Journal of Pharmaceutical Investigation (2012) 42:171–176 DOI 10.1007/s40005-012-0025-4
RESEARCH ARTICLE
Effect of poloxamer on physicochemical properties of tacrolimus solid dispersion improving water solubility and dissolution rate Jung-Myung Ha • Seung-Yeob Kang • Chun-Woong Park Sung-A Bin • Yun-Seok Rhee • Jeong-Woong Seo • Su-Heon Kim • Sang-Cheol Chi • Eun-Seok Park
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Received: 1 May 2012 / Accepted: 30 May 2012 / Published online: 14 June 2012 Ó The Korean Society of Pharmaceutical Sciences and Technology 2012
Abstract Tacrolimus (TCR; also FK-506 and trade name prografÒ), an antibiotic of macrolide family and a novel immunosuppressive agent, is a natural product of actinomycete Streptomyces tskubaensis. But TCR is poorly soluble in water (0.012 mg/mL), so its bioavailability is low and irregular. The aim of this study is to characterize physicochemical properties of TCR and investigate the improvement of solubility and dissolution rate of TCR solid dispersion (SD) with poloxamer. TCR SDs, consisting of various grades and ratios of poloxamer were prepared by hot-melting method and were characterized by DSC, PXRD, and FT-IR. The dissolution profile and solubility of TCR from the SDs were evaluated. SD of TCR prepared with poloxamer 188 at the ratio of 1:1 by the hotmelting method resulted in a significant increase in TCR solubility and enhanced dissolution profile over the TCR crystalline powder. Keywords Tacrolimus Solubility improvement Solid dispersion Poloxamer Hot-melting method Introduction Tacrolimus (TCR), an antibiotic of macrolide family is a novel immunosuppressive agent to be mainly used for the J.-M. Ha S.-Y. Kang C.-W. Park S.-A. Bin J.-W. Seo S.-H. Kim S.-C. Chi E.-S. Park (&) School of Pharmacy, Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon, Gyeonggi-do 440-746, Republic of Korea e-mail:
[email protected] Y.-S. Rhee College of Pharmacy, Gyeongsang National University, Jinju 660-751, Republic of Korea
reduction of the patient’s immune system after allogeneic organ transplant, which is a natural product of the actinomycete S. tsukubaensis (Wong 2001). TCR has the low solubility in water (0.012 mg/mL) and showed relatively low bioavailability of around 20 % (Honbo et al. 1987). Because TCR is a substrate for CYP3A5 and MDR1 genes, there are the large inter-individual variations in the pharmacokinetic parameters of TCR by the polymorphisms of the genes are major reasons and it was reported as an important factors in TCR-induced toxicity (Haufroid et al. 2004; Thervet et al. 2003; Yamauchi et al. 2002). In order to enhance the oral absorption of TCR, (Honbo et al. 1987) reported that oily ethanol formulation and solid dispersion (SD) formulation were most potent among many different formulations of TCR. Dissolution rate could thus become the rate-limiting step for gastrointestinal absorption after an oral administration of TCR, and quick release of TCR in the gastrointestinal tract following the oral administration is desirable. There have been various kinds of formulations tried as PLGA-PEG nanoparticles (Shin et al. 2010), selfmicro emulsifying drug delivery systems (Borhade et al. 2008), pH-sensitive microspheres (Lamprecht et al. 2004), and SDs (Joe et al. 2010; Park et al. 2009; Yamashita et al. 2003). The SDs could be usually prepared by solvent evaporating method or hot-melting method and solvent evaporating method has been used for a long time in the preparation of SDs or mixed crystals of organic compounds. They are prepared by dissolving a physical mixture of two solid components in a common solvent, followed by evaporation of the solvent. However, some disadvantages associated with this method are the higher cost of preparation, the difficulty in completely removing liquid solvent, the possible adverse effect of the supposedly negligible amount of the solvent on the chemical stability of the drug, the selection of a common
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volatile solvent, and the difficulty of reproducing crystal forms (Sharma et al. 2009). The other process, the hot-melting or fusion method, was first proposed by Sekiguchi and Obi (1961) to prepare fastrelease SD dosage forms and one of the most widely used methods. The melted mixture of drug and a water-soluble carrier was heated directly until it melted. The melted mixture was then cooled and solidified rapidly in an ice bath under rigorous stirring. The final solid mass was crushed pulverized, and sieved. The main advantages of this direct hot-melting method are its simplicity and economy. The SD using hot-melting method especially has several advantages such as no necessity of organic solvent, being an echo-friendly method and effortlessness for the scale-up step in industrial production (Patel et al. 2010). Furthermore, it could enhance the water solubility and bioavailability of poorly water-soluble drugs following as changing of solid state from long-range-ordered crystalline state to short-range-ordered amorphous state (Janssens and Van den Mooter 2009). In this study, TCR was used as a model drug, and poloxamer were used as a hydrophilic carrier in various ratios. SD is prepared by hot-melting method to improve solubility of TCR and avoid the problems related to organic solvents. The effect of grade of polymers and drug: polymer ratios on physicochemical properties of formulations was investigated.
Experimental Materials TCR was from Molcan Biopharma Co. (China). HPLC grade acetonitrile and anhydrous ethanol were from J.T. Baker Chemical Co. (USA). HPLC grade 2-propanol and tetrahydrofuran were from Fisher Chemical Co. (USA). Poloxamer 188, 237, 338, and 407 (PluronicÒ F-68, F-87, F-108, F-127) were from BASF Wyandotte Co. (Germany). Preparation of SDs by hot-melting method TCR SDs, contsisting of various ratios of poloxamer were prepared. The weight ratios of TCR and poloxamer were varied as 1:1, 1:2, 1:5, 1:10, 1:20 and 1:100 (w/w), respectively. Appropriate weights of TCR powder and poloxamer were weighed separately and mixed by geometric dilution. The physical mixtures were heated on a heating block to 130 °C until all the mixture had completely dissolved and was uniformly dispersed. The mixture was allowed to cool down slowly to room temperature. The solidified melts were milled and sieved using a 100 mesh sieve.
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Solubility measurement An excess amount of TCR powder or SDs prepared were weighed and added into 2 ml distilled water in screwcapped glass tubes. The suspension was magnetically shaken at 100 rpm in a thermostatic water bath maintained at 25 ± 0.5 °C. After 72 h, 1.2 mL of supernatant was withdrawn, centrifuged at 3,000 rpm for 10 min and mixed with 0.3 mL of acetonitrile. The mixed sample was analyzed by validated HPLC method. Differential scanning calorimetry (DSC) DSC studies were carried out using Pyris 1 DSC thermal analyzer system (Perkin-Elmer Inc., USA). Indium and zinc standards were used to calibrate and validate daily the DSC temperature scale; enthalpy response was calibrated and validated daily with indium. Data were treated mathematically using the Pyris software version 2.0 (PerkinElmer Inc.). The samples were hermetically sealed in pans and heated at constant rate of 10 °C/min over a temperature of 30–150 °C. Purging nitrogen gas was introduced for inert atmosphere. X-ray diffraction (XRD) The TCR crystalline powder, SD and physical mixture of TCR with poloxamers were recorded using an X-ray diffraction method with Ni filter and Cu-Ka radiation, a voltage of 40 kV and a current of 40 mA. The diffraction data were collected at room temperature. For studies, the angular range 0–40° 2h was scanned with a step size of 5° 2h and a dwell time of 8 min at each step. The 2h values and the intensities of the peaks were compared for both pure ingredients and SD system. Fourier-transform infrared spectroscopy (FT-IR) The evaluation of changes in the chemical structure of TCR was performed using IFS-66/S FT-IR spectrometer (Bruker corp., Germany). TCR crystalline powder, poloxamer 188, physical mixture and SD were evaluated to confirm the structure of TCR in each condition. Data were collected over a spectral region from 4,000 to 400 cm-1 with an instrument resolution of 4 cm-1. Dissolution study The dissolution profiles of TCR from various SD formulations were determined at 37 ± 0.5 °C with a paddle speed at 100 rpm using the USP apparatus II (Dissolution Tester DST-600A, Fine Scientific Instruments, Korea). Dissolution medium was 900 mL of distilled water. In each
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dissolution test, a quantity of samples corresponding to 1 mg of TCR was put into 900 mL of the dissolution medium. Aliquots (10 mL) were collected at 30, 45, 60, 75 and 90 min and centrifuged at 2,000 rpm for 5 min. 4 mL of centrifuged sample was mixed with 1 mL acetonitrile. The concentration of TCR released at the predetermined sampling time was assayed by validated HPLC method.
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TCR is highly soluble in various organic solvents, such as chloroform (594 mg/mL), methanol (588 mg/mL), acetone (542 mg/mL), ethanol (370 mg/mL). However TCR is poorly soluble in water (0.012 mg/mL). Due to the low water-solubility, TCR shows low bioavailability. Several ways to increase its bioavailability have been described in the prior reports (Nassar et al. 2009; Wang et al. 2011) SD is one of the simple methods to increase the solubility of the drugs. Figure 1 showed the effect of different grade of poloxamer on solubility and dissolution profiles of TCR from SD. The solubility profiles of TCR SDs with various grades of poloxamer at the ratio of 1:20 were shown in Fig. 1a. All of the TCR SDs prepared with poloxamer showed largely increased solubility. Increased solubility of TCR SDs was due to increased wettability and decreased crystallinity of TCR dispersed in poloxamers. In case of SD with poloxamer 407, TCR interacted with many propylene oxide hydrophobic segments of poloxamer 407, so the solubility of SD with poloxamer because of the interaction between TCR and poloxamer 407 by many propylene oxide segments, hydrophobic parts, the solubility of SD with poloxamer 407 was higher than other poloxamers. Figure 1b illustrates the dissolution profiles of TCR from SDs prepared with various grades of poloxamer. The dissolution rates of TCR released at 90 min from the SD with various poloxamers at ratio of 1:20 was ranged from 83.95 to 71.77 %. It was found from the result that SDs prepared with four different kinds of poloxamers at the ratio of 1:20 increased their dissolution rate significantly in compared with dissolution rates of TCR powder. The improvement of dissolution rate of solid dispersion SD with poloxamer 407 at 90 min is significantly higher than that with poloxamer 188 however the gap between them is much smaller than that in solubility test. Figure 2 showed the effect of different ratio of TCR and poloxamer on solubility and dissolution profile in SD. The solubility of TCR SD with poloxamer 188 at the various ratios (1:1, 1:2, 1:5, 1:10, 1:20, 1:50, and 1:100 w/w) in distilled water was shown in Fig. 2a. All of the tested
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Fig. 1 Effect of different grade of poloxamer on a solubility of TCR solid dispersions prepared with TCR and poloxamer at the ratio of 1:20 in water at 25 °C (mean ± SD, n = 3). b Dissolution profiles of TCR from solid dispersions prepared with TCR and poloxamer (1:20): filled square TCR powder, filled circle poloxamer 188, open circle poloxamer 237, inverted filled triangle poloxamer 338, open triangle poloxamer 407 (mean ± SD, n = 3)
samples showed increase in drug solubility. This phenomenon can be ascribed to a higher wettability of TCR in the presence of poloxamer 188. Poloxamer 188 shows a significant solubilization effect on TCR. When the amount of poloxamer 188 decreased in the SD, the solubility of TCR was increased. The solubility of TCR SD with poloxamer 188 at the ratio of 1:1 increased up to 30-fold than the solubility of TCR in distilled water. Figure 2b illustrates the dissolution profiles of TCR from SDs prepared with different amounts of poloxamer 188. The percentage of TCR released at 90 min from the SD with poloxamer 188 at the various ratios was ranged from 105.95 to 37.59 %. In 1:50, and 1:100 samples, there is no significant differences in percentage released compared with the TCR powder. It seemed that SDs with the ratio of 1:50 and 1:100 didn’t have effect on the dissolution rate of TCR. Among SDs prepared, the dissolution rates of TCR with poloxamer 188 (1:1) seems to be most greatly enhanced. Similar
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Fig. 3 DSC thermograms of TCR solid dispersion prepared with poloxamer 188: TCR powder, poloxamer 188, physical mixture of TCR: poloxamer 188 = 1:1 w/w, solid dispersions of TCR: poloxamer 188 = 1:1 w/w, 1:2 w/w, 1:5 w/w, 1:10 w/w, 1:20 w/w, 1:50 w/w, 1:100 w/w
Fig. 2 Effect of poloxamer 188 ratio on a solubility of TCR solid dispersion in water at 25°C (mean ± SD, n = 3). b Dissolution profiles of TCR from solid dispersions prepared by the melting method: open diamond TCR powder, filled circle solid dispersion of TCR: poloxamer 188 = 1:1 w/w, open circle 1:2 w/w, inverted filled triangle 1:5 w/w, open triangle 1:10 w/w, filled square 1:20 w/w, open square 1:50 w/w, filled diamond 1:100 w/w (mean ± SD, n = 3)
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findings have been reported with several SDs such as sulfathiazole with PVP, ciprofloxacin with PEG 6000 and alkyl p-aminobenzoate with PEG 6000. (France´s et al. 1991; Saers and Craig 1982; Simonelli et al. 1969) In order to explain these phenomena many mechanisms were proposed: formation of continuous drug layer (Dubois and Ford 1985) and release of intact particles, from which dissolution occurs over a large area (Saers and Craig 1982).
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DSC curves for pure TCR crystalline powder, poloxamer 188, their physical mixtures of the both (1:1 w/w) were shown in Fig. 3. The thermograms of TCR and poloxamer 188 showed intense peaks at temperatures near their melting point, 130–133 and 52–57 °C, respectively. Physical mixture (1:1 w/w) of TCR and poloxamer 188 showed a peak originated from TCR on its thermogram. It was confirmed that the crystallinity of TCR does not change in
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Fig. 4 XRD patterns of TCR solid dispersion with poloxamer 188: TCR powder, poloxamer 188, physical mixture of TCR: poloxamer 188 = 1:1 w/w, solid dispersion of TCR: poloxamer 188 = 1:1 w/w, 1:2 w/w, 1:5 w/w, 1:10 w/w, 1:20 w/w, 1:50 w/w, 1:100 w/w
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The FT-IR studies were carried out to investigate the interaction between TCR and poloxamer 188. The FT-IR spectra of pure TCR crystalline powder, poloxamer 188, the physical mixture of TCR and poloxamer 188 (1:1 w/w) and the SD with poloxamer 188 (1:1 w/w) prepared by the hotmelting method were shown in Fig. 5. In the FT-IR spectra of TCR crystalline, absorption bands of O–H stretching vibration at 3,450 cm-1, C = O (ester and ketone) stretching vibrations at 1740, 1725 and 1693 cm-1, C = O (ketoamide) and C = C stretching vibration at 1,637 cm-1, C–O (ester) stretching vibration at 1,194 cm-1, C–O–C (ether) stretching vibrations at 1,176 and 1,094 cm-1 were observed. In case of poloxamer 188, huge polymer with a molecular weight of 8,500, absorption bands of C–O (ester) stretching vibration at 1,100 cm-1, aliphatic bond at 3,000 cm-1, O–H stretching vibration at 3,600 cm-1 were observed. These bands were also observed for the physical mixture of TCR and poloxamer 188 with the same absorbance. From these results, it was confirmed that there was no interaction between TCR and poloxamer 188 in the physical mixture (Hirasawa et al. 1999; Kushida et al. 2002; Perng et al. 1998; Tantishaiyakul et al. 1999).
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Fig. 5 FT-IR spectra: TCR crystalline powder, poloxamer 188, physical mixture of TCR: poloxamer 188 = 1:1 w/w, and solid dispersion of TCR: poloxamer 188 = 1:1 w/w
the physical mixtures with carrier, poloxamer 188. Figure 1 also showed the thermo grams of the SDs with various ratios (1:1, 1:2, 1:5, 1:10, 1:20, 1:50, and 1:100 w/w) of TCR and poloxamer 188. The intense peaks of TCR in the thermo grams of the SDs were disappeared. The disappearance on the thermogram indicated that the drug is dispersed or melted in polymer homogeneously (Kaur et al. 1980; Law et al. 1992). The XRD patterns of TCR crystalline powder, poloxamer 188, physical mixture of them (1:1 w/w) and the SDs with various ratios of poloxamer 188 were shown in Fig. 4. Numerous distinctive peaks indicated a high crystallinity of TCR crystalline powder. XRD pattern of the physical mixture was similar to that obtained for the pure TCR powder. However, TCR SDs prepared with various ratios of poloxamer 188 by hot-melting method did not show the intensive peak at the specific 2-theta of TCR, which suggested that TCR exists in an amorphous state in the SD. The amorphous form of the drug has a higher thermodynamic activity than its crystalline form. The higher thermodynamic energy level of the drug could lead to the rapid dissolution property (Betageri and Makarla 1995; Jung et al. 1999).
TCR SD prepared with poloxamer by the hot-melting method resulted in significant increases in drug solubility and enhanced dissolution profiles of TCR over its pure crystalline powder. The drug converted to amorphous form in SD prepared with poloxamer 188. In the SD powder, various grades of poloxamer enhanced the solubility and the dissolution rate of TCR significantly. Poloxamer 407 was the most effective carrier to improve the solubility and the dissolution rate of TCR. In case of poloxamer 188, the best ratio of TCR and poloxamer 188 was 1:1. The SD technique employed this study involves relatively simple preparation steps and may be utilized in preparing granules, tablets, capsules, and other oral dosage forms. Although the direct filling of SD into hard gelatin capsules is a relatively simple process, there are very limited reports on the scaleup of the technology. Further studies on scale-up and validation of the process will be essential. Many problems and challenges still remain with SD systems. Nevertheless, with further investigation, it can be one of the promising ways developing advanced formulation (Serajuddin 1999).
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