Inclusion complex of tamibarotene with hydroxypropyl-β-cyclodextrin: Preparation, characterization, in-vitro and in-vivo evaluation

Graphical AbstractUnlabelled image


Introduction
Tamibarotene, 4-[ (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2naphthalenyl) carbamoyl] benzoic acid (Am80, the formula shown in Fig. 1), is a synthetic retinoid drug mainly used for relapsed or refractory acute promyelocytic leukemia (APL). Tamibarotene can be optionally combined with retinoic acid receptor α [1]; this combination recovers the normal function of primary marrow cells (PML) and the retinoic acid receptor gene leukemia α (RARα) by releasing the fusion of PML/RARα gene, which promotes the differentiation of promyelocytic cells, so that a therapeutic effect for APL can be achieved. Compared with other retinoid used for the treatment of APL, Am80 has a higher efficacy, produces less adverse reactions [2], and hardly has any drug resistance [3]. In addition, Am80 is less reactive to light, heat, oxidation, and other physical and chemical factors. It is especially suitable for relapsed or refractory APL patients who are resistant to all trans retinoic acid (ATRA) [4]. In 2005, tamibarotene tablet was first approved for use against acute promyelocytic leukemia in Japan, with the trade name Amnolake and specifications of 2 mg. As deeper research, tamibarotene is being investigated for various disease, studies have shown that tamibarotene can be used for multiple myeloma [5], Crohn's disease [5], chronic obstructive pulmonary disease [6], liver cancer [7][8][9][10], solid tumors [5] and Alzheimer [3]. However, tamibarotene has a poor solubility; when administrated orally, the first-pass effect is obvious, the bioavailability is relatively low (50%-70%) and the absorption is poor. In addition, Am80 has no injection available because of its poor water solubility. Therefore, improving the solubility of Am80 and increasing its bioavailability for clinical use are problems need to be solved urgently.
Several methods can be used to enhance the solubility of insoluble drugs, such as the use of suitable solubilizers, making poorly soluble drugs into soluble prodrugs, the use of nano technology and inclusion technique, etc. Among them, inclusion technique can significantly improve the stability and solubility of insoluble drugs, so as to improve its bioavailability [3,5]. The preparation of an inclusion complex is simpler and requires less additive materials, making it more suitable for commercial production. A dozen cyclodextrin-containing drugs have already been approved and marketed [11]. Furthermore, inclusion complex can be used as an intermediate of various dosage forms. Therefore, this study intends to adopt the inclusion technology to prepare a water-soluble tamibarotene inclusion complex with improved bioavailability.
Cyclodextrin and its derivatives are widely used as pharmaceutical materials for preparing inclusion complexes due to its non-toxicity, high biodegradability and various other advantages [11]. They can be used to improve the water-solubility, chemical stability and bioavailability of insoluble drugs, as well as reducing drug toxicity [12]. Among three types of cyclodextrin, β-CD is the least soluble, making it the easiest to be crystallized from water. Thus, β-CD is the most commonly used cyclodextrin. HP-β-CD is the ether derivative of β-CD. Compared with β-CD, HP-β-CD is more soluble and less toxic [13]. When administered via injection, HP-β-CD has almost no stimulation to muscle and mucosal. It has been approved by the United States Food and Drug Administration (FDA) to be used as a drug solubilizing agent and penetration enhancer. Thus, HP-β-CD has great potential applied in pharmaceutical excipients [14].
The aim of this study was to prepare a tamibarotene hydroxypropyl-β-cyclodextrin inclusion complex (Am80-HPβ-CD) through a freeze-drying method so as to improve the solubility and bioavailability of tamibarotene. To observe the formation of the inclusion complex, Am80-HP-β-CD was characterized by differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR). Next, the in-vitro dissolution characteristics of Am80-HP-β-CD and Am80 were verified with a dissolution method. Furthermore, the pharmacokinetics characteristics and the improvements in bioavailability were verified through the in-vivo experiments performed on rats. These results suggest that HP-β-CD will be potentially useful in the delivery of water-insoluble anticancer agents such as tamibarotene.

Preparation of Am80 -HP-β-CD inclusion complex
The Am80-HP-β-CD inclusion complex was prepared via a freeze-drying method. The Am80 was first dissolved in ethanol (3 ml), and then dispersed the Am80 solution into HP-β-CD aqueous solution at the molar ratio of 1:1. The obtained suspension was then stirred continually for 4 h at room temperature. The ethanol was then entirely removed by rotary evaporation for 15 min at 65°C, −0.08 MPa and then 10 ml distilled water was added and the solution was filtrated with a 0.45 μm microporous membrane. The solution was then cooled to room temperature and freeze-dried for 24 h. Finally, the inclusion complex was obtained.

Differential scanning calorimetry (DSC). The thermal
characteristics of the Am80, HP-β-CD physical mixture and the Am80-HP-β-CD inclusion complex were determined using a differential scanning calorimeter (DSC 204, Netzsch, Germany). Samples were accurately weighed in an aluminum pan, with an empty aluminum pan used as reference. The samples were then heated over a temperature range of 35-500°C, at the heating rate of 10°C/min under flowing nitrogen gas.

Fourier transform infrared spectroscopy (FT-IR). The FT-
IR spectra of Am80, HP-β-CD, physical mixture, and the Am80-HP-β-CD inclusion complex were obtained using a Fourier transform infrared spectrometer (Nicolet 6700 FT-IR Spectrometer, USA). Samples were prepared as KBr disks. The spectra were recorded from 4000 cm −1 to 400 cm −1 with 64 scans at the resolution of 4 cm −1 .

Study of in-vitro dissolution
The study of in-vitro dissolution of Am80-HP-β-CD (equivalent to 2 mg of Am80) and Am80 (2 mg

Pharmacokinetic study. The study of in-vivo
bioavailability was carried out on six adult Wistar rats (female or male) weighing 200 ± 20 g. The rats used for this study were housed individually under normal conditions and fasted overnight before the experiment with free access to water. Six rats were randomly assigned into two groups. One group was given tamibarotene solution through intragastric administration, while the other was given Am80-HP-β-CD inclusion complexes, both with dosage of 20 mg/kg. At specified time intervals (0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12, 24 h after intragastric administration), 300 μl blood samples was collected from jugular venous sinus. Then the blood samples were processed according to 2.2.4.2.

Data analysis and statistics.
The plasma concentration versus time curves of the Am80 solution and Am80-HPβ-CD inclusion complex were plotted. Pharmacokinetic parameters were calculated by DAS 2.0 software. The corresponding sampling time (tmax) and the maximum peak concentration (Cmax) were directly obtained from the curves.
The powder was white and smooth. It is also soluble in distilled water. The aqueous solution is quite stable, it can be kept at 4°C for at least one month.

Differential scanning calorimetry (DSC)
Change of sample temperature in the DSC test occurred due to a phase transition, or endothermic and exothermic effect of the samples. Generally speaking, the phase transition and dehydrogenation reduction will produce an endothermic effect of reducing dehydrogenation, while crystallization and oxidation will produce an exothermic effect. As shown in Fig. 3, tamibarotene has a typically sharp endothermic peak at 231°C, HP-β-CD has a large characteristic absorption peak at about 360°C; the absorption peak of the physical mixture of HP-β-CD and tamibarotene is slightly different from that of the superimposed peaks of tamibarotene and HP-β-CD. The interactions of Am80 and HP-β-CD may potentially be the causes. The Am80-HP-β-CD inclusion complex has formed a new absorption peak at 320°C, without the typical peaks at around 231°C (Am80) and 360°C (HP-β-CD). This is caused by significantly weakened crystallization degree of tamibarotene when it formed an inclusion complex with HP-β-CD, which stops the thermal analysis from detecting the endothermic peak of the tamibarotene crystals (231°C). Comparing the absorption peak of tamibarotene (231°C) and HP-β-CD (360°C), we can conclude that the Am80-HP-β-CD inclusion complex has been synthesized successfully.

Fourier transform infrared spectroscopy (FT-IR)
The FT-IR spectra of Am80, HP-β-CD, Am80-HP-β-CD inclusion complex, and their physical mixture are shown in Fig. 4. These changes are relatable to HP-β-CD cavities of similar size, benzene, and cyclohexane group of Am80, inferring that the benzene and cyclohexane group of Am80 entered the HP-β-CD cavity, causing Am80 to be entrapped in the cavity of HP-β-CD.

In vitro dissolution study
The dissolution profiles of the Am80 solution and Am80-HPβ-CD inclusion complex were shown in Fig. 5. The cumulative dissolution percentage of Am80-HP-β-CD inclusion complex was  Fig. 4 -Spectrum of A(Am80), B (HP-β-CD), C (Am80-HP-β-CD) and D (the physical mixture).
significantly higher than that of Am80. As for Am80-HP-β-CD, the cumulative dissolution percentage was over 97% in the first 3 min, and stayed stable after 3 min, while for the Am80, the cumulative dissolution percentage was only 4.8% in the first 3 min, after 45 min, the cumulative dissolution percentage reached about 60% and stayed stable. The dissolution speed and the dissolution percentage of Am80-HP-β-CD were significantly higher than that of Am80. Thus, it can be concluded that the inclusion complex can significantly improve the dissolution efficiency. The cause could be that the Am80 crystal disappeared after the formation of Am80-HP-β-CD inclusion complex.

Analysis method validation results
The typical chromatograms of Am80, blank plasma and plasma contained Am80 were shown in Fig. 6. The retention time for Am80 was about 7 min. It was shown that the endogenous components of plasma have no interference with Am80. The standard curve showed a good linearity within the range of 0.1~9.6 μg/ml. Typical mean linear equation of standard curves for Am80 was A = 26.352C-0.5541, r = 0.999, where A represented the peak area of Am80 and C was the concentration of Am80. The RSD values of inter-day and intra-day precision at three concentrations were less than 5% and 10%. The RSD values of the extraction yields of Am80 at three different concentration levels were less than 15%. The results showed that the method had good accuracy and precision. The extraction recovery would satisfy the requirements of the pharmacokinetic study.

Pharmacokinetics and bioavailability study
Mean plasma concentration versus time profiles of Am80-HPβ-CD inclusion complex and Am80 were illustrated in Fig. 7. Pharmacokinetic parameters were calculated by using the DAS 2.0 software; the results were shown in Table 1. By comparing the results of the two formulations, Am80-HP-β-CD inclusion    To be specific, the AUC0-∞ of Am80-HP-β-CD inclusion complex was about 3.79 times compared with that of the Am80 after administration. Meanwhile, the Cmax of Am80-HP-β-CD inclusion complex was about 5.37 times compared with the Am80. The increase of AUC0-∞ and Cmax indicated the improvement of bioavailability. Evidently, the relative oral bioavailability of Am80-HP-β-CD inclusion complex increased about 2.79 times compared with Am80. Moreover, the tmax of Am80-HP-β-CD decreased 7 times compared with Am80, the decrease of tmax indicated Am80 can be absorbed into blood faster, which may be caused by the increase of solubility after the formation of the inclusion complex. The result of the in vivo studies was consistent with the in vitro studies. These results proved that the Am80-HP-β-CD inclusion complex could significantly enhance the oral bioavailability of various medications.
The poor absorption of Am80 may result from its low solubility and dissolution rate. The improvement of oral bioavailability for Am80 could be ascribed to the increase of its dissolution and water-solubility after Am80 entrapped into the HP-β-CD cavity.

Conclusion
In this study, the Am80-HP-β-CD inclusion complex was successfully prepared through a freeze-drying method at the mole ratio of 1:1. The DSC and FT-IR characteristics have proved the formation of the Am80-HP-β-CD inclusion complex. The invitro dissolution showed the dissolution efficiency of Am80-HP-β-CD inclusion complex was significantly higher than that of Am80. An improved dissolution with approximately 97% drug release in 3 min was observed, in comparison with Am80 with approximately 60% release in 45 min. In vivo studies indicated that the AUC0-∞ has increased 2.79 times and the Cmax 4.37 times after the formation of inclusion complex. Further, the decrease of Cl/F and tmax indicated the Am80-HP-β-CD inclusion complex can be absorbed into blood faster as well as being cleaned out slower. In short, the bio-availability of Am80 was notably increased by complexing it with HP-β-CD.