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Effects of Dose, pH, and Osmolarity on Nasal Absorption of Secretin in Rats II: Histological Aspects of the Nasal Mucosa in Relation to the Absorption Variation Due to the Effects of pH and Osmolarity
Effects of Dose, pH, and Osmolarity on Nasal Absorption of Secretin in Rats II: Histological Aspects of the Nasal Mucosa in Relation to the Absorption Variation Due to the Effects of pH and Osmolarity TAKAYUKIOHWAKI‘, HIDENOBU ANDO, FUMIOKAKIMOTO, KEIZOUUESUGI, YASUOMIYAKE, AND MASANORI OVANO
SUM10 WATANABE,
Received December 2, 1986, from the Research Laboratories of Pharmaceutical Development, Eisai Co., Ltd., Kawashima-cho, Hashima-gun, Accepted for publication June 18, 1987 Gifu 483, Japan. -.
~.-
-
-
Abstract 9Nasal absorptionof secretin in rats was enhanced in an acid solution, and the maximum absorption was observed at a sodium chloride solution molarity of 0.462. In order to examine reasons for the variation of absorbability caused by the change of pH and osmolarity in secretin preparations, both a pretreatment study, in which the nasal rnucosa was treated with placeboprior to the administrationof a secretin preparation, and a histological study were conducted in rats. The nasal absorption of secretin was determined by measuring the increased secretion of pancreatic juice. Similar profiles of nasal absorption, both after intranasal administrationof secretin preparations and as a result of pretreatment effects, were obtained in studies of the effects of pH and osmolarity. However, in the pH-effect study, the absorptionwith the use of active preparations was observed to be significantly larger than that with the pretreatment effect below a pH of 4.79, and significantlysmaller than that with the pretreatment effect at a pH of 7 to 8. The results of histological studies revealed structural changes of the epithelial cells of the nasal mucosa at pH 2.94, and shrinkage of epithelial cells was observed at a sodium chloride solution molarity of 0.462. - - ~- -. -
In a previous study,’ a n investigation on t h e intranasal administration o f secretin, a hormone secreted in the digest i v e tract and used clinically for t h e treatment of duodenal ulcer,”s was conducted in rats to reveal the feasibility of a nasal dosage form o f secretin. The absorbability o f secretin through the nasal mucosa was observed to be one-tenth of the bioavailability after intravenous administration, and to be affected by the pH and osmolarity o f secretin preparations. A number o f investigations o n the nasal absorption of peptides have been reported,’o-l* but few reports have attempted to elucidate w h y the variation of nasal absorption o f peptides was brought about by pH and osmolarity. For the present paper, by conducting the pretreatment study and histological study o f nasal mucosa, we examined reasons w h y the nasal absorption o f secretin decreased w i t h increasing pH and w h y the m a x i m u m absorption o f secretin
pretreatment of the nasal mucosa was prepared with the same buffer solution, containing no secretin, and physiological saline (0.154 M NaCl, pH 6.4) containing 10 CHR unitd50 pL of secretin was prepared for intranasal administration after pretreatment. To examine the effect of osmolarity on nasal absorption, secretin, adjusted to a concentration of 10 CHR units/50 pL,was dissolved in sodium chloride solutions ranging in molar concentration from 0 to 1.078, and in sorbitol solutions ranging in molar concentration from 0 to 2.156. The placebo used for the pretreatment of the nasal mucosa was prepared with the same concentration of sodium chloride solution without secretin, and 10 CHR units150 rJ, of secretin was dissolved in physiological saline (0.154 M NaCI, pH 6.4) as used for the intranaeal administration after pretreatment. The osmolarity of each solution was measured with an osmometer (model 3W, Advanced Instruments, Inc., MA) operating on the principle of freezing-point depression. Surgical Preparations of Rats-Rats were prepared surgically by the following procedure for the administration of secretin preparations and for the determination of their pharmacological responses. Male Sprague-Dawley rats weighing from 250 to 300 g, and fasted previously for 24 h, were anesthetized with an intraperitoneal injection of 170 mg/100 g of urethane (Wako) -30 min before the surgical procedures were performed. For intranasal administration directly into the nasal cavity, the surgical operation described by Hussain et al.13 was employed; for intravenous administration, the surgical operation described in the previous study’ was employed. The amount of secretin absorbed was measured by the biological assay described in the previous study.’ It has been reported14 that the secreted volume of pancreatic juice plotted against the logarithm of the intravenous dose of secretin was linear from 0.5 to 8 CHR units. This indicates that the amount of secretin absorbed through the nasal mucosa can be calculated by comparing the amount absorbed on intravenous administration. In this study, the volume of pancreatic juice secreted as a result of the absorption of secretin was calculated by subtracting the amount computed from the amount of secretin during the predose period from the amount of secretion during the administration period.
Experimental Section Materials-A 24 000 Crick, Happer and Raper (CHR) unitdmg preparation of synthetic secretin (Eisai Co., Ltd., Tokyo) was used in this study. The other chemicals employed were of analytical or reagent grade. Secretin Preparations-For intravenous administration, secretin was dissolved in physiological saline (0.154 M NaCI, pH 6.4). adjusted to a concentration of lCHR unit150 pL. For the effect of pH on nasal absorption, secretin, adjusted to a concentration of 10 CHR units/50 pL, was dissolved in isotonic 0.236 M citric acid:0.123 M disodium phosphate buffer (Wako Pure Industries, Ltd., Osaka), ranging in pH from 2.1 to 6.33, and isotonic 0.171 M potassium phosphate:O.144 M sodium acid carbonate buffer (Wako), ranging in pH from 7 to 8. The placebo that was used for
OO22-3549/87/09OO-0695$0 1.OO/O 0 1987, American Pharmaceutical Association
l n t rave nous
Retreatment
lntranasal
Flgure 1--Protocol for pretreatment study. Key: (a) measurement of the secreted volume of pancreatic juice during the predose period (basal secretion); (b) measurement of the secreted volume of pancreatic juice after intravenous administration;(c) period for washing out secretin; (d) period for pretreatment of nasal mucosa; (e) measurement of the secreted volume of pancreatic juice after intranasal administration. Journal of Pharmaceutical Sciences / 695 Vol. 76, No. 9, September 1987
Administration Procedure-Intravenous and intranasal administrations of secretin preparations followed the protocol of administration described in the previous study.' Pretreatment Study I'rocedure-Pretreatment was conducted following the protocol shown in Figure 1. A 100-pL aliquot ofplacebo solution was administered intranasally for the pretreatment of the nasal mucosa. As soon as 90 min of pretreatment had been completed, 10 mL of physiological saline (0.154 M NaCI, pH 6.4) was poured into the nasal cavity with a syringe to wash away the placebo solution. Then, 10 CHR unitsi50 pL of secretin dissolved in physiological saline (0.154 M NaCI, pH 6.4) was administered. Histological Examination of the Nasal Mucosa in Rats-After pretreatment of the nasal mucosa, the rats were killed, decapitated, and the nasal passages were flushed with 10% buffered formalin. After fixation, the heads were decalcified and four cross sections of the nasal cavity of each rat were stained with Hematoxylin-Eosin and examined microscopically.'s
Results and Discussion In our previous study,' in which the nasal absorption of secretin was studied in rats, the relative bioavailability of secretin on intranasal administration was revealed to be approximately one-tenth of that on intravenous administration, as estimated from the pharmacological response. The bioavailability was also enhanced in acid solution and reached a maximum in a 0.462 M sodium chloride solution. A pretreatment study and a histological study were conducted to investigate why the nasal absorption of secretin decreased with increasing pH and why the maximum absorption was observed at a sodium chloride solution molarity of 0.462. The total volume of pancreatic juice secreted during 90 min (TSVgo),the maximum volume of pancreatic juice secreted during each 15 min (SV,,,), the time to SV,,, (t,,,), and the absorption ratio of each dose of intranasally administered secretin to the 1 CHR unit given intravenously ( r ) for the pretreatment study of the pH effect on nasal absorption of secretin are presented in Table I. For intranasal administration of secretin dissolved in isotonic buffers, t,,, was varied according to the change of pH and was most retardative a t pH 3.81. The SV,,, value a t neutral pH values was smaller than that in an acidic environment.' On the other hand, for the pretreatment study, t,,, was the same a t all pH values, and SV,,, at neutral pH values was a little smaller than a t acidic ones. Moreover, SV,,, was almost the same in both studies a t acidic pH values. The pH dependency curves of TSVgofor the intranasal administration of the active preparation of secretin dissolved in buffer solution and the results of the pretreatment study are shown in Figure 2. Similar pH profiles were obtained in both studies, but it
was observed that the TSVgOvalues for the administration of active preparations were significantly larger than those for the pretreatment effect below a pH of 4.79, and were significantly smaller than those for the pretreatment effect a t a pH of 7 to 8. In order to examine whether the enhancement of nasal absorption of secretin a t acidic pH values was caused by damage of the nasal mucosa, a histological study was conducted. Typical microscopic specimens of exposed rat nasal mucosa are shown in Figure 3, which presents a photograph of untreated nasal mucosa and one of nasal mucosa treated with an isotonic buffer a t pH 2.94. Structural changes of the epithelial cells were observed in the nasal mucosa treated with the buffer, in comparison with the untreated control. In Figure 2, it could be supposed, from the observation of microscopic specimens of rat nasal mucosa, that the level of TSVgo in the pretreatment study would be the same or higher a t acidic pH values and the same a t a pH of 7 to 8, in comparison with that after the administration of active preparations. Figure 4 shows osmolarity dependency curves after intranasal administration of the secretin preparation dissolved in sodium chloride solutions and those dissolved in sorbitol solutions. A maximum value of TSVgowas observed at a sodium chloride solution osmolarity of 859 mOsm, while TSVgo was decreased as the osmolarity of the sorbitol solution increased. It was reported that the intestinal absorption of sulfisoxasole16 and quinine17 decreased with increasing
3
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v
0
40 -
Q,
>
In 30 I-
20 10 v---
~
2
~
3
4
5
6
7
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PH Figure 2-The pH-dependency curves after intranasal administrationof secretin in rats. Key: ((3)administration of active preparations (n = 4-8); (0) result ofpretreatment study (n = 3-7); (a)significantly different (p < 0.05) versus pretreatment study. The data are expressed as mean ? SE.
Table I-Results of Pretreatment Study of pH Effect in Rat Preparations'
PH 6.40d 2.10" 2.94" 3.81 " 4.798 6.33" 7.Oo6 8.0O8
Dosage Unit, CHR Unit
1 10 10 10 10 10 10 10
T S b , d-
15.67f 3.111 46.00 ? 8.179 (2.67 2 2.596)' 29.93 f 3.163 (-1.50 2 1.179) 24.60 f 4.229 (1.67 2 1.440) 24.80 2 3.064 (-0.67 ? 0.892) 17.40 4 2.304 (0.67 2 1.656) 18.33 f 3.153 (0.33 ? 0.491) 22.33 5 4.061 (-1.67 C 3.569)
SVm,, FL
11.96 ? 13.83 2 8.14 -t 10.40 ? 9.60 2 5.70 2 7.67 ? 11.50 2
1.359 2.520 1.068 1.757 1.014 0.921 0.847 2.134
Lax,
min
0-1 5 15-30 15-30 15-30 15-30 15-30 15-30 15-30
P
0.294 0.191 0.157 0.158 0.1 1 1 0.117 0.143
"The data are expressed as mean 2 SEM; n = 3-7; total secreted volume of pancreatic juice during 90 min (TSV,,, pL); maximum secreted volume of pancreatic juice during 15 min (SV,,,,, pL); time to SV,,, (I,,, rnin). 'The value r indicates the absorption ratio of intranasal administration to intravenous administration and is expressed as r = TSV, (intranasal):TSV, (intravenous) x 1 /dosage unit of intranasal administration. 'The data enclosed in parentheses are control data (n = 3). dlntravenous. Intranasal. 696 / Journal of Pharmaceutical Sciences Vol. 76, No. 9, September 1987
osmotic pressure, irrespective of the ionization of the osmoregulatory agents. Our results suggest that there was an optimum molar concentration of NaCl a t which secretin was most absorbed through the nasal mucosa, and that the nasal absorption of secretin would be affected not only by the osmolarity but also the osmoregulatory agents. A pretreatment study and histological study were conducted to discover why the maximum absorption of secretin through the nasal mucosa was observed a t a sodium chloride solution molarity of 0.462. Figure 5 shows the dependency curves of sodium chloride molarity on TSVgo after intranasal administration of active preparations of secretin dissolved in sodium chloride solutions, together with the results of the pretreatment study. Figure 6 shows typical microscopic specimens of exposed rat nasal mucosa. Figure 5 shows that similar profiles were obtained in both studies. At a sodium chloride molarity of 0, the larger TSVgOvalue in the pretreatment study, compared with that following administration of the active preparation, would be caused by hydration of the nasal mucosa. In microscopic specimens of exposed rat nasal mucosa treated with 0.924 M of sorbitol solution (Figure 6a), no significant lesion was observed, as compared with the control
Flgure 3-Photomicrographs of exposed rat nasal mucosa: (a) untreated control; (b) specimen treated with an isotonic buffer at pH 2.94.
rat shown in Figure 3a. On the other hand, in the microscopic specimen of exposed rat nasal mucosa treated by 0.462 M of sodium chloride solution (Figure 6b), shrinkage of epithelium cells was observed compared with the control rat. Although there was almost the same osmolarity in the 0.462 M sodium chloride solution a s in the 0.924 M sorbitol solution, structural changes of the epithelial cells were observed in rat nasal mucosa treated with this solution, and it was suggested that the ionic species of NaCl acted on the epithelial cells more strongly than did the sorbitol. As a result of the pH-effect study, it can be assumed that the effect of pH on the nasal absorption of secretin is mainly due to structural changes of the epithelial membrane, but also due to the self-association and conformational changes of secretin1a2° or to the change of electric charge on the epithelial surface. However, the results of the osmolarityI
Figure 5- Dependency curves of sodium chloride molarity affer intranasal administration of secretin in rats. Key: (a)administration of active preparations (n = 5-7); (0) result of pretreatment study (n = 3-8). The data are expressed as mean 5 SEM.
A 1
-20 -
g
> m I-
10-
0
1 XI03 Osmotic Pressure (mOsm)
Flgure 4-Osmolarify-dependency curves affer intranasal administration of secretin in rats. Key: (a)NaCl solution (n = 5-7); (0) sorbitol solutions (n = 3-5). The data are expressed as mean f SEM.
Figure 6-Photomicrographs of exposed rat nasal mucosa: (a) treated with 0.924 M sorbitol solution; (b) treated with 0.462 M sodium chloride solution. Journal of Pharmaceutical Sciences I697 Vol. 76, No. 9, September 1987
effect study revealed that the nasal absorption of secretin i n rats decreased i n proportion to the increase of molar concentration of sorbitol solution. The maximum absorption of secretin i n rats w a s observed at a sodium chloride solution molarity of 0.462. T h i s would result from the difference of the influence of sodium chloride and sorbitol on the nasal mucoea.
References and Notes 1. Ohwaki, T.;Ando, H.; Watanabe, S.; Miyake, Y. J. Pharm. Sci. 1985,74,550-552. 2. Matauo, H.; Seki, A.; Ishikawa, T. Hormon to Rinsho 1975,23, m7-fil2. - - . - - -.
3. Grossman, M.1. Gastroenterolo y 1966,50,912-913. 4. Glass, G. Gastroenterolo y l96t 51,580-581. 5. Miyoshi, A.; Fujii, K.; Otuhara, T.; Suyama, T.; Kai, T.; Kawaoe, T.; Ina awa, T. Shinryo to Shinyaku 1974,11, 1833-1837. 6. konturek. J. Gut 1973.14., 842-846. -----7. Konturek; S. J.; Radecki,'T.; Thor, P.; Kwiecien, N. Am. J. Dig. Dis. 1973,18,135-141. 8. Kawai, K.; Misaki, F.; Miyaoka, T.; Kimoto, K.; Shimamoto, K.; Takebayashi, M.; Nakajima, M.; Mitauyoshi, Y.; Fukumoto, K. Gendai no Rinsho 1974.8.177-183. 9. Nakamura, K. Shinryo 'to'shinyaku 1974,11, 1929-1933.
l.
698 /Journal of Pharmaceutical Sciences Vol. 76, No. 9, September 1987
10. Hirai, S.;Ikenaga, T.; Matsuzawa, T. Diabetes 1977,27, 296299. 11. Anik, S.T.; McRae, G.; Nerenberg, C.; Worden, A.; Foreman, J.; Hwang, J.; Kushinsky, S.; Jones, R. E.; Vickery, B. J. Phurm. Scr. 1984,73, 684-685. 12. Su, K. S. E.; Campanale, K. M.; Mendelsohn, L. G.; Kerchner, G. A.; Gries, C. L. J . Pharm. Sci. 1985,74,394-398. 13. Hussain, A.; Hirai, S.; Bawarshi, R. J. Pharm. Sci. 1980,69, 1411. _
14. Tachibana, S. Jpn. J. Phurmacol. 1971,21,325436. 15. Su,K. S. E.; Campanale, K. M.; Gries, C. L. J. Phurm. Sci. 1984, 73,1251-1254. 16. Morvola, M.;Reinikaine, A.; Heliovaara, M.; Huikari, A. J. Phurm. Phrmacol. 1979.31. 615-618. 17. Sakiya, Y.;Miyauchi, Y . ; Tsuemura, Y. Chem. Phurm. Bull. 1981,29,1470-1472;Chem. Abstr. 1981,95,67904~. 18. Bodanszky, A.; Ondetti, M. A.; Mutt, V.; Bodanszky, M. J. A m . Chem. Soc. 1969,12,944-949. 19. Bodanszkv. M.; Fink. M. L.: Funk. K. W.; Said. S. I. Clcn. Endocrinol. 1976,5,'195%-200s. 20. Patel, D.J.; Bodanszky, M.; Ondetti, M. A. Macromolecules 1970,3,694-698.
Acknowledgments The authors
atefull thank Mr. Motooka and Mr. Fukuda a t the Department o f g m g S a i t y Research in Eisai Co., Ltd., for providing the microscopic specimens of nasal mucorn.
SUM10 WATANABE,
Received December 2, 1986, from the Research Laboratories of Pharmaceutical Development, Eisai Co., Ltd., Kawashima-cho, Hashima-gun, Accepted for publication June 18, 1987 Gifu 483, Japan. -.
~.-
-
-
Abstract 9Nasal absorptionof secretin in rats was enhanced in an acid solution, and the maximum absorption was observed at a sodium chloride solution molarity of 0.462. In order to examine reasons for the variation of absorbability caused by the change of pH and osmolarity in secretin preparations, both a pretreatment study, in which the nasal rnucosa was treated with placeboprior to the administrationof a secretin preparation, and a histological study were conducted in rats. The nasal absorption of secretin was determined by measuring the increased secretion of pancreatic juice. Similar profiles of nasal absorption, both after intranasal administrationof secretin preparations and as a result of pretreatment effects, were obtained in studies of the effects of pH and osmolarity. However, in the pH-effect study, the absorptionwith the use of active preparations was observed to be significantly larger than that with the pretreatment effect below a pH of 4.79, and significantlysmaller than that with the pretreatment effect at a pH of 7 to 8. The results of histological studies revealed structural changes of the epithelial cells of the nasal mucosa at pH 2.94, and shrinkage of epithelial cells was observed at a sodium chloride solution molarity of 0.462. - - ~- -. -
In a previous study,’ a n investigation on t h e intranasal administration o f secretin, a hormone secreted in the digest i v e tract and used clinically for t h e treatment of duodenal ulcer,”s was conducted in rats to reveal the feasibility of a nasal dosage form o f secretin. The absorbability o f secretin through the nasal mucosa was observed to be one-tenth of the bioavailability after intravenous administration, and to be affected by the pH and osmolarity o f secretin preparations. A number o f investigations o n the nasal absorption of peptides have been reported,’o-l* but few reports have attempted to elucidate w h y the variation of nasal absorption o f peptides was brought about by pH and osmolarity. For the present paper, by conducting the pretreatment study and histological study o f nasal mucosa, we examined reasons w h y the nasal absorption o f secretin decreased w i t h increasing pH and w h y the m a x i m u m absorption o f secretin
pretreatment of the nasal mucosa was prepared with the same buffer solution, containing no secretin, and physiological saline (0.154 M NaCl, pH 6.4) containing 10 CHR unitd50 pL of secretin was prepared for intranasal administration after pretreatment. To examine the effect of osmolarity on nasal absorption, secretin, adjusted to a concentration of 10 CHR units/50 pL,was dissolved in sodium chloride solutions ranging in molar concentration from 0 to 1.078, and in sorbitol solutions ranging in molar concentration from 0 to 2.156. The placebo used for the pretreatment of the nasal mucosa was prepared with the same concentration of sodium chloride solution without secretin, and 10 CHR units150 rJ, of secretin was dissolved in physiological saline (0.154 M NaCI, pH 6.4) as used for the intranaeal administration after pretreatment. The osmolarity of each solution was measured with an osmometer (model 3W, Advanced Instruments, Inc., MA) operating on the principle of freezing-point depression. Surgical Preparations of Rats-Rats were prepared surgically by the following procedure for the administration of secretin preparations and for the determination of their pharmacological responses. Male Sprague-Dawley rats weighing from 250 to 300 g, and fasted previously for 24 h, were anesthetized with an intraperitoneal injection of 170 mg/100 g of urethane (Wako) -30 min before the surgical procedures were performed. For intranasal administration directly into the nasal cavity, the surgical operation described by Hussain et al.13 was employed; for intravenous administration, the surgical operation described in the previous study’ was employed. The amount of secretin absorbed was measured by the biological assay described in the previous study.’ It has been reported14 that the secreted volume of pancreatic juice plotted against the logarithm of the intravenous dose of secretin was linear from 0.5 to 8 CHR units. This indicates that the amount of secretin absorbed through the nasal mucosa can be calculated by comparing the amount absorbed on intravenous administration. In this study, the volume of pancreatic juice secreted as a result of the absorption of secretin was calculated by subtracting the amount computed from the amount of secretin during the predose period from the amount of secretion during the administration period.
Experimental Section Materials-A 24 000 Crick, Happer and Raper (CHR) unitdmg preparation of synthetic secretin (Eisai Co., Ltd., Tokyo) was used in this study. The other chemicals employed were of analytical or reagent grade. Secretin Preparations-For intravenous administration, secretin was dissolved in physiological saline (0.154 M NaCI, pH 6.4). adjusted to a concentration of lCHR unit150 pL. For the effect of pH on nasal absorption, secretin, adjusted to a concentration of 10 CHR units/50 pL, was dissolved in isotonic 0.236 M citric acid:0.123 M disodium phosphate buffer (Wako Pure Industries, Ltd., Osaka), ranging in pH from 2.1 to 6.33, and isotonic 0.171 M potassium phosphate:O.144 M sodium acid carbonate buffer (Wako), ranging in pH from 7 to 8. The placebo that was used for
OO22-3549/87/09OO-0695$0 1.OO/O 0 1987, American Pharmaceutical Association
l n t rave nous
Retreatment
lntranasal
Flgure 1--Protocol for pretreatment study. Key: (a) measurement of the secreted volume of pancreatic juice during the predose period (basal secretion); (b) measurement of the secreted volume of pancreatic juice after intravenous administration;(c) period for washing out secretin; (d) period for pretreatment of nasal mucosa; (e) measurement of the secreted volume of pancreatic juice after intranasal administration. Journal of Pharmaceutical Sciences / 695 Vol. 76, No. 9, September 1987
Administration Procedure-Intravenous and intranasal administrations of secretin preparations followed the protocol of administration described in the previous study.' Pretreatment Study I'rocedure-Pretreatment was conducted following the protocol shown in Figure 1. A 100-pL aliquot ofplacebo solution was administered intranasally for the pretreatment of the nasal mucosa. As soon as 90 min of pretreatment had been completed, 10 mL of physiological saline (0.154 M NaCI, pH 6.4) was poured into the nasal cavity with a syringe to wash away the placebo solution. Then, 10 CHR unitsi50 pL of secretin dissolved in physiological saline (0.154 M NaCI, pH 6.4) was administered. Histological Examination of the Nasal Mucosa in Rats-After pretreatment of the nasal mucosa, the rats were killed, decapitated, and the nasal passages were flushed with 10% buffered formalin. After fixation, the heads were decalcified and four cross sections of the nasal cavity of each rat were stained with Hematoxylin-Eosin and examined microscopically.'s
Results and Discussion In our previous study,' in which the nasal absorption of secretin was studied in rats, the relative bioavailability of secretin on intranasal administration was revealed to be approximately one-tenth of that on intravenous administration, as estimated from the pharmacological response. The bioavailability was also enhanced in acid solution and reached a maximum in a 0.462 M sodium chloride solution. A pretreatment study and a histological study were conducted to investigate why the nasal absorption of secretin decreased with increasing pH and why the maximum absorption was observed at a sodium chloride solution molarity of 0.462. The total volume of pancreatic juice secreted during 90 min (TSVgo),the maximum volume of pancreatic juice secreted during each 15 min (SV,,,), the time to SV,,, (t,,,), and the absorption ratio of each dose of intranasally administered secretin to the 1 CHR unit given intravenously ( r ) for the pretreatment study of the pH effect on nasal absorption of secretin are presented in Table I. For intranasal administration of secretin dissolved in isotonic buffers, t,,, was varied according to the change of pH and was most retardative a t pH 3.81. The SV,,, value a t neutral pH values was smaller than that in an acidic environment.' On the other hand, for the pretreatment study, t,,, was the same a t all pH values, and SV,,, at neutral pH values was a little smaller than a t acidic ones. Moreover, SV,,, was almost the same in both studies a t acidic pH values. The pH dependency curves of TSVgofor the intranasal administration of the active preparation of secretin dissolved in buffer solution and the results of the pretreatment study are shown in Figure 2. Similar pH profiles were obtained in both studies, but it
was observed that the TSVgOvalues for the administration of active preparations were significantly larger than those for the pretreatment effect below a pH of 4.79, and were significantly smaller than those for the pretreatment effect a t a pH of 7 to 8. In order to examine whether the enhancement of nasal absorption of secretin a t acidic pH values was caused by damage of the nasal mucosa, a histological study was conducted. Typical microscopic specimens of exposed rat nasal mucosa are shown in Figure 3, which presents a photograph of untreated nasal mucosa and one of nasal mucosa treated with an isotonic buffer a t pH 2.94. Structural changes of the epithelial cells were observed in the nasal mucosa treated with the buffer, in comparison with the untreated control. In Figure 2, it could be supposed, from the observation of microscopic specimens of rat nasal mucosa, that the level of TSVgo in the pretreatment study would be the same or higher a t acidic pH values and the same a t a pH of 7 to 8, in comparison with that after the administration of active preparations. Figure 4 shows osmolarity dependency curves after intranasal administration of the secretin preparation dissolved in sodium chloride solutions and those dissolved in sorbitol solutions. A maximum value of TSVgowas observed at a sodium chloride solution osmolarity of 859 mOsm, while TSVgo was decreased as the osmolarity of the sorbitol solution increased. It was reported that the intestinal absorption of sulfisoxasole16 and quinine17 decreased with increasing
3
a 50 -
v
0
40 -
Q,
>
In 30 I-
20 10 v---
~
2
~
3
4
5
6
7
0
PH Figure 2-The pH-dependency curves after intranasal administrationof secretin in rats. Key: ((3)administration of active preparations (n = 4-8); (0) result ofpretreatment study (n = 3-7); (a)significantly different (p < 0.05) versus pretreatment study. The data are expressed as mean ? SE.
Table I-Results of Pretreatment Study of pH Effect in Rat Preparations'
PH 6.40d 2.10" 2.94" 3.81 " 4.798 6.33" 7.Oo6 8.0O8
Dosage Unit, CHR Unit
1 10 10 10 10 10 10 10
T S b , d-
15.67f 3.111 46.00 ? 8.179 (2.67 2 2.596)' 29.93 f 3.163 (-1.50 2 1.179) 24.60 f 4.229 (1.67 2 1.440) 24.80 2 3.064 (-0.67 ? 0.892) 17.40 4 2.304 (0.67 2 1.656) 18.33 f 3.153 (0.33 ? 0.491) 22.33 5 4.061 (-1.67 C 3.569)
SVm,, FL
11.96 ? 13.83 2 8.14 -t 10.40 ? 9.60 2 5.70 2 7.67 ? 11.50 2
1.359 2.520 1.068 1.757 1.014 0.921 0.847 2.134
Lax,
min
0-1 5 15-30 15-30 15-30 15-30 15-30 15-30 15-30
P
0.294 0.191 0.157 0.158 0.1 1 1 0.117 0.143
"The data are expressed as mean 2 SEM; n = 3-7; total secreted volume of pancreatic juice during 90 min (TSV,,, pL); maximum secreted volume of pancreatic juice during 15 min (SV,,,,, pL); time to SV,,, (I,,, rnin). 'The value r indicates the absorption ratio of intranasal administration to intravenous administration and is expressed as r = TSV, (intranasal):TSV, (intravenous) x 1 /dosage unit of intranasal administration. 'The data enclosed in parentheses are control data (n = 3). dlntravenous. Intranasal. 696 / Journal of Pharmaceutical Sciences Vol. 76, No. 9, September 1987
osmotic pressure, irrespective of the ionization of the osmoregulatory agents. Our results suggest that there was an optimum molar concentration of NaCl a t which secretin was most absorbed through the nasal mucosa, and that the nasal absorption of secretin would be affected not only by the osmolarity but also the osmoregulatory agents. A pretreatment study and histological study were conducted to discover why the maximum absorption of secretin through the nasal mucosa was observed a t a sodium chloride solution molarity of 0.462. Figure 5 shows the dependency curves of sodium chloride molarity on TSVgo after intranasal administration of active preparations of secretin dissolved in sodium chloride solutions, together with the results of the pretreatment study. Figure 6 shows typical microscopic specimens of exposed rat nasal mucosa. Figure 5 shows that similar profiles were obtained in both studies. At a sodium chloride molarity of 0, the larger TSVgOvalue in the pretreatment study, compared with that following administration of the active preparation, would be caused by hydration of the nasal mucosa. In microscopic specimens of exposed rat nasal mucosa treated with 0.924 M of sorbitol solution (Figure 6a), no significant lesion was observed, as compared with the control
Flgure 3-Photomicrographs of exposed rat nasal mucosa: (a) untreated control; (b) specimen treated with an isotonic buffer at pH 2.94.
rat shown in Figure 3a. On the other hand, in the microscopic specimen of exposed rat nasal mucosa treated by 0.462 M of sodium chloride solution (Figure 6b), shrinkage of epithelium cells was observed compared with the control rat. Although there was almost the same osmolarity in the 0.462 M sodium chloride solution a s in the 0.924 M sorbitol solution, structural changes of the epithelial cells were observed in rat nasal mucosa treated with this solution, and it was suggested that the ionic species of NaCl acted on the epithelial cells more strongly than did the sorbitol. As a result of the pH-effect study, it can be assumed that the effect of pH on the nasal absorption of secretin is mainly due to structural changes of the epithelial membrane, but also due to the self-association and conformational changes of secretin1a2° or to the change of electric charge on the epithelial surface. However, the results of the osmolarityI
Figure 5- Dependency curves of sodium chloride molarity affer intranasal administration of secretin in rats. Key: (a)administration of active preparations (n = 5-7); (0) result of pretreatment study (n = 3-8). The data are expressed as mean 5 SEM.
A 1
-20 -
g
> m I-
10-
0
1 XI03 Osmotic Pressure (mOsm)
Flgure 4-Osmolarify-dependency curves affer intranasal administration of secretin in rats. Key: (a)NaCl solution (n = 5-7); (0) sorbitol solutions (n = 3-5). The data are expressed as mean f SEM.
Figure 6-Photomicrographs of exposed rat nasal mucosa: (a) treated with 0.924 M sorbitol solution; (b) treated with 0.462 M sodium chloride solution. Journal of Pharmaceutical Sciences I697 Vol. 76, No. 9, September 1987
effect study revealed that the nasal absorption of secretin i n rats decreased i n proportion to the increase of molar concentration of sorbitol solution. The maximum absorption of secretin i n rats w a s observed at a sodium chloride solution molarity of 0.462. T h i s would result from the difference of the influence of sodium chloride and sorbitol on the nasal mucoea.
References and Notes 1. Ohwaki, T.;Ando, H.; Watanabe, S.; Miyake, Y. J. Pharm. Sci. 1985,74,550-552. 2. Matauo, H.; Seki, A.; Ishikawa, T. Hormon to Rinsho 1975,23, m7-fil2. - - . - - -.
3. Grossman, M.1. Gastroenterolo y 1966,50,912-913. 4. Glass, G. Gastroenterolo y l96t 51,580-581. 5. Miyoshi, A.; Fujii, K.; Otuhara, T.; Suyama, T.; Kai, T.; Kawaoe, T.; Ina awa, T. Shinryo to Shinyaku 1974,11, 1833-1837. 6. konturek. J. Gut 1973.14., 842-846. -----7. Konturek; S. J.; Radecki,'T.; Thor, P.; Kwiecien, N. Am. J. Dig. Dis. 1973,18,135-141. 8. Kawai, K.; Misaki, F.; Miyaoka, T.; Kimoto, K.; Shimamoto, K.; Takebayashi, M.; Nakajima, M.; Mitauyoshi, Y.; Fukumoto, K. Gendai no Rinsho 1974.8.177-183. 9. Nakamura, K. Shinryo 'to'shinyaku 1974,11, 1929-1933.
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698 /Journal of Pharmaceutical Sciences Vol. 76, No. 9, September 1987
10. Hirai, S.;Ikenaga, T.; Matsuzawa, T. Diabetes 1977,27, 296299. 11. Anik, S.T.; McRae, G.; Nerenberg, C.; Worden, A.; Foreman, J.; Hwang, J.; Kushinsky, S.; Jones, R. E.; Vickery, B. J. Phurm. Scr. 1984,73, 684-685. 12. Su, K. S. E.; Campanale, K. M.; Mendelsohn, L. G.; Kerchner, G. A.; Gries, C. L. J . Pharm. Sci. 1985,74,394-398. 13. Hussain, A.; Hirai, S.; Bawarshi, R. J. Pharm. Sci. 1980,69, 1411. _
14. Tachibana, S. Jpn. J. Phurmacol. 1971,21,325436. 15. Su,K. S. E.; Campanale, K. M.; Gries, C. L. J. Phurm. Sci. 1984, 73,1251-1254. 16. Morvola, M.;Reinikaine, A.; Heliovaara, M.; Huikari, A. J. Phurm. Phrmacol. 1979.31. 615-618. 17. Sakiya, Y.;Miyauchi, Y . ; Tsuemura, Y. Chem. Phurm. Bull. 1981,29,1470-1472;Chem. Abstr. 1981,95,67904~. 18. Bodanszky, A.; Ondetti, M. A.; Mutt, V.; Bodanszky, M. J. A m . Chem. Soc. 1969,12,944-949. 19. Bodanszkv. M.; Fink. M. L.: Funk. K. W.; Said. S. I. Clcn. Endocrinol. 1976,5,'195%-200s. 20. Patel, D.J.; Bodanszky, M.; Ondetti, M. A. Macromolecules 1970,3,694-698.
Acknowledgments The authors
atefull thank Mr. Motooka and Mr. Fukuda a t the Department o f g m g S a i t y Research in Eisai Co., Ltd., for providing the microscopic specimens of nasal mucorn.