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Does Increasing the Lipophilicity of Peptides Enhance Their Nasal Absorption?
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Does Increasing the Lipophilicity of Peptides Enhance Their Nasal Absorption? To the Editor: Previous communications from our laboratory and others concluded that the low bioavailabilities of nasally administered peptides are due, in art, to peptidase-catalyzed hydrolysis in the nasal cavity."We and others have demonstrated the existence of peptidase enzyme systems in the nasal cavities of both animals and humans,14 and we have shown that the types of hydrolyses produced by these systems may vary greatly among species6 On the other hand, it has been demonstrated that the small amounts of peptides that cross the nasal membrane in rats do so at a very rapid rate.'s8 Thus, the systemic bioavailability of a nasally administered peptide is the result of two rapid and competing kinetic processes: absorption and hydrolysis.' It should be possible to minimize hydrolysis and improve bioavailability by accelerating the absorption process so that a greater fraction of the peptide follows the absorption pathway. Some workers have attempted to accomplish this through the use of absorption e n h a n c e r ~ , ~but * l ~many of these compounds are irritating to the nasal mucosa.ll Another approach is to increase the lipophilicity of the peptide so as to improve its penetration into the nasal membrane. This communication reports on the comparative partition coefficients, in vitro hydrolysis rates, and the rates of disappearance from rat nasal cavity of the small peptide L-tyrosylL-tyrosine (LTLT) and its methyl ester (LTLTME). The analytical method was similar to those described previously,'r5v6 and the rates of hydrolysis of the compounds were determined in washings from the rat nasal cavity using a technique described previously.' Rates of disappearance of the compounds from the rat nasal cavity were determined using the in vivo-in situ technique described previously.' The oil-water partition coefficientsin the n-octanol: pH 7.4 phosphate buffer system were 0.02 for LTLT and 3.2 for LTLTME. The rates of the hydrolyses of LTLT and LTLTME
in rat nasal washings were very similar (see Figure 11, suggesting that the peptidase enzyme systems of the rat nasal cavity have about the same affinity for the two compounds. This also suggests that hydrolysis could be responsible for significant loss of the compounds from the rat nasal cavity during the in vivo-in situ experiments. It has previously been shown that the rates of hydrolysis of peptides, in particular LTLT, in rat nasal enzyme systems are concentration d e ~ e n d e n t . ~Therefore, .~' we studied the disappearance of LTLTME from the rat nasal cavity at various concentrations to determine the most advantageous concentration(s) a t which to compare the absorption rates of the two compounds. The results shown in Figure 2 indicate that the disappearance of LTLTME from the rat nasal cavity was slowest at 40 mM and was almost as slow at 8 mM. At 40 mM, the fraction of LTLTME that was converted to tyrosine (by hydrolysis of the peptide bond) was 0.1 times that a t 2 mM. There was no evidence of hydrolysis of the methyl ester a t any concentration. These results suggest that the rat nasal peptidases become saturated a t -8 mM LTLTME. Therefore, to minimize hydrolysis during the in vivo-in situ experiments, it was decided to study both compounds a t concentrations at which the nasal peptidases were saturated. For LTLT, the concentration was limited to 13.4mM by its solubility; however, this concentration exceeded the concentration at which the peptidase enzymes were saturated. Figure 3 shows the comparative rates of overall disappearance of LTLTME and LTLT from the rat nasal cavity using the in vivo-in situ technique. Since the absorption rate constant for passive diffusion is concentration independent, whereas rat nasal peptidases are inhibited at the high concentrations employed (see Figure 21, it can be safely
lo
I
L-tyrosyl-L-tyrosine (LTLT)
-.
. 0
I
20
40
60
so
Time (min) L-tyrosyl-L-tyrosine Methyl Ester (LTLTME)
1180 I Journal of Pharmaceutical Sciences 80, No. 12, December 1991
Figure 1-Hydrolysis of LTLTME(0)and LTLT (0)in rat nasal washhgs in vitro. Data represent mean concentrations SDs (n = 4), and the initial concentration was 2 rnM.
*
0022-3549/91/1200-1 180$02.50/0 0 1991, American Pharmaceutical Association
. 1 /
0
.
, 1
.
-
,
2
.
,
.
3
, 4
.
, 5
,
I
6
Time (min)
Flgure 2-Concentration dependence of the overall disappearance of LTLTME from the rat nasal cavity usingthe in vivo-in situ technique. Data represent mean concentration 2 SD (n = 4), and the initial concentrations were 40, 8, and 2 mM. The percents of tyrosine recovered after 2 rnin were 2.4, 8.7, and 24.0% for 40, 8, and 2 mM, respectively. assumed that virtually all the disappearance from this system was due t o passive absorption. These results show t h a t the rates o f passive absorption of these two similar peptides are identical, despite the fact that there i s a 160-fold difference in their partition coefficients. These preliminary results suggest that enhancing the lipophilicity of a peptidase-labile peptide would probably not have any great effect o n i t s bioavailability from the nasal cavity. Thus, costly product development efforts u t i l i z i n g this approach may have l i t t l e chance o f success.
References and Notes 1. Hussain, A.; Faraj, J.; Aramaki, Y.; Truelove, J. E. Biochm. Biophys. Res. Comm. 1985,133,923-928. 2. Hirai, S.;Yashiki, T Mima, H. Znt. J.Pharm. 1981,9,165-172. 3. Hirai, S.;Yashiki, T; Mima, H. Znt.J.Pharm. 1981,9,173-184. 4. Hussain, M.A.;Shenvi, A. B.; Rowe, S. M.; SheRer, E. Pharm. Res., 1989,6,186-189. 5. Fara', J. A.; Hussain, A. A.; Aramaki, Y.; Iseki, K.; Kagoshima, M.; dittert, L. W. J.Pharm. Sci.,1990,79,69%702. 6. Hussain, A. A,; Iseki, K.; Kagoshima, M.; Dittert, L. W. J. Pharm. Sci., 1990,79,947-948. 7. Anik, S.;McRae, G.; Nerenberg, C.; Worden, A.; Foreman, J.;Yu,
T i m e (rnin)
Flgure &Overall disappearanceof LTLTME (0)and LTLT (0)from the rat nasal cavity using the in vivo-in situ technique. Data represent mean concentrations SDs (n = 4), and the initial concentration of LTLTME was 40 mM and that of LTLT was 13.4 mM.
*
H. J.; Kushinsky, S.;Jones, R.; Vickery, V. J.Pharm. Sci.1984, 73,684-685. 8. Harris, A. S.; Ohlin, M.; Lethagen, S.; Nilsson, I. M. J . Pharm.
Sci..1988.77.337-339. 9. Hirai, S.; Ikenaga, T.; Matsuzawa, T.Diabetes, 1978,27,296-299. 10. Moses, A.C.; Flier, J. S.;Gordon, G. S.; Silver, R. D.; Carey, M. C. Clin. Res., 1984,32,254A-260A. 11. Ennis, R. D.;Borden, L.; Lee, W. A. Pharm. Res., 1990, 7, 468475. 12. Huang, C-H. Ph.D. Thesis, University of Kentucky, 1983. ANWARHUSSAIN SALIM HAMADI MASATOYO KAGASHIMA KEN ISEKI LEWISDIITERT' College of Pharmacy University of Kentucky Lexington, KY 40536 Received July 2, 1990. Accepted for publication February 25.1991.
Journal of Pharmaceutical Sciences I 1181 80, No. 72, December 1997
Does Increasing the Lipophilicity of Peptides Enhance Their Nasal Absorption? To the Editor: Previous communications from our laboratory and others concluded that the low bioavailabilities of nasally administered peptides are due, in art, to peptidase-catalyzed hydrolysis in the nasal cavity."We and others have demonstrated the existence of peptidase enzyme systems in the nasal cavities of both animals and humans,14 and we have shown that the types of hydrolyses produced by these systems may vary greatly among species6 On the other hand, it has been demonstrated that the small amounts of peptides that cross the nasal membrane in rats do so at a very rapid rate.'s8 Thus, the systemic bioavailability of a nasally administered peptide is the result of two rapid and competing kinetic processes: absorption and hydrolysis.' It should be possible to minimize hydrolysis and improve bioavailability by accelerating the absorption process so that a greater fraction of the peptide follows the absorption pathway. Some workers have attempted to accomplish this through the use of absorption e n h a n c e r ~ , ~but * l ~many of these compounds are irritating to the nasal mucosa.ll Another approach is to increase the lipophilicity of the peptide so as to improve its penetration into the nasal membrane. This communication reports on the comparative partition coefficients, in vitro hydrolysis rates, and the rates of disappearance from rat nasal cavity of the small peptide L-tyrosylL-tyrosine (LTLT) and its methyl ester (LTLTME). The analytical method was similar to those described previously,'r5v6 and the rates of hydrolysis of the compounds were determined in washings from the rat nasal cavity using a technique described previously.' Rates of disappearance of the compounds from the rat nasal cavity were determined using the in vivo-in situ technique described previously.' The oil-water partition coefficientsin the n-octanol: pH 7.4 phosphate buffer system were 0.02 for LTLT and 3.2 for LTLTME. The rates of the hydrolyses of LTLT and LTLTME
in rat nasal washings were very similar (see Figure 11, suggesting that the peptidase enzyme systems of the rat nasal cavity have about the same affinity for the two compounds. This also suggests that hydrolysis could be responsible for significant loss of the compounds from the rat nasal cavity during the in vivo-in situ experiments. It has previously been shown that the rates of hydrolysis of peptides, in particular LTLT, in rat nasal enzyme systems are concentration d e ~ e n d e n t . ~Therefore, .~' we studied the disappearance of LTLTME from the rat nasal cavity at various concentrations to determine the most advantageous concentration(s) a t which to compare the absorption rates of the two compounds. The results shown in Figure 2 indicate that the disappearance of LTLTME from the rat nasal cavity was slowest at 40 mM and was almost as slow at 8 mM. At 40 mM, the fraction of LTLTME that was converted to tyrosine (by hydrolysis of the peptide bond) was 0.1 times that a t 2 mM. There was no evidence of hydrolysis of the methyl ester a t any concentration. These results suggest that the rat nasal peptidases become saturated a t -8 mM LTLTME. Therefore, to minimize hydrolysis during the in vivo-in situ experiments, it was decided to study both compounds a t concentrations at which the nasal peptidases were saturated. For LTLT, the concentration was limited to 13.4mM by its solubility; however, this concentration exceeded the concentration at which the peptidase enzymes were saturated. Figure 3 shows the comparative rates of overall disappearance of LTLTME and LTLT from the rat nasal cavity using the in vivo-in situ technique. Since the absorption rate constant for passive diffusion is concentration independent, whereas rat nasal peptidases are inhibited at the high concentrations employed (see Figure 21, it can be safely
lo
I
L-tyrosyl-L-tyrosine (LTLT)
-.
. 0
I
20
40
60
so
Time (min) L-tyrosyl-L-tyrosine Methyl Ester (LTLTME)
1180 I Journal of Pharmaceutical Sciences 80, No. 12, December 1991
Figure 1-Hydrolysis of LTLTME(0)and LTLT (0)in rat nasal washhgs in vitro. Data represent mean concentrations SDs (n = 4), and the initial concentration was 2 rnM.
*
0022-3549/91/1200-1 180$02.50/0 0 1991, American Pharmaceutical Association
. 1 /
0
.
, 1
.
-
,
2
.
,
.
3
, 4
.
, 5
,
I
6
Time (min)
Flgure 2-Concentration dependence of the overall disappearance of LTLTME from the rat nasal cavity usingthe in vivo-in situ technique. Data represent mean concentration 2 SD (n = 4), and the initial concentrations were 40, 8, and 2 mM. The percents of tyrosine recovered after 2 rnin were 2.4, 8.7, and 24.0% for 40, 8, and 2 mM, respectively. assumed that virtually all the disappearance from this system was due t o passive absorption. These results show t h a t the rates o f passive absorption of these two similar peptides are identical, despite the fact that there i s a 160-fold difference in their partition coefficients. These preliminary results suggest that enhancing the lipophilicity of a peptidase-labile peptide would probably not have any great effect o n i t s bioavailability from the nasal cavity. Thus, costly product development efforts u t i l i z i n g this approach may have l i t t l e chance o f success.
References and Notes 1. Hussain, A.; Faraj, J.; Aramaki, Y.; Truelove, J. E. Biochm. Biophys. Res. Comm. 1985,133,923-928. 2. Hirai, S.;Yashiki, T Mima, H. Znt. J.Pharm. 1981,9,165-172. 3. Hirai, S.;Yashiki, T; Mima, H. Znt.J.Pharm. 1981,9,173-184. 4. Hussain, M.A.;Shenvi, A. B.; Rowe, S. M.; SheRer, E. Pharm. Res., 1989,6,186-189. 5. Fara', J. A.; Hussain, A. A.; Aramaki, Y.; Iseki, K.; Kagoshima, M.; dittert, L. W. J.Pharm. Sci.,1990,79,69%702. 6. Hussain, A. A,; Iseki, K.; Kagoshima, M.; Dittert, L. W. J. Pharm. Sci., 1990,79,947-948. 7. Anik, S.;McRae, G.; Nerenberg, C.; Worden, A.; Foreman, J.;Yu,
T i m e (rnin)
Flgure &Overall disappearanceof LTLTME (0)and LTLT (0)from the rat nasal cavity using the in vivo-in situ technique. Data represent mean concentrations SDs (n = 4), and the initial concentration of LTLTME was 40 mM and that of LTLT was 13.4 mM.
*
H. J.; Kushinsky, S.;Jones, R.; Vickery, V. J.Pharm. Sci.1984, 73,684-685. 8. Harris, A. S.; Ohlin, M.; Lethagen, S.; Nilsson, I. M. J . Pharm.
Sci..1988.77.337-339. 9. Hirai, S.; Ikenaga, T.; Matsuzawa, T.Diabetes, 1978,27,296-299. 10. Moses, A.C.; Flier, J. S.;Gordon, G. S.; Silver, R. D.; Carey, M. C. Clin. Res., 1984,32,254A-260A. 11. Ennis, R. D.;Borden, L.; Lee, W. A. Pharm. Res., 1990, 7, 468475. 12. Huang, C-H. Ph.D. Thesis, University of Kentucky, 1983. ANWARHUSSAIN SALIM HAMADI MASATOYO KAGASHIMA KEN ISEKI LEWISDIITERT' College of Pharmacy University of Kentucky Lexington, KY 40536 Received July 2, 1990. Accepted for publication February 25.1991.
Journal of Pharmaceutical Sciences I 1181 80, No. 72, December 1997