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Evaluation of gelatin microspheres for nasal and intramuscular administrations of salmon calcitonin
European Journal of Pharmaceutical Sciences 13 (2001) 179–185 www.elsevier.nl / locate / ejps
Evaluation of gelatin microspheres for nasal and intramuscular administrations of salmon calcitonin a,
b
b
b
Kazuhiro Morimoto *, Hideyuki Katsumata , Toshiyuki Yabuta , Kazunori Iwanaga , Masawo Kakemi b , Yasuhiko Tabata c , Yoshito Ikada c b
a Department of Pharmaceutics, Hokkaido College of Pharmacy, 7 -1 Katsuraoka-cho, Otaru-city, Hokkaido 047 -0264, Japan Department of Pharmaceutics, Osaka University of Pharmaceutical Sciences, 4 -20 -1 Nasahara, Takatsuki-city Osaka 569 -1094, Japan c Institute of Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho Shogoin, Sakyo-ku, Kyoto 606 -8507, Japan
Received 22 May 2000; received in revised form 12 December 2000; accepted 15 December 2000
Abstract The suitability of gelatin microspheres for nasal and intramuscular delivery of salmon calcitonin (sCT) was examined. Negatively and positively charged gelatin microspheres were prepared using acidic gelatin [isoelectric point (IEP) value of 5.0] and basic gelatin (IEP59.0), respectively. The average diameters of positively charged gelatin microspheres in their dried state were 3.4, 11.2, 22.5 and 71.5 mm, while that of negatively charged gelatin microspheres was 10.9 mm. Both types of gelatin microspheres were capable of adhering to the nasal mucosa. The mucoadhesion of positively charged gelatin microspheres was significantly higher than that of their negatively charged counterparts. The absorption of sCT after intranasal and intramuscular administration was evaluated by calculating the area above the hypocalcemic–time curve (AAC) in rats. The AAC values after nasal administration of sCT in positively and negatively charged gelatin microspheres were significantly greater than that in pH 7.0 PBS. Therefore, the nasal absorption of sCT was enhanced by both types of gelatin microspheres. The hypocalcemic effect after administration of sCT in positively charged gelatin microspheres of 11.2 mm was significantly greater than that of negatively charged gelatin microspheres of the same size. On the other hand, AAC values were not affected by their particle sizes. The AAC values after the intramuscular administration of sCT in positively and negatively charged gelatin microspheres were significantly increased compared to that in PBS. Furthermore, the time-courses of the plasma calcium levels differed between positively and negatively charged gelatin microspheres. The hypocalcemic effect of the negatively charged gelatin microspheres tended to appear more slowly and last longer compared to that of positively charged gelatin microspheres. The hypocalcemic effects after intramuscular administration of sCT in gelatin microspheres were not affected by their particle sizes as well as those after intranasal administration. In conclusion, the gelatin microspheres have been shown to be a useful vehicle for nasal or intramuscular delivery of sCT. 2001 Elsevier Science B.V. All rights reserved. Keywords: Gelatin, Microspheres; Nasal delivery; Intramuscular administration; Salmon calcitonin; Peptide drug
1. Introduction Calcitonin, which is a cyclic polypeptide of 32 amino acids [molecular weight (Mw )53500 Da], has a physiological role in the regulation of calcium homeostasis and is a potent inhibitor of osteoclastic bone resorption (Camilleri et al., 1991). Salmon calcitonin (sCT) is widely used because it is the most potent of the calcitonins available, it is well tolerated and is clinically effective (Stevenson and Evans, 1981). Like other peptides, sCT is easily degraded by proteolytic enzymes in the gastrointestinal tract and, *Corresponding author. Tel.: 181-134-62-1848; fax: 181-134-621848. E-mail address: [email protected] (K. Morimoto).
therefore, has a low bioavailability when administered via oral route (Sakuma et al., 1998). In addition, sCT has a short half-life (approximately 15|20 min) after parenteral administration (Huwyler et al., 1979). Thus frequent administration is required to maintain its pharmacological effects. Intranasal and pulmonary delivery of sCT has been shown to be effective (Kobayashi et al., 1994; Morimoto et al., 1995). In particularly, it has been reported that once daily dosing of sCT via the nasal route has decreased the osteoclastic bone resorption in humans (Thamsborg et al., 1996). However, the nasal and pulmonary bioavailability of sCT is lower than that following intramuscular and subcutaneous injection. Such systems may remove the need for multiple administrations during long term treatment. Development of formulations for the delivery of sCT
0928-0987 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0928-0987( 01 )00094-X
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is important for more effective and safer therapy. The absorption of sCT has to be enhanced by using a special formulation. Acidic and basic gelatins are denatured, biodegradable proteins obtained by the alkaline and acidic treatment of collagen, respectively (Tabata and Ikada, 1998). This treatment affects the electrical nature of collagen, yielding gelatin with different isoelectric point (IEP) values. The negatively and positively charged gelatin microspheres are prepared using acidic gelatin (IEP55.0) and basic gelatin (IEP59.0), respectively. We have previously reported the characteristics of sCT release from positively and negatively charged gelatin microspheres and the high bioavailability after pulmonary administration of sCT in the gelatin microsphere (Morimoto et al., 2000). In the present study, to solve the delivery problem of sCT, the negatively and positively charged gelatin microspheres of various particle sizes as suspension forms of hydrogel by swelling in pH 7.0 PBS were designed for nasal and intramuscular delivery. The absorption of sCT after nasal and intramuscular administration in gelatin microspheres was evaluated in rats.
2. Experimental procedures
2.1. Materials sCT (24.0 U / mg, purity598.1%) was obtained from Bachem Feinchemikalien AG (Switzerland). Acidic and basic gelatins (Mw 599 kDa) with IEPs of 5.0 and 9.0, respectively, were kindly supplied from Nitta Gelatin Co., Ltd (Osaka, Japan). Rhodamine isothiocyanate (RITC; purity599%) was purchased from Wako Pure Chemical Industries (Osaka, Japan). All other chemicals were of the highest purity available commercially.
2.2. Preparation of gelatin microspheres Gelatin microspheres with diameters ranging from 20 to 100 mm were prepared by glutaraldehyde crosslinking of an aqueous gelatin solution dispersed in an oil phase in the absence of a surfactant according to the modified method by Tabata and Ikada (1989). Briefly, aqueous acidic or basic gelatin solution (10%) preheated at 408C was added drop-wise to olive oil and the mixture was stirred at 420 rev / min at 408C for 10 min to yield a water-in-oil (w / o) emulsion. Stirring was continued for a further 10 min at 158C, and the microspheres were washed three times with acetone and isopropanol, respectively, by centrifugation (3000 rev / min, 5 min, 48C). After being air-dried, the microspheres were sized by passing them through sieves of different apertures. Gelatin microspheres with diameters less than 20 mm were prepared using sonication to reduce the size of the w / o emulsion (Tabata et al., 1999). Briefly, the mixture of
10% aqueous gelatin solution (0.2 ml) and olive oil (5 ml) was preheated and agitated with a vortex mixer at 408C for 1 min, followed by ultrasonication at 3.0 W/ cm for different time periods up to 40 s. The emulsion was agitated for 15 min at 48C. Then, as described above, the resulting microspheres were washed and dried. The non-crosslinked and dried gelatin microspheres were dispersed in 5 ml of aqueous glutaraldehyde solution (7.5 mg / ml; 25%) at 48C for 15 h to allow crosslinking. The microspheres were further agitated in a 10 mM aqueous glycine solution at 378C for 1 h to block residual aldehyde groups of unreacted glutaraldehyde. The resulting microspheres were finally washed three times with doubledistilled water by centrifugation and freeze-dried. The water content of the prepared gelatin microspheres was calculated from their volume before and after swelling in phosphate buffered saline (pH 7.0 PBS) for 24 h at 378C. At least 200 of the freeze-dried microspheres were examined by light microscopy to measure their diameters from which the volume of the respective microspheres was calculated. The average diameters of the positively charged gelatin microspheres in the dried state were 3.460.06, 11.260.20, 22.560.41 or 71.561.59 mm, while that of the negatively charged gelatin microspheres was 10.960.24 mm. For the incorporation of sCT into the freeze-dried gelatin microspheres for nasal and intramuscular administrations, 0.1 ml of sCT solutions in pH 7.0 PBS (2250 and 150 U / ml) were dropped onto 10.5 mg of freeze-dried gelatin microspheres, respectively. These were left at 48C for 3 h to allow the solution to penetrate into the microspheres. In this study, approximately 100% of the sCT aqueous solution was allowed to impregnate in the microspheres during the swelling process because the solution volume was less than that which would theoretically impregnate into each microsphere. In fact, the adsorbed amounts of sCT into the surface of the microsphere were less than 2.0% and were not significant between the positively and negatively charged gelatin microspheres. sCT incorporated gelatin microspheres were suspended in 1.4 ml of PBS. The final concentration of gelatin and sCT in the gelatin microsphere preparation for nasal administrations was 7 mg / ml and 150 U / ml, respectively. For intramuscular administrations, the final concentrations were 7 mg / ml and 5.0 U / ml, respectively.
2.3. Nasal and intramuscular administration Male Wistar rats weighing 210–300 g, were fasted for 20 h prior to an experiment, but allowed free access to water. The rats were anesthetized by an intraperitoneal injection of sodium pentobarbital (7.0 mg / kg) with additional doses given intraperitoneally as necessary during the experiments. Nasal administration of sCT preparation was performed according to the method of Hirai et al. (1981).
K. Morimoto et al. / European Journal of Pharmaceutical Sciences 13 (2001) 179 – 185
The sCT gelatin microsphere preparations as the suspension form of hydrogel by swelling in pH 7.0 PBS and the sCT solution in pH 7.0 PBS (dosage volume50.1 ml / kg body weight) were administered to the nasal cavity through a tube by means of micropipette inserted into the nostril at a sCT dose of 15.0 U / kg. In addition, the sCT gelatin microsphere preparations as the suspension form of hydrogel by swelling in pH 7.0 PBS or the sCT solution in pH 7.0 PBS (dosage volume50.2 ml / kg body weight) were administered intramuscularly (i.m.) to separate groups of rats at a sCT dose of 1.0 U / kg. The blood samples were periodically withdrawn from the jugular vein. The absorption of sCT was evaluated by monitoring the hypocalcemic effects. Plasma calcium levels were determined using the Calcium C test Wako (Wako Pure Chemical Industries, Osaka, Japan). The area surrounding the hypocalcemic effect–time curve (0–6 h) and the line showing 100% of plasma calcium level (% of initial) was calculated by means of trapezoidal integration. This area was defined as the area above the hypocalcemic effect– time curve (AAC) and was used for evaluating the sCT absorption.
2.4. Adhesion of gelatin microspheres to nasal mucosa Gelatin microspheres labeled with RITC were prepared to measure the mucoadhesion of gelatin microspheres to the nasal mucosa as previously described (Morimoto et al., 2000). An in situ nasal perfusion experiment was performed according to the method previously described (Iwanaga et al., 2000). Briefly, under pentobarbital anesthesia, the trachea and esophagus of rats (body weight5 250–300 g) were incised and nasal cavity was cannulated from its posterior site. The RITC-labeled gelatin microspheres suspended in PBS were administered (dosage volume50.1 ml / kg body weight) to the nasal cavity through the nostrils. Thirty minutes after nasal administration, the nasal cavity was perfused with pH 7.0 PBS from its posterior site through the cannula for 10 min (378C, flow-rate50.2 ml / min). The effluent from the nostrils was collected for 10 min. The drug amount recovered in this effluent was measured and determined as the ‘non-adhering fraction’. Immediately after perfusion, the rats were decapitated and the nasal mucosa was exposed. The surface of the nasal mucosa was washed with 20 ml of PBS and the amount of drug recovered in this medium was measured and determined as the ‘adhering fraction’. The concentration of RITC in the samples was measured by fluorospectrometry after being dissolved with 1 N HCl. Preliminarily, we confirmed the reasonability of this experimental procedure by using fluorescein isothiocyanate dextran (Mw 540,000) solution as a non-absorbable and non-adhering solution. In each experiment, the total amount of RITC recovered (non-adhering fraction plus adhering fraction) was approximately 100%.
181
2.5. Data analysis All data were expressed as mean6S.E. (standard errors of the mean). Comparisons between the group means were evaluated by the unpaired t-test. The statistical significance of differences among more than two groups were determined by one-way ANOVA. When F-tests from ANOVA indicated significant differences (P,0.05), a multiple comparison was performed to compare group means by the Bonferroni t-procedure.
3. Results
3.1. Nasal administration of sCT gelatin microspheres Nasal absorption of sCT from the positively and negatively charged gelatin microspheres of various particle sizes suspended in pH 7.0 PBS was estimated in the anesthetized rat model. Fig. 1 shows the time-course of the hypocalcemic effects in rats after nasal administration of sCT (15.0 U / kg) in positively and negatively charged gelatin microspheres (particle sizes510.9 and 11.2 mm, respectively). The hypocalcemic effects after administration of sCT in positively and negatively charged gelatin microspheres were significantly greater than that following administration in the aqueous solution (in pH 7.0 PBS). The nasal administration of sCT in the positively charged gelatin microspheres has significantly greater hypocalcemic effects than that after administration in the negatively charged gelatin microspheres as shown in Table 1. Fig. 2 shows the time-course of hypocalcemic effects in
Fig. 1. Time-course of hypocalcemic effects after nasal administration of sCT (15 U / kg) in positively and negatively charged gelatin microspheres (particle sizes510.9 and 11.2 mm, respectively) in rats. Solution (j, N56); positively charged microspheres of 11.2 mm (n; N55); negatively charged microspheres of 10.9 mm (m, N54). Each point represents the mean6S.E.
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Table 1 Area above the hypocalcemic effect–time curve (AAC) after nasal and intramuscular administration of sCT in rats a Dosage formulations
Nasal administration Solution Negatively charged gelatin microsphere Positively charged gelatin microsphere
Intramuscular administration Solution Negatively charged gelatin microsphere Positively charged gelatin microsphere
Particle size (mm)
–
N
AAC (% of initial level3min)
6
1933.06417.5
rats after nasal administration of sCT (15.0 U / kg) in positively charged gelatin microspheres of various particle sizes (3.4, 11.2, 22.5 and 71.2 mm). The hypocalcemic effects after nasal administration of sCT in the gelatin microspheres were unaffected by the particle size (Table 1).
3.2. Adhesion of gelatin microspheres to nasal mucosa b
10.9
4
4075.66782.6
3.4 11.2 22.5 71.2
5 6 5 6
6467.161614.0 b 5906.361356.4 b,c 5687.661154.1 b 5988.961308.7 b
–
6
1999.96338.9
10.9
7
3056.46577.9 d
3.4 11.2 22.5 71.2
4 4 4 4
3764.16690.7 d 2791.56210.5 d 2561.26351.0 d 2936.36414.8 d
Mucoadhesion of RITC labeled negatively and positively charged gelatin microspheres (particle sizes510.9 and 11.2 mm, respectively) was examined by in situ nasal perfusion experiments in rats. As shown in Table 2, the amount of positively charged gelatin microspheres recovered in the adhering fraction was significantly higher than that of negatively charged gelatin microspheres, indicating that the mucoadhesion ability of positively charged gelatin microspheres was greater than that of negatively charged ones.
3.3. Intramuscular administration of sCT gelatin microspheres
a
Doses of sCT of nasal and intramuscular administration were 15.0 and 1.0 U / kg, respectively. No significant differences were observed between the negatively (10.9 mm) and positively (11.2 mm) charged gelatin microspheres after i.n. and i.m. administration. No significant differences were observed when compared among gelatin microspheres of various sizes after i.n. and i.m administration. Each value represents the mean6S.E. b P,0.05 vs. nasal administration of sCT in solution. c P,0.05 vs. nasal administration of negatively charged gelatin microspheres. d P,0.05 vs. i.m. administration of sCT in solution.
The hypocalcemic effects after intramuscular administration of sCT in positively and negatively charged gelatin microspheres of various particle sizes suspended in pH 7.0 PBS were estimated in the anesthetized rat model. Fig. 3 shows the time-course of hypocalcemic effects in rats after intramuscular administration of sCT (1.0 U / kg) in negatively and positively charged gelatin microspheres (particle sizes510.9 and 11.2 mm, respectively). The hypocalcemic effects after intramuscular administration of sCT in positively and negatively charged gelatin microspheres were significantly greater than that produced following administration in the aqueous solution (in pH 7.0 PBS). The administration of sCT in negatively charged gelatin microspheres tended to appear more slowly and lasted longer compared to that in positively charged microspheres. Fig. 4 shows the time-course of the hypocalcemic effects in rats after intramuscular administration of sCT (1.0 U / kg) in positively charged gelatin microspheres of
Table 2 Comparison of adhesion ability to nasal mucosa between positively and negatively charged gelatin microspheres a Dosage formulations
Fig. 2. Time-course of hypocalcemic effects after nasal administration of sCT (15 U / kg) in positively charged gelatin microspheres of various particle sizes (3.4, 11.2, 22.5 and 71.2 mm) in rats. Solution (j, N56); positively charged microspheres: diameters of 3.4 mm (s, N55), 11.2 mm (n, N56), 22.5 mm (h, N55), 71.5 mm (d, N56). Each point represents the mean6S.E.
Negatively charged gelatin microsphere Positively charged gelatin microsphere a *
Particle size (mm)
N
Recovered amount of RITC from adhering fraction (% of total recovered)
10.9
5
46.062.47
11.2
5
58.662.17*
Each value represents the mean6S.E. P,0.01 vs. negatively charged gelatin microspheres.
K. Morimoto et al. / European Journal of Pharmaceutical Sciences 13 (2001) 179 – 185
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4. Discussion
Fig. 3. Time-course of hypocalcemic effects after intramuscular administration of sCT (1.0 U / kg) in negatively and positively charged gelatin microspheres (particle sizes510.9 and 11.2 mm, respectively) in rats. Solution (j, N56); positively charged microspheres of 11.2 mm (n, N54); negatively charged microspheres of 10.9 mm (m, N57). Each point represents the mean6S.E.
various particle sizes (3.4, 11.2, 22.5 and 71.2 mm). The hypocalcemic effect after intramuscular administration of sCT in gelatin microspheres was unaffected by the particle size.
Fig. 4. Time-course of hypocalcemic effects after intramuscular administration of sCT (1.0 U / kg) in positively charged gelatin microspheres of various particle sizes (3.4, 11.2, 22.5 and 71.2 mm) in rats. Solution (j, N56); positively charged microspheres: diameters of 3.4 mm (s, N54), 11.2 mm (n, N54), 22.5 mm (h, N54), 71.5 mm (d, N54). Each point represents the mean6S.E.
The negatively and positively charged gelatin microspheres of a wide range of particle sizes (3.4|71.2 mm) were prepared using acidic and basic gelatin for nasal and intramuscular delivery of sCT as a peptide drug. The feature of the particular formulations as a drug delivery system including microspheres is defined by two characteristics, the rate of drug release and the degradation profile. Gelatin is a biodegradable and biocompatible polymer and gelatin microspheres are degraded by enzymatic (trypsin, pepsin and collagenase) hydrolysis in the body (Tabata and Ikada, 1998). We previously reported that the gelatin microspheres slightly underwent enzymatic degradation, when incubated in the bronchoalveolar lavage fluid (Morimoto et al., 2000). Thus, the degradation profile of the formulation is generally determined by the materials composed of the formulation. On the other hand, the release rate of a drug from particular formulations is thought vary with their composition, size, charge, interaction with the drug incorporated and so on. In our previous study, sCT was rapidly (within 2 h) released from gelatin microspheres in pH 7.0 PBS, thereafter, the release of sCT was leveled off and no sustained release was observed. And the release rate was not affected by the size of the microspheres. Furthermore, we clarified that the release rate of sCT from positively charged gelatin microspheres was much higher than that from negatively charged ones and this difference in release rate was caused by the electrostatic interactions between the microspheres and sCT (Morimoto et al., 2000). In this study, the absorption enhancing effects of sCT after intranasal and intramuscular administration of gelatin microspheres were evaluated in the anesthetized rat model. The AAC values after nasal administration of sCT in gelatin microspheres were significantly higher than that of sCT in PBS in regardless of their charges, indicating that the gelatin microspheres enhanced the nasal absorption of sCT compared to sCT solution (Fig. 1 and Table 1). However, this enhancing effect of gelatin microspheres was only observed within 2 h of administration and the enhancing effect of positively charged gelatin microspheres tended to be greater than that of the negatively charged ones. These results were consistent with the release profile of sCT from gelatin microspheres in PBS. Furthermore, the fact that the size of microspheres did not affect the hypocalcemic effect (Fig. 2) also agreed with the result obtained from in vitro release experiments. Therefore, the high concentration of sCT at the mucosal surface of the nasal membrane, which was induced by the release from the microspheres, may be one of the important factors responsible for increased sCT absorption produced by gelatin microspheres. Besides the release rate of the drug, the mucoadhesion ability is also thought to be an important factor in defining the potential mucosal dosage formulation. The mucoadhe-
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sion of positively charged gelatin microspheres with a mean particle size of 11.2 mm was significantly higher than that of negatively charged gelatin microspheres of the approximately same size (Table 2). It has been reported that some mucoadhesive microspheres improved the nasal delivery of poorly absorbed drugs such as peptide drugs (Illum et al., 1987; Edman et al., 1992; Morath, 1998). However, these microspheres were the dry type and the absorption enhancing mechanism by these microspheres was different from that of our microspheres of the full swollen type. We also reported that mucoadhesive preparations made with Carbopol (Morimoto et al., 1984) and hyaluronate (Morimoto et al., 1991) increased the nasal and rectal absorption of peptide drugs. It is generally accepted that mucoadhesive preparations can increase the drug concentration in the vicinity of the mucosal cells by increasing contact between drugs and the mucosa; consequently, enhancing the drug absorption (Nakamura et al., 1996; Sakuma et al., 1998). Therefore, the high concentrations of sCT in the superficial mucosa of the nasal cavity, which was induced by the initial release from the microspheres, are also important factors in enhancing sCT absorption by gelatin microspheres. However, the mucociliary clearance is important problem to solve because it is normally less than 20 min and decreases with anesthesia and surgical operation. Therefore, we are now investigating the effect of mucociliary clearance on the absorption enhancing effects of the gelatin microspheres using conscious or non-operated conditions. We also considered that absorption of sCT after nasal administration might be attributable to avoiding enzymatic degradation in the nasal cavity. sCT was degraded by trypsin-like enzymes when intranasally administered as previously reported (Morimoto et al., 1995). The gelatin microspheres may have a stabilizing effect on the enzymatic degradation of sCT. As the enzymatic degradation of gelatin microspheres in the rats bronchoalveolar fluids was slight (Morimoto et al., 2000), it could be speculated that the gelatin microspheres may stabilize sCT against the enzymes in the nasal cavity. However, it is unclear which factor contributes to enhancing the nasal absorption of sCT – the high concentration of sCT in the vicinity of nasal mucosa or the stabilizing sCT – from these data. We are now conducting investigations to clarify this point. Intramuscular administration of sCT in both gelatin microspheres enhanced the absorption of sCT compared to that in PBS in regardless of both their charges and sizes (Figs. 3 and 4 and Table 1). When sCT was intramuscularly injected as PBS solution, sCT would spread widely from the injected site. On the other hand, when sCT was injected as microsphere formulations, the distribution of sCT was considered to be limited regardless of their charges and sizes because the microspheres formed a depot at the injected site. Therefore, the stability of sCT to enzymes after intramuscular administration of microspheres formulations was considerably improved compared
to that of the PBS formulation, resulting in enhanced sCT absorption. However, the hypocalcemic effects after intramuscular administration of sCT in negatively charged gelatin microspheres were slower to appear than with the positively charged one, although no significant differences in AAC values were observed between these two microspheres (Fig. 3 and Table 2). The difference in the pattern of hypocalcemic effects between the positively and negatively charged gelatin microspheres might be caused by the difference in the release profile of sCT in the muscular tissue between these gelatin microspheres. However, this phenomenon was not observed in the case of the intranasal administration of microspheres. Therefore, further investigations on both the release and degradation profile of sCT in the nasal cavity and muscular tissue are needed to clarify these points. Since gelatin has been extensively used in pharmaceutics and medicine in general, the safety of gelatin in the body is well established by its widespread clinical use. In conclusion, the gelatin microspheres have been shown to be a useful vehicle for nasal or intramuscular delivery of sCT.
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K. Morimoto et al. / European Journal of Pharmaceutical Sciences 13 (2001) 179 – 185 Nakamura, F., Ohta, R., Machida, Y., Nagai, T., 1996. In vitro and in vivo nasal mucoadhesion of some water-soluble polymers. Int. J. Pharm. 134, 173–181. Sakuma, S., Sudo, R., Suzuki, N., Kikuchi, H., Akashi, M., Hayashi, M., 1998. Mucoadhesion of polystyrene nanoparticles having surface hydrophilic polymeric chains in the gastrointestinal tract. Int. J. Pharm. 177, 161–172. Stevenson, J.C., Evans, I.M.A., 1981. Pharmacology and therapeutic use of calcitonin. Drugs 21, 257–273. Tabata, Y., Ikada, Y., 1998. Protein release from gelatin matrixes. Adv. Drug Deliv. Rev. 31, 287–301.
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Tabata, Y., Ikada, Y., 1989. Synthesis of gelatin microsphere containing interferon. Pharm. Res. 6, 422–427. Tabata, Y., Ikada, Y., Morimoto, K., Katsumata, H., Yabuta, T., Iwanaga, K., Kakemi, M., 1999. Surfactant-free preparation of biodegradable hydrogel microspheres for protein release. J. Bioactive Compat. Polymers 14, 371–384. Thamsborg, G., Jensen, J.E., Kollerup, G., Hauge, E.M., Melsen, F., Sorensen, O.H., 1996. Effect of nasal salmon calcitonin on bone remodeling and bone mass in postmenopausal osteoporosis. Bone 18, 207–212.
Evaluation of gelatin microspheres for nasal and intramuscular administrations of salmon calcitonin a,
b
b
b
Kazuhiro Morimoto *, Hideyuki Katsumata , Toshiyuki Yabuta , Kazunori Iwanaga , Masawo Kakemi b , Yasuhiko Tabata c , Yoshito Ikada c b
a Department of Pharmaceutics, Hokkaido College of Pharmacy, 7 -1 Katsuraoka-cho, Otaru-city, Hokkaido 047 -0264, Japan Department of Pharmaceutics, Osaka University of Pharmaceutical Sciences, 4 -20 -1 Nasahara, Takatsuki-city Osaka 569 -1094, Japan c Institute of Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho Shogoin, Sakyo-ku, Kyoto 606 -8507, Japan
Received 22 May 2000; received in revised form 12 December 2000; accepted 15 December 2000
Abstract The suitability of gelatin microspheres for nasal and intramuscular delivery of salmon calcitonin (sCT) was examined. Negatively and positively charged gelatin microspheres were prepared using acidic gelatin [isoelectric point (IEP) value of 5.0] and basic gelatin (IEP59.0), respectively. The average diameters of positively charged gelatin microspheres in their dried state were 3.4, 11.2, 22.5 and 71.5 mm, while that of negatively charged gelatin microspheres was 10.9 mm. Both types of gelatin microspheres were capable of adhering to the nasal mucosa. The mucoadhesion of positively charged gelatin microspheres was significantly higher than that of their negatively charged counterparts. The absorption of sCT after intranasal and intramuscular administration was evaluated by calculating the area above the hypocalcemic–time curve (AAC) in rats. The AAC values after nasal administration of sCT in positively and negatively charged gelatin microspheres were significantly greater than that in pH 7.0 PBS. Therefore, the nasal absorption of sCT was enhanced by both types of gelatin microspheres. The hypocalcemic effect after administration of sCT in positively charged gelatin microspheres of 11.2 mm was significantly greater than that of negatively charged gelatin microspheres of the same size. On the other hand, AAC values were not affected by their particle sizes. The AAC values after the intramuscular administration of sCT in positively and negatively charged gelatin microspheres were significantly increased compared to that in PBS. Furthermore, the time-courses of the plasma calcium levels differed between positively and negatively charged gelatin microspheres. The hypocalcemic effect of the negatively charged gelatin microspheres tended to appear more slowly and last longer compared to that of positively charged gelatin microspheres. The hypocalcemic effects after intramuscular administration of sCT in gelatin microspheres were not affected by their particle sizes as well as those after intranasal administration. In conclusion, the gelatin microspheres have been shown to be a useful vehicle for nasal or intramuscular delivery of sCT. 2001 Elsevier Science B.V. All rights reserved. Keywords: Gelatin, Microspheres; Nasal delivery; Intramuscular administration; Salmon calcitonin; Peptide drug
1. Introduction Calcitonin, which is a cyclic polypeptide of 32 amino acids [molecular weight (Mw )53500 Da], has a physiological role in the regulation of calcium homeostasis and is a potent inhibitor of osteoclastic bone resorption (Camilleri et al., 1991). Salmon calcitonin (sCT) is widely used because it is the most potent of the calcitonins available, it is well tolerated and is clinically effective (Stevenson and Evans, 1981). Like other peptides, sCT is easily degraded by proteolytic enzymes in the gastrointestinal tract and, *Corresponding author. Tel.: 181-134-62-1848; fax: 181-134-621848. E-mail address: [email protected] (K. Morimoto).
therefore, has a low bioavailability when administered via oral route (Sakuma et al., 1998). In addition, sCT has a short half-life (approximately 15|20 min) after parenteral administration (Huwyler et al., 1979). Thus frequent administration is required to maintain its pharmacological effects. Intranasal and pulmonary delivery of sCT has been shown to be effective (Kobayashi et al., 1994; Morimoto et al., 1995). In particularly, it has been reported that once daily dosing of sCT via the nasal route has decreased the osteoclastic bone resorption in humans (Thamsborg et al., 1996). However, the nasal and pulmonary bioavailability of sCT is lower than that following intramuscular and subcutaneous injection. Such systems may remove the need for multiple administrations during long term treatment. Development of formulations for the delivery of sCT
0928-0987 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0928-0987( 01 )00094-X
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is important for more effective and safer therapy. The absorption of sCT has to be enhanced by using a special formulation. Acidic and basic gelatins are denatured, biodegradable proteins obtained by the alkaline and acidic treatment of collagen, respectively (Tabata and Ikada, 1998). This treatment affects the electrical nature of collagen, yielding gelatin with different isoelectric point (IEP) values. The negatively and positively charged gelatin microspheres are prepared using acidic gelatin (IEP55.0) and basic gelatin (IEP59.0), respectively. We have previously reported the characteristics of sCT release from positively and negatively charged gelatin microspheres and the high bioavailability after pulmonary administration of sCT in the gelatin microsphere (Morimoto et al., 2000). In the present study, to solve the delivery problem of sCT, the negatively and positively charged gelatin microspheres of various particle sizes as suspension forms of hydrogel by swelling in pH 7.0 PBS were designed for nasal and intramuscular delivery. The absorption of sCT after nasal and intramuscular administration in gelatin microspheres was evaluated in rats.
2. Experimental procedures
2.1. Materials sCT (24.0 U / mg, purity598.1%) was obtained from Bachem Feinchemikalien AG (Switzerland). Acidic and basic gelatins (Mw 599 kDa) with IEPs of 5.0 and 9.0, respectively, were kindly supplied from Nitta Gelatin Co., Ltd (Osaka, Japan). Rhodamine isothiocyanate (RITC; purity599%) was purchased from Wako Pure Chemical Industries (Osaka, Japan). All other chemicals were of the highest purity available commercially.
2.2. Preparation of gelatin microspheres Gelatin microspheres with diameters ranging from 20 to 100 mm were prepared by glutaraldehyde crosslinking of an aqueous gelatin solution dispersed in an oil phase in the absence of a surfactant according to the modified method by Tabata and Ikada (1989). Briefly, aqueous acidic or basic gelatin solution (10%) preheated at 408C was added drop-wise to olive oil and the mixture was stirred at 420 rev / min at 408C for 10 min to yield a water-in-oil (w / o) emulsion. Stirring was continued for a further 10 min at 158C, and the microspheres were washed three times with acetone and isopropanol, respectively, by centrifugation (3000 rev / min, 5 min, 48C). After being air-dried, the microspheres were sized by passing them through sieves of different apertures. Gelatin microspheres with diameters less than 20 mm were prepared using sonication to reduce the size of the w / o emulsion (Tabata et al., 1999). Briefly, the mixture of
10% aqueous gelatin solution (0.2 ml) and olive oil (5 ml) was preheated and agitated with a vortex mixer at 408C for 1 min, followed by ultrasonication at 3.0 W/ cm for different time periods up to 40 s. The emulsion was agitated for 15 min at 48C. Then, as described above, the resulting microspheres were washed and dried. The non-crosslinked and dried gelatin microspheres were dispersed in 5 ml of aqueous glutaraldehyde solution (7.5 mg / ml; 25%) at 48C for 15 h to allow crosslinking. The microspheres were further agitated in a 10 mM aqueous glycine solution at 378C for 1 h to block residual aldehyde groups of unreacted glutaraldehyde. The resulting microspheres were finally washed three times with doubledistilled water by centrifugation and freeze-dried. The water content of the prepared gelatin microspheres was calculated from their volume before and after swelling in phosphate buffered saline (pH 7.0 PBS) for 24 h at 378C. At least 200 of the freeze-dried microspheres were examined by light microscopy to measure their diameters from which the volume of the respective microspheres was calculated. The average diameters of the positively charged gelatin microspheres in the dried state were 3.460.06, 11.260.20, 22.560.41 or 71.561.59 mm, while that of the negatively charged gelatin microspheres was 10.960.24 mm. For the incorporation of sCT into the freeze-dried gelatin microspheres for nasal and intramuscular administrations, 0.1 ml of sCT solutions in pH 7.0 PBS (2250 and 150 U / ml) were dropped onto 10.5 mg of freeze-dried gelatin microspheres, respectively. These were left at 48C for 3 h to allow the solution to penetrate into the microspheres. In this study, approximately 100% of the sCT aqueous solution was allowed to impregnate in the microspheres during the swelling process because the solution volume was less than that which would theoretically impregnate into each microsphere. In fact, the adsorbed amounts of sCT into the surface of the microsphere were less than 2.0% and were not significant between the positively and negatively charged gelatin microspheres. sCT incorporated gelatin microspheres were suspended in 1.4 ml of PBS. The final concentration of gelatin and sCT in the gelatin microsphere preparation for nasal administrations was 7 mg / ml and 150 U / ml, respectively. For intramuscular administrations, the final concentrations were 7 mg / ml and 5.0 U / ml, respectively.
2.3. Nasal and intramuscular administration Male Wistar rats weighing 210–300 g, were fasted for 20 h prior to an experiment, but allowed free access to water. The rats were anesthetized by an intraperitoneal injection of sodium pentobarbital (7.0 mg / kg) with additional doses given intraperitoneally as necessary during the experiments. Nasal administration of sCT preparation was performed according to the method of Hirai et al. (1981).
K. Morimoto et al. / European Journal of Pharmaceutical Sciences 13 (2001) 179 – 185
The sCT gelatin microsphere preparations as the suspension form of hydrogel by swelling in pH 7.0 PBS and the sCT solution in pH 7.0 PBS (dosage volume50.1 ml / kg body weight) were administered to the nasal cavity through a tube by means of micropipette inserted into the nostril at a sCT dose of 15.0 U / kg. In addition, the sCT gelatin microsphere preparations as the suspension form of hydrogel by swelling in pH 7.0 PBS or the sCT solution in pH 7.0 PBS (dosage volume50.2 ml / kg body weight) were administered intramuscularly (i.m.) to separate groups of rats at a sCT dose of 1.0 U / kg. The blood samples were periodically withdrawn from the jugular vein. The absorption of sCT was evaluated by monitoring the hypocalcemic effects. Plasma calcium levels were determined using the Calcium C test Wako (Wako Pure Chemical Industries, Osaka, Japan). The area surrounding the hypocalcemic effect–time curve (0–6 h) and the line showing 100% of plasma calcium level (% of initial) was calculated by means of trapezoidal integration. This area was defined as the area above the hypocalcemic effect– time curve (AAC) and was used for evaluating the sCT absorption.
2.4. Adhesion of gelatin microspheres to nasal mucosa Gelatin microspheres labeled with RITC were prepared to measure the mucoadhesion of gelatin microspheres to the nasal mucosa as previously described (Morimoto et al., 2000). An in situ nasal perfusion experiment was performed according to the method previously described (Iwanaga et al., 2000). Briefly, under pentobarbital anesthesia, the trachea and esophagus of rats (body weight5 250–300 g) were incised and nasal cavity was cannulated from its posterior site. The RITC-labeled gelatin microspheres suspended in PBS were administered (dosage volume50.1 ml / kg body weight) to the nasal cavity through the nostrils. Thirty minutes after nasal administration, the nasal cavity was perfused with pH 7.0 PBS from its posterior site through the cannula for 10 min (378C, flow-rate50.2 ml / min). The effluent from the nostrils was collected for 10 min. The drug amount recovered in this effluent was measured and determined as the ‘non-adhering fraction’. Immediately after perfusion, the rats were decapitated and the nasal mucosa was exposed. The surface of the nasal mucosa was washed with 20 ml of PBS and the amount of drug recovered in this medium was measured and determined as the ‘adhering fraction’. The concentration of RITC in the samples was measured by fluorospectrometry after being dissolved with 1 N HCl. Preliminarily, we confirmed the reasonability of this experimental procedure by using fluorescein isothiocyanate dextran (Mw 540,000) solution as a non-absorbable and non-adhering solution. In each experiment, the total amount of RITC recovered (non-adhering fraction plus adhering fraction) was approximately 100%.
181
2.5. Data analysis All data were expressed as mean6S.E. (standard errors of the mean). Comparisons between the group means were evaluated by the unpaired t-test. The statistical significance of differences among more than two groups were determined by one-way ANOVA. When F-tests from ANOVA indicated significant differences (P,0.05), a multiple comparison was performed to compare group means by the Bonferroni t-procedure.
3. Results
3.1. Nasal administration of sCT gelatin microspheres Nasal absorption of sCT from the positively and negatively charged gelatin microspheres of various particle sizes suspended in pH 7.0 PBS was estimated in the anesthetized rat model. Fig. 1 shows the time-course of the hypocalcemic effects in rats after nasal administration of sCT (15.0 U / kg) in positively and negatively charged gelatin microspheres (particle sizes510.9 and 11.2 mm, respectively). The hypocalcemic effects after administration of sCT in positively and negatively charged gelatin microspheres were significantly greater than that following administration in the aqueous solution (in pH 7.0 PBS). The nasal administration of sCT in the positively charged gelatin microspheres has significantly greater hypocalcemic effects than that after administration in the negatively charged gelatin microspheres as shown in Table 1. Fig. 2 shows the time-course of hypocalcemic effects in
Fig. 1. Time-course of hypocalcemic effects after nasal administration of sCT (15 U / kg) in positively and negatively charged gelatin microspheres (particle sizes510.9 and 11.2 mm, respectively) in rats. Solution (j, N56); positively charged microspheres of 11.2 mm (n; N55); negatively charged microspheres of 10.9 mm (m, N54). Each point represents the mean6S.E.
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Table 1 Area above the hypocalcemic effect–time curve (AAC) after nasal and intramuscular administration of sCT in rats a Dosage formulations
Nasal administration Solution Negatively charged gelatin microsphere Positively charged gelatin microsphere
Intramuscular administration Solution Negatively charged gelatin microsphere Positively charged gelatin microsphere
Particle size (mm)
–
N
AAC (% of initial level3min)
6
1933.06417.5
rats after nasal administration of sCT (15.0 U / kg) in positively charged gelatin microspheres of various particle sizes (3.4, 11.2, 22.5 and 71.2 mm). The hypocalcemic effects after nasal administration of sCT in the gelatin microspheres were unaffected by the particle size (Table 1).
3.2. Adhesion of gelatin microspheres to nasal mucosa b
10.9
4
4075.66782.6
3.4 11.2 22.5 71.2
5 6 5 6
6467.161614.0 b 5906.361356.4 b,c 5687.661154.1 b 5988.961308.7 b
–
6
1999.96338.9
10.9
7
3056.46577.9 d
3.4 11.2 22.5 71.2
4 4 4 4
3764.16690.7 d 2791.56210.5 d 2561.26351.0 d 2936.36414.8 d
Mucoadhesion of RITC labeled negatively and positively charged gelatin microspheres (particle sizes510.9 and 11.2 mm, respectively) was examined by in situ nasal perfusion experiments in rats. As shown in Table 2, the amount of positively charged gelatin microspheres recovered in the adhering fraction was significantly higher than that of negatively charged gelatin microspheres, indicating that the mucoadhesion ability of positively charged gelatin microspheres was greater than that of negatively charged ones.
3.3. Intramuscular administration of sCT gelatin microspheres
a
Doses of sCT of nasal and intramuscular administration were 15.0 and 1.0 U / kg, respectively. No significant differences were observed between the negatively (10.9 mm) and positively (11.2 mm) charged gelatin microspheres after i.n. and i.m. administration. No significant differences were observed when compared among gelatin microspheres of various sizes after i.n. and i.m administration. Each value represents the mean6S.E. b P,0.05 vs. nasal administration of sCT in solution. c P,0.05 vs. nasal administration of negatively charged gelatin microspheres. d P,0.05 vs. i.m. administration of sCT in solution.
The hypocalcemic effects after intramuscular administration of sCT in positively and negatively charged gelatin microspheres of various particle sizes suspended in pH 7.0 PBS were estimated in the anesthetized rat model. Fig. 3 shows the time-course of hypocalcemic effects in rats after intramuscular administration of sCT (1.0 U / kg) in negatively and positively charged gelatin microspheres (particle sizes510.9 and 11.2 mm, respectively). The hypocalcemic effects after intramuscular administration of sCT in positively and negatively charged gelatin microspheres were significantly greater than that produced following administration in the aqueous solution (in pH 7.0 PBS). The administration of sCT in negatively charged gelatin microspheres tended to appear more slowly and lasted longer compared to that in positively charged microspheres. Fig. 4 shows the time-course of the hypocalcemic effects in rats after intramuscular administration of sCT (1.0 U / kg) in positively charged gelatin microspheres of
Table 2 Comparison of adhesion ability to nasal mucosa between positively and negatively charged gelatin microspheres a Dosage formulations
Fig. 2. Time-course of hypocalcemic effects after nasal administration of sCT (15 U / kg) in positively charged gelatin microspheres of various particle sizes (3.4, 11.2, 22.5 and 71.2 mm) in rats. Solution (j, N56); positively charged microspheres: diameters of 3.4 mm (s, N55), 11.2 mm (n, N56), 22.5 mm (h, N55), 71.5 mm (d, N56). Each point represents the mean6S.E.
Negatively charged gelatin microsphere Positively charged gelatin microsphere a *
Particle size (mm)
N
Recovered amount of RITC from adhering fraction (% of total recovered)
10.9
5
46.062.47
11.2
5
58.662.17*
Each value represents the mean6S.E. P,0.01 vs. negatively charged gelatin microspheres.
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4. Discussion
Fig. 3. Time-course of hypocalcemic effects after intramuscular administration of sCT (1.0 U / kg) in negatively and positively charged gelatin microspheres (particle sizes510.9 and 11.2 mm, respectively) in rats. Solution (j, N56); positively charged microspheres of 11.2 mm (n, N54); negatively charged microspheres of 10.9 mm (m, N57). Each point represents the mean6S.E.
various particle sizes (3.4, 11.2, 22.5 and 71.2 mm). The hypocalcemic effect after intramuscular administration of sCT in gelatin microspheres was unaffected by the particle size.
Fig. 4. Time-course of hypocalcemic effects after intramuscular administration of sCT (1.0 U / kg) in positively charged gelatin microspheres of various particle sizes (3.4, 11.2, 22.5 and 71.2 mm) in rats. Solution (j, N56); positively charged microspheres: diameters of 3.4 mm (s, N54), 11.2 mm (n, N54), 22.5 mm (h, N54), 71.5 mm (d, N54). Each point represents the mean6S.E.
The negatively and positively charged gelatin microspheres of a wide range of particle sizes (3.4|71.2 mm) were prepared using acidic and basic gelatin for nasal and intramuscular delivery of sCT as a peptide drug. The feature of the particular formulations as a drug delivery system including microspheres is defined by two characteristics, the rate of drug release and the degradation profile. Gelatin is a biodegradable and biocompatible polymer and gelatin microspheres are degraded by enzymatic (trypsin, pepsin and collagenase) hydrolysis in the body (Tabata and Ikada, 1998). We previously reported that the gelatin microspheres slightly underwent enzymatic degradation, when incubated in the bronchoalveolar lavage fluid (Morimoto et al., 2000). Thus, the degradation profile of the formulation is generally determined by the materials composed of the formulation. On the other hand, the release rate of a drug from particular formulations is thought vary with their composition, size, charge, interaction with the drug incorporated and so on. In our previous study, sCT was rapidly (within 2 h) released from gelatin microspheres in pH 7.0 PBS, thereafter, the release of sCT was leveled off and no sustained release was observed. And the release rate was not affected by the size of the microspheres. Furthermore, we clarified that the release rate of sCT from positively charged gelatin microspheres was much higher than that from negatively charged ones and this difference in release rate was caused by the electrostatic interactions between the microspheres and sCT (Morimoto et al., 2000). In this study, the absorption enhancing effects of sCT after intranasal and intramuscular administration of gelatin microspheres were evaluated in the anesthetized rat model. The AAC values after nasal administration of sCT in gelatin microspheres were significantly higher than that of sCT in PBS in regardless of their charges, indicating that the gelatin microspheres enhanced the nasal absorption of sCT compared to sCT solution (Fig. 1 and Table 1). However, this enhancing effect of gelatin microspheres was only observed within 2 h of administration and the enhancing effect of positively charged gelatin microspheres tended to be greater than that of the negatively charged ones. These results were consistent with the release profile of sCT from gelatin microspheres in PBS. Furthermore, the fact that the size of microspheres did not affect the hypocalcemic effect (Fig. 2) also agreed with the result obtained from in vitro release experiments. Therefore, the high concentration of sCT at the mucosal surface of the nasal membrane, which was induced by the release from the microspheres, may be one of the important factors responsible for increased sCT absorption produced by gelatin microspheres. Besides the release rate of the drug, the mucoadhesion ability is also thought to be an important factor in defining the potential mucosal dosage formulation. The mucoadhe-
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sion of positively charged gelatin microspheres with a mean particle size of 11.2 mm was significantly higher than that of negatively charged gelatin microspheres of the approximately same size (Table 2). It has been reported that some mucoadhesive microspheres improved the nasal delivery of poorly absorbed drugs such as peptide drugs (Illum et al., 1987; Edman et al., 1992; Morath, 1998). However, these microspheres were the dry type and the absorption enhancing mechanism by these microspheres was different from that of our microspheres of the full swollen type. We also reported that mucoadhesive preparations made with Carbopol (Morimoto et al., 1984) and hyaluronate (Morimoto et al., 1991) increased the nasal and rectal absorption of peptide drugs. It is generally accepted that mucoadhesive preparations can increase the drug concentration in the vicinity of the mucosal cells by increasing contact between drugs and the mucosa; consequently, enhancing the drug absorption (Nakamura et al., 1996; Sakuma et al., 1998). Therefore, the high concentrations of sCT in the superficial mucosa of the nasal cavity, which was induced by the initial release from the microspheres, are also important factors in enhancing sCT absorption by gelatin microspheres. However, the mucociliary clearance is important problem to solve because it is normally less than 20 min and decreases with anesthesia and surgical operation. Therefore, we are now investigating the effect of mucociliary clearance on the absorption enhancing effects of the gelatin microspheres using conscious or non-operated conditions. We also considered that absorption of sCT after nasal administration might be attributable to avoiding enzymatic degradation in the nasal cavity. sCT was degraded by trypsin-like enzymes when intranasally administered as previously reported (Morimoto et al., 1995). The gelatin microspheres may have a stabilizing effect on the enzymatic degradation of sCT. As the enzymatic degradation of gelatin microspheres in the rats bronchoalveolar fluids was slight (Morimoto et al., 2000), it could be speculated that the gelatin microspheres may stabilize sCT against the enzymes in the nasal cavity. However, it is unclear which factor contributes to enhancing the nasal absorption of sCT – the high concentration of sCT in the vicinity of nasal mucosa or the stabilizing sCT – from these data. We are now conducting investigations to clarify this point. Intramuscular administration of sCT in both gelatin microspheres enhanced the absorption of sCT compared to that in PBS in regardless of both their charges and sizes (Figs. 3 and 4 and Table 1). When sCT was intramuscularly injected as PBS solution, sCT would spread widely from the injected site. On the other hand, when sCT was injected as microsphere formulations, the distribution of sCT was considered to be limited regardless of their charges and sizes because the microspheres formed a depot at the injected site. Therefore, the stability of sCT to enzymes after intramuscular administration of microspheres formulations was considerably improved compared
to that of the PBS formulation, resulting in enhanced sCT absorption. However, the hypocalcemic effects after intramuscular administration of sCT in negatively charged gelatin microspheres were slower to appear than with the positively charged one, although no significant differences in AAC values were observed between these two microspheres (Fig. 3 and Table 2). The difference in the pattern of hypocalcemic effects between the positively and negatively charged gelatin microspheres might be caused by the difference in the release profile of sCT in the muscular tissue between these gelatin microspheres. However, this phenomenon was not observed in the case of the intranasal administration of microspheres. Therefore, further investigations on both the release and degradation profile of sCT in the nasal cavity and muscular tissue are needed to clarify these points. Since gelatin has been extensively used in pharmaceutics and medicine in general, the safety of gelatin in the body is well established by its widespread clinical use. In conclusion, the gelatin microspheres have been shown to be a useful vehicle for nasal or intramuscular delivery of sCT.
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