284
reviews 46 Zabriskie, D. W., Warehdm, D. A. and Polansky, M. J. (1987) J. Ind. Microbiol.2, 85-87 47 Tsai, B., Mann, M., Morris, F., Rotgers, C. and Fenton, D. (1987)J. Ind. Microbiol.2, 181-187 48 Horn:. U., Krug, M. and Sawistowski, J. (1990) Biotechnol. Leu. 12, 191-196 49 Aiba, S. and Koizumi, J. (1984) Biotedmol. Bioeng. 26, 1026-1031
50 Son, K. H., Jang, J. H. and Kim, J. H. (1987) Biotechnol. Leu. 9, 821-824 51 Hopkins, D. J., Betenbangh, M. J. and Dhurjati, P. (1987) Biotechnol. Bioeng. 29, 85-91 52 Ryn, W., Paulekar, S. J. and Stark, B. C. (1989) BiotechnoL Bioeng, 34, 309-319 53 Caunt, P., lmpoolsup, A. and Greenfidd, P. F. (1989) Biotechnol. Left. 11, 5-10
The nasal delivery of peptides and proteins Lisbeth lUum Many drugs of the future will be therapeutically active peptides and proteins developed through recombinant-DNA technology. A major factor limiting their exploitation is the current lack of appropriate non-parenteral delivery systems. Nasal systems incorporating absorption enhancers may provide a convenient, efficient means of administering protein and peptide therapeutics. Most peptide drugs are poorly absorbed when administered orally due to degradation in the gastrointestinal tract or inefficient transport across the epithelial barrier: the majority are therefore administered parenteraUy. However, the inconvenience of this route of administration and the need to design replacement therapies able to mimic the often pulsatile endogenous secretion pattern of peptides, have led, in recent years, to the extensive investigation of the nasal route for delivery of these drugs. An important advantage of nasal delivery is the ease of administration and the large surface area available for absorption. The subepithelial layer is highly vascularized and the venous blood from the nose passes directly into the systemic circulation, avoiding the first pass effect of the liver (i.e. metabolism of drugs by the liver). However, in contrast to most generic drugs, peptides and proteins generally show low bioavailabilities (in the order of 1-2%) 1 when administered nasally. A common feature of commercially available, nasally administered peptide drugs, such as luteinizing hormone releasing hormone (LHRH), oxytocin, vasopressin and calcitonin, is their relatively low molecular weights and high potencies: only small quantities are needed systemically to obtain clinically relevant plasma levels. For most other peptide and
protein drugs, some form of delivery system and/ or absorption enhancement system is necessary in order to obtain a sufficient bioavailability. Recent reviews and books, by O'Hagan and lllum 1, Fisher2 and Chien et al. 3, cover the nasal administration of drugs, including therapeutic peptides and proteins. Barriers to nasal absorption The normal low systemic absorption of peptides from the nasal cavity can be attributed to a range of factors. These include (1) the difficulty of transporting large hydrophilic molecules across the epithelial membrane, (2) the possibility of degradation by extraceUular and intracellular enzymes present in the nasal cavity and nasal membranes, and (3) the rapid clearance of the drug away from the site of absorption by the mucociliary clearance system in the nasal cavity. The importance of these factors to nasal absorption is discussed below.
Deposition and clearanceof deliverFsystems The physiology of the nasal cavity has been described expertly by Mygind 4. The three major regions of the nasal cavity are the nasal vestibule, the olfactory region and the respiratory region, the latter comprising the largest surface area in the cavity (Fig. 1). The respiratory region is lined by a L. Ilium is at the Department of Pharmaceutical Sciences, pseudostratified columnar epithelium that is Nottingham University, University Park, Nottingham NG7 covered with microvilli and cilia in the main part 2RD, UK; and DanBioSyst UK Ltd, 6 William Lee of the region, whereas in the anterior part this Buildings, Highfields Science Park, Nottingham N G 7 2RQ, epithelium is naked (squamous and transitional UK. epithelium). The respiratory epithelium is TIBTECHAUGUST1991(VOL9)
~) 1991, ElsevierSciencePublishersLtd (UK) 0167-9430/91/$2.00
285
reviews covered by a two-component mucus layer: a bottom layer consisting of a low viscosity sol surrounding the cilia and the microvilli, and, on the surface, a more viscous and bioadhesive gel layer. The cilia beat continuously in the sol layer with a frequency of 1000 strokes per minute thereby moving the surface gel layer towards the pharynx. The function of this mucociliary dearance mechanism is to remove any inhaled dust or foreign partides. In theory, inhaled particles or drops should be cleared from the nasal cavity within about 15-20 min by this mucociliary clearance mechanism4. This relatively short time which is available for the absorption process to take place, is an important limitation which needs to be considered in the absorption of peptides and proteins from a nasal delivery system, particularly those that can be characterized as difficult molecules (i.e. molecules with non-optimal absorption characteristics). It is still a matter of discussion as to which regions of the nasal cavity are optimal for the absorption of drugs, but in general, the respiratory and the olfactory epithelia are considered to play an important role in drug transport. Direct pathways from the nasal olfactory epithelium to the central nervous system including the cerebrospinal fluid have been found. These may be of importance for providing enhanced delivery to the brain, through areas unprotected by the blood-brain barrier, of poorly_ absorbed solutes such as peptides and proteins ~',. Nasal clearance will very much depend on the particle size of the formulation and the site of deposition. Droplets deposited in the sparsely ciliated or non-ciliated anterior region ofthe nasal cavity are cleared at a slower rate due to a very slow drag from the contiguous mucus. Nasal sprays are mainly deposited in the atrium of the nose while nasal drops spread more extensively and are therefore cleared more rapidly6'7. A very close relationship has also been shown to exist between the rate of clearance (and therefore the deposition of the drug), and the degree of absorption sq°. In these studies, a vasopressin analogue, desmopressin, as a nasal spray, was found to deposit in the anterior part of the nose, to be cleared slowly and to be absorbed to a greater extent as compared with desmopressin administered as nasal drops using a rhinyle system (Ferring Pharmaceuticals), or a conventional pipette s-l°. Most drops were deposited in the posterior of the cavity and were cleared very rapidly. It was also possible to influence, to a certain extent, the clearance time from the nasal cavity using nasal spray solutions with higher viscosities. This was probably due to the formation of larger particles which would be more likely to deposit anteriorly in the nasal cavity. Ilium et al. 11 showed that microspheres with a diameter of about 50 pm made from bioadhesive
- ~
w - -
__
Figure ! Schematic representation of the lateral wall of the nasal cavity. A, nasal vestibule; B, internal ostium; C, inferior concha; D, middle concha; E, superior concha; hatched area, olfactory region. Reproduced, with permission, from Ref. 5.
gelling materials such as starch, albumin and DEAE-sephadex and administered as freeze-dried powders were deposited in the anterior part of the nose of human volunteers and cleared very slowly from the nasal cavity. The time for 50% clearance was up to three hours as compared to 15 min for solutions and normal pharmaceutical powders. As shall be discussed later, this principle for a delivery system has been exploited successfully to deliver systemically active peptides of different molecular weights via the nasal route.
Transport through the epithelial membrane The epithelial membrane constitutes a highly efficient barrier to drug absorption. In order to pass the membrane, the drug has to pass through the membrane cells (transcellular transport) by means of passive or concentration-gradient diffusion, by active transport or by transport in vesicles t2. Alternatively, the drug has to pass between the cells (i.e. through the tight junctions). The transport of molecules across the membrane is dependent upon several factors such as pH, molecular weight and lipophilic/hydrophilic balance. In several studies, it has been shown that the absorption of small molecules across the nasal membrane is dependent on the pH, the greatest absorption taking place at the pH where the molecule is non-ionized 13'14. However, even at pH values where there was partial ionization of the molecules, absorption was still significant. These studies suggested that transport occurred through the transcellular route for more lipophilic TIBTECHAUGUST1991 (VOL9)
286
reviews Table 1. Chemical modifications of peptides which may reduce degradation in vivo Olefinsubstitution D-aminoacid substitution Carbonylreduction Dehydroaminoacid substitution Retm-inversomodification S-S bridgesubstitution N-terminalto C-terminalcyclization Thiomethylenemodification N-e~-methylsubstitution C-o
reviews 46 Zabriskie, D. W., Warehdm, D. A. and Polansky, M. J. (1987) J. Ind. Microbiol.2, 85-87 47 Tsai, B., Mann, M., Morris, F., Rotgers, C. and Fenton, D. (1987)J. Ind. Microbiol.2, 181-187 48 Horn:. U., Krug, M. and Sawistowski, J. (1990) Biotechnol. Leu. 12, 191-196 49 Aiba, S. and Koizumi, J. (1984) Biotedmol. Bioeng. 26, 1026-1031
50 Son, K. H., Jang, J. H. and Kim, J. H. (1987) Biotechnol. Leu. 9, 821-824 51 Hopkins, D. J., Betenbangh, M. J. and Dhurjati, P. (1987) Biotechnol. Bioeng. 29, 85-91 52 Ryn, W., Paulekar, S. J. and Stark, B. C. (1989) BiotechnoL Bioeng, 34, 309-319 53 Caunt, P., lmpoolsup, A. and Greenfidd, P. F. (1989) Biotechnol. Left. 11, 5-10
The nasal delivery of peptides and proteins Lisbeth lUum Many drugs of the future will be therapeutically active peptides and proteins developed through recombinant-DNA technology. A major factor limiting their exploitation is the current lack of appropriate non-parenteral delivery systems. Nasal systems incorporating absorption enhancers may provide a convenient, efficient means of administering protein and peptide therapeutics. Most peptide drugs are poorly absorbed when administered orally due to degradation in the gastrointestinal tract or inefficient transport across the epithelial barrier: the majority are therefore administered parenteraUy. However, the inconvenience of this route of administration and the need to design replacement therapies able to mimic the often pulsatile endogenous secretion pattern of peptides, have led, in recent years, to the extensive investigation of the nasal route for delivery of these drugs. An important advantage of nasal delivery is the ease of administration and the large surface area available for absorption. The subepithelial layer is highly vascularized and the venous blood from the nose passes directly into the systemic circulation, avoiding the first pass effect of the liver (i.e. metabolism of drugs by the liver). However, in contrast to most generic drugs, peptides and proteins generally show low bioavailabilities (in the order of 1-2%) 1 when administered nasally. A common feature of commercially available, nasally administered peptide drugs, such as luteinizing hormone releasing hormone (LHRH), oxytocin, vasopressin and calcitonin, is their relatively low molecular weights and high potencies: only small quantities are needed systemically to obtain clinically relevant plasma levels. For most other peptide and
protein drugs, some form of delivery system and/ or absorption enhancement system is necessary in order to obtain a sufficient bioavailability. Recent reviews and books, by O'Hagan and lllum 1, Fisher2 and Chien et al. 3, cover the nasal administration of drugs, including therapeutic peptides and proteins. Barriers to nasal absorption The normal low systemic absorption of peptides from the nasal cavity can be attributed to a range of factors. These include (1) the difficulty of transporting large hydrophilic molecules across the epithelial membrane, (2) the possibility of degradation by extraceUular and intracellular enzymes present in the nasal cavity and nasal membranes, and (3) the rapid clearance of the drug away from the site of absorption by the mucociliary clearance system in the nasal cavity. The importance of these factors to nasal absorption is discussed below.
Deposition and clearanceof deliverFsystems The physiology of the nasal cavity has been described expertly by Mygind 4. The three major regions of the nasal cavity are the nasal vestibule, the olfactory region and the respiratory region, the latter comprising the largest surface area in the cavity (Fig. 1). The respiratory region is lined by a L. Ilium is at the Department of Pharmaceutical Sciences, pseudostratified columnar epithelium that is Nottingham University, University Park, Nottingham NG7 covered with microvilli and cilia in the main part 2RD, UK; and DanBioSyst UK Ltd, 6 William Lee of the region, whereas in the anterior part this Buildings, Highfields Science Park, Nottingham N G 7 2RQ, epithelium is naked (squamous and transitional UK. epithelium). The respiratory epithelium is TIBTECHAUGUST1991(VOL9)
~) 1991, ElsevierSciencePublishersLtd (UK) 0167-9430/91/$2.00
285
reviews covered by a two-component mucus layer: a bottom layer consisting of a low viscosity sol surrounding the cilia and the microvilli, and, on the surface, a more viscous and bioadhesive gel layer. The cilia beat continuously in the sol layer with a frequency of 1000 strokes per minute thereby moving the surface gel layer towards the pharynx. The function of this mucociliary dearance mechanism is to remove any inhaled dust or foreign partides. In theory, inhaled particles or drops should be cleared from the nasal cavity within about 15-20 min by this mucociliary clearance mechanism4. This relatively short time which is available for the absorption process to take place, is an important limitation which needs to be considered in the absorption of peptides and proteins from a nasal delivery system, particularly those that can be characterized as difficult molecules (i.e. molecules with non-optimal absorption characteristics). It is still a matter of discussion as to which regions of the nasal cavity are optimal for the absorption of drugs, but in general, the respiratory and the olfactory epithelia are considered to play an important role in drug transport. Direct pathways from the nasal olfactory epithelium to the central nervous system including the cerebrospinal fluid have been found. These may be of importance for providing enhanced delivery to the brain, through areas unprotected by the blood-brain barrier, of poorly_ absorbed solutes such as peptides and proteins ~',. Nasal clearance will very much depend on the particle size of the formulation and the site of deposition. Droplets deposited in the sparsely ciliated or non-ciliated anterior region ofthe nasal cavity are cleared at a slower rate due to a very slow drag from the contiguous mucus. Nasal sprays are mainly deposited in the atrium of the nose while nasal drops spread more extensively and are therefore cleared more rapidly6'7. A very close relationship has also been shown to exist between the rate of clearance (and therefore the deposition of the drug), and the degree of absorption sq°. In these studies, a vasopressin analogue, desmopressin, as a nasal spray, was found to deposit in the anterior part of the nose, to be cleared slowly and to be absorbed to a greater extent as compared with desmopressin administered as nasal drops using a rhinyle system (Ferring Pharmaceuticals), or a conventional pipette s-l°. Most drops were deposited in the posterior of the cavity and were cleared very rapidly. It was also possible to influence, to a certain extent, the clearance time from the nasal cavity using nasal spray solutions with higher viscosities. This was probably due to the formation of larger particles which would be more likely to deposit anteriorly in the nasal cavity. Ilium et al. 11 showed that microspheres with a diameter of about 50 pm made from bioadhesive
- ~
w - -
__
Figure ! Schematic representation of the lateral wall of the nasal cavity. A, nasal vestibule; B, internal ostium; C, inferior concha; D, middle concha; E, superior concha; hatched area, olfactory region. Reproduced, with permission, from Ref. 5.
gelling materials such as starch, albumin and DEAE-sephadex and administered as freeze-dried powders were deposited in the anterior part of the nose of human volunteers and cleared very slowly from the nasal cavity. The time for 50% clearance was up to three hours as compared to 15 min for solutions and normal pharmaceutical powders. As shall be discussed later, this principle for a delivery system has been exploited successfully to deliver systemically active peptides of different molecular weights via the nasal route.
Transport through the epithelial membrane The epithelial membrane constitutes a highly efficient barrier to drug absorption. In order to pass the membrane, the drug has to pass through the membrane cells (transcellular transport) by means of passive or concentration-gradient diffusion, by active transport or by transport in vesicles t2. Alternatively, the drug has to pass between the cells (i.e. through the tight junctions). The transport of molecules across the membrane is dependent upon several factors such as pH, molecular weight and lipophilic/hydrophilic balance. In several studies, it has been shown that the absorption of small molecules across the nasal membrane is dependent on the pH, the greatest absorption taking place at the pH where the molecule is non-ionized 13'14. However, even at pH values where there was partial ionization of the molecules, absorption was still significant. These studies suggested that transport occurred through the transcellular route for more lipophilic TIBTECHAUGUST1991 (VOL9)
286
reviews Table 1. Chemical modifications of peptides which may reduce degradation in vivo Olefinsubstitution D-aminoacid substitution Carbonylreduction Dehydroaminoacid substitution Retm-inversomodification S-S bridgesubstitution N-terminalto C-terminalcyclization Thiomethylenemodification N-e~-methylsubstitution C-o
