December 2012 Regular Article Chem. Pharm. Bull. 60(12) 1479–1486 (2012) 1479 Formulation and Evaluation of Thienorphine Hydrochloride Sublingual Delivery System Fei Liu,a,b Yumei Zhao, a Jianxu Sun,a Yongliang Gao,*,a and Zhenqing Zhang*,a a Beijing Institute of Pharmacology and Toxicology; No. 27 Taiping Road, Beijing 100850, P. R. China: and b Department of Pharmacy, The First Affiliated Hospital of PLA; No. 51 Fucheng Road, Beijing 10048, P. R. China. Received June 7, 2012; accepted September 4, 2012 Thienorphine hydrochloride (ThH) is a highly insoluble and readily metabolized partial-opioid agonist. It is used for the treatment of pain and heroin addiction. This study aimed to formulate and evaluate sublin- gual delivery systems containing ThH. Dimethyl-β-cyclodextrin (DM-β-CD) can enhance the solubility and permeability of hydrophobic drugs. In this paper, ThH cyclodextrin inclusion complexes were prepared and administrated sublingually with the objective of improving the drug’s aqueous solubility, in vitro permeation rate, and in vivo absorption rate. The formulation was prepared with DM-β-CD using the freeze-dried meth- od and characterized using phase solubility, differential scanning calorimetry (DSC), X-ray and NMR analy- ses. The results of each test indicated the formation of dynamic inclusion complexes between ThH and DM- β-CD. The inclusion complexes also showed significant increases in in vitro aqueous solubility and mucosal permeability. According to the pharmacokinetic study of the complex in rats, the AUC and Cmax values of the sublingual delivery group were 40 and 46 times higher than those of the gastrointestinal group, whereas tmax was shorter, which proved that in vivo absorption and metabolism had been improved. It can therefore be concluded that the inclusion technology and sublingual delivery system were suitable for ThH development. Key words bioavailability; sublingual drug delivery; absorption; solubility; cyclodextrin; thienorphine Thienorphine (21-cyclopropyl-7-[1-(R)-1-hydroxy-1-methyl- network and finally the oral membrane. In addition, perme- 3-(thien-2-yl) propyl]-6,14-endoethano-6,7,8,14-tetra hydro- ation across the sublingual mucosa can be easily achieved oripavine, Fig. 1, is a new type of partial-opioid agonist, was when the concentration of active ingredient is relatively high. synthesized by the chemists in our institute.1) Meanwhile, Therefore, to develop a preparation of ThH suitable for sublin- ThH, a hydrochloride salt with a crystal water of thienor- gual delivery, increasing solubility is the primary concern.6,7) phine, has been developed as drug candidate. It has fared Cyclodextrins (CDs), cyclic oligosaccharides consisting comparatively well with respect to aqueous solubility and of 6–8 glucopyanose units, can trap the lipophilic drug in a pharmacodynamic action compared to its parent compound cage-like meshwork.8) So far, CDs have been reported to in- and its other salts.2) The drugs currently in clinical use for crease the solubility, stability, and bioavailability of drugs in a the treatment of opioid dependence are either full-opioid few different delivery systems.9,10) Many studies have reported agonists or antagonists. Partial-opioid agonists could be more that β-cyclodextrin (β-CD, Fig. 2) is a good natural cyclodex- widely used because they provide certain advantages. They trin because of its efficient drug complexation and availability −2 have unique pharmacological properties that allow them to in pure form. However, its low water solubility (1.5×10 M) prevent the addiction that can occur with full-opioid agonists and toxicity limit their application in pharmaceutical formula- and there has been a lack of subjective side effects.3) Although tions.11,12) Recently, various kinds of chemically modified CD ThH tablets have already passed through clinical testing in derivatives have been prepared to extend the physicochemical China, further development of ThH has been restricted by its properties and inclusion capacity of parent CDs. One of the very low oral bioavailability. This has two causes. First, ThH derivatives is dimethyl-β-cyclodextrin (DM-β-CD, Fig. 2), is poorly soluble, which causes a great deal of dissolution in which has more pronounced solubilization (0.2 M) and com- the gastrointestinal (GI) tract and significant enteric excretion. plexation than its parent compound. It also has some features Second, it is extensively metabolized by the liver, as previ- that make it safer for use in clinical settings.13) The DM-β-CD ously indicated.4) in particular has been shown to be excellent absorption en- Oral mucosal drug delivery is an alternative dosage form hancers in mucosal drug delivery.14) These all indicate that for thienorphine. It has been suggested that the sublingual they are well suited to sublingual administration. route has the following advantages: sufficient blood supply, This study aimed to formulate and evaluate a sublingual avoidance of the liver and GI, rapid absorption, and high bio- availability. Buccal and sublingual sectors are commonly used routes for drug delivery. The sublingualis more permeable and thinner than the buccal mucosa, with higher blood flow.5) This suggests that one way of improving ThH bioavailability may be the development of a sublingual delivery system. How- ever, sublingual delivery has strict requirements. The com- pound must first dissolve and disperse into the saliva. Then it must cross the unstirred water layer consisting of the mucin The authors declare no conflict of interest. Fig. 1. Chemical Structure of Thienorphine * To whom correspondence should be addressed. e-mail: [email protected]; © 2012 The Pharmaceutical Society of Japan [email protected] 1480 Vol. 60, No. 12 prepared with water saturated with oil phase. One milliliter of each solution was then transferred to 10 mL centrifuge tubes containing 1 mL of oil phase saturated with water. The tubes were vortex-mixed for 2 h at 37°C and centrifuged at 3000×g for 5 min. After centrifugation, 100 µL was withdrawn from the water phase and assayed by HPLC at time zero (C0) and after shaking to ensure partition (Cw). The partition coefficient was Koil/water=(C0−Cw)/Cw. The experiments were performed in triplicate. HPLC analysis of thienorphine was performed on a Shimadzu 10A HPLC system (Shimadzu, Japan). The de- tection wavelength was set at 220 nm. Chromatographic separation was carried out with a Varian ODS-C18 column (4.6 mm×250 mm, 5 µm; Varian, U.S.A.) with mobile phase consisted of methanol and phosphoric acid solution (pH= 3) in Fig. 2. Chemical Structure of β-Cyclodextrin (R=H) and Dimethyl- a ratio of 40 : 60 (v/v), and the column temperature was 35°C. β-CD (R=CH ) 3 The flow rate was 1.0 mL·min−1. Phase Solubility Test The phase solubility studies were delivery system for ThH-DM-β-CD inclusion complexes. performed according to the method of Higuchi and Connors.15) Based on the in vitro results, a suitable formulation was con- A schematic explanation of the principle of this technique is structed and administered to test rats by a sublingual route. given below, via Eq. 1: Pharmacokinetic parameters were compared to those of rats K given the drug via the gastrointestinal tract (GIT). The results ThH+ DM-ββ -CDr ThH:DM- -CD (1) K of each test indicate that the inclusion complexes prepared by d the co-lyophilization method were satisfactory with respect to Here Kr is the combination rate constant for the complex complexing and increasing solubility, permeability, and bio- formation and Kd is the dissociation rate constant of the com- availability. plex. Excess ThH was added into aqueous solutions of DM-β-CD Experimental at a serial concentrations in capped tubes and shaken at 25°C Materials ThH (>99%) was synthesized in the Institute for 48 h. After reaching equilibrium, the suspensions were fil- of Pharmacology and Toxicology (Beijing, China). Buprenor- tered through a 0.45 µm Millipore filter followed by the quan- phine standard (>99%) n-octanol (>99%) was purchased tification of the drug by HPLC. Phase solubility curves were from the Sigma-Aldrich Chemical Co. (U.S.A.). DM-β-CD represented as the total dissolved drug concentration relative (>98%) was purchased from the Li Quan Co., Ltd. (Shanxi, to the concentration of DM-β-CD. The stable constant (Ks) China). Methanol, acetonitrile and other materials were of was calculated from the initial straight portion of the phase analytical grade. solubility diagram using Eq. 215): Preparation of ThH-DM-β-CD Complexes The bulk slope ThH-DM-β-CD complexes were carried out according to the Ks = (2) S (1 slope) freeze-dried method as follows: ThH and the corresponding 0 amount of DM-β-CD (molar ratio 1 : 2) were dissolved in etha- Here S0 is the solubility of ThH in the absence of DM-β-CD nol and water, respectively. The resulting aqueous mixtures (equal to the intercept of the diagram), slope is the slope of were stirred for 2 h at constant temperature of 60°C. The the experimental phase solubility diagram for ThH-DM-β-CD, above preparation was then frozen and freeze-dried for 24 h, and Ks is the stability constant (Kr/Kd). using a lyophilizer (MDF-382E, Sihuan, China) at −80°C for Differential Scanning Calorimetry (DSC) Test Thermal 24 h. properties of the powder samples were investigated with a Aqueous Solubility The aqueous solubility of the ThH, differential scanning calorimeter (CDR-4P, Shanghai, China). ThH/DM-β-CD physical mixture and its DM-β-CD complexes Approximately 10 mg of sample was analyzed in an open were compared using a constant-temperature shaker (THX-82, aluminium pan, and heated at scanning rate of 10°C·min−1 Shanghai, China). The suspensions with excess amounts of from room temperature to 500°C.