Enhanced solubility, dissolution, and absorption of lycopene by a solid dispersion technique: The dripping pill delivery system

    Enhanced solubility, dissolution, and absorption of lycopene by a solid dispersion technique: The dripping pill delivery system Chih-Wei Chang, Chih-Yuan Wang, Yu-Tse Wu, Mei-Chich Hsu PII: DOI: Reference:

S0032-5910(16)30405-3 doi: 10.1016/j.powtec.2016.07.013 PTEC 11775

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Powder Technology

Received date: Revised date: Accepted date:

1 February 2016 31 May 2016 3 July 2016

Please cite this article as: Chih-Wei Chang, Chih-Yuan Wang, Yu-Tse Wu, Mei-Chich Hsu, Enhanced solubility, dissolution, and absorption of lycopene by a solid dispersion technique: The dripping pill delivery system, Powder Technology (2016), doi: 10.1016/j.powtec.2016.07.013

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ACCEPTED MANUSCRIPT Enhanced solubility, dissolution, and absorption of lycopene by a solid dispersion technique: the dripping pill delivery

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system

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Chih-Wei Chang1, Chih-Yuan Wang1, Yu-Tse Wu1,*, Mei-Chich Hsu2,** 1

School of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan

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Department of Sports Medicine, Kaohsiung Medical University, Kaohsiung 807,

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Taiwan

*

Corresponding author

Yu-Tse Wu, Ph.D

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Assistant Professor

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School of Pharmacy, Kaohsiung Medical University 100, Shih-Chuan 1st Road, Kaohsiung, 80708, Taiwan

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Telephone: 886-7-312-1101 ext. 2254 Fax: 886-7-312-0683

E-mail: [email protected] **

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Corresponding author

Mei-Chich Hsu, Ph.D Professor

Department of Sports Medicine, Kaohsiung Medical University 100, Shih-Chuan 1st Road, Kaohsiung, 80708, Taiwan Telephone: 886-7-312-1101 ext. 2793 ext. 613 Fax: 886-7-313-8359 E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract Lycopene, a natural, hydrophobic compound derived from tomatoes, has attracted considerable attention due to its various benefits such as being an

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anti-oxidant, anti-cancer, and anti-hyperlipidemic agent. The insolubility of lycopene

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is a major obstacle that leads to poor bioavailability. We aimed to optimize a

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straightforward, promising approach utilizing a solid dispersion technique, named the dripping pill delivery system, for improving the absorption of lycopene. The polyethylene glycol 6000-based dripping pills were prepared by the hot melt method with rapid cooling. A 32 full factorial design was employed to optimize the effects of

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the independent variables, Cremophor EL and Tween 80, on the solubility, dissolution, and stability tests. The physicochemical characteristics and interaction were

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investigated by the disintegration test, scanning electron microscope, differential scanning calorimetry, and Fourier transform infrared spectroscopy. The in vivo experiment was carried out to verify the usefulness of the developed formulae. The

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optimal dripping pill formulae elevated the solubility and dissolution of lycopene,

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which was confirmed to be formed in an amorphous state. Furthermore, the optimal

Keywords

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dripping pill exhibited an approximate 6-fold improvement in bioavailability.

hot melt method; tomato; oral bioavailability; factorial design; solubilization

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technique

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ACCEPTED MANUSCRIPT 1. Introduction Lycopene, a hydrophobic compound derived from tomatoes and other red fruits and vegetables, such as red carrots, watermelons, pink guavas, and papayas [1],

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occurs naturally in the all-trans form. Lycopene has attracted considerable attention

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due to its various benefits. First, lycopene has a unique structural feature, a long chain

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with conjugated double bonds. It possesses a superior capacity for free radical scavenging. Second, lycopene plays an important role in cell apoptosis, which may give it a protective property against prostate cancer [2] and colon cancer [3, 4]. Third, lycopene

markedly

attenuates

hyperlipidemia

[5]

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the

inhibition

of

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3-hydroxy-3-methyl-glutaryl-CoA reductase [1]. However, lycopene is insoluble and possesses extremely low bioavailability (BA), which is the major limitation of its

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application.

Design of pharmaceutical preparations is a promising way to improve upon the absorption of active ingredients with low solubility. The BA of lycopene is higher in

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processed tomato products than in unprocessed, fresh tomatoes. For instance, tomato

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paste, one of the lipid-based preparations, has been demonstrated to yield 3.8-fold higher area under the curve responses of lycopene compared with fresh tomatoes [6];

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nonetheless, it remains intolerable for regular consumption. To the best of our knowledge, some formulations of lycopene have been developed, such as liposomes [7], micelles, chylomicrons [8], and lipid-based solid dispersion [9]. However, these

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methods carry some notable disadvantages. Liposomes carry the disadvantage of insufficient stable loading of drugs [10], while lipid-based formulations may have poor solvent capacity and are less easily digested [11]. For decades, solid dispersions have provided a very fruitful approach to improve the release rate and oral bioavailability of poorly soluble compounds. Dispersing of solid compound in water soluble carrier results in increase of the surface area and improvement of the wettability of the compound; hence, it elevates the dissolution rate and bioavailability. Polymer carriers are particularly likely to form amorphous solid solutions for further improving an active ingredient's dissolution properties [12]. The dripping pill is prepared according to the principle of a solid dispersion, which enhances the solubility and dissolution of the target ingredient via modification of the size, wettability, porosity, and crystalline structure [13]. Using the hot melt method, the molten mixture containing the active ingredients and excipients 3

ACCEPTED MANUSCRIPT (e.g., polymer) is gravitationally extruded to produce drops, and solidified by rapid cooling to form spherical pills [14]. Dripping pills can be taken sublingually, buccally, or by swallowing; moreover, the method draws attention because of its simple

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fabrication, ease of dispensing, high stability, and the equal content of each pill.

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We aimed to optimize a straightforward, promising approach utilizing the solid dispersion technique, namely the dripping pill delivery system, for improving the BA

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of lycopene. A 32 full factorial design was used to optimize the effects of formulations on the solubility of contents, dissolution, and stability. The relationship of the optimized formulation between the in vitro and in vivo tests was further confirmed by

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the pharmacokinetic parameters in rats.

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ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. Chemicals and reagents Lycopene standard was obtained from Sigma–Aldrich (purity >90%, St. Louis,

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MO, USA), and tomato extract (TE, lycopene >18%) was obtained from Compson

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(Taichung, Taiwan). Analytical grade methanol, n-hexane, and tetrahydrofuran were

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supplied by E. Merck (Darmstadt, Germany). Dichloromethane and ethyl acetate were supplied by Honeywell (Morris Plains, New Jersey, USA). Polyethylene glycol (PEG) 6000 was obtained from Koch-Light (Haverhill, Suffolk, UK), polyoxyethylene castor oil (Cremophor EL; HLB = 14) was obtained from Sigma-Aldrich (St. Louis, MO,

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USA), and polyoxyethylene sorbitan fatty acid ester (Tween 80; HLB = 15) was obtained by Shimakyu (Osaka, Japan). Other chemicals used in the work were all of

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analytical grade. Pure water was prepared using the Milli-Q system (Millipore, Bedford, MA, USA).

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2.2. HPLC conditions for lycopene determination The HPLC system (Hitachi, Tokyo, Japan) consisted of an L-7100 pump, an

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L-7200 autosampler, and an L-7455 PDA detector. Samples were separated on a reversed-phase C18 column (LiChrocart Purospher STAR RP-18, 250  4.0 mm, i.d.,

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5 m, Merck). The mobile phase, methanol and tetrahydrofurane (75:25, v/v), was degassed using a Millipore membrane filter (0.22 μm, Bedford, MA, USA) and delivered at 1.0 mL/min. The sample injection volume was 20 L, and the detection

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wavelength was 475 nm. A stock solution of lycopene (200 g/mL) was prepared in ethyl acetate:dichloromethane:n-hexane (80:16:4) [15]. Linearity was confirmed by the coefficient of determination (r2) of 0.9996 over the range of 5.0–200 g/mL, and the regression equation was y = 25654x + 81858. Precision was assessed as the intraand inter-day variation in lycopene, as indicated by the relative standard deviation, which ranged from 0.5% to 1.7%. The intra- and inter-day accuracies of lycopene ranged from 99.7% to 100.2%.

2.3. Preparation of tomato extract-loaded dripping pills Tomato extract-loaded dripping pills (TEDPs) were prepared according to the principles of the hot melt method coupled with a dripping pill machine (Model: DWJ-2000S5; Yantai Kangde Medical Company, Shandong, China) (Fig 1). In the first beaker, PEG 6000 (245 g), melted at 85°C in a water bath, served as a carrier of

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ACCEPTED MANUSCRIPT the dripping pill preparation. A particular advantage of PEGs for the formation of solid dispersions is that PEG 6000 may be useful to solve various problems such as stability, solubility, dissolution, and BA; PEGs 4000–6000 are the most frequently

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used PEGs in manufacturing [12]. The second beaker consisted of a mixture of TE (30

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g), Cremophor EL (10 g), and Tween 80 (15 g). The solution in the first beaker was gently poured into the second beaker and mixed thoroughly, making the total mass

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300 g. The mixed solution was then transferred into the machine’s drug container with continuous stirring. The oil bath heater and drug dripper were maintained at 85°C. The solution was extruded through a multi-outlet drug drip manifold by gravity; it

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dripped down into cold dimethylpolysiloxane (10°C) and gradually cooled and solidified to pills. Finally, when the pills reached the bottom reservoir, the oil pump

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brought the formed dripping pills to the collector. The residual oil on the pill’s surface was drained via a stainless steel filter, centrifuged in a centrifugal oil purifier, and wiped out with Kimtech Science wipers (Kimberly-Clark Corporation, Irving, TX,

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USA).

factorial design

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2.4. Formulation of tomato extract-loaded dripping pills using 32 full

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A full factorial design (32) was employed in the formulation of TEDPs for the screening of the influence and optimization of the two studied factors, the emulsifiers Cremophor EL (A) and Tween 20 (B), each at three levels (Table 1). The observed

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responses were solubility (g/mL), dissolution at pH 1.2 at 2 h (%), dissolution at pH 6.8 at 2 h (%), and stability at 6 months (%) (Y1, Y2, Y3, and Y4, respectively). Nine formulae were suggested and randomly arranged (F1–F9) by Design-Expert 6.0.3 software (StatEase Inc., Minneapolis, MN, USA) (Table 2).

2.5. Solubility study The solubility study was previously described [14]. The prepared TEDPs were pulverized and placed in 0.1 N hydrochloric acid solution (pH 1.2). The samples were shaken at 50 rpm for 24 h at 37 ± 0.5°C, then the supernatant was collected and filtered through 0.45 μm syringe filters (Millex-GV, Millipore, Bedford, MA, USA) for lycopene analysis.

2.6. In vitro dissolution study The in vitro dissolution study was previously described [14]. The release of lycopene from the prepared TEDPs was determined using the paddle method with an 6

ACCEPTED MANUSCRIPT SR8 PLUS dissolution test station (Hanson Research, Chatsworth, CA, USA). The dissolution medium was a 0.1 N hydrochloric acid solution (pH 1.2, 500 mL) and a phosphate buffer solution (pH 6.8, 500 mL) for acid and base, respectively. The

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dissolution fluid was maintained 37 ± 0.5°C at a rotation speed of 50 rpm. Samples

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were withdrawn at 5, 10, 20, 30, 40, 60, and 120 min, and filtered through 0.45 μm syringe filters (Millex-GV, Millipore, Bedford, MA, USA). The initial volume of the

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dissolution fluid was maintained by adding the same volume of fresh dissolution fluid after each withdrawal. In total, 6 replicate samples were analyzed by HPLC and the cumulative release (%) was calculated.

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2.7. Stability study

The stability study was performed according to the guideline of International

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Conference on Harmonization of Technical Requirements for Registration of Pharmaceutical for Human Use [16]. The stability of lycopene from the prepared TEDPs was determined in a temperature/humidity test chamber (HRM-80B, Prema,

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Taiwan). Samples were subjected to accelerated conditions (40 ± 2°C and 75 ± 5%

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RH) and were withdrawn after 1, 2, 3, 4, 5, and 6 months for lycopene analysis.

2.8. Physicochemical characteristics of tomato extract-loaded

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dripping pills

2.8.1. Determination of the content, weight variation, particle diameter, and roundness of tomato extract-loaded dripping pills

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The prepared TEDPs were pulverized and 100 mg were weighed out for lycopene content determination. The lycopene content of each pill was analyzed by HPLC developed for each formulation. The weight variation, particle diameter, and roundness of TEDPs were tested for 20 randomly selected pills of each formulation. Weight variation was tested using an electronic balance (XS3250C, Precisa, Dietikon, Switzerland); it was considered acceptable if the masses of all pills were within the range of 85%–115% of the average mass. Particle size was determined using an electronic Vernier caliper (resolution: 0.01 mm; TD-6615, Tenda, Taiwan). Roundness was assessed using an optical microscope (Olympus, Tokyo, Japan) and presented as elongation. The elongation was calculated as the ratio of the short diameter and long diameter of pills; the tendency to approach one was considered as round.

2.8.2. Disintegration test Disintegration time was determined according to Pharmacopoeia of the People’s 7

ACCEPTED MANUSCRIPT Republic of China [17], using a DG-2B disintegration tester (Hsiangtai Machinery Industry, New Taipei City, Taiwan) with 1000 mL of pure water, which was maintained at 37 ± 1°C. The reciprocating distance of the basket was 55 ± 2 mm, and

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the frequency was 30–32 rpm. The disintegration time was recorded when the pills

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were completely disintegrated and passed through the mesh (i.d.; 0.42 mm). Six pills per formulation were tested. The disintegration time within 30 min is considered as

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immediate release solid dispersion.

2.8.3. Scanning electron microscopy

The surface morphology of each prepared TEDP was visualized using scanning

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electron microscopy (SEM) (SU8010, Hitachi, Tokyo, Japan). The samples were prepared by sticking the pills on a double adhesive tape, which adhered to an

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aluminum stub. The stubs were then coated with platinum for 2 min. TEDPs were observed under 30× magnification for spherical appearance and 1000×, 3000×, and 5000× for sectional appearance.

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2.8.4. Differential scanning calorimetry

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Differential scanning calorimetry (DSC) analysis was performed using the DSC 7 (PerkinElmer, Waltham, Massachusetts, USA) to evaluate any possible

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drug-excipient interaction, such as the generation of crystallinity and polymorphism. For DSC scanning, TE first underwent recrystallization. Thermograms of TE, PEG 6000, the physical mixture, and each prepared TEDP were obtained. Samples of 10

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mg were placed in aluminum pans and sealed. The pans were heated from 25°C to 200°C at a rate of 10°C/min under a nitrogen atmosphere.

2.8.5. Fourier transform infrared spectroscopy Fourier transform infrared spectroscopy (FT-IR) spectra of TE, PEG 6000, and TEDPs were obtained using a PerkinElmer 2000 spectrophotometer (Waltham, Massachusetts, USA). Each sample was dispersed in KBr, ground in a mortar and pestle, and made into a disk. Spectra were collected over a range of 4000–400 cm−1.

2.9. In vivo study 2.9.1. Experimental design Animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (No. IACUC-103008) of Kaohsiung Medical University Hospital (Kaohsiung, Taiwan). Experiments were executed in a parallel study design, with three rats (Sprague-Dawley, weight 240 ± 10 g) in each group. Rats 8

ACCEPTED MANUSCRIPT were fasted overnight with free access to water for at least 12 h and dosed orally by gavage, either with optimized TEDP (150 mg lycopene/kg rat) or TE (500 mg lycopene/kg rat). A jugular vein catheterization model [18] was applied for repeated

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sampling in the unrestrained conscious rats. Blood samples (200 μL) were collected in

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heparinized tubes before dosing (0 h) and at 1, 2, 3, 4, 6, 8, 10, 12, 24, 36, and 48 h post-dosing, and the removed blood after each sampling was replaced with an equal

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volume of heparinized normal saline. The samples were immediately placed on ice and centrifuged at 12,000 rpm for 10 min. The plasma was separated and stored at −70°C for further analysis.

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2.9.2. Sample preparation

An aliquot of 100 μL of absolute ethanol was added to 100 μL of plasma sample

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to precipitate the proteins. The extraction was performed by adding an aliquot of 300 L of n-hexane into the mixture followed by vortexing for 1 min and the supernatant organic phase was collected. The extraction step was performed twice. The collected

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liquid then underwent evaporation under nitrogen at room temperature. The residue

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was reconstituted in 50 μL CH2Cl2 and 50 μL mobile phase for HPLC.

2.9.3. HPLC validation

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HPLC method was validated to examine the selectivity, linearity, accuracy, and precision. Selectivity was confirmed by inspecting the interference that appeared at the retention time of lycopene. A working standard solution of lycopene (10 μL) was

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spiked with blank rat plasma (90 μL) to construct the calibration curve over a range of 0.05–10.0 μg/mL. Linearity was confirmed by the coefficient of determination (r2) of 0.9999, and the regression equation was y = 32113x + 385. Precision was assessed as the intra- and inter-day variation in lycopene, as indicated by the relative standard deviations, which ranged from 3.7% to 17.7%. The intra- and inter-day accuracies of lycopene determination ranged from 96.5% to 117.8%.

2.10. Data analysis The data are expressed as mean ± standard deviation. The full factorial design (32) for the optimization of TEDP formulation was computed and analyzed by analysis of variance (ANOVA) using the Design-Expert 6.0.3 software (StatEase Inc., Minneapolis, MN, USA). Pharmacokinetic data were fitted to a noncompartmental model according to [19]. The pharmacokinetic parameters t1/2, Cmax/Dose, AUC0–48/Dose, and AUC0–∞/Dose were compared between treatments statistically by 9

ACCEPTED MANUSCRIPT the independent sample t-test using the SPSS 19.0 software (International Business Machines Corporation, Armonk, NY, USA). A p < 0.05 was considered to indicate

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statistical significance.

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ACCEPTED MANUSCRIPT 3. Results and discussion 3.1. Analysis of factorial design 3.1.1. Formulation design of TEDPs

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Design of experiment is a combination of mathematical and statistical

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approaches for the optimization of the processes, it provides an alternative to assess

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the impacts more efficiently than the one-factor-at-a-time experiments [20]. The application of a factorial design in pharmaceutical formulation development has played a crucial role in the relationship between independent factors and responses.

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Regarding the solid dispersion, full factorial design has been widely applied to optimization of prepared formulations. The amounts of surfactants, carriers, polymer ratios, cooling temperatures, kneading times were commonly chosen as independent

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factors; whereas solubility, dissolution rates, and yields were commonly chosen as responses [21-25]. In the present study, we created a thoroughgoing design by

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selecting decisive responses in pharmaceutical development, namely solubility, dissolution at pH 1.2 at 2 h, dissolution at pH 6.8 at 2 h, and stability at 6 months. A

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32 full factorial design was used, and the observed responses of the 9 formulations produced with variable levels of independent factors are shown in Table 3.

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In our preliminary test, 10% active ingredient loading exhibited a faster release, which was in accordance with previous study [16]. If the percentage of the active ingredient is too high, it will form small crystals, rather than remaining dispersed.

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Therefore, in the present study, we kept the ratio of active ingredient loading in constant (10%), and studied to effect of changing the amounts of emulsifiers in the TEDP formulations. The two factors, Cremophor EL (HLB = 14) and Tween 80 (HLB = 15) were selected according to previous study [13]. Cremphor and Tween 80, listed in Food Additives Permitted for Direct Addition to Food for Human Consumption in federal regulation of Food and Drug Administration [26], are commonly used in solid dispersion preparation. Their nonionic, surfactant features are due to their hydrophilic and hydrophobic moieties. In aqueous solutions, they form colloidal particles, spherical micelles with lipophilic interiors, when present above the critical micellar concentration. Because of their amphiphilic nature, they can reduce the interfacial tension of solvent systems and disperse various immiscible materials [27]. They are mainly used as solubilizing agents, particularly in aqueous preparations containing volatile oils, fat-soluble vitamins, and other hydrophobic substances [28]. 11

ACCEPTED MANUSCRIPT 3.1.2. Effect of progress factors on solubility In the solubility test (Fig 2), the solubility of lycopene in TE approached zero. The insolubility of lycopene in water, ethanol, and methanol as well as its solubility in

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chloroform, hexane, benzene, carbon disulfide, acetone, petroleum ether, and oil has

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been demonstrated [29]. The dripping pill preparation increased the solubility of lycopene; moreover, the model of factors was shown to be significant (p = 0.0041).

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Both Cremophor EL and Tween 80 exerted significant effects on solubility (p = 0.0049 and p = 0.0056 for Cremophor EL and Tween 80, respectively). In particular, condition F1, containing the highest levels of both factors, displayed the best

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solubility (189.82 g/mL) while F9, containing the lowest levels of both factors, exhibited the worst solubility (83.67 g/mL). These results corresponded to the

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masses of the two emulsifiers used.

3.1.3. Effect of progress factors on dissolution In the dissolution test, as expected, TE did not dissolve in either acidic or basic

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medium (Fig 3). The solid dispersion system, the dripping pill, prepared by the hot

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melt method with rapid cooling, might allow active ingredient formation in an amorphous phase; therefore, the release was elevated. Regarding dissolution at pH 1.2

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(Fig 3a), F1 showed an initial burst release (almost reaching a maximum within 20 min). Other formulations exhibited a relatively slow initial release (reaching a maximum within 40 min). Conditions F1, F2, F4, and F7 produced similar results at

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the 2-h time point; however, F7 was regarded as the highest (69.57%). F9 produced the lowest (3.75%) dissolution in acidic media. The model of factors was demonstrated to be nonsignificant (p = 0.2817). Neither Cremophor EL nor Tween 80 had significant effects (p = 0.2148 and p = 0.3461 for Cremophor EL and Tween 80, respectively). Regarding dissolution at pH 6.8 at 2 h (Fig 3b), most formulations completely released within 20 min. F1 and F4 produced similar results at the 2-h time point; however, F4 was regarded as the highest (64.44%). Similarly, F9 produced the lowest (4.17%) dissolution in basic media. The model of factors was demonstrated to be significant (p = 0.0208). Tween 80 played an effective role (p = 0.0105) while Cremophor EL did not (p = 0.1192). The dripping pill preparation in water-soluble carriers improved lycopene release. Two possible mechanisms of enhanced drug release from solid dispersions have been studied [30]. First, the solubility or dissolution rate of the drug can be 12

ACCEPTED MANUSCRIPT increased, which is firmly supported by our present observations. Second, the particle size and agglomeration can be reduced, though our data do not support this aspect.

3.1.4. Effect of progress factors on stability

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On the other hand, the stability of formulations during storage is another major

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concern. The instability renders formulations unsuitable as products. Under the accelerated storage conditions of the long-term stability test (Fig 4), preparations F5

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and F9 displayed similarly high stabilities; however, F5 retained the highest lycopene content after 6 months (64.26%). F1, with the highest content of both emulsifiers with attractive solubility and dissolution, was also the lease stable, retaining the lowest

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lycopene content after 6 months (5.36%). Adding too much emulsifier may have decreased the viscosity of the polymeric matrix, thereby facilitating more rapid

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recrystallization. In addition, Tween 80 may have superior stabilization properties to Cremophor EL [31], which is constant with our results. The model of factors was shown to be significant (p = 0.0766). Cremophor EL exerted significant effects on

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stability (p = 0.0401) while Tween 80 did not (p = 0.2769).

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Thus, when comprehensively considering all of the aforementioned responses (i.e., solubility, dissolution at pH 1.2 at 2 h, dissolution at pH 6.8 at 2 h, and stability

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at 6 months), a “biggest-is-best” criterion was applied for each response in the Design Expert software. F4 (i.e., the content of Cremophor EL and Tween 80 in the TEDP preparation were 10 and 15 g, respectively), with the highest desirability (0.87), was

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therefore selected as the optimal TEDP. A small residual between observed and expected values of the studied responses was obtained (Table 4).

3.2. Physicochemical characteristics 3.2.1. The content, weight variation, particle diameter, roundness, and disintegration time of tomato extract-loaded dripping pills The prepared formulations 0.37 ± 0.03 mg consisted of lycopene in each pill (Table 5). The TEDPs had a mean particle diameter of 3.55 ± 0.05 mm and a mean weight of 30.3 ± 0.7 mg with a small weight variation. All formulations were considered round in shape because the elongation factors were all >0.96. In addition, the disintegration time of an active ingredient was positively correlated with its BA [32]. The mean disintegration time of TEDPs was 287.8 ± 31.8 s. In particular, our data also showed that F9 had the longest disintegration time (360.5 s); however, it was unsuitable as an optimal TEDP candidate because it possessed poor solubility and 13

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3.2.2. Scanning electron microscopy SEM micrographs are shown in Fig 5. The dripping pills produced by the

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optimized formula were spherical, showing a smooth surface (Fig 5a–d). The natural

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form of TE exhibited a crystal (Fig 5e–h). Our results suggested that the dripping pill

3.2.3. Differential scanning calorimetry

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preparation dispersed the target ingredient and formed in an amorphous phase.

DSC provided information about melting, crystallization, decomposition, and physicochemical status of the active ingredient as well as its interaction with different

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excipients [33]. TE exhibited a endothermic peak at 172°C (Fig 6), corresponding to the melting point of lycopene [29]. PEG 6000 showed an endothermic peak at 63°C,

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corresponding to its melting point. In a physical mixture, two endothermic peaks remained, located at 63C and 175°C. In each prepared TEDP formulation, a single

6000 and no crystals existed.

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endothermic peak at 63°C indicated that the lycopene was fully dispersed in the PEG

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3.2.4. Fourier transform infrared spectroscopy To gain further insight into the molecular interactions, FT-IR measurements

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were taken (Fig 7). In TE, peaks were observed in the region of 1600–1700 cm−1, which can be assigned to lipids contained in tomatoes [34]. A sharp peak obtained at a frequency of 957 cm−1 can be attributed to the presence of the trans CH out-of-plane

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deformation vibration of lycopene [34]. No peak shift was observed in the final formulation. This observation supported the nonexistence of drug-excipient interactions in the prepared TEDPs.

3.3. In vivo study An animal study was conducted to harmonize the understanding of the in vitro and in vivo situations. The correlation between enhanced dissolution rates and absorption rates from solid dispersions was not entirely straightforward; therefore, the in vivo study was crucial. Mean plasma concentrations on the lycopene-time profile achieved after single administration of both the optimal TEDP (150 mg lycopene/kg rat) and TE (500 mg lycopene/kg rat) are shown in Fig 8. The mean pharmacokinetic parameters are shown in Table 6. Compared with rats receiving TE, the Cmax/dose, AUC0–48/dose, and AUC0–∞/dose of lycopene were significantly improved, increasing by 523.9% (p < 0.001), 543.4% (p < 0.001), and 495.4% (p < 0.001), respectively, in 14

ACCEPTED MANUSCRIPT rats receiving the optimal TEDP. The Tmax of lycopene was 8 h and 6 h for the TEDP and TE. The t1/2 of lycopene did not differ between the two groups (p > 0.05), indicating that the metabolism and excretion of rats were not altered by the TEDP.

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The relative BA was calculated to be 595.4%. These data provided strong evidence of

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an improvement in lycopene absorption under optimal TEDP conditions. Faisal et al. [9] developed a novel lipid-based solid dispersion prepared by a

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solvent evaporation method, which exhibited a 237% improvement of relative BA compared with Lycovit®, a commercial lycopene product encapsulated in gelatine beadlets. No differences in Tmax or t1/2 of lycopene were revealed. However, we could

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not compare the pharmaceutical parameters with their study owing to different animal species. Another study aimed to develop an osmotically controlled asymmetric

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membrane capsules for the delivery of solid dispersion of lycopene. The solid dispersion was also prepared by a solvent evaporation method [35]. Till date, there are two methods available for preparing solid solutions: the hot melt method and the

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solvent evaporation method [12]. The solvent evaporation method became popular in

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the 1970s and 1980s because it offered the possibility of forming solid dispersions from thermolabile substances. Recently, however, the ecological and subsequent

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economic aspects associated with the use of organic solvent-based methods have become more problematic. In our study, the proposed dripping pill preparation progresses in a simple, rapid, low-cost manner. Moreover, it provides a simple approach to dose adjustment.

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In practical application, there are numerous advantages of solid dispersion preparation; however, it usually required larger amounts of materials usage. As it is currently difficult to obtain the pure lycopene, we cannot utilize the pure compound as model. In addition, the hot melt method is only applicable to thermostable substances. Regarding lycopene, high heating (to 120°C and 140°C) promoted its isomerization from the all-trans form to cis-isomers and further oxidation of cis-isomers [36]; however, Hsu revealed that hot-break processing (92°C for 2 min) did not alter the lycopene content of tomato juice [37]. Similarly, the hot melt method employed in our preparation took only 2 min for TE, mixed thoroughly with melted PEG 6000 (85°C), to form TEDP by rapid cooling. The advantages of the dripping pill preparation compensated for the possible instability of lycopene. Nonetheless, the application of this platform to thermolabile substances remains debatable.

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ACCEPTED MANUSCRIPT 4. Conclusion The insolubility of lycopene is a major obstacle to its various therapeutic effects, which leads to poor BA. This is overcome by applying a solid dispersion technique,

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the dripping pill delivery system. Dripping pills, prepared by a straightforward hot

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melt method with rapid cooling and formed in an amorphous phase, improve upon the

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absorption of lycopene. Remarkable ameliorations of lycopene properties were demonstrated in both in vitro and in vivo experiments. The solubility and dissolution rates of lycopene were significantly increased. The optimal TEDP also possessed good stability. SEM and DSC measurement indicated that lycopene was dispersed in

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the matrix of polymer in amorphous state. The FT-IR observation supported the nonexistence of drug-excipient interactions. At last, the in vivo experiment was

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carried out to verify the usefulness of the formulae. The optimal TEDP exerted an approximate 6-fold improvement in lycopene absorption. The dripping pill delivery system is a promising and practical platform, thereby considered an effective

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approach to the enhancement of BA of a poorly soluble component.

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ACCEPTED MANUSCRIPT Acknowledgments Funding

for

this

study

was

provided

in

part

by

research

grant

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MOST103-2113-M-037-004-MY2 from the Ministry of Science and Technology,

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Taiwan.

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[24] P. Habeeb, N. Madhavan, K. Gladis, Y. Anitha, S. Mohammed, P. Raghunath,

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Formulation, Optimization And Evaluation Of Solid Dispersion Tablets Of Aceclofenac Using Kollidon 30, Int. J. Biopharm. 4 (2013) 10-17.

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[25] J. Shah, S. Vasanti, B. Anroop, H. Vyas, Enhancement of dissolution rate of valdecoxib by solid dispersions technique with PVP K 30 & PEG 4000: preparation and in vitro evaluation, J. Incl. Phenom. Macrocycl. Chem. 63 (2009) 69-75.

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[28] R.C. Rowe, P.J. Sheskey, M.E. Quinn, A.P. Association, P. Press, Handbook of pharmaceutical excipients, Pharmaceutical press, 2009.

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[31] D. Vidanovic, J. Milic Askrabic, M. Stankovic, V. Poprzen, Effects of nonionic surfactants on the physical stability of immunoglobulin G in aqueous solution during mechanical agitation, Die. Pharmazie. 58 (2003) 399-404. [32] H.N. Bhagavan, B.I. Wolkoff, Correlation between the disintegration time and the bioavailability of vitamin C tablets, Pharm. Res. 10 (1993) 239-242. [33] S.M. Abouelatta, A.A. Aboelwafa, R.M. Khalil, O.N. ElGazayerly, Floating lipid beads for the improvement of bioavailability of poorly soluble basic drugs: In-vitro optimization and in-vivo performance in humans, Eur. J. Pharm. Biopharm. 89 (2015) 82-92. [34] I. Bunghez, M. Raduly, S. Doncea, I. Aksahin, R. Ion, LYCOPENE DETERMINATION IN TOMATOES BY DIFFERENT SPECTRAL TECHNIQUES (UV-VIS, FTIR AND HPLC), Dig. J. Nanomater. Biostruct. 6 (2011). [35] N. Jain, R. Sareen, N. Mahindroo, K. Dhar, Development and optimization of 19

ACCEPTED MANUSCRIPT osmotically controlled asymmetric membrane capsules for delivery of solid dispersion of lycopene, Scientific. World. J. 2014 (2014). [36] J. Chen, J. Shi, S.J. Xue, Y. Ma, Comparison of lycopene stability in water-and

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oil-based food model systems under thermal-and light-irradiation treatments,

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LWT-Food. Sci. and Technol. 42 (2009) 740-747.

[37] K.-C. Hsu, Evaluation of processing qualities of tomato juice induced by thermal

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and pressure processing, LWT-Food. Sci. and Technol. 41 (2008) 450-459.

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ACCEPTED MANUSCRIPT Tables Table 1. 32 full factorial design layout: factors and responses. Level 0

A: Cremophor EL

15 g

10 g

B: Tween 80

15 g

10 g

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+

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Factor

− 5g 5g

Y1: Solubility (g/mL); Y2: dissolution at pH 1.2 at 2 h (%); Y3: dissolution at pH 6.8

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at 2 h (%); Y4: stability at 6 month (%)

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ACCEPTED MANUSCRIPT Table 2. The composition of the prepared formulations.

F9

30

260

15 15 15 10 10 10 5 5

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240 245 250 245 250 255 250 255

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30 30 30 30 30 30 30 30

5

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F1 F2 F3 F4 F5 F6 F7 F8

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Formulation Tomato extract (g) PEG 6000 (g) Cremophor EL (g) Tween 80 (g)

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TE

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Note: The total mass of prepared formulations is 300 g.

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15 10 5 15 10 5 15 10 5

ACCEPTED MANUSCRIPT Table 3. The observed responses of the prepared formulations produced with variable levels of independent factors. Factor

Response

Formulation B

Y2

Y3

Y4

F1

+

+

189.8 ± 4.01 68.65 ± 0.91 63.59 ± 1.55 5.36 ± 0.34

F2

+

0

136.46 ± 1.43 68.92 ± 1.47 47.42 ± 0.10 19.25 ± 0.21

F3

+

−

120.34 ± 2.59 65.46 ± 2.75 38.57 ± 0.92 41.83 ± 0.76

F4

0

+

170.13 ± 0.48 68.62 ± 1.01 64.44 ± 0.03 58.00 ± 0.63

F5

0

0

146.96 ± 1.27 42.36 ± 2.02 38.13 ± 0.26 64.26 ± 0.92

F6

0

−

98.92 ± 2.49 64.85 ± 2.88 26.70 ± 0.06 53.07 ± 0.27

F7

−

+

112.81 ± 5.26 69.57 ± 1.58 55.81 ± 0.96 40.93 ± 1.51

F8

−

0

97.94 ± 1.17 29.88 ± 0.70 36.77 ± 2.21 44.84 ± 0.05

F9

−

−

83.67 ± 1.06 3.75 ± 2.05

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Y1

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A

4.17 ± 2.16 63.26 ± 1.72

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Data are expressed as the mean ± standard deviation. A: Cremophor EL; B: Tween 80; Y1: Solubility (g/mL); Y2: dissolution at pH 1.2 at 2

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h (%); Y3: dissolution at pH 6.8 at 2 h (%); Y4: stability at 6 month (%)

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ACCEPTED MANUSCRIPT Table 4. The observed and predicted levels for optimized formulation. Optimized level

A: Cremophor EL

10 g

B: Tween 80

15 g

Response

Expected

Y1: Solubility (g/mL)

167.65

Y2: Dissolution at pH 1.2 at 2 h (%)

68.95

68.62

0.33

Y3: Dissolution at pH 6.8 at 2 h (%)

62.64

64.44

−1.8

Y4: Stability at 6 month (%)

49.78

58.00

−8.22

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Residual

170.13

−2.48

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Observed

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Residual = expected value–observed value

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ACCEPTED MANUSCRIPT Table 5. Characteristics of the prepared formulations: content, weight variation, particle diameter, roundness, and disintegration. Content

Weight

Particle

(mg/pill)

(mg)

diameter

Elongation

Disintegration time (s)

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Formulation

0.39 ± 0.02

30.8 ± 0.7

3.51 ± 0.06

0.97 ± 0.02

258.8 ± 12.3

F2

0.36 ± 0.02

31.3 ± 0.7

3.52 ± 0.07

0.96 ± 0.02

262.0 ± 7.8

F3

0.39 ± 0.01

30.8 ± 1.5

3.51 ± 0.09

0.96 ± 0.02

273.7 ± 20.3

F4

0.42 ± 0.01

29.9 ± 1.0

3.59 ± 0.09

0.96 ± 0.02

277.8 ± 11.9

F5

0.35 ± 0.02

29.5 ± 1.4

3.48 ± 0.12

0.96 ± 0.02

275.2 ± 33.4

F6

0.37 ± 0.02

30.4 ± 1.3

3.63 ± 0.07

0.97 ± 0.02

312.0 ± 6.2

F7

0.39 ± 0.01

29.2 ± 1.2

3.56 ± 0.06

0.96 ± 0.01

274.3 ± 17.1

F8

0.32 ± 0.02

30.6 ± 1.6

3.56 ± 0.08

0.96 ± 0.02

295.7 ± 20.1

F9

0.35 ± 0.02

30.5 ± 1.5

3.56 ± 0.09

0.97 ± 0.02

360.5 ± 43.9

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F1

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(mm)

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Data are expressed as the mean ± standard deviation.

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ACCEPTED MANUSCRIPT Table 6. Pharmacokinetic parameters of optimal TEDP and TE. Parameter

TEDP

TE

(150 mg lycopene/kg rat) (500 mg lycopene/kg rat) AUC0–48 (μg·h/mL)

33.2 ± 1.4

AUC0–∞ (μg·h/mL)

38.7 ± 1.3

1.17 ± 0.02

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1.46 ± 0.03

25.8 ± 1.1

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Cmax (μg/mL)

32.5 ± 2.7

8

t1/2 (h)

16.5 ± 2.3

20.7 ± 3.6

14.6 ± 0.3***

2.34 ± 0.0

***

51.6 ± 2.2

Cmax/Dose (ng rat/mL)

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Tmax (h)

6

332 ± 13.5

AUC0–∞/Dose (ng rat·h/mL)

387 ± 13.5***

65 ± 5.4

595.4

-

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AUC0–48/Dose (ng rat·h/mL)

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Relative BA (%)

Data are expressed as the mean ± standard deviation. indicates significant difference compared to the TE (p < 0.001). 0-∞ T DP 0-∞ T

T DP T

× 100%.

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Relative BA =

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***

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ACCEPTED MANUSCRIPT Figure captions Fig. 1. Dripping pill machine.

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Fig. 2. Solubility profiles of 9 formulae of TEDPs and TE. Data are expressed as the

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mean ± standard deviation.

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Fig. 3. In vitro dissolution profiles of 9 formulae of TEDPs and TE in (a) pH 1.2 acidic medium and (b) pH 6.8 basic medium. Data are expressed as the mean ± standard deviation.

Fig. 4. Stability profiles of 9 formulae of TEDPs and TE. Data are expressed as the

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mean ± standard deviation.

Fig. 5. Scanning electron micrographs of optimal TEDP and TE. (a) optimal TEDP at

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30× magnification, (b) optimal TEDP at 1000× magnification, (c) optimal TEDP at 3000× magnification, (d) optimal TEDP at 5000× magnification, (e) TE at 30×

TE at 5000× magnification.

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magnification, (f) TE at 1000× magnification, (g) TE at 3000× magnification, and (h)

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Fig. 6. Differential scanning calorimetry thermograms of 9 formulae of TEDPs, TE, PEG 6000, and physical mixture.

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Fig. 7. Fourier transform infrared spectroscopy spectra of TE, PEG 6000, and 9 formulae of TEDPs

Fig. 8. Plasma lycopene concentration-time curve of optimal TEDP and TE. Data are

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expressed as the mean ± standard deviation.

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Fig. 2

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Fig. 3

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Fig. 4

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Fig. 5

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Fig. 6

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Fig. 7

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Fig. 8

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Graphical abstract

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ACCEPTED MANUSCRIPT

Highlights

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 

We developed a tomato extract-loaded dripping pill delivery system. The optimal formulae led to amelioration of solubility and dissolution of lycopene. The optimal formulae was confirmed to be formed in an amorphous state. The optimal formulae exhibited a 6-fold improvement in bioavailability of lycopene.

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 

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