Approaches to overcome barriers to nasal absorption

In general, the nasal route is suitable for delivery of molecules with a molecular weight of less than about 1000. For these molecules, the nasal bioavailability may be sufficient to achieve therapeutic levels without the use of any adjuvants. For small peptides, the intranasal bioavailability may approach that of an intravenous injection. Serum levels of metkephamid, a pentapep-tide, following intranasal administration in the rat have been reported to be similar to those resulting after intravenous injection.99 Similarly, desmo-pressin, a nonapeptide, is adequately absorbed in humans.100 Although most peptides will be absorbed by passive diffusion, an active transport system may exist for amino acids and di- or tripeptides. The absorption of L-tryp-tophan across the rat nasal mucosa has been shown to involve a saturable, active transport mechanism.101 For molecules with a molecular weight above about 1000, the use of adjuvants is required.102 Because the major barriers to nasal absorption are the penetration and enzymatic barriers, these adjuvants will often be penetration enhancers or protease inhibitors, respectively. In addition, the dosage form design may be important. These approaches are discussed in the following sections. Penetration enhancers The use of absorption enhancers to facilitate the nasal delivery of peptides and proteins has been widely investigated. Commonly used enhancers include bile salts, surfactants, fusidates, phospholipids, and cyclodextrins. Lysophosphatidylcholine, a lysophospholipid, has been reported to enhance the nasal absorption of biosynthetic hGH in the rat to achieve a relative bioavailability of 25.8%. In this study, a mucolytic agent, N-acetyl-L-cysteine, and a transmembrane fatty acid transporter, palmitoyl-DL-carnitine, were also found to promote the nasal absorption of hGH, with relative bioavail-abilities of 12.2 and 22.1%, respectively.103 A mucoadhesive and cationic polysaccharide, chitosan, has been reported to enhance the absorption of insulin across the nasal mucosa of rat and sheep. Although mucoadhesion may be partly responsible, it is also possible that the cationic nature of chitosan has a transient effect on the gating function of tight junctions.104 Alkylglycosides, a family of nonionic detergents, have also been shown to effectively enhance the nasal absorption of insulin in rats.105 The use of fusidates, bile salts, and cyclodextrins as enhancers is discussed after a brief discussion of the mechanisms by which these enhancers exert their effects.

Penetration enhancers may exert their effect by opening the tight junctions between epithelial cells, by altering the properties of the mucous membrane or mucus layer, or by a combination of different mechanisms.106 In a study by Donovan et al.107 using a series of PEGs, it was found that the greatest absorption enhancement resulted when changes in the cell-to-cell adhesion in the mucosa were observed. This suggests that the paracellular route may play an important role in intranasal absorption. Enhancers or other adjuvants may affect membrane permeability as well as ciliary activity. Sodium cholate, a surface-active bile salt, and aprotinin, a protease inhibitor, have been reported to be highly ciliotoxic in the rat.108

The effect of adjuvants on nasal mucociliary clearance can be evaluated by studying their effect on ciliary beat frequency (CBF) in in vitro systems using chicken embryo tracheal tissue and human adenoid tissue. However, the inhibitory effects of enhancers on CBF may be higher under these in vitro studies compared to actual in vivo situations. This is because the in vitro ciliated tissue is directly exposed to the adjuvants tested, and the in vivo cilia are protected by the mucus layer. Also, when a formulation is administered in vivo, it is diluted by mucus and slowly eliminated by mucociliary clear-ance.106 Extended clinical experience to predict any long-term toxicity to the nasal mucosa following chronic use of formulations containing penetration enhancers is generally lacking.90 Sodium tauro-24,25-dihydrofusidate. A fusidic acid derivative, STDHF, has been widely used in preclinical and clinical studies to enhance the nasal absorption of peptides and proteins. It was found to enhance the bioavailability of salmon calcitonin in human volunteers following intranasal administration.109 Intranasal administration of a powder formulation of insulin and STDHF in a sheep model resulted in systemic absorption of insulin, which resulted in a hypoglycemic effect. The bioavail-ability of the powder formulation increased as the mole ratio of STDHF to insulin increased.110

The intranasal administration of hGH to sheep, rabbits, and rats in the presence of STDHF has been compared. Although there were differences based on species and mode of delivery, all three species showed rapid absorption followed by rapid clearance. Formulations of hGH with STDHF increased the hGH bioavailability by 11-fold in rats and rabbits and by 21-fold in sheep.95 The comparison is for hGH formulations containing 0.5% STDHF with those without STDHF. Concentrations of STDHF at or slightly higher than the critical micelle concentration were required. Intranasal administration of hGH in this way could achieve pulsatile kinetics similar to that of endogenous secretion, unlike the kinetics achieved following subcutaneous administration. Similar results were seen in a human study using hGH-STDHF intranasal formulations. In the presence of STDHF, hGH was easily absorbed through the nasal mucosa, with plasma profiles similar to the normal endogenous peaks of GH. Compared to subcutaneous injections, the hGH peaks following intranasal delivery were of shorter duration, and Cmax values were lower. However, the uptake measured as AUC24h was only about 1.5 to 3% of that obtained with subcutaneous administration.111 Bile salts. Bile salts and bile salt-fatty acid mixed micelles have also been widely investigated as penetration enhancers.112 When insulin was administered as an aerosol with 1% deoxycholate to normal and diabetic subjects, it was found to traverse the nasal mucosa and rapidly appear in the circulation. Nasal insulin absorption achieved in this study was approximately 10% as efficient as intravenous insulin.113

Gordon et al.114 investigated the absorption of insulin by the nasal mucosa of humans by using a series of bile salts. Therapeutically useful amounts of insulin could be absorbed, and the absorption positively correlated with increasing hydrophobicity of the bile salts. Absorption began at the critical micellar concentration of bile salts. It seems that solubilization of insulin in mixed bile salt micelles presents a high insulin concentration for absorption. Also, the bile salts may form reverse micelles, so that the insulin is solubilized in the hydrophilic interior, and the hydrophobic surfaces face outward. This will facilitate the transport of reverse micelles through the lipid environment.

Bile salts and bile salt-fatty acid mixed micelles have also been investigated to enhance the intranasal transport of a dipeptide115 and recombinant human a-interferon.116 Sodium glycocholate (1%) has been reported to enhance by a factor of 2.7 (p < .05) the absorption of angiopeptin across rabbit nasal tissue mounted in the Ussing chamber. Angiopeptin, a synthetic octapeptide, has potential use in the inhibition of restenosis of coronary arteries after angioplasty or heart transplantation.117

A nasal formulation containing a nona- or decapeptide with LHRH agonist or antagonist activity has been described in the patent literature. The peptide was dissolved in 0.02 M acetate buffer (pH 5.2) along with a bile salt surfactant such as sodium glycocholate. The surfactant acted as a penetration enhancer and increased the solubility of the LHRH analogue. Studies in monkeys and beagle dogs suggested that the surfactant was able to enhance absorption of the peptide without inducing toxicity in the nasal mucosa.118 Cyclodextrins. Cyclodextrins have also been evaluated as absorption enhancers for nasal delivery of peptides and proteins.119 The most promising cyclodextrin in this regard appears to be dimethyl p-cyclodextrin (DMpCD). This and other cyclodextrins have been reported to enhance the absorption of recombinant human granulocyte colony-stimulating factor following intranasal administration in rabbits.120121 Similarly, DMpCD enhanced the absorption of corticotropin (ACTH) and insulin through the nasal mucosa of rats.97

Several other studies have also investigated the potential of DMpCD to enhance the nasal absorption of insulin.96 122 124 It was found that the coadministration of 5% w/v DMpCD with an insulin solution resulted in high bioavailability (108.9 +/- 36.4%) from the nasal mucosa of rats compared to intravenous administration. The other cyclodextrins evaluated in this study did not have a significant effect on nasal insulin absorption.122 The mechanism for the effectiveness of DMpCD as a nasal enhancer is not clear. Studies done by my group have shown that cyclodextrin can prevent or reduce the formation of insoluble aggregates in insulin solution.125

Shao et al.123 reported that cyclodextrins are capable of dissociating insulin hexamers into smaller aggregates, and this dissociation may provide an additional mechanism for enhancement of insulin diffusivity across nasal mucosa. However, the primary mechanism may be solubilization of nasal membrane components. Thus, cyclodextrins may have damaging effects on the nasal mucosa, and this needs to be evaluated. However, DMpCD has been reported to have only a mild and reversible effect on the nasal muco-ciliary clearance, as measured by its effects on CBF of chicken embryo tracheal and human adenoid tissue.97 It has also been suggested that cyclodex-trins may actually protect against enhancer damage in nasal delivery systems.126,127 Protease inhibitors. As discussed in Section 9.2, nasal delivery avoids the hepatic first-pass effects. However, the enzymatic activity in the nasal mucosa creates a pseudo-first-pass effect for the intranasal delivery of drugs. These enzymes include cytochrome P-450, aldehyde dehydroge-nases, epoxide hydroxylase, carboxylesterase, carbonic anhydrase, glu-tathione transferase, and various exo- and endopeptidases. For the intranasal delivery of peptides and proteins, the principal enzymatic barrier appears to be the aminopeptidase activity of the nasal mucosa.128 Thus, protease inhibitors, especially aminopeptidase inhibitors, have been used to prevent the enzymatic breakdown of peptides and proteins on intranasal administration. Many absorption enhancers, such as bile salts, may also have inhibitory effects on aminopeptidases, and this may partly explain their mechanism of action. For example, sodium glycocholate can reduce the enzymatic degradation of TRH in a homogenate of rabbit nasal mucosa.129 Because bile salts and some other enhancers may damage the mucous membrane, their chronic use in humans may not be feasible.

For these reasons, enzymatic inhibitors offer a promising alternate approach to enhance the intranasal delivery of peptides and proteins. Some of the most promising aminopeptidase inhibitors are puromycin, p-chlo-romercuribenzoate, and the even-more-potent bestatin and amastatin. The class of a-aminoboronic acid derivatives includes boroleucine, borovaline, and boroalanine, which are also potent and reversible inhibitors of ami-nopeptidases.

These a-aminoboronic acid derivatives have been shown to inhibit the degradation of leucine-enkephalin (Leu-Enk) in the nasal perfusate of rats. In this study, boroleucine was found 100 times more effective in enzyme inhibition than bestatin and 1000 times more effective than puromycin.130 Boroleucine has also been reported to completely inhibit the metabolism of LHRH when perfused through the isolated rat nasal cavity.131

Other studies have also investigated the hydrolysis of Leu-Enk in nasal mucosa and its stabilization by the use of protease inhibitors.132 133 Other protease inhibitors such as the trypsin inhibitors, aprotinin, and soybean trypsin inhibitor have also been investigated but failed to enhance the nasal absorption of vasopressin and desmopressin in rats. However, camostat mesilate, an aminopeptidase and trypsin inhibitor, was successfully used in this study. Coadministration of the peptides with camostat mesilate significantly increased the antidiuretic effect and thus the nasal absorption of vasopressin and desmopressin in rats.134

Protease inhibitors can be successfully used in intranasal peptide formulations only if their effects are reversible and no long-term toxicity results from their usage. Hussain et al.135 investigated the recovery of peptide hydro-lytic activity after exposure of the rat nasal cavity to various aminopeptidase inhibitors. Boroleucine (0.1 ^m) was a potent inhibitor of Leu-Enk metabolism, but when it was washed out, the Leu-Enk metabolism rate returned to control levels. Thus, aminoboronic acid derivatives can enhance nasal delivery reversibly and at very low concentrations. In contrast, other aminopep-tidase inhibitors needed much higher concentrations (0.1 mM for bestatin and 1 mM for puromycin), and their effects were not reversible. A rebound of aminopeptidase activity higher than control levels was seen when these inhibitors were washed out. The mechanism for such effect is not known but may involve membrane disruption. Another approach to reduce pro-teolytic degradation can be protein pegylation (see Chapter 6 ). Pegylated salmon calcitonin was reported to show strong resistance to enzymatic degradation in rat nasal mucosa.136 Formulation or delivery devices. The choice of formulation or delivery device can also have an effect on the extent and efficiency of drug absorption from the nasal cavity. Powder dosage forms may be more effective than liquid dosage forms for intranasal delivery. When insulin was administered to the nasal cavity of beagle dogs, absorption from a powder form was better than that from the liquid form. Sustained absorption can be achieved by adding Carbopol 934 as an excipient to the powder. Because Carbopol 934 can form a gel in an aqueous environment, it allows insulin to stay on the nasal mucosa for a prolonged period.137 Carbopol gel has also been directly used to enhance the absorption of insulin and calcitonin from the nasal cavity of rats.138 Powder formulations of insulin containing DMCD were found to be effective following nasal administration in rabbits, but liquid formulations under the same conditions were not effective.9697

Another formulation approach to enhance the intranasal delivery of peptides and proteins is the use of microspheres. It has been shown that uptake and translocation of solid particles can take place through the nasal epithelial lining through the nasal-associated lymphatic tissue.139 Hyaluronic acid ester microspheres have been reported to produce a large and significant increase in the nasal absorption of insulin in sheep, similar to that obtained with bioadhesive starch microspheres.140 In concept, another way to improve nasal absorption of peptides is to increase their lipophilicity. However, this may or may not be true in actual practice. In a study that compared the rates of disappearance of a small peptide and its methyl ester in rat nasal cavity, the lipophilicity did not affect the bioavailability of the peptide.141

The mode of delivery can also have an influence on drug absorption from the nasal cavity. The drug can be administered as nasal drops or as a nasal spray. Drops have been shown to spread more extensively than spray, with three drops sufficient to cover most of the nasal mucosa in human subjects.142 However, the use of an intranasal spray device can deposit well-controlled doses within the nasal cavity. Studies with the nasal administration of desmopressin in humans have shown that the plasma levels of the peptide achieved with the spray were two- to threefold higher than those achieved with drops.100 Two types of spray devices are commonly used: a metered-dose nebulizer (MDN) and a metered-dose aerosol (MDA). The MDN device operates by mechanical actuation and releases a fixed dose each time the actuator is pressed. The MDA device uses propellants and operates by pressurized actuation. The use of propellants may or may not be a disadvantage for peptide stability, and selection of some suitable device needs to be done on a case-by-case basis.89

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