AMPB powder was purchased from Tokyo Chemical Industry Co., Ltd, Japan, Starch-based food thickener granules was Toromerin, purchased from NUTRI Co., Ltd, Japan, xantham-based food thickener was Tsururinko Quickly, Clinico Co., Ltd, Japan, swallowing-aid jelly was Ryukakusan Co., Ltd, Japan.

Starch-based food thickener granules (Toromerin, purchased from NUTRI Co., Ltd, Japan) were mixed well with purified water and allowed to stand for 2 minutes before preparing 2.4, 4.7 and 7.1 (w/v) % solutions according to the method described in the package insert. AMPB (6.93 mg) was mixed into each of these solutions. Xantham-based food thickener (Tsururinko Quickly, Clinico Co., Ltd, Japan) was mixed well with purified water and allowed to stand for 2 minutes before preparing 1.0, 2.0 and 3.0 (w/v) % solutions according to the method described in the package insert. AMPB (6.93 mg) was mixed into each of these solutions. As reference, 6.93 mg of AMPB was mixed with (wrapped in) 5 g swallowing-aid jelly (Ryukakusan Co., Ltd, Japan).

The thickness of the two kinds of food thickener was performed according to The Japanese Society of Dysphagia Rehabilitation recommendations [10]. The samples tested were: starch-based food thickener solutions (2.4, 4.7 and 7.1 (w/v) %), xanthan-based food thickener solutions (1.0, 2.0 and 3.0 (w/v) %) and 5 g swallowing-aid jelly. The test was performed as follows: an empty cylindrical ring (30 mm diameter) was placed in the centre of concentric circles drawn on a horizontal sheet. A sample of food thickener or swallowing-aid jelly (20 mL) was poured into the inside of the ring and held there for 30 seconds. The ring was then lifted vertically and the spread of the sample on the sheet with the concentric circles was evaluated after 30 seconds on a 6-point scale. The distance (mm) was determined as a mean value ± S.D. (n = 3). The standard criteria for high, medium and low concentrations were 32 - 30, 36 - 32 and 43 - 36 mm, respectively.

TPA was carried out to evaluate the physical properties of the food thickener solutions using TA. XT plusC texture analyzer (Stable Micro Systems, Surrey, UK) [11]. The samples tested were: starch-based food thickener solutions (2.4, 4.7 and 7.1 (w/v) %), xanthan-based food thickener solutions (1.0, 2.0 and 3.0 (w/v) %) and 5 g swallowing-aid jelly. The TPA was performed according to the method used to determine the acceptability criteria for persons with difficulty swallowing, as published by the Japanese Consumers Affairs Agency [12]. The texture parameters were hardness, adhesiveness and cohesiveness. Textural properties were analyzed in two sequential “two-bite” compression tests with a cylindrical-shaped probe, diameter 20 mm, height 100 mm. All samples were compressed to 70% of their original length. The test speed was set to 10 mm/s. The values of hardness, adhesiveness and cohesiveness were calculated from the obtained profiles using software provided by Stable Micro Systems. The stress-strain curves for the two types of thickener and the swallowing-aid jelly were drawn using the data from the TA. XT plusC texture analyzer.

Aliquots of 6.93 mg powdered AMPB were precisely weighed and mixed with the starch-based food thickener solutions (2.4, 4.7 and 7.1 (w/v) %), xanthan gum-based food thickener solutions (1.0, 2.0 and 3.0 (w/v) %), and 5 g swallowing -aid jelly for 30 seconds. Portions weighing 5.0 g of each of the above mixtures, were wrapped in nylon mesh (N-No.255HD; material: nylon66, nylon fiber width 43 μm, mesh opening size 57 μm) and dipped in 20 mL water contained in a 50 mL glass beaker. A small bar magnet (4 × 10 mm) which rotated at 150 rev/min, was put in the bottom of the beaker. The water temperature was kept at 37˚C. Samples (20 mL) were taken from the mixture in the beaker after 5, 15, 30 and 60 seconds and the concentration of AMP in the dissolved medium was determined by HPLC (LC-2010C, Shimadzu Corp., Japan). The HPLC method was based on the Japanese Pharmacopoeia and a previous report [13]. An integrator (LC solution; Shimadzu Corp., Japan) and reverse-phase column (CAPCELL PAK C18 UG120 S5: 150 × 4.6 mm i.d.; OSAKA SODA Co., Ltd, Japan) were used for HPLC. The column temperature was kept at 25˚C. The mobile phase composition was methanol: potassium dihydrogen phosphate (41 → 10,000) (13:7, v/v) and the flow rate was 1.0 mL/min. Run time was 10 minutes. The ultraviolet detection wavelength was set at 237 nm. First-order dissolution rate constants for the food thickeners and swallowing-aid jelly were calculated from the dissolved AMP concentrations.

Taste sensor measurements of the dissolved solutions were performed as follows. The bitterness of the dissolved solutions was measured by the change in the membrane electric potential caused by adsorption (CPA) using taste sensor SA501 (Intelligent Sensor Technology Inc., Atsugi, Japan). In this study, the CPA of membrane AN0 was used, which is suitable for the evaluation of bitterness of basic substances. The taste sensor measurements were performed as described in previous articles [13] [14] [15] [16] [17].

In vitro dissolution tests of AMP from two types of thickener and swallowing-aid jelly were carried out using a conventional dissolution tester (PJ-62N, Riken’s, Miyamoto Riken Ind., Co., Ltd., Osaka, Japan) according to the Japanese Pharmacopoeia, 17th edn. The test was done using the paddle method at 37˚C and 50 rev/min using 900 mL of both the first fluid (pH 1.2) and/or diluted Mcllvaine buffer (pH 4.5) [18]. At various time intervals, 2 mL aliquots were withdrawn and replaced by the same volume of fresh medium. The concentration of AMP was determined by HPLC as described above. First-order dissolution rates from the food thickeners and swallowing-aid jelly were calculated from the dissolved AMP concentrations.

Regarding to LST evaluation of two kinds of food thickener solution and swallow ing-aid jelly, only 4.7% solution of starch-based food thickener fell within these criteria. This suggests that the LST criteria are determined based on the use of xanthan gum-based thickener solutions [19] [20].

The data of TPA suggest that patients may need considerable powers of chewing and swallowing when 7.1 (w/v) % starch-based food thickeners are used and that more dilute starch-based food thickeners may be more suitable for patients with

Data are presented as mean (n = 3) ± S.D. *p < 0.05, **p < 0.01 vs. Starch-based thickener 2.4 (w/v) % (Tukey’s test) or vs. Xanthan gum-based thickener 1.0 (w/v) % (Tukey’s test).

low powers of chewing and swallowing. The cohesiveness of swallowing-aid jelly was the lowest of all the thickeners. Swallowing-aid jelly is considered to have physical properties that make it easy to swallow for dysphasia.

In related to in vitro conventional dissolution test of AMPB powder mixed with two types of thickener solution and swallowing-aid jelly, xanthan gum-based thickeners show very low dissolution of AMP, approximating zero order dissolution, as shown in Figure 1(c) and Table 3. This may be caused by the xanthan-based thickener acting as a protective wetting agent for AMPB powder, as reported previously [21]. The AMP dissolution % from AMPB powder mixed with the starch-based thickener solutions and their first-order dissolution rate constants decreased as the concentration of starch-based thickener increased. This suggests that the hydrophilic starch molecular network might work as a barrier to diffusion of AMP into the medium. Whereas the fast dissolution and high dissolved % of AMP were obtained in AMPB powder mixed with swallowing-aid jelly. This is probably due to the low cohesiveness of swallowing-aid jelly, as shown in Table 2.

Regarding the taste sensor data shown Figure 2. The taste sensor value ranking coincided with the ranking of dissolution concentrations of AMP, as shown in Figure 1. This easily expects that much dissolution AMP from each of food thickener or swallow-aid jelly, give rise to enhance the bitterness in oral cavity. None of the three concentrations of xanthan gum-based thickeners (1.0, 2.0 and 3.0 (w/v) % showed any bitterness for the first 60 seconds. The 7.1 (w/v) % starch-based thickener solution did show any bitterness suppression effect for at least 30 seconds, while swallowing-aid jelly and 2.4 and 4.7 (w/v) % starch-based thickener solutions can be expected to show bitterness inhibition in the oral cavity.

In addition, the lack of AMP dissolution from xanthan gum-based thickeners under conditions mimicking the oral cavity may have a risk of less dissolution under gastric conditions, thereby decreasing the bioavailability of the drug.

In related to dissolution test of AMPB powder mixed with food thickeners, as shown in Figure 3, the mixture of AMPB with starch-based thickener or swallowing-aid jelly showed 100% dissolution, and therefore seem not limit bioavailability in patients. The smallest dissolution rate constant was observed for xanthan gum-based thickener, as shown in Table 4. As mentioned above, this could be due to a protective effect of the xanthan gum-based thickener’s act on the AMPB powder. Alternatively, the xanthan gum-based thickener may act as a diffusion barrier limiting AMP dissolution into the medium. It has been reported that xanthan gum adsorbs various drugs [21] [22]. It is postulated that the reason for this is the excellent acid resistance, heat resistance and drought resistance of the gum [23] [24], and the intermolecular associations of xanthan gum wrapping around the drug and preventing it from getting wet, even though xanthan gum-based thickeners are said safety and their effect in modifying bolus rheology in the therapeutic medical for dysphagia [25].