratory coating unit (TB-1, Kaikai Co. Ltd., Shanghai, China) was used to prepare patches. The obtained formulation was coated onto a silicone-coated release liner (Shanghai Fupeng Adhesive Products Co. Ltd, China) at a thickness of ~ 80 μm. The coated release liner was oven-dried at 60 °C for 10 min and after removal of the solvent, it was then laminated with a fabric backing film (Hangzhou Xiaoshan Johnson Cloth Factory, Zhejiang, China). The 5 cm 2 patch (n = 3) was weighed accurately for drug content determination. The method of drug content determination was in accordance with our previous work [20]. 2.5. In vitro permeation experiments

Fig. 1. The DSC curves (a, b) and FTIR spectra (c, d) for TEF, LOX and their salts. Melting points of TEF, LOX and their salts were presented in (a) and (b), respectively.

2.3. Synthesis of organic amine salts TEF or LOX was adequately dispersed in CHCl3 and an equimolar amount of each organic amine was added under stirring. The solutions were subjected to an ultrasound treatment for 1 h at room temperature and then dried by vacuum rotary evaporation. Subsequently, the amine salts obtained were dried in a vacuum for 12 h. These amine salts have been confirmed by DSC (DSC1 STAR e System, Mettler-Toledo International Inc., Schwerzenbach, Switzerland) and FTIR (Spectrum 100, PerkinElmer Inc., Massachusetts, USA).

Rabbit excised skin was used to evaluate the skin permeation of TEF, LOX and their amine salts from transdermal patches. The process of skin preparation was conducted in accordance with our previous report [20]. After anesthesia, hair on the abdomen of the rabbits was carefully removed with clippers (model 900, TGC, Japan) and an electrical shaver (BRAUN 190, Germany). Fresh samples of full thickness skin (epidermis with SC and dermis) were excised after the animals were sacrificed. The integrity of the skin was carefully checked by microscopic observation, and any skin which was not uniform was rejected. The sub-dermal tissue was surgically removed and the skin was washed immediately with physiological saline, and then wrapped in aluminum foil. The skin samples were then stored at −70 °C until required (used within 1 week after preparation). In vitro permeation experiments were performed using a twochamber side-by-side glass diffusion cell (cell capacity of 3.0 mL, effective diffusion area = 0.95 cm 2) with a water jacket connected to a water bath at 32 °C. The excised skin was mounted between the cell halves by clamping them so that the dermal side of the skin faced the receiver solution. A circular transdermal patch was pressed on the skin with the adhesive side facing the stratum corneum. Phosphate buffered saline (pH 7.4) was used as the receptor medium. The duration of the exposure was set up for 12 h. At each predetermined time point, 2.0 mL of receptor medium was withdrawn and the same volume of a fresh receptor medium was added into the receiver to maintain sink condition. 200 μL of the withdrawn samples was mixed with 200 μL of internal standard solution (125 μg/mL) for HPLC analysis. The cumulative amount of TEF and LOX passing across the rabbit skin was calculated based on the measured drug concentrations in the receiver medium. All in vitro experiments were carried out in triplicate or quadruplicate. The permeation profiles were yielded by the cumulative amount of drug permeated per unit area (Q) versus time. The steady-state flux (J) was obtained from the slope of the linear portion of the plot which was dependent on the R2 value of the linear fit (R2 > 0.99). The lag time (Tlag) was determined by extrapolation of the linear portion of the permeation curve to the abscissa. The enhancing effect of organic amines or chemical enhancers was calculated as follows:

2.4. Preparation of patches The drug-in-adhesive transdermal patches were prepared by dissolving the drug, DURO-TAK® 87-4098 PSA, and enhancers in ethanol

ð2Þ

ER ¼ Q 12 h ðamine salts or with enhancersÞ =Q 12 h ðparent drug or without enhancersÞ:

Table 2 The permeation parameters of TEF, LOX and their salts across the rabbit skin (n = 3). Permeants

Parent compound TEtA DEtA NP TEA DEA

TEF-amine salts

LOX-amine salts

Q12 h (μg/cm2)

J (μg/cm2/h)

Tlag (h)

ER

Q12 h (μg/cm2)

J (μg/cm2/h)

Tlag (h)

ER

8.58 82.40 39.94 19.11 16.40 2.03

0.53 6.23 2.74 1.71 1.11 0.16

0 0 0 0 0 0

1 9.60 4.66 2.23 1.91 0.24

0.02 8.10 4.21 4.58 4.40 2.00

0 0.46 0.29 0.30 0.32 0.12

0 0 0 0 0 0

1 405 211 229 220 100

± ± ± ± ± ±

0.91 18.12 14.55 7.00 7.07 0.73

± ± ± ± ± ±

0.33 0.87 1.46 0.70 0.60 0.02

± ± ± ± ± ±

0.02 1.39 0.70 0.20 1.63 0.39

± ± ± ± ±

0.11 0.04 0.04 0.14 0.02

76

H. Xi et al. / Journal of Controlled Release 169 (2013) 73–81

Table 3 The effect of chemical enhancers on TEF-TEtA and LOX-TEtA (n = 3). Permeants

Control AZ OA IPM MT NMP TP a

Log P

a

– 6.58 7.70 7.43 3.20 −0.40 −0.62

OH number

– 0 1 0 1 0 1

TEF-TEtA

LOX-TEtA

Q12 h (μg/cm2)

J (μg/cm2/h)

Tlag (h)

ER

Q12 h (μg/cm2)

J (μg/cm2/h)

Tlag (h)

ER

82.40 147.87 144.01 111.40 38.15 11.65 36.53

6.23 11.25 9.91 7.78 3.71 0.62 2.94

0 0 0 0 0 0 0

1 1.79 1.75 1.35 0.46 0.14 0.44

8.10 118.63 5.42 23.12 4.85 7.25 4.69

0.46 10.61 0.50 2.08 0.42 0.69 0.46

0 0 0 0 0 0 0

1 14.65 0.67 2.85 0.60 0.90 0.58

± ± ± ± ± ± ±

18.12 38.52 62.04 41.97 9.66 4.55 19.34

± ± ± ± ± ± ±

0.87 2.76 2.91 3.86 1.17 0.05 2.36

± ± ± ± ± ± ±

1.39 45.22 1.03 10.48 1.10 0.68 1.11

± ± ± ± ± ± ±

0.11 2.41 0.14 0.80 0.12 0.09 0.04

Data were obtained from the SciFinder database.

2.6. The formulation optimization of compound transdermal patches The in vitro permeation experiments were performed for the optimization of formulation. The optimization process was based on determining the steady-state fluxes of TEF and LOX for different formulations. Firstly, the patch formulations containing different amine salts of TEF or LOX were studied by single factor test. In order to make the permeation results comparable, the loading of a salt was equal to 2% of the parent drug (w/w, based on solid adhesive weight). Subsequently, the effect of the chemical enhancer was evaluated for the largest permeability salts of TEF and LOX, respectively, and the loading of a chemical enhancer was 15% (w/w). The enhancer providing the best enhancement for both aforementioned salts was chosen as the formulation ingredient. A central composite design (CCD) was used to optimize the formulation of compound patches containing TEF and LOX. The optimal formulation was defined as a compound patch with isochronous permeation rates of TEF (J1) and LOX (J2), the maximum value of J1 and J2 and the minimum loading of chemical enhancer. The independent variables in CCD were the loadings of TEF amine salt, LOX amine salt and chemical enhancer, respectively, and the response variables were J1 and J2, respectively. The experiments were designed by Design Expert® software (7.0.0 version, Stat-Ease Inc., Minnesota, USA). Using Matlab (R2009a version, The MathWorks Inc., Massachusetts, USA), multidimensional response surfaces (3D and 2D) were individually fitted to each response variable for estimating the fitting mathematical models of J1 and J2, respectively. P-value and lack of fit were used to evaluate each equation and identify the fitting models. According to these fitting models, once the optimal solution was obtained, we evaluated its reliability using two random formulations. 2.7. Local disposition study in rabbit knees Prior to dosing, the left knee hairs of 20 male rabbits were removed with electric hair clippers and a shaver. The rabbits whose knee skin was found to be broken by visual inspection were discarded. Each rabbit received an optimized compound patch (20 cm 2; equal to 11 mg TEF and 24 mg LOX) once. The transdermal

patches were applied to left knee joints and then removed immediately before sampling (for rabbits sacrificed at 72 h, patches were removed at 24 h). The residual adhesive on the skin surface was carefully wiped off using cotton soaked in ethanol. Plasma and the local tissues of bilateral knee joints (including skin, ligament, fat pad and synovial fluid) were obtained from 4 rabbits at every sampling point (2, 6, 12, 24 and 72 h postdose). Approximately 1 mL of blood was drawn from the ear vein and was immediately placed into a heparinized tube, and then plasma was separated by centrifugation. After sacrifice, knee joints on both the application site and the nonapplication site were punctured for the collection of synovial fluid and other tissue samples were also collected surgically. Wet weights of tissue samples were measured exactly and all of the samples were stored at −70 °C until further analysis. 2.8. Sample extraction procedure A plasma sample was mixed with 10 μL of internal standard solution (125 μg/mL). The mixture was extracted with 1 mL of ethyl acetate by vortex-mixing for 3 min and centrifuged at 7000 g for 5 min at 4 °C [21,22]. The organic layer was transferred into a clean test-tube and then evaporated to dryness under nitrogen at 40 °C. The residues were reconstituted in 100 μL mobile phase by vortexmixing for 2 min, and centrifuged at 7000 g for 5 min at 4 °C. The supernatants were analyzed using the HPLC system. Each of the weighed skin samples (0.05 g) was placed in a test tube. Internal standard solution (150 μL, 1250 μg/mL) and methanol (850 μL) were added to the skin sample. These samples were extracted for 10 min with ultrasonication and centrifuged at 7000 g for 5 min at 4 °C. The supernatants were analyzed using the HPLC system. For ligament and fat pad samples, these samples were mixed with 15 μL of internal standard solution (125 μg/mL) and extracted with 1 mL of ethyl acetate under ultrasonic treatment. The rest of the procedure was in accordance with that of plasma samples. Because of the limited collection of synovial fluid, each sample was mixed with 500 μL normal saline prior to processing. Samples were prepared by adding 7.5 μL of internal standard solution (125 μg/mL) and then mixing with 2 mL of ethyl acetate. The rest of

Table 4 Variables in the central composite design. Factor

Independent variables

Responses Constraints

X1 = TEF-TEtA loading (%, w/w) X2 = LOX-TEtA loading (%, w/w) X3 = AZ loading (%, w/w) J1 = flux of TEF (μg/cm2/h) J2 = flux of LOX (μg/cm2/h) Maximize J1 and J2, and J1 ≈ J2, minimize X3

Levels used, actual (coded) Low (−1.414)

Medium (0)

High (1.414)

2 2 2

6 6 11

10 10 20

H. Xi et al. / Journal of Controlled Release 169 (2013) 73–81 Table 5 Experimental design and experimental data of the responses (n = 4). Formula

X1 (%)

X2 (%)

X3 (%)

J1 (μg/cm2/h)

J2 (μg/cm2/h)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

3.62 8.38 3.62 8.38 3.62 8.38 3.62 8.38 2.00 10.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00

3.62 3.62 8.38 8.38 3.62 3.62 8.38 8.38 6.00 6.00 2.00 10.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00

5.65 5.65 5.65 5.65 16.35 16.35 16.35 16.35 11.00 11.00 11.00 11.00 2.00 20.00 11.00 11.00 11.00 11.00 11.00 11.00

2.14 12.23 6.98 12.23 13.35 21.51 13.52 30.00 4.94 24.03 21.70 17.95 1.15 22.65 18.03 14.53 15.01 13.38 18.65 15.07

1.38 3.24 7.83 6.99 7.02 6.67 13.98 20.33 7.92 7.10 5.29 13.38 0.56 17.54 8.22 7.73 8.85 5.72 9.64 7.89

the procedure was in accordance with that of plasma samples except that the residues were reconstituted in 50 μL mobile phase. 2.9. Quantitative analysis The concentrations of TEF and LOX in the receptor medium, plasma and joint tissues were determined using a HPLC method. Chromatography was performed with a Hitachi HPLC apparatus (Hitachi High-Technologies Corporation, Tokyo, Japan) consisting of an L-2130 pump, an L-2200 automatic injector and an L-2400 ultraviolet absorbance detector. Separation of the analytes was accomplished using a Kromasil® C18 column (200 mm × 4.6 mm, 5 μm particle size) from Eka Chemicals (Bohus, Sweden). For in vitro and in vivo studies, the mobile phase was composed of 0.5% acetic acid in water–methanol (60:40), adjusted to pH 6.8 with TEtA. The flow-rate of the mobile phase was kept at 1 mL/min and the wavelength was set at 292 nm. Methyl paraben was used as the internal standard for all of the studies and the injection volume was 20 μL. All of the analytical methods have been validated. 2.10. Assessment of anti-inflammatory activity

77

The dose for rats is translated from that for rabbits using the body surface area normalization method [24]. Before injection of CFA, the volumes (V) of bilateral hind paws of each rat were measured by a water plethysmometer (YLS–7B, Jinan Yiyan Technology Co. Ltd., Shangdong, China) as the baseline value. The bilateral hind paws of each rat from the control group were injected with 0.1 mL of saline respectively and the bilateral hind paws of rats from the other groups were injected with 0.1 mL of CFA. After the injection of CFA, the paw volumes were measured at 0.5, 3, 5, 7, 9, 11 and 13 days (d). A transdermal patch was applied to the left knee joint at 5 d. The paw swelling degree (S) was calculated for each group using Eq. (3): S% ¼

V t −V n  100 Vn

ð3Þ

where Vn and Vt were the paw volumes before and after the injection of CFA respectively. To further compare the anti-inflammatory activity difference of applying transdermal patches on the different sites, the same protocol was carried out except that transdermal patches were applied on the abdominal skin which was a non-inflammatory area. 2.11. Pain behavioral tests Acetic acid induced writhing was used to evaluate the analgesic effect of test transdermal patches in mice. Prior to dosing, the abdomen hairs of 60 mice were removed carefully. These mice were divided into 6 groups each consisting of 10 animals and treated in accordance with the scheme in Section 2.10 except dose (5 cm 2). Similarly, the dose for mice is translated from that for rabbits. Before inducing the writhing, the mice are administrated with patches for 4 h. After removing the patches, the writhing was induced by intraperitoneal injection of acetic acid in distilled water (0.6%, v/v). Numbers of writhing (W) were recorded after discarding the first 5 min for a period of 20 min and pain inhibition ratio (PIR) was calculated for each group using Eq. (4): PIR% ¼

W c −W t  100 Wc

ð4Þ

where Wc and Wt were the writhing numbers of the control group and the test groups respectively. 2.12. Statistical data analysis

The adjuvant arthritis (AA) model induced in rats using CFA, which is frequently used to study the immunological aspects of RA [23], was used to evaluate the anti-inflammatory effect of test transdermal patches. Male rats weighing 200–220 g were divided into 6 groups each consisting of 6 animals and treated as follows:

For all the studies, the data were calculated and presented as mean ± SD. Statistical analyses of the data were performed using ANOVA and the Student's t-test. The level of significance was taken as P ≤ 0.05. 3. Results and discussion

• Control group — no dose administration; • Positive group — Ketotop® ketoprofen patch (10 cm 2, 4 mg of ketoprofen); • Negative group — blank patch (10 cm 2, only without any drug); • TEF group — TEF-TEtA patch (10 cm 2, equal to 6 mg of TEF); • LOX group — LOX-TEtA patch (10 cm 2, equal to 12 mg of LOX); • TEF–LOX group — the optimized compound patch (10 cm 2, equal to 6 mg of TEF and 12 mg of LOX).

3.1. The formation of organic amine salts Our recent works [25,26] proved that the organic amines DEtA, TEtA, DEA, TEA and NP could enhance the skin permeation of weak acidic TEF and LOX by ion pair formation. Therefore, the salts of TEF and LOX with these organic amines were prepared for the formulation optimization. The DSC curves and FTIR spectra for TEF,

Table 6 Summary of results of reduced quadratic models for regression analysis of responses J1 and J2. Response J1 J2

Equation 0.06X32

J1 = −16.56 + 2.22X1 + 2.41X3 − J2 = −1.69 + 0.24X2 + 0.17X3 + 0.10X2X3

R2

Adj R2

Pred R2

Adeq precision

Lack of fit prob F (P-value)

0.8769 0.8869

0.8538 0.8657

0.8181 0.7901

19.294 19.823

Not significant (0.1993) Not significant (0.2015)

78

H. Xi et al. / Journal of Controlled Release 169 (2013) 73–81

LOX and their salts were shown in Fig. 1. The DSC curves showed that the melting points of their salts were lower than that of the corresponding parent compound, but no clear relationship was observed between the melting point changes of these salts and their physicochemical properties. In FTIR spectra, the absorptions at ν3303 cm −1 and ν3067 cm −1 were assigned to the strongly stretching vibrations of the OH groups from TEF and LOX, respectively, but after formation of organic amine salts, the corresponding peaks became weaker or even disappeared. These results indicated the formation of the strong intermolecular interaction between the acidic OH group from the parent compound and the basic N atom from organic amines. 3.2. Optimization of the formulation

Fig. 2. 3D response surfaces and 2D projections for TEF flux (J1) and LOX flux (J2).

The skin permeation data of TEF, LOX and their corresponding salts from the transdermal patch system were presented in Table 2. The results showed that the skin permeations of TEF and LOX from this system were quite poor, while all the salts of TEF and LOX except TEF-DEA markedly increased the skin penetration of TEF and LOX. Obviously, the salts TEF-TEtA and LOX-TEtA provided the best enhancement for TEF and LOX among these salts, which was in accordance with the skin permeation results in the solution system reported in our recent works [25,26]. Therefore, TEF-TEtA and LOX-TEtA were used as the drug ingredients for the formulation design. Six chemical enhancers (AZ, OA, IPM, MT, NMP and TP) were used to evaluate the enhancing effect on TEF-TEtA and LOX-TEtA, respectively. The permeation data were shown in Table 3. The relatively lipophilic enhancers AZ, OA and IPM had enhancing effects on TEF-TEtA, while only AZ and IPM had enhancing effects on LOX-TEtA. It has been widely accepted that the intercellular lipid domain in the stratum corneum (SC) is the main pathway of transdermal drug delivery [27]. Therefore, this suggests that the lipophilic enhancers can distribute well into the modified stratum corneum. In addition, the enhancers with OH groups may disturb the intermolecular interaction between TEF or LOX and organic amines, and consequently decrease the skin penetration of the salts, especially LOX-TEtA. Overall, AZ with the best enhancement on TEF-TEtA and LOX-TEtA was chosen as the enhancer for the formulation design. Oral dose per day and bioavailability of TEF and LOX are similar in clinical trials (Table 1), thus we expect that the skin permeation rates of TEF and LOX from compound patches are also comparable on the application site. The loadings of drug and enhancers in transdermal products are important factors for the skin permeation of drug. Therefore, a 3-factor, 2-level CCD was suitable for optimization of the formulation as shown in Table 4. The experimental result of each formulation is given in Table 5. The experiment data in Table 5 were fitted to the reduced quadratic polynomial models using the Design Expert® software (Table 6). Other algebraic terms in quadratic models were ignored in that their P-values were greater than 0.05. The pred R2 was in reasonable agreement with the adj R2 and non-significant lack of fit was good. Therefore, the models were used to navigate the design space. The equation of J1 was a monotone increasing function that depended on X1 and X3 when X3 was not more than 20.08, and the equation of J2 was a monotone increasing function that depended on X2 and X3. Indeed, there was an interaction between X1 and X2 by means of X3. The response surfaces of J1 and J2 (Fig. 2) were obtained according to the model equations in Table 6. In Fig. 2(c) and (d), J1 showed an optimal value in the broad area (4 ≤ X1 ≤ 10, 10 ≤ X3 ≤ 18) and J2 showed an optimal value in the narrow area (9 ≤ X2 ≤ 10, 10 ≤ X3 ≤ 18). Unfortunately, the formulation with 20% of AZ presented a bad physical appearance (some oily substances diffused into the backing layer) after being stored for one month at room temperature, but the formulations with 15% of AZ were still good. Therefore, the upper limit of AZ loading was reset to 15%. According to the equations of J1 and J2, when X3 was set to 10 or 15, there was a

H. Xi et al. / Journal of Controlled Release 169 (2013) 73–81 Table 7 Composition of check formulations and the experimental data of response (n = 4). Formula X1 (%)

X2 (%)

X3 (%)

5

10

10

5

10

15

Response

Experimental value (μg/cm2/h)

Predicted value (μg/cm2/h)

Prediction error (%)

J1 J2 J1 J2

13.35 14.13 17.03 18.08

12.64 12.41 17.19 18.26

5.62 13.86 0.93 0.99

± ± ± ±

3.22 1.22 0.65 0.69

relationship (X2 = 1.23 + 1.79X1 or X2 = 3.01 + 1.28X1) between X1 and X2 for J1 ≈ J2. Consequently, when X2 ≤ 10, X1 was defined between 4.90 and 5.46. Based on the prediction, two formulas were selected and the responses of flux of drugs were evaluated (Table 7). The experimental values of drug fluxes were comparable with the corresponding predicted values which suggested a good prediction ability of the model equations. Therefore, the optimal formulation was composed of 5% of TEF-TEtA, 10% of LOX-TEtA and 15% of AZ. 3.3. Intra-articular drug delivery Synovium is the primary target of the RA process. In practice, drug concentration in the synovium can be predict by that in the synovial fluid, because the extracellular fluid of synovium is continuous with synovial fluid [28]. Therefore, the drug disposition in the synovial fluid is more significant for RA treatment. Local tissue concentrations of TEF and LOX in two knee joints of rabbits were measured after topical application of the optimized compound patch to only one knee joint. The results were showed in Fig. 3. A drug after being released from transdermal patches can be transferred into the articular cavity via two pathways, direct diffusion on the application site and systemic circulation. In Fig. 3(b), on the application site, TEF and LOX were absorbed into the skin with maximum values of 135.41 ± 41.93 μg/g and 145.78 ± 44.92 μg/g at 6 h, respectively. Moreover, there was no significant difference in the drug concentrations of TEF and LOX in the skin at each time point. Namely, TEF and LOX diffused into the skin with a similar rate from the optimized compound patch which suggested that they penetrated through the skin with the isochronous rates. This result was in agreement with that of the in vitro experiment. Drugs had been detected in the contralateral skin after 2 h and the drug concentrations were obviously lower than that on the application site, which indicated that drugs were transported quickly via blood and then diffused into the skin. After drug delivery to the dermis, TEF and LOX were absorbed into the blood because of the capillary network in the dermis. Fig. 3(c) showed that TEF continued to accumulate in plasma before removing the patches (24 h) because of its longer half-life while the LOX concentration in plasma rose to a peak value (0.51 ± 0.08 μg/mL) at 6 h. As it was next to the skin of the knee joint, the ligament was considered as the tissue in which a drug could diffuse as soon as it penetrates through the skin. In Fig. 3(d), the maximum concentrations of TEF and LOX in the ligament were brought out on the bilateral sites at 2 h and 6 h, respectively. Thereafter, on the application site, the TEF concentration tended to level off from 6 h to 24 h but the LOX concentration went down. On the non-application site, the drug concentrations in the ligament were lower than that on the application site and the absorption behaviors of TEF and LOX in the ligament, in general, followed that in plasma. As shown in Fig. 3(e), the TEF concentration in the fat pad presented a continuous accumulation on the bilateral sites before removing the patches but the TEF concentration on the application site was higher. In addition, the LOX concentration in the fat pads below the application site did not show a significant change between 2 h and 6 h and LOX was not detected at 24 h. However, on the non-application site, LOX in the fat pad was not detected in the whole experiment. From Fig. 3(f), both TEF and LOX appeared to have a matching drug

79

absorption in the synovial fluids of the bilateral sites, which was similar with that in plasma. At 72 h (after removing transdermal patches), TEF was eliminated at different degrees in plasma and joint tissues but was not significantly different between the bilateral joint tissues, while LOX was not detected. Based on the above results, TEF and LOX from transdermal patches were transferred into the skin, ligament and fat pad on the application site by direct diffusion and on the non-application site by the redistribution of systemic blood supply before removing the transdermal patches. TEF and LOX were transferred only into a certain depth via direct diffusion on the application site. Not like in the other joint tissues, TEF and LOX in synovial fluid originated from systemic blood supply because synovial fluid was regarded as a plasma ultrafiltrate [28]. This suggested that the TEF and LOX from transdermal patches played therapeutic effects for RA mainly by systemic blood supply instead of a direct diffusion on the local site. 3.4. Anti-inflammatory and analgesic effects Following the optimized compound patches on only one knee or on the abdominal skin, anti-inflammatory effects on primary arthritis in bilateral hind paws were evaluated in the RA rat model (Fig. 4). The alterations of swelling on the bilateral paws were observed from 0.5 d after injection and leveled off from 3 d to 5 d. Although these patches were applied on only one knee or on the abdominal skin, after dosing,

Fig. 3. Local tissue disposition of TEF and LOX in two knee joints of rabbits after topical application to only one knee joint (the patch was removed at 24 h) (n = 4). (a) Schematic diagram of knee joint; TEF and LOX concentrations in (b) skin, (c) plasma, (d) ligament, (e) fat pad, and (f) synovial fluid. *P b 0.05; **P b 0.01; ***P b 0.001.

80

H. Xi et al. / Journal of Controlled Release 169 (2013) 73–81

(18.40 ± 2.80 μg/cm2/h and 18.07 ± 2.31 μg/cm2/h, respectively) but also a similar cumulative amount of drug permeated (198.20 ± 27.82 μg/cm2 and 194.89 ± 12.83 μg/cm2, respectively) at 12 h. As the drug action is dependent on the dose absorbed into the body, the lower response of TEF relative to LOX is mainly related with their different action mechanisms. In addition, the cumulative amount of drug permeated from the marketed ketoprofen patch at 12 h was 298.45 ± 65.85 μg/cm2, thereby the optimized compound patch system and the LOX-TEtA patch system were advantageous over the marketed ketoprofen patch based on their anti-inflammatory and analgesic effects. Although LOX played a leading role in the anti-inflammatory and analgesic effects, it was generally accepted that DMARDs could slow the progression of RA and actually modified the disease but NSAIDs could not. Therefore, it was daringly inferred that the combined administration of TEF-TEtA and LOX-TEtA could provide a better RA treatment in the long term management than the administration of LOX alone. In other words, the effect of TEF from the compound patches could not be ignored in controlling the RA activity. Fig. 4. (a, b) After CFA injection, the profiles of swelling degree (%) of hind paws of rats with applied transdermal patches on the left hind paw at 5 d; (c, d) after CFA injection, the profiles of swelling degree (%) of hind paws of rats with applied transdermal patches on abdominal skin at 5 d (n = 6).

the positive, TEF, LOX and TEF–LOX groups presented an obvious paw swelling inhibition at 7 d or 9 d, compared with the negative group. As shown in Fig. 4, the different onsets of response that appeared after applying on two different sites might be caused by an experimental error. This indicated that the drug which penetrated across the skin started to exert a pharmacologic action after 2 d to 4 d following transdermal administration. Although an exact onset of response was not obtained for all the formulations, compared with the positive group, the administration of LOX and TEF–LOX presented a better totality of response (P b 0.05) after 11 d which might lead to a preferable RA therapeutic effect. In addition, the swelling inhibition observed in the TEF group with the applied TEF patches on different sites was entirely comparable with that of the positive group after 11 d. The results indicated that the compound patches had an outstanding therapeutic effect for RA and LOX played a main antiinflammatory effect. There was no significant difference between the swelling alterations of bilateral hind paws in all the groups. It also further confirmed that the transdermal patches applied topically exerted a RA therapeutic activity by drug delivery via system circulation. As shown in Table 8, significant analgesic effects were observed in the positive, LOX and TEF–LOX groups compared with the control group and there was no difference between the positive group and the TEF–LOX group which proved that the compound patches had an excellent analgesic effect. The TEF group hardly presented any analgesic effect which was comparable with the control group. As similar as the permeation results from the optimized compound patch system, the permeations of TEF-TEtA and LOX-TEtA from their own patch systems had not only a similar permeation rate Table 8 The analgesic effect of TEF and LOX on the pain induced by acetic acid in mice after transdermal administration (n = 10). Group

Dose (cm2)

Numbers of writhing

PIR (%)

Control Negative Positive TEF LOX TEF–LOX

– 5 5 5 5 5

38 37 11 32 13 6

– 2.63 71.05 15.79 65.79 84.21

⁎⁎⁎ P b 0.001 vs. control.

± ± ± ± ± ±

18 21 6⁎⁎⁎ 10 6⁎⁎⁎ 4⁎⁎⁎

4. Conclusion Using the amine salts of TEF and LOX and the optimization of CCD, the optimized compound patches successfully displayed maximizing and isochronous permeation rates between TEF and LOX across rabbit skin, which was a bright prospect for compound transdermal patches containing TEF and LOX. Although transdermal administration applied topically can provide a local enhanced drug delivery for the superficial joint tissues by direct diffusion, it seemed unlikely to do that for the deeper tissue synovial fluid. Namely, transdermal products may achieve the same RA therapeutic effect applied on a non-inflammatory area. If so, the risk of skin irritation as a result of frequent administration on the inflammatory area may be avoided. References [1] G.S. Firestein, Evolving concepts of rheumatoid arthritis, Nature 423 (2003) 356–361. [2] J.R. O'Dell, Therapeutic strategies for rheumatoid arthritis, N. Engl. J. Med. 350 (2004) 2591–2602. [3] J.M. van den Hoven, S.R. Van Tomme, J.M. Metselaar, B. Nuijen, J.H. Beijnen, G. Storm, Liposomal drug formulations in the treatment of rheumatoid arthritis, Mol. Pharm. 8 (2011) 1002–1015. [4] J.R. O'Dell, Combination DMARD therapy for rheumatoid arthritis: a step closer to the goal, Ann. Rheum. Dis. 55 (1996) 781–783. [5] M.C. Claussen, T. Korn, Immune mechanisms of new therapeutic strategies in MS: teriflunomide, Clin. Immunol. 142 (2012) 49–56. [6] R. Nicholas, P. Giannetti, A. Alsanousi, T. Friede, P.A. Muraro, Development of oral immunomodulatory agents in the management of multiple sclerosis, Drug Des. Devel. Ther. 5 (2011) 255–274. [7] S. Radhofer-Welte, X. Rabasseda, Lornoxicam, a new potent NSAID with an improved tolerability profile, Drugs Today (Barc.) 36 (2000) 55–76. [8] Australian Rheumatology Association, Leflunomide, http://www.rheumatology. org.au/downloads/leflunomide230811_000.pdf, (accessed 14/6/2012, 2012). [9] M.T. Nurmohamed, B.A. Dijkmans, Efficacy, tolerability and cost effectiveness of disease-modifying antirheumatic drugs and biologic agents in rheumatoid arthritis, Drugs 65 (2005) 661–694. [10] E.M. Homdrum, R. Likar, G. Nell, Xefo® rapid: a novel effective tool for pain treatment, Eur. Surg. 38 (2006) 342–352. [11] The U.S. Food and Drug Administration, FDA drug safety communication: new boxed warning for severe liver injury with arthritis drug Arava (leflunomide), http://www.fda.gov/Drugs/DrugSafety/PostmarketDrugSafetyInformationforPatients andProviders/ucm218679.htm, (accessed 14/6/2012, 2010). [12] C.M. Lee, H.I. Maibach, Deep percutaneous penetration into muscles and joints, J. Pharm. Sci. 95 (2006) 1405–1413. [13] R.O. Day, A.J. McLachlan, G.G. Graham, K.M. Williams, Pharmacokinetics of nonsteroidal anti-inflammatory drugs in synovial fluid, Clin. Pharmacokinet. 36 (1999) 191–210. [14] N. Shinkai, K. Korenaga, H. Mizu, H. Yamauchi, Intra-articular penetration of ketoprofen and analgesic effects after topical patch application in rats, J. Control. Release 131 (2008) 107–112. [15] N. Shinkai, K. Korenaga, Y. Okumura, H. Mizu, H. Yamauchi, Microdialysis assessment of percutaneous penetration of ketoprofen after transdermal administration to hairless rats and domestic pigs, Eur. J. Pharm. Biopharm. 78 (2011) 415–421.

H. Xi et al. / Journal of Controlled Release 169 (2013) 73–81 [16] J. Radermacher, D. Jentsch, M.A. Scholl, T. Lustinetz, J.C. Frolich, Diclofenac concentrations in synovial fluid and plasma after cutaneous application in inflammatory and degenerative joint disease, Br. J. Clin. Pharmacol. 31 (1991) 537–541. [17] W.F. Elmquist, K.K. Chan, R.J. Sawchuk, Transsynovial drug distribution: synovial mean transit time of diclofenac and other nonsteroidal antiinflammatory drugs, Pharm. Res. 11 (1994) 1689–1697. [18] X. Niu, L. Zhao, Y. Jiang, R. Ma, L. Fang, Effect of amines on the percutaneous absorption of ketoprofen in a lipophilic system consisting of ethanol–isopropyl myristate, J. Shenyang Pharm. Univ. 25 (2008) 856–865. [19] M. Rinaudo, Y. Rozand, P. Mathieu, T. Conrozier, Role of different pre-treatments on composition and rheology of synovial fluids, Polymers 1 (2009) 16–34. [20] H. Xi, Y. Yang, D. Zhao, L. Fang, L. Sun, L. Mu, J. Liu, N. Zhao, Y. Zhao, N. Zheng, Z. He, Transdermal patches for site-specific delivery of anastrozole: in vitro and local tissue disposition evaluation, Int. J. Pharm. 391 (2010) 73–78. [21] J.M. Parekh, R.N. Vaghela, D.K. Sutariya, M. Sanyal, M. Yadav, P.S. Shrivastav, Chromatographic separation and sensitive determination of teriflunomide, an active metabolite of leflunomide in human plasma by liquid chromatography– tandem mass spectrometry, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 878 (2010) 2217–2225.

81

[22] Y. Zhang, D. Zhong, D. Si, Y. Guo, X. Chen, H. Zhou, Lornoxicam pharmacokinetics in relation to cytochrome P450 2C9 genotype, Br. J. Clin. Pharmacol. 59 (2005) 14–17. [23] W. van Eden, J.P.A. Wagenaar-Hilbers, M.H.M. Wauben, Adjuvant arthritis in the rat, Current Protocols in Immunology, John Wiley & Sons, Inc., 2001 [24] S. Reagan-Shaw, M. Nihal, N. Ahmad, Dose translation from animal to human studies revisited, FASEB J. 22 (2008) 659–661. [25] H. Xi, D. Cun, L. Shang, L. Mu, W. Song, L. Fang, Effect of the stability of hydrogen-bonded ion pairs with organic amines on transdermal penetration of teriflunomide, Int. J. Pharm. 436 (1–2) (2012) 857–861. [26] H. Xi, Z. Wang, Y. Chen, W. Li, L. Sun, L. Fang, The relationship between hydrogen-bonded ion-pair stability and transdermal penetration of lornoxicam with organic amines, Eur. J. Pharm. Sci. 47 (2) (2012) 325–330. [27] H. Trommer, R.H. Neubert, Overcoming the stratum corneum: the modulation of skin penetration. A review, Skin Pharmacol. Physiol. 19 (2006) 106–121. [28] P. Knox, J. Levick, J. McDonald, Synovial fluid—its mass, macromolecular content and pressure in major limb joints of the rabbit, Exp. Physiol. 73 (1988) 33–45.