2023 Volume 46 Issue 12 Pages 1805-1809
Transdermal scopolamine applied to the postauricular area is used to treat drooling. We investigated the duration of action of scopolamine ointment and the effect of the application site on drug efficacy and concentration in the salivary glands of rats. Scopolamine ointment was applied to the skin over the salivary glands (SSG) and back (SB). Saliva volume was measured after intraperitoneal administration of pilocarpine. Blood and salivary glands were collected after scopolamine ointment application, and scopolamine concentrations in the plasma and salivary glands were measured. Saliva volume after application in the SSG group was significantly lower at all time points than in the non-treated group, and the change in saliva volume in the SSG group was greater than that in the SB group at all time points. This suggests that applying scopolamine ointment to the SSG strongly suppresses salivary secretion. Scopolamine concentration in the salivary glands of the SSG group was significantly higher at 9 h. The change in the efficacy of scopolamine ointment depending on the application site was due to the difference in transfer to the salivary glands. Transdermal administration of scopolamine to the skin over the salivary glands may have high efficiency in treating drooling.
Drooling is caused by the hypersecretion of saliva due to oral diseases, adverse drug reactions, and deterioration of oral actions and swallowing function in diseases.1–3) In particular, drooling is a common symptom in patients with cerebral palsy (CP) and neuromuscular diseases, and occurs in 22–50% of patients with amyotrophic lateral sclerosis,4,5) 10–81% of patients with Parkinson's disease,6–8) and 22–40% of pediatric patients with CP.9,10) Drooling not only reduces the QOL of patients owing to discomfort and poor appearance11–13) but can also lead to death as a result of aspiration pneumonia caused by the inflow of saliva into the trachea.2,14–16)
Therapeutic strategies for drooling involve drug therapy with anticholinergics and botulinum toxin, radiation, and surgery.1,17–19) Oral anticholinergic drugs are commonly used as an initial treatment, but some patients are unable to continue taking these drugs because of systemic adverse effects such as constipation,20,21) urinary retention,21,22) and hallucinations.21,23)
Scopolamine patches, which are transdermal scopolamine dosage forms developed as prophylactic antiemetics, have been used for drooling, and their efficacy has been reported.22,24,25) In Japan, scopolamine ointments, which are hospital preparations, are used as alternatives, because scopolamine patches are not approved and are not commercially available.26–28) Scopolamine patches and ointments are applied to the posterior auricle, which is the most absorbent area,29) and the patches are used once every 3 d and the ointments once or twice a day.26–28) However, the duration of action of scopolamine ointment has not yet been scientifically verified. Moreover, it has been speculated in Japan that the application area of these drugs is involved in the manifestation of drug efficacy and these drugs act locally on the salivary glands because the auricular skin is close to the salivary glands, which secrete saliva. Therefore, scopolamine ointment is sometimes applied to the skin near the salivary glands.27) However, the relationship between the application site of transdermal scopolamine and therapeutic efficacy has not been investigated.
The purpose of this study was to verify the duration of action of scopolamine ointment, which is one of the transdermal scopolamine dosage forms, and to investigate the effect of application site on drug efficacy and drug concentration in salivary glands in rats.
Adult male Sprague–Dawley rats (200–300 g) were obtained from Japan SLC (Shizuoka, Japan). The rats were housed in the institutional animal facility in a controlled environment (temperature 25 ± 1 °C and 12 h light/dark cycle) with access to food and water ad libitum and were acclimatized to the study environment for at least 1 week. Animals were maintained and experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals at Kanazawa University. This study is reported in accordance with the ARRIVE Statement for animal studies and was approved by the Committee on Animal Experimentation of Kanazawa University (Kanazawa, Japan; Approval No. AP-204178).
Preparation and Administration of DrugsScopolamine hydrobromide n-hydrate was purchased from Nacalai Tesque (Kyoto, Japan) and hydrophilic cream was purchased from NIKKO Pharmaceutical (Gifu, Japan). The scopolamine ointment contained 5% scopolamine hydrobromide n-hydrate in a hydrophilic cream, the concentration of which was the same as that used clinically.
The rats were fasted for a minimum of 18 h with ad libitum access to water. An adhesive plaster (Injection pad M size: 13 × 13 mm [NICHIBAN, Tokyo, Japan]) containing 0.02 g of 5% scopolamine ointment was applied on the skin above salivary glands (SSG) or the back (SB) after completely removing hair using a razor. In addition, the peeling off of the scopolamine ointment was prevented by covering the site of application with tape. In the intravenous administration (IV) group, scopolamine hydrobromide n-hydrate was dissolved in 0.9% (w/v) NaCl and administered intravenously at a volume of 0.5 mg/kg body weight.
Measurement of Saliva VolumeIn the non-treated (NT), SSG, and SB groups (n = 9), saliva volume was measured before application and 3, 6, 9, and 24 h after application. In the IV group (n = 9), the measurements were performed before administration and 3, 6, and 9 h after administration. All the experiments were started between a.m. 8 and 9 o’clock to eliminate the influence of diurnal variations in saliva secretion. At each measurement time point, after anesthesia with an intraperitoneal injection of medetomidine hydrochloride (0.15 mg/kg body weight), midazolam (2.0 mg/kg body weight), and butorphanol (2.5 mg/kg body weight), the rats were placed at a 45-degree angle, with their heads down and the ventral surface facing upwards. The lower jaw was lifted upward to open the mouth, and a preweighed dry swab was placed in the mouth and kept for 15 min for saliva absorption. Saliva was collected for 30 min starting 10 min after pilocarpine injection (1.0 mg/kg body weight). After saliva collection, the weight of the wet swab was determined, and the difference between the wet and dry weights was calculated to obtain the weight of saliva produced.
Measurement of Drug Concentration in the Blood and TissueAfter anesthesia with an intraperitoneal injection of medetomidine hydrochloride, midazolam, and butorphanol, blood and submandibular glands were collected at 3, 6, 9, and 24 h after ointment application (n = 8 at each collect point). Blood was drawn from the jugular vein and centrifuged immediately, and the plasma was separated and stored at −30 °C until use. Submandibular gland tissues were collected, washed with chilled normal saline below 4 °C, and stored at −30 °C before measurement. The collected tissue was homogenized in phosphate-buffered saline (PBS) (tissue : PBS = 1 : 5). The homogenate was centrifuged at 15000 × g for 15 min at 4 °C and the supernatant was used for determination of scopolamine concentration in the tissue. The drug concentration was determined using an LC-MS-8050 triple quadrupole LC-MS/MS (Shimadzu, Kyoto, Japan) coupled to an LC-30A system (Shimadzu). Chromatography was performed on a CAPCELL PAK C18 MG III (ID 2.0 × 50 mm; Osaka Soda, Osaka, Japan) at 40 °C using step-gradient elution (flow rate, 0.4 mL/min) as follows: 0 to 1.0 min, 95% A/5% B; 1.0 to 2.0 min, 95% A/5% B to 80% A/20% B; 2.0 to 2.5 min, 80% A/20% B to 20% A/80% B; 2.5 to 5.5 min, 20% A/80% B; 5.5 to 6.0 min, 20% A/80% B to 95% A/5% B, and 6.0 to 8.0 min, 95% A/5% B (A, water containing 0.1% formic acid; B, acetonitrile containing 0.1% formic acid). D3-scopolamine was used as an internal standard. The mass numbers of the molecular and product ions for each compound were as follows: scopolamine (304.1/138.1, CE −22 eV) and d3-scopolamine (307.1/141.1, CE −17 eV). Lab Solution software (version 5.89) was used for data manipulation. The detection limit for each compound was 1 nM and values below the limit of measurement were excluded from the analysis. The tissue-to-plasma partition coefficients in the salivary glands (Kp) were determined using the following formula:
 |
Data are shown as mean ± standard deviation (S.D.). The sample size was determined based on a pilot study. The sample size for the measurement of saliva volume was nine (n = 9) and that for the measurement of drug concentration was eight (n = 8). Statistical analysis was performed using the Mann–Whitney U test, Friedman test, and Kruskal–Wallis test adjusted with Bonferroni correction. Differences with p < 0.05 were considered significant. All statistical analyses were performed using the SPSS version 25 software (IBM, Armonk, NY, U.S.A.).
Figure 1 shows changes in saliva volume measured before and 3, 6, 9, and 24 h after application of scopolamine ointment in the NT and SSG groups, and before and 3, 6, and 9 h after intravenous administration of scopolamine in the IV group. In the NT group, the saliva volume before and at any time point after the application was not different. The saliva volume decreased significantly at 6 and 9 h (6 h: 1.8 ± 2.2 mg [p = 0.01]; 9 h: 2.1 ± 1.9 mg [p < 0.01]) after application compared with that before application (263.0 ± 166.4 mg) in the SSG group. In the IV group, the saliva volume decreased significantly at 3 h after administration (1.4 ± 1.3 mg) compared with that before administration (271.8 ± 146.7 mg [p < 0.01]), and the difference was negligible at 9 h (272.7 ± 126.6 mg).
Circles, triangles, and squares represent SSG, IV, and NT groups, respectively. Each point is the mean ± S.D. * p < 0.05 for each point versus before application and administration in the SSG group. †p < 0.05 for each point versus before application and administration in the IV group. IV, scopolamine administered intravenously; NT, non-treated; SSG, scopolamine ointment applied to the skin over the salivary glands.
Changes in salivary volume measured before and after the application of scopolamine ointment in the NT, SSG, and SB groups are shown in Fig. 2. The saliva volume decreased significantly at 6 and 9 h in the SSG group (pre: 263.0 ± 166.4 mg; 6 h: 1.8 ± 2.2 mg; 9 h: 2.1 ± 1.9 mg) and at 6 h in the SB group compared with that before application (pre: 363.4 ± 183.7 mg; 6 h: 106.9 ± 121.1 mg). On the contrary, the saliva volume in the SSG group was significantly lower than that in the NT group at all time points from 3 to 24 h after application, whereas in the SB group, it was significantly lower only at 6 and 24 h. No significant difference was observed in the saliva volume between the SSG and SB groups.
Closed circles, open circles, and squares represent SSG, SB, and NT groups, respectively. Each point is mean ± S.D. * p < 0.05 for each point versus before application. †p < 0.05 for SSG or SB group versus NT group. NT, non-treated; SB, scopolamine ointment applied to the skin on the back; SSG, scopolamine ointment applied to the skin over the salivary glands.
Figure 3 shows the changes in drug concentrations in the plasma and salivary gland tissue after the administration of scopolamine in the SSG and SB groups. There was no difference in the plasma scopolamine concentration between the SSG and SB groups at any time point. The scopolamine concentration in salivary glands tended to be higher in the SSG group than in the SB group at 6 h (SSG: 309.5 ± 665.5 ng/g; SB: 9.1 ± 8.2 ng/g, p = 0.09) and was significantly higher at 9 h (SSG: 27.9 ± 33.1 ng/g; SB: 1.2 ± 0.6 ng/g, p < 0.01). Kp values in the SSG group (3 h: 44.6 ± 98.3; 6 h: 47.0 ± 84.7, 9 h: 5.0 ± 4.9) tended to be higher than those in the SB group at 6 and 9 h (3 h: 1.7 ± 1.3; 6 h: 2.5 ± 2.2; 9 h: 1.6 ± 0.8) (Table 1).
(A) Scopolamine concentration in the plasma. (SSG: 3 h, n = 5; 6 h, n = 6; 9 h, n = 7; 24 h, n = 7. SB: 3 h, n = 6; 6 h, n = 5; 9 h, n = 3; 24 h, n = 5). (B) Scopolamine concentration in the salivary glands (SSG: 3 h, n = 7; 6 h, n = 8; 9 h, n = 8; 24 h, n = 7. SB: 3 h, n = 5; 6 h, n = 5; 9 h, n = 4; 24 h, n = 6). The closed and open circles represent the SSG and SB groups, respectively. Each point is mean ± S.D. * p < 0.05 for SSG group versus SB group. SB, scopolamine ointment applied to the skin on the back; SSG, scopolamine ointment applied to the skin over the salivary glands.
| 3 h | 6 h | 9 h | 24 h | |
|---|---|---|---|---|
| SSG | 44.6 ± 98.3 | 47.0 ± 84.8 | 5.1 ± 4.9 | 3.6 ± 2.1 |
| SB | 1.7 ± 1.3 | 2.5 ± 2.2 | 1.6 ± 0.8 | 3.0 ± 3.0 |
| p-Value | 0.69 | 0.11 | 0.18 | 0.76 |
Data are shown as mean ± standard deviation. SB, scopolamine ointment applied to the skin on the back; SSG, scopolamine ointment applied to the skin over the salivary glands.
Most studies on the use of transdermal scopolamine for drooling have focused on its efficacy.26–28) There has been almost no verification of the effect of application site for transdermal scopolamine dosage forms. Furthermore, information on the duration of action of scopolamine ointment used in hospital preparations in Japan is insufficient. In this study, we investigated the duration of action of scopolamine ointment and the effect of its application site on its efficacy and drug concentration in rats. Our results showed that scopolamine ointment had a long-lasting effect, which was strengthened by applying it to the skin over the salivary glands.
Furthermore, the effect of scopolamine ointment was sustained for a longer duration than that of its intravenous administration; however, the effect was shorter than that of the scopolamine patch and did not last for 1 d. In a previous clinical study in healthy subjects, scopolamine ointment was effective for up to 6 h after application.29) In the same report, saliva volume tended to decrease 9–24 h after the application of scopolamine ointment; however, there was no significant difference before and after application.29) Thus, the results of the report are similar to those of the present study. In contrast, another study reported that the scopolamine patch reduces drooling for up to 72 h after application.24) Therefore, in a future study of transdermal scopolamine forms, the difference in the duration of the effect between the scopolamine ointment and patch should be considered.
A scopolamine patch, a transdermal agent, was applied behind the auricle, and scopolamine ointment was applied to the same area. The postauricular area was chosen as the patch site because it has the highest skin permeability for scopolamine; the permeability of the back and chest skin was half that of the postauricular area, while that of the thigh skin was 10–20 times lower.30) However, because the postauricular area is close to the salivary glands, which are the tissues that secrete saliva, it is inferred in Japan that scopolamine ointment has local effects on the salivary glands, and there have been reports on application of scopolamine ointment to the skin near the salivary glands.27) To substantiate this hypothesis, we investigated the effect of scopolamine ointment application site on drug efficacy and drug concentration in rats. Scopolamine ointment reduced the salivary volume in rats when applied to the skin above the salivary glands and back (Fig. 2). Saliva volume in the rats treated with the formulation applied to the back was significantly lower only at 6 and 24 h compared with that in untreated rats; however, the salivary volume in rats treated with the formulation applied to the skin over the salivary gland was significantly lower at all time points after application. Moreover, the change in saliva volume when the formulation was applied to the skin over the salivary glands was greater than that when it was applied to the back skin at all time points. These results suggest that applying scopolamine ointment to areas over the salivary glands leads to a stronger salivary-lowering effect than that achieved with application over other areas. However, there was no significant difference in the saliva volume between the SSG and SB groups at all time points. We believe that this was due to the fact that almost no saliva was secreted because of the effect of scopolamine ointment on rats in the SSG groups, and the saliva-lowering effect peaked out.
To investigate the cause of the differences in drug efficacy depending on the application site of the scopolamine ointment, we measured the concentrations of scopolamine in the blood and salivary glands after its application. Plasma concentrations of scopolamine tended to be higher in the SSG group than in the SB group; however, these concentrations were not statistically different. The skin permeability of drugs is affected by skin thickness.31) In rats, the skin on the back is the thickest; however, the difference in skin thickness at different body sites is smaller than in humans. Therefore, we surmised that the blood concentration of scopolamine did not depend on the application site in this study. On the contrary, concentrations in the salivary glands were higher in rats where scopolamine was applied to the skin over the salivary glands compared with that when it was applied to the skin on the back (Fig. 3). Furthermore, the Kp values of the salivary gland tissues were higher when the ointment was applied to the skin over the salivary glands than when applied to the back skin (Table 1). However, no significant differences were observed because of the large variability in the data at any time point. We considered that the reason for this variation was that the salivary glands of the rats were small, and the application site and salivary glands tended to be misaligned. Moreover, we considered the possibility that variation in scopolamine concentration in salivary glands could be related to interindividual differences in skin permeability and the effects of skin damage.32) Our results suggest that scopolamine applied to the skin over the salivary glands reaches the salivary glands without passing through the blood. Pilocarpine, a muscarinic agonist, is known to act locally on the salivary glands through the oral mucosa when administered intraorally.33) Sprays,34) gargles,35,36) and oral mucosa patches37) of pilocarpine have been developed. In this study, we demonstrate that scopolamine can be applied topically to the skin over the salivary glands and suggest a new route for reaching the salivary glands via transdermal administration. Application to the skin over the salivary glands may have both beneficial effects and reduced systemic side effects.
Nevertheless, our study has some limitations. First, our experiments were performed in rats, not in humans. The muscarinic receptor subtype of salivary glands is similar in rats and humans, but the structures of the salivary glands differ. Our present findings may differ in humans and thus further studies are needed to verify this. Second, there was a wide variability in the results in rats in which the ointment was applied to the skin over the salivary glands. As mentioned above, we consider that this was caused by a misalignment between the application site and the salivary glands because of the small salivary glands of the rats. In future studies, we plan to reduce this variation by adjusting the application area and concentration.
In conclusion, we found that scopolamine ointment, which is one of the transdermal scopolamine dosage forms, persistently inhibited salivary secretion when applied to the skin over the salivary glands of rats. Furthermore, application of the scopolamine ointment to the skin over the salivary glands enhanced its effect, and the scopolamine concentration in the salivary glands increased without affecting the blood scopolamine concentration. Our study suggests that the application of scopolamine to the skin over the salivary glands increases the transferability of scopolamine to the salivary gland tissue and suppresses saliva secretion more effectively. Transdermal administration of scopolamine to the skin over the salivary glands may be a treatment option for salivation with combined efficacy and safety.
This work was supported by JSPS KAKENHI, Grant Numbers JP20K16072 and JP23K06232.
The authors declare no conflict of interest.
