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Immunological effects of microneedle-mediated insulin delivery: Preliminary rat studies
International Journal of Pharmaceutics 444 (2013) 103–105
Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Note
Immunological effects of microneedle-mediated insulin delivery: Preliminary rat studies Yuqin Qiu, Yunhua Gao ∗ , Suohui Zhang, Lei Guo, Jianmin Chen, Bai Xu Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China
a r t i c l e
i n f o
Article history: Received 26 October 2012 Accepted 11 January 2013 Available online 20 January 2013 Keywords: Insulin Microneedles Immunogenicity Intradermal delivery
a b s t r a c t The objective of the present study is to compare the immunogenicity of insulin by microneedle-mediated intradermal delivery or subcutaneous injection. Female and male rats were treated with insulin by either microneedle-mediated intradermal delivery or subcutaneous injection twice a week for 4 weeks. A control group without insulin administration was also studied. Human anti-insulin antibody (AIA) levels were measured by radioimmunoassay (RIA) before starting insulin treatment and after that within 20 weeks. The male rats did not induce positive AIA levels, and there was no significant difference between all male groups. In contrast, in female rats the AIA levels were significantly higher in microneedle group compared with injection group, and lasted from 6 to 20 weeks after starting insulin treatment. The increased immunogenicity of insulin delivery by microneedle might be due to the large number of immune cells in skin. © 2013 Elsevier B.V. All rights reserved.
Microneedles are needle-like structures with diameters in the size order of microns and lengths up to 1 mm. These structures are used to pierce the upper layer of the skin in a non-invasive and painless way to enable (trans)dermal drug delivery. Recently, microneedles have become the promising route for the (trans)dermal delivery of macromolecules, such as vaccines and therapeutic proteins (van der Maaden et al., 2012). The skin is an excellent organ for vaccination because of the large number of immune cells (Amorij et al., 2012). Not surprisingly, microneedle-based intradermal immunization has shown to be more dose effective than conventional intramuscular and subcutaneous immunization in both human and animal studies (Chen et al., 2011; Van Damme et al., 2009). Whereas vaccines should be targeted to immune cells of skin, for therapeutic proteins the immunological effects are unwanted. Unwanted immunogenicity can lead to total loss of the therapeutic effect of a protein by neutralizing antibodies, and may even lead to depletion of endogenous or breaking the immune-tolerance to selfantigens (Singh, 2011). Since the skin is a potent immune organ, the unwanted protein immunogenicity is perhaps one of the biggest safety concerns. However, to our knowledge, still little is known about the unwanted immunogenicity of therapeutic protein via the skin surface by means of microneedle delivery technology. Recombinant DNA human insulin is a model protein which has been frequently studied in microneedle-based delivery (McAllister
∗ Corresponding author. Tel.: +86 10 82543581; fax: +86 10 82543581. E-mail address: [email protected] (Y. Gao). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.01.020
et al., 2003). It is composed of 51 amino acids, and has a molecular weight of 5808 Da. Insulin is stable between pH 5 and 8 as a hexamer, which means it is stored in the body as a unit of six insulin molecules. In our previous studies, sustained release of insulin through rat skin was obtained by microneedle pretreatment (Wu et al., 2010). In this work, we tested the immunogenicity of the insulin by microneedle mediated delivery compared with subcutaneous injection. Rats were used as the animal model here. Although animal models are currently not able to predict immunogenicity of biologics in humans, the characterization of the antibody responses could provide a valuable database for making judgments about the relative immunogenicity of different administration routes (Baumann, 2009; Wierda et al., 2001). What we first wanted to assess here was the potential for insulin to induce antibody production by subcutaneous injection or microneedle-based intradermal administration. Sprague Dawley rats weighing approximately 230 g at the beginning of the study were tested. Three groups were studied: control group without insulin, microneedle group with insulin solution and SC injection group, and each group contained 5 males and 5 females. Regular insulin solution named Novolin® R Penfill (100 IU/ml) was used. In the injection group, insulin diluted to 1.0 IU/ml with sterile water for injection was injected subcutaneously with a hypodermic needle. Insulin dose for subcutaneous injection was 1.5 IU/kg body weight. In the microneedle groups, rat abdominal skin was treated by microneedles as reported before (Wu et al., 2010), the treated area was 1 cm × 1 cm. A patch (cotton pad) soaked with insulin solution (50 IU/ml) was applied on the treated skin, protected with an occlusive cover. After 3 h, the patch or hydrogel was removed.
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Y. Qiu et al. / International Journal of Pharmaceutics 444 (2013) 103–105
10
(a) Control Subcutaneous injection Microneedle-solution
70
Microneedle-CP gel Microneedle-solution
60
Binding percentage %
Binding percentage %
(a)
5
50 40 30 20 10 0 0
0 0
2
4
6
2
4
6
8
8
Week Week
(b)
Control Subcutaneous injection Microneedle-solution
70 60
25
Cumulated amount of FITC-insulin (g/cm2)
Bingding percentage %
(b)
50 40 30 20 10
CP gel Solution
20
15
10
5
0 0
2
4
6
8
Week Figure 1. Antibody levels in rats immunized with insulin by microneedles or injection: (a) males and (b) females.
The doses for the injection group and microneedle group were set based on the similar effects of lowering blood glucose as previously reported (Wu et al., 2010). Two immunizations were performed every week, and the immunization lasted for 4 weeks. Three hundred microliters of blood were sampled by tail bleeding every 2 weeks since the first immunization. Anti-insulin antibodies (AIA) in the rat serum were determined by radioimmunoassay using an Iodine [125 I] insulin antibody radioimmunoassay kit (Beijing Chemclin Biotech Co., Ltd., China). The insulin antibody concentration was expressed as the percent of radioactive insulin bound to immunoglobulins compared with the total amount of radioactivity. According to the kit introduction, the binding percentage of 5% was fixed as positive threshold for humans. The Statistical Package for the Social Sciences (SPSS) software was used for the statistical analysis of the results. Specifically, the one-way ANOVA with Bongerroni’t test was used for a confidence level of 95%. The AIA levels obtained in each group are shown in Figure 1a and b. After the immunization, in the first 4 weeks, AIA levels of all groups were below the specific binding threshold both in male and female rats. After 6 weeks, in the male rats, the AIA levels were still below the threshold. Although the levels of 4-week time point of the injection group and 6-, 8-week time point of microneedle group were slightly higher than the control group, it is hard to be defined as significant. In contrast, in the female rats, the AIA levels in the microneedle group were significantly higher than the injection group and the control group (P < 0.005), and there was no significant difference between the injection group and control
0 0
1
2
3
4
5
6
Time (h) Figure 2. (a) Antibody levels in rats with microneedle delivered insulin loaded in a CP gel or solution and (b) in vitro permeation of FITC-insulin from CP gel or solution through microneedle treated rat skin.
group. Results obtained indicated more potential for insulin to induce antibody production by microneedle-based intradermal delivery compared with subcutaneous injection. Recently, Pennell et al. reviewed that sex differences affect both the innate and adaptive immune responses and females have usually higher antigenic response than males. The results in this work are consistent with the literature (Pennell et al., 2012). Further, we evaluated the effect of excipients in the formulation administered to rats after treated by microneedles to the potential of AIA induction. It was studied in 5 female rats, and all the experiment procedures were the same with solution application, except for the cotton patch soaked with insulin solution (50 IU/ml) was replaced with a carbopol 971 hydrogel (CP gel, 0.5%) loaded with insulin (50 IU/g). Figure 2a showed that after 6 weeks, the AIA levels of solution group were higher than CP gel group, the difference at 8-week time point was significant (P < 0.05). Also, we evaluated the in vitro delivery of FITC-insulin from CP gel and solution through microneedle treated rat skin, using the method described in our previous studies (Wu et al., 2010). Figure 2b shows that the penetration of FITC-insulin from CP gel was slower than that from solution. It indicated the carbopol gel blocked FITC-insulin delivery. These results indicated that the AIA levels induced by microneedle mediated insulin delivery might be dose-dependant. Carbopol 971 was used as a novel adjuvant in several reports (Dey et al., 2012). However, the large molecular size and cross-linking structure made it
Y. Qiu et al. / International Journal of Pharmaceutics 444 (2013) 103–105
might be due to the large number of immune cells in epidermis of skin. This study also suggests that it is important to evaluate immunogenicity of biological macromolecules delivered by microneedle technology.
females
70
males
Binding percentage %
105
60 50
Acknowledgments
40 30
This work was supported by the Science Foundation of the Chinese Academy of Sciences.
20
References
10
Amorij, J.P., Kersten, G.F., Saluja, V., Tonnis, W.F., Hinrichs, W.L., Slutter, B., Bal, S.M., Bouwstra, J.A., Huckriede, A., Jiskoot, W., 2012. Towards tailored vaccine delivery: needs, challenges and perspectives. J. Control. Release 161, 363–376. Baumann, A., 2009. Nonclinical development of biopharmaceuticals. Drug Discov. Today 14, 1112–1122. Chen, X., Fernando, G.J., Crichton, M.L., Flaim, C., Yukiko, S.R., Fairmaid, E.J., Corbett, H.J., Primiero, C.A., Ansaldo, A.B., Frazer, I.H., Brown, L.E., Kendall, M.A., 2011. Improving the reach of vaccines to low-resource regions, with a needle-free vaccine delivery device and long-term thermostabilization. J. Control. Release 152, 349–355. Dey, A.K., Burke, B., Sun, Y.D., Hartog, K., Heeney, J.L., Montefiori, D., Srivastava, I.K., Barnett, S.W., 2012. Use of a polyanionic carbomer, Carbopol971P, in combination with MF59, improves antibody responses to HIV-1 envelope glycoprotein. Vaccine 30, 2749–2759. McAllister, D.V., Wang, P.M., Davis, S.P., Park, J.H., Canatella, P.J., Allen, M.G., Prausnitz, M.R., 2003. Microfabricated needles for transdermal delivery of macromolecules and nanoparticles: fabrication methods and transport studies. Proc. Natl. Acad. Sci. U.S.A. 100, 13755–13760. Pennell, L.M., Galligan, C.L., Fish, E.N., 2012. Sex affects immunity. J. Autoimmun. 38, J282–J291. Singh, S.K., 2011. Impact of product-related factors on immunogenicity of biotherapeutics. J. Pharm. Sci. 100, 354–387. Van Damme, P., Oosterhuis-Kafeja, F., Van der Wielen, M., Almagor, Y., Sharon, O., Levin, Y., 2009. Safety and efficacy of a novel microneedle device for dose sparing intradermal influenza vaccination in healthy adults. Vaccine 27, 454–459. van der Maaden, K., Jiskoot, W., Bouwstra, J., 2012. Microneedle technologies for (trans)dermal drug and vaccine delivery. J. Control. Release 161, 645–655. Wierda, D., Smith, H.W., Zwickl, C.M., 2001. Immunogenicity of biopharmaceuticals in laboratory animals. Toxicology 158, 71–74. Wu, Y., Gao, Y., Qin, G., Zhang, S., Qiu, Y., Li, F., Xu, B., 2010. Sustained release of insulin through skin by intradermal microdelivery system. Biomed. Microdevices 12, 665–671.
0 0
2
4
6
8
10
12
14
16
18
20
Week Figure 3. Kinetics of AIA levels in rats immunized with insulin delivery by microneedles.
difficult to penetrate through the skin. Therefore, the dose effect was the dominant factor here. The kinetics of antibody production has been studied with the microneedle- solution group, as it shown in Figure 3. Insulin was administered for 4 weeks, during this period, the AIA levels were below the specific binding threshold. There was a rapid increase within 4–8 weeks in the female rats, and reached the maximum value (57.3 ± 13.3)% at the 8-week time point. After that, the level rapidly declined to (19.6 ± 3.2)% at the 10-week time point. The levels fell back toward the normal within 20 weeks. In conclusion, the results of our studies suggest that the production of insulin antibodies depends on the administration route. Compared with subcutaneous injection, the microneedle delivery provided more potential to produce AIA. Further, the production of AIA by microneedle delivery might be dose-dependant. The increased immunogenicity of insulin delivery by microneedles
Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Note
Immunological effects of microneedle-mediated insulin delivery: Preliminary rat studies Yuqin Qiu, Yunhua Gao ∗ , Suohui Zhang, Lei Guo, Jianmin Chen, Bai Xu Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China
a r t i c l e
i n f o
Article history: Received 26 October 2012 Accepted 11 January 2013 Available online 20 January 2013 Keywords: Insulin Microneedles Immunogenicity Intradermal delivery
a b s t r a c t The objective of the present study is to compare the immunogenicity of insulin by microneedle-mediated intradermal delivery or subcutaneous injection. Female and male rats were treated with insulin by either microneedle-mediated intradermal delivery or subcutaneous injection twice a week for 4 weeks. A control group without insulin administration was also studied. Human anti-insulin antibody (AIA) levels were measured by radioimmunoassay (RIA) before starting insulin treatment and after that within 20 weeks. The male rats did not induce positive AIA levels, and there was no significant difference between all male groups. In contrast, in female rats the AIA levels were significantly higher in microneedle group compared with injection group, and lasted from 6 to 20 weeks after starting insulin treatment. The increased immunogenicity of insulin delivery by microneedle might be due to the large number of immune cells in skin. © 2013 Elsevier B.V. All rights reserved.
Microneedles are needle-like structures with diameters in the size order of microns and lengths up to 1 mm. These structures are used to pierce the upper layer of the skin in a non-invasive and painless way to enable (trans)dermal drug delivery. Recently, microneedles have become the promising route for the (trans)dermal delivery of macromolecules, such as vaccines and therapeutic proteins (van der Maaden et al., 2012). The skin is an excellent organ for vaccination because of the large number of immune cells (Amorij et al., 2012). Not surprisingly, microneedle-based intradermal immunization has shown to be more dose effective than conventional intramuscular and subcutaneous immunization in both human and animal studies (Chen et al., 2011; Van Damme et al., 2009). Whereas vaccines should be targeted to immune cells of skin, for therapeutic proteins the immunological effects are unwanted. Unwanted immunogenicity can lead to total loss of the therapeutic effect of a protein by neutralizing antibodies, and may even lead to depletion of endogenous or breaking the immune-tolerance to selfantigens (Singh, 2011). Since the skin is a potent immune organ, the unwanted protein immunogenicity is perhaps one of the biggest safety concerns. However, to our knowledge, still little is known about the unwanted immunogenicity of therapeutic protein via the skin surface by means of microneedle delivery technology. Recombinant DNA human insulin is a model protein which has been frequently studied in microneedle-based delivery (McAllister
∗ Corresponding author. Tel.: +86 10 82543581; fax: +86 10 82543581. E-mail address: [email protected] (Y. Gao). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.01.020
et al., 2003). It is composed of 51 amino acids, and has a molecular weight of 5808 Da. Insulin is stable between pH 5 and 8 as a hexamer, which means it is stored in the body as a unit of six insulin molecules. In our previous studies, sustained release of insulin through rat skin was obtained by microneedle pretreatment (Wu et al., 2010). In this work, we tested the immunogenicity of the insulin by microneedle mediated delivery compared with subcutaneous injection. Rats were used as the animal model here. Although animal models are currently not able to predict immunogenicity of biologics in humans, the characterization of the antibody responses could provide a valuable database for making judgments about the relative immunogenicity of different administration routes (Baumann, 2009; Wierda et al., 2001). What we first wanted to assess here was the potential for insulin to induce antibody production by subcutaneous injection or microneedle-based intradermal administration. Sprague Dawley rats weighing approximately 230 g at the beginning of the study were tested. Three groups were studied: control group without insulin, microneedle group with insulin solution and SC injection group, and each group contained 5 males and 5 females. Regular insulin solution named Novolin® R Penfill (100 IU/ml) was used. In the injection group, insulin diluted to 1.0 IU/ml with sterile water for injection was injected subcutaneously with a hypodermic needle. Insulin dose for subcutaneous injection was 1.5 IU/kg body weight. In the microneedle groups, rat abdominal skin was treated by microneedles as reported before (Wu et al., 2010), the treated area was 1 cm × 1 cm. A patch (cotton pad) soaked with insulin solution (50 IU/ml) was applied on the treated skin, protected with an occlusive cover. After 3 h, the patch or hydrogel was removed.
104
Y. Qiu et al. / International Journal of Pharmaceutics 444 (2013) 103–105
10
(a) Control Subcutaneous injection Microneedle-solution
70
Microneedle-CP gel Microneedle-solution
60
Binding percentage %
Binding percentage %
(a)
5
50 40 30 20 10 0 0
0 0
2
4
6
2
4
6
8
8
Week Week
(b)
Control Subcutaneous injection Microneedle-solution
70 60
25
Cumulated amount of FITC-insulin (g/cm2)
Bingding percentage %
(b)
50 40 30 20 10
CP gel Solution
20
15
10
5
0 0
2
4
6
8
Week Figure 1. Antibody levels in rats immunized with insulin by microneedles or injection: (a) males and (b) females.
The doses for the injection group and microneedle group were set based on the similar effects of lowering blood glucose as previously reported (Wu et al., 2010). Two immunizations were performed every week, and the immunization lasted for 4 weeks. Three hundred microliters of blood were sampled by tail bleeding every 2 weeks since the first immunization. Anti-insulin antibodies (AIA) in the rat serum were determined by radioimmunoassay using an Iodine [125 I] insulin antibody radioimmunoassay kit (Beijing Chemclin Biotech Co., Ltd., China). The insulin antibody concentration was expressed as the percent of radioactive insulin bound to immunoglobulins compared with the total amount of radioactivity. According to the kit introduction, the binding percentage of 5% was fixed as positive threshold for humans. The Statistical Package for the Social Sciences (SPSS) software was used for the statistical analysis of the results. Specifically, the one-way ANOVA with Bongerroni’t test was used for a confidence level of 95%. The AIA levels obtained in each group are shown in Figure 1a and b. After the immunization, in the first 4 weeks, AIA levels of all groups were below the specific binding threshold both in male and female rats. After 6 weeks, in the male rats, the AIA levels were still below the threshold. Although the levels of 4-week time point of the injection group and 6-, 8-week time point of microneedle group were slightly higher than the control group, it is hard to be defined as significant. In contrast, in the female rats, the AIA levels in the microneedle group were significantly higher than the injection group and the control group (P < 0.005), and there was no significant difference between the injection group and control
0 0
1
2
3
4
5
6
Time (h) Figure 2. (a) Antibody levels in rats with microneedle delivered insulin loaded in a CP gel or solution and (b) in vitro permeation of FITC-insulin from CP gel or solution through microneedle treated rat skin.
group. Results obtained indicated more potential for insulin to induce antibody production by microneedle-based intradermal delivery compared with subcutaneous injection. Recently, Pennell et al. reviewed that sex differences affect both the innate and adaptive immune responses and females have usually higher antigenic response than males. The results in this work are consistent with the literature (Pennell et al., 2012). Further, we evaluated the effect of excipients in the formulation administered to rats after treated by microneedles to the potential of AIA induction. It was studied in 5 female rats, and all the experiment procedures were the same with solution application, except for the cotton patch soaked with insulin solution (50 IU/ml) was replaced with a carbopol 971 hydrogel (CP gel, 0.5%) loaded with insulin (50 IU/g). Figure 2a showed that after 6 weeks, the AIA levels of solution group were higher than CP gel group, the difference at 8-week time point was significant (P < 0.05). Also, we evaluated the in vitro delivery of FITC-insulin from CP gel and solution through microneedle treated rat skin, using the method described in our previous studies (Wu et al., 2010). Figure 2b shows that the penetration of FITC-insulin from CP gel was slower than that from solution. It indicated the carbopol gel blocked FITC-insulin delivery. These results indicated that the AIA levels induced by microneedle mediated insulin delivery might be dose-dependant. Carbopol 971 was used as a novel adjuvant in several reports (Dey et al., 2012). However, the large molecular size and cross-linking structure made it
Y. Qiu et al. / International Journal of Pharmaceutics 444 (2013) 103–105
might be due to the large number of immune cells in epidermis of skin. This study also suggests that it is important to evaluate immunogenicity of biological macromolecules delivered by microneedle technology.
females
70
males
Binding percentage %
105
60 50
Acknowledgments
40 30
This work was supported by the Science Foundation of the Chinese Academy of Sciences.
20
References
10
Amorij, J.P., Kersten, G.F., Saluja, V., Tonnis, W.F., Hinrichs, W.L., Slutter, B., Bal, S.M., Bouwstra, J.A., Huckriede, A., Jiskoot, W., 2012. Towards tailored vaccine delivery: needs, challenges and perspectives. J. Control. Release 161, 363–376. Baumann, A., 2009. Nonclinical development of biopharmaceuticals. Drug Discov. Today 14, 1112–1122. Chen, X., Fernando, G.J., Crichton, M.L., Flaim, C., Yukiko, S.R., Fairmaid, E.J., Corbett, H.J., Primiero, C.A., Ansaldo, A.B., Frazer, I.H., Brown, L.E., Kendall, M.A., 2011. Improving the reach of vaccines to low-resource regions, with a needle-free vaccine delivery device and long-term thermostabilization. J. Control. Release 152, 349–355. Dey, A.K., Burke, B., Sun, Y.D., Hartog, K., Heeney, J.L., Montefiori, D., Srivastava, I.K., Barnett, S.W., 2012. Use of a polyanionic carbomer, Carbopol971P, in combination with MF59, improves antibody responses to HIV-1 envelope glycoprotein. Vaccine 30, 2749–2759. McAllister, D.V., Wang, P.M., Davis, S.P., Park, J.H., Canatella, P.J., Allen, M.G., Prausnitz, M.R., 2003. Microfabricated needles for transdermal delivery of macromolecules and nanoparticles: fabrication methods and transport studies. Proc. Natl. Acad. Sci. U.S.A. 100, 13755–13760. Pennell, L.M., Galligan, C.L., Fish, E.N., 2012. Sex affects immunity. J. Autoimmun. 38, J282–J291. Singh, S.K., 2011. Impact of product-related factors on immunogenicity of biotherapeutics. J. Pharm. Sci. 100, 354–387. Van Damme, P., Oosterhuis-Kafeja, F., Van der Wielen, M., Almagor, Y., Sharon, O., Levin, Y., 2009. Safety and efficacy of a novel microneedle device for dose sparing intradermal influenza vaccination in healthy adults. Vaccine 27, 454–459. van der Maaden, K., Jiskoot, W., Bouwstra, J., 2012. Microneedle technologies for (trans)dermal drug and vaccine delivery. J. Control. Release 161, 645–655. Wierda, D., Smith, H.W., Zwickl, C.M., 2001. Immunogenicity of biopharmaceuticals in laboratory animals. Toxicology 158, 71–74. Wu, Y., Gao, Y., Qin, G., Zhang, S., Qiu, Y., Li, F., Xu, B., 2010. Sustained release of insulin through skin by intradermal microdelivery system. Biomed. Microdevices 12, 665–671.
0 0
2
4
6
8
10
12
14
16
18
20
Week Figure 3. Kinetics of AIA levels in rats immunized with insulin delivery by microneedles.
difficult to penetrate through the skin. Therefore, the dose effect was the dominant factor here. The kinetics of antibody production has been studied with the microneedle- solution group, as it shown in Figure 3. Insulin was administered for 4 weeks, during this period, the AIA levels were below the specific binding threshold. There was a rapid increase within 4–8 weeks in the female rats, and reached the maximum value (57.3 ± 13.3)% at the 8-week time point. After that, the level rapidly declined to (19.6 ± 3.2)% at the 10-week time point. The levels fell back toward the normal within 20 weeks. In conclusion, the results of our studies suggest that the production of insulin antibodies depends on the administration route. Compared with subcutaneous injection, the microneedle delivery provided more potential to produce AIA. Further, the production of AIA by microneedle delivery might be dose-dependant. The increased immunogenicity of insulin delivery by microneedles